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Paolo Colombo - One of the best experts on this subject based on the ideXlab platform.
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expression and characterization of recombinant par j 1 and par j 2 resembling the allergenic epitopes of Parietaria judaica pollen
Scientific Reports, 2019Co-Authors: Yulia Dorofeeva, Paolo Colombo, Rudolf Valenta, Miguel Blanca, Adriano Mari, Roman Khanferyan, Margarete FocketejklAbstract:The weed wall pellitory, Parietaria judaica, is one the most important pollen allergen sources in the Mediterranean area causing severe symptoms of hay fever and asthma in allergic patients. We report the expression of the major Parietaria allergens, Par j 1 and Par j 2 which belong to the family of lipid transfer proteins, in insect cells. According to circular dichroism analysis and gel filtration, the purified allergens represented folded and monomeric proteins. Insect cell-expressed, folded Par j 2 exhibited higher IgE binding capacity and more than 100-fold higher allergenic activity than unfolded Escherichia coli-expressed Par j 2 as demonstrated by IgE ELISA and basophil activation testing. IgE ELISA inhibition assays showed that Par j 1 and Par j 2, contain genuine and cross-reactive IgE epitopes. IgG antibodies induced by immunization with Par j 2 inhibited binding of allergic patients IgE to Par j 1 only partially. IgE inhibition experiments demonstrated that insect cell-expressed Par j 1 and Par j 2 together resembled the majority of allergenic epitopes of the Parietaria allergome and therefore both should be used for molecular diagnosis and the design of vaccines for allergen-specific immunotherapy of Parietaria allergy.
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multiple ige recognition on the major allergen of the Parietaria pollen par j 2
Molecular Immunology, 2015Co-Authors: Valeria Longo, Maria Assunta Costa, Fabio Cibella, Giuseppina Cuttitta, Stefania La Grutta, Paolo ColomboAbstract:The interaction between IgE antibodies and allergens is a key event in triggering an allergic reaction. The characterization of this region provides information of paramount importance for diagnosis and therapy. Par j 2 Lipid Transfer Protein is one of the most important allergens in southern Europe and a well-established marker of sensitization in Parietaria pollen allergy. The main aim of this study was to map the IgE binding regions of this allergen and to study the pattern of reactivity of individual Parietaria-allergic patients. By means of gene fragmentation, six overlapping peptides were expressed in Escherichia coli, and their IgE binding activity was evaluated by immunoblotting in a cohort of 79 Parietaria-allergic patients. Our results showed that Pj-allergic patients display a heterogeneous pattern of IgE binding to the different recombinant fragments, and that patients reacted simultaneously against several protein domains spread all the over the molecule, even in fragments which do not contain structural features resembling the native allergen. Our results reveal the presence of a large number of linear and conformational epitopes on the Par j 2 sequence, which probably explains the high allergenic activity of this allergen.
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cloning expression in e coli and immunological characterization of par j 3 0201 a Parietaria pollen profilin variant
Molecular Immunology, 2014Co-Authors: Angela Bonura, Rudolf Valenta, A Trapani, L Gulino, Valeria Longo, R Asero, Paolo ColomboAbstract:Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767 bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201.
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innate and adaptive immune responses to the major Parietaria allergen par j 1 in healthy subjects
Immunobiology, 2013Co-Authors: Angela Bonura, Mario Melis, Francesco Gervasi, Sonia Quaratino, C Di Sano, Paolo ColomboAbstract:Abstract In this study we wanted to analyse the pattern of the immune response to the Parietaria major allergen Par j 1 in freshly purified peripheral blood mononuclear cell (PBMC) from healthy subjects. We observed that Par j 1 was capable of inducing IFN-γ production by CD3− and CD16+/CD56+ cells exclusively in healthy individuals. Furthermore, a multiparametric analysis allowed us a better definition of two IFN-γ-Par j 1 specific populations (IFN-γdim and IFN-γhigh) characterized by the presence of different proportions of NKT and NK cells. We also identified the concomitant presence of a subset of IL-10+ NK cells. Moreover, CFSE staining showed that the Par j 1 preferentially induced the proliferation of CD3−/CD56+/CD335+ cells. Finally, a subset of CD4+/CD25+/FoxP3+/IL-10− T cells was identified. The result of this pilot study suggest that during a tolerogenic response, the major allergen of the Parietaria pollen works as an activator of both the innate and the adaptive human immune system.
