Biomedical subjects

W Astrid van Leeuwen

Publications and source records attributed to W Astrid van Leeuwen.

3 recordsLinked to original sources

PubMed2005

Lipid transfer proteins from fruit: cloning, expression and quantification.

BACKGROUND: Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs. METHODS: cDNA for Mal d 3 and Pru p 3 was cloned, expressed in the yeast Pichia pastoris and the resulting proteins were purified via cation exchange chromatography. The immune reactivity of rMal d 3 was compared to nMal d 3 by RAST (inhibition), immunoblotting and basophil histamine release testing. To obtain monoclonal and monospecific polyclonal antibodies, mice and rabbits were immunized with purified nMal d 3. RESULTS: The deduced amino acid sequence of Mal d 3 was identical to the published sequence, Pru p 3 differed at two positions (S9A and S76H). The rMal d 3 had an IgE-binding potency and biological activity close to its natural counterpart. One sandwich ELISA selectively detecting apple LTP and another cross-reactive with cherry, nectarine and hazelnut LTP were developed. In addition, a competitive RIA was developed with polyclonal rabbit antiserum and labeled nMal d 3. CONCLUSION: rMal d 3 (as shown before for rPru p 3) may be a useful tool for application in component-resolved diagnosis of food allergy. Assays for the measurement of LTP will increase the traceability of this potentially dangerous allergen.

Allergens↗

PubMed2002

Maturation of Pichia pastoris-derived recombinant pro-Der p 1 induced by deglycosylation and by the natural cysteine protease Der p 1 from house dust mite.

The mature cysteine protease from Dermatophgoides pteronyssinus, Der p 1, is a major house dust mite allergen. Its enzymatic activity has been shown to have pro-inflammatory effects that could also negatively influence efficacy of allergen-specific immunotherapy. The aim of this study was to express recombinant pro-Der p 1 (rpro-Der p 1) in the yeast Pichia pastoris and to study its maturation. Expression was achieved at a concentration ranging from 45 mg.L-1 (methanol-induced expression) to 168 mg.L-1 (constitutive expression). No significant spontaneous maturation of the secreted proenzyme was observed. rpro-Der p 1 with a sequence-based molecular mass of 34 kDa was hyperglycosylated by the yeast, migrating at 50-60 kDa on SDS/PAGE. Compared with its natural counterpart (nDer p 1), the recombinant proenzyme demonstrated decreased IgE reactivity, resulting in a 30-fold lower capacity to induce histamine release from human basophils. Decreased immunoreactivity was also shown by competitive RIA and sandwich ELISA with Der p 1-specific antibody reagents. CD spectra of rpro-Der p 1 and nDer p 1 revealed significant structural differences. Deglycosylation of rpro-Der p 1 with endoglycosidase H resulted in a decrease in apparent molecular mass from 50 kDa to 34 kDa, but did not affect nDer p 1. On removal of N-glycans from rpro-Der p 1, which harbours two putative N-glycosylation sites in both propeptide and mature sequence, the mature rDer p 1 appeared. This suggests that hyperglycosylation hampers spontaneous maturation. Maturation of the recombinant pro-enzyme was also achieved by addition of the active natural cysteine protease, nDer p 1. In conclusion, high-level expression of rpro-Der p 1 in P. pastoris results in a stable hypoallergenic proenzyme with potential for use in allergen-specific immunotherapy.

Allergens↗

PubMed2002

IgE to Bet v 1 and profilin: cross-reactivity patterns and clinical relevance.

BACKGROUND: Individuals with pollen allergy often have IgE against plant-derived foods. This can be due to cross-reactive IgE against Bet v 1 and homologues, profilins, and/or cross-reactive carbohydrate determinants. OBJECTIVE: The aim of this study was to correlate sensitization to Bet v 1 and profilin with individual recognition patterns to plant foods and clinical relevance. METHODS: Fifty-two patients with pollen allergy and IgE against at least one plant-derived food were included in the study. Adverse reactions to plant-derived foods were documented by using standardized interviews. Skin prick tests were performed for pollen (grass, birch, and mugwort) and 14 plant-derived foods. In addition, recombinant (r) Bet v 1 and rBet v 2 (profilin) were tested intracutaneously. Specific IgE against the abovementioned allergens were determined by means of RAST. Cross-reactivity was studied by means of RAST inhibition. RESULTS: Eighty-five percent of patients were sensitized to Bet v 1, and 71% were sensitized to profilin. Profilin was associated with a higher number of positive RAST results to plant-derived foods than Bet v 1. In contrast, Bet v 1 was associated with more positive skin prick test responses and more food-related symptoms. Sensitization to Bet v 1 was associated with IgE against apple, hazelnut, and peach, whereas sensitization to profilin was associated with positive RAST results to all investigated plant-derived foods except apple, peach, and melon. CONCLUSIONS: IgE antibodies against Bet v 1 have a more limited spectrum of cross-reactivity than those against profilin, but they frequently give rise to clinically relevant cross-reactivities to food. In analogy to anticarbohydrate IgE, cross-reactive IgE against food profilins have no or very limited clinical relevance.

Adolescent↗