Allergenic differences between the domestic mites Blomia tropicalis and Dermatophagoides pteronyssinus

BACKGROUND: House dust mite allergens are the most important indoor allergens associated with asthma and rhinitis in Singapore and the tropics. Recent data to suggest that besides the Dermatophagoides spp., the domestic mite Blomia tropicalis (Bt) is also an important source of allergens in these regions. OBJECTIVE: To evaluate the degree of allergenic cross-reactivity between Bt and D. pteronyssinus (Dp). METHODS: Cross-reactivity between extracts of Bt and Dp was evaluated by fluorescent allergosorbent (FAST) inhibition studies and cross enzyme immunoelectrophoresis. Additionally, the major Dp allergens - Der p 1, Der p 2 and Der p 5, were also compared with the Bt extract by dot blot inhibition. Skin prick and intradermal end-point titration were then carried out to compare the homologous allergens of the mite species, Blo t 5 and Der p 5. RESULTS: FAST inhibition studies showed low to moderate cross-reactivity between the two dust mite extracts (maximum cross-inhibition, 60%). Native allergens studied by cross enzyme immunoelectrophoresis using mite allergic sera also showed similar results but with at least four cross-reactive IgE binding antigens. Dot blot inhibition studies using allergens of Dp, Der p 1, Der p 2, and Der p 5, showed little cross-reactivity between these allergens with components of the crude Bt extracts. Further, evaluation of a recombinant allergen of Bt, Blo t 5, showed low levels of cross-reactivity even with its homologous Dp counterpart, Der p 5. CONCLUSION: These results provide evidence that Bt allergens are distinct and have relatively low to moderate cross-reactivity with Dermatophagoides spp. allergens. Bt allergens should therefore be included in the diagnostic panel for the evaluation of allergic disorders in the tropics, and the development of new diagnostic and therapeutic strategies should include allergens of Bt.     

Crossreactivity between Dermatophagoides siboney and other house dust mite allergens in sensitized asthmatic patients

Background The role of Pyroglyphid mites in IgE-mediated serisitizalion has been well eslahlished. Although mites belonging to the genus Dermatophagoidess dominate in the acarofauna of domestic dust. non-pyroglyphid mites might also be of clinical importance. In Cuba, Dermatophagoides siboney is found in dust coexisting with D. pteronyssiuus and Blomia tropicalis. Dermatophagoides forinae is not found. Storage mites, such as Acorus siro, Lepidoglyphus destructor and Tyrophagus putrescentiae might also be present. Objective The aim of this study was to investigate the allergenic relationships among the mites present in the environment of allergic patients in Cuba. Methods The crossreactivialty between an extract of D. siboney and the above mentioned six mites was studied by specific IgE inhibition, using sera of mite-sensitive patients after incubation with serial dilutions of D. sibaney. Results The inhibitory effect of D. sidiney extract was more pronounced against D. farinae. 91%, than against D. pyteronyssinus, 62%. Specific IgE to B. tropicali, A. siro, L. destructor and T. putrescentiae were inhibited to a much lesser extent, i.e. between 47 and 58%. The correlation between IgE to D. siboney and IgE to D. pteronyssinus and D. farinae was of 0.92 and 0.90. respectively. A weaker correlation was found between D. siboney and L. destructor (0.7), A. siro (0.67), B. tropicalis (0.54) and T. putrescentiae (0.51). Conclusion Thus. D. siboney seems to be most closely related to D. farinae.     

