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Background: Probiotics are used as a management strategy for allergic diseases, but their effects on allergic responses in sensitized allergic individuals remain unclear. This study explored the immunomodulatory effects of probiotics on allergen-specific allergic reactions in an allergy mouse model., Methods: C57BL/6 mice were presensitized by epicutaneous patching with recombinant Der p 2, and were subsequently administered orally with either heat-killed wild-type Lactobacillus casei or NaHCO(3) buffer for 5 weeks (n=6 per group). All mice then received 2 subcutaneous immunizations of Der p 2 to mimic allergen immunotherapy, followed by aerosol challenge with Der p 2 a week later., Results: The Der p 2-sensitized mice fed L. casei showed significantly lower Der p 2-specific IgE and IgG1 after subcutaneous immunizations and airway challenge with Der p 2 compared to the control, unfed group. Splenic T cells of mice fed L. casei showed suppression of Th-2 (IL-5, IL-13, IL-10 and IL-4) and proinflammatory (TNF-alpha, IFN-gamma) cytokines. Following airway allergen challenge, the mice fed L. casei showed histological evidence of attenuation of lung inflammation, as well as reductions in the total cell count and Th2 and proinflammatory cytokines in bronchoalveolar lavage fluid, compared to controls. Taken together, these results suggest that the oral administration of heat-killed L. casei could effectively downregulate the pre-existing Th-2 allergic responses and pulmonary inflammatory responses upon subcutaneous and airway allergen challenge., Conclusions: L. casei has intrinsic adjuvant and immunomodulatory properties that could potentially be exploited for secondary prevention or treatment of allergic respiratory diseases., (Copyright 2008 S. Karger AG, Basel.)

Background: Blomia tropicalis is an important domestic dust mite in the tropics and subtropics. This study describes cDNA cloning of the group 4 allergen of B. tropicalis, and the evaluation of the sensitization of this allergen in atopic populations from 2 geographic regions., Methods: cDNA cloning was carried out using the Smart RACE cDNA amplification kit. The full-length Blo t 4 cDNA was isolated by cDNA library screening, 5' and 3' rapid amplification of cDNA ends and long-distance PCR. Sequence analysis was performed with a combination of the Clustal W, CGC and Blast program packages. The cDNA was expressed in Pichia pastoris yeast. The skin prick test was used to evaluate the sensitization profile of recombinant Blo t 4, crude dust mite allergen extracts and major B. tropicalis recombinant allergen Blo t 5., Results: The cloned Blo t 4 had a molecular weight of 56 kDa and had 68% amino acid homology with group 4 allergens of Dermatophagoides pteronyssinus and 65% with those of Euroglyphus maynei. A sensitization profile to the expressed recombinant Blo t 4 allergen (28%) showed an unusually higher frequency than to the major allergen Blo t 5 (22%) in allergic subjects from Chengdu, PR China. In comparison, the subjects from Singapore showed very low sensitization to Blo t 4 (4%) compared with Blo t 5 (84%)., Conclusions: Group 4 allergens of B. tropicalis may be an important dust mite allergen in certain distinct populations., (2008 S. Karger AG, Basel.)

Background: Recent studies have demonstrated differences in the composition of gut microbiota in infants with and without allergic diseases, particularly eczema., Methods: A case-control study involving 21 toddlers (age 3.0 +/- 0.5 years) with and 28 age-matched toddlers without eczema was conducted. Four groups of aerobic gut microbiota were identified and quantitated in stool samples grown on selective media. Three groups of anaerobes were enumerated by fluorescent in situ hybridization followed by quantitative flow cytometry. We also performed molecular typing of lactic-acid-producing bacteria (LAB) and enterococcal isolates to facilitate detailed analysis at species level by bacterial 16S rDNA sequencing., Results: Toddlers with eczema harbored significantly lower counts of Bifidobacterium [(median 0.14 (25th and 75th percentile: 0.04 and 0.47) vs. 0.71% (0.16, 1.79) of cells acquired, p = 0.003)] and Clostridium [(0.28 (0.09, 0.78) vs. 0.83% (0.35, 1.82) of cells acquired, p = 0.012)] but significantly higher counts of total LAB [7.3 (6.1, 8.5) vs. 5.7 (4.4, 7.3) log CFU/g, p = 0.006] in particular enterococci [6.3 (4.8, 7.4) vs. 5.0 (3.4, 6.4) log CFU/g, p = 0.018]. There was no significant correlation between eczema severity score and bifidobacterial counts., Conclusion: The results further confirm previous reports that the gut microecosystem differs between children with and without eczema and extend them beyond infancy.

