Pollen and fungal spores are associated with seasonal and perennial allergies. However, most scientific literature thus far suggests that pollen allergy is more clinically relevant than fungal allergy. Several environmental and biological factors and the difficulty in producing reliable fungal extracts account for this. Biodiversity, taxonomy, and meteorology are responsible for the types and levels of pollen and fungal spores, their fragments, and the presence of free airborne allergens. Therefore, it is difficult to accurately measure both pollen and fungal allergen exposure. In addition, understanding the enzymatic nature of fungal and some pollen allergens, the presence of allergenic and nonallergenic substances that may modulate the allergic immune response, and allergen cross-reactivity are all necessary to appropriately evaluate both sensitivity and exposure. The raw materials and manufacturing processes used to prepare pollen versus fungal extracts differ, further increasing the complexity to properly determine allergic sensitivity and degrees of exposure. The pollen extracts used for diagnosis and treatment are relatively consistent, and some have been standardized. However, obtaining clinically relevant fungal extracts is more difficult. Doing so will allow for the proper selection of such extracts to more appropriately diagnose and treat both pollen- and fungal-induced allergic diseases.

antibodies, or a recombinant human high-affinity IgE epsilon receptor fragment as primary tracer molecules [4][5][6][7][8].Many of these assays incorporated HRP as the reporter enzyme while others used alkaline phosphatase conjugates.Comparative evaluations of these different tests demonstrated substantial variance of results for the various assays [9-14], but no efforts have been put forth to identify why such dramatic differences might exist.To address this issue more thoroughly, we opted to comparatively evaluate the responses of results evident in ELISA that incorporate either HRP or alkaline phosphatase (AP).We hypothesize that the differences in responses evident in the two assays resides in the binding differences of the reporter enzyme conjugates. Materials and Methods SeraThe serum samples used throughout were derived from dogs suspected of clinical allergy and had previously been submitted by veterinarians for evaluation using Stallergenes Greer macELISA for detection of allergen-specific IgE.The sole criterion for selecting

This chapter reviews the taxonomy, ecology, and habitat of allergenic grasses belonging to the family Poaceae and the molecular characteristics of the 13 grass pollen allergen groups recognized by the International Union of Immunological Societies Allergen Nomenclature Sub-Committee. The chapter describes how various techniques of molecular biology and protein chemistry are used to define allergenically important and cross-reactive IgE-binding epitopes, to develop specific assays for allergen detection and quantitation, to delineate determinants involved in B- and T-cell recognition, and to design novel approaches for allergen immunotherapy. The clinical relevance of the observed cross-reactivities among grass pollen allergens and those between homologous proteins derived from other pollen sources, fruits, and vegetables is discussed.

Pollen and fungal raw materials are utilized to produce allergen extracts for diagnosis and treatment of allergies in most areas of the world. Because these materials are natural products, they are subjected to large qualitative and quantitative variations of their allergenic characteristics, which are addressed during manufacturing. For example, because pollen is collected from the outdoors, environmental contaminants may impact this product before and during collection. The fungi utilized to produce raw materials are derived from particular strains grown in a laboratory. The culture medium and growth conditions are relevant parameters partially responsible for the allergenic properties of the fungal raw materials. While manufacturers of allergen extracts use different raw materials and manufacturing techniques, they follow analogous steps to produce allergen extracts. Another consideration is associated with the diverse regulations in place in different areas of the world. Harmonization is needed to produce consistent pollen and fungal allergen extracts. This chapter provides an overview of the steps involved in obtaining pollen and fungal raw materials and in manufacturing the associated allergen extracts. A brief comparison between the general regulations in place in the United States and Europe is also provided. This information will assist the clinician to properly select the best allergen extracts commercially available.

Allergen immunotherapy (AIT) is thought to be clinically effective and safe in treating allergic rhinitis, asthma, and stinging insect allergy in Europe and North America. However, there are intercontinental differences in AIT therapeutic products in terms of their application and regulation. In North America unmodified standardized and nonstandardized aqueous aeroallergen extracts are approved and used almost exclusively for subcutaneous immunotherapy, whereas more product options are available in Europe, including adsorbed allergens, chemically modified allergens, or both. Both liquid extracts and tablets are approved for sublingual immunotherapy in Europe. Nevertheless, within the European Union, there are major differences in AIT products approved and used in individual countries. There are major differences in the clinical approach to subcutaneous immunotherapy in polysensitized patients; in the United States mixed extracts containing multiple aeroallergens are used, whereas European allergists preferably administer separate injections of single allergen sources or homologous groups deemed to be clinically relevant. Moreover, the regulatory approach differs between the European Union and United States. In contrast to the United States, where common allergen standards exist based on biologic activity, no common standards exist in Europe. In terms of development of new investigational products, the United States has followed the European example for phase II and III studies; no formal US Food and Drug Administration guidance has been issued.

