Asthma is a complex and heterogeneous respiratory disorder characterized by chronic airway inflammation. It has generally been associated with allergic mechanisms related to type 2 airway ...inflammation. Nevertheless, between 10 and 33% of asthmatic individuals have nonallergic asthma (NA). Several targeted treatments are in clinical development for patients with Th2 immune response, but few biomarkers are been defined for low or non-Th2-mediated inflammation asthma. We have recently defined by gene expression a set of genes as potential biomarkers of NA, mainly associated with disease severity: IL10, MSR1, PHLDA1, SERPINB2, CHI3L1, IL8, and PI3. Here, we analyzed their protein expression and specificity using sera and isolated peripheral blood mononuclear cells (PBMCs). First, protein quantification was carried out using ELISA (in sera) or Western blot (proteins extracted from PBMCs by Trizol procedure), depending on the biomarker in 30 healthy controls (C) subjects and 30 NA patients. A receiver operating characteristic curve analysis was performed by using the R program to study the specificity and sensitivity of the candidate biomarkers at a gene- and protein expression level. Four kinds of comparisons were performed: total NA group vs C group, severe NA patients vs C, moderate-mild NA patients vs C, and severe NA patients vs moderate-mild NA patients. We found that all the single genes showed good sensitivity vs specificity for some phenotypic discrimination, with CHI3L1 and PI3 exhibiting the best results for C vs NA: CHI3L1 area under the curve (AUC) (CI 95%): 0.95 (0.84-1.00) and PI3 AUC: 0.99 (0.98-1.00); C vs severe NA: PI3 AUC: 1 (0.99-1.00); and C vs moderate-mild NA: CHI3L1 AUC: 1 (0.99-1.00) and PI3 AUC: 0.99 (0.96-1.00). However, the results for discriminating asthma disease and severity with protein expression were better when two or three biomarkers were combined. In conclusion, individual genes and combinations of proteins have been evaluated as reliable biomarkers for classifying NA subjects and their severity. These new panels could be good diagnostic tests.
Endo-β-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein ...family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic β-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that β-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen β-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long β-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of β-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.
Asthma is a complex disease comprising various phenotypes and endotypes, all of which still need solid biomarkers for accurate classification. In a previous study, we defined specific genes related ...to asthma and respiratory allergy by studying the expression of 94 genes in a population composed of 4 groups of subjects: healthy control, nonallergic asthmatic, asthmatic allergic, and nonasthmatic allergic patients. An analysis of differential gene expression between controls and patients revealed a set of statistically relevant genes mainly associated with disease severity, i.e.,
, and
. Here, we analyzed whether these genes and their proteins could be potential asthma biomarkers to distinguish between nonallergic asthmatic and asthmatic allergic subjects. Protein quantification was determined by ELISA (in serum) or Western blot (in protein extracted from peripheral blood mononuclear cells or PBMCs). Statistical analyses were performed by unpaired
-test using the Graph-Pad program. The sensitivity and specificity of the gene and protein expression of several candidate biomarkers in differentiating the two groups (and the severity subgroups) was performed by receiver operating characteristic (ROC) curve analysis using the R program. The ROC curve analysis determined single genes with good sensitivity and specificity for discriminating some of the phenotypes. However, interesting combinations of two or three protein biomarkers were found to distinguish the asthma disease and disease severity between the different phenotypes of this pathology using reproducible techniques in easy-to-obtain samples. Gene and protein panels formed by single biomarkers and biomarker combinations have been defined in easily obtainable samples and by standardized techniques. These panels could be useful for characterizing phenotypes of asthma, specifically when differentiating asthma severity.
