Hidenobu Hoshi

Characterization of dog allergens Can f 1 and Can f 2. 1. Preparation of their recombinant proteins and antibodies.

Background: Recombinant dog allergens, rCan f 1 and rCan f 2, and their antibodies are good tools for the characterization of dog allergens in order to develop modern therapeutic and preventive methods for dog allergy. Methods: In this study, cDNA was synthesized from the mRNA of dog salivary glands and cloned into the pGEX4T vector. rCan f 1 and rCan f 2 containing glutathione S-transferase were prepared by an Escherichia coli expression system. The antibodies against the recombinant allergens were prepared in rabbit. The serum of patients with dog allergy was evaluated by ELISA and immunoblot, using the recombinant allergens, goat anti-human immunoglobulin (Ig) E (Ε) labeled with biotin, and enzyme-labeled streptavidin. The binding of IgE in the serum of patients with dog allergy to dog saliva as a natural antigen was determined in the presence or absence of dog saliva, rCan f 1 and rCan f 2 as competitors. The anaphylactic potential of rCan f 1 and rCan f 2 was evaluated. The body temperature of the mice sensitized with rCan f 1 and rCan f 2 was monitored after intravenous injection of the allergens. The passive cutaneous anaphylaxis reaction was examined for rCan f 1 and rCan f 2. Dog salivary glands, dog saliva and dog hair/dander extracts were analyzed with antibodies by means of an immunoblot assay. The expression of the mRNA of Can f 1 and Can f 2 was verified in various dog tissues by reverse transcription polymerase chain reaction. Results: The E. coli expression system revealed the yield of rCan f 1 and rCan f 2 in 36 and 30 mg/l of culture. The molecular weights of rCan f 1 and rCan f 2 were 18 and 20 kDa in SDS-PAGE, respectively. rCan f 1 and rCan f 2 were found to bind to specific IgE in the serum of dog allergy patients. The binding of IgE in the patient serum for dog saliva was partially inhibited in the presence of rCan f 1 and rCan f 2. These recombinant allergens showed positive signals in passive cutaneous anaphylaxis reaction and induced anaphylactic shock in the mouse model, resulting in a decrease in body temperature. The polyclonal rabbit antibody for rCan f 1 bound to a protein of 20 kDa in the salivary gland, saliva and hair/dander extracts of dogs. The rabbit antibody for rCan f 2 bound to proteins in the saliva and the hair/dander extracts. The proteins possessed a molecular weight of 22/ 23 kDa. Reverse transcription polymerase chain reaction showed the presence of mRNA expression of Can f 1 and Can f 2 not only in the salivary gland but also in dog skin. A clear expression of Can f 2 mRNA was observed in dog skin. Conclusions: The recombinant allergens and antibodies for Can f 1 and Can f 2 are available for immunological and biochemical characterization of dog allergens. The molecular weight of the natural Can f 1 and Can f 2 in dog saliva and hair/dander extracts showed a higher molecular weight than that of rCan f 1 and rCan f 2. The significance of dog skin as the tissue producing dog allergens, especially Can f 2, should be considered in further studies.

Characterization of dog allergens Can f 1 and Can f 2. 2. A comparison of Can f 1 with Can f 2 regarding their biochemical and immunological properties.

Background: The major dog allergens, Can f 1 and Can f 2, are members of the lipocalin protein family. The characterization of both dog allergens is still not complete. Their deduced amino acid sequences indicate the presence of three cysteine residues, probably connected with a disulfide bridge. We compared the biochemical and immunological properties of Can f 1 with those of Can f 2 using gel filtration, electrophoresis, and immunological assays. Methods: The rCan f 1, rCan f 2 and dog salivary proteins containing natural Can f 1 and Can f 2 were analyzed by HPLC gel filtration. The recombinant Can f 1 (rCan f 1) and rCan f 2 were analyzed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) with or without reduction. The binding ability of rabbit IgG purified by protein G affinity chromatography from the antiserum against rCan f 1 and rCan f 2 was examined after a reduction in the recombinant allergens. The immunological cross-reaction between rCan f 1 and rCan f 2 was examined by an enzyme-linked immunosorbent assay (ELISA) using the rabbit IgG against rCan f 1 and rCan f 2. The cross-reaction of human IgE in the serum of a patient with dog allergy between rCan f 1 and rCan f 2 was also analyzed by competitive ELISA. Results: The molecular weights of rCan f 1 and of rCan f 2 were 18 and 21 kDa, respectively, using SDS-PAGE under reducing conditions, but the natural Can f 1 and Can f 2 were separated by HPLC gel filtration into fractions containing proteins of 31 and 34 kDa, respectively. rCan f 1 and rCan f 2 migrated as multiple bands (30–100 kDa) in native PAGE in the presence or absence of a reductant. The molecular weights of natural Can f 1 and of Can f 2 were 20 and 23 kDa, respectively, in SDS-PAGE under reducing conditions. The ability of rabbit IgG to bind to rCan f 1 and rCan f 2 increased after the reduction of the recombinant allergens. The rabbit IgG against rCan f 1 bound to rCan f 2. Cross-reaction of human IgE was observed between rCan f 1 and rCan f 2. Conclusions: In the native and recombinant forms, Can f 1 and Can f 2 possessed a dimer structure under natural (non-reduced) condition. The dimers of Can f 1 and of Can f 2 were not built with a disulfide bridge but by non-covalent association. Cleavage of a disulfide bond of rCan f 1 and rCan f 2 increased the ability of binding of rabbit IgG to the allergens. The cross-reactivity of rabbit IgG and human IgE between rCan f 1 and rCan f 2 indicates that the same epitope(s) was present in Can f 1 and Can f 2.

In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production

Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity.