Biomedical subjects

J Leiro

Publications and source records attributed to J Leiro.

At least 37 records · Page 2Linked to original sources

PubMed2002

Antiprotozoals effective in vitro against the scuticociliate fish pathogen Philasterides dicentrarchi.

The histophagous ciliate Philasterides dicentrarchi causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus and sea bass Dicentrarchus labrax. The present study screened 52 candidate antiprotozoals for activity against this pathogen in vitro. Of these compounds, 14 were effective (i.e. killed all ciliates within a 24 h assay period). In descending order of efficacy (minimum lethal concentration 100 to 0.8 ppm), these were niclosamide, oxyclozanide, bithionol sulfoxide, toltrazuril, N-(2'-hydroxy-5'-chloro-benzoyl) 2-chloro-4-nitroaniline, furaltadone, doxycycline hyclate, formalin, albendazole, carnidazole, pyrimethamine, quinacrine hydrochloride and quinine sulfate. Administration in filtered seawater rather than phosphate-buffered saline inactivated doxycycline hyclate and albendazole, and markedly reduced that of bithionol sulfoxide and toltrazuril, suggesting that these compounds may not be effective in bath administration. In view of these findings, we discuss the potential utility of chemotherapy as a strategy for the control of scuticociliatosis in farmed turbot and sea bass.

Animals↗

PubMed2002

Resveratrol modulates rat macrophage functions.

This study investigated the effects of trans-resveratrol (trans-3,4',5-trihydroxystilbene, RESV), a natural polyphenol from grapes with known antioxidant activity, on the respiratory-burst responses and phagocytic activity of rat macrophages. RESV at concentrations of 1-10 microM significantly and dose-dependently inhibited (a) the extracellular production of reactive oxygen intermediates (ROls) by resident peritoneal macrophages stimulated with phorbol 12-myristate 13-acetate (PMA) (a potent activator of protein kinase C, PKC) and (b) intracellular production of ROIs after opsonin-independent phagocytosis of Kluyveromyces lactis cells. Over the 10-100 microM concentration ranges, RESV likewise inhibited the production of reactive nitrogen intermediates (RNIs) by macrophages stimulated with thioglycollate. RESV concentrations above 10 microM also dose-dependently inhibited the phagocytosis of K. lactis cells. The results obtained demonstrate that RESV is a potent inhibitor of the antipathogen responses of rat macrophages and, thus, suggest that this agent may have applications in the treatment of diseases involving macrophage hyperresponsiveness.

Adjuvants, Immunologic↗

PubMed2002

Modulation of rat macrophage function by the Mangifera indica L. extracts Vimang and mangiferin.

Vimang is an aqueous extract of Mangiferia indica L., traditionally used in Cuba as an anti-inflammatory, analgesic and antioxidant. In the present study, we investigated the effects of Vimang and of mangiferin (a C-glucosylxanthone present in the extract) on rat macrophage functions including phagocytic activity and the respiratory burst. Both Vimang and mangiferin showed inhibitory effects on macrophage activity: (a) intraperitoneal doses of only 50-250 mg/kg markedly reduced the number of macrophages in peritoneal exudate following intraperitoneal injection of thioglycollate 5 days previously (though there was no significant effect on the proportion of macrophages in the peritoneal-exudate cell population); (b) in vitro concentrations of 0.1-100 microg/ml reduced the phagocytosis of yeasts cells by resident peritoneal and thioglycollate-elicited macrophages; (c) in vitro concentrations of 1-50 microg/ml reduced nitric oxide (NO) production by thioglycollate-elicited macrophages stimulated in vitro with lipopolysaccharide (LPS) and IFNgamma; and (d) in vitro concentrations of 1-50 microg/ml reduced the extracellular production of reactive oxygen species (ROS) by resident and thioglycollate-elicited macrophages stimulated in vitro with phorbol myristate acetate (PMA). These results suggest that components of Vimang, including the polyphenol mangiferin, have depressor effects on the phagocytic and ROS production activities of rat macrophages and, thus, that they may be of value in the treatment of diseases of immunopathological origin characterized by the hyperactivation of phagocytic cells such as certain autoimmune disorders.

Adjuvants, Immunologic↗

PubMed2002

Effect of (-)-epigallocatechin-3-gallate on respiratory burst of rat macrophages.

