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J Leiro

Publications and source records attributed to J Leiro.

At least 55 records · Page 3Linked to original sources
PubMed1997

Effects of chitinolytic and proteolytic enzymes on in vitro phagocytosis of microsporidians by spleen macrophages of turbot, Scophthalmus maximus L.

The serum of many teleosts, including turbot, contains chitinolytic and proteolytic enzymes. In the present study, the possible role of these enzymes in nonspecific immune responses to microsporidian infection was investigated. The rate of phagocytosis of Glugea caulleryi spores by turbot splenic macrophages was significantly reduced after pretreatment of spores with proteolytic or chitinolytic enzymes, suggesting that alteration of surface glycoproteins affects spore recognition. However, intracellular superoxide production by macrophages was significantly higher after stimulation with protease-treated spores, or with untreated spores plus normal turbot serum (NTS), than after stimulation with untreated spores in the absence of NTS. These results support the view that the chitinolytic and proteolytic activities in teleost serum may play a role in defence against microsporidian infection.

Animals↗
PubMed1997

Monoclonal antibodies against diagnostic Anisakis simplex antigens.

Five monoclonal antibodies (UA2, UA3, UA5, UA6, and UA8) specific for Anisakis simplex are described. All are IgG1/kappa monoclonal antibodies, except for UA2, which is an antibody IgM/kappa. The molecular weights of the major components recognized in immunoblotting are 48 and 67 kDa (UA2); 139 kDa (UA3 and UA5; same epitope); 35, 38, and 139 kDa (UA6); and 205 kDa (UA8). UA2 was the only monoclonal antibody to recognize both components of an excretion-secretion antigen preparation and antigens in the excretory cell and esophageal glands of third-stage A. simplex larvae; antigens in the excretory cell were also recognized by UA3 and UA6. Cross-reactivity studies using a hyperimmune polyclonal rabbit serum reacting with various ascaridoid nematodes indicated that the antigens captured by our monoclonal antibodies were specific for A. simplex. Finally, comparative studies of our monoclonal antibodies and An2 (the only monoclonal antibody currently available for serodiagnosis of human anisakiasis), based on the calculation of multiples of normal activity for human anisakiasis sera, indicated that our monoclonal antibodies (and particularly UA3) recognized antigens that are good candidates for serodiagnostic purposes.

Animals↗
PubMed1996

Antigenic cross-reactivity in mice between third-stage larvae of Anisakis simplex and other nematodes.

We used ELISA and immunoblotting to investigate antigenic cross-reactivity in mice between third-stage larvae of Anisakis simplex and five other nematodes: the ascaridoids Ascaris suum, Toxocara canis and Hysterothylacium aduncum, and the nonascaridoids Trichinella spiralis and Trichuris muris. Two sera were raised against each species (including A. simplex, but excluding A. suum), by infection or by immunization with somatic antigens. Serum against A. suum was raised by immunization only. The reactivities of each serum with A. simplex somatic antigens (SA), excretion-secretion antigens (ES), pseudocoelomic fluid antigens (PF) and cuticular antigens (CA) were investigated. The results of ELISA indicated high antigenic cross-reactivity between A. simplex and the remaining ascaridoid nematodes, confirming that there is extensive antigenic similarity within this group of nematode parasites. Immunoblotting again confirmed the high degree of cross-reactivity between the SA of A. simplex and SAs of the other ascaridoids, although several A. simplex SA components in the 11-18 kDA range were only recognized by sera from mice infected with A. simplex. In addition, two A. simplex PF components of 22 and 27 kDA, were recognized only by sera from mice infected with, or immunized with the SA of, A. simplex. Finally, the anti-phosphorylcholine monoclonal antibody BH8 recognized only a small number of A. simplex antigens, indicating that phosphorylcholine epitopes are not significant contributors to the observed cross-reactivity with the other nematodes.

Animals↗
PubMed1996

Free and bound biotin molecules in helminths: a source of artifacts for avidin biotin-based immunoassays.