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identification of cross reactive and genuine Parietaria judaica pollen allergens
The Journal of Allergy and Clinical Immunology, 2003Co-Authors: Sabine Stumvoll, Kerstin Westritschnig, Jonas Lidholm, Susanne Spitzauer, Paolo Colombo, Giovanni Duro, Dietrich Kraft, Domenico Geraci, Rudolf ValentaAbstract:Abstract Background: The weed Parietaria judaica is one of the most important pollen allergen sources in the Mediterranean area. Objective: We sought to identify P judaica pollen allergen, which might be used to serologically distinguish genuine Parietaria sensitization and cross-reactivity to allergens from other weed species (eg, mugwort and ragweed). Methods: The allergen profile of P judaica IgE-reactive sera from weed pollen-sensitized allergic individuals from the Mediterranean region (n = 36) with high Parietaria pollen exposure and from weed pollen-allergic patients with little or no Parietaria exposure (Austria, n=42; Scandinavia, n=8; United States, n=19) was established by CAP FEIA measurements and by IgE immunoblot inhibition experiments with recombinant allergens. Results: The majority (83%) of the Mediterranean weed pollen-allergic patients mounted high IgE antibody levels (mean specific IgE, 20.89 kUA/L) against recombinant (r) Par j 2, whereas only 7% of the non-Mediterranean weed-allergic patients showed low IgE reactivity to rPar j 2 (mean specific IgE, 1.03 kUA/L). The cytoskeletal protein profilin and a 2-EF-hand calcium-binding allergen were identified as cross-reactive Parietaria allergens, which were recognized preferentially by Parietaria -positive, non-Mediterranean weed pollen-allergic patients. Conclusion: rPar j 2 might be used as a diagnostic marker allergen to identify weed pollen-allergic patients who are genuinely sensitized against Parietaria pollen and thus would be particularly suited for specific immunotherapy with Parietaria pollen extract. (J Allergy Clin Immunol 2003;111:974-9.)
Angela Bonura - One of the best experts on this subject based on the ideXlab platform.
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modulating allergic response by engineering the major Parietaria allergens
The Journal of Allergy and Clinical Immunology, 2017Co-Authors: Angela Bonura, Daniela Di Blasi, Mario Melis, C Uasuf, Cibella Fabio, Cinzia Butteroni, Francesco Gervasi, Bianca Barletta, Silvia Corinti, Gabriella Di FeliceAbstract:Capsule Summary An engineered recombinant hybrid composed of the two major Parietaria allergens (PjEDcys) with reduced allergenicity, retained immunogenicity but immunomodulatory activity for Allergen Immunotherapy of Parietaria allergic patients.
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modulating allergic response by engineering the major Parietaria allergens
The Journal of Allergy and Clinical Immunology, 2017Co-Authors: Angela Bonura, Daniela Di Blasi, Mario Melis, C Uasuf, Cibella Fabio, Cinzia Butteroni, Francesco Gervasi, Bianca Barletta, Silvia Corinti, Gabriella Di FeliceAbstract:Capsule Summary An engineered recombinant hybrid composed of the two major Parietaria allergens (PjEDcys) with reduced allergenicity, retained immunogenicity but immunomodulatory activity for Allergen Immunotherapy of Parietaria allergic patients.
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cloning expression in e coli and immunological characterization of par j 3 0201 a Parietaria pollen profilin variant
Molecular Immunology, 2014Co-Authors: Angela Bonura, Rudolf Valenta, A Trapani, L Gulino, Valeria Longo, R Asero, Paolo ColomboAbstract:Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767 bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201.
