C. Cuéllar

Recidivous acute urticaria caused by Anisakis simplex

This study aimed to determine the cause of acute recidivous urticaria in patients who usually eat fish or other seafood. Twenty‐five patients were studied. The skin prick test with larval Anisakis simplex extract was performed; total and specific IgE against A. simplex was measured with the CAP System; specific antibodies to A. simplex were determined by ELISA; and immunorecognition patterns of the sera were studied by Western blot. Nineteen patients showed specific IgE to A. simplex, but specific IgE to Ascaris was demonstrated in only two patients. No patients reacted to Toxocara canis or Echinoccocus granulosus antigens with the same test. The skin prick test was positive in 16 patients, in two of them persisting for 48 h. Five patients showed neither skin reaction nor specific IgE to A. simplex. Sera showed specific immunoglobulin levels against A. simplex larval crude extract, by both ELISA and Western blot. Likewise, specific immunoglobulin levels against excretory‐secretory antigen were also measured by ELISA. Only one patient showed sensitization to fish. A. simplex was found to be the main cause of acute recidivous urticaria in patients who usually eat fish and are not sensitized to it.

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.

Gastro‐allergic anisakiasis as a consequence of simultaneous primary and secondary immune response

Gastro‐allergic anisakiasis has been reported as an entity in which an acute parasitism by Anisakis simplex is accompanied by an immunoglobulin (Ig)E‐mediated systemic allergic reaction. Serum samples were obtained from 24 patients within 24 h after the onset of symptoms (day 0) and after 1 month (day 30) and in 13 patients after 6 months. Total IgE was assessed by the Imx method. Specific IgE was assessed by CAP‐FEIA. Specific IgM, IgG, IgG4 and IgA antibodies were assessed by enzyme‐linked immunosorbent assay against crude extract (CE) and excretory–secretory products (ESP). IgE immunoblotting (IB) was directed against CE or ESP (day 0 and day 30). We found a rise of total IgE, specific IgE, number of bands in IgE‐IB, IgG and IgG4 between day 0 and day 30 with a fall to near basal levels after 6 months. IgM levels were highest at day 0, falling over the next 6 months and IgA levels remained almost unchanged. Correlation studies revealed a parallel stimulation of nearly all Ig isotypes, except IgM anti‐ESP, whose antibody levels correlated negatively with specific IgG levels. We found an extension of the IgE antibody repertoire in IB. We conclude that the allergic IgE‐mediated reaction in the course of gastro‐allergic anisakiasis involves a parallel secondary Th2 type memory response and a primary immunologic stimulation of both Th2 and Th1 lymphocyte subsets against previously unrecognized antigens.

The Hookworm Tissue Inhibitor of Metalloproteases (Ac-TMP-1) Modifies Dendritic Cell Function and Induces Generation of CD4 and CD8 Suppressor T Cells

Hookworm infection is a major cause of disease burden for humans. Recent studies have described hookworm-related immunosuppression in endemic populations and animal models. A Tissue Inhibitor of Metalloproteases (Ac-TMP-1) has been identified as one of the most abundant proteins released by the adult parasite. We investigated the effect of recombinant Ac-TMP-1 on dendritic cell (DC) and T cell function. Splenic T cells from C57BL/6 mice injected with Ac-TMP-1 showed reduced proliferation to restimulation with anti CD3 or bystander antigens such as OVA. Incubation of bone marrow-derived DCs with Ac-TMP-1 decreased MHC Class I and, especially, Class II expression but increased CD86 and IL-10 expression. Co-incubation of splenic T cells with DCs pulsed with Ac-TMP-1 induced their differentiation into CD4+ and, particularly, CD8+ CD25+Foxp3+ T cells that expressed IL-10. These cells were able to suppress proliferation of naïve and activated CD4+ T cells by TGF-Β-dependent (CD4+ suppressors) or independent (CD8+ suppressors) mechanisms. Priming of DCs with non-hookworm antigens, such as OVA, did not result in the generation of suppressor T cells. These data indicate that Ac-TMP-1 initiates the development of a regulatory response through modifications in DC function and generation of suppressor T cells. This is the first report to propose a role of suppressor CD8+ T cells in gastrointestinal helminthic infections.