Progress has been made in the harmonization of efficacy and safety outcome measures for allergen immunotherapy (AIT) trials, but unresolved issues still remain. Furthermore, there are discrepancies in recommendations from professional medical societies and regulatory agencies regarding requirements for AIT trials. In this article, we reviewed published recommendations and current data from recent clinical trials, as well as the criteria applied by regulatory authorities for approval of AIT products, to provide updated considerations for conducting phase 3 AIT trials. Topics discussed include analysis of outcomes and trial designs for pediatric and asthma indications, as well as trial designs for perennial allergic rhinoconjunctivitis. In addition, the need for harmonization of safety reporting is emphasized. Considerations presented in this article may further effort to find common ground among professional medical societies and government agencies in developing future recommendations for AIT trial design.

Objective: To review the topic of fungal raw materials used for the production of allergen extracts and the associated challenges and highlight candidate areas for development before standardized fungal allergen extracts can be commercially produced.Data Sources: A PubMed search was performed using focused keywords and combined with a review of regulatory documents and industry guidelines. Several books on mycology also were consulted.Study Selections: The information obtained through the literature, books, and industry was scrutinized and combined with personal experience and expertise to write this article.Results: Fungi are complex ubiquitous organisms on Earth. They are beneficial and detrimental for humans. Fungi can cause hypersensitivity reactions, including types I, III, and IV. The procurement of fungal raw materials to prepare allergen extracts for diagnosis and possible allergen immunotherapy is complex owing to the intrinsic nature of fungi and their complex genome. Allergen manufacturers produce allergen extracts with variable qualitative and quantitative compositions, which can lead to unpredictable clinical outcomes.Conclusion: The clinician should be aware of the factors responsible for the qualitative and quantitative compositions of fungal allergen extracts and the reasons that currently preclude their standardization. Scientific advances and collaboration and cooperation between allergen manufacturing companies and regulatory agencies are necessary to improve the quality and consistency of fungal extracts. Moreover, clinicians should understand the limitations of currently available fungal extracts. (C) 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The biological purity of pollen collected to prepare allergen extracts (AE) is currently based on particle counts, measured by optical microscopy. Acceptable levels of biologic contaminants (plant parts, fungal spores, foreign pollen) are internally proposed by manufacturers. The study objectives are: 1) examine a number of pollen lots from different species to identify the commonest biological contaminants difficult to recognize during the completion of pollen purity assessments (PPA), and 2) propose recommendations on how to count and interpret their presence. 2,547 pollen lots comprising multiple tree, weed, and grass species, obtained for the production of AE, were analyzed over three years. Pollen samples were stained with Calberla's solution and examined by optical microscopy under 400X and 1,000X magnifications. The commonest structures that could interfere in the completion of PPA and the associated interpretation are: Pollen grains with uncommon morphologies and disruptions, large numbers of miscellaneous fungal spores, presence of aggregated small (<4mm) spores, unusual plant parts, presence of contaminants like parasitic mites and insects, and presence of active fungal growth. The presence of large number of plant parts and miscellaneous spores indicates the pollen needs further cleaning. Single aggregations of small spores should be counted as one particle. Mites/insects should be counted as individual particles. The suitability of disrupted pollen to prepare AE should be re-evaluated. If fungal growth is present, the pollen should be discarded. An atlas illustrating examples of the structures mentioned and others is necessary to properly train individuals to perform PPA. Volumetric counts should be considered.

OBJECTIVE:To provide physicians, researchers, and other interested health care professionals with information about how mite source materials and allergen extracts are manufactured, including the critical process parameters that can affect the final composition of allergenic extracts available for clinical use.DATA SOURCES:A PubMed search was performed using focused keywords combined with relevant regulatory documents and industry guidelines.STUDY SELECTIONS:The information obtained through literature and specialized books was evaluated and combined with the personal expertise and experience of the authors.RESULTS:Dermatophagoides farinae and Dermatophagoides pteronyssinus are the primary species responsible for allergen sensitizations and allergy symptoms in genetically predisposed individuals. Storage mites belonging to the families Glycyphagidae, Echimyopodidae, and Acaridae can also be relevant sources of indoor mite allergens. The cultivation and purification processes used to produce mite raw materials play a critical role in the final composition of mite allergen extracts. Mite extract standardization in the United States is based on total allergenic activity with respect to a single national standard, whereas in Europe consistency is ensured by in-house standards and international references. Because of the limitation of allergen avoidance and pharmacotherapy for patients with severe allergic rhinitis and asthma, house dust mite subcutaneous immunotherapy or sublingual immunotherapy can be an invaluable treatment option for them.CONCLUSION:Differences in manufacturing processes and extract standardization approaches may lead to differences in extract quality and potency. Physicians should be aware of these potential sources of mite extract variability. Use of well-standardized house dust mite extracts would be critical for success in the diagnosis and treatment of house dust mite allergy.

