Mugwort-allergic Patients Research Articles

Art v 1 IgE epitopes of patients and humanized mice are conformational

Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and thecorresponding HLA have been made. We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

T Cell Epitope-Containing Domains of Ragweed Amb a 1 and Mugwort Art v 6 Modulate Immunologic Responses in Humans and Mice.

BackgroundRagweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) are the major cause of pollen allergy in late summer. Allergen-specific lymphocytes are crucial for immune modulation during immunotherapy. We sought to generate and pre-clinically characterise highly immunogenic domains of the homologous pectate lyases in ragweed (Amb a 1) and mugwort pollen (Art v 6) for immunotherapy.MethodsDomains of Amb a 1 (Amb a 1α) and Art v 6 (Art v 6α) and a hybrid molecule, consisting of both domains, were designed, expressed in E. coli and purified. Human IgE reactivity and allergenicity were assessed by ELISA and mediator release experiments using ragweed and mugwort allergic patients. Moreover, T cell proliferation was determined. Blocking IgG antibodies and cytokine production in BALB/c mice were studied by ELISA and ELISPOT.ResultsThe IgE binding capacity and in vitro allergenic activity of the Amb a 1 and Art v 6 domains and the hybrid were either greatly reduced or abolished. The recombinant proteins induced T cell proliferative responses comparable to those of the natural allergens, indicative of retained allergen-specific T cell response. Mice immunisation with the hypoallergens induced IL-4, IL-5, IL-13 and IFN-γ production after antigen-specific in vitro re-stimulation of splenocytes. Moreover, murine IgG antibodies that inhibited specific IgE binding of ragweed and mugwort pollen allergic patients were detected.ConclusionAccumulation of T cell epitopes and deletion of IgE reactive areas of Amb a 1 and Art v 6, modulated the immunologic properties of the allergen immuno-domains, leading to promising novel candidates for therapeutic approach.

Mugwort-Sensitized Individuals from North Europe, South Europe and North America Show Different IgE Reactivity Patterns

Background: Sensitization to weed pollen allergens at the molecular level is not fully understood. We studied IgE reactivity to the major mugwort allergen Art v 1 in relation to allergens from other weed pollen and cross-reactive components in mugwort-sensitized subjects. Methods: Art v 1 and Amb a 1 were affinity purified and coupled to experimental ImmunoCAP® tests. Samples from North Europe (n = 50), South Europe (n = 19) and North America (n = 41) were analyzed for IgE against mugwort pollen, weed allergen components, pan-allergens and cross-reactive carbohydrate determinants (CCDs). Results: The prevalence of IgE reactivity (>0.35 kU_A/l) to Art v 1 was significantly higher in samples from North Europe than in those from North America. IgE to Amb a 1 was more common in North America than in North and South Europe, while IgE to Par j 2 was common in South Europe, less common in North America, and absent in North Europe. IgE to Art v 3 in mugwort-allergic patients was more common in North Europe than in South Europe and North America, while IgE to Sal k 1 was similar between the areas. Subjects with an Art v 1/mugwort-specific IgE ratio <0.5 had more often IgE to Amb a 1, profilin, polcalcin and CCDs than subjects with a ratio >0.5. Conclusions: Mugwort-sensitized subjects have different IgE reactivity profiles to weed allergens, reflecting their exposure to various pollens. Subjects with a low ratio between the IgE levels to Art v 1 and mugwort have a diverse IgE reactivity profile, indicating a role for cross-reactive allergens in their mugwort sensitization.

A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29-31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more alpha-arabinofuranosyl residues with some beta-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked beta-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation

Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4(+) cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.

Untersuchung zur Kreuzreaktivität zwischen short, western, giant und false Ragweed

By EAST, SDS-PAGE, Westemblot and especially EAST inhibition we could show that there is a high degree of crossreactivity between short-, giant-, western and false ragweed. Nevertheless, we recommend that EAST measurements should be made with short ragweed allergen discs. This covers the most sensitivities in sera of ragweed-allergic patients. Short ragweed is the most common ragweed especially in Europe. Although we could not prove crossreactivity between ragweed and mugwort, we recommend that sera of mugwort-allergic patients should also be investigated using ragweed allergen disks. In 18.2% of mugwort-allergic patients we could also measure class > 1 using ragweed allergen discs.

Reduction of Substance P in Nasal Lavage Fluid after Allergen Challenge of Mugwort Allergic Patients during Allergen Immunotherapy

