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.     

The upper and lower airway responses to nasal challenge with house-dust mite Blomia tropicalis

BACKGROUND: The house dust mite Blomia tropicalis (B. tropicalis) was found to be the most prevalent domestic mite in Singapore. However, its pathogenicity in allergic airway diseases remains to be investigated. METHODS: Twenty adults with persistent allergic rhinitis (PAR) were studied. Five had a history of asthma, and all were asymptomatic except one who was under treatment with low-dose inhaled corticosteroid. Nasal challenge was carried out by nasal spray with phosphate-buffered saline (PBS) and with increasing concentrations of crude B. tropicalis extracts (0.6, 6.0 and 60 micro g/ml) at 15 min intervals. Subjective symptom scores and absolute number of sneezes were recorded together with objective measurements of spirometry (forced expiratory volume in 1 s, FEV1) and acoustic rhinomanometry (volume of the nasal cavity). These were performed at baseline, 5 min after each incremental challenge, and 30 min, 1 h, 3 h, 5 h and 7 h after the last challenge. Meanwhile, concentrations of mediators in nasal secretions (tryptase, leukotriene C4 (LTC4) and eosinophil cationic protein (ECP)) were measured in nasal aspirate samples at similar time intervals. An identical (control) challenge procedure with PBS alone was repeated in seven patients after a washout period of at least 2 weeks. RESULTS: Significant increases in the subjective and objective nasal symptoms, together with a significant increase of tryptase and LTC4 concentrations in nasal secretion, were found 5 min after each challenge with B. tropicalis, but not with PBS. There was no definitive pattern of the late-phase nasal response in either subjective symptoms or objective measurements. Three patients (3/5) with a history of asthma showed a fall in FEV1 readings (33%, 22% and 11% from baseline, respectively) at 7 h post challenge with concomitant mild wheezing in the night. CONCLUSIONS: Our study demonstrates direct evidence of allergic nasal response to B. tropicalis in sensitized adults. It shows that nasal provocation may also provoke concomitant asthmatic symptoms during the late-phase reaction, especially in people with a history of asthma.     

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.     

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.     

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.     

Sensitization to Dermatophagoides siboney, Blomia tropicalis, and other domestic mites in asthmatic patients

Mite species adapted to warm, humid climates are commonly found in house dust in the tropics. In Cuba, Dermatophagoides pteronyssinus, D. siboney , and Blomia tropicalis are the most common and abundant mite species in house dust. To investigate the pattern of Sensitization of Cuban asthmatic patients to common mite species, we skin-prick-tested (SPT) 148 patients with a clinical history of asthma and possible mite allergy, and determined specific IgE antibodies against mite allergens ( D. pteronyssinus, D. farinae, D. siboney, B. tropicalis, Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae , and Glycyphagus domesticus ). The prevalence of positive SPT was high to D. siboney (88%), D. pteronyssinus (87%), A. siro (85%), B. tropicalis (85%), and D. farinae (83%). The largest skin reactions were obtained with D. siboney and B. tropicalis extracts. The skin test response to the D. siboney extract correlated to those of D. farinae, D. pteronyssinus, B. tropicalis , and A. siro. The highest IgE levels were found to Dermatophagoides species and B. tropicalis. IgE to D. siboney and B. tropicalis were found in 97% and 96% of the patients, respectively. The prevalence of specific IgE to the other mites studied varied from 46 to 65%. D. siboney and B. tropicalis are important sensitizers among asthmatic patients in Cuba.     

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。     

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.     

Blomia tropicalis allergen 5 (Blo t 5) T‐cell epitopes and their ability to suppress the allergic immune response

Blomia tropicalis is the major asthma allergen in the tropics comparable to Dermatophagoides pteronyssinus. However, little is known about the B. tropicalis epitopes recognized by T cells. Our aim was to identify the T‐cell epitopes in the major B. tropicalis allergen, Blo t 5, and investigate the potential of the corresponding peptides to inhibit the allergic inflammatory lung response. C57BL/6 mice were immunized with plasmid DNA encoding Blo t 5 and T‐cell epitopes identified using the interferon‐γ ELISPOT assay with 15‐mer overlapping peptides. C57BL/6 mice were sensitized with bone‐marrow‐derived dendritic cells (BMDC) pulsed with Blo t 5 allergen followed by intranasal Blo t 5 challenge. Two H‐2^b restricted epitopes (Bt5_76–90 and Bt5_106–115) were recognized by CD4 T cells specific for Blo t 5, but no CD8 epitopes were identified. In mice sensitized with Blo t 5‐pulsed BMDC and challenged with intranasal Blo t 5 Bt5_76–90 and Bt5_106–115, peptide‐specific CD4 T cells were found to secrete the T helper type 2 cytokines interleukin‐5 and interleukin‐13. Intradermal administration of synthetic peptides encoding the identified T‐cell epitopes suppressed allergic airway inflammation to further allergen challenges. Hence, we have identified novel CD4 T‐cell epitopes specific for Blo t 5 and demonstrated that these peptides could be employed therapeutically to suppress the T‐cell response in a murine model of allergic airway inflammation.     

