Characterization of the inflammatory infiltrate during IgE‐mediated late phase reactions in the skin of normal and atopic dogs
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In canine and human atopic patients, the intracutaneous injection of offending allergens is followed by the development of both immediate and late‐phase reactions. The present study was performed to expand on the characterization and dynamics of inflammatory cell subsets during IgE‐mediated late‐phase reactions in canine skin. Three normal dogs and three Dermatophagoides farinae ‐allergic dogs were selected for this experiment. All dogs were challenged intradermally with mite allergen, purified anticanine IgE antibodies (positive control) or phosphate‐buffered saline (negative control). Skin biopsies were obtained before and 6, 12 and 24 h post‐injection. Sections were stained with metachromatic and eosinophil‐specific histological stains. Additionally, we used an immunohistochemical method with antibodies specific for canine leukocyte antigens. This study confirmed the occurrence of a late‐phase reaction in atopic skin following allergen challenge, and in normal and atopic canine skin after intradermal injection of IgE‐specific antibodies. Whereas early emigrating dermal cells were composed chiefly of neutrophil and activated eosinophil granulocytes, there was an influx of αβ T‐lymphocytes and dermal dendritic cells in later stages of the late‐phase reactions. Because IgE‐mediated late‐phase reactions resemble spontaneous atopic canine skin lesions, both at macroscopic and microscopic levels, we propose the use of similar challenges to study the anti‐inflammatory effects of anti‐allergic drugs in a pre‐clinical setting.Keywords:
Intradermal injection
The natural production of <i>Der p1</i>, the major faecal allergen from the house dust mite <i>(Dermatophagoides pteronyssinus),</i> was investigated. Mite cultures grown on radiolabelled substrate were examined at intervals over a period of 21 days and incorporation of radiolabel into the mite proteins measured over the time course. Mite proteins were extracted, purified by chromatofocussing and characterized by immunodiffusion, SDS-PAGE, electroblotting. and autoradiography. The faecal allergen, <i>Der p1</i>, did not incorporate a significant level of label until the 14th day (p < 0.05). The results suggest that <i>Der p1</i> is a protein synthesized and excreted by the house dust mite.
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Acaridae
Immunodiffusion
Electroblotting
Radial immunodiffusion
Ouchterlony double immunodiffusion
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A major house dust mite allergen, <i>Der p</i> I, was isolated from spent growth medium and physico-chemically characterized. These studies show that the allergen is monomeric, contains approximately 216 residues and 4 intra-chain disulphide bonds. The N-terminal amino acid is threonine. Circular dichroism studies show that the allergen contains 10% <i>α</i>-helical, 50% <i>β</i>-pleated sheet and 40% random structures.
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Allergen from the house dust mite ( Dermatophagoides sp.) is a major trigger factor of allergic disorders, and its characterization is crucial for the development of specific diagnosis or immunotherapy. Here we report the identification of a novel dust mite ( Dermatophagoides farinae ) antigen whose primary structure belongs to the gelsolin family, a group of actin cytoskeleton‐regulatory proteins. Isolated mite cDNA, termed Der f 16, encodes 480 amino acids comprising a four‐repeated gelsolin‐like segmental structure, which is not seen in conventional gelsolin family members. Enzyme immunoassay indicated that recombinant Der f 16 protein, prepared using an Escherichia coli expression system, bound IgE from mite‐allergic patients at 47% (8/17) frequency. This is the first evidence that the gelsolin family represents a new class of allergen recognizable by atopic patient IgE.
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Allergen-specific IgE production is the central event in the pathogenesis of atopic disorders and increases in specific IgE serum antibodies are an indicator of immediate hypersensitivity responses in humans and in animal models of allergy. Consequently, accurate and user-friendly methods are needed to measure serum levels of allergen-specific IgE. This review examines historical and recent developments in in vivo and in vitro methods for the detection of allergen-specific IgE in humans and in animal models. Routinely, in vitro methods such as enzyme-linked immunosorbant assays or radioallergosorbant tests and in vivo methods such as the skin prick test (SPT) for humans and the passive cutaneous anaphylaxis assay (PCA) used in animals are utilized to detect allergen-specific IgE. While in vivo assays are usually more accurate than in vitro assays since they provide a functional readout of IgE activity, they are relatively costly and require considerable expertise. On the other hand in vitro assays are limited by the fact that the amount of allergen-specific serum IgG exceeds IgE antibody by several orders of magnitude, resulting in competition for allergen binding. Consequently, methods that use allergen as a direct capture step are limited by the availability of free allergen binding sites for IgE. In order to circumvent this problem, in vitro methods usually require prior depletion of IgG or use high amounts of allergen in order to facilitate availability of free binding sites for IgE detection. Clearly, these approaches are limited for small sample volumes and allergens that are in short supply. New methods such as protein microarray could potentially overcome this problem by providing high allergen concentrations in a relatively small reaction volume. Currently, in vitro methods are rarely used in isolation for prognosis but are used primarily to complement the information obtained from in vivo assays. With the emergence of new technologies it is conceivable that in vitro assays may in the future replace in vivo assays, however until then in vivo assays remain the gold standard of allergen-specific IgE detection.
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A method for the detection of specific IgE in human serum is described, using a modified radioimmune assay termed the Inhibition Assay Technique (IAT) The technique is based on the ability of specific IgE to bind with allergen. The difference in total IgE before incubation with the allergen and after incubation with the allergen gives a relative concentration of specific IgE to that particular allergenic substance. The assay has been shown to correlate with both the intradermal skin test as well as the standard RAST now used to determine specific IgE.
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There was a significant amount of non-specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)-specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain-, but not mite feces- or pollen-specific IgE+ cells and an i.n. injection of papain induced papain-specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces-specific IgE+ cells in the lymph nodes nor mite feces-specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen-specific IgE+ small B cells in the lymph nodes on Day 10, when non-specific IgE Ab titers reached a peak in the serum, implying induction of related allergen-specific IgE+ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen-specific IgE Abs in the serum. These results indicate that when firstly-sensitized non-specific IgE+ small B cells in mouse lymph nodes include some secondly-sensitized allergen-specific ones, mice produce IgE Abs specific for the secondly-injected allergen.
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