Tandem Mass Tagging (TMT) Reveals Tissue-Specific Proteome of L4 Larvae of Anisakis simplex s. s.: Enzymes of Energy and/or Carbohydrate Metabolism as Potential Drug Targets in Anisakiasis.

Anisakis simplex s. s. is a parasitic nematode of marine mammals and causative agent of anisakiasis in humans. The cuticle and intestine of the larvae are the tissues most responsible for direct and indirect contact, respectively, of the parasite with the host. At the L4 larval stage, tissues, such as the cuticle and intestine, are fully developed and functional, in contrast to the L3 stage. As such, this work provides for the first time the tissue-specific proteome of A. simplex s. s. larvae in the L4 stage. Statistical analysis (FC ≥ 2; p-value ≤ 0.01) showed that 107 proteins were differentially regulated (DRPs) between the cuticle and the rest of the larval body. In the comparison between the intestine and the rest of the larval body at the L4 stage, 123 proteins were identified as DRPs. Comparison of the individual tissues examined revealed a total of 272 DRPs, with 133 proteins more abundant in the cuticle and 139 proteins more abundant in the intestine. Detailed functional analysis of the identified proteins was performed using bioinformatics tools. Glycolysis and the tricarboxylic acid cycle were the most enriched metabolic pathways by cuticular and intestinal proteins, respectively, in the L4 stage of A. simplex s. s. The presence of two proteins, folliculin (FLCN) and oxoglutarate dehydrogenase (OGDH), was confirmed by Western blot, and their tertiary structure was predicted and compared with other species. In addition, host-pathogen interactions were identified, and potential new allergens were predicted. The result of this manuscript shows the largest number of protein identifications to our knowledge using proteomics tools for different tissues of L4 larvae of A. simplex s. s. The identified tissue-specific proteins could serve as targets for new drugs against anisakiasis.

Shotgun Proteomics for L3 and L4 Anisakis simplex Development Stages.

Anisakis simplex s.s. is a parasitic nematode that causes anisakiasis in humans. L3 stage larvae, which are present in many fish species and cephalopods all over the globe, might be consumed and develop occasionally into the L4 stage but cannot reproduce. Anisakiasis is an emerging health problem and economic concern. In recent years, proteomic methods have gained greater acceptance among scientists involved in parasitology and food science. According to that, here, we present tandem mass tag (TMT)-based shotgun proteomics to define differences in proteomic composition between L3 and L4 development stages of A. simplex s.s.

Intrinsic properties of anisakid nematode larvae as a potential tool for the detection in fish products.

Anisakid nematode larvae (NL) in fish products comprise a risk to human health and, if visible, lead to the rejection of these products by consumers. Therefore, great efforts are being made for the identification of these anisakid larvae to estimate the potential consumer health risk as well as to develop effective detection methods in order to prevent the introduction of heavily infected fish products into the market. The tasks of national reference laboratories include the improvement of detection methods and to promote their further development. As a prerequisite for improved detection, it is important to understand the structural properties of anisakid NL and compounds produced during host-parasite interactions. This review provides an overview of the intrinsic properties of anisakid NL and reports the latest detection methods in published literature. First, in order to define the potentially interesting intrinsic properties of anisakid nematodes for their detection, anatomy and compounds involved in host-parasite interactions are summarised. These can be used for various detection approaches, such as in the medical field or for allergen detection in fish products. In addition, fluorescence characteristics and their use as both established and promising candidates for detection methods, especially in the field of optical sensing technologies, are presented. Finally, different detection and identification methods applied by the fish processing industries and by control laboratories are listed. The review intends to highlight trends and provide suggestions for the development of improved detection and identification methods of anisakid NL in fish products.

Proteomic and Bioinformatic Investigations of Heat-Treated Anisakis simplex Third-Stage Larvae.

Anisakis simplex third-stage larvae are the main source of hidden allergens in marine fish products. Some Anisakis allergens are thermostable and, even highly processed, could cause hypersensitivity reactions. However, Anisakis proteome has not been studied under autoclaving conditions of 121 °C for 60 min, which is an important process in the food industry. The aim of the study was the identification and characterization of allergens, potential allergens, and other proteins of heat-treated A. simplex larvae. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify 470 proteins, including allergens-Ani s 1, Ani s 2, Ani s 3, Ani s 4, Ani s 5-and 13 potential allergens that were mainly homologs of Anisakis spp., Ascaris spp., and Acari allergens. Ani s 2, Ani s 3, Ani s 5, and three possible allergens were found among the top 25 most abundant proteins. The computational analysis allowed us to detect allergen epitopes, assign protein families, and domains as well as to annotate the localization of proteins. The predicted 3D models of proteins revealed similarities between potential allergens and homologous allergens. Despite the partial degradation of heated A. simplex antigens, their immunoreactivity with anti-A. simplex IgG antibodies was confirmed using a Western blot. In conclusion, identified epitopes of allergenic peptides highlighted that the occurrence of Anisakis proteins in thermally processed fish products could be a potential allergic hazard. Further studies are necessary to confirm the IgE immunoreactivity and thermostability of identified proteins.

