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.

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.

New shrimp IgE-binding proteins involved in mite-seafood cross-reactivity.

SCOPE: Shrimp is a seafood consumed worldwide and the main cause of severe allergenic reactions to crustaceans. Seafood allergy has been related to mite sensitization, mainly mediated by tropomyosin, but other proteins could be involved. The aim of the study was to identify new shrimp allergens implicated in mite-seafood cross-reactivity (CR) in two different climate populations: dry and humid climates. METHODS AND RESULTS: Shrimp and mite IgE-binding profiles of patients from continental dry and humid climates were analyzed by immunoblotting, and the most frequently recognized Solenocera melantho shrimp proteins were identified by MS as α-actinin, ß-actin, fructose biphosphate aldolase, arginine kinase, sarcoplasmic calcium-binding protein, and ubiquitin. Using inhibition immunoblot assays, we demonstrate that tropomyosin and ubiquitin were responsible for mite-seafood CR from both climates; but also α-actinin and arginine kinase are implicated in dry- and humid-climate populations, respectively. Reciprocal inhibition assays demonstrated that mites are the primary sensitizer in humid-climate, as shrimp is in continental dry-climate population. CONCLUSION: Several new shrimp allergens have been identified and should be considered in the diagnosis and treatment of shrimp allergy and mite-seafood CR. Differences in mite-seafood CR were founded to be based on the climate.

Pediatric allergy and immunology in Spain.

The data of the ISAAC project in Spain show a prevalence of childhood asthma ranging from 7.1% to 15.3%, with regional differences; a higher prevalence, 22.6% to 35.8%, is described for rhinitis, and atopic dermatitis is found in 4.1% to 7.6% of children. The prevalence of food allergy is 3%. All children in Spain have the right to be visited in the National Health System. The medical care at the primary level is provided by pediatricians, who have obtained their titles through a 4-yr medical residency training program. The education on pediatric allergy during that period is not compulsory and thus very variable. There are currently 112 certified European pediatric allergists in Spain, who have obtained the accreditation of the European Union of Medical Specialist for proven skills and experience in pediatric allergy. Future specialists in pediatric allergy should obtain their titles through a specific education program to be developed in one of the four accredited training units on pediatric allergy, after obtaining the title on pediatrics. The Spanish Society of Pediatric Allergy and Clinical Immunology (SEICAP) gathers over 350 pediatric allergists and pediatricians working in this field. SEICAP has a growing activity including yearly congresses, continued education courses, elaboration of technical clinical documents and protocols, education of patients, and collaboration with other scientific societies and associations of patients. The official journal of SEICAP is Allergologia et Immunophatologia, published every 2 months since 1972. The web site of SEICAP, http://www.seicap.es, open since 2004, offers information for professionals and extensive information on pediatric allergic and immunologic disorders for the lay public; the web site is receiving 750 daily visits during 2011. The pediatric allergy units are very active in clinical work, procedures as immunotherapy or induction of oral tolerance in food allergy, contribution to scientific literature, and collaboration in international projects.

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.

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.

Occupational respiratory allergy to roe deer.

BACKGROUND: Although scattered reports have been published on roe deer allergenicity, no systematic studies of allergenicity or possible cross-reactions have appeared. OBJECTIVES: To describe 2 patients with occupational roe deer allergy, demonstrated by positive skin and conjunctival provocation test results, and to note cross-reactions to other animal (mainly cow) allergens. METHODS: Two workers at animal rehabilitation centers were sensitized to roe deer. One patient had a history of rhinoconjunctivitis and the other a history of rhinoconjunctivitis and probable asthma. Both patients underwent skin testing with a standard battery of inhaled and epithelial allergens and with roe deer hair and dander extract and conjunctival provocation tests with roe deer hair extract. Immunodetection for IgE (both patients) and IgE immunoblot inhibition tests to determine inhibitory effect (1 patient) were also performed. RESULTS: The results of skin tests and conjunctival provocation tests showed that both patients were sensitized to roe deer allergens. In one patient specific IgE to roe deer extract was detected, and this extract completely inhibited IgE binding to cow hair and dander extract in immunoblotting tests. Specific IgE to roe deer proteins could not be demonstrated in the other patient. CONCLUSIONS: Our results suggest that roe deer epidermal derivatives can cause occupational respiratory disease in exposed workers and that allergy to this species should be considered in individuals who present with similar symptoms and exposure histories. Immunoblot inhibition studies suggested the possibility of cross-reaction between roe deer proteins and cow proteins.

Mite immunotherapy.

