Cloning of a Schistosoma japonicum gene encoding a major immunogen recognized by hyperinfected rabbits.

A library of randomly sheared Schistosoma japonicum genomic DNA fragments was constructed in the bacteriophage expression vector lambda gt11. A portion of the library was screened with sera collected from rabbits 8 weeks after they were infected with 1000 cercariae. Four clones whose recombinant gene products react with the rabbit sera were purified to homogeneity. Clone SjIR-12A was chosen for detailed study because of its very intense reaction with the rabbit sera. SjIR-12A was found to encode part of a 70 kDa protein (Sj70) that is present in both soluble egg antigen (SEA) and soluble worm antigen preparations (SWAP). Western blot analysis suggests that Sj70 is the only SWAP component that is strongly immunoreactive with the rabbit sera. Rabbit antibodies that react with the SjIR-12A fusion protein were immunoaffinity purified and used to localize immunoreactive product to the nervous tissue of male and female adult worms, the dorsal and lateral tegument of male adult worms, and in eggs to the miracidial tegument and the area between the eggshell and miracidium. Southern hybridization analysis suggests there are approximately four copies of the Sj70 gene per haploid genome.

Characterization of a large gene family in Schistosoma japonicum that encodes an immunogenic miracidial antigen.

Three of eleven clones isolated from a genomic expression library of Schistosoma japonicum DNA using chronically infected human sera also react with chronically infected mouse sera. Characterization of these three clones showed that they contain different members of the same gene family. One clone contains two members of the gene family approximately 2 kb apart and in opposite orientation to each other. DNA sequence homologies between pairs of genes range from 98% to 99.5%. Southern hybridization results indicate there are approximately 40 copies of these genes per haploid genome. Sera from mice immunized with purified fusion protein detected immunoreactive products in the central ganglion and ciliated epidermal cells of miracidia.

Proteolysis of human hemoglobin by schistosome cathepsin D.

Schistosomes feed on human blood. They employ proteases to degrade hemoglobin from ingested erythrocytes, using the residues released for amino acid metabolism. However, the identity and the role of the participating protease(s) are unclear and controversial. Confocal microscopy localized schistosomal cathepsin D to the parasite gastrodermis, and revealed elevated protease expression in females. At sub-cellular level, cathepsin D was localized to superficial digestive vacuoles of the gut and to cisternae of the gastrodermal rough endoplasmic reticulum. Schistosome cathepsin D, expressed in insect cells, autoactivated at pH 3.6 to a approximately 40 kDa form that cleaved the substrates o-aminobenzoyl-Ile-Glu-Phe-nitroPhe-Arg-leu-NH(2) and hemoglobin. The NH(2)-terminal residues of mature cathepsin D of Schistosoma japonicum and Schistosoma mansoni were Asn1 and Gly1, respectively, revealing that the proregion peptide was comprised of 35 residues. The proteases cleaved hemoglobin at pH 2.5--4.6, releasing numerous fragments. S. Japonicum cathepsin D cleaved at 13 sites, S. mansoni cathepsin D at 15 sites. Early cleavage sites were alpha Phe33-Leu34 and beta Phe41-Phe42, while others included alpha Leu109-Ala-110 and beta Leu14-Trp15, demonstrating a preference for bulky hydrophobic residues at P1 and P1'. Most of the schistosomal cathepsin D cleavage sites were discrete from those of human cathepsin D. The gastrodermal location, elevated expression in females, acidic pH optima, similar substrate preferences in two species, and the discrete substrate preferences compared with human cathepsin D together provide compelling support for the hypothesis that schistosomal cathepsin D plays an integral role in hemoglobin proteolysis, and might be selectively targeted by drugs based on protease inhibition.

The cytochemical localization of succinic dehydrogenase in the sperm mitochondria of the gastropod Biomphalaria glabrata. A comparison of two methods.