Larval distribution of Toxocara canis in BALB/c mice at nine weeks and one year post-inoculation.

The migratory route of Toxocara canis in the mouse includes two phases: a hepato-pulmonary phase and a myotropic-neurotropic phase. A study was made of the migratory behaviour of larvae one year post-inoculation (p.i.) comparing the results to those obtained at the end of an initial experiment (day 63 p.i). The larval distribution per organ was already definitive at 9 weeks p.i. The total parasitic load showed a reduction when the experiment was prolonged for one year.

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 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 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.

Cross-reactions of sera from Toxascaris leonina and Ascaris suum infected mice with Toxocara canis, Toxascaris leonina and Ascaris suum antigens

The ELISA method using larval ES products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worms extract, was used to determine the possible cross-reactions in BALB/c and C57BL/10 mice inoculated with embryonated eggs or adult worms extract of T. leonina of T. leonina or A. suum in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. leonina against different heterologous Ag, no cross-reactions against T. canis ES and A. suum ES Ag were observed using a single dose. Similarly, in multiple doses no response against T. canis ES Ag was observed. In mice inoculated with adult worms extract of T. leonina cross-reactions with T. canis ES and A. suum ES Ag did not occur. Sera from BALB/c mice infected with embryonated eggs of A. suum, was tested using ES Ag from both A. suum and T. canis and no reactions were observed. This fact confirmed the resistance of this murine strain to A. suum embryonated eggs. When we used sera of susceptible C57BL/10 mice infected against different heterologous Ag, we observed no cross-reactions against T. canis ES Ag. In the case of both BALB/c and C57BL/10 and C57BL/10 mice immunized with a single dose of A. suum adult crude extract no cross-reactions were seen against ES T. canis Ag and with sera from C57BL/10 mice against ES T. leonina. These facts confirmed the specificity of the ES T. canis Ag.

Enzyme-linked immunosorbent assay, immunoblot analysis and RAST fluoroimmunoassay analysis of serum responses against crude larval antigens of Anisakis simplex in a Spanish random population

The results obtained in a study of seroprevalence by means of ELISA and immunoblot with crude larval extracts of Anisakis simplex using 1008 human sera from Spanish people showing no clinical suspicion of anisakidosis are given. For the evaluation of the results obtained by ELISA the Diagnostic Index (DI) was used, as the ratio between the optical density resulting from the test serum and the optical density of the negative control. Forty-seven sera showed DIs between 1.5 and 2, and 14 sera were greater than 2. After comparison of the immunoblot analysis with the immunorecognition pattern of a human anisakidosis reference serum, a diagnostic criterion could be established for those sera that, at a 1/100 dilution, showed a DI by ELISA greater than 1.5. Seven of 14 selected sera with DIs in ELISA higher than 1.3 showed anti-Anisakis specific IgE antibodies by RAST fluoroimmunoassay.

Comparative study of assays detecting circulating immune complexes and specific antibodies in patients infected with Toxocara canis

A sandwich ELISA method using previously described E/S antigen-specific monoclonal antibodies has been developed to detect circulating immune complexes in patients infected with Toxocara canis. This technique could be used for the study of the dynamics of the parasite-host relationship, as we believe the detection of immune complexes and/or soluble antigen to be an improvement over detection of antibodies only. In this parasitosis, antibodies may be present in residual levels for prolonged periods after active infection.

Seroprevalence of toxocariasis in children and adults in Madrid and Tenerife, Spain

A study on the seroprevalence of toxocariasis, using ELISA with Toxocara larval excretory-secretory antigens, was carried out on human populations in two regions of Spain. Sera from a population of 195 children from Madrid and 143 children from Santa Cruz de Tenerife (Canary Isles), showed a prevalence of 0% and 4.2% respectively. Sera from a population of 272 adults from Madrid and 803 adults from Santa Cruz de Tenerife showed a prevalence of 3.6% and 17.4%. Reasons for these differences in the seroprevalence of Toxocara in the different age groups from the two regions are discussed.