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.     

IgE, IgA, and IgG responses to common yeasts in atopic patients

This study was undertaken to analyze the differences in exposure and sensitization to five common environmental yeasts. The responses of IgG, IgA. and IgE to Candida albicans. C. utilis, Cryptococcus albidus, Rhodotorula rubra, and Saccharomyces cerevisiae and purified S. cerevisiae enolase were analyzed by immunoblotting (IgE-1B), and the cross-reactivity of their IgE-binding components by IgE-1B inhibition. Twenty atopic subjects, with asthma, allergic rhinitis, or atopic dermatitis were included. In skin prick tests (SPT), 12 of the patients showed simultaneous reactivity to at least two of the five yeasts, four reacted to one of the yeasts, and four had no responses. Antigens run in SDS-PAGE and transferred to nitrocellulose were probed with enzyme-labeled IgA-, IgG-, and IgE-specific antibodies. The IgE immunoblotting revealed most IgE-binding bands in C. albicans (11 bands) followed by C. utilis (eight bands), S. cerevisiae (five bands), R. rubra (five bands), and Cr. albidus (four bands). Six of the IgE-binding bands of C. albicans and C. utilis shared molecular weight, and only two bands shared molecular weight with other yeasts. These were the 46-kDa band, shared by all five yeasts, and a 13-kDa band shared by four yeasts. Prominent IgE binding was seen to a 46-kDa band of C. albicans (seven patients), C. utilis (five patients), and S. cerevisiae (one patient) and to corresponding weak bands of Cr. albidus and R. rubra (one patient). The possible cross-reactivity of the 46-kDa band was analyzed by IgE-IB inhibition and densitometry, revealing clear C albicans inhibition of C. utilis (80%) and enolase (98%) (autoinhibition 100%). The strongest IgG responses were seen against S. cerevisiae and C albicans. The responses were mainly against mannans of C. albicans and S. cerevisiae, suggesting that most of the exposure is to these yeasts. Yeasts with different types of exposure, from saprophytic growth on human mucous membranes to exposure by air and food, were shown to cross-react at the allergenic level. Atopic patients primarily sensitized by C albicans and S. cerevisiae may develop allergic symptoms by exposure to other environmental yeasts due to cross-reacting IgE antibodies.     

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.     

Immunological characterization of Echinococcus granulosus cyclophilin, an allergen reactive with IgE and IgG4 from patients with cystic echinococcosis

By immunological screening of a cDNA library derived from protoscoleces of Echinococcus granulosus with IgE from patients with cystic echinococcosis (CE) and allergic manifestations, we isolated a protein identical to E. granulosus cyclophilin. The protein, named EA21, has close homology with Malassezia furfur cyclophilin allergen (Mal f 6) and with human cyclophilin. Using immunoblotting (IB) with a polyclonal antibody specific to EA21, we identified E. granulosus cyclophilin both in protoscoleces and in sheep hydatid fluid. Of the 58 sera from patients with CE, 29 (50%) were IgE positive to EA21, whereas, despite the high sequence homology, none were IgE positive to Mal f 6 or human cyclophilin. Only 26 of the 58 patients (45%) had IgG specific to EA21, whereas all patients (100%) had IgG specific to Mal f 6 and human cyclophilin. IB analysis showed that serum IgE-binding reactivity to EA21 differed significantly in patients with and without allergic reactions (20 of 25, 80% versus nine of 33, 27%; P < 10(-4)). Conversely, five of the 25 patients who had CE-related allergic manifestations (20%) and 21 of the 33 who did not (63%) had specific IgG4 (P = 10(-3)) and total IgG to EA21. EA21 induced a proliferative response in 15 of 19 (79%) patients' PBMC regardless of the allergic manifestations, but it induced no IL-4 production. Overall, these findings suggest that E. granulosus cyclophilin is a conserved, constitutive, parasite protein that does not cross-react with cyclophilins from other organisms and is involved in the allergic symptoms related to CE.     

Comparison of IgE and IgG antibody responses of atopic individuals with sensitization to tree and grass pollens

Sera of atopic individuals with predominant sensitization to either tree pollen (TAs) or tree and grass pollens (TGAs) as well as of nonatopic subjects (NAs) were analyzed for IgE, IgG, and IgG4 antibodies specific for grass pollen allergens. Of 600 atopic individuals with serum IgE antibodies specific for birch pollen allergens, 54% also had serum IgE antibodies specific for grass pollen. The mean titers of IgG antibodies specific for grass pollen proteins were about 10 times higher in the sera of TGAs than those in the TAs and NAs. SDS-PAGE immunoblotting analysis of grass pollen proteins using sera of TGAs, TAs, and NAs with respect to the binding of these proteins with IgE and IgG antibodies in these sera exhibited a similar pattern of variation. Quantitation by enzyme immunoassay of the antibody binding to a recombinant grass pollen allergen, rKBG8.3, further demonstrated that elevated IgG antibody levels in TGAs are mainly due to a broader range of specificities, and not to high specific binding to the individual protein. Statistically significant correlation was found between IgE and IgG4 antibodies specific for the Kentucky bluegrass (KBG) extract, but not for the isolated recombinant allergen. These results indicate that the grass pollens elicit a complex array of antibody specificities in both atopics and nonatopics, and that the profile of antibodies specific to the pollen extract and pure allergens differs, suggesting that single grass allergens may be inadequate for replacing grass pollen extracts for immunotherapy.     

