Luda Bardina - Academia.edu (original) (raw)
Papers by Luda Bardina
Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 2014
Food protein-induced enterocolitis syndrome (FPIES) is a gastrointestinal hypersensitivity disord... more Food protein-induced enterocolitis syndrome (FPIES) is a gastrointestinal hypersensitivity disorder with a poorly understood pathophysiology and no biomarkers to aid in diagnosis. To investigate humoral and cellular responses to casein in children with milk-FPIES, including the role of casein-specific (cs) IgA and T-cell mediated TGF-β responses. Thirty-one children previously diagnosed with milk-FPIES were challenged with milk. Twelve age-matched children with FPIES to other foods and 6 milk-tolerant children without a history of FPIES were used as controls. Casein-specific IgE, IgG, IgG4, and IgA were measured in serum and TGF-β levels in supernatants of casein-stimulated PBMCs. Twenty-six children with milk-FPIES reacted (active milk-FPIES) and five tolerated milk (milk-FPIES resolved) during food challenge. All of them had significantly lower levels of csIgG, csIgG4, and csIgA than control children (p-value<0.001). There were no TGF-β responses in supernatants of active milk-...
Pediatric Allergy and Immunology, 2013
Background-Decreased serum food-specific-IgA antibodies have been associated with allergic diseas... more Background-Decreased serum food-specific-IgA antibodies have been associated with allergic disease in cross-sectional, case-control studies. The purpose of this study was to prospectively compare egg-white-(EW)-specific-IgA and IgA2 levels between egg-allergic children and children tolerating egg. Methods-Seventeen egg allergic children were followed prospectively. Total IgA, EW-specific-IgA and EW-specific-IgA2 levels were measured in their sera with a sensitive ELISA. As negative controls were used children with no previous history of egg allergy. Egg-allergic children with or without concomitant milk allergy were evaluated as additional controls with measurement of casein-specific-IgA. Results-After 2.5±0.9 years, 9 out of 17 allergic children became tolerant and 8 remained allergic to baked egg. Baseline EW-specific-IgA2 levels were significantly lower in the eggallergic subjects (median 23.9ng/ml) compared with the negative control subjects (99.4ng/ml) and increased significantly by 28% over the study time period in 8 out of the 9 allergic children that became tolerant to baked egg. There was no significant change over time in EW-specific-IgA in any of the study groups. Non-milk-allergic subjects with concomitant egg allergy had almost 3fold higher casein-specific-IgA levels than the milk-and egg-allergic subjects (P=0.025). Conclusions-These results suggest a potential role for allergen-specific-IgA2 antibodies in the induction of food tolerance. Furthermore, they support the hypothesis that immature or impaired production of allergen-specific-IgA2 may be associated with the pathophysiology of food allergy, a defect that seems to be selective for the culprit allergen.
Journal of Agricultural and Food Chemistry, 2011
Among tree nut allergens, pecan allergens remain to be identified and characterized. The objectiv... more Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20À22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118À132, 208À219, and 238À249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.
Regulatory Toxicology and Pharmacology, 2006
Cry1F protein, derived from Bacillus thuringiensis, is eVective at controlling lepidopteran pests... more Cry1F protein, derived from Bacillus thuringiensis, is eVective at controlling lepidopteran pests and a synthetic Cry1F transgene was transferred into maize. For the safety assessment of genetically modiWed food crops, the allergenic potential of the introduced novel trait(s) is evaluated. Because no single parameter is currently predictive of allergic potential, a 'weight of evidence' approach has been proposed. As part of this assessment, the amino acid (aa) sequence of the Cry1F protein was compared to a database of known allergens using recommended criteria. The Cry1F protein did not show signiWcant similarity or a match of eight contiguous identical aa with any allergen. However, a single six contiguous aa match was identiWed between Cry1F and the Der p7 protein of the dust mite, Dermatophagoides pteronyssinus. To investigate whether Cry1F was cross-reactive with Der p7, sera from 10 dust mite allergic patients containing Der p 7-speciWc IgE antibody were used to compare IgE-speciWc binding. No evidence of cross-reactivity was observed between Cry1F and Der p7. This study provides in vitro IgE sera screening data, that when considered in the context of other bioinformatic data [
The Lancet, 1984
A membrane-coated virus having a diameter of 85-90 nm and containing a 40-45 nm core was found to... more A membrane-coated virus having a diameter of 85-90 nm and containing a 40-45 nm core was found to replicate in cell cultures derived from chimpanzee liver after inoculation of serum containing infective non-A, non-B (NANB) hepatitis viruses from two independent sources. Replication of this agent was not observed when the same cells were inoculated with a chloroform-extracted inoculum or were left uninoculated. Replication involves assembly of virus cores on tubular structures similar to those seen in liver cells of chimpanzees infected with most isolates of NANB virus.