Anisakis simplex products impair intestinal epithelial barrier function and occludin and zonula occludens-1 localisation in differentiated Caco-2 cells.

BACKGROUND: Anisakis spp. are nematode parasites found in a wide range of marine organisms. Human beings may accidentally become infected, showing the symptoms of anisakiasis and allergic responses. There has been evidence of increased intestinal permeability in A. simplex-sensitized subjects and that specific IgE titres increase in some allergic patients when fishery products are re-introduced into their diet. The aims of this work were to study the effect of A. simplex crude extract on the intestinal integrity and permeability by using Caco-2 cell monolayer. To analyse the capacity of Ani s 4 allergen to cross the epithelial barrier. METHODOLOGY/PRINCIPAL FINDINGS: Cellular bioenergetics, transepithelial electrical resistance, viability, permeability, reactive oxygen species generation and immunofluorescent staining of tight junction proteins were analysed. A. simplex crude extract compromises the Caco-2 cell monolayer integrity in a dose-dependent manner. This effect is detected at 1 hour of culture and integrity is recovered after 24 hours of culture. The epithelial barrier disruption is accompanied by an increase in paracellular permeability and reactive oxygen species production and by a delocalization of occludin and zonula occludens-1. Finally, Ani s 4, a thermostable and resistant to digestion allergen with cystatin activity, is able to cross the epithelial barrier in Caco-2 monolayer and reach a cumulative mean percentage of 22.7% of total concentration in the basolateral side after 24 hours of culture. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that A. simplex induces an early and reversible alteration of integrity and permeability of Caco-2 cell monolayer and that an underlying mechanism of this effect would involve the oxidative stress and disruption of epithelial tight junctions. Additionally, it has been shown that Ani s 4 allergen is able to cross the epithelial barrier. These findings could explain the increased intestinal permeability observed in Anisakis-sensitized patients, the changes over time in IgE sensitization to A. simplex allergens, and the specific IgE persistence in Anisakis allergy.

Anisakis spp. as etiological agent of zoonotic disease and allergy in European region ­ an overview

Nematodes of the genus Anisakis are worldwide distributed marine species parasitized many fish and cephalopod species as larvae and sea mammals as adult form. Anisakiosis as food-borne disease is an important public health problem worldwide. Human become infected by eating raw or undercooked fish or squids. Well documented are gastrointestinal response to infection but increasingly allergic symptoms were observed also after eating well cooked fish. This is because some of allergens of Anisakis are thermostable and resistant to pepsin treatment. Due to a significant increase in human mobility and global transport of fresh products like fish on ice, food-borne diseases require educational campaigns that pay attention to threats in various parts of the world.

Recombinant vs native Anisakis haemoglobin (Ani s 13): Its appraisal as a new gold standard for the diagnosis of allergy.

Recombinant allergens are currently the best option for serodiagnosis of human anisakiasis in terms of sensitivity and specificity. However, previous reports showed high rates of anisakiasis patients who were negative to Ani s 7 and especially to Ani s 1. Recently, Anisakis haemoglobin was described as a major allergen (Ani s 13). Although Ani s 13 belongs to a conserved protein family, it seems not to be a cross-reacting antigen because of the absence of IgE recognition against Ascaris haemoglobin in Anisakis patients. The aim of this study is to develop a more sensitive and specific diagnosis tool for Anisakis based on the recently discovered allergen Ani s 13. We obtained and purified recombinant Anisakis haemoglobin (rAni s 13) and the native form (nAni s 13). The recognition of both recombinant and native haemoglobins by anti-haemoglobin IgE from patients' sera was assessed by indirect ELISA and immunoblotting using 43 Anisakis sensitised patients and 44 non-Anisakis sensitised patients. Native Ani s 13 was also treated with periodate to study if oxidation of glycans destroys antibody binding. Furthermore, it was structurally characterised by negative staining electron microscopy and analytical ultracentrifugation. Recombinant Ani s 13 was only recognised by four patients with gastro-allergic anisakiasis (GAA) and immunoblotting analyses showed no bands. However, nAni s 13 was detected by 72.1% of Anisakis sensitised patients measured by indirect ELISA. Particularly, 18 (90%) out of 20 GAA patients were positive. Tetramers and octamers were the most abundant homomers of nAni s 13 but octamers had higher content of bound heme. None of the non-Anisakis sensitised patients were positive. Combined use of purified native form of Ani s 13 with current gold standards would improve the sensitivity and specificity for diagnosing anisakiasis.