Background: The house dust mites Dermatophagoides pteronyssinus (Der p) and Blomia tropicalis (Blo t) are the most common house dust mite species in Southeast Asia. To date, there have only been a few studies on the sensitization profile of the general populations in Southeast Asia to house dust mites. The aim of this study was to determine the profiles of Der p and Blo t sensitization among Singaporean and Malaysian subjects., Methods: Enzyme-linked immunosorbent assay was used to detect specific IgE to Der p and Blo t mite crude extracts as well as purified Der p 1, Der p 2 and Blo t 5 allergens. Sera used were from 229 Singaporean subjects (124 with rhinitis, 105 without rhinitis) and 143 Malaysian subjects (94 adults and 49 children with asthma)., Results: The sensitization profile of rhinitis subjects to the dust mite allergens used in this study was as follows: Blo t extract positive: 91/124 (73%); Blo t 5 positive: 62/124 (50%); Der p extract positive: 61/124 (49%); Der p 1 positive: 53/124 (43%); Der p 2 positive: 45/124 (36%). The nonrhinitis subjects' sensitization profile was as follows: Blo t extract positive: 60/105 (57%); Blo t 5 positive: 24/105 (23%); Der p extract positive: 38/105 (36%); Der p 1 positive: 14/105 (13%); Der p 2 positive: 17/105 (16%). The study of Malaysian asthmatic adults showed that 39% of them were sensitized to Der p 1, 32% to Der p 2 and 37% to Blo t 5. Among the asthmatic children, sensitization to Blo t 5, Der p 1 and Der p 2 was 90, 57 and 39%, respectively., Conclusion: This study clearly revealed that dual sensitization to B. tropicalis and D. pteronyssinus is common in the general populations of Singapore and Malaysia. Sensitization to Blo t 5 is more prevalent than to Der p 1 and Der p 2., (Copyright 2003 S. Karger AG, Basel)

Blomia tropicalis is an important mite species in many parts of the world and the most predominant mite species in tropical countries. The prevalence of sensitization to this species has probably been underestimated because commercial extracts are largely unavailable. Identification and characterization of B. tropicalis allergens is an important step toward understanding the role of this species in allergic sensitization and could provide appropriate reagents for diagnostic and therapeutic procedures. This paper describes the isolation, sequence analysis, expression and allergenicity of a cDNA gene coding for a B. tropicalis allergen with homology to paramyosin, a high-molecular-weight allergen previously identified in Dermatophagoides farinae. The full-length Blo t 11 cDNA gene was isolated by cDNA library screening, 5'-rapid amplification of cDNA ends and long-distance PCR. Sequence analysis was performed with a combination of CLUSTAL W, CGC and BLAST program packages. The cDNA gene was expressed as a GST fusion protein in Escherichia coli and purified by affinity chromatography using the glutathione Sepharose column. Allergenicity of the rBlo t 11 was tested by human IgE dot blot immunoassay. Blo t 11 is a 3,111-bp cDNA gene with a 2,625-bp open reading frame coding for an 875-amino acid protein, exhibiting significant homology with different invertebrate paramyosins. The human IgE dot blot immunoassay showed that the rBlo t 11 reacted positively to 52% (33/63) of sera from asthmatic patients. Blo t 11 is the homolog of Der f 11 exhibiting potentially important allergenic activity., (Copyright 2002 S. Karger AG, Basel)

