Parietaria major allergens vs pollen in the air we breathe.

BACKGROUND: Parietaria and Urtica are the genera from the Urticaceae family more frequent in Mediterranean and Atlantic areas. Moreover, both genera share pollination periods, and their pollen (of the main species) is so similar that there is no aerobiological evidence of the proportion of each of them in the airborne pollen identification, except in the case of U. membranacea. However, Parietaria is one of the most important causes of pollinosis and Urtica is not. Our aim is determine if airborne Urticaceae pollen concentrations show the aerodynamics of the two major allergens of Parietaria (Par j 1 and Par j 2) as well as the allergen distribution in the different-sized particles. METHODS: The air was sampled during the pollination period of Urticaceae using Hirst Volumetric Sampler and Andersen Cascade Impactor in two cities of Southern Spain (Córdoba and Granada). The samples were analysed by the methodology proposed by the Spanish Aerobiology Network (REA) and the minimum requirements of the European Aeroallergen Society (EAS) for pollen, and by ELISA immunoassay for allergens. RESULTS: The patterns of airborne pollen and Par j 1-Par j 2 were present in the air during the studied period, although with irregular oscillations. Urticaceae pollen and Par j 1-Par j 2 allergens located in PM2.5 showed positive and significant correlation during the period with maximum concentrations (March to April). CONCLUSION: Parietaria aeroallergens show similar pattern of Urticaceae airborne pollen. Urticaceae pollen calendar is as a good tool for allergy prevention. On the other hand, important concentrations of Par j 1 and Par j 2 were located in the breathable fraction (PM2.5), which could explain the asthmatic symptoms in the allergic population to Parietaria.

Evaluation of methods for the estimation of threshold concentrations by the skin prick test.

BACKGROUND: The allergen dose-response curve is flat; thus, small changes in wheal size reflect large differences in skin sensitivity. The sensitivity as measured by provocation tests is given by the threshold concentration that causes symptoms and/or objective signs. The threshold concentrations differ by several magnitudes between the most and the least sensitive individuals clinically allergic to the same allergen. Variation in technique can be minimized by relating allergen responses to that to histamine. The aim here is to present and validate simple methods for estimation of the skin sensitivity given as the concentration inducing a wheal of the same size as that with the positive reference, 10 mg/ml of histamine HCl, in the same patient. METHODS: Data from previously reported trials on the biological equilibration of allergen extracts were used to document a method to calculate the concentration of allergen required to induce a wheal of the same size as that with 10 mg/ml of histamine dihydrochloride in the same patient, and to validate the methods using the parallel line bioassay as the gold standard. RESULTS: The validated methods correlated well with the results obtained using the gold standard method and provide results of skin prick testing based on threshold concentrations of allergen. CONCLUSIONS: The validated methods reduce the error of differences in testing techniques and make it possible to report skin sensitivity at threshold concentrations. A simple method to be used in clinical practice and a method suitable to describe changes in skin reactivity over time or during treatment are proposed.

Serum IL-9 levels and sublingual immunotherapy: preliminary report.

Th9 is a new T cell subset characterized by IL-9 production. It has been reported that serum IL-9 levels are related with symptom severity in patients with allergic rhinitis (AR). This study is aimed at investigating whether serum IL-9 may be modulated by sublingual immunotherapy (SLIT) in patients with persistent AR due to Parietaria allergy. Twenty-one AR patients (9 males, median age 41 years) successfully treated with SLIT and 52 AR patients (25 males, median age 34 years) treated only with drugs were evaluated during the pollen season. Serum IL-9 was dosed in all patients. SLIT-treated patients showed significantly lower serum IL-9 levels than untreated AR patients (p <0.0001). In conclusion, this preliminary study shows that a single pre-seasonal SLIT course might modulate serum IL-9.

Proteomic analysis of Parietaria judaica pollen and allergen profiling by an immunoproteomic approach.