Anisakis pegreffii (Nematoda: Anisakidae) products modulate oxidative stress and apoptosis-related biomarkers in human cell lines.

BACKGROUND: In countries with elevated prevalence of zoonotic anisakiasis and high awareness of this parasitosis, a considerable number of cases that associate Anisakis sp. (Nematoda, Anisakidae) and different bowel carcinomas have been described. Although neoplasia and embedded larvae were observed sharing the common site affected by chronic inflammation, no association between the nematode and malignancy were directly proved. Similarly, no data are available about the effect of secretory and excretory products of infecting larvae at the host's cellular level, except in respect to allergenic interaction. METHODS: To test the mechanisms by which human non-immune cells respond to the larvae, we exposed the fibroblast cell line HS-68 to two Anisakis products (ES, excretory/secretory products; and EC, crude extract) and evaluated molecular markers related to stress response, oxidative stress, inflammation and apoptosis, such as p53, HSP70, TNF-α, c-jun and c-fos, employing cell viability assay, spectrophotometry, immunoblotting and qPCR. RESULTS: Both Anisakis products led to increased production of reactive oxygen species (ROS), especially in EC-treated cells. While the ES treatment induces activation of kinases suggesting inflammation and cell proliferation (or inhibition of apoptosis), in EC-treated cells, other signaling pathways indicate the inhibition of apoptosis, marked by strong upregulation of Hsp70. Elevated induction of p53 in fibroblasts treated by both Anisakis products, suggests a significantly negative effect on the host DNA. CONCLUSIONS: This study shows that in vitro cell response to Anisakis products can result in at least two different scenarios, which in both cases lead to inflammation and DNA damage. Although these preliminary results are far from proving a relationship between the parasite and cancer, they are the first to support the existence of conditions where such changes are feasible.

Development of liquid chromatography-tandem mass spectrometry methods for the quantitation of Anisakis simplex proteins in fish.

The parasite Anisakis simplex is present in many marine fish species that are directly used as food or in processed products. The anisakid larvae infect mostly the gut and inner organs of fish but have also been shown to penetrate into the fillet. Thus, human health can be at risk, either by contracting anisakiasis through the consumption of raw or under-cooked fish, or by sensitisation to anisakid proteins in processed food. A number of different methods for the detection of A. simplex in fish and products thereof have been developed, including visual techniques and PCR for larvae tracing, and immunological assays for the determination of proteins. The recent identification of a number of anisakid proteins by mass spectrometry-based proteomics has laid the groundwork for the development of two quantitative liquid chromatography-tandem mass spectrometry methods for the detection of A. simplex in fish that are described in the present study. Both, the label-free semi-quantitative nLC-nESI-Orbitrap-MS/MS (MS1) and the heavy peptide-applying absolute-quantitative (AQUA) LC-TripleQ-MS/MS (MS2) use unique reporter peptides derived from anisakid hemoglobin and SXP/RAL-2 protein as analytes. Standard curves in buffer and in salmon matrix showed limits of detection at 1µg/mL and 10µg/mL for MS1 and 0.1µg/mL and 2µg/mL for MS2. Preliminary method validation included the assessment of sensitivity, repeatability, reproducibility, and applicability to incurred and naturally-contaminated samples for both assays. By further optimization and full validation in accordance with current recommendations the LC-MS/MS methods could be standardized and used generally as confirmative techniques for the detection of A. simplex protein in fish.

Antigenicity of Anisakis simplex s.s. L3 in parasitized fish after heating conditions used in the canning processing.

BACKGROUND: Some technological and food processing treatments applied to parasitized fish kill the Anisakis larvae and prevent infection and sensitization of consumers. However, residual allergenic activity of parasite allergens has been shown. The aim here was to study the effect of different heat treatments used in the fish canning processing industry on the antigen recognition of Anisakis L3. Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) were experimentally infected with live L3 Anisakis. After 48 h at 5 ± 1 °C, brine was added to the muscle, which was then canned raw (live larvae) or heated (90 °C, 30 min) (dead larvae) and treated at 113 °C for 60 min or at 115 °C for 90 min. Anisakis antigens and Ani s 4 were detected with anti-crude extract and anti-Ani s 4 antisera respectively. RESULTS: Ani s 4 decreased in all lots, but the muscle retained part of the allergenicity irrespective of the canning method, as observed by immunohistochemistry. Dot blot analysis showed a high loss of Ani s 4 recognition after canning, but residual antigenicity was present. CONCLUSION: The results indicate that heat treatment for sterilization under the conditions studied produces a decrease in Ani s 4 and suggest a potential exposure risk for Anisakis-sensitized patients.