The avidin-biotin molecular recognition system is widely used in parasite immunology. However, the presence of biotin and/or biotin-containing molecules (BCMs) in samples may lead to erroneous results. In the work reported herein we investigated the extent to which biotin and BCMs present in helminth extracts may interfere in avidin/biotin-based immunoassays and developed an enzyme-linked immunosorbent assay (ELISA) for quantification of these components. In avidin-based ELISA using antinematode monoclonal antibodies, an extract of the nematode Anisakis simplex showed very high background reactivity due to biotin/BCMs, whereas the background reactivity in an extract of the nematode Trichinella spiralis was negligible. To investigate interspecies differences further, we performed Western-blot analyses (with avidin as the detector) of extracts from seven nematodes (A. simplex, Ascaris suum, Toxocara canis, Hysterothylacium aduncum, T. spiralis, and Trichuris muris) and the cestode Bothriocephalus scorpii. Even within superfamilies there was considerable variation in the banding patterns obtained. The above-mentioned results confirm that biotin and BCMs may be a significant source of interference in ELISA and immunoblotting, two of the techniques most widely used in parasitological immunodiagnosis. A competition ELISA designed to allow accurate quantification of biotin and BCMs in helminth extracts likewise indicated very considerable interspecies variation. Both A. simplex and H. aduncum had very high biotin/BCM contents. Microdialysis of extracts in the presence of dimethylsulfoxide to remove free biotin prior to ELISA indicated that the high biotin/BCM content of the H. aduncum extract (but not the A. simplex extract) was very largely due to free biotin. Taken together, these results indicate that extreme caution should be exercised in the use of avidin/biotin-based immunoassays for the detection of helminth antigens and that in many cases it may be better to use an alternative recognition system.

Animals↗
PubMed1996

The role of opsonization by antibody and complement in in vitro phagocytosis of microsporidian parasites by turbot spleen cells.

We investigated the role played by opsonization by antibody and complement in in vitro phagocytosis of microsporidian spores by turbot adherent phagocytes. Most turbot adherent cells displaying phagocytic activity are probably macrophages. Phagocytosis of yeast cells and polystyrene beads was greatly enhanced in the presence of both the Ig and the non-Ig (i.e. complement-containing) fractions of normal turbot serum, but phagocytosis of Glugea caulleryi or Tetramicra brevifilum spores was not affected by either fraction. Neither anti-G. caulleryi immune serum, nor anti-T. brevifilum immune serum (which cross-reacted considerably with G. caulleryi antigens), enhanced phagocytosis of G. caulleryi spores. Finally, spores treated with sodium m-periodate (to modify the structure of surface-borne sugars) were less effectively ingested than untreated spores, suggesting that phagocytosis of microsporidian spores involves recognition of such sugars by the phagocytic cell. The results of this study support the hypothesis that microsporidian parasites of fish in some way modulate the host phagocytic responses.

Animals↗
PubMed1996

The humoral immune response of turbot to recently isolated pathogenic Enterococcus strains. Cross-reactivity with other Gram-positive bacteria.

An Enterococcus sp. causing severe mortalities among farmed turbot (Scophthalmus maximus) has recently been detected in northwest Spain. We found that specific turbot serum antibodies raised against isolate RA-99.1 of the new pathogen by intraperitoneal immunization, did not cross react in enzyme-linked immunosorbent assay (ELISA) with other enterococcal or non-enterococcal Gram-positive bacteria. In immunoblotting, antibodies raised against strain RA-99.1 recognized the same components in the homologous total soluble antigen preparation (TSA) as in TSA of two other isolates of the pathogen obtained from different farms. Anti-RA-99.1 serum also recognized some components of the TSA of several other Gram-positive bacteria. Immunogold labelling indicated that the antigens which provoke the humoral immune response to this pathogen are located mainly on the bacterial surface.