Biologic activity of Dermatophagoides siboney and Blomia tropicalis allergens in exposed and unexposed mite‐allergic individuals. Effect of patient selection on the biologic standardization of mite extracts

BACKGROUND: This study aimed to investigate the influence of patient selection criteria, i.e., mite-allergic individuals exposed and not exposed to Dermatophagoides siboney and Blomia tropicalis, on the biologic activity of mite extracts. Determination of the potency of mite extracts in vivo requires selection of patients with a clinical history of mite allergy. In Scandinavia, there are some anamnestic criteria for mite allergy, whereas in the tropics, where patients are continuously exposed to high levels of mites, selection of patients with mite allergy by clinical history is difficult. METHODS: A total of 210 Cuban asthmatics with continuous symptoms, and 43 Swedes with a clinical history of mite allergy were investigated. Skin prick tests were performed with D. siboney, D. pteronyssinus, D. farinae, B. tropicalis, Acarus siro, Lepidoglyphus destructor, and Tyrophagus putrescentiae extracts. For analysis of the biologic activity of mite extracts, Cuban patients were divided into four groups: 1) all patients skin-test-positive to mites 2) patients positive to mites, but not to other inhalant allergens 3) patients reacting most to the mite species analyzed 4) patients reactive only to mites and reacting most to the mite species analyzed. The biologic potency was calculated according to the Nordic Guidelines. RESULTS: Due to cross-reactivity between mites, Swedish mite-sensitive patients, with a clear clinical history of mite allergy, but not exposed to D. siboney and B. tropicalis, were more skin reactive to these mites than were Cubans. The estimated potency increased gradually to >200% in group 4. In group 1 Cubans, the reactivity to all mites but B. tropicalis was lower than that in mite-sensitive Swedes. CONCLUSIONS: According to the influence of patient selection criteria on the estimation of the potency of mite extracts, the determination of the biologic activity of allergenic extracts in subjects without a clear-cut clinical history should be replaced by new methods when available.     

Allergenic composition of the mite Suidasia medanensis and cross-reactivity with Blomia tropicalis

BACKGROUND: Mites of the genus Suidasia are commonly found in house dust and may play an allergenic role in exposed populations. However, the allergenic potential and clinical impact of this genus has not been well established. The main objective of this project was to evaluate the allergenic role of the mite Suidasia medanensis. METHODS: An extract of S. medanensis was prepared and the allergen composition determined by immunoblot. Specific IgE antibody levels to S. medanensis and Blomia tropicalis were evaluated by radioallergosorbent (RAST) in the sera of 97 allergic asthmatic patients and 50 nonallergic subjects. Cross-reactivity between S. medanensis and the mite species B. tropicalis and Dermatophagoides farinae was investigated by RAST and immunoblot inhibitions. RESULTS: Seventy-one asthmatic patients sera (73.2%) had positive IgE reactivity to S. medanensis; 14 allergens with molecular weights ranging from 7.5 to 105 kDa were detected. The most frequently detected had molecular weights of 30-31 (54.8%), 24.5 (42%), 21 (38.7%), 47 (35%) and 58 kDa (35.5%). Blomia tropicalis extract inhibited IgE binding to nine of these identified allergens. Four B. tropicalis allergens were inhibited by S. medanensis extract. RAST inhibition results demonstrated a high degree of inhibition by B. tropicalis (87.2%) and D. farinae (90.9%) than by S. medanensis (32%). CONCLUSIONS: Sensitization to S. medanensis is common in asthmatic allergy patients in Cartagena. An important degree of cross-reactivity was established between S. medanensis and B. tropicalis, and D. farinae.     

Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus

BACKGROUND: Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates. The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens. OBJECTIVES: This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite. Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated. METHODS: Blo t 10 was isolated using mouse anti-Der p 10 antibodies. Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA). Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10. Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10. RESULTS: The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites. SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects. Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10. IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule. The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal. CONCLUSION: Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist. The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.     

