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Biomedical subjects

I M Cesari

Publications and source records attributed to I M Cesari.

At least 19 recordsLinked to original sources
PubMed1992

Experimental chemotherapy of Schistosoma mansoni with praziquantel and oxamniquine: differential effect of single or combined formulations of drugs on various strains and on both sexes of the parasite.

The susceptibility of two Venezuelan (YT and SM) and one Brazilian (BH) strain of Schistosoma mansoni to single oral doses of praziquantel (Pz; 250 or 500 mg/kg), oxamniquine (Ox; 40, 60, or 100 mg/kg) or to low-dose combinations of both drugs (33 mg/kg Pz and 25 mg/kg Ox; 66 mg/kg Pz and 12.5 mg/kg Ox; 250 mg/kg Pz and 40 mg/kg Ox) was experimentally evaluated in mice. At lower doses of either drug, adult worms of the SM isolate were less susceptible than those of the BH and YT isolates. However, no difference in liver or intestinal egg counts (IECs) could be detected among the isolates after this treatment. At such doses, Pz was better than Ox at reducing IECs. In spite of lowered IECs, eggs continued to accumulate in the liver after Ox treatment. At higher individual doses or following treatment with low-dose combinations of both drugs, no difference in susceptibility could be detected among the parasite isolates. Under such conditions, oviposition was drastically reduced in all three isolates. We confirm that Ox preferentially kills male parasites and present for the first time evidence for the preferential killing of female worms by Pz. We propose that the synergistic effect obtained in the present study and in other investigations using low-dose combinations of both drugs may be due to the preferential cytotoxicity of each drug against a different parasite sex.

Animals↗
PubMed1992

Isoenzyme studies in one Brazilian and two Venezuelan strains of Schistosoma mansoni.

1. Enzyme polymorphism, analyzed by starch gel electrophoresis, was found to be zero for acid phosphatase, phosphoglucomutase, phosphoglucose isomerase, glucose 6-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase and malic enzyme, in one Brazilian and two Venezuelan strains of Schistosoma mansoni. 2. All loci studied were monomorphic within strains, but the isoenzymic patterns were, however, different among the strains. 3. Results suggest a drastic loss of the genetic variability usually found in natural populations.

Animals↗
PubMed1992

Immunocapture assay for quantification of human IgA antibodies to parasite antigenic enzymes. Application with the alkaline phosphatase of Schistosoma mansoni.

Conditions are described for using solid phase adsorbed jacalins in an immunocapture assay for IgA antibodies to the alkaline phosphatase of Schistosoma mansoni. Microtiter plates were activated with polylysine and jacalins were covalently adsorbed by means of glutaraldehyde. From three different jacalins, the one purified from seeds of Artocarpus tonkinensis showed the lowest non-specific adsorption and was used for further studies. Comparing solutions of bovine serum albumin, ovalbumin and Tween 20, it was shown that the latter was most successful in blocking non-specific adsorption. Low serum dilutions resulted in a less efficient IgA capture by the adsorbed jacalin than higher dilutions. Under optimal working conditions, a high correlation could be shown between the presence of specific anti-alkaline phosphatase antibodies of IgA isotype and IgG isotype.

Alkaline Phosphatase↗
PubMed1992

Parasite enzymes as a tool to investigate immune responses.

Previous evidences reported by us and by other authors revealed the presence of IgG in sera of Schistosoma mansoni-infected patients to immunodominant antigens which are enzymes. Besides their immunological interest as possible inductors of protection, several of these enzyme antigens might be also interesting markers of infection in antibody-detecting immunocapture assays which use the intrinsic catalytic property of these antigens. It was thus thought important to define some enzymatic and immunological characteristics of these molecules to better exploit their use as antigens. Four different enzymes from adult worms were partially characterized in their biochemical properties and susceptibility to react with antibodies of infected patients, namely alkaline phosphatase (AKP, Mg2+, pH 9.5), type I phosphodiesterase (PDE, pH 9.5), cysteine proteinase (CP, dithiothreitol, pH 5.5) and N-acetyl-beta-D-glucosaminidase (NAG, pH 5.5). The AKP and PDE are distinct tegumental membrane-bound enzymes whereas CP and NAG are soluble acid enzymes. Antibodies in infected human sera differed in their capacity to react with and to inhibit these enzyme antigens. Possibly, the specificity of the antibodies related to the extent of homology between the parasite and the host enzyme might be in part responsible for the above differences. The results are also discussed in view of the possible functional importance of these enzymes.

Africa↗
PubMed1992

New approaches for the control and eradication of schistosomiasis in Venezuela.

