Biomedical subjects

F M Ubeira

Publications and source records attributed to F M Ubeira.

At least 37 records · Page 2Linked to original sources

PubMed2001

Non-isotopic detection of Tetramicra brevifilum (Microspora) DNA in turbot tissues.

A non-isotopic in situ hybridization (ISH) method was developed for detection of Tetramicra brevifilum, a commercially important parasite in farmed turbot Scophthalmus maximus. The probe relies on sequences from the small-subunit rRNA gene (SSUrDNA) of Tetramicra brevifilum and was obtained by polymerase chain reaction then labeled with digoxigenin. The results obtained demonstrate that the probe hybridizes well with genomic DNA of the spores; thus, it is an effective method for detecting multiorgan infections of turbot by T. brevifilum.

Animals↗

PubMed2000

Non-specific responses of turbot (Scophthalmus maximus L.) adherent cells to microsporidian spores.

We investigated non-specific responses of turbot spleen- and pronephros-resident adherent cells to spores of fish microsporidians, and the effects of the glucocorticoid dexamethasone (DX) on these responses. On average, 65% of adherent cells from the spleen and pronephros showed esterase activity (as characteristic of macrophages); 32% showed peroxidase activity (as characteristic of neutrophils), and 19% of peroxidase-positive cells were capable of phagocytosing microsporidian spores. A significantly higher proportion of adherent cells showed phagocytic activity when viable spores were the target than when non-viable spores were the target. Microsporidian spores stimulated adherent cells to produce reactive oxygen and nitrogen intermediates (ROIs and RNIs), though less effectively than the other stimulants tested. Adherent cells exposed to viable spores produced significantly less intracellular superoxide than adherent cells exposed to non-viable spores. Daily injection of fish with DX over 6 days significantly inhibited both phagocytosis of microsporidian spores and spore-induced ROI production, and similar effects were observed when adherent cells were exposed to DX in vitro.

Animals↗

PubMed2000

RFLP analysis of PCR-amplified small subunit ribosomal DNA of three fish microsporidian species.

The phylogenetic relationships of the microsporidian species Microgemma caulleryi, Pleistophora finisterrensis and Tetramicra brevifilum were investigated on the basis of restriction fragment length polymorphism (RFLP) analysis of PCR-amplified small-subunit rDNA (SSUrDNA). Using PCR primers specific for microsporidian SSUrDNA, a single product was obtained from each species, and heteroduplex analysis indicated a high degree of sequence homology among the 3 products. In RFLP analysis of the PCR-amplified SSUrDNA, the enzymes AluI and DdeI gave restriction patterns that differed among all 3 species. Phylogenetic analysis using restriction patterns as differential characters indicated that Microgemma caulleryi and Tetramicra brevifilum are more closely related to each other than to Pleistophora finisterrensis.

Animals↗

PubMed2000

Usefulness of currently available methods for the diagnosis of Anisakis simplex allergy.

BACKGROUND: Serodiagnosis of anisakiosis and Anisakis allergy is difficult since many Anisakis antigens show cross-reactivity complications. In the present study, we assess the usefulness of the major immunologic methods currently available for the diagnosis of Anisakis allergy. METHODS: Four tests (skin prick test, CAP-FEIA system, Western blotting, and an antigen-capture ELISA using O-deglycosylated antigen bound by the monoclonal antibody UA3) were applied to Anisakis-free subjects and subjects with confirmed Anisakis allergy. RESULTS: The skin prick test, CAP-FEIA, and the antigen-capture ELISA identified Anisakis allergy sera with 100% sensitivity, while Western blotting showed 96%/ sensitivity. The antigen-capture ELISA also showed 100% specificity, but CAP-FEIA showed a specificity of only 50%. In Western blotting, none of the bands detected were specific for either Anisakis-free or Anisakis allergy subjects. The skin prick test was not applied to the Anisakis-free subjects, so its specificity could not be determined. CONCLUSIONS: On the basis of these results, we suggest that the most appropriate procedure for diagnosis of Anisakis allergy suspected on clinical grounds is the antigen-capture ELISA using UA3, or, alternatively, a preliminary skin prick test with a positive result subsequently confirmed by UA3-ELISA.

