Specific IgG4: Possible Role in the Pathogenesis and a New Marker in the Diagnosis of Anisakis‐associated Allergic Disease

IgG₄ and IgE are immunoglobulin isotypes which are mediated by the same Th2‐mediated mechanism. The postulated pathogenic and protective function of IgE or IgG_4, respectively, in allergic disease is opposite in parasitic infection. The possible role of IgG₄ against recombinant major allergens on the appearance of different forms of Anisakis simplex‐associated allergic disease was studied. Gastro‐allergic anisakiasis (GAA) and Anisakis‐sensitization‐associated chronic urticaria (CU+) were compared for specific IgE, IgG₄ and the respective recognition of Ani s 1 and Ani s 7. Gastro‐allergic anisakiasis showed higher IgE and IgG₄ levels against crude extract and both recombinant allergens. Whereas IgE recognition of Ani s 7 did not differ and supports both clinical entities to be associated with previous acute parasitism, the IgE recognition rates of Ani s 1 and IgG₄ recognition of both Ani s 1 and Ani s 7 were higher in GAA. IgG₄ levels were associated with IgE, but also with age, time to last parasitic episode and frequency of fish intake. Logistic regression analysis showed that the presence of specific IgG₄ against Ani s 7 was an independent marker associated with GAA. In the diagnosis of Anisakis‐associated allergic disease phenotypes (GAA versus CU+), measurement of specific IgG₄ against recombinant allergens could be useful. Further, evaluation of specific IgE and IgG₄ facilitates more insight into the protective versus pathogenic potential of IgE and IgG_4.     

Prevalence of and risk factors for IgE sensitization to Anisakis simplex in a Spanish population

BACKGROUND: The number of allergic reactions to A. simplex reported in Spain has increased dramatically in the last decade. Nevertheless, there have been no studies of the prevalence of and possible risk factors for IgE sensitization to this parasite, possibly because suitably specific diagnostic methods have only recently become available. The objective was to investigate the prevalence of and risk factors for IgE sensitization to A. simplex in Galicia, a region of northwestern Spain with a population of about 3 million and high average fish consumption (78.5 g/person per day). METHODS: The study was performed with a random sample of 2801 healthy blood donors distributed in 53 geographic areas, proportional to the density of donors. IgE sensitization to A. simplex was tested by a capture ELISA method that has proved to be the most specific method currently available. RESULTS: The results showed a total of only 12 positive subjects, of whom five also showed IgG1 sensitization. All positive subjects and 101 randomly selected seronegative subjects were then included in a case-control study of risk factors for sensitization to A. simplex, based on a telephone interview about fish consumption (especially raw and undercooked fish). All seropositive subjects (but only 25% of seronegative subjects) reported consumption of undercooked fish or homemade raw-fish products. CONCLUSIONS: Our results strongly suggest that sensitization to A. simplex is caused only by live larvae, and not by allergens contained in fish tissues, and that ingestion of homemade boquerones (anchovies [Engraulis encrasicholus] in vinegar), and to a much lesser extent of undercooked fish, are the main risk factors for IgE sensitization to Anisakis in this region.     

Different Fish-Eating Habits and Cytokine Production in Chronic Urticaria with and without Sensitization against the Fish-Parasite Anisakis simplex

BackgroundAnisakis simplex sensitization has been associated with acute, but also with chronic urticaria. The objective of this study is to characterize chronic urticaria with (CU +) and without sensitization (CU-) against the ubiquitous fish parasite A. simplex in a transversal and longitudinal evaluation.Methods16 CU + and 22 CU- patients were included and assessed for Urticaria activity score (UAS), fish-eating habits by standardized questionnaire and cytokine production (assessed by flow cytometric bead-based array) of peripheral blood mononuclear cells after stimulation with A. simplex extract or Concanavalin A (Con A). Patients were randomly put on a fish-free diet for three months and UAS, as well as cytokine production were again assessed. A difference of ≥ 1 in UAS was defined as improvement.ResultsThere was no difference in UAS in both groups. Anisakis induced IL-2, IL-4 and IFN-γ production was higher in CU +. Con A induced IL-6 and IL-10 production was higher in CU +. CU + was associated with higher total fish intake, whereas CU- was associated with oily fish intake. The correlation of UAS was positive with oily fish, but negative with total fish intake.There was a better UAS-based prognosis in CU + without diet. Improvement was associated with higher Con A induced IL-10/IFN-γ as well as IL-10/IL-6 ratios. Further, previous higher oily fish intake was associated with improvement.ConclusionsOur data confirm the different clinical and immunological phenotype of CU +. Our results show a complex relationship between fish-eating habits, cytokine production and prognosis, which could have important consequences in dietary advice in patients with CU. When encountering A. simplex sensitization, patients should not be automatically put on a diet without fish in order to reduce contact with A. simplex products.     

Human immunoglobulin isotype profiles produced in response to antigens recognized by monoclonal antibodies specific to Anisakis simplex

BACKGROUND: Anisakis simplex is a medically important pathogen which not only causes anisakiasis but may provoke allergy reactions, ranging from mild urticaria to anaphylactic shock. OBJECTIVE: To investigate anti-Anisakis isotype profiles in anisakiasis and Anisakis allergy patients. METHODS: Capture ELISA techniques were used to investigate the isotype profiles of antibodies specific for two defined Anisakis simplex antigens, in serum from Japanese patients with confirmed anisakiasis and from Spanish patients with allergy to Anisakis. The antigens were 'UA2R antigens' (two proteins with MW of 48 and 67 kDa, recognized by our monoclonal antibody UA2) and 'UA3R antigens' (two proteins with MW of 139 and 154 kDa, recognized by our monoclonal antibody UA3). RESULTS: Considering IgG, the two most frequent isotypes in the response to the UA2R antigens were IgG1 and IgG2, with IgG4 detected in only one case; in response to the UA3R antigens, by contrast, the two most frequent isotypes were IgG1 and IgG4 (though IgG2 remained reasonably frequent). As regards potential utility for serodiagnosis, 95% of the Japanese anisakiasis patients and 84% of the allergy patients showed detectable IgG1 antibodies to the UA3R antigens. Furthermore, all allergy patients showed IgE antibodies to these antigens. CONCLUSION: Anisakis simplex contains antigens that induce responses which are differentially regulated. Because of their immunogenicity, immunodominance and allergenic nature, we consider that the 139/154-kDa antigens recognized by our MoAb UA3 are good candidates for use in tests for the diagnosis of anisakiasis and of the allergy caused by this parasite.     

Comparison of recombinant cathepsins L1, L2, and L5 as ELISA targets for serodiagnosis of bovine and ovine fascioliasis

Parasitology Research - Infections caused by Fasciola hepatica are of great importance in the veterinary field, as they cause important economic losses to livestock producers. Serodiagnostic...     

Using entropy of drug and protein graphs to predict FDA drug-target network: theoretic-experimental study of MAO inhibitors and hemoglobin peptides from Fasciola hepatica

There are many drugs described with very different affinity to a large number of receptors. In this work, we selected Drug-Target pairs (DTPs/nDTPs) of drugs with high affinity/non-affinity for different targets like proteins. Quantitative Structure-Activity Relationships (QSAR) models become a very useful tool in this context to substantially reduce time and resources consuming experiments. Unfortunately, most QSAR models predict activity against only one protein. To solve this problem, we developed here a multi-target QSAR (mt-QSAR) classifier using the MARCH-INSIDE technique to calculate structural parameters of drug and target plus one Artificial Neuronal Network (ANN) to seek the model. The best ANN model found is a Multi-Layer Perceptron (MLP) with profile MLP 32:32-15-1:1. This MLP classifies correctly 623 out of 678 DTPs (Sensitivity = 91.89%) and 2995 out of 3234 nDTPs (Specificity = 92.61%), corresponding to training Accuracy = 92.48%. The validation of the model was carried out by means of external predicting series. The model classifies correctly 313 out of 338 DTPs (Sensitivity = 92.60%) and 1411 out of 1534 nDTP (Specificity = 91.98%) in validation series, corresponding to total Accuracy = 92.09% for validation series (Predictability). This model favorably compares with other LDA and ANN models developed in this work and Machine Learning classifiers published before to address the same problem in different aspects. These mt-QSARs offer also a good opportunity to construct drug-protein Complex Networks (CNs) that can be used to explore large and complex drug-protein receptors databases. Finally, we illustrated two practical uses of this model with two different experiments. In experiment 1, we report prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of 10 rasagiline derivatives promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, SEC and 1DE sample preparation, MALDI-TOF MS and MS/MS analysis, MASCOT search, MM/MD 3D structure modeling, and QSAR prediction for different peptides of hemoglobin found in the proteome of the human parasite Fasciola hepatica; which is promising for anti-parasite drug targets discovery.     

Minor interspecies variations in the sequence of the gp53 TSL-1 antigen of Trichinella define species-specific immunodominant epitopes

Among the Trichinella TSL-1 antigens, whose antigenicity is generally due mainly to tyvelose-containing epitopes, gp53 is unusual in that its antigenicity is due mainly to protein epitopes. In the present study we mapped two of these epitopes, recognized by monoclonal antibodies (mAbs) that specifically recognize gp53 from all encysting Trichinella species (mAb US9), or gp53 from Trichinella spiralis alone (mAb US5). Based on previously published sequences of this glycoprotein [Mol. Biochem. Parasitol. 72 (1995) 253], in this study, we cloned the full gp53 cDNA from a new strain, Trichinella britovi (ISS 11; AN: ), and from another T. spiralis isolate (ISS 115; AN: ). The gp53 sequence comprised an ORF of 1239bp, coding for 412 amino acids, with 61 nucleotide differences (resulting in 38 residue changes) between the two species. Mapping of US5- and US9-recognized epitopes was undertaken through the construction and expression in the pGEX4T vector of truncated gp53 peptides, and by the construction of peptides derived from the antigenic regions. The epitope recognized by mAb US9 was a linear peptide of 8 residues, 33Met- 40Ser, located in the amino-terminal region, while the corresponding epitope recognized by mAb US5 was a 47-amino acid sequence containing two alpha-helix regions flanked by random coils, 290Thr- 336Lys. Molecular modeling of these peptides seems to indicate that recognition of the US9 epitope depends on the presence of two available hydroxyl groups provided by one methionine and one serine on T. spiralis gp53 (not present on Trichinella pseudospiralis gp53). Additionally, the stability of the US5 epitope seems to depend on correct folding of the 47-amino acid sequence (only present in T. spiralis). The relevance of these findings for understanding the antigenic recognition of Trichinella TSL-1 antigens, and for further studies to investigate possible function(s) of gp53 in Trichinella, is discussed.     

The Anisakis simplex Ani s 7 major allergen as an indicator of true Anisakis infections

Ani 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 CX_17–25CX_9–22CX₈CX₆ 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 cross‐react 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.