M. Rodero

The Anisakis allergy debate: does an evolutionary approach help?

Allergic phenomena share common pathways with the immune response against helminth parasites. The definitions regarding allergens and their related concepts have their roots in the area of allergy research. The experience with the fish parasite Anisakis simplex-associated allergic features still nurtures an open debate on the necessity of larvae being alive to induce allergic reactions such as urticaria or anaphylaxis. Conceptual definitions of allergen, major allergen, as well as putatively crossreacting antibodies, as are used in food allergy, depend on the clinical relevance of specific IgE and deserve careful interpretation in the various forms of A. simplex-associated allergic features. Conversely, an evolutionary based interpretation of the presence of specific IgE depends on the viability of A. simplex.

Different serum cytokine levels in chronic vs. acute Anisakis simplex sensitization-associated urticaria.

The knowledge on immune mechanisms of chronic urticaria (CU) at the cytokine level is widely scarce. We compared pro‐ and anti‐inflammatory as well as Th1‐ and Th2‐associated serum cytokine levels in two phenotypes of CU: associated with (CU+) and without (CU−) sensitization against Anisakis simplex, a ubiquitous fish parasite, that has been associated with acute urticaria in gastro‐allergic anisakiasis (GAA) and with CU+. Thirteen CU+ and 19 CU− patients were compared with 13 GAA patients and 15 control subjects for cytokines, measured by cytometric bead array. Urticaria activity score was positively correlated with IL‐6 in CU−. Serum levels of IL‐10 were lower in CU+ and CU− with respect to the control group. Median IFN‐γ was lower in all urticaria groups. Patients with previous parasitism by A. simplex displayed higher TGF‐β levels than subjects without previous parasitism. The main finding was lower levels of IL‐17 in CU+ with respect to GAA or controls, with a further tendency to even lower levels in CU−. Different urticaria phenotypes are associated with distinct serum cytokine levels.

Follow-up of antibody avidity in BALB/c mice infected with Toxocara canis.

In human Toxocara canis infection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through a T. canis infection in BALB/c mice. Infection with T. canis embryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies against T. canis (IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40-80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDa T. canis proteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.

Seroprevalence of anti- Kudoa sp. (Myxosporea: Multivalvulida) antibodies in a Spanish population

A majority of Kudoa species infect the somatic muscle of fish establishing cysts. Because there is no effective method to detect infected fish without destroying them, these parasitised fish reach the consumer. The elevated humoral responses detected previously by us in BALB/c mice immunised with Kudoa sp. pseudocyst extracts and the high IgG1 and IgE levels induced by the oral administration of Kudoa pseudocysts to BALB/c mice showed the possible immunopathological effects in man from the ingestion of Kudoa-infected fish. In this work, we investigated the seroprevalence of anti-Kudoa sp. antibodies in a Spanish healthy population and the possible association between the manifestation of allergic reactions after fish consumption and the humoral responses to Kudoa sp. antigens. Specific anti-Kudoa sp. antibody levels in sera of patients diagnosed with several digestive pathologies were also determined, studying their possible association with the alteration of analytic parameters in these patients.

Cellular immune responses in mice immunized with Anisakis simplex larval antigens

Cellular immune responses to Anisakis simplex L3 antigens were investigated in BALB/c mice injected subcutaneously with a homologous crude extract (CE). Popliteal lymph nodes (PLN) were found to be increased in size and weight after A. simplex CE footpad injection. The effects of A. simplex CE in vitro proliferation were assayed with non-fractionated PLN cells or nylon-wool purified T cells derived from pooled lymph node cells of mice subcutaneously injected with CE. Spleen cells from immunized animals (antigen alone, or larva alone, or antigen plus larva) were studied by flow cytometry. The immunization induced a high proportion of CD4+ and TCRαβ+ T cells. The number of B cells (CD45+ and TCRαβ–) in pre-immunized and infected mice was lower than that observed in animals subjected to infection only. The number of CD4+ T cells increased in the infected and in the pre-immunized and infected mice. In the latter, a decrease of CD8a+ T cells was noted. The greatest increase in CD8a+ and TCRαβ– T cells was found in mice that had been subjected to infection only. Histological analysis showed that the most prominent lesions were gastric and intestinal in animals infected orally with one larva.