The toxic effects derived from overproduction of oxygen radicals [reactive oxygen species (ROS)] by immune cells can be partially abolished by the antioxidant activities of plant polyphenols. In the present study, we investigated the antioxidant action of a catechin, (-)-epigallocatechin-3-gallate (EGCG), on the respiratory-burst responses of rat peritoneal macrophages. EGCG at concentrations of 50-200 microM blocked the production of nitric oxide by macrophages stimulated in vivo with sodium thioglycollate then 5 days later in vitro with lipopolysaccharide and gamma-interferon. At 1-100 microM, EGCG also inhibited the extracellular liberation of oxygen radicals by resident peritoneal macrophages stimulated with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). At low concentrations (1-5 microM), EGCG increased the reduction of nitro blue tetrazolium (NBT) by the superoxide anions generated in the non-enzymatic system NADH/PMS, acting as a pro-oxidant agent, while at concentrations above 10 microM, EGCG acts as a scavenger of superoxide anions. These results show that EGCG is capable of modulating ROS production during the respiratory burst of rat peritoneal macrophages by acting as a superoxide anion scavenger. EGCG may therefore be useful in the prevention and treatment of diseases due to increased free radical production.

Animals↗

PubMed2002

PCR detection of Tetramicra brevifilum (Microspora) infection in turbot (Scophthalmus maximus L.) musculature.

This study investigated the spatial distribution of Tetramicra brevifilum spores in the musculature of infected turbot Scophthalmus maximus, with the aim of identifying the most appropriate body locations for diagnostic assays. A PCR protocol optimized for the detection of T. brevifilum spores in turbot muscle is also described. In fish showing low- and moderate-intensity infection, the spatial distribution of spores was best fitted by a negative binomial distribution, indicating a clumped spatial pattern; the negative binomial coefficient k was lower for fish with low-intensity infection, indicating a more markedly clumped pattern in these fish. In fish with high-intensity infection, the spatial distribution of spores was best fitted by the Poisson distribution, indicating a random pattern. In both low- and moderate-intensity infection, spores were present at highest density in the musculature adjoining the dorsal fins. Samples for PCR were therefore obtained from this location. PCR amplification was of the small subunit ribosomal DNA (SSUrDNA), using a pair of species-specific primers that amplify the 1250 bp product. The PCR protocol developed showed better sensitivity than microscopical techniques (detection rate by microscopy 25%, versus 42% by PCR), suggesting that it may be useful for routine screening for Tetramicra brevifilum infection in cultured turbot.

Animals↗

PubMed2001

Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain).

Two outbreaks of scuticociliatosis affecting farmed turbot Scophthalmus maximus in Galicia are described. Moribund fish showed cutaneous ulcers, darkened skin, swimming behaviour alterations, exophthalmos, and/or abdominal distension as a result of accumulation of ascitic fluid in the body cavity. Ciliates were detected in fresh mounts of practically all organs and tissues, including the blood and ascitic fluid. Histopathological studies revealed severe encephalitis and meningitis (associated with different degrees of softening or liquefaction of the brain), necrosis of the hepatic parenchyme, severe oedema of the intestinal wall, degeneration of muscle fibres, hyperplasia of the branchial epithelium, and/or vascular and perivascular inflammation. In some cases, parasites are surrounded by abundant monocytic and lymphocytic infiltrate. We report the morphological and biometric characteristics of this ciliate, which allow identification as Philasterides dicentrarchi. We discuss possible routes of entry into the host, and environmental factors possibly facilitating infection.

Animals↗

PubMed2001

Effects of bacteria on the growth of an amoeba infecting the gills of turbot.

We analysed the influence of various bacteria on the in vitro growth of trophozoites of a Platyamoeba strain isolated from diseased gill tissues of cultured turbot. Little or no growth was shown by amoebae cultured in the presence of (1) the turbot-pathogenic bacteria Vibrio anguillarum, Aeromonas salmonicida or Streptococcus sp., (2) Pasteurella piscicida or Vibrio vulnificus (pathogenic for some fishes but not turbot), or (3) the non-pathogenic 'environmental' bacteria Vibrio campbelli, Vibrio fluvialis or Pseudomonas dondorofii. The only bacteria which were successfully utilized as food sources were Aeromonas hydrophila (pathogenic for some fishes but not turbot) and the non-pathogens Vibrio natriegens, Pseudomonas nautica and Escherichia coli. These results suggest that the colonization of the gills of cultured turbot by the epizoic amoeba Platyamoeba may be an indicator of faecal contamination.

Amebiasis↗

PubMed2001

Respiratory burst responses of rat macrophages to microsporidian spores.