Differential effects of gangliosides on human IgE and IgG4 production

The effects of gangliosides on human IgE and IgG4 production were studied. Of the various gangliosides tested, only GM2 and GM3 inhibited the IgE and IgG4 production induced by interleukin (IL)-4 plus hydrocortisone (HC), or that induced by IL-13 plus HC, in human surface IgE- and IgG4-negative (sIgE^?, sIgG4^?) B cells without affecting the production of IgG1, IgG2, IgG3, IgM, IgA1 or IgA2. In contrast, GM1, GD1a, GD1b, GD3, GT1b and GQ1b were without effects. The GM2- and GM3-mediated inhibition was specific, since each was blocked by a corresponding antibody. Of the various factors tested, IL-6, IL-10, and tumor necrosis factor (TNF)-α enhanced the IgE and IgG4 production induced by IL-4 plus HC or by IL-13 plus HC, while IL-8 and transforming growth factor (TGF)-β inhibited these responses. However, only TNF-α counteracted the GM2- and GM3-mediated inhibition of IgE and IgG4 production, while IL-6, IL-10, anti-IL-8 monoclonal antibody and anti-TGF-β antibody failed to do so. Anti-TNF-α monoclonal antibody, but not control IgG1, not only inhibited IgE and IgG4 production in the absence of TNF-α but also blocked the counteraction of inhibition by TNF-α. In cultures containing IL-4 plus HC or IL-13 plus HC. GM2 and GM3 specifically inhibited TNF-α production without affecting TNF-α receptors, IL-6 production or IL-6 receptors. These results indicate that GM2 and GM3 inhibit IgE and IgG4 production by inhibiting endogenous TNF-α production.     

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.     

Lack of human IgE cross-reactivity between mite allergens Blo t 1 and Der p 1

BACKGROUND: The group 1 mite allergens are the most significant indoor allergens and they belong to the papain-like cysteine protease family. To date there is only one published report on the isolation and characterization of group 1 allergens from Blomia tropicalis mites. The aims of the study are to determine the cross-reactivity between group 1 allergens and to evaluate their clinical importance in allergic patients. METHODS: The full-length Blo t 1 gene was obtained by SMART RACE cDNA amplification method using gene-specific primers. The sequence alignment was performed using LOOK followed by three-dimensional homology modeling. The cDNA was expressed in Pichia pastoris as a secretory protein. Identification of native Blo t 1 in crude mite and spent mite medium extracts was done by Western immunoblot using monoclonal antibody. Allergenicity of recombinant Blo t 1 and native Der p 1 was examined by human IgE ELISA with 80 asthmatic sera. RESULTS: The cDNA sequence consists of 1105 base pairs, including 5'- and 3'-untranslating regions, encoding an open reading frame of 330 amino acid residues. The predicted molecular weight of the deduced protein was approximately 38 kDa. Blo t 1 shared 53 and 34% nucleotide and amino acid, respectively, sequence homology with Der p 1. Native Blo t 1 was detected in both crude mite and spent mite medium extracts, and its estimated molecular weight was about 26 kDa. The recombinant Blo t 1 reacted positively with IgE in 90 and 65% of sera from asthmatic children and adults, respectively, indicating that it is a major allergen. The correlation of human IgE reactivity between Blo t 1 and Der p 1 was low in these sera. CONCLUSION: The full-length cDNA encoding group 1 Blomia tropicalis mite allergen (designated as Blo t 1) has been characterized and expressed from local mites in Singapore. This fecal allergen showed high frequency of human IgE reactivity with asthmatic sera in the tropics and there was a low correlation of IgE reactivity between Blo t 1 and Der p 1.     

IgG1 and IgG4 antibody responses to the dust mite Lepidoglyphus destructor in a naturally exposed farming population

The natural humoral immune response to the dust mite Lepidoglyphus destructor was assessed by comparing the IgG1 and IgG4 responses elicited in allergic ( n = 44) and healthy ( n = 16) individuals in a farming population chronically exposed to this allergen. With the aid of an immunoblotting technique and ELISA, the sera were analyzed for anti- L. destructor antibodies. While the majority of sera from the allergy group displayed several bands for both IgG1 and IgG4, the nonatopic healthy group was negative as analyzed in immunoblotting. When they were analyzed in ELISA, there was a significantly higher response in the allergy group than in the healthy group for IgG4, but not for IgG1. Taken together, these results imply that the immune system of individuals spared from allergic reactions to L. destructor not only lacks IgE antibodies but also seems largely to "ignore" these allergens/antigens despite exposure.