Dermatophagoides pteronyssinus and D. farinae are the most common house dust mites and are among the most common sources of indoor allergens worldwide. These species are very common in humid regions, where most allergic individuals are sensitized to house dust mites. Specific immunotherapy with mite extracts has demonstrated clinical benefits in several double-blind, placebo-controlled trials that are included in recent reviews of subcutaneous immunotherapy, including pediatric and adult patients with rhinoconjunctivitis and or asthma. Most successful studies of mite immunotherapy have used native allergen extracts adsorbed onto aluminum hydroxide, or chemically modified mite-allergen extracts. Several studies have also shown efficacy using sublingual immunotherapy in pediatric and adult patients with asthma and/or rhinitis. Additionally, the efficacy of subcutaneous immunotherapy has been demonstrated in patients with atopic dermatitis, although more double-blind, placebo-controlled studies are needed. Based on several studies, it cannot be concluded that mite immunotherapy is more dangerous or safer than immunotherapy with grasses, epithelia, or animal epithelia. Because the delivery of high doses of allergen carries with it the risk for immunoglobulin E (IgE)-mediated events, several methods have been developed to reduce specific IgE binding to mite-allergen extracts. An important challenge for future mite immunotherapy modalities is the delivery of relatively high doses without a significant risk for severe reactions.

In vivo and in vitro allergenicity of the domestic mite Chortoglyphus arcuatus.

BACKGROUND: Chortoglyphus arcuatus is frequently identified in mattress dust samples in coastal areas of northern Spain. OBJECTIVES: To establish the prevalence of positive skin test reactions to C. arcuatus and to analyze its allergenicity and cross-reactivity. METHODS: One hundred thirty-eight consecutive patients entered the study. The main referred symptoms were rhinoconjunctivitis and asthma. Skin tests were performed with extracts of Dermatophagoides pteronyssinus, Lepidoglyphus destructor, Tyrophagus putrescentiae, C. arcuatus, and Glycyphagus domesticus. Enzyme-linked immunosorbent assay inhibition experiments were conducted with the different mite species and immunoblots with serum samples from 31 sensitized patients. Conjunctival provocation tests were performed in 10 patients with C. arcuatus-positive skin test reactions and in 9 with negative results; all the patients had positive skin test reactions to D. pteronyssinus. RESULTS: The prevalence of positive skin test reactions to D. pteronyssinus was 94.2% and to C. arcuatus was 58%. There was a significant correlation between the number of mites to which patients were sensitized and the time of disease evolution (P = .02). Results of enzyme-linked immunosorbent assay inhibition experiments suggested minimal cross-reactivity between C. arcuatus and D. pteronyssinus. Immunoblot results confirmed specific IgE binding to several bands. Conjunctival test results were positive in 9 of 10 C. arcuatus-positive individuals and slightly positive in 2 of 9 C. arcuatus-negative, D. pteronyssinus-positive individuals. CONCLUSIONS: There is a high prevalence of sensitization to C. arcuatus in northern Spain. Sensitization to this species should be considered of clinical significance. There is minimal cross-reactivity between C. arcuatus and D. pteronyssinus.

Patterns of latex allergen recognition in children sensitized to natural rubber latex.

Single recombinant latex allergens permit the study of the pattern of sensitization to individual allergens. We aimed to quantify the IgE-response to individual latex allergens in children sensitized to latex. The study group included 31 latex-sensitized children: 26 operated at least twice, 20 of them with spina bifida; two children with one operation and three atopic non-operated children. IgE antibodies to rHev b 1, rHev b 3, rHev b 5, rHev b 6.01, rHev b 7.02 and rHev b 8, coupled to ImmunoCAPs, were measured in each serum. IgE responses to rHev b 1, rHev b 5 and rHev b 6.01 were found in 17 children each, and their mean +/- s.d. levels were 5 +/- 7.4, 16.8 +/- 14 and 10 +/- 18 kU/l, respectively. IgE responses to rHev b 3 (4 +/- 5.4 kU/l) were found in eight children. Two children had IgE to rHev b 7 (1.7 and 3.2 kU/l), and none to rHev b 8. Four sera were negative to all tested recombinant allergens. We divided the patients in three groups: sensitized only to rHev b 1, sensitized only to rHev b 5 and/or rHev b 6.01, and sensitized to both rHev b 1 and to rHev b 5 and/or rHev b 6.01. The three groups had the same profile of clinical features. Hev b 5 induces the quantitatively higher IgE responses in children with multiple surgeries sensitized to latex. Responses to Hev b 6.01 equal those of Hev b 1.