No abstract

No abstract

A 659 bp cDNA clone** coding for an allergen of Pj pollen has been isolated from a lambda gt 11 library, and its DNA sequence determined. The cDNA insert showed an open reading frame of 429 bp coding for an allergenic protein of 14,866 Da and a deduced amino acid sequence containing 143 residues. The expressed recombinant protein represented the major allergen Par j I since it reacted with 95% of the sera from Pj-allergic patients (n = 22) and with two Par j I-specific monoclonal antibodies. No similarity with other known DNA and protein sequences has been detected.

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 Mediterranean area. Species belonging to this genus induce IgE responses in approximately 10 million people. Identification of allergens by means of independent strategies suggest that the allergens of the two more common species, Parietaria judaica and Parietaria officinalis, show molecular weights ranging between 10 and 14 kD and that the allergens of the two extracts are highly cross-reactive. Biochemical analysis and molecular cloning allowed the isolation and immunological characterization of the two major allergens of the P. judaica pollen, Par j 1 and Par j 2. Sequence comparison suggests that the P j major allergens of P. judaica belong to the nonspecific lipid transfer protein family, and three-dimensional modeling by homology has revealed that both proteins present a very conserved structural motif composed of four α-helices. Immunological analysis has shown that Par j 1 and Par j 2 are able to bind most of the P. judaica-specific IgE and some of their IgE determinants have been mapped. Recombinant Par j 1 and Par j 2 allergens have been shown to possess immunological properties equivalent to their natural counterpart and their availability represents a fundamental tool for the diagnosis and therapy of Parietaria pollen allergy.

No abstract

Background: Par j 1 represents a major allergenic component of Parietaria judaica (Pj) pollen, since it is able to induce an immunoglobulin E (IgE) response in 95% of Pj-allergic patients. It belongs to the non-specific lipid transfer protein family, sharing with them a common three-dimensional structure. Methods: Disulphide bond variants of the recombinant Par j 1 (rPar j 1) allergen were generated by site-directed mutagenesis, and the immunological activity of rPar j 1 and its conformational mutants was compared with the use of the skin prick test (SPT). The ability to bind IgE antibodies was evaluated by Western blot, ELISA and ELISA inhibition. T cell reactivity was measured by peripheral blood mononuclear cell proliferation assay. Results: The disruption of Cys14–Cys29 and Cys30–Cys75 bridging (PjA mutant) caused the loss of the majority of specific IgE-binding activity. Additional disruption of the Cys4–Cys52 bridge (PjC mutant) and the latter Cys50–Cys91 bridge (PjD mutant) led to the abolition of IgE-binding activity. On the SPT, PjB (lacking the Cys4–Cys52 and Cys50–Cys91 bridges) was still capable of triggering a type I hypersensitive reaction in 9 out of 10 patients, and PjA in 3 out of 10 patients, while PjC and PjD did not show any SPT reactivity. All the mutants preserved their T cell reactivity. Conclusion: Recombinant hypoallergenic variants of the rPar j 1 allergen described herein may represent a useful tool for improved immunotherapy.

The relationship between maternal hormones and factors secreted by the implanting embryo is still controversial. We have analysed the in-vitro effect of oestradiol and human embryo-derived histamine-releasing factor (EHRF) on histamine release from rat uterine mast cells. Rat uterine mast cells which were preincubated with oestradiol and then challenged with human EHRF gave histamine release values two-to threefold higher than those without preincubation. The enhancement observed was time-and temperaturedependent. A similar enhancement was obtained with human sensitized basophils but not with rat peritoneal mast cells. Oestradiol, used as a direct challenge, did not induce any histamine release from either rat uterine or peritoneal mast cells, or from human sensitized basophils. Oestradiol preincubation also enhanced the histamine release induced by anti-IgE but did not enhance the histamine release induced by substance P or compound 48/80, two secretagogues that are not mediated by IgE. Moreover, uterine fragments derived from rats at various oestrus phases, with different amounts of endogenous oestrogen, were challenged in vitro with EHRF. The release of histamine by mast cells was higher at the proestrus and preimplantation phases than at dioestrus. All these findings suggest that the interaction of oestradiol with rat uterine mast cells was capable of enhancing in vitro the histamine releasing effect of EHRF.

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 antibodies from a pool of sera (n = 23) of patients allergic to Pj. Sequence analysis showed open reading frames of 176 and 138 amino acids. Both clones contain a putative signal peptide giving two mature processed proteins named Par j 1.0102 of 14,726 D and Par j 1.0201 of 10,677 D. These proteins represent isoallergenic forms of the major Pj allergen Par j 1.0101 (clone P5) previously reported. The Par j 1.0102 shared 98% amino acid sequence homology with the P5, while the Par j 1.0201 shared 89% homology. Since P1, P5 and P9 clones were expressed in Escherichia coli, and since the three allergenic proteins shared a very high degree of sequence identity and comparable binding to the Pj-specific IgE, we decided to analyze in more detail the immunological properties of only one allergen, the recombinant Par j 1.0101. The allergenic activity determined by the histamine release assay ranged between 9 and 56%, depending on the allergic patient analyzed, while it blocked approximately 40% of all the Pj-specific IgE antibodies, as detected after ELISA and cross-absorption analysis.