Background: An emerging paradigm holds that resistance to the development of allergic diseases, including allergic rhinoconjunctivitis, relates to an intact epithelial/epidermal barrier during early childhood. Conceivably, the immunologic and genomic footprint of this resistance is preserved in nonatopic, nonallergic adults and is unmasked during exposure to an aeroallergen.Objective: The aim of this study was to obtain direct support of the epithelial/epidermal barrier model for allergic rhinoconjunctivitis.Methods: Twenty-three adults allergic to house dust mites (HDMs) (M+) and 15 nonsensitive, nonallergic (M-) participants completed 3-hour exposures to aerosolized HDM (Dermatophagoides pteronyssinus) powder on 4 consecutive days in an allergen challenge chamber. We analyzed: (1) peripheral blood leukocyte levels and immune responses; and (2) RNA sequencing-derived expression profiles of nasal cells, before and after HDM exposure.Results: On HDM challenge: (1) onlyM+ persons developed allergic rhinoconjunctivitis symptoms; and (2) peripheral blood leukocyte levels/responses and gene expression patterns in nasal cells were largely concordant between M+ and M- participants; gross differences in these parameters were not observed at baseline (pre-exposure). Two key differences were observed. First, peripheral blood CD4(+) and CD8(+) T-cell activation levels initially decreased in M- participants versus increased in M+ participants. Second, in M-compared with M+ participants, genes that promoted epidermal/epithelial barrier function (eg, filament- aggregating protein [filaggrin]) versus inflammation (eg, chemokines) and innate immunity (interferon) were upregulated versus muted, respectively.Conclusion: An imprint of resistance to HDM challenge in nonatopic, nonallergic adults was muted T-cell activation in the peripheral blood and inflammatory response in the nasal compartment, coupled with upregulation of genes that promote epidermal/epithelial cell barrier function.