Importance of including Blomia tropicalis in the routine diagnosis of Venezuelan patients with persistent allergic symptoms

Background:  Blomia tropicalis is a common mite found in the house dust of many tropical countries including Venezuela. The prevalence of skin test and specific serum immunoglobulin (Ig)E reactivity to B. tropicalis in Venezuela has not been previously evaluated.
Methods:  In the present study we evaluated the skin reactivity by skin prick test and specific IgE by a multiple antigen blot assay, against B. tropicalis and Dermatophagoides pteronyssinus , in a group of 115 subjects who attended the Allergy Clinic of the Institute of Biomedicine, Caracas, Venezuela, and we studied possible cross reactions between similar proteins of these two mites.
Results:  One hundred and six patients with persistent allergic respiratory symptoms showed a positive skin prick test to at least one of the mite extracts, with the frequency of positive reactions to B. tropicalis being as high as to D. pteronyssinus . Twelve patients reacted only to D. pteronyssinus and 13 different patients only to B. tropicalis . Specific IgE to each of the mite extracts was found with similar frequency, and the results coincided with the skin test reactivity.
Conclusions:  The study indicated the importance of including B. tropicalis in routine diagnostic testing in tropical and sub-tropical situations.     

Prevalence of sensitization to the storage mites Acarus siro, Tyrophagus putrescentiae, and Lepidoglyphus destructor in allergic patients with different degrees of sensitization to the house-dust mite Dermatophagoides pteronyssinus

The prevalence of sensitization to the storage mites Acarus siro (AS), Tyrophagus putrescentiae (TP), and Lepidoglyphus destructor (LD) was studied in 250 sera of patients with different degrees of sensitization to the house-dust mite Dermatophagoides pteronyssinus (DP) by measuring IgE binding to extracts of the storage mites. Additionally, allergenic cross-reactivity between DP and the storage mite species was studied by RAST inhibition with five individual sera (and a pool of these sera) with moderate IgE levels to all three storage mites and to DP. Increased serum IgE to storage mites was found in 46% of the 200 patients sensitized to DP. Increased prevalence rates of IgE titers to storage mites were associated with higher IgE levels to DP In 50 sera without sensitization to DP, only five sera showed increased IgE to one of the storage mites. Extracts of TP almost completely inhibited the IgE binding to AS, and vice versa. DP inhibited IgE binding to all storage mites up to 60%, whereas IgE binding to DP was only minimally inhibited by extracts of storage mites. In conclusion, cosensitization to storage mites is a frequent finding in patients sensitized to DP. Although this is largely the result of cross-reactivity between different mite species, it may nevertheless be of clinical significance in patients exposed to storage mites.     

Sensitization to Blomia kulagini in a general population of a subtropical region of Spain (Canary Islands)

García-Robaina JC, Eraso E, Martínez J, Martinez A, de la Torre Morin F, Hernández-Nieto L, Pineda-Algorta J, Guisantes J. Sensitization to Blomia kulagini in a general population of a subtropical region of Spain (Canary Islands). Allergy 1997; 52: 727–731.
The Blomia genus has been described as allergenic in man. The present study aimed to assess the prevalence of B. kulagini sensitization in a large population of allergic subjects without occupational exposure in a subtropical region (Canary Islands, Spain). Secondarily, a new standardized B. kulagini extract was evaluated. The study population comprised 207 patients. RAST for B. kulagini was positive in 76.2% of patients, and 47 of them were selected for the biologic standardization. When the prick test was performed with the nonstandardized extracts, results were positive in 76.6%, whereas when the test was repeated with the standardized extract, sensitivity rose to 95.7%. The conjunctival provocation test was positive in 78.3% of 46 evaluated patients. The bronchial provocation test was positive in 18 sensitized patients and negative in five controls. In conclusion, B. kulagini is an important cause of sensitization among the occupationally unexposed population of the studied area and should be included in allergy diagnostic tests. For reliable prick tests, the use of standardized extracts is mandatory.     