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innate and adaptive immune responses to the major Parietaria allergen par j 1 in healthy subjects
Immunobiology, 2013Co-Authors: Angela Bonura, Mario Melis, Francesco Gervasi, Sonia Quaratino, C Di Sano, Paolo ColomboAbstract:Abstract In this study we wanted to analyse the pattern of the immune response to the Parietaria major allergen Par j 1 in freshly purified peripheral blood mononuclear cell (PBMC) from healthy subjects. We observed that Par j 1 was capable of inducing IFN-γ production by CD3− and CD16+/CD56+ cells exclusively in healthy individuals. Furthermore, a multiparametric analysis allowed us a better definition of two IFN-γ-Par j 1 specific populations (IFN-γdim and IFN-γhigh) characterized by the presence of different proportions of NKT and NK cells. We also identified the concomitant presence of a subset of IL-10+ NK cells. Moreover, CFSE staining showed that the Par j 1 preferentially induced the proliferation of CD3−/CD56+/CD335+ cells. Finally, a subset of CD4+/CD25+/FoxP3+/IL-10− T cells was identified. The result of this pilot study suggest that during a tolerogenic response, the major allergen of the Parietaria pollen works as an activator of both the innate and the adaptive human immune system.
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characterization of a par j 1 par j 2 mutant hybrid with reduced allergenicity for immunotherapy of Parietaria allergy
Clinical & Experimental Allergy, 2012Co-Authors: Angela Bonura, Mario Melis, Cinzia Butteroni, Bianca Barletta, Silvia Corinti, Maria Assunta Costa, Rosa Passantino, Giovanna Montana, Luisa M Bondi, G Di FeliceAbstract:SummaryBackground Parietaria pollen is one of the major cause of pollinosis in the southern Europe. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease restoring a normal immunity against allergens. Methods We designed a recombinant molecule (PjEDloop1) comprised of genetic-engineered variants of the major allergens of the Parietaria pollen (Par j 2/Par j 1). Purity and chemical–physical properties of the derivative were analysed by RP-HPLC chromatography and Photon Correlation Spectroscopy. Immunological activity was evaluated by means of Western blotting, ELISA inhibition and PBMC proliferation assay in 10 Parietaria allergic patients. Basophil activation was studied in six subjects. The immunogenicity of the hybrid was studied looking at the immune responses induced in a mouse model of sensitization. Results The PjEDloop1 hybrid was produced as a purified recombinant protein with high stability in solution. Western blot, ELISA inhibition and basophil activation test showed that the PjEDloop1 displays a remarkable reduced IgE binding and anaphylactic activity. CD3 reactivity was conserved in all patients. Mice immunization with the rPjEDloop1 induced antibodies and T cell responses comparable to that obtained by the wild type allergens. Such antibodies shared the specificities to rPar j 1 and rPar j 2 with human IgE antibodies. Conclusion Our results demonstrated that a mutant hybrid expressing genetically engineered forms of the major P. judaica allergens displayed reduced allergenicity and retained T cell reactivity for the induction of protective antibodies in vaccination approaches for the treatment of Parietaria pollinosis.
Domenico Geraci - One of the best experts on this subject based on the ideXlab platform.
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isolation expression and immunological characterization of a calcium binding protein from Parietaria pollen
Molecular Immunology, 2008Co-Authors: Angela Bonura, Kerstin Westritschnig, Domenico Geraci, Rudolf Valenta, G Di Felice, Raffaella Tinghino, A Trapani, L Gulino, Saverio Amoroso, Enrico ScalaAbstract:The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480 bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.