Two-dimensional polyacrylamide gel electrophoresis (2DE) and Western immunoblotting have proven t... more Two-dimensional polyacrylamide gel electrophoresis (2DE) and Western immunoblotting have proven to be invaluable and fundamental techniques for comprehensive characterization of food allergens. Here, we describe and discuss detailed protocols used in our studies on identification of allergenic proteins in shrimp, sesame, hazelnut, and pistachio.
Allergy
Although most of cow&amp;#39;s milk (CM) allergic children will outgrow their allergy, th... more Although most of cow&amp;#39;s milk (CM) allergic children will outgrow their allergy, the pathomechanism of the natural development of tolerance remains poorly understood. It has been suggested that the balance between milk specific IgE and IgG4 plays a major role. We aimed to investigate differences in IgE and IgG4 antibody binding to cow&amp;#39;s milk (CM) epitopes between patients with persistent CM allergy (CMA) and those that naturally became tolerant. Sera from thirty-five children with proven CMA (median age at inclusion of 10 months) were analysed retrospectively; 22 patients have become tolerant (median age at tolerance acquisition of 51 months) during the study period as confirmed by a negative oral food challenge. IgE and IgG4 binding to sequential epitopes derived from 5 major CM proteins were measured with a peptide microarray-based immunoassay. At baselines, greater intensity and broader diversity of IgE and IgG4 binding have been found in children with persistent CMA beyond 5 years of age compared to patients with transient CMA. Moreover, children with transient CMA had IgE and IgG4 antibodies that more often recognized the same epitopes, compared to those with persistent CMA. From baseline to the time of tolerance development, both IgE and IgG4 binding intensity decreased significantly, particularly in areas of α-s- and β-casein (p&amp;lt;0.01, FDR&amp;lt;0.1). Interestingly, differences between IgE and IgG4 binding intensity to CM peptides decreased when the patients became tolerant. Our results suggest that the overlap between IgE and IgG4 might be important in natural tolerance acquisition. Further studies are needed to confirm our data and can eventually lead to development of more targeted treatment of food allergy. This article is protected by copyright. All rights reserved.
Journal of Agricultural and Food Chemistry, Apr 27, 2011
Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain... more Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.
Journal of Agricultural and Food Chemistry
Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain... more Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.
Children, 2015
Kiwifruit allergy has been described mostly in the adult population, but immunoglobulin (Ig)E-med... more Kiwifruit allergy has been described mostly in the adult population, but immunoglobulin (Ig)E-mediated allergic reactions to kiwifruit appear to be occurring more frequently in children. To date, 13 allergens from kiwifruit have been identified. Our aim was to identify kiwifruit allergens in a kiwifruit allergic-pediatric population, describing clinical manifestations and patterns of recognition. Twenty-four children were included. Diagnosis of kiwifruit allergy was based on compatible clinical manifestations and demonstration of specific IgE by skin prick test (SPT) and/or serum-specific IgE determination. SDS-PAGE and immunoblotting were performed with kiwifruit extract, and proteins of interest were further analyzed by mass spectrometry/mass spectrometry. For component-resolved in vitro diagnosis, sera of kiwifruit-allergic patients were analyzed by an allergen microarray assay. Act d 1 and Act d 2 were bound by IgE from 15 of 24 children. Two children with systemic manifestations recognized a protein of 15 kDa, homologous to Act d 5. Act d 1 was the allergen with the highest frequency of recognition on microarray chip, followed by Act d 2 and Act d 8. Kiwifruit allergic children develop systemic reactions most frequently following ingestion compared to adults. Act d 1 and Act d 2 are major allergens in the pediatric age group.
Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 2014
Heating destroys many conformational epitopes and reduces allergenicity of some foods. IgE-epitop... more Heating destroys many conformational epitopes and reduces allergenicity of some foods. IgE-epitope binding has been shown to be different among patients who outgrew their cow's milk or hen's egg allergy and those who did not. A significant proportion of milk- or egg-allergic children are tolerant to these foods in their baked forms. We sought to explore the effects of heating on milk and egg proteins and to evaluate for differences in immunolabeling among children with regard to reactivity to heated milk or egg. Sera from participants in clinical dietary intervention trials were utilized. Milk and egg samples were variably heated and prepared (at times within a wheat matrix). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), protein transfer, and Western blot were completed. Sera from 20 milk-allergic and 24 egg-allergic children were utilized. Gel electrophoresis showed strongly staining casein bands that persisted for up to 60 min of heating. In contr...
Journal of Allergy and Clinical Immunology, 2010
Background-Lentils are oftentimes responsible for allergic reactions to legumes in Mediterranean ... more Background-Lentils are oftentimes responsible for allergic reactions to legumes in Mediterranean children. Though the primary sequence of the major allergen, Len c 1 is known, the location of the IgE binding epitopes remains undefined. Objective-We sought to identify IgE-binding epitopes of Len c 1 and relate epitope binding to clinical characteristics. Methods-135 peptides corresponding to the primary sequence of Len c 1 were probed with sera from 33 lentil-allergic individuals and 15 non-atopic controls by means of microarray immunoassay. Lentil-specific IgE, Skin Prick Tests and clinical reactions to lentil were determined. Epitopes were defined as overlapping signal above inter-and intra-slide cutoffs and confirmed by inhibition assays using a peptide from the respective region. Hierarchical clustering of microarray data was used to correlate binding patterns with clinical findings. Results-The lentil-allergic patients specifically recognized IgE-binding epitopes located in the Cterminal region, between peptide 107 and 135. Inhibition experiments confirmed the specificity of IgE binding in this region, identifying different epitopes. Linkage of cluster results with clinical data and lentil specific IgE levels displayed a positive correlation between lentil-specific IgE levels, epitope recognition and respiratory symptoms. Modeling based on the three-dimensional structure of a homologous soy vicilin suggests that the Len c 1 epitopes identified are exposed on the surface of the molecule. Conclusion-Several IgE-binding sequential epitopes of Len c 1 have been identified. Epitopes are located in the C-terminal region, and are predicted to be exposed on the surface of the protein. Epitope diversity is positively correlated with IgE levels, pointing to a more polyclonal IgE response.
Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 2014
Food protein-induced enterocolitis syndrome (FPIES) is a gastrointestinal hypersensitivity disord... more Food protein-induced enterocolitis syndrome (FPIES) is a gastrointestinal hypersensitivity disorder with a poorly understood pathophysiology and no biomarkers to aid in diagnosis. To investigate humoral and cellular responses to casein in children with milk-FPIES, including the role of casein-specific (cs) IgA and T-cell mediated TGF-β responses. Thirty-one children previously diagnosed with milk-FPIES were challenged with milk. Twelve age-matched children with FPIES to other foods and 6 milk-tolerant children without a history of FPIES were used as controls. Casein-specific IgE, IgG, IgG4, and IgA were measured in serum and TGF-β levels in supernatants of casein-stimulated PBMCs. Twenty-six children with milk-FPIES reacted (active milk-FPIES) and five tolerated milk (milk-FPIES resolved) during food challenge. All of them had significantly lower levels of csIgG, csIgG4, and csIgA than control children (p-value<0.001). There were no TGF-β responses in supernatants of active milk-...