Olive-pollen allergy is one of the leading causes of respiratory allergy in Mediterranean countries and some areas of North America. Currently, allergen-specific immunotherapy is the only ...etiophatogenic treatment. However, this approach is not fully optimal, safe, or effective. Thus, efforts continue in the search for novel immunotherapy strategies, being one of the most promising the use of peptides derived from major allergens. This work tries to determine the therapeutic potential and safety of 5 dodecapeptides derived from the main allergen of olive-pollen allergy, Ole e 1. The immunomodulatory capacity of these peptides was studied using peripheral blood mononuclear cells (PBMCs) obtained from 19 olive-pollen-allergic patients and 10 healthy controls. We determined the capacity of these peptides to inhibit the proliferative response toward olive-pollen allergenic extract and to induce the regulatory cytokines, IL-10 and IL-35. To test the safety and absence of allergenicity of the peptides, the basophil activation was analyzed by flow-cytometry, using peripheral blood. The results showed that two of five peptides inhibited near to 30% the proliferative response against the total olive-pollen allergenic extract in olive-pollen-allergic patients. Inhibition increased to nearly 35% when the 5 peptides were used in combination. In both cases, a statistically significant induction of IL-10 and IL-35 secretion was observed in the supernatants of allergic patients PBMCs cultures. None of the 5 peptides induced basophil activation and cross-link inflammatory cell-bound IgE. In conclusion, these results open up new possibilities in the treatment of olive-pollen allergy, which could solve some of the problems facing current therapy approaches.
•Gene-profiles able to differentiate asthma from allergic diseases have been defined.•These groups of molecular biomarkers have been studied in non-invasive samples.•Beside, we have described new ...potential biomarkers of severity in asthma.•New tools to be assessed as diagnostic and/or therapeutic targets are provided.
Asthma is a complex, chronic respiratory disease with a wide clinical spectrum. Use of high-throughput technologies has generated a great deal of data that require validation. In this work the objective was to validate molecular biomarkers related to asthmatic disease types in peripheral blood samples and define their relationship with disease severity. With this purpose, ninety-four previously described genes were analyzed by qRT-PCR in 30 healthy control (HC) subjects, 30 patients with nonallergic asthma (NA), 30 with allergic asthma (AA), and 14 patients with allergy (rhinitis) but without asthma (AR). RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the TRIzol method. After data normalization, principal component analysis (PCA) was performed, and multiple approaches were used to test for differential gene expression. Relevance was defined by RQ (relative quantification) and corrected P value (<0.05). Protein levels of IL-8 and MSR1 were determined by ELISA and Western blot, respectively.
PCA showed 4 gene expression clusters that correlated with the 4 clinical phenotypes. Analysis of differential gene expression between clinical groups and HCs revealed 26 statistically relevant genes in NA and 69 in AA. Protein interaction analysis revealed IL-8 to be a central protein. Average levels of IL-8 were higher in the asthma patients’ sera (NA: 452.28±357.72, AA: 327.46±377pg/ml) than in HCs (286.09±179.10), but without reaching statistical significance. Nine genes, especially MSR1, were strongly associated with severe NA.
In conclusion, several molecular biomarkers of asthma have been defined, some of which could be useful for the diagnosis or prognosis of disease severity.
Olive pollen and yellow mustard seeds are major allergenic sources with high clinical relevance. To aid with the identification of IgE-reactive components, the development of sensitive methodological ...approaches is required. Here, we have combined T7 phage display and protein microarrays for the identification of allergenic peptides and mimotopes from olive pollen and mustard seeds. The identification of these allergenic sequences involved the construction and biopanning of T7 phage display libraries of mustard seeds and olive pollen using sera from allergic patients to both biological sources together with the construction of phage microarrays printed with 1536 monoclonal phages from the third/four rounds of biopanning. The screening of the phage microarrays with individual sera from allergic patients enabled the identification of 10 and 9 IgE-reactive unique amino acid sequences from olive pollen and mustard seeds, respectively. Five immunoreactive amino acid sequences displayed on phages were selected for their expression as His6-GST tag fusion proteins and validation. After immunological characterization, we assessed the IgE-reactivity of the constructs. Our results show that protein microarrays printed with T7 phages displaying peptides from allergenic sources might be used to identify allergenic components -peptides, proteins or mimotopes- through their screening with specific IgE antibodies from allergic patients.
•The development of methodological approaches is required to identify IgE-reactive components.•The combination of T7 phage display and protein microarrays approach is here proposed.•The new approach permitted the identification of IgE-immunodominant allergenic peptides and mimotopes.
To determine whether the β-lactam allergy delabeling was safe and cost-saving in Primary Care (PC) patients.
We have conducted a retrospective chart review of PC patients with β-lactam allergy label ...evaluated in our Allergy Unit between 2017 and 2022.
