[COMPONENT ANALYSIS OF THREE ANISAKIASIS ALLERGY CASES IN OUR DEPARTMENT].

Reactivity to an anisakis allergen component was examined in three patients with a history of an anisakiasis anaphylaxis. Case 1, a 38-year-old man, allergic symptoms appeared 0.5 hours after ingestion, and the component Ani s 1 and 3 were positive. Case 2, a 44-year-old woman, allergic symptoms appeared 4 hours after ingestion, and components Ani s 3 and 12 were positive. Case 3, a 36-year-old woman, developed allergic symptoms 7 hours after ingestion of fish and shellfish, and tested positive for Ani s 1, 4, and 12. Case 3 reacted strongly to both heated and unheated Anisakis extract, while cases 1 and 2 reacted weakly to heated Anisakis extract. The most common allergen was Ani s 12, followed by Ani s 1, when analyzed in conjunction with existing reports on 10 cases. Anisakis IgE was class 3 or higher in all cases. Analysis of 13 cases showed 2 cases sensitized to Ani s 4 and moderate or higher anaphylaxis, while Ani s 4-sensitized patients were reported to be more likely to develop severe disease. It is possible that the patients sensitized to Ani s 4 need to be careful about the severity of their allergic symptoms.

IgE reactivity to fish allergens from Pacific cod (Gadus macrocephalus) in atopic dogs.

BACKGROUND: IgE reactivity to fish allergens in atopic dogs, which are used as models for food allergy, has not been elucidated to date. We investigated IgE reactivity to crude extracts and purified allergens derived from the Pacific cod (Gadus macrocephalus) in atopic dogs to identify the allergenic proteins of cod. RESULTS: The levels of specific IgE to crude cod extracts were measured in the sera of 179 atopic dogs, including 27 dogs with cod allergy, using enzyme-linked immunosorbent assay (ELISA). Specific IgE to crude cod extracts were present in 36 (20%) of the 179 atopic dogs and in 12 (44%) of the 27 dogs with cod allergy. The allergens in crude cod extracts were analyzed by ELISA, immunoblotting, and liquid chromatography-tandem mass spectrometry. In allergen component analysis, IgE reactivity to tropomyosin and enolase was observed in the sera of dogs with cod allergy. IgE reactivity to parvalbumin, collagen, and tropomyosin was evaluated using the sera of atopic dogs that tested positive for specific IgE to crude cod extracts. Among the 36 dogs with IgE reactivity to crude cod extracts, 9 (25%), 14 (39%), and 18 (50%) dogs tested positive for specific IgE to parvalbumin, collagen, and tropomyosin, respectively. CONCLUSIONS: The IgE reactivity to cod allergens observed in dogs was similar to that in humans, and this finding further supports the use of atopic dogs with fish allergy as a model for fish allergy in humans.

AN ADULT CASE OF ANAPHYLAXIS CAUSED BY ALLERGY TO JELLYFISH.

A 35-year-old female, professional diver, reported nausea, vomiting, and systemic hives 20 to 30 minutes after ingestion of antipasto made with jellyfish. Patient reported prior episodes of swelling after stings from several different creatures, including jelly fish. She also developed a systemic allergic reaction after sting from an unknown creature while diving. On the initial visit to our hospital, serum total IgE level was 545IU/ml. We extracted crude allergen from jellyfish and evaluated allergen specific IgE antibody levels using ELISA. Patient samples showed higher levels of jellyfish-derived allergen specific IgE than healthy control samples. Basophils were isolated from the peripheral blood of patient. Stimulation with jellyfish-derived allergen showed expression of surface antigens on basophils increased in a concentration-dependent manner. Methods using sodium dodecyl sulfate poly acrylamide gel electrophoresis and immunoblotting showed acid-soluble collagen fraction from jellyfish contained above 250kDa weighed protein that may have caused this current event. A provocation test using jellyfish samples was not performed due to risk of anaphylactic shock. The patient was diagnosed with a jellyfish allergy due to IgE mediated anaphylaxis after ingestion. She was asked to refrain from consuming any food containing jellyfish. IgE-mediated food allergy caused by jellyfish is rare worldwide. Collagen was speculated to be an allergen in this study. Additional study to detect specific allergens related to jellyfish allergy would be particularly useful to specify disease phenotypes and individual care in future.

