Laser-facilitated epicutaneous immunotherapy with depigmented house dust mite extract alleviates allergic responses in a mouse model of allergic lung inflammation.

BACKGROUND: Skin-based immunotherapy of type 1 allergies has recently been re-investigated as an alternative for subcutaneous injections. In the current study, we employed a mouse model of house dust mite (HDM)-induced lung inflammation to explore the potential of laser-facilitated epicutaneous allergen-specific treatment. METHODS: Mice were sensitized against native Dermatophagoides pteronyssinus extract and repeatedly treated by application of depigmented D pteronyssinus extract via laser-generated skin micropores or by subcutaneous injection with or without alum. Following aerosol challenges, lung function was determined by whole-body plethysmography and bronchoalveolar lavage fluid was analyzed for cellular composition and cytokine levels. HDM-specific IgG subclass antibodies were determined by ELISA. Serum as well as cell-bound IgE was measured by ELISA, rat basophil leukemia cell assay, and ex vivo using a basophil activation test, respectively. Cultured lymphocytes were analyzed for cytokine secretion profiles and cellular polarization by flow cytometry. RESULTS: Immunization of mice by subcutaneous injection or epicutaneous laser microporation induced comparable IgG antibody levels, but the latter preferentially induced regulatory T cells and in general downregulated T cell cytokine production. This effect was found to be a result of the laser treatment itself, independent from extract application. Epicutaneous treatment of sensitized animals led to induction of blocking IgG, and improvement of lung function, superior compared to the effects of subcutaneous therapy. During the whole therapy schedule, no local or systemic side effects occurred. CONCLUSION: Allergen-specific immunotherapy with depigmented HDM extract via laser-generated skin micropores offers a safe and effective treatment option for HDM-induced allergy and lung inflammation.

In vitro evidence of efficacy and safety of a polymerized cat dander extract for allergen immunotherapy.

BACKGROUND: Allergy to cat epithelia is highly prevalent, being the major recommendation for allergy sufferers its avoidance. However, this is not always feasible. Allergen specific immunotherapy is therefore recommended for these patients. The use of polymerized allergen extracts, allergoids, would allow to achieve the high allergen doses suggested to be effective while maintaining safety. RESULTS: Cat native extract and its depigmented allergoid were manufactured and biochemically and immunochemically characterized. Protein and chromatographic profiles showed significant modification of the depigmented allergoid with respect to its corresponding native extract. However, the presence of different allergens (Fel d 1, Fel d 2, Fel d 3, Fel d 4 and Fel d 7) was confirmed in the allergoid. Differences in IgE-binding capacity were observed as loss of biological potency and lower stability of the IgE-allergen complex on surface plasmon resonance. The allergoid induced production of IgG antibodies able to block IgE-binding to native extract. Finally, studies carried out with peripheral-blood mononuclear cells from cat allergic patients showed that the allergoid induced IFN-γ and IL-10 production similar to that induced by native extract. CONCLUSIONS: Cat depigmented allergoid induced production of cytokines involved in a Th1 and Treg response, was able to induce production of IgG-antibodies that blocks IgE-binding to cat native extract, and showed reduced interaction with IgE, suggesting greater safety than native extract while maintaining in vitro efficacy.

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.

Different sensitization to storage mites depending on the co-exposure to house dust mites.

BACKGROUND: Co-sensitization to house dust mites (HDMs) and storage mites (SMs) is very frequent, although the clinical relevance is not well established. OBJECTIVE: To describe the pattern of sensitization and immunologic characterization of patients with positive skin prick test reactions to HDMs and SMs in 4 areas of Spain, selected according to high exposure to HDMs and variable exposure to SMs. METHODS: One hundred sixty-nine individuals with positive skin prick test reactions to HDMs and SMs were included. Specific IgE levels to different mite species and to Der p 1, Der p 2, and Der p 10 were determined. Immunoblots to Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae extracts were carried out, and allergograms were obtained. Results of different areas were compared. RESULTS: A high rate of polysensitization to SMs was observed, although 12% of participants did not have specific IgE to any SM species. Sensitization to Dermatophagoides species, Der p 2, and L destructor were predominant, although significant differences were observed among areas depending on the grade of exposure to SMs. In areas with high exposure, the SM allergogram showed greater recognition of group 2 allergen. CONCLUSION: Sensitization patterns to SMs in patients sensitized to HDMs and SMs differ depending on the exposure to SMs. Sensitization, mainly to L destructor, seems to exist in areas with high exposure, possibly with group 2 allergens mainly involved. However, in areas with low SM populations, sensitizations observed by skin prick testing appear to be related to HDM exposure.