Animals↗
PubMed1996

The humoral immune response of turbot, Scophthalmus maximus L., to spore-surface antigens of microsporidian parasites.

Experiments based on enzyme-linked immunosorbent assay (ELISA) revealed considerable antigenic homology in turbot between two species of microsporidian, Tetramicra brevifilum (a parasite of the turbot, Scophthalmus maximus) and Glugea caulleryi (a parasite of the lesser sand-eel, Ammodytes tobianus). We next investigated whether G. caulleryi is able to suppress the turbot immune response. Intraperitoneal inoculation of turbot with G. caulleryi spores (whether heat-killed or viable) did not suppress the humoral immune response to injection of G. caulleryi spores plus adjuvant 15 days later; in fact, specific serum antibody levels (as revealed by ELISA) reached maximum levels by about Day 30 post re-exposure. Similar results were obtained with cellular enzyme-linked immunosorbent assay: 15 days after injection with G. caulleryi spores plus adjuvant, specific antibody secretion rate was higher in turbot which had been pre-exposed to G. caulleryi spores than in turbot which had not been pre-exposed.

Animals↗
PubMed1995

A sandwich immunoassay to quantify low levels of turbot (Scophthalmus maximus) immunoglobulins.

We have developed an enzyme-linked immunosorbent assay for the quantification of turbot (Scophthalmus maximus) immunoglobulin (Ig). The capture antibody is a rabbit polyclonal antiserum to turbot Ig, and the detector antibody a monoclonal antibody (UR3) to the turbot Ig heavy chain. Both antibodies bind nearly 100% of turbot Ig. The assay allows detection of turbot Ig in serum at concentrations as low as 0.16 micrograms ml-1 and takes less than 4 h. Precision is satisfactory, with intra-assay coefficients of variation (CVs) ranging from 2.1 to 16.6%, and inter-assay CVs ranging from 5.8 to 24.6%. We used the assay to determine Ig concentrations in the sera of healthy turbot of different weights. Mean serum Ig concentration was 3.35 +/- 0.74 mg ml-1 for fish weighing 15-25 g and 11.14 +/- 1.87 mg ml-1 for fish weighing 1000-2000 g.

Animals↗
PubMed1995

Anisakis simplex: stage-specific antigens recognized by mice.

To investigate antigenic differences between the developmental stages of Anisakis simplex, somatic, excretion-secretion and detergent-solubilized surface antigen preparations of third- and fourth-stage A. simplex larvae, and a somatic antigen preparation of adults, were characterized by SDS-PAGE and immunoblotting using mouse immune serum containing antibodies against the two larval stages. Excretion-secretion and surface antigen preparations from third-stage larvae behaved very differently from those of fourth-stage larvae, in both SDS-PAGE and immunoblotting; this suggests that certain excretion-secretion and surface antigens are highly stage-specific. By contrast, somatic components of third- and fourth-stage larvae were found to have very similar banding patterns, suggesting conservation of these components during the development of the parasite in mice. The SDS-PAGE and immunoblotting results for adult somatic components seem to support this hypothesis.

Animals↗
PubMed1994

Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays.

Five monoclonal antibodies (mAbs) to immunoglobulins (Igs) of the turbot Scophthalmus maximus were produced and characterized. All the mAbs (denominated UR1, UR3, UR4, UR6 and UR7) are of isotype IgG1/kappa and show good anti-turbot Ig reactivity in enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results of competitive ELISA and immunoblotting analysis indicate that these five mAbs react with at least three different epitopes on the turbot Ig H chain. Except in the case of UR1, reactivity with periodate-treated purified turbot Ig was much lower than with the untreated Ig, suggesting that carbohydrate residues are involved in epitope recognition. All the mAbs showed reactivity with sera from the closely related species Scophthalmus rhombus but not with sera from species of other flatfish genera. One of these mAbs (UR3) has been successfully applied for the detection of antibodies against Vibrio anguillarum in ELISA.