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

BACKGROUND Anisakis 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. METHODS 16 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. RESULTS There 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. CONCLUSIONS Our 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.

Humoral immune responses induced by Kudoa sp. (Myxosporea: Multivalvulida) antigens in BALB/c mice

The majority of Kudoa species infect the somatic muscle of fish establishing cysts. As there is no effective method to detect infected fish without destroying them these parasited fish reach the consumer. This work was developed to determine whether this parasite contains antigenic compounds capable of provoking an immune response in laboratory animals, in order to consider the possible immunopathological effects in man by the ingestion of Kudoa infected fish. BALB/c mice were injected by the subcutaneous route with the following extracts suspended in aluminium hydroxide: group 1 (black Kudoa sp. pseudocyst extract), group 2 (white Kudoa sp. pseudocyst extract), and group 3 (non-infected hake meat extract). Specific antibody levels were measured by ELISA against homologous and heterologous antigens. The highest responses were obtained from the black Kudoa sp. pseudocyst extract (group 1). The low optic density levels detected in group 3 proved that the results obtained in groups 1 and 2 were a consequence of the parasitic extract injection. The IgG1 was the predominant subclass. IgE detected in groups 1 and 2 showed the possible allergenic nature of some of the components of the parasitic extract. High IgA levels and medium IgG2a and IgG3 levels were obtained in groups 1 and 2. Low IgG2b responses were shown. No cross-reactions between Kudoa sp. pseudocyst extracts and the non-infected hake meat extract were observed.

New insights into the allergenicity of tropomyosin: a bioinformatics approach

The invertebrate panallergen tropomyosin is a protein with an extremely simple folding. This makes it a perfect target for investigating structural differences between invertebrate and vertebrate tropomyosins, which are not considered allergenic. Phylogenetic and sequence analyses were conducted in order to explore the differences in primary structure between several tropomyosins and to promote an experimental development in the field of food allergy, based on the study of tropomyosin. The phylogenetic analyses showed that tropomyosin is a useful evolutionary marker. The phylogenetic trees obtained with tropomyosin were not always phylogenetically correct, but they might be useful for allergen avoidance by tropomyosin allergic individuals. Sequence analyses revealed that the probability of alpha helix folding in invertebrate tropomyosins was lower than in all the studied vertebrate ones, except for the Atlantic bluefin tuna Thunnus thynnus tropomyosin. This suggested that the lack of alpha helix folding may be involved in the immunogenicity of tropomyosins. More specifically, the regions adjacent to the positions 133–135 and 201 of the invertebrate tropomyosins, presented lower probability of alpha helix folding than those of vertebrates and are candidates to be responsible for their allergenicity.

Mast cell inhibition by ketotifen reduces splanchnic inflammatory response in a portal hypertension model in rats

Experimental early prehepatic portal hypertension induces an inflammatory exudative response, including an increased infiltration of the intestinal mucosa and the mesenteric lymph nodes by mast cells and a dilation and tortuosity of the branches of the superior mesenteric vein. The aim of this study is to verify that the prophylactic administration of Ketotifen, a stabilizing drug for mast cells, reduces the consequence of splanchnic inflammatory response in prehepatic portal hypertension. Male Wistar rats were used: Sham-operated and with Triple Partial Portal Vein Ligation, which were subcutaneously administered poly(lactide-co-glycolide) acid microspheres with vehicle 24h before the intervention and SO and rats with Triple Partial Portal Vein Ligation, which were administered Ketotifen-loaded microspheres. Around 48h after surgery, the portal pressure was measured; the levels of chymase (Rat Mast Cell Protease-II) were assayed in the superior mesenteric lymph complex and granulated and degranulated mast cells in the ileum and cecum were quantified. Prophylactic administration of Ketotifen reduced portal pressure, the incidence of dilation and tortuosity of the superior mesenteric vein branches, the amount of Rat Mast Cell Protease-II in the superior mesenteric lymph complex and the number of activated mast cells in the cecum of rats with portal hypertension. In summary, the administration of Ketotifen reduces early splanchnic inflammatory reaction in the rat with prehepatic portal hypertension.

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.