Pediatric Allergy and Immunology, 2013
Background-Decreased serum food-specific-IgA antibodies have been associated with allergic diseas... more Background-Decreased serum food-specific-IgA antibodies have been associated with allergic disease in cross-sectional, case-control studies. The purpose of this study was to prospectively compare egg-white-(EW)-specific-IgA and IgA2 levels between egg-allergic children and children tolerating egg. Methods-Seventeen egg allergic children were followed prospectively. Total IgA, EW-specific-IgA and EW-specific-IgA2 levels were measured in their sera with a sensitive ELISA. As negative controls were used children with no previous history of egg allergy. Egg-allergic children with or without concomitant milk allergy were evaluated as additional controls with measurement of casein-specific-IgA. Results-After 2.5±0.9 years, 9 out of 17 allergic children became tolerant and 8 remained allergic to baked egg. Baseline EW-specific-IgA2 levels were significantly lower in the eggallergic subjects (median 23.9ng/ml) compared with the negative control subjects (99.4ng/ml) and increased significantly by 28% over the study time period in 8 out of the 9 allergic children that became tolerant to baked egg. There was no significant change over time in EW-specific-IgA in any of the study groups. Non-milk-allergic subjects with concomitant egg allergy had almost 3fold higher casein-specific-IgA levels than the milk-and egg-allergic subjects (P=0.025). Conclusions-These results suggest a potential role for allergen-specific-IgA2 antibodies in the induction of food tolerance. Furthermore, they support the hypothesis that immature or impaired production of allergen-specific-IgA2 may be associated with the pathophysiology of food allergy, a defect that seems to be selective for the culprit allergen.
Journal of Agricultural and Food Chemistry, 2011
Among tree nut allergens, pecan allergens remain to be identified and characterized. The objectiv... more Among tree nut allergens, pecan allergens remain to be identified and characterized. The objective was to demonstrate the IgE-binding ability of pecan 11S legumin and characterize its sequential IgE-binding epitopes. The 11S legumin gene was amplified from a pecan cDNA library and expressed as a fusion protein in Escherichia coli. The native 11S legumin in pecan extract was identified by mass spectrometry/mass spectrometry (MS/MS). Sequential epitopes were determined by probing the overlapping peptides with three serum pools prepared from different patients' sera. A three-dimensional model was generated using almond legumin as a template and compared with known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot blot, 16 (57%) bound to 11S legumin, designated Car i 4. MS/MS sequencing of native 11S legumin identified 33 kDa acidic and 20À22 kDa basic subunits. Both pecan and walnut seed protein extracts inhibited IgE binding to recombinant Car i 4, suggesting cross-reactivity with Jug r 4. Sequential epitope mapping results of Car i 4 revealed weak, moderate, and strong reactivity of serum pools against 10, 5, and 4 peptides, respectively. Seven peptides were recognized by all three serum pools, of which two were strongly reactive. The strongly reactive peptides were located in three discrete regions of the Car i 4 acidic subunit sequence (residues 118À132, 208À219, and 238À249). Homology modeling of Car i 4 revealed significant overlapping regions shared in common with other tree nut legumins.
Regulatory Toxicology and Pharmacology, 2006
Cry1F protein, derived from Bacillus thuringiensis, is eVective at controlling lepidopteran pests... more Cry1F protein, derived from Bacillus thuringiensis, is eVective at controlling lepidopteran pests and a synthetic Cry1F transgene was transferred into maize. For the safety assessment of genetically modiWed food crops, the allergenic potential of the introduced novel trait(s) is evaluated. Because no single parameter is currently predictive of allergic potential, a 'weight of evidence' approach has been proposed. As part of this assessment, the amino acid (aa) sequence of the Cry1F protein was compared to a database of known allergens using recommended criteria. The Cry1F protein did not show signiWcant similarity or a match of eight contiguous identical aa with any allergen. However, a single six contiguous aa match was identiWed between Cry1F and the Der p7 protein of the dust mite, Dermatophagoides pteronyssinus. To investigate whether Cry1F was cross-reactive with Der p7, sera from 10 dust mite allergic patients containing Der p 7-speciWc IgE antibody were used to compare IgE-speciWc binding. No evidence of cross-reactivity was observed between Cry1F and Der p7. This study provides in vitro IgE sera screening data, that when considered in the context of other bioinformatic data [
The Lancet, 1984
A membrane-coated virus having a diameter of 85-90 nm and containing a 40-45 nm core was found to... more A membrane-coated virus having a diameter of 85-90 nm and containing a 40-45 nm core was found to replicate in cell cultures derived from chimpanzee liver after inoculation of serum containing infective non-A, non-B (NANB) hepatitis viruses from two independent sources. Replication of this agent was not observed when the same cells were inoculated with a chloroform-extracted inoculum or were left uninoculated. Replication involves assembly of virus cores on tubular structures similar to those seen in liver cells of chimpanzees infected with most isolates of NANB virus.
