Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy

BACKGROUND: Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.     

Cloning and sequencing of a gene encoding a 2S albumin seed storage protein precursor from English walnut (Juglans regia), a major food allergen

Background: Walnuts rank third in per capita consumption of tree nuts in the United States and can be associated with systemic IgE-mediated reactions in some individuals. Objective: The objectives of the study were to clone a gene encoding one of the major food allergens in the walnut kernel and to characterize the recombinant allergen. Methods: A cDNA expression library in the lambda vector Uni-ZAP, which was prepared from walnut somatic embryos, was screened by using a patient's sera that reacted with multiple protein bands on immunoblotting. Results: A cDNA clone containing an insert of 663 bp was identified and named Jug r 1. DNA sequence analysis of this clone revealed that it encoded a protein 142 amino acids in length. Comparison of the encoded protein sequences with protein databases revealed that this clone exhibits a 46.1% identity with the Brazil nut (Bertholletia excelsa) methionine-rich 2S albumin seed storage protein precursor, Ber e 1. Jug r 1 appears to be an important walnut food allergen; 12 of 16 sera from patients allergic to walnuts demonstrated IgE binding to the 2S albumin seed storage protein precursor fusion protein. An IgE-binding inhibition study suggests that the walnut 2S protein precursor undergoes posttranslational modification into a large and small subunit that is similar to castor seed, cottonseed, mustard seed, and Brazil nut 2S seed storage protein allergens. Interestingly, the gene encoding this allergenic protein in Brazil nuts has recently gained notoriety because of its experimental use as a transgene to enhance the nutritional quality of legumes. Conclusion: This is now the sixth definitive 2S albumin seed storage protein demonstrated to bind IgE, suggesting that this class of proteins is inherently allergenic. (J Allergy Clin Immunol 1998;101:807-814.)     

Purification and characterization of a soybean hull allergen responsible for the Barcelona asthma outbreaks. II. Purification and sequencing of the Gly m 2 allergen

Background A low MW allergen from soybean hull, Gly m 1, with two isoallergens, Gly m 1 A and Gly m 1 B, was associated with the asthma outbreaks that occurred in Cartagena, Spain. Using sera of asthmatic epidemic patients (AEP) from Barcelona, three main soybean hull allergens, two of them with MWs atid pIs identical to those reported for Gly m 1 A and Gly m 1 B, were identified. Objective The purpose of this study was to purify and to study the N-terminal amino acid sequence of the third allergen, which has a MW of 8 kDa. Method The purification procedure combined the double dialysis method and preparative isoelectofocusing (IEF). Specific IgE determination to the fractions obtained demonstrated three peaks, one of them corresponding to the 8 kDa allergen. The pooled fractions containing this allergen were studied by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-PAGEAVestern blot and IEF/Westem blot. Only a band with a MW of 8 kDa and a pI of 6 was obtained. Its allergenic activity was measured and it was demonstrated that the allergenicity of soybean hull correlates with the presence of the 8 kDa allergen. The N-terminal amino acid sequence of the first 20 amino acids, which was registered at the PIR Data Submission as the N-terminal partial sequence of Gly m 2, was determined according the Edman degradation method. Results Gly m 2 N-terminal amino acid sequence lacks homology with that reported for the allergen Gly m 1 but has a homology of 71% with a storage protein from cotyledon of Vigna radiata (cow pea) and 64% with a‘disease response protein’ from Pisum sativum (green pea). These results suggest that Gly m 2 in soybeans could protect against diseases which affect soybean plants. Conclusion This study demonstrates the existence of another soybean hull allergen, Gly m 2, partially responsible for the soybean asthma outbreaks that occurred in Barcelona, Spain.     

Ana o 3, an important cashew nut (Anacardium occidentale L.) allergen of the 2S albumin family

BACKGROUND: Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. We have previously cloned and characterized major cashew allergens belonging to the vicilin and legumin families of seed storage proteins. OBJECTIVE: Here we set out to describe a third major cashew allergen, a 2S albumin. METHODS: The recombinant cashew 2S albumin was amplified from a cDNA library by means of PCR, sequenced, and expressed in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition immunoblotting was used to identify the corresponding native cashew nut proteins. The mass of affinity-purified native allergen was determined by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy. Patients' sera were used to probe solid-phase 2S albumin peptides to identify linear epitopes. RESULTS: The cloned allergen, designated Ana o 3, was identified as 2S albumin. MALDI-TOF mass spectroscopy of native Ana o 3 yielded a molecular mass of 12,598 d. Immunoblot analysis showed 21 (81%) of 26 sera from patients with cashew allergy were reactive. Three native Ana o 3 large-subunit isoforms with molecular weights ranging from approximately 6 to 10 kd were identified. Probing of overlapping synthetic Ana o 3 peptides with patients' sera identified 16 reactive peptides, 4 of which gave strong signals and one of which positionally overlaps linear epitopes in mustard and walnut allergenic 2S albumins. The overlapping cashew and walnut epitopes also share considerable homology. CONCLUSIONS: We conclude that this 2S albumin protein is a major allergen in cashew nut and demonstrates a possible basis for cross-reactivity with walnut 2S albumin.     