Animals↗
PubMed1993

Requirements for the induction of cross-reactive anti-Trichinella IgE antibodies in mice.

Mice primed with Trichinella spiralis or T. pseudospiralis and reinfected with either the homologous or the heterologous species produced high levels of IgE antibodies that cross-reacted with the non-inducing strain in passive cutaneous anaphylaxis assays. Cross-reactive antibodies were not induced by primary infection. Cross-reactivity persisted for more than 6 months following secondary infection or destruction of encysted larvae with mebendazole. Both the prevention of larvi-position by thiabendazole and the interruption of infection using naphthalophos indicated that the presence of the pre-adult stage alone provided sufficient priming for the induction of detectable levels of cross-reactive IgE by subsequent reinfection. These results suggest the existence of two sets of Trichinella allergens, one comprising species-specific major allergens (MAs) and the other comprising minor allergens (mAs) evoking a cross-reactive IgE response that occurs to a detectable extent only when the response to MAs has reached its ceiling. These findings are relevant to the design of experiments investigating the role played by IgE antibodies in protection against reinfection in rodents.

Animals↗
PubMed1993

Anisakis simplex: antigen recognition and antibody production in experimentally infected mice.

The kinetics of antibody response to intraperitoneal infection of mice with third stage larvae of Anisakis simplex was investigated by ELISA. Maximum antibody response to excretion-secretion (ES) antigens was reached before maximum response to somatic (SA) antigens. Total immunoglobulin (Ig) production (consisting mainly of IgM and IgG1 isotypes) was very similar in both cases. Immunoblotting was used to characterize the antigens recognized by the host in the presence or absence of the metabolic products released by the parasite in vivo. Sera from mice infected with live larvae (anti-L3 L serum) and immunized with dead larvae (anti-L3 D serum) recognized a similar pattern of bands in immunoblots of ES and SA antigen preparations. In the latter, however, three bands at 14, 17 and 18 kD were only recognized by the anti-L3 L serum. A possible explanation is that these low molecular weight antigens are ES products released only in vivo. Finally, the immune response in mouse was compared using ELISA and immunoblotting with the response of a human anisakiasis reference serum, and was found to display considerable similarities. This suggests that the mouse may be a useful model for studying the immunobiology of A. simplex in man.

Animals↗
PubMed1991

Anatomical location of phosphorylcholine and other antigens on encysted Trichinella using immunohistochemistry followed by Wheatley's trichrome stain.

This work investigated the location on the parasite of Trichinella antigens recognized by the mouse immune system and the question as to which of them bear the epitope phosphorylcholine (PC). Wheatley's trichrome stain (initially developed for faecal smears) proved to be excellent for visualization of Trichinella structures, enabling four types of stichocyte to be distinguished. By applying this stain on infected muscle sections after immunocytochemistry using (a) anti-PC BH8 monoclonal antibodies, (b) serum from mice that had been infected twice in the presence of 0.05% thiabendazole (to prevent reproduction by adult females) and then bled on day 7 post-reinfection, (c) serum from infected mice that were bled on day 14 postinfection, or (d) serum from infected mice that were bled on day 42 postinfection, we found (1) that PC is an abundant structural epitope on the hypodermis/muscle, genital primordium and intestinal tract but is absent from the cuticle and stichosome; (2) that the principle secretory cells of adult worms are delta- and beta-stichocytes, whereas those of migrating and encysted L1 larvae are alpha-stichocytes; and (3) that Trichinella antigens recognized in the encysted phase of the parasite's life cycle are present in parasitized myofibres in the sarcoplasmic matrix and in the nucleoplasm of hypertrophic nuclei. The significance of these findings is discussed.

Animals↗
PubMed1991

Specific immunosuppression by Trichinella: fine specificity and effect on lymphocyte function in vivo.