Schistosomiasis in America with the exception of Brazil, behaves as a chronic mild disease with few clinical manifestations due to low parasite burden. These features restrict the clinical and parasitological diagnosis. The most commonly used stool examination method, Kato-Katz, becomes insensitive when the majority of individuals excrete less than 100 eggs/g of feces. In view that antigen-detecting techniques have not been able to reveal light infections, the antibody detecting assays remain as a very valuable diagnostic tool for epidemiological surveillance. The Venezuelan Schistosomiasis Research Group (CECOICE) has designed a mass chemotherapy strategy based on sero-diagnosis. Since blood sampling is one of the important limiting factors for large seroepidemiological trials we developed a simple capillary technique that successfully overcame most of the limitations of blood drawing. In this sense, ELISA seems to be the most adequate test for epidemiological studies. Soluble egg Schistosoma mansoni antigen (SEA) has been largely used in Venezuela. The sensitivity of ELISA-SEA in our hands is 90%, moreover its specificity reach 92% when populations from non-endemic areas but heavily infected with other intestinal parasites are analyzed. The Schistosomiasis Control Program is currently carrying out the surveillance of endemic areas using ELISA-SEA as the first screening method, followed by the Circumoval Precipitin test for validation assay. The results with these two serological techniques allowed us to defined the criteria of chemotherapy in populations of the endemic areas. On the search of better diagnostic technique, Alkaline Phosphatase Immunoenzyme Assay (APIA) is being evaluated in field surveys.

Algorithms↗
PubMed1990

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni.

This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complement-dependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule.

Alkaline Phosphatase↗
PubMed1990

Antigenicity of adult Schistosoma mansoni alkaline phosphatase.

Antibodies to the alkaline phosphatase (AP) of Schistosoma mansoni in infected human and mice sera were evaluated by a direct solid-phase AP immunoadsorption assay (APIA) and by Western blot and immunostaining. APIA consisted of (a) solid-phase capture of immunoglobulins from infected human or mice, (b) immunoadsorption of the enzyme antigen by the antibodies, and (c) detection of the enzymatic activity. By this procedure the appearance of the anti-AP response in mice was detected around 50 days post-infection; this response was not specific for an AP of a given schistosome strain and it was not induced by an autoimmunity phenomenon. Fourteen out of 15 sera from infected people tested by APIA showed a clear antibody response against this enzyme. Immunoblots in non-reducing conditions supported APIA results indicating that the parasite AP was specifically recognized by the antibodies present in infected human and mice sera. These results suggest the possible usefulness of the schistosome AP as a marker for S. mansoni infection.

Alkaline Phosphatase↗
PubMed1986

A simplified methodology for purification of peanut (Arachis hypogaea) agglutinin.

The agglutinin from peanut (Arachis hypogaea) was readily isolated by affinity chromatography on acid-treated Sepharose 6B. The recovered lectin (50 mg/100 g seeds) appeared as a single band of Mr 32,000 on gel electrophoresis and its specific haemagglutination titre on desialylated human A red blood cells was very high (2(15)).

Chromatography, Affinity↗
PubMed1983

Schistosoma mansoni: surface membrane isolation by polycationic beads.

The Schistosoma mansoni surface membrane complex was isolated by binding polycationic beads to the worm surface in a sucrose- or sorbitol-acetate buffer, pH 5.0, at 4 C. The ratio of incorporation [3H]cholesterol/[14C]arachidonic acid was measured as well as the specific activities of the alkaline phosphatase (EC 3.1.3.1), Type I phosphodiesterase (EC 3.1.4.1), and Ca2+-adenosine triphosphatase (EC 3.6.1.3). The results indicated that membranes isolated on beads were of comparable or greater purity than membranes isolated by sucrose gradient centrifugation. The isolation procedure was rapid (30 min) and produced membrane fractions whose cytoplasmic surfaces were probably exposed.

Alkaline Phosphatase↗
PubMed1983

Enhancement of eosinophil effector function by soluble factors released by S. mansoni: role of proteases.

Schistosomulum-released products (SRP) have been shown to enhance both expression of rat and human eosinophil Fc receptors and IgG-dependent cytotoxicity. The present work provides additional evidence of the secretion of eosinophil-enhancing factors by schistosomula and other developmental stages of schistosomes, including adult worms. The heat lability, as well as the strong inhibition of the stimulating activity of SRP by the protease inhibitor Trasylol, suggest that thermolabile proteases secreted by the parasite are involved in this mechanism. The purification of the schistosome proteases by preparative isoelectric focusing and gel filtration demonstrated that neutral proteases able to hydrolyze the collagenase substrates Azocoll and Z-Gly-Pro-Leu-Gly-Pro are able to significantly enhance eosinophil effector functions. Purified Clostridium histolyticum collagenase was also able to mimic the enhancing effect of schistosome proteases, suggesting involvement of a collagenase-like activity of the enzymes in the eosinophil stimulation.

Animals↗
PubMed1983

Aminopeptidase activity in adult Schistosoma mansoni.