Control Process for Manufacturing and Standardization of Allergenic Molecules.

It is widely accepted that the success of the allergen immunotherapy (AIT), beyond clinical parameters such as dose, dosage regimen, or compliance, depends on the quality and composition of the final products used in the vaccines. Allergenic vaccines are pharmaceutical preparations derived from the natural sources which contain the allergenic components responsible for allergic sensitization. The selection of the appropriate allergenic sources must be a requirement. They suffer a dramatic transformation during the manufacturing process which renders a biologically standardized final product. The inclusion of the appropriate control analyses in the manufacturing process has demonstrated to be an efficient method to guarantee the quality and homogeneity of the final product as well as being a very useful tool for saving time and money. In this context, in the last years, the Regulatory Agencies have released specific guidelines to guarantee the manufacturing of the most appropriate products for the treatment of patients.

Enzymatic activity of allergenic house dust and storage mite extracts.

Proteases are involved in the pathogenicity of allergy, increasing epithelial permeability and acting as adjuvants. Enzymatic activity is therefore important for the allergenicity of an extract and also affects its stability and safety. However, the enzymatic activity of extracts is not usually evaluated. The objective of this study was to evaluate the enzymatic activity of the most allergenic mite extracts and to investigate their allergenic properties. Extracts from nine allergenic mite species (Dermatophagoides pteronyssinus, Dermatophagoides farinae Hughes, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae (Schrank), Glycyphagus domesticus (DeGeer), Acarus siro L., Chortoglyphus arcuatus, and Blomia tropicalis) were characterized. Protein and allergen profiles were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot, respectively. Gelatinolytic activity was evaluated with a zymogram and the activity of other enzymes (cysteine, serine proteases, and esterases) was evaluated individually or with the API-ZYM system. The main differences in protease activity were found between house dust mites and storage mites. House dust mites presented higher cysteine protease activity while storage mites presented higher serine protease activity. These differences are in line with their trophic specialization. A wide range of different activities was found in all the extracts analyzed, reflecting the fact that the extracts preserve the activity of many enzymes, this being necessary for a correct diagnosis. However, enzymes may act as adjuvants and, therefore, could lead to undesirable effects in immunotherapies, making this activity not suitable for treatment products. Modified extracts with lower enzymatic activity could be more appropriate for immunotherapy.

Arginine kinase from the cellar spider (Holocnemus pluchei): a new asthma-causing allergen.

BACKGROUND: We report a 31-year-old farmer whose work consists in handling cereal and vegetables, who consulted our clinic because of asthma symptoms after inhalation of dust during manipulation of the deposited material, usually inside the warehouse. METHODS AND RESULTS: Skin prick tests and specific immunoglobulin E (IgE) determinations were negative with common aeroallergens. The patient noted the presence of many spiders in the warehouse, which were identified as the cellar spider Holocnemus pluchei and the common house spider, Tegenaria domestica. Extracts of spider bodies brought in by the patient were obtained and used to perform in vivo and in vitro studies. Molecular characterization of IgE-binding bands was performed by mass spectrometry. We obtained positive prick tests to the extracts of the bodies of both spiders. Immunoblotting displayed different bands in both spider extracts, in a range of 20-70 kDa. All were hemocyanins, except for a 17-kDa protein of Holocnemus identified as an arginine kinase (AK). Bronchial challenge was positive with the extract of the cellar spider and with the AK, but was negative with the domestic house spider. CONCLUSION: We present the first case of respiratory allergy due to sensitization to AK from a common spider, confirmed by bronchial provocation tests.

Seafood hypersensitivity in mite sensitized individuals: is tropomyosin the only responsible allergen?