Oral tolerance induction to Dermatophagoides pteronyssinus and Blomia tropicalis in sensitized mice: occurrence of natural autoantibodies to immunoglobulin E

BACKGROUND: The dust mites Dermatophagoides pteronyssinus (Dp) and Blomia tropicalis (Bt) are important sources of indoor allergens in tropical and subtropical countries. Murine models allow the analysis of the immune response and regulation of IgE production to Dp and Bt allergens. Oral tolerance induces unresponsiveness in naive animals, but its application in sensitized animals can provide useful information to improve allergy therapy. OBJECTIVE: To study the profile of IgE and IgG subclasses antibody upon oral administration with Bt and Dp extract in previously sensitized mice. Further, the occurrence of autoantibodies IgG anti-IgE in the immunization and in the oral tolerance was investigated. METHODS: A/Sn mice were immunized with Bt or Dp extract in alum, orally administrated with 0.25 mg of Bt or Dp extract or PBS at the 6th, 7th and 8th days after immunization and boosted twice with their respective allergens. To analyse the mice groups, specific IgE antibodies were measured by passive anaphylaxis reaction and specific IgG subclasses and anti-IgE IgG autoantibody by ELISA assay. RESULTS: IgE levels were markedly increased in Bt-immunized mice compared with Dp-immunized mice. A distinct profile of the specific isotypes was verified in Bt-immunized mice with a preferential production of IgG3 and IgA antibodies, whereas Dp-immunized mice developed high titres of anti-Dp IgG1, IgG2a and IgG2b antibodies. The antigen feeding inhibited IgE response in both fed-mice groups but only Dp-fed mice presented decreased levels of IgG antibodies. Free anti-IgE IgG autoantibodies were detected mainly in the Dp-immunization and they correlated with the antibody isotypes found against the allergen. CONCLUSIONS: This is the first time that the murine-type I hypersensitivity is employed to study Bt-immunization, showing a marked IgE production, associated with IgG response, which is at least in part driven by T-independent antigens. The oral tolerance protocol in previously sensitized animals was able to down-modulate IgE response and points out this route as a strategy for allergy therapy.     

Purification and IgE binding capacity of Der s 3, a major allergen from Dermatophagoides siboney

Background Sensitization to the house dust mite Dermatophagoides siboney has been demonstrated in asthmatic patients. Previously, Dermatophagoides siboney group 1 and group 2 allergens, named Der s 1 and Der s 2, respeetively, have been purified. Objectives The aim of this study was to purify and to study the IgE reactivity of a 30 kDa component, suspected to correspond to group 3 allergens. Methods The protein was purified by affinity chromatography using anti-Der f 3 monoclonal antibodies and semi-preparative SDS-PAGE. The IgE binding capacity of the purified fractions was tested with sera from 106 mite- sensitive asthmatic patients using a modified chemiluminiscent method. Results Affinity chromatography resulted in fractions containing the 30 kDa component which was further purified to homogeneity by SDS-PAGE. Seventythree per cent of the sera showed IgE reactivity to this protein, indicating that it is a major allergen. The protein also reacted with anti Der f 3 polyclonal antibodies and had tryptic activity. There were differences in the reaetivity to Der s 3 according to the age of the patients. Conclusion Based on the frequency of IgE reactions and the reactivity with antibodies directed to Der f 3, it is proposed to name this 30 kDa allergen from D. siboney, Der s 3.     

热带剥爪螨变应原Blot 5的克隆表达、纯化及免疫学鉴定

目的:克隆热带剥爪螨主要变应原Blot 5基因,表达纯化该蛋白,并检测其免疫活性。方法:提取热带剥爪螨总RNA,采用RT-PCR的方法扩增Blot 5编码基因,将其连入原核表达载体pET-19b,将重组质粒转化入大肠杆菌BL21 Star(DE3)pLysS,IPTG诱导表达后,通过Ni2+亲和层析纯化重组变应原Blot 5。用Dot blot、Western blot和ELISA等方法检测重组变应原Blot 5免疫活性。结果:Dot blot和Western blot结果表明重组变应原Blot 5和热带剥爪螨粗提液都能和Blot 5鼠单克隆抗体结合。重组变应原Blot 5检测69份热带剥爪螨过敏患者血清和21份户尘螨过敏患者血清中的特异性IgE,阳性率分别为29.0%和33.3%。结论:表达和纯化了具有与天然蛋白相似的免疫活性的重组热带剥爪螨变应原Blot 5,可用于热带剥爪螨变应原的标准化,为标准化抗原的临床特异性诊断与治疗奠定基础。     