This study investigated the respiratory burst responses of rat resident peritoneal macrophages and of peritoneal macrophages stimulated 5 days previously with viable spores of the fish infecting microsporidian Microgemma caulleryi. Nitric oxide production by resident macrophages and prestimulated macrophages in response to viable microsporidian spores was significantly lower than in response to Escherichia coli lipopolysaccharide (LPS) (nitrite concentration in medium 57 +/- 1 microM for resident macrophages stimulated with LPS versus 31 +/- 1 microM for resident macrophages stimulated with microsporidian spores and 36 +/- 4 microM for M. caulleryi prestimulated macrophages; P < 0.05). Extracellular release of reactive oxygen species (ROS) by resident macrophages in response to microsporidian spores was similar to that in response to Kluyveromyces lactis yeast cells and to that in response to phorbol myristate (a stimulator of protein C kinase). Intracellular ROS production by resident macrophages in response to microsporidian spores was similar to that produced in response to yeast cells. Both extracellular ROS production and intracellular ROS production (in response to all stimuli) were significantly lower after in vivo prestimulation of macrophages with microsporidian spores. These results demonstrate that microsporidian spores of species other than those that habitually infect mammals are capable of modulating the respiratory burst of rat peritoneal macrophages. Such modulation may contribute to avoidance by the microsporidian of cytotoxic responses associated with the respiratory burst.

Animals↗

PubMed2001

Effect of Tetramicra brevifilum (Microspora) infection on respiratory-burst responses of turbot (Scophthalmus maximus L.) phagocytes.

In vitro assays were performed to investigate microsporidian-induced intracellular and extracellular production of reactive oxygen species (ROS) by peritoneal-exudate adherent (PEA) cells from turbot. ROS production was quantified using the fluorescent reagents OxyBURST Green H2HFF BSA (extracellular) and OxyBURST Green H2DCFDA succinimidyl ester (intracellular). Five days before assay, the cells had been elicited in vivo by intraperitoneal injection of sodium thioglycollate or spores of Tetramicra brevifilum. Elicitation with spores led to a marked increase in the proportion of neutrophils among PEA cells. PEA cells from normal turbot showed considerable extracellular and intracellular ROS production in response to microsporidian spores. By contrast, PEA cells from microsporidian-infected turbot showed considerably reduced extracellular and intracellular ROS production in response to microsporidian spores. Extracellular ROS production was affected by the addition of infected turbot serum to the assay medium, regardless of whether the PEA cells had been obtained from normal or infected fish. The presence of microsporidian-infected turbot serum significantly reduced intracellular ROS production by PEA cells elicited with microsporidian spores. These results suggest that (a) microsporidian spores partially suppress the repiratory-burst response of turbot phagocytes; and (b) infected turbot serum contains substances capable of modulating the respiratory-burst response of turbot phagocytes to microsporidian spores.

Animals↗

PubMed2001

Characterization of two monoclonal antibodies raised in Btk(xid) mice that recognize phosphorylcholine-bearing antigens from Trichinella and other helminths.

This study investigated the binding properties of two monoclonal antibodies (mAbs US1 and US2) raised in (CBA/n x BALB/c)F1 (NBF1) Btk(xid) male mice. Both mAbs show unusual specificity for phosphorylcholine (PC)-containing TSL4 antigens of Trichinella. Specifically, and in contrast to mAbs raised in normal mice, US1 and US2 mAbs do not bind to artificial PC-protein conjugates and are not inhibited by either free PC or NPPC, although US2 was partially inhibited by NPPC at high concentration (10(-2) M). However, both mAbs completely abrogate the binding to Trichinella antigens of other anti-PC mAbs (e.g. BH8 and Mab-2). These results suggest that both US1 and US2 recognize complex PC-containing epitopes. The patterns of recognition of PC-bearing antigens from different helminths by US1, US2, Mab-2 and BH8 were broadly correlated with phylogenetic proximity. The closest similarities were observed between the members of the Trichinelloidea superfamily (Trichinella spiralis and Trichuris muris) and among the ascarids (Toxocara canis, Anisakis simplex, Hysterothylacium aduncum and Ascaris lumbricoides). However, US1 did not react with the filarial nematode Onchocerca volvulus and reacted only weakly with Onchocerca gibsoni, while US2 reacted only weakly with both species. Only BH8 recognized PC-bearing antigens from the trematode Fasciola hepatica and the cestode Bothriocephalus scorpii. These results suggest that PC is attached to identical or very similar structures on most different nematode species, although major differences exist with respect to helminth species from groups such as the trematodes and cestodes that are phylogenetically distant from the nematodes.

Animals↗

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↗

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↗