BACKGROUND: Seafood allergy has been related to mite sensitization, mainly mediated by the muscle protein tropomyosin. OBJECTIVES: To determine the correlation between seafood hypersensitivity and mite sensitization (Dermatophagoides pteronyssinus and Chortoglyphus arcuatus, a highly prevalent storage mite in Spain) and to investigate the implication of tropomyosin in cross-reactivity. METHODS: Patients from Northwest Spain were divided into 2 groups. The mite-seafood group contained 30 allergic mite individuals with a clinical history of food hypersensitivity. The mite group contained 40 individuals with positive skin prick test results to D pteronyssinus and C arcuatus but negative seafood test results. Specific IgE (sIgE) to whole mite and shrimp extracts, mite tropomyosin (rDer p 10), and shrimp tropomyosin (rPen a 1) were determined in each serum sample. Allergenic profiles were analyzed by immunoblot. Cross-reactivity studies were investigated by enzyme-linked immunosorbent assay and immunoblot inhibition studies. RESULTS: In the mite-seafood group, 71% of patients had positive sIgE results to shrimp and 55% of them to shrimp tropomyosin. A strong correlation was found between sIgE to shrimp tropomyosin and mite tropomyosin. Positive correlation was observed between sIgE to shrimp tropomyosin and severity of symptoms. In the mite group, none of the 20% of patients with sIgE to shrimp tested positive to shrimp tropomyosin. In the immunoblot inhibition experiment, the shrimp extract was totally inhibited by mite extract. These data suggest that primary sensitization is related to mite sensitization. CONCLUSION: Tropomyosin does not seem to be the main allergen involved in mite-seafood sensitization in mite sensitized individuals. High levels of sIgE to tropomyosin seem to be related to severity of symptoms.

Gregarines in Dermatophagoides spp. (Acari: Pyroglyphidae): light microscopy observation.

Recently there has been an increasing interest in studying arthropods that live close to humans, such as cockroaches and mites, for their potential as vectors. Gregarines observed under light microscopy in intestinal extracts of house dust mites (Dermatophagoides spp.) are described for the first time in scientific literature.

Oral mite anaphylaxis by Thyreophagus entomophagus in a child: a case report.

Sensitization to Thyreophagus entomophagus, a storage mite, is uncommon and might produce occupational respiratory disorders in farmers. We present the first case of a child suffering anaphylaxis produced by ingestion of contaminated flour with Thyreophagus entomophagus.

Enzymatic activity of Dermatophagoides pteronyssinus extracts after acidic treatment.

BACKGROUND: Mite extracts contain potent enzymes. These enzymes, especially Der p 1, may affect the bronchial homeostasis and the amplification of the allergic response. The objectives of this study were to determine how depigmentation affects the enzymatic activity of allergen extracts of Dermatophagoides pteronyssinus and to verify if these depigmented extracts retain their in vitro allergenic properties. METHODS: Four native extracts were manufactured from 4 different batches of raw material of D. pteronyssinus. Once extracted, native extracts were reconstituted and modified by adding increasing quantities of 2 M HCl to the solution and dialyzed against double-distilled water. The enzymatic activity of these 8 extracts (4 native and 4 depigmented) was evaluated using in vitro methods. The allergenic potency was evaluated by human specific IgE and IgG ELISA inhibition experiments. The major allergen content (Der p 1 and Der p 2) was measured with monoclonal antibodies. RESULTS: Protease, phosphatase, lipase and glycosidase activity was detected in native extracts. After depigmentation, all the enzymatic activities showed a significant decrease. SDS-PAGE reveals the same protein profile in both types of extracts. The results of ELISA inhibition confirmed that depigmented extracts preserved their antigenic and allergenic capacity. Der p 2 levels increased in depigmented extracts, while the detection capacity of Der p 1 decreased. CONCLUSIONS: The depigmentation process significantly reduced the enzymatic activity of these mite extracts, while preserving their allergenicity and antigenicity. No significant differences were observed in the antigenic profile of native and depigmented extracts.

Enzymatic activity in body and fecal extracts of the storage mite Chortoglyphus arcuatus.