Muscle-phase Trichinella larvae depress the immune response of mice to the phosphorylcholine (PC)-bearing Trichinella antigen FCp without affecting responses to other PC-bearing or non-PC antigens. The depressive activity is independent of antigen dose and Trichinella species and, in adoptive cell transfer experiments with lethally irradiated recipient mice, depended on the state of the recipient (infected recipients had a depressed response even a month after their encysted larvae had been killed and regardless of whether the donor had been exposed to FCp) but not on the state of the transferred cells. We conclude that lymphocytes are not permanently altered by the depressive action, that the agent responsible persists in the host at least a month after the death of the encysted Trichinella larvae, and that the alteration does not eliminate lymphocyte immunological memory.

Animals↗
PubMed1988

Immunomodulation by Trichinella spiralis: primary versus secondary response to phosphorylcholine-containing antigens.

The present work was designed to determine in detail the capacity of the nematode Trichinella to modulate the plaque-forming cell (PFC) response of BCF1 mice to the parasite's own antigens. To this end, we studied the PFC responses shown by infected and non-infected BCF1 mice using as the target antigen phosphorylcholine, an epitope which is found in the parasite. From the results presented here, the following conclusions can be drawn: i) Trichinella spiralis is capable of modulating the immunoresponse to thymus-dependent (TD), but not to thymus-independent (TI), parasite antigens; ii) Trichinella spiralis suppresses the PFC response to the parasite-derived TD antigen FCp1 (a particulate antigen containing PC) during the muscle stage of its life cycle, but does not affect the responses to other parasite-derived PC-bearing antigens; this seems to indicate that the suppressive activity exerted by Trichinella is highly specific; iii) anti-PC PFC production in the secondary response was also suppressed by the parasite. Finally, the inability of the FCp1 antigen to induce detectable anti-PC PFC, other than IgM, is discussed.

Animals↗
PubMed1988

The effect of the intestinal worms and migrating L1 larvae of Trichinella spiralis on the production of antiparasitic IgE antibodies.

The effect of the adult worms and migrating L1 larvae of Trichinella spiralis on the production of specific IgE antibodies was determined in BCF1 mice. To achieve this, we combined the effect of two anthelminthics: thiabendazole, to produce chemosterilization of adult females, and napthalophos, to expel adult worms from the intestine of infected mice on the desired day. Our results demonstrate that when the natural route of infection is used the production of IgE antibodies is not dependent on the infection dose or the number of migrating L1 larvae, and that both intestinal worms and migrating L1 larvae contribute to the production of reaginic antibodies. In addition to this, an extended period of antigenic stimulation (10-12 days) is required for the induction of a detectable, specific IgE response by adult worms. Finally, our results seem to indicate that although the effects of adult worms and migratory L1 larvae on the IgE production are not additive, the presence of adult worms in the intestine of mice may stimulate a secondary exposure to common antigens released by the migrating L1 larvae.

Animals↗
PubMed1988

Allergenic cross-reactivity of several strains of Trichinella in mice and rats by the passive cutaneous anaphylaxis technique.

The cross-reactivity of antiparasite IgE antibody responses induced by allergenic antigens obtained from Trichinella spiralis and Trichinella pseudospiralis was determined by the passive cutaneous anaphylaxis (PCA) technique in BCF1 mice infected with T. spiralis, Trichinella nelsoni, Trichinella nativa, and T. pseudospiralis and in rats infected with T. spiralis and T. pseudospiralis. Our results demonstrate that when the antigen used for the PCA challenge is derived from muscular L1 larvae of T. spiralis, high IgE antibody titres can be detected from sera of animals infected with T. spiralis, T. nelsoni, or T. nativa, but not with T. pseudospiralis, during the entire life cycle of the parasite. However, when homologous antigens are used in the PCA test, we obtained comparable values of IgE titres in rats and mice infected with both T. spiralis and T. pseudospiralis strains. These results suggest the existence of a high degree of immunologic identity between the allergenic antigens of T. spiralis, T. nelsoni, and T. nativa strains, but not T. pseudospiralis.

Allergens↗