Thien Van Do

The major allergen (parvalbumin) of codfish is encoded by at least two isotypic genes: cDNA cloning, expression and antibody binding of the recombinant allergens.

The major allergen (parvalbumin) from cod, designated Allergen M Gad c 1, has been intensively studied both from the structural and immunological sides. In the present study, transcripts of two isotypic parvalbumin genes in Atlantic cod were identified and characterized. Subsequently, subfragments were inserted into the expression vector pET-19b, generating plasmids with coding capacity for complete parvalbumin polypeptides fused to an N-terminal his(10) tag. Most of the recombinant products were found in the soluble fraction of the expression host Escherichia coli. The target proteins showed to react with polyclonal antibodies raised against Allergen M and demonstrated binding to specific IgE from 12 sera of patients allergic to cod in ELISA inhibition experiments. Sera with classes 4 and 5 CAP FEIA exhibited also strong binding to recombinant parvalbumins in immunoblots.

Evaluation of the Allergenicity and Antigenicity of Bovine‐Milk αs1‐Casein Using Extensively Purified Synthetic Peptides

αs1‐Casein (CAS1_BOVIN), the major allergen of cows milk (CM), is widely used as hydrolysates in infant diet formulae and additive to other processed food items. To date, most of the reported B‐cell epitope mapping were performed on polyethylene pins or cellulose‐derivative membrane. We sought to locate the motifs critical for human‐specific IgE and rabbit polyclonal IgG binding using extensively purified CAS1_BOVIN, synthetic peptides and derivatives. Thirteen overlapping peptides covering the whole CAS1_BOVIN encompassing 17 : 20 amino acid (AA) were synthesized by f‐moc AA solid‐phase polyamide peptide synthesis. In addition, six cyanogen bromide (CNBr) cleavage fragments were prepared. Limited hydrolysis, oxidized and reduced/alkylated derivatives were also produced. The preparations were purified by ion exchange, gel filtration chromatography, reversed phase and high‐performance liquid chromatography. The homogeneity was visualized by sodium dodecyl sulfate (SDS) and poly acryl amide gel electrophoresis (PAGE) followed by IgE and IgG immunoblotting. IgE binding was measured by Biotin Streptavidin (Bio/strep) fluoro enzyme immuno assay (FEIA) or ELISA‐inhibition. Eighteen CM allergy (CMA) sera from 45 clinically examined children (Melbourne) and five adults (Bergen) were selected. Individual sera and pools were used for mapping IgE‐binding epitopes. Rabbit IgG sera and pools were used for locating the antigenic sites of the molecule. Results indicated that all the individual CMA sera and pools recognized the intact molecule and three of the CNBr fragments as major antibody‐binding allergens. The N‐ and C‐terminal peptides (CAS 16‐35; CAS 136‐155) showed high IgE‐binding affinity. CAS 1‐18 and CAS 181‐199 showed high IgG bindings. Considering the diversity of the antibody specificities, a reasonable agreement between IgE and IgG epitopes were found at the N‐ and C‐terminals of CAS1_BOVIN. Mapping IgE B‐cell epitopes by direct Bio/strep FEIA allowed the development of a sensitive modified technique for detecting unlabelled, casein immune dominant peptides in food products.

Effects of industrial processing on the immunogenicity of commonly ingested fish species.

Background: Food-processing techniques may induce changes in fish protein immunogenicity. Allergens from >100 fish species have been identified, but little is known on the effects of processing on fish protein immunogenicity. Methods: IgE binding of sera of patients allergic to fresh and processed (smoked, salted/sugar-cured, canned, lye-treated and fermented) cod, haddock, salmon, trout, tuna, mackerel and herring and of hydrolysates based on salmon and whiting was investigated using immunoblot and inhibition ELISA. Results: Parvalbumin oligomers were identified using monoclonal and polyclonal antibodies. IgE binding was seen in most sera at 12–14 kDa (parvalbumin), and at 17–60 kDa for all fish except tuna. Changes in IgE binding appeared to reflect altered parvalbumin monomers and oligomers. Smoked haddock, salmon and mackerel had increased IgE binding and novel bands at 30 kDa. Chemically processed cod, salmon, trout and pickled herring had reduced or abolished IgE binding. The serum of 1 subject, however, had increased IgE binding to these products and also inhibition of binding by both fish hydrolysates to their constituent fish species. Conclusion: Process-induced changes in fish protein immunogenicity were more dependent on process rather than species, although individual responses varied. Changes in the allergenicity of a product may depend on the net effect of processing on parvalbumin oligomerization patterns, which may also vary in different species. Chemical processes generally caused loss in IgE-binding activity, though sensitization may occur to modified or degraded rather than intact peptides as shown by increased binding by chemically processed fish and hydrolysates in 1 subject. The clinical significance of these findings remains to be established.