The diagnostic accuracy of specific IgE to Ara h 6 in adults is as good as Ara h 2

Specific IgE (sIgE) to Ara h 2 is useful in diagnosing peanut allergy. Our aim was to assess the diagnostic value of sIgE to Ara h 6, another 2S albumin, in an adult population suspected of peanut allergy. Subjects with suspected peanut allergy between 2002 and 2013 were included if a diagnostic double‐blind, placebo‐controlled food challenge with peanut was performed. sIgE to Ara h 2 and Ara h 6 was measured by ImmunoCAP ISAC 112. Of 107 challenged subjects, 65 had a positive challenge (61%). The discriminative ability of sIgE to Ara h 2 and Ara h 6 was comparable: AUC 0.81 vs 0.82. Positive predictive value for both tests was 95% using a cutoff value ≥1 ISU/l with poor corresponding sensitivity values (58% for Ara h 2, 62% for Ara h 6), but good specificity values (95% for both tests). In conclusion, the diagnostic value of sIgE to Ara h 6 on population level was as good as sIgE to Ara h 2. On individual level, however, 5% of the subjects showed contradicting results between both tests using a cutoff of 0.3 ISU/l, leading to a risk of misdiagnosis if only one of both tests is used.     

Characterization of IgE epitopes of Cuc m 2, the major melon allergen,and their role in cross‐reactivity with pollen profilins

Background Plant profilins are described as minor allergens, although with some exceptions in foods such as melon, watermelon or orange. In fact, they could be responsible for many cross‐reactions among distantly related species. This is likely to be a consequence of the presence of common epitopes. Objective To characterize the B epitopes of Cuc m 2, a model of plant food profilin, using phage display techniques and to compare with other profilins, such as those of timothy grass and birch pollen, and human I profilin, to understand the mechanism of cross‐reaction among members of this family. Methods IgE of melon‐allergic patients was used to select clones from a phage display 12 mer peptide library. After two rounds of screening, Cuc m 2‐specific clones were eluted and the DNA insertion sequenced. The residues of each clone were mapped on the Cuc m 2 surface to define a mimotope, which was also localized on the three‐dimensional surfaces of other profilins. Results Seventeen melon‐allergic patients were selected. Sera from each of them recognized the melon profilin, Cuc m 2, but the majority also recognized Phl p 12 or Bet v 2, timothy grass‐, and birch‐pollen profilins, respectively. A Cuc m 2 mimotope was defined and mapped onto its surface giving the following sequence: S₂W₃A₅Y₆D₉H₁₀T₁₁₁P₁₁₂G₁₁₃Q₁₁₄N₁₁₆M₁₁₇R₁₂₁L₁₂₂. The homologous residues in Phl p 12 and Bet v 2 had almost identical sequences. By contrast, the homologous sequence in human profilin showed many differences. Conclusions The identified mimotope could be involved in cross‐reactions among food and pollen profilins. Many of these cross‐reactions observed in the clinical realm could be explained by the presence of a common epitope found in food and pollen allergens. A new strategy of immunotherapy based on this IgE region could be used in alternative immunotherapy strategies. Cite this as: L. Tordesillas, L. F. Pacios, A. Palacín, J. Cuesta‐Herranz, M. Madero and A. Díaz‐Perales, Clinical & Experimental Allergy, 2010 (40) 174–181.     

Cot/tpl2 activity is required for TLR-induced activation of the Akt p70 S6k pathway in macrophages: Implications for NO synthase 2 expression

LPS stimulation activates IKK and different MAP kinase pathways, as well as the PI3K‐Akt‐mTOR‐p70 S6k pathway, a negative regulator of these MyD88‐dependent intracellular signals. Here, we show that Cot/tpl2, a MAP3K responsible for the activation of the MKK1‐Erk1/2, controls P‐Ser473 Akt and P‐Thr389 p70 S6k phosphorylation in LPS‐stimulated macrophages. Analysis of the intracellular signalling in Cot/tpl2 KO macrophages versus WT macrophages reveals lower IκBα recovery and higher phosphorylation of JNK and p38α after 1 h of LPS stimulation. Moreover, Cot/tpl2 deficiency increases LPS‐induced NO synthase 2 (NOS2) expression in macrophages. Inhibition of the PI3K pathway abolishes the differences in IκBα and NOS2 expression between Cot/tpl2 KO and WT macrophages following LPS administration. Furthermore, in zymosan‐ and polyI:C‐stimulated macrophages, Cot/tpl2 mediates P‐Ser473 Akt phosphorylation, increases IκBα levels and decreases NOS2 expression. In conclusion, these data reveal a novel role for the Cot/tpl2 pathway in mediating TLR activation of the Akt‐mTOR‐p70 S6k pathway, allowing Cot/tpl2 to fine‐control the activation state of other signalling pathways.