Parietaria judaica pollen is a common cause of airway allergic disease in the Mediterranean area. Proteome analysis of mature Parietaria judaica pollen by two-dimensional gel electrophoresis (2-DE) and mass spectrometry has established the first reference proteome map of this weed. Proteins involved in a variety of cellular functions as well as the occurrence of allergens were detected. By using 2-DE and immunoblotting with sera from Parietaria judaica allergic patients we obtained a more detailed characterization of Parietaria judaica allergen profile so to improve our comprehension of the pathogenesis of pollen-induced allergic reaction.

Antibacterial, Anticandidal, Phytochemical, and Biological Evaluations of Pellitory Plant.

Pellitory plant (Parietaria judaica (PJ)) is one of the most widely used Arabian traditional medicinal plants due to its ability to cure several infectious diseases and other illnesses. The current study is aimed at assessing the phytoconstituents, antilipase, antiamylase, antimicrobial, and cytotoxic characters of the Pellitory plant (Parietaria judaica (PJ)). Phytochemical screening and procyanidin detection were conducted according to the standard phytochemical procedures. Porcine pancreatic lipase and α-amylase inhibitory activities were carried out using p-nitrophenyl butyrate and dinitrosalicylic acid assays, respectively. In addition, antimicrobial activity was determined utilizing a microdilution assay against several bacterial and fungal strains. Besides, the cytotoxic effect against HeLa cell line was tested employing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The quantitative test results revealed that the methanol fraction of PJ contains 18.55 ± 0.55 mg of procyanidin and has a potential α-amylase inhibitory activity compared with the antidiabetic drug Acarbose with IC50 values of 15.84 ± 2.25 and 28.18 ± 1.22 µg/ml, respectively. Also, it has a potential antilipase activity compared to the commercial antiobesity drug, Orlistat, with IC50 values of 38.9 ± 0.29 and 12.3 ± 0.35 µg/ml, respectively. The acetone, hexane, and methanol fractions have broad-spectrum antibacterial activity against the screened bacterial strains, while the acetone fraction has shown anticandidal activity with a MIC value of 0.195 mg/ml. The PJ hexane and acetone fractions decreased HeLa cell viability significantly (p value < 0.0001) by approximately 90% at the concentration of 0.625 mg/ml. The revealed outcomes showed that the methanol fraction has strong α-amylase and lipase inhibitory characters. Besides, acetone, hexane, and methanol fractions have broad-spectrum antibacterial activity, while the acetone fraction revealed potent antifungal activity against Candida albicans. Moreover, at low concentrations, hexane and acetone fractions have potent cytotoxic and antiproliferative activity against HeLa cancer cells. Nevertheless, PJ acetone, hexane, and methanol fractions can serve as an effective source of natural products to develop new antiobesity, antidiabetic, antimicrobial, and anticancer agents.

Production of an egg yolk antibody against Parietaria judaica 2 allergen.

Specific antibodies are essential tools for studying proteins as well as for diagnostic research in biomedicine. The egg yolk of immunized chicken is an inexpensive source of high-quality polyclonal antibodies. The 12-kDa Parietaria judaica 2 allergen was expressed as a fusion protein and was used to immunize Leghorn chickens. In this paper, we show, using 2-dimensional gel electrophoresis and immunoblotting, that chicken antibodies raised against a recombinant allergen can be used to recognize similar proteins from a pollen raw extract. Allergen identity was confirmed by nanoLC-nanospray-tandem mass spectrometry analysis. Our data demonstrate for the first time that a synergistic combination of molecular biology, 2-dimensional PAGE, and use of nonmammalian antibodies represents a powerful tool for reliable identification of allergens.

Multiple IgE recognition on the major allergen of the Parietaria pollen Par j 2.

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.

Diagnosis of Parietaria judaica pollen allergy using natural and recombinant Par j 1 and Par j 2 allergens.

