Analysis of a cDNA sequence encoding the immunoglobulin heavy chain of the Antarctic teleost Trematomus bernacchii.

A spleen cDNA library was constructed from the Antarctic teleost Trematomus bernacchii and immunoscreened with rabbit IgG specific for T. bernacchii Ig heavy chain. Eleven cDNA clones, varying in size and encoding the entire heavy chain or parts of it, were isolated. Here the complete nucleotide and deduced amino acid sequences of clone 2C2 encoding the secretory IgH chain form are reported. Comparison of the amino acid sequence of the entire constant region of the T. bernacchii Ig heavy chain with those from other teleosts and two holostean fish showed percent identity ranging 53.6-60.6%, with the highest values found for Salmoniformes. The multiple sequence alignment revealed the presence of two remarkable insertions: one at the VH-CH1 boundary and a second one, not found in any other IgM heavy chain, localised at the CH2-CH3 boundary. The latter occurred in the region proposed to act as a 'hinge', and resulted in a CH2-CH3 hinge peptide longer than any other IgM hinge. Differences were also found in the number and position of putative N-glycosylation sites of the compared sequences. It is suggested that the unusual features found in the T. bernacchii Ig heavy chain might contribute to the flexibility of the Ig molecule and help understand more about the adaptation of Ig molecules to the polar sea environment.

Plasma and bile antibodies of the teleost Trematomus bernacchii specific for the nematode Pseudoterranova decipiens.

We investigated the occurrence of antibodies against protein antigens of the nematode parasite Pseudoterranova decipiens in the plasma and bile of the Antarctic teleost Trematomus bernacchii. Three different P. decipiens protein solutions were prepared: excreted/secreted proteins from live larvae (ESP); surface-associated proteins obtained by mild extraction of larval bodies (SAP); and cuticular soluble proteins recovered by extraction in strong reducing conditions (CSP). Using different immunoassays, these 3 preparations were tested for their ability to bind fish antibody. As determined by ELISA, the specific antibody binding activity was higher in SAP than in CSP. As determined by dot-blot immunoassay, the specific antigen binding activity versus SAP was higher in bile than in plasma antibodies. A different number of antigenic components of SAP and ESP were identified by immunoblotting performed with plasma or bile antibodies. These results led to the conclusion that T. bernacchii parasitism by nematodes involves plasma and bile anti-parasite antibodies. Furthermore bile antibodies were found to be more reactive and more heterogeneous than plasma.

Association of responsiveness to the major pollen allergen of Parietaria officinalis with HLA-DRB1* alleles: a multicenter study.

Parietaria, a plant belonging to the family of Urticaceae, is a major source of allergenic pollen in Europe. In the context of a multinational study, we investigated whether in allergic subjects antibody response towards Par o 1, the major allergen from P. officinalis, was associated with defined HLA-DRB1* alleles. The study population consisted of 234 allergic patients: 65 from Bulgaria, 30 from Israel, 99 from Italy, and 40 from Spain. In the Italian study group, the prevalence of ST positivity to Parietaria was 77%. In Parietaria ST-positive subjects, the prevalences of IgG and IgE serum Ab towards Par o 1 were 91% and 75%, respectively. HLA-DRB1*1101 and/or 1104 were significantly positively associated with the presence of IgG Ab and with high levels of IgE Ab towards this allergen (p = 0.0007 and p = 0.012, respectively). In the Spanish study group, the positive association of DR1100 with responsiveness to Par o 1 was confirmed (p = 0.02, RR = 4, and p = 0.002, RR = 7, for IgG and IgE Ab, respectively). None of the Bulgarian patients had IgE Ab to Par o 1, whereas IgG Ab response was observed in 7 out of 65 subjects and was positively associated with DRB1*1101 and/or 1104 (p = 0.025). In the Israeli study group, responsiveness to Par o 1 was not associated with specific HLA-DRB1* alleles. In conclusion, this study shows that in allergic patients from three European populations antibody response to the major allergen from the pollen of Parietaria is associated with HLA-DRB1*1101 and/or 1104. Our data suggest that this association is stronger in subjects monosensitized to Parietaria.

Characterization of a dominant antigenic determinant of Par o I encoded by recombinant DNA.

BACKGROUND: The pollens from Parietaria judaica and Parietaria officinalis are a major cause of pollinosis in Europe. Par o I (13.5 kDa) and Par j I (12 kDa), the major allergens from these species, are highly crossreactive. METHODS: We have immunoscreened a P. judaica pollen cDNA expression library with a rabbit antiserum specific for Par j I and with a serum pool from allergic patients. An immunopositive clone containing a 26 bp insert was further characterized. The insert sequence was determined and the beta-galactosidase fusion protein was partially purified by electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. RESULTS: This fusion protein specifically and extensively inhibited Par o I and Par j I binding of a rabbit antiserum and of a serum pool obtained from allergic patients. The antifusion-protein antiserum obtained in a rabbit (anti 6a) specifically precipitated radioiodinated purified Par o I in the double antibody radioimmunoassay (DARIA) and competed with antibodies of sera from allergic patients for the binding to Parietaria pollen extract allergens by enzyme linked immunosorbent assay (ELISA). We investigated the prevalence of antibody response towards the 6a epitope in patients naturally sensitized to Parietaria. The presence of 6a specific IgE antibodies was assessed in the sera of 33 patients using inhibition assays. All sera had antibodies with this specificity: the extensive percentage of inhibition reached suggested that they dominated individual ab response. CONCLUSION: In conclusion, the antibody response induced by natural exposure to the pollen of Parietaria appears to be higly focused on a single linear antigenic determinant of the major allergens which may play a relevant role in the development of clinical allergy. This report is, to our knowledge, the first description of a dominant linear epitope of a major allergen.