Allergy Department. Hospital Virgen del Rocio (Sevilla).
A total of 391 patients labeled for β-lactam allergy in PC were studied.
(a) Outcome evaluation of a β-lactam allergy delabeling procedure. (b) A ratio between the total e-prescribed antibiotic cost and the number of treatment days (the experimental daily antibiotic cost or EDAC) before and after delabeling was analyzed in delabeled and truly allergic patients.
The results of skin testing were positive in 9.2% of the reported cases (36 of 391 patients). The reactions to oral provocation challenge (OPC) occurred in 2.14% of the patients who underwent negative skin testing to offending β-lactam (in 15 of 699 OPC). A total of 307 patients (78.5%) were delabeled; 70 (17.9%) had a β-lactam selective response and 14 (3.59%) reacted to both penicillin and cephalosporin. The EDAC before and after the procedure in delabeled patients was significantly lower (0.88 € vs 0.62 €, p<10−3), than that observed in truly allergic group (0.87 € vs. 0.76 €, p=not significant).
To delabel β-lactam allergy in Primary Care patients is safe in most patients, cost-saving in antibioticotherapy, and allows identify the main clinical β-lactam allergy phenotypes that benefit from this procedure.
Evaluar la seguridad del desetiquetado de alergia a β-lactámicos y su impacto económico en la prescripción antibiótica en atención primaria (AP).
Estudio observacional retrospectivo en situación de práctica clínica habitual.
Unidad de Alergología, Hospital Virgen del Rocío, Sevilla.
391 pacientes etiquetados de alergia a β-lactámicos en AP.
a) Evaluación de un procedimiento de desetiquetado de alergia a β-lactámicos. b) Se analizó la relación entre el coste total del antibiótico e-prescrito y el número de días de tratamiento (el coste diario antibiótico experimental the experimental daily antibiotic cost, EDAC).
Las pruebas cutáneas a β-lactámicos fueron positivas en el 9,2% de casos (36 de 391 pacientes). Las reacciones durante la provocación oral controlada ocurrieron en el 2,14% de casos con pruebas cutáneas negativas a β-lactámicos (en 15 de 699 provocaciones). Un total de 307 pacientes (78,5%) fueron desetiquetados; 70 (17,9%) tuvieron una respuesta selectiva a un β-lactámico y 14 (3,59%) reaccionaron tanto a penicilina como a cefalosporina. El EDAC antes y después del procedimiento en los pacientes desetiquetados fue significativamente menor (0,88€ versus 0,62€, p<10−3), al observado en el grupo de pacientes alérgicos (0,87€ versus 0,76€, p no significativo).
Desetiquetar la alergia a β-lactámicos en pacientes de AP es seguro en la mayoría de los pacientes, ahorra costes en antibioterapia y permite identificar los principales fenotipos clínicos de alergia a β-lactámicos que se benefician de este procedimiento.
The allergenic non-specific lipid transfer protein Ole e 7 from olive pollen is a major allergen associated with severe symptoms in areas with high olive pollen levels. Despite its clinical ...importance, its cloning and recombinant production has been unable by classical approaches. This study aimed at determining by mass-spectrometry based proteomics its complete amino acid sequence for its subsequent expression and characterization. To this end, the natural protein was in-2D-gel tryptic digested, and CID and HCD fragmentation spectra obtained by nLC-MS/MS analyzed using PEAKS software. Thirteen out of the 457 de novo sequenced peptides obtained allowed assembling its full-length amino acid sequence. Then, Ole e 7-encoding cDNA was synthesized and cloned in pPICZαA vector for its expression in Pichia pastoris yeast. The analyses by Circular Dichroism, and WB, ELISA and cell-based tests using sera and blood from olive pollen-sensitized patients showed that rOle e 7 mostly retained the structural, allergenic and antigenic properties of the natural allergen. In summary, rOle e 7 allergen assembled by de novo peptide sequencing by MS behaved immunologically similar to the natural allergen scarcely isolated from pollen.