BACKGROUND: Parietaria judaica pollen is one of the main causes of allergic diseases in the Mediterranean area and contains two major allergens, called Par j 1 and Par j 2. OBJECTIVE: To evaluate the diagnostic potential of natural and recombinant forms of Par j 1 and Par j 2 in comparison with standardized P. judaica pollen extract. METHODS: Thirty patients allergic to P. judaica pollen and 15 control patients were investigated. Skin prick tests and determination of specific IgE levels were performed with commercial P. judaica extract, natural Par j 1 and Par j 2, and recombinant forms of both allergens expressed in P. pastoris. RESULTS: The whole group of patients with allergy to P. judaica had a positive skin test reaction to purified nPar j 1-Par j 2 and rPar j 2 at 5 microg/mL, and no false-positive reactions were detected. Natural and recombinant Par j 1 and Par j 2 showed no significantly different responses in skin tests compared with P. judaica extract. A high correlation was found between the serum-specific IgE levels to P. judaica extract vs. natural (R=0.996; P<0.001) and recombinant allergens (R=0.887 and 0.982 for rPar j 1 and rPar j 2, respectively; P<0.001). rPar j 2 displayed a 100% sensitivity and specificity among P. judaica-allergic patients. CONCLUSIONS: In vivo and in vitro diagnosis of P. judaica pollen allergy could be simplified using rPar j 2. This protein showed comparable IgE response and skin prick reactivity with those produced by P. judaica pollen extract.

Detection and release of allergenic proteins in Parietaria judaica pollen grains.

Rapid diffusion of allergenic proteins in isotonic media has been demonstrated for different pollen grains. Upon contact with stigmatic secretion or with the mucosa of sensitive individuals, pollen grains absorb water and release soluble low-molecular-weight proteins, these proteins enter in the secretory pathway in order to arrive at the cell surface. In this study we located allergenic proteins in mature and hydrated-activated pollen grains of Parietaria judaica L. (Urticaceae) and studied the diffusion of these proteins during the first 20 min of the hydration and activation processes. A combination of transmission electron microscopy and immunocytochemical methods was used to locate these proteins in mature pollen and in pollen grains after different periods of hydration and activation processes. Activated proteins reacting with antibodies in human serum from allergic patients were found in the cytoplasm, wall, and exudates from the pollen grains. The allergenic component of these pollen grains changes according to the pollen state; the presence of these proteins in the exine, the cytoplasm, and especially in the intine and in the material exuded from the pollen grains, is significant in the hydrated-activated studied times, whereas this presence is not significant in mature pollen grains. The rapid activation and release of allergenic proteins of P. judaica pollen appears to be the main cause of the allergenic activity of these pollen grains.

Purification of a novel aminopeptidase from the pollen of Parietaria judaica that alters epithelial integrity and degrades neuropeptides.

BACKGROUND: Parietaria judaica pollen is a common cause of pollinosis in the Mediterranean area. OBJECTIVE: This study sought to purify and characterize the peptidase responsible for the majority of proteolytic activity present in the pollen extract of P judaica, and to investigate its contribution to the allergic response. METHODS: A serial of chromatographic steps was applied to isolate the peptidase from P judaica's pollen, and its biochemical properties were determined. Bioactive peptides present in the airways were incubated with the peptidase, and their degradation was visualized by direct protein sequencing. In addition, we measured the cellular detachment, by methylene blue binding assay, of an airway-derived epithelial cell line (A549) in the presence of the peptidase, and visualized, by Western blot, the degradation of proteins from intercellular junctions. RESULTS: We purified a 98-kDa peptidase from the pollen of P judaica that was classified as an aminopeptidase on the basis of its biochemical properties and internal amino acid sequence. The aminopeptidase was able to degrade bioactive peptides. Moreover, the aminopeptidase caused cellular detachment of A549 cell line and degradation of occludin and E-cadherin. CONCLUSION: Our results suggest that the P judaica aminopeptidase can alter the integrity of the epithelium barrier by degrading occludin as well as E-cadherin. In addition, P judaica aminopeptidase can degrade bioactive peptides, which can exacerbate the overall bronchoconstrictive effect detected in asthmatic lungs. CLINICAL IMPLICATIONS: The novel aminopeptidase described here could constitute a relevant therapeutic target in the treatment of allergic disorders induced by the pollen of P judaica.