Basic isoforms of Par o 1, the major allergen of Parietaria officinalis pollen.

We describe a group of basic isoforms of Par o 1 (cumulatively referred to as Par o 1b), purified by anion-exchange chromatography. The allergenic activity of Par o 1b was compared with that of the acidic isoform (Par o 1a) by RAST inhibition. Par o 1b showed a cathodic mobility in crossed immunoelectrophoresis. It was found to be homogeneous in SDS-PAGE and SE-HPLC (14.5 kDa), and heterogeneous in PAG-IEF, yielding five IgE-binding bands with pI ranging between 7.9 and 9.6 PAG-IEF individual components were isolated by cation-exchange HPLC. The N-terminal amino acid sequence of the main component (pI 8.8) was determined and found to be similar to that of Par o 1a.

Purification and characterization of Par o I, major allergen of Parietaria officinalis pollen.

Par o I, a major allergen of Parietaria officinalis, was purified from the pollen extract. The purified allergen was obtained by ultrafiltration, Sephadex gel filtration and DE-52 ion exchange chromatography: the purified preparation yields a single band in polyacrylamide gel isoelectric focusing (PAG-IEF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, a single immunoprecipitation arc in crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a single peak in size exclusion high-performance liquid chromatography (HPLC). Par o I is a glycoprotein with a protein to carbohydrate ratio of 100:21. The molecular weight, determined by SDS-PAGE, Sephadex G-50 gel filtration and size exclusion HPLC, varied between 13.5 and 14.5 kDa according to the method employed. The isoelectric point was 4.6. The amino acid composition and the sequence of the first twelve N-terminal residues were determined. The allergenicity was assayed in vivo and in vitro. 29/29 Parietaria-allergic patients were skin positive to Par o I and possessed high level of specific serum IgE antibody as it determined by radioallergosorbent test (RAST). Par o I contained dominant epitopes for human IgE as inhibited to 85% the pollen extract RAST performed with a pool of sera of allergic patients. The RAST inhibitory activity was not abolished by deglycosylation.

Isolation and in vitro translation of mRNA from inflorescences of Parietaria judaica.

The aqueous extract of inflorescences of Parietaria judaica contains an allergen homologous to the major pollen allergen Par o I (14 kD), as shown by radio-allergosorbent test (RAST) inhibition and immunoblot analysis. Poly(A)+ RNA was obtained from inflorescences and was shown to be able to code in vitro for a protein homologous to Par o I with respect to sodium dodecylsulphate polyacrylamide gel electrophoretic mobility and to antigenic specificity as defined by the binding, in affinity chromatography, to solid-phase IgG of rabbit anti-Par o I antisera, and in RAST inhibition, to IgE antibodies of human reaginic serum pool.

Low molecular weight allergens of the pollen of Parietaria officinalis.

The allergenic composition of a low mol. wt fraction of the pollen extract of Parietaria officinalis (PO) was investigated. Fraction C, that was eluted after oxytocin (mol. wt 1040) when the pollen extract was gel filtered on Sephadex or on Biogel, was cross-reactive in the RAST with the major allergen P015 and was capable of eliciting histamine release from leukocytes of sensitive donors. RAST inhibition (RAST I) analysis of the eluate of gel filtration on Sephadex G-10 revealed several peaks of IgE binding activity. Analysis of fine specificity of response of individual patients carried out by skin-prick tests and by RAST I, revealed individual patterns of reactivity, indicating that allergens contained in fraction C were minor allergens.

Effect of the time of extraction on the potency and allergenic composition of the extract of Parietaria officinalis pollen.

Five Parietaria officinalis pollen extracts were obtained after respective 0.5, 4, 16, 48, 96 h of extraction in 0.0125 M NH4HCO3 pH 8.7 containing 0.05% NaN3 and 5.7 X 10(-4) M phenylmethylsulphonylfluoride. Conductivity, pH, absorption coefficient at 280 and 360 nm and content in protein and carbohydrates were determined in the extracts. Allergenic potency, evaluated by RAST inhibition was higher for the shorter-time extracts. The allergenic composition of the extracts was analyzed by a combination of gel filtration of the extracts and RAST inhibition of the eluted fractions: low molecular weight allergens were recovered only in longer-time extracts.

Detection of complexed IgE in sera of patients allergic to the pollen of Parietaria officinalis.

34 sera of patients allergic to the pollen of Parietaria officinalis were analyzed by ultracentrifugation on sucrose gradient (40-10%) in 0.9% NaCl at 24,000 rpm with a SW 27.1 rotor for complexed circulating IgE. Total and specific anti-PO IgE content of the gradient's fractions were determined by radioimmunoassay. IgE presence was demonstrated in at least two peaks; one corresponding to monomeric IgE, another corresponding to IgE with a sedimentation constant higher than that of the monomers. The ratio between monomeric and complexed IgE was different for each serum. Discordant results of PRIST and RIST analyses suggest that complexed IgE has fewer sites available for reaction with anti-IgE than the monomers.

Genetics of a low-density lipoprotein allotype in a cattle.

The paper describes a cattle serum antigen (LdlA1) located on a low-density lipoprotein and detected by single radial diffusion. The specificity is inherited in a simple Mendelian manner and the gene controlling its synthesis is inherited independently from the one controlling the synthesis of the alpha 2 macroglobulin McA1 antigen.