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SummaryAni s 7 is currently the most important excretory/secretory (ES) Anisakis simplex allergen, as it is the only one recognized by 100% of infected patients. The allergenicity of this molecule is due mainly to the presence of a novel CX17-25CX9-22CX8CX6 tandem repeat motif not seen in any previously reported protein. In this study we used this allergen as a model to investigate how ES allergens are recognized during Anisakis infections, and the usefulness of a recombinant fragment of Ani s 7 allergen (t-Ani s 7) as a marker of true Anisakis infections. The possible antigenic relationship between native Ani s 7 (nAni s 7) from Anisakis and Pseudoterranova decipens antigens was also investigated. Our results demonstrate that nAni s 7 is secreted and recognized by the immune system of rats only when the larvae are alive (i.e. during the acute phase of infection), and that this molecule is not present in, or is antigenically different from, Pseudoterranova allergens. The t-Ani s 7 polypeptide is a useful target for differentiating immunoglobulin E antibodies induced by true Anisakis infections from those induced by other antigens that may crossreact with Anisakis allergens, including P. decipiens. The results also support the hypothesis that the Ani s 7 major allergen does not participate in maintaining the antigenic stimulus during chronic infections.

Diagnosis in gastro-allergic anisakiasis (GAA) is straightforward, when clinical history is combined with further allergological evaluation of specific IgE by means of skin prick test and serum specific IgE. In Anisakis simplex sensitisation associated chronic urticaria (CU+), clinical evaluation of possible previous parasitism is difficult, and positive serum specific IgE could be due to cross-reactivity or other unknown factors. In this study, we evaluated the association between IgE seropositivity to the recombinant allergens Ani s 1 and Ani s 7 and several A. simplex-associated allergic disorders. Twenty-eight patients with GAA and 40 patients with CU+ were studied and their IgE responses were compared with a control group composed of patients with chronic urticaria not sensitized to A. simplex (CU-) according to the skin prick test, as well as a group of 15 healthy subjects not referring urticaria or currently A. simplex associated symptoms. 82.1% of GAA patients and 42.5% of CU+ patients were positive for Ani s 1 (P < 0.001), while the Ani s 7 allergen was recognized by 92.9 and 92.5% of sera from patients with GAA and CU+, respectively. The combined positivity obtained for both allergens reached 100% in GAA, and 95% in CU+. IgE determinations to Ani s 1 and Ani s 7 allergens are useful to diagnose the Anisakis infections and to differentiate among several A. simplex-associated allergic disorders. The IgE responses to Ani s 1 are mainly associated with GAA, while this molecule cannot be considered a major allergen in CU+ patients.

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.

BackgroundThe aim of this study was to investigate the relationship between prior Anisakis infections and upper gastrointestinal bleeding (UGIB), and its interaction with non-steroidal anti-inflammatory drug (NSAID) intake.Methods/Principal FindingsWe conducted a hospital-based case-control study covering 215 UGIB cases and 650 controls. Odds ratios (ORs) with their confidence intervals (95% CIs) were calculated, as well as the ratio of the combined effects to the sum of the separate effects of Anisakis allergic sensitization and NSAIDs intake. Prior Anisakis infections were revealed by the presence of anti-Anisakis IgE antibodies specific to the recombinant Ani s 1 and Ani s 7 allergens used as the targets in indirect ELISA. Prior Anisakis infections (OR 1.74 [95% CI: 1.10 to 2.75]) and the intake of NSAIDs (OR 6.63 [95% CI: 4.21 to 10.43]) increased the risk of bleeding. Simultaneous NSAIDs intake and Anisakis allergic sensitization increased the risk of UGIB 14-fold (OR = 14.46 [95% CI: 6.08 to 34.40]). This interaction was additive, with a synergistic index of 3.01 (95% CI: 1.18–7.71).ConclusionsPrior Anisakis infection is an independent risk factor for UGIB, and the joint effect with NSAIDs is 3 times higher than the sum of their individual effects.