The proceedings of a National Institutes of Health workshop and a meeting of an Advisory Committee of the US Food and Drug Administration,1U.S. Food and Drug Administration Center for Biologics Evaluation and Research: Allergenic Products Advisory Committee, May 12, 2011. Available at: http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/AllergenicProductsAdvisoryCommittee/UCM258587.pdf. Accessed May 28, 2014.Google Scholar, 2Togias A. Asthma, Allergy, and Inflammation Branch, Division of Allergy, Immunology and Transplantation, NIAID/NIHEnvironmental exposure units: clinical trial design for validation. National Institute for Allergy and Infectious Diseases/National Institutes of Health, Bethesda2010: 1-12Google Scholar as well as related commentaries,3Bernstein J.A. Correlation between a pollen challenge chamber and a natural allergen exposure study design for eliciting ocular and nasal symptoms: early evidence supporting a paradigm shift in drug investigation?.J Allergy Clin Immunol. 2012; 130: 128-129Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 4Devillier P. Le Gall M. Horak F. The allergen challenge chamber: a valuable tool for optimizing the clinical development of pollen immunotherapy.Allergy. 2011; 66: 163-169Crossref PubMed Scopus (48) Google Scholar highlight the utility of an allergen challenge chamber (ACC) for conducting clinical trials for allergic rhinoconjunctivitis (AR). Mitigation of factors that might confound the design, analysis, and interpretation of these trials requires a systematic comparison of symptoms present in the natural setting versus those elicited after exposure to aeroallergens in an ACC. In prior studies we conducted out-of-season challenges with 3 separate pollens in patients with seasonal allergy.5Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Responses to ragweed pollen in a pollen challenge chamber versus seasonal exposure identify allergic rhinoconjunctivitis endotypes.J Allergy Clin Immunol. 2012; 130: 122-127.e8Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Effect of confounding cofactors on responses to pollens during natural season versus pollen challenge chamber exposure.J Allergy Clin Immunol. 2014; 133 (e1-7): 1340-1346Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Although there was high correlation in symptom scores in the natural pollination seasons and ACC, competing environmental influences (eg, mold) in the natural setting blunted the responsiveness to these pollens, whereas this confounder was not present in the ACC.5Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Responses to ragweed pollen in a pollen challenge chamber versus seasonal exposure identify allergic rhinoconjunctivitis endotypes.J Allergy Clin Immunol. 2012; 130: 122-127.e8Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Effect of confounding cofactors on responses to pollens during natural season versus pollen challenge chamber exposure.J Allergy Clin Immunol. 2014; 133 (e1-7): 1340-1346Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar However, most patients with allergy are polysensitized to outdoor (eg, pollens) and indoor (eg, house dust mite [HDM]) aeroallergens. HDM exposure is common, associated with perennial allergy, and a significant risk factor for AR and asthma. Here we compared the symptom dynamics in the natural setting versus the ACC in HDM-sensitive (M+) and nonsensitive (M−) participants meeting the inclusion/exclusion criteria shown in Table E1 in this article's Online Repository at www.jacionline.org. M− subjects lacked both a history of AR and skin prick test (SPT) wheal reactivity (≥5 mm) to 17 allergens, including HDM (see Table E2 in this article's Online Repository at www.jacionline.org). The study comprised 4 study phases: a 4-day run-in phase followed by 2 ACC exposure phases (ACC-I and ACC-II) with an intervening 38-day observation phase (Fig 1, A). This design allowed for evaluation of the reproducibility of symptom responses and factors that could potentially confound clinical trials in the ACC, including mediators of nocebo effects,7Bingel U. Avoiding nocebo effects to optimize treatment outcome.JAMA. 2014; 312: 693-694Crossref PubMed Scopus (141) Google Scholar as discussed in the Methods section in this article's Online Repository at www.jacionline.org. The study was conducted in the early fall, when only weed pollens were detected in San Antonio, Texas (Fig 1, A and B).6Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Effect of confounding cofactors on responses to pollens during natural season versus pollen challenge chamber exposure.J Allergy Clin Immunol. 2014; 133 (e1-7): 1340-1346Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar To mitigate this confounding factor, we selected M+ participants with negative SPT responses for weed pollens (see Table E2). All participants were allergy drug free throughout the study (see Table E3 in this article's Online Repository at www.jacionline.org). Each ACC phase comprised exposure for 3 hours on 4 consecutive days to a purified mite body powder of Dermatophagoides pteronyssinus (Fig 1, A and B, and see the Methods section in this article's Online Repository). The end point we targeted was an increase in instantaneous total symptom scores (iTSSs) of at least 6 units in 50% of participants from baseline levels. To achieve this goal, the ACC was calibrated to deliver 70 to 110 ng/m3 HDM Der p 1 antigen, as measured by means of ELISA (see Table E4 and the Methods section in this article's Online Repository at www.jacionline.org). iTSSs in the ACC and reflective total symptom scores (rTSSs) in the natural setting were recorded by using a 5-point Likert scale (see Table E5 in this article's Online Repository at www.jacionline.org). Of the 40 participants meeting inclusion criteria, 35 (21 M+ and 14 M− participants) completed all 4 study phases, and of these, 13 M+ participants and 1 M− participant had detectable (≥0.35 kU/L) serum specific IgE (ssIgE) to D pteronyssinus. This dichotomy between SPT reactivity but undetectable ssIgE has been reported for many allergens (see the Discussion section in this article's Online Repository at www.jacionline.org).8de Vos G. Skin testing versus serum-specific IgE testing: which is better for diagnosing aeroallergen sensitization and predicting clinical allergy?.Curr Allergy Asthma Rep. 2014; 14: 430Crossref PubMed Scopus (50) Google Scholar There were no differences in the key demographic characteristics between M+ and M− participants (see Table E6 in this article's Online Repository at www.jacionline.org). Five participants withdrew from the study for nonmedical reasons. M− participants had minimal symptoms in the ACC (mean iTSS, <1; Fig 1, B). In contrast, M+ participants experienced a mean increase of 3 units in the iTSS within 30 minutes of HDM challenge, and iTSSs reached a plateau after approximately 120 minutes (Fig 1, B). There was a high degree of concordance in symptom responses in M+ participants in the ACC recorded by using the Likert and visual analog scales (see Fig E1 in this article's Online Repository at www.jacionline.org). Eleven episodes of bronchospasm occurred in 5 M+ participants, a rate consistent with prior findings.9Horak F. Toth J. Marks B. Stubner U.P. Berger U.E. Jager S. et al.Efficacy and safety relative to placebo of an oral formulation of cetirizine and sustained-release pseudoephedrine in the management of nasal congestion.Allergy. 1998; 53: 849-856Crossref PubMed Scopus (43) Google Scholar These participants exhibited slightly higher total symptom scores (TSSs; see Fig E2 in this article's Online Repository at www.jacionline.org), had greater than 15% improvement in FEV1.0 after treatment with nebulized albuterol, and returned to the ACC without additional exacerbations (see the Discussion section in this article's Online Repository). These findings suggest that M+ participants with mild intermittent asthma can be safely evaluated within ACCs. The concordance in symptom responses during and between ACC-I and ACC-II was high (Fig 1, C, and see Table E7 in this article's Online Repository at www.jacionline.org). In contrast, the correlations between rTSSs recorded in the run-in versus observation phases or between rTSSs versus iTSSs were much lower (see Fig E3 in this article's Online Repository at www.jacionline.org). During ACC-I and ACC-II, an increase in iTSSs of 6 or greater from baseline (pre-exposure) was experienced in greater than 55% of participants (Fig 1, D). While less than 10% of participants had iTSSs of 15 or greater at baseline, 67% and 57% of M+ participants achieved iTSSs of 15 or greater in ACC-I and ACC-II, respectively (Fig 1, E). Substantial data indicate that ssIgE levels to allergens might serve as biomarkers for symptom severity.10Ciprandi G. Tosca M.A. Silvestri M. The practical role of serum allergen-specific IgE as potential biomarker for predicting responder to allergen immunotherapy.Expert Rev Clin Immunol. 2014; 10: 321-324Crossref PubMed Scopus (11) Google Scholar Accordingly, TSSs were greater in M+ participants with a detectable ssIgE level for D pteronyssinus in the ACC (Fig 2, A and B). The failure to detect such an association in the natural setting (Fig 2, A and B) might relate to variable HDM levels measured in dust from mattresses in the participants' homes (see Fig E4 and the Methods section in this article's Online Repository at www.jacionline.org). Pollen SPT reactivity (P+) stratified TSSs, with M+P+ participants having higher rTSSs and iTSSs when compared with those of M+P− participants (Fig 2, C). Levels of T-cell activation were greater in M+P+ participants compared with those seen in M+P− participants before and during ACCs (Fig 2, D, and see Fig E5 and the Methods section in this article's Online Repository at www.jacionline.org). Notably, T-cell activation has been associated with symptom responses during allergy.11Majori M. Piccoli M.L. Melej R. Pileggi V. Pesci A. Lymphocyte activation markers in peripheral blood before and after natural exposure to allergen in asthmatic patients.Respiration. 1997; 64: 45-49Crossref PubMed Scopus (7) Google Scholar Thus exposure to pollens in the months preceding the ACC exposures might have rendered M+P+ participants constitutively "primed," serving as a basis for the higher T-cell activation and symptoms in the natural and ACC settings (Fig 2, C and D). In the ACC phases the effects of pollen sensitization and ssIgE status were additive, with M+IgE+P+ participants manifesting maximal responsiveness after HDM exposure (Fig 2, E). The trigger for the constitutive priming could be winter and spring tree pollens because all M+P+ participants were reactive based on SPT reactivity to tree pollens (see Table E2). Moreover, the extended tree pollination season, which terminated a few months before the start of the ACC exposures, is typically associated with intense symptoms.6Jacobs R.L. Harper N. He W. Andrews C.P. Rather C.G. Ramirez D.A. et al.Effect of confounding cofactors on responses to pollens during natural season versus pollen challenge chamber exposure.J Allergy Clin Immunol. 2014; 133 (e1-7): 1340-1346Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar At the 2 transition points from the natural settings to the ACC (run-in → ACC-I and observation phase → ACC-II), there was a decrease in TSSs (Fig 1, Fig 2, A). This decrease was greater in those with more symptoms (ie, higher in M+P+ participants than in M+P− participants [Fig 2, F] and higher in M+IgE+ participants than in M+IgE− participants [data not shown]). This decrease might relate to (1) differences in how TSSs were recorded in the natural versus ACC settings (reflective vs instantaneous scoring, respectively); (2) learned responses secondary to increased emphasis/education by research staff in the ACC on how to accurately record TSSs; and (3) the controlled environment in the ACC (see the Discussion in this article's Online Repository). Inspection of the overall TSS trajectory indicated that after initiation of ACC exposures, there was a downward shift in the TSS (Fig 1, B). The baseline (pre-ACC) iTSS recorded before commencing challenge 1 was higher than the baseline iTSS recorded before challenges 2 and 3, and the baseline iTSS before challenges 4 through 8 were similar (Fig 1, B). Furthermore, the rTSS in the observation phase was lower than the rTSS in the run-in phase (Fig 1, B). This downward shift in baseline iTSS would give the mistaken impression that responsiveness was greater in challenges 2 and 3 and lower thereafter, when in fact the responsiveness in exposures 1 and 4 through 8 was similar (Fig 2, G). These downward shifts in TSSs did not differ by pollen SPT or ssIgE status (data not shown), and we surmise this might relate to a combination of factors: learned responses (secondary to education in the ACC) and partial clinical tolerance akin to what has been observed after repetitive exposure to allergens, including HDM (see the Discussion section in this article's Online Repository).12Woodfolk J.A. High-dose allergen exposure leads to tolerance.Clin Rev Allergy Immunol. 2005; 28: 43-58Crossref PubMed Google Scholar, 13Liu L.Y. Swenson C.A. Kelly E.A. Kita H. Jarjour N.N. Busse W.W. Comparison of the effects of repetitive low-dose and single-dose antigen challenge on airway inflammation.J Allergy Clin Immunol. 2003; 111: 818-825Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar In this study exposure to HDM concentrations used in the ACC was associated with reliable and reproducible elicitation of symptoms. SPT reactivity to pollen, which was associated with increased inflammatory status, and ssIgE levels to HDM were biological markers that correlated with symptom responses in the ACC. These 2 biomarkers stratified M+ participants as higher versus lower responders in the ACC, whereas this stratification is obscured in the natural settings. Therefore we suggest that out-of-pollination season challenges with HDM in the ACC might help mitigate the confounding of factors present in the natural setting: variable or low exposure to HDM, lack of association of ssIgE levels with rTSSs, and effects of competing environmental influences in a primed polysensitized subject. We also suggest that the other factors that could potentially confound clinical trials in an ACC (and natural setting) are nocebo7Bingel U. Avoiding nocebo effects to optimize treatment outcome.JAMA. 2014; 312: 693-694Crossref PubMed Scopus (141) Google Scholar and placebo14Enck P. Bingel U. Schedlowski M. Rief W. The placebo response in medicine: minimize, maximize or personalize?.Nat Rev Drug Discov. 2013; 12: 191-204Crossref PubMed Scopus (476) Google Scholar effects (expectations, learning process, and participant-physician communication). Confounding could occur by (1) misattribution of positive therapy effects to decrease symptom scores related to more precise symptom scoring (because of learned behavior) and/or partial clinical tolerance (because of repetitive exposure) and (2) imbalance in the proportion of high versus low responders in the treatment versus placebo arms. An example that highlights the potential for this imbalance is the observation that the effectiveness of anti-IgE therapy for asthma differed by the overall sensitization status of the trial participants.15Busse W.W. Morgan W.J. Gergen P.J. Mitchell H.E. Gern J.E. Liu A.H. et al.Randomized trial of omalizumab (anti-IgE) for asthma in inner-city children.N Engl J Med. 2011; 364: 1005-1015Crossref PubMed Scopus (732) Google Scholar Therapies might be more effective in patients with greater responsiveness to allergen exposure, a trait that can be readily identified in an ACC. Thus we surmise that mindfulness of the abovementioned confounders and use of an ACC might together facilitate detection of differences in the effects of placebo versus therapy in clinical trials, especially in exploratory studies with novel therapeutic agents when both the participant numbers and therapy effect sizes might be modest. Download .docx (.23 MB) Help with docx files Online Repository Data Download .pdf (.39 MB) Help with pdf files Fig E1 Download .pdf (.16 MB) Help with pdf files Fig E2 Download .pdf (.4 MB) Help with pdf files Fig E3 Download .pdf (.37 MB) Help with pdf files Fig E4 Download .pdf (.68 MB) Help with pdf files Fig E5