Relationships between IgE/IgG4 epitopes, structure and function in Anisakis simplex Ani s 5, a member of the SXP/RAL-2 protein family.

BACKGROUND: Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies. The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown. The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG4 binding regions. METHODOLOGY/PRINCIPAL FINDINGS: The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG4 epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients. The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG4. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera. CONCLUSIONS/SIGNIFICANCE: This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG4 binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.

Molecular characterization and comparison of four Anisakis allergens between Anisakis simplex sensu stricto and Anisakis pegreffii from Japan.

It remains unclear whether allergens are the same among the sibling species of Anisakis simplex sensu lato. This study was carried out to compare the amino acid sequences of three major (Ani s 1, 2 and 12) and one minor (Ani s 9) Anisakis allergens between A. simplex s.s. and Anisakis pegreffii. We found 2 (out of 163), 1 (out of 869), and 29 (out of 266) amino acid variable sites for Ani s 1, 2, and 12, respectively. However, as both intra- and inter-species variations were present at the same amino acid positions, no amino acid variations clearly distinguished the two sibling species. IgE-binding epitopes (Ani s 1) and a binding motif of human leukocyte antigen (Ani s 2 and 9) demonstrated by previous studies were conserved. The similarities of the amino acid sequences of the allergens indicate possible similar allergy-associated health risks in humans infected with or accidentally ingesting either Anisakis species.

[Role of helminth antigens in the abnormal mitosis of bone marrow cells in laboratory animals].

The intraabdominal administration of somatic extracts of the cestodes Hydatigera taeniaformis Batsch 1786, Lamarck, 1816 and Diphyllobothrium latum Linnaeus, 1758 and the nematodes Anisakis simplex larva Rudolphi 1809, Toxocara canis Railliet et Henry, 1912 in albino mice proved that these helminths had a karyopathic effect on the bone marrow cells of the animals. The antigenic composition of these extracts was investigated using the agar gel immunodiffusion test. The antigenic composition of the parasites was ascertained to affect their karyopathic properties. The amount of antigens and their foreignness caused a marked karyopathic effect on the bone marrow cells of laboratory animals during intraabdominal administration.

MISS-Prot: web server for self/non-self discrimination of protein residue networks in parasites; theory and experiments in Fasciola peptides and Anisakis allergens.

Infections caused by human parasites (HPs) affect the poorest 500 million people worldwide but chemotherapy has become expensive, toxic, and/or less effective due to drug resistance. On the other hand, many 3D structures in Protein Data Bank (PDB) remain without function annotation. We need theoretical models to quickly predict biologically relevant Parasite Self Proteins (PSP), which are expressed differentially in a given parasite and are dissimilar to proteins expressed in other parasites and have a high probability to become new vaccines (unique sequence) or drug targets (unique 3D structure). We present herein a model for PSPs in eight different HPs (Ascaris, Entamoeba, Fasciola, Giardia, Leishmania, Plasmodium, Trypanosoma, and Toxoplasma) with 90% accuracy for 15 341 training and validation cases. The model combines protein residue networks, Markov Chain Models (MCM) and Artificial Neural Networks (ANN). The input parameters are the spectral moments of the Markov transition matrix for electrostatic interactions associated with the protein residue complex network calculated with the MARCH-INSIDE software. We implemented this model in a new web-server called MISS-Prot (MARCH-INSIDE Scores for Self-Proteins). MISS-Prot was programmed using PHP/HTML/Python and MARCH-INSIDE routines and is freely available at: . This server is easy to use by non-experts in Bioinformatics who can carry out automatic online upload and prediction with 3D structures deposited at PDB (mode 1). We can also study outcomes of Peptide Mass Fingerprinting (PMFs) and MS/MS for query proteins with unknown 3D structures (mode 2). We illustrated the use of MISS-Prot in experimental and/or theoretical studies of peptides from Fasciola hepatica cathepsin proteases or present on 10 Anisakis simplex allergens (Ani s 1 to Ani s 10). In doing so, we combined electrophoresis (1DE), MALDI-TOF Mass Spectroscopy, and MASCOT to seek sequences, Molecular Mechanics + Molecular Dynamics (MM/MD) to generate 3D structures and MISS-Prot to predict PSP scores. MISS-Prot also allows the prediction of PSP proteins in 16 additional species including parasite hosts, fungi pathogens, disease transmission vectors, and biotechnologically relevant organisms.

