Serum IgE cross-reactivity between fish and chicken meats in dogs.

BACKGROUND: In humans, a cross-reactive clinical allergy has been reported between three chicken and fish meat proteins: beta-enolase, aldolase A and parvalbumin. OBJECTIVE: To evaluate if IgE cross-reactivity between chicken and fish also existed in the dog. ANIMALS: Sera from dogs with suspected allergic skin disease and with IgE against chicken and fish. METHODS AND MATERIALS: Sera were analysed by ELISA and immunoblotting with chicken, white fish (haddock and cod) and salmon extracts. Reciprocal inhibition ELISAs and inhibition immunoblots were then performed. Protein sequencing of bands identified on multiple extracts was determined by mass spectrometry. RESULTS: Out of 53 archived canine sera tested by ELISA against chicken, white fish or salmon, 15 (28%), 12 (23%) and 26 (49%), respectively, had elevated IgE against one, two or all three of these extracts. Seven of the triple-reactive sera were subjected to reciprocal inhibition ELISAs. A >50% inhibition was found between chicken-fish, chicken-salmon and fish-salmon in seven, four and five of seven dogs, respectively. Immunoblotting identified multiple IgE-binding proteins of identical molecular weights in the three extracts; these were partially to fully cross-reactive by inhibition immunoblotting. Mass spectrometry identified nine cross-reactive proteins as: pyruvate kinase, creatine kinase, alpha-actin, glyceraldehyde-3-phosphate dehydrogenase, beta-enolase, aldolase, malate dehydrogenase, lactate dehydrogenase and triose-phosphate isomerase 1. All of these have been reported previously as fish, shellfish and/or chicken allergens for humans. CONCLUSIONS AND CLINICAL IMPORTANCE: Whether any of these newly identified IgE cross-reactive chicken-fish allergens is the cause of clinical allergy needs to be determined in dogs reacting to at least two of these common food sources.

Is the 32-kDa fragment the functional enamelin unit in all species?

Enamelin is an extracellular enamel matrix protein essential for normal amelogenesis. After secretion, porcine enamelin is processed to generate several enamelin-degradation products. The cumulative 32-kDa enamelin is the most abundant enamelin present, and various roles for this molecule have been suggested. However, the proteolytic cleavage sites in porcine enamelin that generate the 32-kDa enamelin are not conserved across species, and the 32-kDa enamelin analogue may not be present in all species. To explore this we studied rat enamelin biochemistry using western blotting with anti-peptide IgGs to porcine 32-kDa enamelin and to the putative rat 32-kDa enamelin analogue. The dominant enamelins in secretory-stage rat enamel migrated at around 60-70 kDa. In contrast, the dominant enamelins in secretory-stage porcine enamel migrated at around 32 kDa. In contrast, secretory-stage porcine-enamel enamelins were dominated by the 32-kDa enamelin. Rat enamelin was completely removed from maturation-stage enamel without any accumulation of 32-kDa enamelin. We suggest that a discrete 32-kDa enamelin is not essential for normal amelogenesis in all species, and in pig it may be a processing product of a larger functional enamelin molecule. The pig may be an atypical model in terms of enamelin biochemistry and function, and caution should be exercised when assigning functional roles to the 32-kDa enamelin as a discrete enamel matrix entity.

A mutation in the mouse Amelx tri-tyrosyl domain results in impaired secretion of amelogenin and phenocopies human X-linked amelogenesis imperfecta.

Amelogenesis imperfecta (AI) describes a broad group of clinically and genetically heterogeneous inherited defects of dental enamel bio-mineralization. Despite identification of a number of genetic mutations underlying AI, the precise causal mechanisms have yet to be determined. Using a multi-disciplinary approach, we describe here a mis-sense mutation in the mouse Amelx gene resulting in a Y --> H substitution in the tri-tyrosyl domain of the enamel extracellular matrix protein amelogenin. The enamel in affected animals phenocopies human X-linked AI where similar mutations have been reported. Animals affected by the mutation have severe defects of enamel bio-mineralization associated with absence of full-length amelogenin protein in the developing enamel matrix, loss of ameloblast phenotype, increased ameloblast apoptosis and formation of multi-cellular masses. We present evidence to demonstrate that affected ameloblasts express but fail to secrete full-length amelogenin leading to engorgement of the endoplasmic reticulum/Golgi apparatus. Immunohistochemical analysis revealed accumulations of both amelogenin and ameloblastin in affected cells. Co-transfection of Ambn and mutant Amelx in a eukaryotic cell line also revealed intracellular abnormalities and increased cytotoxicity compared with cells singly transfected with wild-type Amelx, mutant Amelx or Ambn or co-transfected with both wild-type Amelx and Ambn. We hypothesize that intracellular protein-protein interactions mediated via the amelogenin tri-tyrosyl motif are a key mechanistic factor underpinning the molecular pathogenesis in this example of AI. This study therefore successfully links phenotype with underlying genetic lesion in a relevant murine model for human AI.