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identification of cross reactive and genuine Parietaria judaica pollen allergens
The Journal of Allergy and Clinical Immunology, 2003Co-Authors: Sabine Stumvoll, Kerstin Westritschnig, Jonas Lidholm, Susanne Spitzauer, Paolo Colombo, Giovanni Duro, Dietrich Kraft, Domenico Geraci, Rudolf ValentaAbstract:Abstract Background: The weed Parietaria judaica is one of the most important pollen allergen sources in the Mediterranean area. Objective: We sought to identify P judaica pollen allergen, which might be used to serologically distinguish genuine Parietaria sensitization and cross-reactivity to allergens from other weed species (eg, mugwort and ragweed). Methods: The allergen profile of P judaica IgE-reactive sera from weed pollen-sensitized allergic individuals from the Mediterranean region (n = 36) with high Parietaria pollen exposure and from weed pollen-allergic patients with little or no Parietaria exposure (Austria, n=42; Scandinavia, n=8; United States, n=19) was established by CAP FEIA measurements and by IgE immunoblot inhibition experiments with recombinant allergens. Results: The majority (83%) of the Mediterranean weed pollen-allergic patients mounted high IgE antibody levels (mean specific IgE, 20.89 kUA/L) against recombinant (r) Par j 2, whereas only 7% of the non-Mediterranean weed-allergic patients showed low IgE reactivity to rPar j 2 (mean specific IgE, 1.03 kUA/L). The cytoskeletal protein profilin and a 2-EF-hand calcium-binding allergen were identified as cross-reactive Parietaria allergens, which were recognized preferentially by Parietaria -positive, non-Mediterranean weed pollen-allergic patients. Conclusion: rPar j 2 might be used as a diagnostic marker allergen to identify weed pollen-allergic patients who are genuinely sensitized against Parietaria pollen and thus would be particularly suited for specific immunotherapy with Parietaria pollen extract. (J Allergy Clin Immunol 2003;111:974-9.)
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the allergens of Parietaria
International Archives of Allergy and Immunology, 2003Co-Authors: Paolo Colombo, Angela Bonura, Maria Assunta Costa, Vincenzo Izzo, Giovanni Locorotondo, Rosa Passantino, Saverio Amoroso, Domenico GeraciAbstract:Parietaria is a genus of dicotyledonous weeds of the Urticaceae family including several species and its pollen grain is one of the most important allergenic sources in the Mediterr
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isolation and characterization of two cdna clones coding for isoforms of the Parietaria judaica major allergen par j 1 0101
International Archives of Allergy and Immunology, 1997Co-Authors: Giovanni Duro, Paolo Colombo, Rossana Porcasi, Giovanni Locorotondo, Roberta Cocchiara, Assunta Costa M, Di Fiore R, Domenico GeraciAbstract:Two cDNA clones named P9* and P1* of 794 and 631 bp, respectively, were isolated from a Λ ZAP cDNA expression library using Parietaria judaica (Pj) pollen-specific IgE antibodi
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cdna cloning sequence analysis and allergological characterization of par j 2 0101 a new major allergen of the Parietaria judaica pollen
FEBS Letters, 1996Co-Authors: Giovanni Duro, Paolo Colombo, Maria Assunta Costa, Vincenzo Izzo, Rossana Porcasi, Renata Di Fiore, Giovanni Locorotondo, Mario G Mirisola, Roberta Cocchiara, Domenico GeraciAbstract:A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj-allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23128) of the sera of Pj-allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross-inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10- 14 kDa native allergens and preincubation of sera from Pj- allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10-14 kDa native allergen region, suggesting that these two allergens contributed to the region. A-ey words." cDNA cloning; Allergy; Immunoglobulin E; 1 'arietaria judaica
Oliver Cromwell - One of the best experts on this subject based on the ideXlab platform.
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immunotherapy with an alum adsorbed Parietaria pollen allergoid a 2 year double blind placebo controlled study
Allergy, 1997Co-Authors: M G Tari, Edda Frank, M Mancino, E Ghezzi, Oliver CromwellAbstract:: A double-blind, placebo-controlled study was performed in order to confirm the safety, suitability, and efficacy of an alum-adsorbed Parietaria judaica-pollen allergoid, Allergovit, for allergen-specific immunotherapy. Parietaria pollen is an important cause of pollinosis, particularly in the Mediterranean zone, where it may be encountered for up to 8-9 months of the year. It is an aggressive allergen, and the doses tolerated during immunotherapy are less than those achieved with grass pollen. This factor increases the desirability of using therapeutic preparations with minimal IgE-binding activity, such as allergoids, in order to reduce the risk of side-effects and enable patients to tolerate a higher dose of allergen, thereby increasing the chances of successful specific immunotherapy. Forty patients with rhinitis and/or asthma were allocated at random to active- or placebo-treatment groups at the beginning of the study. All patients received the active preparation during the second year of the study. Immunotherapy was well tolerated by all patients and the incidence of side-effects was low. Treatment resulted in significant reductions in specific cutaneous reactivity and increases in nasal tolerance. A progressive improvement in nasal inspiratory peak flow in association with the immunotherapy indicated a reduction in nasal inflammation. These objective assessments of efficacy endorsed the results from the patients' diary cards, which indicated significant improvements in symptoms and reductions in the use of medication. The immunologic activity of the therapeutic preparation was demonstrated by the induction of a significant specific-IgG antibody response, with increases in IgG4 during the second year of treatment. We conclude that the safety and efficacy of immunotherapy with the Parietaria allergoid make it suitable for consideration in the treatment of patients with Parietaria-pollen-induced rhinitis or asthma.
