Conserved genomic neighborhood is a strong but no perfect indicator for a direct interaction of microbial gene products.

BACKGROUND: The order of genes in bacterial genomes is not random; for example, the products of genes belonging to an operon work together in the same pathway. The cotranslational assembly of protein complexes is deemed to conserve genomic neighborhoods even stronger than a common function. This is why a conserved genomic neighborhood can be utilized to predict, whether gene products form protein complexes. RESULTS: We were interested to assess the performance of a neighborhood-based classifier that analyzes a large number of genomes. Thus, we determined for the genes encoding the subunits of 494 experimentally verified hetero-dimers their local genomic context. In order to generate phylogenetically comprehensive genomic neighborhoods, we utilized the tools offered by the Enzyme Function Initiative. For each subunit, a sequence similarity network was generated and the corresponding genome neighborhood network was analyzed to deduce the most frequent gene product. This was predicted as interaction partner, if its abundance exceeded a threshold, which was the frequency giving rise to the maximal Matthews correlation coefficient. For the threshold of 16%, the true positive rate was 45%, the false positive rate 0.06%, and the precision 55%. For approximately 20% of the subunits, the interaction partner was not found in a neighborhood of ± 10 genes. CONCLUSIONS: Our phylogenetically comprehensive analysis confirmed that complex formation is a strong evolutionary factor that conserves genome neighborhoods. On the other hand, for 55% of the cases analyzed here, classification failed. Either, the interaction partner was not present in a ± 10 gene window or was not the most frequent gene product.

Understanding differences in allergen immunotherapy products and practices in North America and Europe.

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.

Preservation of epithelial cell barrier function and muted inflammation in resistance to allergic rhinoconjunctivitis from house dust mite challenge.

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) only M+ 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.

Fungal raw materials used to produce allergen extracts.

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.

Mite allergen extracts and clinical practice.

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.

Allergen Immunotherapy Clinical Trial Outcomes and Design: Working Toward Harmonization of Methods and Principles.

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.

Randomized, double-blind, placebo-controlled trial of standardized ragweed sublingual-liquid immunotherapy for allergic rhinoconjunctivitis.

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 = 218) or placebo (n = 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.

Immunotherapy preparation guidelines, rules, and regulation.

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.

Allergen stabilities and compatibilities in mixtures of high-protease fungal and insect extracts.

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.

House dust mite sublingual immunotherapy: results of a US trial.

BACKGROUND: Few trials of sublingual immunotherapy (SLIT) in the United States have been reported. OBJECTIVE: This randomized, placebo-controlled feasibility SLIT study compared the safety and physiologic effects of high- versus low-dose Dermatophagoides farinae vaccine. METHODS: Thirty-one D farinae-sensitive adults with allergic rhinitis with or without mild intermittent asthma were eligible for randomization to high-dose maintenance vaccine (n = 10, 4200 allergen units [approximately 70 µg of Der f 1/d]), low-dose maintenance vaccine (n = 10; 60 allergen units [approximately 1 µg of Der f 1/d]), or placebo (n = 11) over 12 to 18 months. Medication-symptom scores and adverse events were monitored, serum D farinae-specific IgE and IgG4 levels were measured, and bronchial reactivity to D farinae was determined at baseline and 6-month intervals. RESULTS: Of the 31 randomized subjects, 6 withdrew because of non-treatment-ascribed events. Four withdrew because of treatment-ascribed effects: high-dose group, 1 of 10 (gastrointestinal symptoms); low-dose group, 1 of 10 (gastrointestinal symptoms); and placebo group, 2 of 11 (headache and increased nasal symptoms). Thus 21 subjects completed the study: high-dose group, 9; low-dose group, 7; and placebo group, 5. Eleven of the 21 subjects experienced mild-to-moderate gastrointestinal symptoms, throat irritation, or both (high-dose group, 5/9; low-dose group, 4/7; and placebo group, 2/5). No severe systemic reactions were noted. No differences in symptom-medication scores were found. High-dose SLIT increased the bronchial threshold to allergen challenge and increased serum D farinae-specific IgG4 levels, whereas low-dose SLIT and placebo had no significant effect. CONCLUSIONS: High-dose D farinae SLIT was generally tolerable, increased serum D farinae-specific IgG4 levels, and improved the bronchial threshold to allergen challenge. Larger US trials are warranted.

Maintenance dosing for sublingual immunotherapy by prominent European allergen manufacturers expressed in bioequivalent allergy units.

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 (Eur1) 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.

Allergen immunotherapy practice in the United States: guidelines, measures, and outcomes.

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.

Sublingual immunotherapy in patients with allergic rhinoconjunctivitis caused by ragweed pollen.