Quantification of major allergen parvalbumin in 22 species of fish by SDS-PAGE.

Fish is an important causative material of food allergy. Although the allergenicity of fish is considered to correlate with the content of parvalbumin, the major fish allergen, available information about the parvalbumin content in fish is limited. In this study, a simple and reliable quantification method for fish parvalbumin by SDS-PAGE was first established. Application of the SDS-PAGE method to 22 species of fish revealed a marked variation in parvalbumin content among fish. Furthermore, the parvalbumin content was found to be higher in dorsal white muscle than in ventral white muscle, in rostral part of white muscle than in caudal part of white muscle and in white muscle than in dark muscle. IgE reactivity of fish was roughly proportional to parvalbumin content. Interestingly, large-sized migratory fish, such as salmon, swordfish and tuna, were commonly very low in both parvalbumin content and IgE reactivity.

Molecular Cloning and Expression of a New Major Allergen, Ani s 14, from Anisakis simplex.

The nematode Anisakis simplex is a representative parasite infecting marine animals. When third stage larvae of A. simplex infecting fish and squids are ingested by humans, individuals previously sensitized by this parasite may experience IgE-mediated allergic reactions. So far, as many as 13 kinds of proteins (Ani s 1-13) have been identified as A. simplex allergens but several more unknown allergens are suggested to exist. In this study, therefore, chemiluminescent immunoscreening of an expression cDNA library constructed from the third stage larvae was conducted to identify a new allergen. As a result, an IgE-positive clone coding for a 23.5 kDa protein (named Ani s 14) composed of 217 amino acid residues was isolated. The regions 4-147 and 34-123 of Ani s 14 share 31% identity with the region 796-940 of Ani s 7 and 32% identity with the region 2-91 of Ani s 12, respectively. Recombinant Ani s 14 was successfully expressed in Escherichia coli as a His-tagged protein and shown to be IgE reactive to 14 (54%) of 26 sera from Anisakis-allergic patients. In conclusion, Ani s 14 is a new major allergen of A. simplex that is specific to Anisakis-allergic patients.

[Comparative analyses of allergens between landlocked and anadromous species of masu salmon Oncorhynchus masou masou].

Although the difference in allergenicity between landlocked and anadromous salmon is little understood, only anadromous salmon are recommended to be labeled in the current allergen labeling system. This study was designed to examine the allergenic potency of landlocked species (yamame) and anadromous species (sakuramasu) of masu salmon Oncorhynchus masou masou, with special reference to parvalbumin, a known major fish allergen. Analysis of the heated extracts by SDS-PAGE suggested that yamame contains parvalbumin in the muscle at considerably higher levels, as compared with sakuramasu. In accordance with this, the parvalbumin content in the muscle of yamame (1.8-7.8 mg/g), determined by visible-light ELISA, was significantly higher than that of sakuramasu (0.28-0.52 mg/g). Furthermore, fluorescence ELISA experiments showed that the heated extract from yamame reacts with serum from fish-allergic patients more strongly than that from sakuramasu. Three parvalbumin isoforms (PA-I, -II and -III) were individually purified from yamame and sakuramasu by gel filtration and reverse-phase HPLC. Based on the retention times in reverse-phase HPLC and the molecular weights estimated by MALDI/TOF-MS, PA-I, -II and -III from yamame were judged to be identical with PA-I, -II and -III from sakuramasu, respectively. Taken together, our data indicate that landlocked masu salmon (yamame) is more allergenic than anadromous salmon (sakuramasu).

Identification of novel three allergens from Anisakis simplex by chemiluminescent immunoscreening of an expression cDNA library.