Allergen specific immunotherapy (ASIT) may impact neuropeptides released into nasal lavage fluid (NLF) including substance P (SP). 15 subjects (8 females and 7 males) mean age 33.0 ± 7.8 years with symptoms during the previous mugwort pollen season were treated with ASIT as pre-seasonal weekly injections of a mugwort pollen allergoid (Allergovit) for 3 years. All had both positive skin prick tests to mugwort pollen allergen and elevated specific IgE to mugwort pollen (Phadia). The control group consisted of 9 untreated subjects. Nasal mugwort allergen challenge with subsequent lavage performed 15 minutes post-challenge was done by the method of Naclerio. The concentration of SP in NLF was determined by RIA (Incstar, USA). Nasal symptoms during the pollen seasons were recorded. Reduction of symptom scores occurred during the three years of ASIT. Levels of SP in NLF after nasal mugwort challenge in both groups prior to therapy were similar: ASIT group - SP 55.1 ± 15.5 pg/ml versus control group- 54.3 ± 10.1 pg/ml. SP levels in NLF 15 minutes after mugwort allergen challenge were significantly less after 1 year of ASIT versus controls (47.4 ± 14.0 pg/ml versus 53.6 ± 6.8 pg/l) with modest further decrease in SP levels in post-challenge NLF after 2 years of ASIT (42.2 ± 9.6 pg/ml versus 51.2 ± 9.2 pg/ml) and 3 years of ASIT (41.6 ± 18.3 pg/ml versus 51.8 ± 7.6 pg/ml). SP may play a role in nasal allergic responses to mugwort pollen. Decreased release of SP upon nasal allergen challenge occurs with each year of ASIT suggesting that ASIT reduces allergen-induced SP release.

Prevalence of IgE-Binding to Art v 1, Art v 4 and Amb a 1 in Mugwort-Allergic Patients

Background: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract. Methods: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA. Results: 91% of the patients’ sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients’ sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera. Conclusions: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe.

Cloning, expression and characterization of mugwort pollen allergen Art v 2, a pathogenesis-related protein from family group 1

Mugwort ( Artemisia vulgaris) belongs to the Compositae family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. Skin prick tests, performed in 19 patients allergic to mugwort and 10 control patients, showed an Art v 2 sensitization prevalence of 58%, whereas none false-positives were detected among control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column and had an apparent molecular mass of 33 and 20 kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Cloned cDNA encoding Art v 2 contains 140 bp that codify for a polypeptide of 15.8 kDa, with a predicted p I value of 5.2, and one potential N-glycosylation site. Protein homology search demonstrated that Art v 2 share 55-42% identical residues with pathogenesis-related protein PR-1 of tomato, potato, rape, wheat and rice. Homology was also found to Ves v 5 (41% identical residues). Bacterial-expressed recombinant Art v 2 was recognized only by 21% of mugwort-allergic patients. In conclusion, Art v 2 from mugwort is the first weed pollen allergen that belongs to the pathogenesis-related protein PR-1 and its recombinant form could help molecular diagnosis of mugwort associated allergy.

Extraction and Purification of Art v 1, the Mugwort Pollen Major Allergen

RATIONALE: Mugwort (Artemisia vulgaris) is widely spread in the temperate and humid climate zones of the northern hemisphere and considered one of the main causes of late summer pollinosis. More than 95% of mugwort allergic patients are sensitized to the glycoallergen Art v 1. Here we investigated extraction conditions from pollen and developed a rapid and efficient method for purification of this major allergen.

Prevalence of sensitization to Artemisia allergens Art v 1, Art v 3 and Art v 60 kDa. Cross-reactivity among Art v 3 and other relevant lipid-transfer protein allergens.

Artemisia vulgaris is a widespread weed in the Mediterranean area and several allergens have been detected in its pollen. One of them, Art v 3, belongs to the lipid-transfer protein (LTP) family and its prevalence in Artemisia-sensitized patients or its relationship with other LTP allergens is not clear. To assess the pattern of sensitization to an array of mugwort allergens in a Mediterranean population, and to study the cross-reactivity of Art v 3 with Pru p 3 and Par j 1, relevant LTP allergens in the area. Skin prick test was performed with whole extracts (A. vulgaris, Parietaria judaica and peach) and pure natural allergens Art v 1, Art v 3, Art v 60 kDa and Par j 1 in 24 mugwort-allergic patients from a Mediterranean area. In vitro assays included measurement of specific IgE and ELISA inhibition among LTP allergens. The three Artemisia allergens elicited a positive skin response in 70-80% of the patients. Seven patients were clearly sensitized to Par j 1 and 11 to Pru p 3. There was no correlation between Par j 1 and Pru p 3 sensitization, but a highly significant correlation was found between peach extract and Art v 3 as regards the skin response. No IgE cross-reactivity was observed between Art v 3/Par j 1 or Pru p 3/Par j 1. In contrast, Art v 3 significantly inhibited the binding to Pru p 3 of IgE from three patients' sera out of six studied, but Pru p 3 was not able to inhibit the IgE binding to Art v 3. Art v 3 is a major mugwort allergen and in some patients with IgE to both Art v 3 and Pru p 3, Art v 3 behaves as the primary sensitizing agent.

Monoclonal antibody-based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1.