Two-dimensional polyacrylamide gel electrophoresis (2DE) and Western immunoblotting have proven t... more Two-dimensional polyacrylamide gel electrophoresis (2DE) and Western immunoblotting have proven to be invaluable and fundamental techniques for comprehensive characterization of food allergens. Here, we describe and discuss detailed protocols used in our studies on identification of allergenic proteins in shrimp, sesame, hazelnut, and pistachio.
Allergy
Although most of cow&amp;#39;s milk (CM) allergic children will outgrow their allergy, th... more Although most of cow&amp;#39;s milk (CM) allergic children will outgrow their allergy, the pathomechanism of the natural development of tolerance remains poorly understood. It has been suggested that the balance between milk specific IgE and IgG4 plays a major role. We aimed to investigate differences in IgE and IgG4 antibody binding to cow&amp;#39;s milk (CM) epitopes between patients with persistent CM allergy (CMA) and those that naturally became tolerant. Sera from thirty-five children with proven CMA (median age at inclusion of 10 months) were analysed retrospectively; 22 patients have become tolerant (median age at tolerance acquisition of 51 months) during the study period as confirmed by a negative oral food challenge. IgE and IgG4 binding to sequential epitopes derived from 5 major CM proteins were measured with a peptide microarray-based immunoassay. At baselines, greater intensity and broader diversity of IgE and IgG4 binding have been found in children with persistent CMA beyond 5 years of age compared to patients with transient CMA. Moreover, children with transient CMA had IgE and IgG4 antibodies that more often recognized the same epitopes, compared to those with persistent CMA. From baseline to the time of tolerance development, both IgE and IgG4 binding intensity decreased significantly, particularly in areas of α-s- and β-casein (p&amp;lt;0.01, FDR&amp;lt;0.1). Interestingly, differences between IgE and IgG4 binding intensity to CM peptides decreased when the patients became tolerant. Our results suggest that the overlap between IgE and IgG4 might be important in natural tolerance acquisition. Further studies are needed to confirm our data and can eventually lead to development of more targeted treatment of food allergy. This article is protected by copyright. All rights reserved.
Journal of Agricultural and Food Chemistry, Apr 27, 2011
Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain... more Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.
Journal of Agricultural and Food Chemistry
Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain... more Although pecans are associated with IgE-mediated food allergies, the allergens responsible remain to be identified and characterized. The 2S albumin gene was amplified from the pecan cDNA library. Dot-blots were used to screen the recombinant protein with pecan allergic patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; serum. The affinity purified native protein was analyzed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS) analysis. Cross-reactivity with walnut was determined by inhibition enzyme-linked immunosorbent assay (ELISA). Sequential epitopes were determined by probing the overlapping peptides with three different patients&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; serum pool. The 3-dimensional homology model was generated, and the locations of the pecan epitopes were compared with those of known sequential epitopes on other allergenic tree nut homologues. Of 28 patients tested by dot-blot, 22 (79%) bound to 2S albumin, designated as Car i 1. Edman sequencing and the MS/MS sequencing of native 2S albumin confirmed the identity of recombinant (r) Car i 1. Both pecan and walnut protein extracts inhibited the IgE-binding to rCar i 1. Sequential epitope mapping indicated weak, moderate, and strong reactivity against 12, 7, and 5 peptides, respectively. Of the 11 peptides recognized by all serum pools, 5 peptides were strongly reactive and located in 3 discrete regions of the Car i 1 (amino acids 43-57, 67-78, and 106-120). Three-dimensional modeling revealed IgE-reactive epitopes to be solvent accessible and share significant homology with other tree nuts providing a possible basis for previously observed cross-reactivity.