Adolescent↗

PubMed2000

O-glycans as a source of cross-reactivity in determinations of human serum antibodies to Anisakis simplex antigens.

BACKGROUND: Anisakis simplex is a seafood-borne parasite that may both infect humans and cause allergy. Serodiagnosis of anisakiasis and allergy caused by this nematode is difficult since most Anisakis antigens show cross-reactivity problems. OBJECTIVE: To analyse the possible role of sugar epitopes contained in Anisakis simplex antigens as causes of false-positive results in serodiagnostic assays. METHODS: The antigens UA2R and UA3R recognized by two anti-Anisakis monoclonal antibodies were used in this study. Capture ELISA techniques were used to compare the reactivities with native or O-deglycosylated antigens of sera from Anisakis-free children (most of them infected by several other parasites) and from Anisakis allergy patients. O-deglycosylation was done by mild alkali treatment with NaOH. SDS-PAGE and immunoblotting were used to characterize the effects of NaOH or N-glycanase F treatment on UA3R. RESULTS: Native UA2R was recognized by IgG1 and IgM antibodies in the sera of both Anisakis-free subjects and allergy patients. Native UA3R was recognized by most sera from allergy patients (92% considering immunoglobulin (Ig) G1, 100% considering IgE), but also by a significant proportion of sera from Anisakis-free subjects (36% considering IgG1, 14% considering IgE). O-deglycosylation of UA3R greatly improved specificity: none of the sera from Anisakis-free patients showed either IgG1 or IgE reactivity with O-deglycosylated UA3R, while the proportion of sera from allergy patients showing IgE reactivity with this antigen was practically unaffected. O-deglycosylation of UA2R did not improve the specificity of assays using this antigen. Our results also show that the protein core of glycoproteins may be altered by even very mild alkali treatment, depending on the nature of the protein. CONCLUSION: Native glycoproteins of A. simplex should not be used for diagnostic purposes. O-deglycosylated UA3R seems to be an excellent candidate for use as target antigen in the serodiagnosis of anisakiasis and A. simplex allergy.

Animals↗

PubMed1999

Analysis of the antigenicity in mice of biotinyl enzymes from Anisakis simplex and other nematodes.

We analyzed the antigenicity of biotinyl enzymes contained in somatic extracts from Anisakis simplex and other parasite nematodes and show in this report that these molecules are an important source of cross-reactivity problems among these nematodes. Cross-reactivity was most pronounced among members of the superfamily Ascaridoidea. These results suggest that the presence of biotinyl enzymes in whole somatic extracts of A. simplex and other parasites may make serodiagnostic assays based on this kind of antigenic preparation unreliable.

Animals↗

PubMed1999

DNA probes for detection of the fish microsporidians Microgemma caulleryi and Tetramicra brevifilum.

DNA probes were developed for the detection and identification of 2 microsporidian parasites of marine fishes, Microgemma caulleryi (infecting the liver of the greater sand-eel, Hyperoplus lanceolatus) and Tetramicra brevifilum (infecting muscle, intestine and liver of the turbot, Scophthalmus maximus, a commercially important species). The probe-development procedure used is fast and straightforward, and readily applicable to the development of probes for other microsporidian species. First, genomic DNA of microsporidian spores was isolated and digested with the restriction enzyme Hind III. The fragments obtained were ligated into the vector pBluescript SK(+) and cloned in Escherichia coli. Appropriate inserts were identified and then amplified by PCR, using primers specific for regions adjacent to the Hind III restriction site in the vector sequence (and thus avoiding the need to develop primers specific for the inserts themselves). The copies were labelled with digoxigenin, for subsequent use as probes, during PCR itself. The specificity of candidate probes was tested in dot-blot hybridization assays, with the target DNA being (a) genomic DNA of the microsporidian from which the probe had been obtained, or of another species, (b) the corresponding genomic DNA in the phagemid, or (c) DNA from the corresponding host tissue. These assays identified a ca 1180 bp probe for M. caulleryi, denominated C38, and a ca 1363 bp probe for T. brevifilum, denominated F9. Similar assays designed to assess sensitivity indicated that F9 showed detectable binding to as little as 500 ng of T. brevifilum genomic DNA, and C38 to as little as 125 ng of M. caulleryi DNA; these results were obtained with detection of DIG by enzyme immunoassay (i.e. using a phosphatase-coupled anti-DIG antibody), and could no doubt be improved if a radioactive labelling and detection system were used. The probes developed in this study will greatly facilitate detection and identification of M. caulleryi and T. brevifilum in fish tissues, and may prove useful for identifying possible intermediate hosts used by these species.