Possible relationship between systemic side effects and sensitization to rPar j 2 in allergic patients submitted to an ultra-rush (20 min) sublingual immunotherapy and selected by component resolved diagnosis.

BACKGROUND: The pollen of Parietaria spp., a weed of the Urticaceae family, is a major cause of respiratory allergy in the Mediterranean area, where the most common species are Parietaria judaica and Parietaria officinalis. In this study, we evaluated the specific serum IgE-binding profiles to individual P. judaica pollen recombinant major allergen, and Phleum pratense cytoskeletal profilin and a 2-EF-hand calcium-binding allergen homologous to cross-reactive Parietaria pollen allergens, in patients allergic to pollen with positive skin test towards Parietaria spp. extract. METHODS: The present observation included 220 patients from the province of Cuneo, north-west Italy, all suffering from rhino-conjunctivitis and/or asthma selected on the basis of skin test positive to P. judaica extract. The sera were evaluated for specific IgE reactivity to P. judaica pollen major recombinant(r) allergen Par j 2, Phleum pratense pollen allergens rPhl p 7 (2-EF-hand calcium binding protein) and rPhl p 12 (profilin), both identified as cross-reactive Parietaria spp. allergens, using Pharmacia CAP System. Out of 220 patients, 37 patients with IgE reactivity to rPar j 2 and 105 patients sensitized to at least one timothy pollen major allergen (i. e. rPhl p 1, rPhl p 2, natural Phl p 4 and rPhl p 6) were submitted to an ultra-rush protocol of sublingual immunotherapy (SLIT). The occurrence of adverse reactions were evaluated in both groups. RESULTS: All 220 patients with pollinosis and positive in vivo skin prick tests had in vitro positive CAP results to P. judaica natural extract. On the contrary, in these patients the prevalence of Par j 2-specific IgE was only 33.2% (73/220). In fact, 116/220 (52.7%) patients with serum specific IgE to crude Parietaria pollen extract had specific IgE to Phl p 12, 18/220 (8.1%) subjects with specific IgE to rPhl p 12 also exhibited specific IgE to Phl p 7 and 26/220 (11.8%) subjects had specific IgE against rPhl p 7. Particularly, geometric mean (25th-75th percentile) of specific IgE to rPar j 2 were as follows: 2.87 kUA/l (1.005-7.465). Out of 73 patients with specific IgE to rPar j 2, 7 subjects (9.6%) had also specific IgE to rPhl p 7, 12 (16.4%) had specific IgE to rPhl p 12 and 4 (4.1%) patients had specific IgE to both recombinant allergens. Of 37 patients under an ultra-rush protocol of SLIT, 3 subjects (8.1%) experienced generalized urticaria, and 1 of them also had diarrhea 3 h after the last dose of Parietaria judaica extract oral-vaccine administration. On the contrary, no systemic reactions were observed in 105 patients after Phleum pratense extract oral intake after a similar ultra-rush SLIT protocol (p = 0.0046). CONCLUSIONS: In the light of present findings, allergen extract-based diagnosis, in vivo and in vitro, cannot discriminate allergic patients that are genuinely sensitized to Parietaria spp. major allergens or to other major allergens to which current immunotherapeutic allergy extracts are standardized. Therefore, in vitro component resolved diagnosis is the unique tool to define the disease eliciting molecule(s). Finally, during sublingual immunotherapy, not only the dose of allergen, but also the biochemical characteristic of the major allergen administered may be an important factor in determining possible systemic reactions.