To compare the relative potency in skin tests of solution and tablet extracts of Timothy grass pollen (TIM) of 2 European manufacturers (ALK-Abelló, Stallergènes), with an FDA approved extract (REF) of 10,000BAU/mL. This is a prospective, multicenter, triple blinded, randomized study in which the in vivoextract potency was determined, based on the wheal size obtained in TIM allergic patients. The four tested TIM extracts were: Soluprick, Staloral 300IR, and Grazax and Oralair 300IR dissolved in 1mL 50% glycerin (under GMP standards). The SPTs were carried out in quadruplicate with the concentrate extracts and three serial half-log dilutions, and +/- controls. The study took place at study sites with different climatologic conditions. To determine if there exists a statistically significant difference between the relative potency of the TIM extracts a parallel line bioassay was carried out using the mean surface of the four wheals of the SPTs per extract and per concentration (Wilcoxon, Asymp. Sig. (2-tailed) at 5% level). Based on the wheal sizes of the concentrate extracts in relation to the REF, BAU values were calculated. Differences in wheal size between concentrate extracts reached statistical significance for all, except Soluprick-REF. The calculated BAU compared to the REF values for both solutions were between 11,300-16,300BAU/mL and the tablets varied between 4200-7300 BAU. Based on SPT whealsizes grass-tablets seem to be more potent than their reported potency of 2800BAU. There is a difference between the allergen concentration as measured in SPT of both tablets.