Occupational allergy to Artemia fish fry feed in aquaculture

BACKGROUND Artemia (brine shrimp) is used as feed for fish fry and shrimp in aquaculture. Two employees in a Norwegian aquaculture research farm reported having chest symptoms when working in an Artemia hatch room. AIMS To determine the presence and prevalence of Artemia sensitization at the farm and the extent of any Artemia-related respiratory and hand skin symptoms and to identify the allergens involved. METHODS Participants completed a questionnaire and structured interview. Skin prick tests (SPTs) were performed, and immunoglobulin E (IgE) antibodies to Artemia, shrimp and recombinant tropomyosin were determined. Gel electrophoresis and immunoblots of Artemia extracts were also carried out. RESULTS Thirty of 42 employees (71%) participated. Among the 24 subjects exposed to Artemia, four (17%) reported chest and/or hand skin symptoms during exposure and three of them were IgE sensitized to Artemia. Five (21%) of those exposed demonstrated IgE antibodies to Artemia and four (17%) had immediate-positive SPTs. A serum pool from these subjects exhibited IgE binding to a protein of approximately 97 kDa in the Artemia extract. CONCLUSIONS Occupational exposure to the Artemia fish fry feed can cause IgE sensitization and allergic symptoms affecting airways and skin.

Nasal indices of eosinophilic and exudative inflammation in bakery-workers

Aims:  Rhinitis symptoms frequently occur in bakery‐workers. Yet, little is known about the pathophysiology of this condition. The objective of the present study was to examine nasal indices of inflammation in relation to occupational dust exposure, occupational rhinitis according to defined criteria, rhinitis symptoms associated to the workplace, and occupational sensitization in bakery‐workers.

Pulmonary illness as a consequence of occupational exposure to shrimp shell powder

OBJECTIVES An employee with no prior history of allergy or asthma, experienced respiratory and flu-like symptoms during production of shrimp shell powder in a seafood savory factory in Norway. We aimed to clarify the diagnosis and to identify the cause of the symptoms by specific inhalation challenge (SIC) and by characterizing the powders biocontaminants, particle size fractions and inflammatory potential. METHODS Respiratory and immunological responses were measured the day before and after each of four challenges with 20-150g shrimp shell powder during three consecutive days. The powder was analyzed for endotoxin, microorganisms and particle size fractions by standardized laboratory methods. Total inflammatory potential was quantified by reactive oxygen species (ROS) production in a granulocyte assay. RESULTS The patient had elevated IgG, but not IgE, towards shrimp shell powder. 20min challenge with 150g shrimp shell powder induced 15% decrease in FVC, 23% decrease in FEV1 and increased unspecific bronchial reactivity by methacholine. Neutrophils and monocytes increased 84% and 59%, respectively, and the patient experienced temperature increase and flu-like symptoms. The shrimp shell powder contained 1118 endotoxin units/g and bacteria including Bacillus cereus, and 57% respirable size fraction when aerosolized. The ROS production was higher for shrimp shell powder than for endotoxin alone. CONCLUSIONS Endotoxin and other bacterial components combined with a high fraction of respirable dust might be the cause of the symptoms. The patients characteristics and response to SIC were best compatible with occupational asthma and organic dust toxic syndrome, while hypersensitivity pneumonitis could not be excluded.

Bronchial responsiveness in bakery workers: relation to airway symptoms, IgE sensitization, nasal indices of inflammation, flour dust exposure and smoking

Background  Bronchial hyperresponsiveness (BHR) is common in bakery workers. The relation between bronchial responsiveness measured with a tidal breathing method and smoking, airway symptoms, IgE‐sensitization, nasal indices of inflammation and flour dust exposure have been studied with bronchial responsiveness expressed as a continuous outcome.