Determination of Blomia tropicalis-specific IgE and IgG subclasses in atopic dermatitis patients

BACKGROUND: To verify the importance of Blomia tropicalis in atopic dermatitis (AD), we determined the cutaneous reactivity and the serum level of B. tropicalis-specific IgE and IgG subclasses in AD patients. METHODS: B. tropicalis-specific IgE and IgG subclasses were determined in AD patients and compared with bronchial asthma (BA) patients and a control group (CG) of nonatopic subjects. Specific IgE was obtained by skin prick test and RAST. B. tropicalis-specific IgG subclasses were determined by ELISA. The data were statistically analyzed by chi-square test (Mantel-Haenszel) and odds ratio (OR). RESULTS: We detected positive skin prick tests in 61.76% of AD and 83.33% of BA patients, and in 12.5% of the CG. RAST was positive in 44.12% of AD and in 61.90% of BA patients, but not in the CG. B. tropicalis-specific IgG1 and IgG2 subclasses showed no significant differences between the three groups. IgG3 subclass positivity was statistically significant in AD patients (41.17%) when compared to BA patients (14.29%) and the CG (16.67%). The determination of B. tropicalis-specific IgG4 was positive in 32.35% of AD patients, 21.43% of BA patients, and 8.33% of the CG. CONCLUSIONS: These results confirm that the storage mite B. tropicalis is an important allergen in AD. It is possible that IgG3 activates the complement in AD patients, releasing vasoactive amines that further amplify the allergic reaction. The positive results of the B. tropicalis-specific IgG4 found in AD and BA were probably due to chronic exposure to this storage mite in the home environment.     

Sensitization to local dust-mite fauna in Singapore

BACKGROUND: Recent studies showed the presence of a unique dust-mite fauna in the indoor environment of Singapore. Immediate hypersensitivity to these dust mites, along with other known indoor allergens, may play a role in the pathogenesis of allergic respiratory diseases. This study evaluated the sensitization rates of the local atopic population to these allergens. METHODS: The skin prick test was performed on a total of 391 individuals (289 patients with asthma and/or allergic rhinitis and 102 healthy controls) using extracts of six species of local dust mites (Austroglycyphagus malaysiensis, Blomia tropicalis, Dermatophagoides pteronyssinus, D. farinae, Sturnophagoides brasiliensis, and Tyrophagus putrescentiae) and 10 other common indoor allergens. Total serum IgE and specific IgE to these dust mites were also quantified with the fluorescence allergosorbent test (FAST). RESULTS: The sensitization rates among patients with asthma and/or allergic rhinitis to dust mites and other inhalant allergens tested (via skin prick tests) were as follow: B. tropicalis (96.2%), D. pteronyssinus (93.4%), D. farinae (92.3%), A. malaysiensis (78.2%), S. brasiliensis (71.6%), T. putrescentiae (71.3%), canary feathers (69.9%), Periplaneta americana (cockroach) (59.5%), Blattella germanica (cockroach) (56.4%), mosquito (Aedes sp.) (46.4%), dog epithelia (mixed breed) (34.3%), kapok seed (31.8%), cat hair (29.1%), Aspergillus fumigatus (20.8%), Penicillium notatum (18.0%), and Candida (Monilia) albicans (9.3%). All patients were observed to react to at least three of the six dust-mite extracts, with 254/289 (87.9%) reacting to at least five or to all six. Skin prick responses to the dust mites were found to correlate with the corresponding specific IgE levels quantified by FAST (P<0.001). In addition, specific IgE levels to D. pteronyssinus and D. farinae were highly correlated (Spearman's rank coefficient = 0.76, P<0.001), as were those to B. tropicalis and A. malaysiensis (r = 0.60, P<0.001). CONCLUSIONS: Asthma and/or allergic rhinitis patients were highly sensitized to the local dust-mite fauna. Thus, these dust mites should be considered important allergenic sources of this region.     

Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin (Blo t 11)

BACKGROUND: The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms. METHODS: Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno-affinity chromatography, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and verified by MALDI-TOF MS. Recombinant full-length Blo t 11 (rFL-Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli. Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme-linked immunosorbent assay (ELISA) and skin prick test. The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies. RESULTS: Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at approximately 66 kD. The nBlo t 11 was confirmed by immunoblot analysis and MALDI-TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11. Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL-Blo t 11, has comparable IgE reactivity with the native counterpart. CONCLUSIONS: This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11. This recombinant peptide, instead of the full-length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.     

Sensitization to dust mites in children with allergic rhinitis in Singapore: does it matter if you scratch while you sneeze?

BACKGROUND: Previously published data established Blomia tropicalis, as the major source of allergic sensitization in asthmatic children in tropical Singapore. Objective To define the prevalence, clinical characteristics and risk factors of species-specific mite sensitization in paediatric allergic rhinitis (AR) patients in this unique environment. METHODS: We performed a prospective evaluation of newly diagnosed AR patients, from 1 May 2003 to 30 April 2004, from the otolaryngology and allergy outpatient clinics of the Kendang Kerbau Children's Hospital in Singapore. Patients included in the study showed evidence of sensitization to at least one respiratory allergen source and completed a detailed questionnaire. Relative risk of sensitization and associated risk factors were calculated using logistic regression analysis with the forward stepwise model. Multivariate regression analysis was performed to adjust for confounding interactions. Continuous values were compared using anova, SPSS 9.0 for Windows (SPSS Inc., 1999). RESULTS: One hundred and seventy-five patients were included, 119 (68%) males, 142 (81%) Chinese, age mean 7.9 years (range 2-16). Sixty-eight patients (39%) reported a concomitant diagnosis and/or clinical complaints of bronchial asthma and 84 (48%) of atopic dermatitis. Skin prick test results were positive for traditional house dust mites (Dermatophagoides pteronyssinus. and D. farinae mix) in 85% of patients and for B. tropicalis in 62%. Overall mite sensitization was 98%, household pets 10%, moulds 9% and food proteins 12%. By far the single most significant factor associated with Dermatophagoides sensitization in this group was the presence of allergic eczema (odds ratio (OR) 31.8%, 95% confidence interval (CI) 3.6-285, P=0.002). Allergic eczema was negatively associated with B. tropicalis sensitization (OR 0.26%, 95% CI 0.14-0.5). CONCLUSIONS: Children with AR and concomitant atopic dermatitis show a preferential sensitization to the Dermatophagoides mites. In our population, B. tropicalis sensitization is more prominent in children with pure respiratory allergy.     

Cloning and expression of Blo t 1, a novel allergen from the dust mite Blomia tropicalis, homologous to cysteine proteases

BACKGROUND: House dust mite allergens have been shown to be a very important stimulus in the causation of asthma and triggers for the exacerbation of symptoms. Therefore, characterization of mite-derived allergens at the molecular level is an important step for the development of effective diagnostic and therapeutic approaches, as well as for epidemiological studies. OBJECTIVE: To clone, express and characterize at the molecular level the cysteine protease from Blomia tropicalis (Bt). METHODS: A full-length cDNA encoding Blo t 1 was cloned from a Bt cDNA library using a PCR and RACE-based strategy. The cDNA was PCR-amplified, sequenced and subcloned into a prokaryotic expression vector. The allergenicity of the recombinant Blo t 1 was evaluated for IgE reactivity by Western blot. RESULTS: Blo t 1 cDNA encodes a 221 amino acids polypeptide with an estimated molecular weight of 25 kDa. The recombinant protein is 35% identical to other mite cysteine proteases. Recombinant Blo t 1 (rBlo t 1) bound IgE from 62% of Bt skin test-positive serum. Dermatophagoides pteronyssinus (Dp) skin test-positive sera did not react with rBlo t 1, indicating the possible presence of unique IgE epitopes on the rBlo t 1 molecule. A three-dimensional image of Blo t 1, constructed based on predicted analysis, showed conserved secondary and tertiary structure with other cysteine proteases. CONCLUSION: We report the cloning, expression and IgE reactivity of Blo t 1, a novel allergen from the domestic mite Blomia tropicalis (Bt), highly homologous to cysteine proteases. This putative cysteine protease, designated Blo t 1, may play a major role as an immunodominant allergen involved in dust mite-specific IgE-mediated hypersensitivity.     