Background: Sublingual immunotherapy with liquid extracts provides an appealing alternative to subcutaneous immunotherapy for the treatment of allergic rhinoconjunctivitis (ARC), but a lack of robust evidence has deterred its use in North America.Objective: To determine the efficacy and tolerability of standardized glycerinated short ragweed sublingual allergen immunotherapy liquid (RW-SAIL) extract in subjects with ragweed-related ARC.Methods: This phase 3, randomized, placebo-controlled trial was conducted in North America. Subjects (age range, 18-55 years) with or without asthma were selected based on ARC symptom severity and erythema skin prick reaction to short ragweed. Subjects self-administered the maximum tolerated dose of RW-SAIL (n 5 218) or placebo (n 5 211) daily beginning approximately 8 to 16 weeks before and through the end of the ragweed pollen season. The primary end point was subject-assessed total combined daily rhinoconjunctivitis symptom and medication scores (TCS).Results: During the entire season, there was a 43% decrease in TCS in subjects treated with RW-SAIL compared with placebo. Similar decreases were observed in TCS between the 2 groups during peak season (42%) and in daily symptom scores during the entire (42%) and peak (41%) seasons. The occurrence of adverse events was similar between the treatment groups; most were mild in severity. Treatment-related oromucosal local application site reactions occurred early and were transient and self-limited. No anaphylaxis occurred.Conclusions: This is the first successful North American confirmatory phase 3 clinical trial to demonstrate the safety and efficacy of a sublingual standardized ragweed allergen immunotherapy liquid extract for the treatment of ARC.

