Characterization of peptidic and carbohydrate cross-reactive determinants in pollen polysensitization.

BACKGROUND: Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. MATERIAL AND METHODS: Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. RESULTS: The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. DISCUSSION: This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.

Influence of thermal processing on the allergenicity of peanut proteins.

Peanuts are one of the most common and severe food allergens. Nevertheless, the occurrence of peanut allergy varies between countries and depends on both the exposure and the way peanuts are consumed. Processing is known to influence the allergenicity of peanut proteins. The aim of this study was to assess the effect of thermal processing on the IgE-binding capacity of whole peanut protein extracts and of the major peanut allergens Ara h 1 and Ara h 2. Whole proteins, Ara h 1, and Ara h 2 were extracted and purified from raw, roasted and boiled peanuts using selective precipitation and multiple chromatographic steps, and were then characterized by electrophoresis and mass spectrometry. The immunoreactivity of whole peanut extracts and purified proteins was analyzed by the enzyme allergosorbent test (EAST) and EAST inhibition using the sera of 37 peanut-allergic patients. The composition of the whole protein extracts was modified after heat processing, especially after boiling. The electrophoretic pattern showed protein bands of low molecular weight that were less marked in boiled than in raw and roasted peanuts. The same low-molecular-weight proteins were found in the cooking water of peanuts. Whole peanut protein extracts obtained after the different processes were all recognized by the IgE of the 37 patients. The IgE-binding capacity of the whole peanut protein extracts prepared from boiled peanuts was 2-fold lower than that of the extracts prepared from raw and roasted peanuts. No significant difference was observed between protein extracts from raw and roasted peanuts. It is noteworthy that the proteins present in the cooking water were also recognized by the IgE of peanut-allergic patients. IgE immunoreactivity of purified Ara h 1 and Ara h 2 prepared from roasted peanuts was higher than that of their counterparts prepared from raw and boiled peanuts. The IgE-binding capacity of purified Ara h 1 and Ara h 2 was altered by heat treatment and in particular was increased by roasting. However, no significant difference in IgE immunoreactivity was observed between whole protein extracts from raw and roasted peanuts. The decrease in allergenicity of boiled peanuts results mainly from a transfer of low-molecular-weight allergens into the water during cooking.

Peanut- and cow's milk-specific IgE, Th2 cells and local anaphylactic reaction are induced in Balb/c mice orally sensitized with cholera toxin.

BACKGROUND: The development of animal models developing specific immunoglobulin (Ig)E presenting the same specificity as human IgE and similar clinical symptoms as those observed in allergic patients are of great interest for the understanding of mechanisms involved in the induction and regulation of food allergy. METHODS: Balb/c female mice were sensitized with whole peanut protein extract (WPPE) by means of intraperitoneal (i.p.) injections with alum or gavages with cholera toxin (CT). The WPPE specific IgE, IgG1 and IgG2a were monitored. Th2 cells activation was analysed assaying interleukin (IL)-4 and IL-5 vs IFNgamma on reactivated splenocytes. Local anaphylactic reaction was evaluated by assaying histamine in faecal samples. The oral sensitization protocol was further extended to cow's milk proteins (CMP). RESULTS: Balb/c mice developed high peanut-specific IgE and IgG1 responses either after i.p. or oral sensitizations. In both cases, antibodies were specific to polymer of glycinin fragments, containing polypeptides from Ara h3/4, and to a lesser extent to Ara h1 and Ara h2. Interleukin-4 and IL-5 production were evidenced. Balb/c mice could also be sensitized to CMP, as demonstrated by CMP-specific IL-4 and IL-5 secretions and induction of IgE specific for whole caseins, beta-lactoglobulin, serum bovine albumin and lactoferrin. Of interest was the occurrence of a local anaphylactic reaction in the peanut and CM models. CONCLUSIONS: In contrast with previous authors, Balb/c mice were sensitized and evidenced an allergic reaction after oral administrations of peanut or CMP plus CT, providing an interesting model for further studies on immunopathogenic mechanisms.