Olive pollen is an important cause of allergy. The non-specific lipid binding protein Ole e 7 is a major allergen with a high incidence and a phenotype associated to severe clinical symptoms. Despite its relevance, its cloning and recombinant expression has been unable by classical techniques. Here, we have inferred the primary amino acid sequence of Ole e 7 by mass-spectrometry. We separated Ole e 7 isolated from pollen by 2DE. After in-gel digestion with trypsin and a direct analysis by nLC-MS/MS in an LTQ-Orbitrap Velos, we got the complete de novo sequenced peptides repertoire that allowed the assembling of the primary sequence of Ole e 7. After its protein expression, purification to homogeneity, and structural and immunological characterization using sera from olive pollen allergic patients and cell-based assays, we observed that the recombinant allergen retained the antigenic and allergenic properties of the natural allergen. Collectively, we show that the recombinant protein assembled by proteomics would be suitable for a better in vitro diagnosis of olive pollen allergic patients.
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•The cloning and recombinant production of the clinically relevant allergen Ole e 7 has been unable by classical approaches.•The de novo peptide sequencing proteomic analysis by PEAKS permitted the assembly of the complete Ole e 7 primary sequence.•Ole e 7 allergen assembled by proteomics behaves similarly to the natural allergen regarding its antigenicity and allergenicity.•rOle e 7 might be used for a more effective clinical diagnosis of olive pollen allergic patients.
Olive tree (Olea europaea) pollen is a main cause of allergy in Mediterranean areas and North America. A novel allergen, Ole e 11, has been detected by proteomic techniques. Protein bands binding IgE ...from allergic sera were excised from a 2D electrophoresis gel and analysed by Edman degradation and MALDI-TOF MS. Four peptides were sequenced and used for designing primers to clone the cDNA codifying the protein. Ole e 11 consists of a 342 amino acid length polypeptide with a molecular mass of 37.4 kDa and a pI of 7.8. The allergen was identified as a pectin methylesterase and showed low identity with other members of this family from foods such as those from carrot (23%), orange (25%) and tomato (24%), and higher identity with those from Arabidopsis thaliana (57%) and Salsola kali (54%) pollen. The protein was overproduced in Pichia pastoris, purified, and characterized as an active enzyme. CD analysis rendered 3%α-helix, 50%β-sheet and 27%β-turns for its secondary structure, which is in agreement with other pectin methylesterase structures. The recombinant protein was demonstrated to be immunologically equivalent to the natural form by immunoblotting, indirect ELISA and inhibition experiments, using polyclonal antiserum and sera from olive pollen allergic patients. The prevalence fluctuated between 55.9% and 75.6% in three different allergic populations. The availability of this new olive pollen allergen could improve the component-resolved diagnosis. Its allergenic relevance is stepped up by the biotechnological use of these enzymes to improve organoleptic properties in processing foods and further confirms the need to include it in an accurate diagnosis.
Cross-reactivity reactions between allergenic polygalacturonases (PGs) from different biological sources, especially foods and pollens from the Oleaceae family, have been described using Salsola kali ...PG (Sal k 6). No PG from olive pollen has been characterized to date, hampering further knowledge about cross-reactions through PGs.
The aim of this work was to determine the potential allergenicity of the PG from olive pollen and clarify its role in cross-reactivity.
A cDNA-encoding olive pollen PG sequence was subcloned into the pET41b vector and used to transform BL21(DE3) Escherichia coli cells to produce a His-tag fusion recombinant protein. The allergenic properties of olive pollen PG were determined by immunoblotting and ELISA in comparison to Sal k 6. The cross-reactivity potential of the protein with other pollen sources was analyzed by inhibition immunoassays.
The existence of other isoforms of Ole e 14 with different allergenicity was confirmed by proteomics and a meta-analysis of the recently reported olive genome. Sal k 6 showed a higher IgE recognition than Ole e 14 regardless of patient sensitization, suggesting the existence of more allergenic Ole e 14 isoforms in olive pollen. IgG and IgE inhibition assays supported the existence of cross-reactions between them and with other PGs from Oleaceae and Poaceae plant families.
A new allergen from olive pollen, Ole e 14, has been identified, produced as a recombinant isoform, and structurally and immunologically characterized. Its role in cross-reactivity has been confirmed and, due to its smaller IgE binding capacity, it could have an important role for therapeutic purposes.
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