Children, 2015
Kiwifruit allergy has been described mostly in the adult population, but immunoglobulin (Ig)E-med... more Kiwifruit allergy has been described mostly in the adult population, but immunoglobulin (Ig)E-mediated allergic reactions to kiwifruit appear to be occurring more frequently in children. To date, 13 allergens from kiwifruit have been identified. Our aim was to identify kiwifruit allergens in a kiwifruit allergic-pediatric population, describing clinical manifestations and patterns of recognition. Twenty-four children were included. Diagnosis of kiwifruit allergy was based on compatible clinical manifestations and demonstration of specific IgE by skin prick test (SPT) and/or serum-specific IgE determination. SDS-PAGE and immunoblotting were performed with kiwifruit extract, and proteins of interest were further analyzed by mass spectrometry/mass spectrometry. For component-resolved in vitro diagnosis, sera of kiwifruit-allergic patients were analyzed by an allergen microarray assay. Act d 1 and Act d 2 were bound by IgE from 15 of 24 children. Two children with systemic manifestations recognized a protein of 15 kDa, homologous to Act d 5. Act d 1 was the allergen with the highest frequency of recognition on microarray chip, followed by Act d 2 and Act d 8. Kiwifruit allergic children develop systemic reactions most frequently following ingestion compared to adults. Act d 1 and Act d 2 are major allergens in the pediatric age group.
Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology, 2014
Heating destroys many conformational epitopes and reduces allergenicity of some foods. IgE-epitop... more Heating destroys many conformational epitopes and reduces allergenicity of some foods. IgE-epitope binding has been shown to be different among patients who outgrew their cow's milk or hen's egg allergy and those who did not. A significant proportion of milk- or egg-allergic children are tolerant to these foods in their baked forms. We sought to explore the effects of heating on milk and egg proteins and to evaluate for differences in immunolabeling among children with regard to reactivity to heated milk or egg. Sera from participants in clinical dietary intervention trials were utilized. Milk and egg samples were variably heated and prepared (at times within a wheat matrix). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), protein transfer, and Western blot were completed. Sera from 20 milk-allergic and 24 egg-allergic children were utilized. Gel electrophoresis showed strongly staining casein bands that persisted for up to 60 min of heating. In contr...
Journal of Allergy and Clinical Immunology, 2010
Background-Lentils are oftentimes responsible for allergic reactions to legumes in Mediterranean ... more Background-Lentils are oftentimes responsible for allergic reactions to legumes in Mediterranean children. Though the primary sequence of the major allergen, Len c 1 is known, the location of the IgE binding epitopes remains undefined. Objective-We sought to identify IgE-binding epitopes of Len c 1 and relate epitope binding to clinical characteristics. Methods-135 peptides corresponding to the primary sequence of Len c 1 were probed with sera from 33 lentil-allergic individuals and 15 non-atopic controls by means of microarray immunoassay. Lentil-specific IgE, Skin Prick Tests and clinical reactions to lentil were determined. Epitopes were defined as overlapping signal above inter-and intra-slide cutoffs and confirmed by inhibition assays using a peptide from the respective region. Hierarchical clustering of microarray data was used to correlate binding patterns with clinical findings. Results-The lentil-allergic patients specifically recognized IgE-binding epitopes located in the Cterminal region, between peptide 107 and 135. Inhibition experiments confirmed the specificity of IgE binding in this region, identifying different epitopes. Linkage of cluster results with clinical data and lentil specific IgE levels displayed a positive correlation between lentil-specific IgE levels, epitope recognition and respiratory symptoms. Modeling based on the three-dimensional structure of a homologous soy vicilin suggests that the Len c 1 epitopes identified are exposed on the surface of the molecule. Conclusion-Several IgE-binding sequential epitopes of Len c 1 have been identified. Epitopes are located in the C-terminal region, and are predicted to be exposed on the surface of the protein. Epitope diversity is positively correlated with IgE levels, pointing to a more polyclonal IgE response.