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immunotherapy with an alum adsorbed Parietaria pollen allergoid a 2 year double blind placebo controlled study
Allergy, 1997Co-Authors: M G Tari, Edda Frank, M Mancino, E Ghezzi, Oliver CromwellAbstract:: A double-blind, placebo-controlled study was performed in order to confirm the safety, suitability, and efficacy of an alum-adsorbed Parietaria judaica-pollen allergoid, Allergovit, for allergen-specific immunotherapy. Parietaria pollen is an important cause of pollinosis, particularly in the Mediterranean zone, where it may be encountered for up to 8-9 months of the year. It is an aggressive allergen, and the doses tolerated during immunotherapy are less than those achieved with grass pollen. This factor increases the desirability of using therapeutic preparations with minimal IgE-binding activity, such as allergoids, in order to reduce the risk of side-effects and enable patients to tolerate a higher dose of allergen, thereby increasing the chances of successful specific immunotherapy. Forty patients with rhinitis and/or asthma were allocated at random to active- or placebo-treatment groups at the beginning of the study. All patients received the active preparation during the second year of the study. Immunotherapy was well tolerated by all patients and the incidence of side-effects was low. Treatment resulted in significant reductions in specific cutaneous reactivity and increases in nasal tolerance. A progressive improvement in nasal inspiratory peak flow in association with the immunotherapy indicated a reduction in nasal inflammation. These objective assessments of efficacy endorsed the results from the patients' diary cards, which indicated significant improvements in symptoms and reductions in the use of medication. The immunologic activity of the therapeutic preparation was demonstrated by the induction of a significant specific-IgG antibody response, with increases in IgG4 during the second year of treatment. We conclude that the safety and efficacy of immunotherapy with the Parietaria allergoid make it suitable for consideration in the treatment of patients with Parietaria-pollen-induced rhinitis or asthma.
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characterization of major allergens of Parietaria officinalis
International Archives of Allergy and Immunology, 1996Co-Authors: Helga Kahlert, Oliver Cromwell, Bernhard H F Weber, M Teppke, R Wahl, H FiebigAbstract:The major allergens of Parietaria officinalis were characterized with a panel of nine monoclonal antibodies (mAbs). The binding of mAbs and patients’ IgE in Western blots revealed t
J A Asturias - One of the best experts on this subject based on the ideXlab platform.
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par j 1 and par j 2 the two major allergens in Parietaria judaica bind preferentially to monoacylated negative lipids
FEBS Journal, 2009Co-Authors: R Gonzalezrioja, A Martinez, J A Asturias, Felix M Goni, Ana Rosa VigueraAbstract:Par j 1 and Par j 2 proteins are the two major allergens in Parietaria judaica pollen, one of the main causes of allergic diseases in the Mediterranean area. Each of them contains eight cysteine residues organized in a pattern identical to that found in plant nonspecific lipid transfer proteins. The 139- and 102-residue recombinant allergens, corresponding respectively to Par j 1 and Par j 2, refold properly to fully functional forms, whose immunological properties resemble those of the molecules purified from the natural source. Molecular modeling shows that, despite the lack of extensive primary structure homology with nonspecific lipid transfer proteins, both allergens contain a hydrophobic cavity suited to accommodate a lipid ligand. In the present study, we present novel evidence for the formation of complexes of these natural and recombinant proteins from Parietaria pollen with lipidic molecules. The dissociation constant of oleyl-lyso-phosphatidylcholine is 9.1 ± 1.2 μm for recombinant Par j 1, whereas pyrenedodecanoic acid shows a much higher affinity, with a dissociation constant of approximately 1 μm for both recombinant proteins, as well as for the natural mixture. Lipid binding does not alter the secondary structure content of the protein but is very efficient in protecting disulfide bonds from reduction by dithiothreitol. We show that Par j 1 and Par j 2 not only bind lipids from micellar dispersions, but also are able to extract and transfer negative phospholipids from bilayers.
