The house dust mite allergen Der p 5 binds lipid ligands and stimulates airway epithelial cells through a TLR2-dependent pathway.

BACKGROUND: Protein crystallographic studies suggest that the house dust mite (HDM) allergen Der p 5 potentially interacts with hydrophobic ligands. Der p 5, in association with its ligand(s), might therefore trigger innate immune signalling pathways in the airway epithelium and influence the initiation of the HDM-allergic response. OBJECTIVE: We investigated the lipid binding propensities of recombinant (r)Der p 5 and characterized the signalling pathways triggered by the allergen in airway epithelial cells. METHODS: rDer p 5 was produced in Pichia pastoris and characterized by mass spectrometry, multi-angle light scattering and circular dichroism. Its interactions with hydrophobic ligands were investigated in fluorescence-based lipid binding assays and in-silico docking simulations. Innate immune signalling pathways triggered by rDer p 5 were investigated in airway epithelial cell activation assays in vitro. RESULTS: Biophysical analysis showed that rDer p 5 was monomeric and adopted a similar α-helix-rich fold at both physiological and acidic pH. Spectrofluorimetry experiments showed that rDer p 5 is able to selectively bind lipid ligands, but only under mild acidic pH conditions. Computer-based docking simulations identified potential binding sites for these ligands. This allergen, with putatively associated lipid(s), triggered the production of IL-8 in respiratory epithelial cells through a TLR2-, NF-kB- and MAPK-dependent signalling pathway. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the fact that Der p 5 represents a HDM allergen of intermediate prevalence, our findings regarding its lipid binding and activation of TLR2 indicate that it could participate in the initiation of the HDM-allergic state.

Characterization of the house dust mite allergen Der p 21 produced in Pichia pastoris.

BACKGROUND: The development of recombinant house dust mite (HDM) allergens opened the way for the in-depth characterization of these molecules but also provided new opportunities to refine the diagnostic procedures of HDM allergy as well as the allergen-specific immunotherapy through tailor-made treatments. OBJECTIVE: In the present study, the HDM allergen Der p 21 was expressed in Pichia pastoris under a secreted form. The physico-chemical as well as the allergenic characterizations of recombinant Der p 21 (rDer p 21) were performed. METHODS: Purified rDer p 21, secreted from recombinant P. pastoris was characterized by CD and MS analysis and the frequency of IgE reactivity was determined by ELISA using 96 sera of HDM-allergic patients from Bangkok. The direct airway epithelial cell activation by rDer p 21 was also evaluated. RESULTS: rDer p 21 was highly expressed under a secreted form in P. pastoris. The physico-chemical characterization of purified rDer p 21 showed that the allergen displayed appropriate α-helix secondary structure content although a two amino acids truncation at the N-terminus of the protein was evidenced by MS. The prevalence of IgE reactivity to rDer p 21 reached 25% in the cohort of the HDM-allergic patients. rDer p 21 could trigger IL-8 production in airway epithelial cells through TLR2-dependent signaling. CONCLUSION: Properly folded rDer p 21 produced in P. pastoris is appropriate for HDM allergy diagnosis as well for future recombinant allergen-based specific immunotherapy.

No abnormalities of reg1 alpha and reg1 beta gene associated with diabetes mellitus.

In order to investigate whether there would be any association between abnormalities of either reg1 alpha or reg1 beta gene and diabetes mellitus in man, these two genes were analyzed in 42 patients with type 1 diabetes mellitus, 12 with fibrocalculous pancreatopathy, 37 with type 2 diabetes mellitus, and 22 normal controls, by PCR-SSCP analysis and nucleotide sequencing technique. Polymorphism in the reg1 alpha gene resulted in three mobility patterns in the PCR-SSCP analysis, due to nucleotide constituents at position -10 before exon 1 being either C/C, T/C or T/T. These three mobility patterns were observed in every group of subjects. The analysis of reg1 beta gene showed nucleotide substitutions in exon 4 in one patient, exon 5 in another patient with type 1 diabetes, and in exon 4 and intron 5 in one patient with fibrocalculous pancreatopathy. The nucleotide substitutions in exon 4 in the patient with type 1 diabetes and that with fibrocalculous pancreatopathy occurred at codons 103 and 84 while that in exon 5 in the patient with type 1 diabetes occurred at codon 141, changing the codons from CAT to CAC, GTG to GCG, and ACT to AAT and resulting in H103H silent, V84A and T141N missense mutations, respectively. In conclusion, using PCR-SSCP and nucleotide sequence analyses, we did not find any association between abnormalities of either reg1 alpha or reg1 beta gene with any type of diabetes studied.

Analysis of the reg1alpha and reg1beta gene transcripts in patients with fibrocalculous pancreatopathy.

Fibrocalculous pancreatopathy is a form of diabetes, associated with tropical chronic calcific pancreatitis, in which islet beta-cell loss and pancreatic stone formation are found. It is likely to be a multifactorial disease with both genetic and environmental components. Regenerating (reg) gene encodes protein that has been involved in pancreatic lithogenesis and the regeneration of islet cells and therefore the abnormality of reg genes could be associated with fibrocalculous pancreatopathy. In this study, regla and reg1beta mRNAs were isolated from peripheral blood lymphocytes obtained from 16 patients with fibrocalculous pancreatopathy, 42 patients with type 1 diabetes, 37 patients with type 2 diabetes, and 22 normal controls. mRNAs were amplified by reverse-transcription polymerase chain reaction (RT-PCR) and analysed by a single strand conformation polymorphism (SSCP) technique. The reg1alpha and reg1beta mRNAs were isolated, indicating the ectopic expression of these genes in peripheral blood lymphocytes; however, variation among mobility patterns was not observed in the SSCP analysis of the RT-PCR products. The results indicated that there was no abnormality of the regla and reg1beta mRNAs obtained from the study groups.

Optimization of a Der p 2-based prophylactic DNA vaccine against house dust mite allergy.

DNA vaccines encoding allergens are promising immunotherapeutics to prevent or to treat allergy through induction of allergen-specific Th1 responses. Despite anti-allergy effects observed in small rodents, DNA-based vaccines are weak immunogens in primates and humans and particularly when administered by conventional injection. The goal of the present study was to improve the immunogenicity of a prophylactic vaccine encoding the major house dust mite allergen Der p 2. In this context, we evaluated the influence of different DNA backbones including notably intron and CpG enriched sequence, the DNA dose, the in vivo delivery by electroporation as well as the heterologous prime boost regimen on the vaccine efficiency. We found that a minimal allergen expression level threshold must be reached to induce the production of specific antibodies but beyond this limit, the intensity of the immune response was independent on the DNA dose and allergen expression. The in vivo DNA delivery by electroporation drastically enhanced the production of specific antibodies but not the IFNg secretion. Vaccination of naïve mice with DNA encoding Der p 2 delivered by electroporation even at very low dose (2µg) prevented the development of house dust mite allergy through Th1-skewed immune response characterized by the drastic reduction of allergen-specific IgE, IL-5 and lung inflammation together with the induction of strong specific IgG2a titers and IFNg secretion. CpG cassette in the DNA backbone does not play a critical role in the efficient prophylaxis. Finally, comparable protective immune responses were observed when using heterologous DNA prime/protein boost or homologous DNA prime/boost. Taken together, these data suggest that the potent Th1 response induced by DNA-based vaccine encoding allergens through electroporation provides the rationale for the evaluation of DNA encoding Der p 2 into HDM allergy clinical trials.