Lipid transfer protein 1 is a possible allergen in Arabidopsis thaliana.

BACKGROUND: The Arabidopsis thaliana genome was recently fully sequenced, and this plant is now considered as the most useful model to study the effects of genetic engineering. The aim of the present study was to identify A. thaliana IgE-binding molecules and to localize their genes in order to evaluate the potential effect of gene insertion on the expression of IgE-binding molecules. METHODS: A. thaliana flower proteins were separated by two-dimensional gel electrophoresis and transferred onto a nitrocellulose sheet. The nitrocellulose sheet was successively incubated with human sera known to contain IgE that binds to rapeseed proteins, alkaline phosphatase-conjugated goat anti-human IgE and 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. One allergen was further identified by N-terminal amino acid microsequencing. RESULTS: The results showed that some individuals possessed IgE that recognized numerous proteins with high molecular masses and various isoelectric points. This binding pattern strongly suggests that the epitopes recognized by these IgE could be, at least partly, sugar residues. Otherwise, out of the 10 sera that possessed IgE to Arabidopsis flower proteins, one serum strongly recognized a unique basic protein with an apparent molecular mass of around 14 kD. This protein was identified by amino acid microsequencing as the lipid transfer protein 1 (LTP1). CONCLUSION: We have demonstrated that A. Thaliana LTP1 is IgE reactive. The gene encoding this protein is located on chromosome 2, but it has been described that family 1 of A. Thaliana LTPs constitutes a multigenic family with genes located on various chromosomes.

Polygalacturonase (pectinase), a new oilseed rape allergen.

BACKGROUND: Type I hypersensitivity to rapeseed pollen allergens was described as the result of a cross-sensitization with various pollens that could constitute an aggravating factor in birch or grass pollen allergies. Recently, a few rapeseed pollen allergens were described. The aim of the present work was to identify new rapeseed pollen allergens by using two-dimensional gel analysis, microsequencing, and mass spectrometry. METHODS: Water extractable proteins from oilseed rape pollen or stamen were separated by two-dimensional gel electrophoresis. The proteins were then electroblotted onto a nitrocellulose (NC) sheet. The NC sheets were successively incubated with (1) individual human sera pre-selected for their immunoglobulin E (IgE) reactivity to rapeseed pollen proteins, (2) alkaline phosphatase (AP)-conjugated goat anti-human IgE and (3) AP substrate. The allergens localized by this method were then identified by microsequencing and MALDI-TOF mass spectrometry analysis. RESULTS: Of the 18 sera studied, five recognized a wide multispot zone with a molecular mass around 43 kD and pIs between 6.5 and 8.5. The results obtained with two representative sera are shown. From this zone, two isoforms of the polygalacturonase enzyme were identified by microsequencing. Confirmation was obtained through MALDI-TOF mass spectrometry analysis. CONCLUSION: The present results allow the identification of a new rapeseed allergen that can be the main allergen for some patients.

Comparison of the IgE titers to bovine colostral G immunoglobulins and their F(ab')2 fragments in sera of patients allergic to milk.

Colostral G immunoglobulins (IgGs) are described in many recent studies as having a beneficial effect for the treatment of viral, bacterial and parasitic diarrhea in animals and humans. The specific IgE titers to bovine colostral IgG, to bovine serum IgG, and to F(ab')2 fragments of IgG were immunoenzymatically quantified in sera of patients allergic to milk, to statistically evaluate and compare their relative immunoreactivity towards these purified antigens. The results clearly indicated that 36% of the population tested was potentially allergic to colostral IgG, and serum IgG globally elicited significantly lower IgE titers. The F(ab')2 fragments lead to a significantly decreased immunoreactivity as compared to colostral IgG. This study shows the interesting use of peptic hydrolysis of IgG in producing fragments with preserved therapeutic immunoactivity and reduced potential allergenicity.