Allergen immunotherapy has been used to treat allergic diseases for more than 100 years. In the U.S., the preparation of diagnostic and therapeutic extracts requires the cooperation of the extract manufacturer, who provides the individual allergen concentrates, and the practicing physician who formulates, dilutes, and administers the final patient-specific treatment extract. The guidelines, rules, and regulations for these activities have been established and continue to be developed as progress is made. The molecular characterization and standardization of allergenic extracts has allowed for improvements in defining the potency of these products. In turn, these advances have led to improved dosing regimens and formulation practices. This review will describe in detail some of these interactions and will identify issues that require more attention.

Exposure to domestic mouse (Mus musculus) is associated with asthma. The purpose of this study was to optimize an ELISA to measure total mouse urine proteins (MUP) using polyclonal antibodies (pAb-ELISA), and compare it with an ELISA to measure Mus m 1 using commercially available monoclonal antibodies (mAb-ELISA). A polyclonal antiserum was obtained from rabbits immunized with MUP, and a portion of it was biotinylated. A pAb-ELISA was designed, and several variables were optimized, including microplate type, concentrations of capture and biotinylated antibodies, and preparation of a standard. Wild mouse and rat urine samples, as well as common allergenic sources present in house dust were tested using both assays. The pAb-ELISA antibodies and assay conditions were compared with mAb using the 4 possible antibody combinations. The dynamic ranges (ng/mL) of pAb-ELISA and mAb-ELISA were 36-0.011 and 25-0.05, respectively. The Mus m1 level detected in domestic mouse urine was 1,500,000 ng/mL (81.4% total proteins). Mus m1 and MUP levels in wild mouse urine were 7.4 and 3.1 ng/mL, respectively (0.06% and 0.03% total proteins, respectively). Mus m1 and MUP levels in rat urine were 5.1 and 1.0 ng/mL, respectively (0.03% and 0.006% total proteins, respectively). The remaining common allergenic sources tested were undetected in both assays. The mAb-ELISA generated greater background than the pAb-ELISA in all 4 possible antibody combinations. A specific pAb-ELISA to measure MUP has been developed. This assay has environmental applications. Additional experiments should be performed to evaluate the potential cross-reactivity among mammal antigens using the reagents developed.

The purpose of this study was to evaluate the reproducibility of results yielded using a monoclonal antibody based ELISA for detection of allergen specific IgE when run in six separate affiliated laboratories. On two separate occasions, duplicate samples of 15 different sera pools were independently evaluated by each laboratory in a single blinded fashion. The average intra-assay variance among reactive assay calibrators in all laboratories was 6.2% (range 2.6–18.2%), while the average intra-laboratory inter-assay variance was 12.1% (range 8.0–17.1%). The overall inter-assay inter-laboratory variance was consistent among laboratories and averaged 15.6% (range 15.1–16.6%). All laboratories yielded similar profiles and magnitudes of responses for replicate unknown samples; dose–response profiles observed in each of the laboratories were indistinguishable. Considering positive/negative results, inter-assay inter-laboratory concordance of results exceeded 95%. Correlation of OD values between and among all laboratories was strong (r > 0.9, p < 0.001). Correlation of OD values between the two separate evaluations was also high for all allergens except olive, which was attributed to lot-to-lot differences of allergen coated wells. Collectively, the results demonstrated that the monoclonal antibody based ELISA for measuring allergen specific canine IgE is reproducible, and documents that consistency of results can be achieved not only in an individual laboratory, but between laboratories using the same monoclonal-based ELISA.