Amelioration of intestinal colitis by macrophage migration inhibitory factor isolated from intestinal parasites through toll-like receptor 2.

In a previous study, we cloned type II MIFs (As-MIF) from Anisakis simplex 3rd stage larva and expressed a recombinant protein that suppressed allergic airway inflammation via regulatory T (CD4(+) CD25(+) Foxp3(+) T; T(reg) )-cell recruitment. In this study, in an effort to evaluate the function of rAs-MIF on another immune disease, we induced intestinal inflammation in mice using dextran sodium sulphate (DSS) with or without the application of rAs-MIF treatment to the mice. As a consequence, weight losses were recovered, and the value of disease activity index (DAI) was reduced by rAs-MIF treatment during the experimental period. The levels of TGF-ß and IL-10 in the spleens and mesenteric lymph nodes (MLN) from the rAs-MIF-treated mice were higher, but the levels of IFN-γ, IL-6 and IL-13 were lower than those of the mice treated with DSS but not with rAs-MIF. Additionally, the T(reg) cells observed were greatly increased in the MLNs of the rAs-MIF-treated mice than those of mice not treated with rAs-MIF. The results of our in vitro experiments showed that the elevated IL-10 production induced by rAs-MIF was generated via toll-like receptor 2. In conclusion, rAs-MIF appears to ameliorate DSS-induced colitis and may prove useful as a therapeutic agent for the treatment of intestinal inflammatory disease.

Evaluation of a real-time polymerase chain reaction (PCR) assay for detection of anisakis simplex parasite as a food-borne allergen source in seafood products.

Anisakis simplex has been recognized as an important cause of disease in humans and as a food-borne allergen source. Actually, this food-borne parasite was recently identified as an emerging food safety risk. An A. simplex -specific primer-probe system based on a real-time polymerase chain reaction (PCR) detection assay has been successfully optimized and validated with seafood samples. In addition, a DNA extraction procedure has been optimized to detect the presence of the nematode in food samples. The assay is a very reliable, specific, and sensitive methodology to detect the presence of traces of this parasite in seafood products, including highly processed samples. As a result, 13 sequences of cytochrome c oxidase II gene were obtained and scrutinized to calculate intra- and interspecific variabilities of 0 and 35-67%, respectively. Finally, an efficiency of 2.07 +/- 0.14 of the assay was calculated, and a limit of detection of 40 ppm parasite in 25 g of sample was also optimized. Actually, the presence of this parasite in several seafood products has been demonstrated, enforcing the necessity of a design for a good manufacturing practice protocol for the processing industry to minimize the presence of this parasite as a food-borne allergen source in seafood products.

Identification of lipids in the cuticle of the parasitic nematode Anisakis simplex and the somatic tissues of the Atlantic cod Gadus morhua.

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex, hexadecanoic acid in the dichloromethane extract of A. simplex, and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode. The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy:(1), CoCoPg and Bu0:0B:(6). The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex. Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite's inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids--ceramides and sphingoid bases. The signals due to N-octanoyl-D-erythro-octasphinganine (m/z 288.3) and N-tetranoyl-D-erythro-tetradecasphinganine (m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.

Glycogen catabolism enzymes and protein fractions in the third and fourth larval stages of Anisakis simplex.

Extracts of Anisakis simplex third (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of alpha-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS-PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: alpha 1, alpha 2 and alpha 3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The alpha-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations of A. simplex larvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.

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.

Anisakis simplex: the activity of larval products on the complement system.

The effects of the larval products (crude extract and excretory-secretory) of Anisakis simplex on the classical and alternative pathways of human complement system were investigated. This could constitute a mechanism to evade host defences, similarly than in other parasitic diseases. The larval products showed a stronger effect on the classical pathway than on the alternative pathway. The most pronounced modulating effects were found for the excretory-secretory products. Chelation of bivalent cations (Ca(2+) or Mg(2+)) by these larval products may be responsible for their mode of action on the alternative pathway, whereas the chelation is not likely to be particularly involved in the anticomplementary activity found on the classical pathway. Detailed studies revealed that the larval products of A. simplex act at the level of the C3 and other complement components. Heating the crude parasite extract led to a notable loss of haemolysis inhibition activity, and the addition of PMSF (a serine protease inhibitor) also cause variation in the activity of the crude extract.