Pollen of Artemisia vulgaris (mugwort) is a relevant cause of pollinosis in temperate and humid regions. Recently, the major allergen of this pollen, Art v 1, has been characterized. To develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) to quantify Art v 1, and to assess the correlation of Art v 1 content with the biological activity of mugwort pollen extracts. Art v 1-specific mAbs were obtained from a BALB/c mouse immunized with high-performance liquid chromatography (HPLC)-purified Art v 1. One of these antibodies (Av 3.7), which recognizes the N-terminal defensin-like domain of Art v 1, was used as the capture antibody in an ELISA method for allergen quantitation. An anti-A. vulgaris rabbit serum was used as the second antibody. Art v 1 was purified by immunoaffinity chromatography and used as the standard in the assay. The purity and identity of the affinity-purified Art v 1 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, amino acid composition, and N-terminal amino acid sequencing. The prevalence of specific IgE against Art v 1, determined by radioallergosorbent test (RAST) in a population of 44 mugwort-allergic patients, was 79%. The Art v 1-ELISA developed displays a detection limit of 0.1 ng/ml, and a practical working range of 0.2-10 ng/ml. The concentration of Art v 1 was measured in 10 A. vulgaris pollen extracts, and a good correlation was observed between the Art v 1 content and the allergenic activity of the extracts. The results prove the usefulness of the Art v 1-ELISA for the standardization of A. vulgaris pollen extracts intended for clinical use.

The T cell response to Art v 1, the major mugwort pollen allergen, is dominated by one epitope.

Mugwort (Artemisia vulgaris) pollen allergens represent the main cause of pollinosis in late summer in Europe. At least 95% of sera from mugwort pollen-allergic patients contain IgE against a highly glycosylated 24- to 28-kDa glycoprotein. Recently, this major allergen, termed Art v 1, was characterized, cloned in Escherichia coli, and produced in recombinant form. In the present study we characterized and compared the T cell responses to natural (nArt v 1) and recombinant Art v 1 (rArt v 1). In vitro T cell responses to nArt v 1 and rArt v 1 were studied in PBMC, T cell lines (TCL), and T cell clones (TCC) established from PBMC of mugwort-allergic patients. Stimulation of PBMC or allergen-specific TCL with either nArt v 1 or rArt v 1 resulted in comparable proliferative T cell responses. Eighty-five percent of the TCC reactive with rArt v 1 cross-reacted with the natural protein. The majority of the CD4(+)CD8(-)TCR alphabeta(+) Art v 1-specific TCC, obtained from 10 different donors, belonged to the Th2 phenotype. Epitope mapping of TCL and TCC using overlapping peptides revealed a single immunodominant T cell epitope recognized by 81% of the patients. Inhibition experiments demonstrated that the presentation of this peptide is restricted by HLA-DR molecules. In conclusion, the T cell response to Art v 1 is characterized by one strong immunodominant epitope and evidently differs from the T cell responses to other common pollen allergens known to contain multiple T cell epitopes. Therefore, mugwort allergy may be an ideal candidate for a peptide-based immunotherapy approach.

Molecular and immunological characterization of profilin from mugwort pollen.

In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No information about the complete molecular structure of any mugwort allergen has been published so far. Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin. Thirty-six percent of the mugwort-allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgE-binding properties of the isoforms. One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions. Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions. Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollen-allergic patients. However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgE-mediated histamine release. Profilin isolated from mugwort pollen also formed multimers. Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen. Inhibition experiments showed extensive IgE cross-reactivity of recombinant mugwort profilin and profilin from various pollen and food extracts.

Allergens in Pollen from Mugwort (&lt;i&gt;Artemisia vulgaris &lt;/i&gt;L.)

A crude and a partly purified extract of mugwort pollen were characterized with particular emphasis on the glycoprotein allergen Ag7. Crossed immunoaffinoelectrophoresis with immobilized Con A in the intermediate gel demonstrated binding of Ag7 to this lectin, indicating that Ag7 contains terminal &lt;i&gt;α&lt;/i&gt;-&lt;i&gt;D&lt;/i&gt;-mannopyranosyl and/or &lt;i&gt;α-D&lt;/i&gt;-glucopyranosyl residues. Crossed immunoaffinoelectrophoresis with free Con A in the first-dimension gel demonstrated microheterogeneity in the carbohydrate moiety of the allergen. Crossed radio-immunoelectrophoresis analysis using sera from 26 mugwort-allergic patients indicated that Ag7 is an important allergen in mugwort pollen. Heat stability experiments demonstrated the presence of at least three heat-resistant allergens, one of these being Ag7. Crossed immunoelectrofocusing indicated a pi value for Ag7 of 4.3.

Immunochemical and biological characterization of a mugwort (Artemisia vulgaris) pollen extract.

A reference extract of mugwort pollen (Artemisia vulgaris) was characterized by crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE) and quantitative skin prick test (QSPT). CIE revealed that the extract contained at least 42 distinct antigens of which 24 migrated towards the anode and 18 towards the cathode at pH 8.6. A CRIE analysis of the crude mugwort pollen extract, performed with sera from 29 mugwort-allergic patients, showed that 10 antigens may be considered allergens; one was classified as a major allergen, five as intermediate allergens, and four as minor allergens. The QSPT performed on the same 29 allergic patients established that 17.4 micrograms lyophilised reference mugwort pollen extract per ml had a biological potency of 1 HEP (histamine equivalent by prick test).