BACKGROUND:Current practice guidelines state that protease-rich fungal and insect extracts can be combined when preparing immunotherapy vaccines, but data supporting the stability of allergens in these mixtures have not been reported. OBJECTIVE:To determine the stabilities and compatibilities of Alternaria alternata and German cockroach allergens in mixtures with other high-protease fungal and insect (cockroach, imported fire ant) extracts at final extract concentrations consistent with injection dose targets for maintenance immunotherapy. METHODS:Mixtures containing Alternaria, German cockroach, and other fungal and insect extracts frequently included in immunotherapy vaccines were analyzed by a combination of quantitative analyses (enzyme-linked immunosorbent assays for multiallergen immunoglobulin E [IgE]-binding potency, major Alternaria allergen Alt a 1, and major German cockroach allergens Bla g 1 and Bla g 2) and qualitative methods (immunoblotting). Mixtures and analogous single-extract controls containing 10 to 50% glycerin were evaluated after storage for up to 12 months at 2°C to 8°C. RESULTS:Mixtures of extracts within the same phylogenetic groups (fungal-fungal, insect-insect) retained favorable Alternaria and German cockroach allergen levels and activities under most conditions examined. For several cross-taxonomic (fungal-insect) extract combinations at 10 to 25% glycerin concentrations, different immunochemical test methods measuring single (major) or multiple allergens yielded threefold to 10-fold variations in allergen recoveries. CONCLUSION:Allergen compatibilities can be compromised in some fungal-insect extract mixtures, contrary to current immunotherapy practice parameter recommendations. Separation of these products into different treatment vials may be required to produce stable mixtures for subcutaneous immunotherapy. Data from assay methodologies with distinct binding specificities provide a critical assessment of allergen activities in high-protease extract mixtures.

Background: Sublingual immunotherapy (SLIT) has become established in Europe, and its efficacy is being evaluated in the United States. The doses used for SLIT in Europe today are difficult to evaluate, because each manufacturer expresses the potency of its extracts differently.Objectives: To compare in vitro European SLIT maintenance solutions against US licensed standardized allergenic extract concentrates and to determine the monthly SLIT doses delivered expressed in bioequivalent allergy units ([B]AU).Methods: We studied Dermatophagoides pteronyssinus, timothy grass pollen, cat (hair) and short ragweed pollen allergen extracts. The SLIT maintenance solutions of 4 leading European manufacturers and standardized concentrate extracts of 3 US manufacturers were analyzed with the following assays: protein content, relative potency (immunoglobulin E [IgE]-binding enzyme-linked immunosorbent assay [ELISA] inhibition) and major allergen content. The relative monthly allergen dose in (B)AU was calculated for each recommended SLIT schedule.Results: Relative potency was approximately 10 times higher for US concentrate standardized extracts which are meant to be diluted than for European SLIT maintenance solutions of D pteronyssinus and timothy grass pollen. For cat (hair) and short ragweed pollen, the difference was less. Measurements of relative potency and major allergen content correlated well. In our assays, European mite extracts contain a very low quantity of Der p 2 compared with US mites.Conclusion: Recommended SLIT doses in Europe vary widely among the manufacturers, but are consistently lower (Eurl) or higher (Eur4) over all four allergens tested. SLIT efficacy probably depends on additional factors apart from the exact dose. SLIT dose finding studies should be done for each product. Ann Allergy Asthma Immunol. 2011;107:448-458.

To discuss recent issues pertinent to allergen immunotherapy practice in the United States. Allergen extract preparation guidelines, updated allergen immunotherapy practice parameter (AIPP) guidelines, and evolving trends in how immunotherapy outcomes will be measured and assessed. Allergen extract preparation guidelines have been established by 2 entities: the US Pharmacopeia and an American Academy of Allergy, Asthma, and Immunology/American College of Allergy, Asthma, and Immunology/Joint Council of Allergy, Asthma, and Immunology Joint Task Force. Minor differences exist between these guidelines, but both focus on aseptic techniques and require that compounding personnel pass a written examination and annual media fill test. The AIPP third update provides new dosing recommendations for Bermuda grass, imported fire ant, and nonstandardized extracts distinguishing between pollen (0.5 mL of a 1:100 or 1:200 vol/vol) and mold/fungi or cockroach (highest tolerated dose) extracts. Because of limited and sometimes conflicting data on high and low proteolytic-containing extract compatibility, the AIPP continues to recommend against mixing these together. Although the AIPP does not specifically recommend a specific diluent, recent evidence suggests normal saline may not be as effective a stabilizer for extract dilutions as glycerin or human serum albumin. Currently, immunotherapy efficacy is determined with subjective assessments that rely on patient reporting, but this may change as health care reform evolves. It will likely become more important for US allergy/immunology practices to demonstrate immunotherapy comparative-effectiveness and report quality measures. Recent comparative-effectiveness studies have demonstrated the cost-effectiveness of immunotherapy compared with symptomatic drug treatment. Ann Allergy Asthma Immunol. 2011;107:289-300.