Anisakis simplex is a representative nematode parasitizing marine organisms, such as fish and squids, and causes not only anisakiasis but also IgE-mediated allergy. Although 10 kinds of proteins have so far been identified as A. simplex allergens, many unknown allergens are considered to still exist. In this study, a chemiluminescent immunoscreening method with higher sensitivity than the conventional method was developed and used to isolate IgE-positive clones from an expression cDNA library of A. simplex. As a result, three kinds of proteins, Ani s 11 (307 amino acid residues), Ani s 11-like protein (160 residues) and Ani s 12 (295 residues), together with three known allergens (Ani s 5, 6 and 9), were found to be IgE reactive. Furthermore, ELISA data showed that both recombinant Ani s 11 and 12 expressed in Escherichia coli are recognized by about half of Anisakis-allergic patients. Ani s 11 and Ani s 11-like protein are characterized by having six and five types of short repetitive sequences (5-16 amino acid residues), respectively. Both proteins share as high as 78% sequence identity with each other and also about 45% identity with Ani s 10, which includes two types of short repetitive sequences. On the other hand, Ani s 12 is also structurally unique in that it has five tandem repeats of a CX(13-25)CX(9)CX(7,8)CX(6) sequence, similar to Ani s 7 having 19 repeats of a CX(17-25)CX(9-22)CX(8)CX(6) sequence. The repetitive structures are assumed to be involved in the IgE-binding of the three new allergens.

Change in tetrodotoxin content of the puffer fish Takifugu rubripes during seed production from fertilized eggs to juveniles.

Changes in tetrodotoxin (TTX) content of the puffer fish Takifugu rubripes during seed production were examined. Two mature female puffer fish T. rubripes (samples 1 and 2) that contained TTX were used. The toxic eggs were artificially fertilized, and hatchlings were reared in an indoor tank for 50 days and then in a netcage at sea for an additional 48 or 38 days. The TTX content of the fertilized eggs of sample 1 was initially 13.0 microg TTX/g, transiently increased to 67.6 microg TTX/g at 4 days after hatching, and then gradually decreased to 0.28 microg TTX/g at 98 days. In contrast, the total TTX content in an individual was 0.016 microg TTX at the fertilization stage and 0.01-0.03 microg TTX at the larval stage until 30 days after hatching. Thereafter, the total TTX content increased remarkably during culture in the netcage at sea, reaching 4.80 microg TTX at 98 days. Change in the TTX content of sample 2 showed a similar tendency to that of sample 1. The present study showed that the TTX content per gram of puffer fish body weight decreased during progression from fertilized eggs to juveniles, whereas the total TTX content increased.

Evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes.

In this study, we investigated the hepatic uptake clearance (CL(uptake)) of tetrodotoxin (TTX) in the marine puffer fish Takifugu rubripes by integration plot analysis after a single bolus injection of 0.25mg TTX/kg body weight into the hepatic vein at 20 degrees C. The blood concentration of TTX decreased over time after the injection, from 1451+/-45 ng/mL at 10 min to 364+/-59 ng/mL at 60 min. TTX concentrations in the spleen and kidney decreased in parallel with the blood concentrations, whereas those in the muscle and skin remained almost the same throughout the experiment. In contrast, the TTX concentration in the liver gradually increased, reaching 1240+/-90 ng/g liver at 60 min after injection. The amount of TTX that had accumulated in the liver 60 min after injection accounted for 63+/-5% of the administered dose. Integration plot analysis indicated a CL(uptake) of 3.1 mL/min/kg body weight in the liver for TTX, a rate far below that of the hepatic portal vein blood flow rate (at most, 9%). This finding is consistent with negligible extraction of TTX by the liver. The results demonstrated conclusively that the liver-specific distribution of TTX in T. rubripes is achieved by removal from the systemic circulation, but not by the hepatic first-pass effect.

Pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique.