Animals↗

PubMed1999

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.

Anaphylaxis↗

PubMed1999

Characterization by enzyme electrophoresis of specimens of the genus Helicometra (Trematoda, Opecoelidae) from fish caught off the coast of northwest Spain.

Species within the genus Helicometra are difficult to distinguish on morphological grounds alone, and are best discriminated with the aid of biochemical techniques. In the work reported here, the electrophoretic mobility of malate dehydrogenase isoenzyme of the Mdh-1 locus was used to characterize 375 individuals of Helicometra obtained from various teleost species (Anguilla anguilla, Conger conger, Gobius niger or Ciliata mustela) caught off the coast of northwest Spain. The results suggest that all specimens belong to only one species, probably H. fasciata. Observed genotype frequencies did not differ from those expected under the assumptions of Hardy-Weinberg equilibrium, even when the genic frequencies differ considerably respecting a Mediterranean population.

Alleles↗

PubMed1998

Carrier-dependent suppression of the anti-phosphorylcholine plaque-forming cell response in Trichinella-infected mice is mediated by anti-hapten IgG1 antibodies.

In normal mice, the phosphorylcholine(PC)-bearing Trichinella spiralis antigen FCp induces PC-specific IgM antibodies. Infection with T. spiralis appears to suppress this response, without affecting the production of anti-PC antibodies in response to other PC-bearing antigens; the suppression can thus be considered carrier-dependent. Previous work in our laboratory has indicated that the observed suppression is due to a soluble factor present in the serum of infected mice. In the work reported here, we investigated the identity of this factor. After in vitro stimulation with FCp, spleen cells from FCp-primed infected mice showed a stronger anti-PC IgM response than spleen cells from FCp-primed uninfected mice, confirming that cell memory for FCp is unimpaired by infection. Passive transfer of serum from infected mice to normal recipients, followed by immunization of recipients with FCp or another thymus-dependent or thymus-independent PC-bearing antigen, confirmed that the suppressive agent is soluble and that its activity is carrier-dependent. The suppressive agent was retained by immunoaffinity chromatography with PC or rabbit anti-mouse Ig as ligand, showing that it is a PC-specific Ig. Gel filtration of the fractions retained by PC-immunoaffinity, and subsequent identification of Ig isotypes by an ELISA-based procedure, indicated that the suppressive Igs are of the IgG1 isotype. These findings may be relevant for understanding antibody-mediated down-regulation of the immune response.

Animals↗

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

Ultrastructural colocalization of phosphorylcholine and a phosphorylcholine-associated epitope in first-stage larvae of Trichinella spiralis.

Although the presence of phosphorylcholine (PC) in Trichinella is well established, the structures of the TSL-4 antigens that bear this epitope are unknown. A subset of TSL-4 antigens (TSL-8 antigens) has been reported to be absent from the surface of first-stage T. spiralis larvae. We report experiments with a monoclonal antibody (mAb US2) developed in mice with a relative inability to produce antibodies to PC. In immunoblotting, mAb US2 and anti-PC mAb (BH8) showed apparently identical binding patterns. In addition, we used an immunogold double-labeling technique to study the anatomical distribution of the epitopes recognized by these mAbs; the results obtained indicate close colocalization of epitopes for BH8 and US2 in tissues of T. spiralis first-stage larvae. On the basis of these results, we suggest that US2 probably binds to all T. spiralis TSL-4 antigens, including TSL-8 antigens. We also clarify some conflicting previous reports on the distribution of PC immunoreactivity in first-stage larvae of T. spiralis.

Animals↗