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a sensitive monoclonal antibody based enzyme linked immunosorbent assay to quantify Parietaria judaica major allergens par j 1 and par j 2
Clinical & Experimental Allergy, 2006Co-Authors: M C Arilla, R Gonzalezrioja, I Ibarrola, Amparo Mir, J Monteseirin, J Conde, A Martinez, J A AsturiasAbstract:Summary Background Parietaria pollen is one of the most important causes of pollinosis in Mediterranean countries. Parietaria judaica pollen extract presents two major allergens, Par j 1 and Par j 2, that belong to the lipid transfer protein family. Objective To develop an ELISA for quantification of both major allergens of P. judaica pollen extracts, and to assert correlation of these allergens content with the allergenic activity of extracts. Methods Natural Par j 1–Par j 2 allergens were purified by gel filtration, ion exchange, and affinity chromatography and identified by mass spectrometry. Rabbit antisera were obtained using this protein preparation as antigen and used for immunoaffinity purification of nPar j 1–Par j 2. BALB/c mice were immunized with the immunopurified nPar j 1–Par j 2 and after fusion and screening by direct ELISA, 5D4 monoclonal antibody was selected as capture antibody to develop a quantitative two-site ELISA. Bound proteins were detected by a biotinylated Par j 1–Par j 2-specific polyclonal antibody. Results The optimized ELISA was developed from 25 to 8000 pg/mL of purified Par j 1–Par j 2, and a linear portion of 200–1000 pg/mL. The intraassay and interassay coefficients of variation were lower than 7% and 14% respectively. The assay was very sensitive and specific as it had a detection limit of 25 pg/mL and did not detect reactivity with the same family plants, as Urtica. Par j 1–Par j 2 allergens content was measured in 14 P. judaica and two P. officinalis pollen extracts showing a significant correlation with their allergenic activity measured by enzyme allergosorbent test inhibition. Conclusions The results proved the usefulness of the two-sandwich ELISA for the standardization of Parietaria pollen extracts intended for clinical use, because of its good correlation with allergenic potency.
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biological characterization of glutaraldehyde modified Parietaria judaica pollen extracts
Clinical & Experimental Allergy, 2004Co-Authors: I Ibarrola, M C Arilla, Amparo Mir, A Martinez, M L Sanz, P M Gamboa, D Benahmed, Angel Ferrer, J A AsturiasAbstract:Summary Background Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts. Objective To assess an accurate methodology for standardization of chemically modified extracts. Methods GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured. Results Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found. Conclusions The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT.
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pcr based cloning and immunological characterization of Parietaria judaica pollen profilin
Journal of Investigational Allergology and Clinical Immunology, 2004Co-Authors: J A Asturias, J L Eseverri, M C Arilla, R Gonzalezrioja, I Ibarrola, A MartinezAbstract:Summary.Profilin has been described as an allergen present in pollen of trees, grasses and weeds. Since Parietaria judaica profilin has a molecular mass similar to other Parietaria allergens (Par j 1 and Par j 2) in the 14-10 kDa range, it is difficult to assess the prevalence of profilin by immunoblotting or to obtain sufficient amounts of purified native profilin for investigation and diagnosis. The aim of this study was to identify P. judaica profilin by PCR-based cDNA cloning and to elucidate its allergenic characteristics. Two cDNA clones encoding P. judaica pollen profilin were isolated by polymerase chain reaction (PCR) amplification using degenerate primers. Sequencing of both clones (Par j 3.0101 and Par j 3.0102) demonstrated a high amino acid sequence homology. Immunodetection of P. judaica pollen after isoelectrofocusing and incubation with rabbit antiserum against profilin indicated the existence of at least 2 iso forms. Expression in Escherichia coli BL21 (DE3) was carried out using a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Cross-reactivity has been found between recombi nant P. judaica pollen profilin and profilins from other botanical unrelated plants.