Marine puffer fish accumulates tetrodotoxin (TTX) in the liver and ovary. In this study, we examined the pharmacokinetics of TTX in Takifugu rubripes by a single administration under general anesthesia at 20 degrees C for 300 min. The blood concentration-time profile showed multiple distinct phases after injection into hepatic portal vein. The area under the blood concentration-time curve (AUC) increased linearly at the dosage of 0.25-0.75 mg TTX/kg body weight, and the total body clearance was 2.06+/-0.17 mL/min/kg body weight. The AUCs following administration into the hepatic portal vein and hepatic vein were closely similar (147+/-33 versus 141+/-1 ng.min/microL), indicating negligible hepatic first-pass effect. Comparison of the AUCs following an administration to the hepatic vein and gastrointestinal tract (0.25 mg TTX/kg body weight) elucidated the bioavailability of TTX to be 62%. There was no significant increase in the AUCs following direct injection into the gastrointestinal tract (0.50 versus 1.0 mg TTX/kg body weight). At the dosage of 0.25 mg TTX/kg body weight into the hepatic vein, hepatic portal vein or gastrointestinal tract, TTX amount in the liver accounted for 84+/-6%, 70+/-9% or 49+/-17% of the total TTX amount applied, respectively. These results demonstrate that TTX is absorbed into the systemic circulation from the gastrointestinal tract by saturable mechanism and finally accumulated in the liver within 300 min.

Sarcoplasmic calcium-binding protein: identification as a new allergen of the black tiger shrimp Penaeus monodon.

BACKGROUND: Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. METHODS: A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. RESULTS: The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. CONCLUSIONS: SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish.

Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii.

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.

Gene expression and distribution of antibacterial L-amino acid oxidase in the rockfish Sebastes schlegeli.

Antibacterial factors in the epidermal mucus of fish have a potential importance in the first line of the host defense response to bacterial pathogens. We previously isolated a novel antibacterial protein termed SSAP (Sebastes schlegeli antibacterial protein) from the skin mucus of the rockfish S. schlegeli and identified it as a new member of the L-amino acid oxidase (LAO) family. In the present study, the localization of SSAP in S. schlegeli was investigated by reverse transcription (RT)-PCR, quantitative real time RT-PCR, Western blotting and measurements of LAO and antibacterial activities. SSAP mRNA was expressed dominantly in skin and gill and weakly in ovary or kidney as shown by RT-PCR and real time RT-PCR. The quantity of SSAP mRNA in skin varied among the individuals, ranging from 1.1 to 13.9 ng microg(-1) total RNA, although no relationship was found between the size of fish and gene expression. SSAP was exclusively detected in skin and gill by Western blotting using a specific anti-SSAP antiserum. In addition, the extracts of both tissues apparently showed LAO activity and antibacterial activity against Photobacterium damselae subsp. piscicida. This study demonstrates that SSAP is predominantly synthesized in skin and gill and probably functions as an antibacterial LAO in both tissues.

Purification and cDNA cloning of a new heat-stable allergen from Anisakis simplex.

The nematode Anisakis simplex is a representative parasite for marine animals and occasionally causes not only anisakiasis but also allergic reactions in sensitized subjects. Besides the known allergens, a number of unidentified allergens have been suggested to still exist in A. simplex. In this study, a new heat-stable allergen of 15kDa (named Ani s 8) was purified from the third stage larvae of A. simplex by gel filtration on Sephacryl S-300, anion-exchange HPLC on Mono Q and reverse-phase HPLC on TSKgel Phenyl-5PW RP. Analysis by fluorescence ELISA showed that 7 of 28 Anisakis-allergic patients had elevated serum levels of IgE to Ani s 8. On the basis of the determined partial amino acid sequence, the complete sequence of Ani s 8 (composed of 150 amino acid residues) was elucidated by cDNA cloning, in which as many as 32 homologs of the cDNA encoding 10 isoforms of Ani s 8 were detected. Ani s 8 shares amino acid sequence homology (up to 36%) with several members of the SXP/RAL-2 protein family, including Ani s 5 (15kDa) previously identified as an A. simplex allergen. Inhibition ELISA data demonstrated the IgE cross-reactivity between Ani s 8 and Ani s 5.