BACKGROUND: Chortoglyphus arcuatus has been described in many countries. Many allergens are potent enzymes, which may promote a Th2 immune response. The aim of this study was to evaluate the enzymatic activity of body and fecal extracts of C. arcuatus. MATERIAL AND METHODS: Feces and bodies of full-grown C. arcuatus cultures were separated by sieving, extracted in PBS, dialyzed and lyophilized. The antigenic profile of both extracts was determined by SDS-PAGE. Immunoblot experiments were conducted using a pool of sera from allergic individuals residing in Galicia, a region of Spain, where this species is abundant. The enzymatic activity of the extracts was evaluated by the zymogram technique. Serine and cysteine protease activity was measured using in vitro methods. The API Zym system was used to determine the enzymatic properties of the extracts. RESULTS: The antigenic profile showed that the body extract contained more and better defined bands than the fecal extract. Allergens were detected in both extracts in a molecular weight range between 14 and 100 kDa. Gelatinolytic gels confirmed that fecal extracts contain more hydrolytic enzymatic activity than body extracts. Serine protease activity in fecal extracts was higher than in body extracts (5.98 vs. 2.701 IU of trypsin/mg of freeze-dried material). No cysteine protease activity was detected. CONCLUSION: C. arcuatus extracts contain several allergens and proteins with high enzymatic activity, especially in the feces. Some of these allergens may be enzymes. Fecal extracts have more enzymatic activity than body extracts.

Allergoids for Allergy Treatment.

BACKGROUND: Chemically modified allergen extracts, known as allergoids, are commonly used for treating allergic patients. In general terms, the concept of allergoids implies allergen extracts with a reduction of their allergenicity maintaining their immunogenicity. Different methods to obtain allergoids have been developed in the past years, opening attractive lines of research. OBJECTIVE: To review the different approaches to allergoid development as well as their characterization, mechanism of action and efficacy and safety issues. METHODS: A revision and analysis of the different types of allergoids has been performed, with special attention to patents submitted and granted in the last years. Additionally, updated information about the mechanism of action and clinical evidence and safety of allergoids has been discussed. RESULTS: Principally, allergoids are obtained by the polymerization of native allergen extracts with aldehydes, including formaldehyde or glutaraldehyde. However, recent patents and publications about different chemical modifications have been presented, as well as about the use of new adjuvants with allergoids. Regarding the characterization, allergoids require more sophisticated analytical methods than native extracts, as a consequence of their properties and characteristics. CONCLUSION: In the last years, the partial understanding of the mechanism of action and the generation of clinical evidence of different types of allergoids, linked to their excellent safety profile and their convenience for a quick build up phase, have made of allergoids an excellent product for allergy treatment.

Preclinical safety and immunological efficacy of Alternaria alternata polymerized extracts.

INTRODUCTION: Alternaria alternata is a widespread fungi whose allergy is a risk factor for asthma development. The use of a polymerized allergen extract (allergoid) may be safer than native extract based treatments while maintaining efficacy. The objective of this study was to characterize biochemically and immunochemically a new Alternaria alternata allergoid. METHODS: Characterization of native and allergoid extracts was performed by determination of protein content, protein and allergenic profile, biological potency, identification of Alternaria allergens, and Alt a 1 quantification. Safety was evaluated in toxicological assays (Ames test, limit test, and fish embryo acute toxicity test in zebrafish, and maximum tolerated dose and Dose-range finding study in rats). Efficacy was evaluated as the capacity to induce IgG antibodies that block IgE-binding to the allergen and cytokine induction (IFN-γ, IL-4, IL-6, IL-10, and TNF-α) in PBMC from atopic donors. RESULTS: Protein and antigenic profiles showed significant modification of the depigmented allergoid with respect to the native extract, inducing a lower IgE binding capacity. Alt a 1, Alt a 3, Alt a 6, and Alt a 8 allergen sequences were identified in the polymer. No toxicological nor genotoxicity effects were observed. The polymer induced IgG antibodies that blocked human IgE binding epitopes, and it induced higher IL-10 levels and similar levels of the other cytokines than native extract in PBMC. CONCLUSIONS: This new A. alternata allergoid could be an effective immunotherapy treatment leading to cytokine stimulation and inducing synthesis of IgG antibodies able to block IgE binding to the allergen. In addition, no toxicological effect was observed, and it may be safer than native extract due to its lower IgE binding capacity and cytokine induction that suggest tolerance induction via T cell shift to Treg (IL-10).

Molecular and biochemical properties of storage mites (except Blomia species).