IgE, IgG1, and IgG4 antibody responses to Blomia tropicalis in atopic patients

BACKGROUND: Allergens from house dust mites (HDMs), Dermatophagoides pteronyssinus and Blomia tropicalis are clinically relevant in atopic respiratory diseases in tropical countries. AIMS OF THE STUDY: To evaluate immunoglobulin (Ig)E, IgG1, and IgG4 antibody responses to B. tropicalis in Brazilian atopic patients. METHODS: About 110 patients with allergic rhinitis with/without asthma and 33 control subjects underwent skin prick testing (SPT) with HDM extracts, and their sera were tested for IgE and IgG subclass antibodies to D. pteronyssinus and B. tropicalis by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Most patients (56%) had positive SPT to B. tropicalis extract (B. tropicalis+ group), although 51% were reactive to both B. tropicalis and D. pteronyssinus and 6% were sensitized to B. tropicalis only. IgE-ELISA detected 43%B. tropicalis positivity with high-specific IgE levels in B. tropicalis+ patients. Specific IgG4 levels were higher in B. tropicalis+ than B. tropicalis- groups and correlated with specific IgE levels. The IgG1 levels to B. tropicalis were higher in patients than controls. The major allergenic B. tropicalis components recognized by B. tropicalis+ patient sera were the 54, 66, and 68 kDa proteins. The IgG4-binding protein profiles closely resembled that of IgE. The IgG1 antibodies recognizing multiple B. tropicalis protein species were detected in sera of all three patient groups. CONCLUSIONS: A large percentage of our allergic patients are B. tropicalis+. They are more frequently sensitized to high-molecular weight (HMW) B. tropicalis components than the major low-molecular weight (11-15 kDa) allergens detected in other studies. The results suggest that HMW B. tropicalis antigenic components are potential candidates for evaluating allergen exposure and sensitization, and for immunotherapy treatment.     

Peptide mapping of immunoglobulin E and immunoglobulin G immunodominant epitopes of an allergenic Blomia tropicalis paramyosin, Blo t 11

BACKGROUND: The identification of immunodominant peptides containing the IgE and IgG epitopes on allergen molecules is an important step in understanding the interaction of the allergen with the immune system and, thus, essential for the development of effective immunotherapeutic and diagnostic reagents. The present study aimed to map the IgE and IgG immunodominant peptides of Blomia tropicalis (Bt) allergen Blo t 11, a high molecular weight allergen homologous to paramyosin, exhibiting important allergenic activity. METHODS: Eleven overlapping fragments of Blo t 11 cDNA gene were expressed as glutathione s-transferase (GST) fusion peptides, which were affinity-purified using the glutathione-Sepharose column. Human IgE and IgG immunodominant peptides were determined by dot blot immunoassay using crude Bt extract-positive sera from asthmatic patients. Evaluation of allergenicity, specific hIgG subclass analysis, and cross- and self-inhibition studies were determined by enzyme-linked immunosorbent assay. RESULTS: Blo t 11 contains multiple IgE and IgG immunodominant peptides scattered throughout the molecule. The dominant IgE and IgG peptides were mapped at amino acid positions 336-557 and 698-875, respectively. An immunodominant peptide (fD) registered a higher percentage of IgE and IgG reactivity compared to the rFL-Blo t 11. Significant serum levels of Blo t 11- and fD-specific IgG1, IgG2 and IgG4, but not IgG3 were detected in the Bt extract-positive sera tested. Cross-inhibition study revealed the rFL-Blo t 11 was significantly inhibited by fD. CONCLUSION: The IgE and IgG immunodominant peptides of Blo t 11 have been mapped. Our data suggest that utilization of Blo t 11 fragment(s) or chimeric fusion fragments containing IgE and IgG epitopes could be a better alternative in the development of diagnostic and therapeutic reagents for mite allergy.     

DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis

BACKGROUND: Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA-based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production. OBJECTIVES: To use a DNA-based immunization protocol for the production and characterization of Blo t 11 monoclonal antibodies (mAbs). METHODS: The 2625 bp cDNA coding for Blo t 11 was cloned into a mammalian expression vector and immunized intramuscularly with electroporation into mice. Monoclonal antibodies to Blo t 11 were generated using a methylcellulose-based hybridoma cloning kit. These mAbs were utilized for native Blo t 11 isolation and the development of sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Six mAbs recognizing the native and recombinant Blo t 11 were generated and characterized. Native Blo t 11 was affinity purified from Bt extract and its identity was confirmed by matrix assisted laser desorption/ionization - time of flight mass spectrometry. The native Blo t 11 showed IgE reactivity with 67% of mite allergic sera. A two-site ELISA developed showed a detection limit of 100 pg/ml of Blo t 11. CONCLUSION: A DNA-based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.     

Skin test reactivity to natural and recombinant Blomia and Dermatophagoides spp. allergens among mite allergic patients in the UK

BACKGROUND: Many asthmatics in tropical and subtropical areas have positive skin prick tests to both Dermatophagoides spp. and to the mite Blomia tropicalis. This may be due to recognition by IgE of cross-reactive allergens between the different mite species or because of sensitization to species-specific allergens. A 14-kDa Blomia tropicalis allergen, Blo t 5, has been cloned and shows 40% sequence homology with Der p 5. The aim of this study was to investigate reactivity to B. tropicalis in patients known to be sensitized to D. pteronyssinus and to assess allergenic activity and cross-reactivity of recombinant (r) Group 5 allergens amongst these patients, who live in the UK and who are not exposed to B. tropicalis in their homes. METHODS: Patients (n = 19) with asthma and/or rhinitis were selected based on clinical history and a positive skin prick test to D. pteronyssinus extract and were compared with non-allergic skin test negative controls (n = 10). IgE antibody responses to Blomia tropicalis, Dermatophagoides pteronyssinus, rDer p 5 and rBlo t 5 were compared by quantitative intradermal skin testing using serial 10-fold dilutions of each allergen. End point titre was the highest dilution giving an 8 x 8 mm wheal at 15 min. IgE antibodies to Blomia tropicalis, Dermatophagoides pteronyssinus, rDer p 5 and rBlo t 5 were measured using RAST, CAP and RIA, respectively. RESULTS: All 19 patients had positive skin tests to D. pteronyssinus at concentrations of 0.001 to 1 AU/ml and 10 were skin test positive to rDer p 5 at concentrations of 10-4 to 5 micro g/ml. Positive intradermal tests to Blomia tropicalis were seen in 12/19 patients at concentrations of 0.002 to 2 micro g/ml. However none of the patients had positive skin tests to rBlo t 5. Non-allergic controls were all skin test negative at the highest concentration of each allergen tested. All subjects had quantifiable specific IgE to D. pteronyssinus, but only two had IgE to B. tropicalis. IgE to Der p 5 was found in six patients, but no patients had IgE to Blo t 5. CONCLUSIONS: This study of patients naturally exposed to D. pteronyssinus but not to Blomia tropicalis, provides evidence for IgE mediated cross-reactivity between allergens produced by both mite species. The results suggest that the Group 5 allergens of D. pteronyssinus and B. tropicalis are species-specific.