Involvement of carrier-mediated transport system in uptake of tetrodotoxin into liver tissue slices of puffer fish Takifugu rubripes.

Although puffer fish contain tetrodotoxin (TTX) at a high concentration mainly in liver, the underlying mechanism remains to be elucidated. In the present study, uptake of TTX into the liver tissue slices of puffer fish Takifugu rubripes was investigated by in vitro incubation experiment. When T. rubripes liver slices were incubated with 0-2000microM TTX at 20 degrees C for 60min, the uptake rates exhibited non-linearity, suggesting that the TTX uptake into T. rubripes liver is carrier-mediated. The TTX uptake was composed of a saturable component (V(max) 47.7+/-5.9pmol/min/mg protein and K(m) 249+/-47microM) and a non-saturable component (P(dif) 0.0335+/-0.0041microL/min/mg protein). The uptake of TTX was significantly decreased to 0.4 and 0.6 fold by the incubation at 5 degrees C and the replacement of sodium-ion by choline in the buffer, respectively, while it was not affected by the presence of 1mM l-carnitine, p-aminohippurate, taurocholate or tetraethylammonium. The TTX uptake by black scraper Thamnaconus modestus liver slices was much lower than that of T. rubripes and independent of the incubation temperature, unlike T. rubripes. These results reveal the involvement of carrier-mediated transport system in the TTX uptake by puffer fish T. rubripes liver slices.

[A case of occupational contact urticaria and oral allergy syndrome due to seafood].

A 20-year-old woman was referred for evaluation after about 2 years of recurrent episodes of localized urticaria during handling of several kinds of raw fish in a sushi shop, where she had worked part-time for 2 years. She had also experienced allergic symptoms such as itching and swelling of her lips, generalized urticaria, laryngeal tightness, stridor and dyspnea immediately after ingestion of raw and cooked seafood, including sole, horse mackerel, sea eel and shellfish, over the previous 1 year before referral. Skin prick tests and blood test for specific IgE antibodies were positive for many kinds of seafood, including sole, horse mackerel, sea eel, eel, crab, and abalone, which belonged to different taxonomic phyla, including Chordata, Arthropoda, and Mollusca. A challenge with a piece of broiled sole induced swelling of the lips, obstruction of the larynx, difficulty with deglutition, and abdominal pain. In addition, serum-specific IgE antibodies to two major fish allergens, parvalbumin and collagen, were detected by ELISA, suggesting that allergic symptoms could be induced by many kinds of seafood in the present patient. She was therefore diagnosed with occupational contact urticaria and oral allergy syndrome due to seafood. At the time of this report, the present patient had been followed for one year and no reactions have occurred since she started to avoid the causative types of seafood.

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli.

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.

Molecular cloning and expression of two new allergens from Anisakis simplex.

The nematode Anisakis simplex is a marine parasite that causes allergy as well as anisakiasis. Although five Anisakis allergens have already been identified, immunoblotting studies suggested that unidentified allergens still exist. In this study, an expression cDNA library constructed from A. simplex was subjected to immunoscreening using an Anisakis-allergic patient serum, and two positive clones coding for allergens (named Ani s 5 and 6) were obtained. Ani s 5 (152 amino acid residues) is homologous with nematode proteins belonging to the SXP/RAL-2 protein family and Ani s 6 (84 amino acid residues) with serine protease inhibitors from various animals. Of the 28 patient sera examined, seven and five reacted to recombinant Ani s 5 and 6 expressed in Escherichia coli, respectively. By inhibition immunoblotting experiments using the recombinant allergens as inhibitors, natural Ani s 5 could be identified as a 15-kDa protein in the crude extract of A. simplex but natural Ani s 6 could not be identified probably due to its low expression. In conclusion, Ani s 5 and 6 are new allergens of A. simplex that are specific to some Anisakis-allergic patients.