BACKGROUND: Specific allergen immunotherapy is most often delivered subcutaneously, but sublingual immunotherapy may confer greater benefit in terms of tolerability and safety, accessibility, and improved antigen delivery. OBJECTIVE: This randomized, double-blind, placebo-controlled trial was conducted to identify a safe and effective maintenance dose range of sublingual standardized glycerinated short ragweed pollen extract in adults with ragweed-induced rhinoconjunctivitis. METHODS: In May 2006, a total of 115 patients with ragweed-induced rhinoconjunctivitis were randomly allocated to placebo (n = 40), medium-dose extract (4.8 microg Amb a 1/d; n = 39), or high-dose extract (48 microg Amb a 1/d; n = 36). In a 1-day (rush) dose-escalation regimen, ragweed pollen extract was administered sublingually in incremental doses until maximum tolerable or scheduled dose was reached and then maintained during the ragweed pollen season. Patient diaries were used to monitor nasal and ocular symptoms and medication. The primary endpoint was symptom score. RESULTS: Both active treatment groups achieved a 15% reduction in total rhinoconjunctivitis symptom scores compared with placebo during the entire ragweed pollen season, but the difference was not statistically significant (P > .10) However, in an analysis of covariance correcting for preseasonal symptoms, both mean daily symptom scores (0.19 +/- 1.16 vs 1.00 +/- 2.30) and medication scores (0.0003 +/- 1.64 vs 0.63 +/- 1.06) for the entire pollen season were significantly reduced in the high-dose versus placebo groups, respectively (P

The Clinical Relevance of Pollen Versus Fungal Spores in Allergic Diseases.

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.

Performance characteristics of a monoclonal antibody cocktail-based ELISA for detection of allergen-specific IgE in dogs and comparison with a high affinity IgE receptor-based ELISA.

The purpose of this study was to define the operational and performance characteristics of a commercially available monoclonal antibody based (mac) ELISA for detection of allergen-specific IgE in dogs. The average intra-assay variance over 1 year was 9.7% (range 2.5-62.7%), while the interassay variance averaged 10.8% (range 8.1-13.8%). The average positive control responses observed for grass, weed, tree and mite allergens during each month remained relatively constant; the average monthly variance was 11.6% (range 8.3-19.2%) for grass pollens, 13.3% (range 9.1-20.4%) for weed pollens, 13.3% (range 9.8-18.2%) for tree pollens and 13.6% (range 8.9-18.7%) for mite allergens. The interlaboratory concordance of results for the macELISA was approximately 91%. The interlaboratory concordance of results comparing the macELISA and a high affinity IgE receptor-based ELISA was approximately 92%. The results demonstrate that the macELISA is reproducible and the results are comparable to the high affinity IgE receptor based ELISA within and between laboratories.

Assessment of cross-reactivity among five species of house dust and storage mites.

In vitro cross-reactivity among two house dust (Dermatophagoides farinae, D. pteronyssinus) and three storage (Acarus siro, Tyrophagus putrescentiae, Lepidoglyphus destructor) mites was examined in 20 mite-sensitive dogs with natural occurring atopic dermatitis (group A), 13 high-IgE beagles experimentally sensitized to D. farinae (group B), and five healthy beagles (group C). Intradermal testing (IDT) and serology for allergen-specific IgE demonstrated that co-sensitization for all possible pairs of the five mites was generally 45% or higher among group A dogs. In the same dogs, enzyme-linked immunosorbent assay cross-inhibition results indicated that each one of D. farinae, A. siro and T. putrescentiae was a strong inhibitor of all the remaining mites, whereas D. pteronyssinus was a strong inhibitor of L. destructor. A high number of positive IDT and serology test results for D. pteronyssinus, A. siro, T. putrescentiae and L. destructor were recorded among group B dogs. No conclusive evidence of exposure to these mites was found upon analysis of dust samples from their environment and their food for the presence of mites and guanine. Also, the number of positive test results was generally higher among group B than among group C dogs. Enzyme-linked immunosorbent assay cross-inhibition revealed that D. farinae was a strong inhibitor of D. pteronyssinus, A. siro and T. putrescentiae. Collectively, these results demonstrated extensive in vitro cross-reactivity among house dust and/or storage mites that can explain false-positive results upon testing of dust mite-sensitive dogs with atopic dermatitis.

Allergens.

What makes an antigen an allergen? This question has been at the core of much research regarding the basic mechanisms of allergy as well as the development of diagnostic and therapeutic allergen products. Great strides have been made since Kjell Aas asked this question in his classic review in Allergy (1), but we appear no closer to the answer. The mechanisms leading to allergic reactions have been well described in recent years and our understanding of it at the molecular level has greatly improved. Conversely, the molecular structures responsible for these allergic reactions are not as well understood. Techniques for the isolation, identification, and epitope mapping of allergens have greatly enhanced our ability to test old and new ideas about what makes an antigen an allergen. We continue to search for "major allergens," and when we identify them, we hope to delineate "allergenic determinants" and "immunodominant epitopes." Even now, unique structural motifs or determinants responsible for allergenicity have not yet been identified, and we will probably never find them.

Mite and cockroach proteases activate p44/p42 MAP kinases in human lung epithelial cells.

BACKGROUND: The mechanisms underlying epithelial cell activation by indoor inhaled antigens are poorly understood. METHODS: In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) in A549 epithelial cells upon exposure to antigens of house dust mite (HDMA), German cockroach (GCA), and American cockroach (ACA). RESULTS: Each of these antigens induced a significant increase in IL-8 levels compared to the medium control. Exposure of A549 cells to these antigens induced the phosphorylation of p44/42 MAPKs within 5 minutes, which reached a peak at 25 minutes later and reached baseline levels at 1 hour after exposure. PD98059, a MEK1 inhibitor, significantly decreased phosphorylation of p44/p42 MAPKs and IL-8 production. Exposure of A549 cells with antigens, which had been preincubated with different protease inhibitors, also resulted in a reduction of both MAPK phosphorylation and IL-8 production. CONCLUSION: Thus, proteolytic antigens present in HDMA, GCA and ACA activate the p44/42 MAPKs airway epithelial cells, which lead to elevated IL-8 production and initiation of the inflammatory cascade.