An aminopeptidase activity capable of hydrolysing leucine 4-nitroanilide and alanine 4-nitroanilide at pH 7.0 was detected in saponin-CaCl2 extracts and homogenates of adult Schistosoma mansoni. The extracts were also capable of acting on synthetic dipeptides at the same pH, preferentially hydrolysing peptide bonds following leucine, alanine, or proline N-terminal residues. Imide bonds were not hydrolysed. The hydrolysis of leucine 4-nitroanilide was apparently stimulated by thiols, strongly inhibited by 1 mM 4-chloromercuric benzene sulfonic acid, and partially inhibited by 1 mM 1,10-phenanthroline.

4-Chloromercuribenzenesulfonate↗
PubMed1982

Neutral protease activities at different developmental stages of Schistosoma mansoni in mammalian hosts.

Neutral protease activities of schistosomula, 20 day old and adult worms of S. mansoni have been studied. 1. The neutral enzymes of the three development stages are able to hydrolyse numerous natural and synthetic substrates. 2. The enzymes of homogenates and saponin CaC12 extracts show significant discrepancies in their specificities, optimum pH activities and in the effects of enzyme inhibitors. 3. A serine protease activity was specific for the schistosomula stage whereas thiol proteinases characterize the later stages of evolution. A metalloaminopeptidase activity was shown at all three stages. 4. Gel filtration chromatography and isoelectric focusing of an adult worm homogenate demonstrated the presence of at least 5 caseolytic and 4 azocollytic neutral activities. Three aminopeptidases, one of which exhibited iminopeptidase specificity were revealed by the same procedure.

Aminopeptidases↗
PubMed1981

Properties of a series of tegumental membrane-bound phosphohydrolase activities of Schistosoma mansoni.

1. Incubation of Schistosoma mansoni for 5 min in a phosphate-buffered medium, pH 7.4, released tegumental material containing the following phosphohydrolase activities: alkaline phosphatase, 5'-nucleotidase, glycerol-2-phosphatase, glucose 6-phosphatase, phosphodiesterase and ATPase. 2. Maximum activity of these enzymes was measured at pH 9.5; however, the phosphodiesterase and ATPase activities were also appreciable at pH 7.0. 3. Solubilization of the released tegumental material in 1% Triton X-100 followed by gel filtration distinguished three peaks of enzyme activity: an ATPase (mol.wt. greater than 1000 000), a phosphodiesterase (mol.wt. 1 000 000) and an alkaline phosphomonoesterase with broad specificity (mol.wt. 232 000). 4. The ATPase activity was highly activated by 10 mM-Mg2+ or 1 mM-Ca2+ and was inhibited by chelating agents. Ouabain, Na+ and K+ had little effect on enzyme activity, whereas activity was increased by 50% in the presence of calmodulin. The phosphodiesterase activity was highest in the presence of 100 mM-Na+ or -K+, and 10 mM-Mg2+ or -Ca2+. Alkaline phosphatase activity was also stimulated by 100 mM-Na+ or -K+, and 10 mM-Mg2+; however Ca2+ inhibited at greater than 1 mM. 5. Surface iodination of parasites followed by detergent solubilization and gel filtration of the released tegumental membranes indicated that these enzymes were not accessible. A major surface component, apparent mol.wt. 80 000, was iodinated. 6. Rabbit anti-(mouse liver 5'-nucleotidase) antibodies did not inhibit the phosphohydrolase activities. However, an immunoglobulin G fraction from sera of mice chronically infected with S. mansoni partially inhibited alkaline phosphatase activity, but was without effect on the phosphodiesterase and ATPase activities. 7. The location of the enzymes in the double membrane of the tegument and their significance in host-parasite interactions is discussed.

Animals↗
PubMed1981

Isolation and partial characterization of the tegumental outer membrane of adult Schistosoma mansoni.

Incubation of adult Schistosoma mansoni at 37 degrees C in chemically defined media caused the shedding of variable amounts of the parasite's tegument. In phosphate-buffered saline, pH 7.4, 37% of 125I-labelled wheat germ agglutinin which was attached to the schistosome surface was released within 5 min. When the released material was collected by centrifugation at 55 000 g for 1 h, the pellet was seen to consist of components of the parasite tegument and contained 1% of the parasite protein and 6.2% of the 5'-AMPase activity. By monitoring the release of the gut enzyme, haemoglobinase, it was shown that predominantly tegumental material was released under these conditions. The material released from the parasite was separated by discontinuous sucrose gradient centrifugation into 4 major fractions and it was shown that a material banding at low density was highly enriched in vesicles of parasite outer membrane. Major polypeptides of apparent molecular weights 135 000, 110 000, 90 000, 65 000, 47 000 and 40 000 and glycoproteins of apparent molecular weights 135 000, 120 000, 65 000, 63 000 and 30-40 000 were identified in this fraction. Comparison of the distribution of 5'-AMPase activity and the contents of each fraction as revealed by ultrastructural examination showed that this enzyme is a good marker for the tegumental outer membrane.

Animals↗