Group 2 allergens are a major cause of sensitization in patients allergic to house dust mites. This study was performed to determine the prevalence of hypersensitivity to group 2 allergens (Der p 2) of Dermatophagoides pteronyssinus (Dp) in asthmatic patients in Taiwan. To facilitate the analysis of Der p 2-specific IgE, we raised a panel of monoclonal antibodies (MoAbs) to Der p 2 antigens. Purified Der p 2 was obtained after MoAb affinity column purification. There were 82 asthmatic patients (41 adults and 41 children) with hypersensitivity to Dp who were analyzed for hypersensitivity to Der p 2. All of them were both skin test- and serology test-reactive to Dp. Using purified Der p 2, 87.8% (72/82) of patients had a skin-test-positive reaction. Six adults (6/41) and 4 children (4/41) had negative skin tests for Der p 2. Ten families (both parents and children were asthmatics) of the 82 patients were selected for Der p 2 skin testing and Der p 2-specific IgE determination using immunoblot analysis. Results showed that 90% (18/20) of patients' skin reactions to Der p 2 and serum contained specific IgE to Der p 2. Because 87.8% (85.4% of adults and 90.2% of children) of the asthmatic patients with Dp hypersensitivity were allergic to Der p 2, its role in the pathogenesis of asthma in Taiwan appears to be important. Purified Der p 2 allergens can be further used for allergen skin testing and immunotherapy., (Copyright 2000 S. Karger AG, Basel)

Background: In tropical climates, sensitization to Bloma tropicalis and Dermatophagoides pteronyssinus is high and mainly directed to species-specific allergens. There is some cross-reactivity between extracts of these mites, probably due to the group 5 allergens that have high sequence homology., Objective and Methods: We used the radioallergosorbent test (RAST), RAST inhibition and immunoblotting inhibition experiments to investigate the cross-reactivity between the recombinant allergens BtM and Der p 5, expressed as glutathione S-transferase fusion proteins, to detect the epitopes involved and to analyze the importance of this cross-reactivity., Results: Seventy-nine percent of 48 patients sera were RAST positive to both recombinants, with a strong correlation (r = 0.8, p<0.0001). BtM inhibited 25 and 21.1% of IgE-binding to B. tropicalis and D. pteronyssinus extracts respectively and Der p 5 inhibited 22 and 24% of IgE-binding to D. pteronyssinus and B. tropicalis extracts. Furthermore, BtM inhibited 74.5% of IgE binding to Der p 5 and Der p 5 inhibited 72.4% of IgE-binding to BtM. RAST inhibition with BtM-derived synthetic peptides showed that peptide 4 (residues 35-50) and peptide 5 (residues 46-61) inhibited 37 and 16% of IgE-binding to BtM while peptides 5 and 2 (residues 14-30) were able to inhibit the IgE binding (32 and 28%, respectively) to Der p 5., Conclusion: There is cross-reactivity between BtM and Der p 5, which explains almost all the cross-reactivity between the two mite extracts. This cross-reactivity seems to be related to epitope(s) at the C-terminal segment of these allergens.

Der p 2, Der p 5 and Der p 7 are three allergens of the house-dust mite Dermatophagoides pteronyssinus that have been cloned and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). We showed that these recombinant allergens produced immediate hypersensitivity skin-test reactions in 70, 60 and 52% respectively of a group of mite-sensitive allergic patients who were strongly positive to whole mite extract (WME). Comparable positivities were found for serum levels of specific IgE antibody against these allergens, as measured by the radioallergosorbant test (RAST). Overall, for the group of allergic patients that we evaluated, the serum IgE antibody concentrations against Der p 2, 5 and 7 were calculated to represent about one third, one quarter and one fifth respectively of the levels measured against the WME. However, for some patients the activity determined against the separate allergens was far higher than that detected against the WME, thus indicating that the concentration of these can be limiting in the WME. We found no significant correlations between the RAST levels against Derp 2 and either Derp 5 or 7, and RAST-inhibition tests indicated a lack of cross-reactivity between Der p 2 and the other two allergens. In contrast, the RAST results revealed the existence of a significant immunological relationship between Der p 5 and 7. Although a certain degree of reactivity against the GST fusion partner was found in the allergic patients studied, this was not a significant influence in determining the positivity against the recombinant allergens. These results confirm the in vivo biological activity of recombinant Der p 2, 5 and 7, and indicate that whilst Der p 2 is undoubtedly a major mite allergen, both Der p 5 and 7 make important contributions toward the overall allergenic activity of house-dust mites.

Sequence information has been obtained from 5 cDNA clones encoding the major house dust mite allergen Der p I, including one which codes for the full length preproenzyme form of the molecule. All translated sequences were unique with 1-3 differences between each clone. 4/5 of the substitutions found were to residues found in Der f I and two positions had a substitution in more than one clone. The polymorphisms included residues already described to be in T cell epitopes, so as well as being necessary to consider them in the construction of synthetic allergens, the substitutions will be important for the interpretation of experimental and genetic studies.