In recent years, the allergological importance of different mite species not belonging to the family Pyroglyphidae has been demonstrated. These mites, commonly named storage mites, include Lepidoglyphus destructor, Glycyphagus domesticus, Tyrophagus putrescentiae, Acarus siro, Aleuroglyphus ovatus, Suidasia medanensis and Thyreophagus entomophagus. Several allergens from these species have been purified, sequenced and cloned. Many of these allergens have shown sequence homology and a biological function similar to those previously described in Blomia tropicalis and the Dermatophagoides spp. The main allergens described in storage mites include fatty acid binding proteins, tropomysin and paramyosin homologues, apoliphorine like proteins, alfa-tubulines and other, such as group 2, 5 and 7 allergens, which definitive biological function has not been described yet. Besides the purification and characterization of allergens, the allergenicity of other species such as Acarus farris, Austroglycyphagus malaysiensis, Blomia kulagini and B. tjibodas, Cheyletus eruditus, Chortoglyphus arcuatus, Gohieria fusca, Thyreophagus entomophagus and Tyrophagus longior has been investigated. Research has also been conducted to identify allergens in parasitic mites, such as Psoroptes ovis, Sarcoptes scabiei, Varroa jacobsoni, Diplaegidia columbae and Hemisarcoptes cooremani. The allergenicity of mites present in agricultural environments has been investigated. Crossreactivity studies have also been performed to elucidate to what extent all these mites share common, or species specific epitopes. Herein we present a comprehensive review of the allergenicity of mite species which have been implicated in human respiratory and/or dermatological diseases.

Role of Predatory Mites in Persistent Nonoccupational Allergic Rhinitis.

Mites can sensitize and induce atopic disease in predisposed individuals and are an important deteriorating factor in patients with allergic rhinitis, asthma, and atopic dermatitis. Although Pyroglyphidae mites have been extensively studied, very scarce reports are available on Cheyletidae spp. especially regarding human respiratory pathology. The main objective of the present study is to investigate the clinical role of this predator mite (Cheyletus eruditus) as a respiratory antigen in a selected sensitized human population. Fifty-two adult patients were recruited from the outpatient allergy clinic to assess their eligibility for the study. The thirty-seven subjects with persistent allergic rhinitis (PAR) who fulfilled the ARIA criteria had a positive IgE response confirmed by skin prick test (SPT) to C. eruditus. Only those individuals (37/47) with a positive SPT to C. eruditus showed a positive nasal provocation test (NPT), while 10 patients with nonallergic mild-to-moderate persistent rhinitis, control group, had a negative NPT with C. eruditus. The present paper describes a new role for the predator mite Cheyletus eruditus as a respiratory allergen in a selected subset of patients in a subtropical environment afflicted with persistent nonoccupational allergic rhinitis.

Cloning and characterization of tropomyosin from the mite Chortoglyphus arcuatus.

Tropomyosin is a pan-allergen that shares a high homology among species. It is involved in cross-reactivity among mites, crustaceans, mollusks and insects. The objectives were to express and purify recombinant tropomyosin from the storage mite Chortoglyphus arcuatus, and to investigate the homology and cross-reactivity with tropomyosin from other invertebrates. Recombinant C. arcuatus tropomyosin (r-Cho a 10) was selected from a library by screening with a pool of patient sera. r-Cho a 10 (UniProt: H2DFL1) was sequenced, expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Polyclonal anti-tropomyosin antibodies were produced in mice. IgE recognition of r-Cho a 10 was checked by immunoblot. Immunoblot inhibition assays were used to identify the native tropomyosin in the complete extract from C. arcuatus and study cross-reactivity between r-Cho a 10 and Der p 10. Identification of tropomyosin in other allergenic sources was performed by immunoblot. r-Cho a 10 showed a high homology (54-96%) with other tropomyosins from different allergenic sources. IgE recognition was observed using a pool of sera from sensitized individuals. Tropomyosins from different extracts were identified not only in the whole C. arcuatus extract but also in Dermatophagoides pteronyssinus, shrimp, mussel, cockroach and Anisakis extracts with polyclonal α-Cho a 10. r-Cho a 10 completely inhibited the recognition of Der p 10. Recombinant C. arcuatus tropomyosin maintained its capacity to recognize IgE. r-Cho a 10 was used to prove cross-reactivity among tropomyosins from other invertebrate species, including mites. This is the first C. arcuatus allergen included in the WHO/IUIS (World Health Organization/International Union of Immunological Societies) Allergen Nomenclature database.