The mitochondrial derivative of the sperm of the gastropod pulmonate Biomphalaria glabrata was studies to ascertain succinic dehydrogenase localization cytochemically. Two techniques were compared. One technique depends on a tetrazolium salt that yields an osmiophilic formazan upon reduction. The other technique is dependent on the reduction of copper ferricyanide. The effects of several electron transport inhibitors were studied. The reaction product observed in the matrix of the mitochondrial derivative using the former technique is sensitive to rotenone and is believed to be nicotinamide adenine dinucleotide-dependent. The reaction product observed in the intracristal spaces using the copper ferricyanide method is insensitive to rotenone and is believed to cytochemically demonstrate succinic dehydrogenase in this material.

Application of the FITC-anti-FITC-gold system to ultrastructural localization of antigens.

We report the application of a fluorescein isothiocyanate (FITC)-anti-FITC method to localize antigens at the ultrastructural level. In the systems studied, the anti-FITC-based detection method displays high specificity and sensitivity. These observations, combined with ease of production and with availability of FITC-protein conjugates, suggest that the FITC-anti-FITC method is a good alternative to presently used methods and is widely applicable to immunochemical and immunocytochemical procedures. The same preparation and protocol can be used for light and electron microscopic studies, thereby reducing possible artifacts introduced if different procedures are used. In the present study, two systems were used to test the method. One system used an FITC-labeled monoclonal antibody (MAb) to schistosome circulating cathodic antigen. In this system, the label was detected in the gut of adult Schistosoma mansoni by an anti-FITC MAb conjugated to 10-nm gold particles. The second system used human IgM antibodies pooled from patients infected with Schistosoma mansoni. In this system detection was accomplished using an anti-human IgM-FITC conjugate followed by the anti-FITC-Au antibody conjugate.

In vitro trypanocidal activity of tetraethylthiuram disulfide and sodium diethylamine-N-carbodithioate on Trypanosoma cruzi.

Tetraethylthiuram disulfide (disulfiram) (TETD) and sodium diethylamine-N-carbodithioate (DECD) were examined for their in vitro trypanocidal activity against Trypanosoma cruzi. Benznidazole (BNZ), the drug used clinically for the chemotherapy of Chagas' disease, was used as a positive control. Inhibition assays included evaluation against the epimastigote, trypomastigote, and amastigote forms. Tetraethylthiuram disulfide and its reductive metabolite DECD inhibited 64.6% and 69.7%, respectively, of epimastigotes at a concentration of 50 micrograms/ml after 72 hr and BNZ caused 69.1% inhibition at the same concentration. Both TETD and DECD were not as effective against tissue culture trypomastigotes as BNZ (TETD = 47.7%,; DECD = 46.1%; BNZ = 88.7%) at 50 micrograms/ml after 24 hr. However, TETD and DECD treatment of amastigote-infected 3T3 fibroblasts yielded 60 and 67% inhibition, respectively, as compared to BNZ with an inhibition of 62%. A possible mechanism of action of TETD and DECD is via interference with the essential metal metabolism of T. cruci. Since toxicity data for both TETD and DECD are available and both drugs are active against the parasite, these drugs are candidates for further study to determine if they are of potential clinical interest as a prophylactic or therapeutic agent against Chagas' disease.

Chelating agent inhibition of Trypanosoma cruzi epimastigotes in vitro.

A number of chelating agents and some of their derivatives are as effective as, or superior to, benznidazole, the compound currently in clinical use, in the suppression of the reproduction of epimastigotes of Trypanosoma cruzi, the protozoa that causes Chagas' disease. All compounds were examined at a culture concentration of 5 micrograms/mL. The most effective compounds included N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, sodium diethylamine-N-carbodithioate, piperidine-N-carbodithioate and several of its analogs, a number of other carbodithioates with two nonpolar groups on the nitrogen, and tetraethylthiuram disulfide, a prodrug of sodium diethylamine-N-carbodithioate and widely used in the treatment of alcoholism. The introduction of additional ionic or nonionic polar groups on the chelating molecule generally results in a loss of tyrpanocidal activity. Common commercially available chelating agents which exhibited no activity included D-penicillamine, meso-2,3-dimercaptosuccinic acid, and triethylenetetramine tetrahydrochloride. Dose-response data on the culture indicated that some of these compounds exhibited inhibition of Trypanosoma cruzi epimastigotes at concentrations as low as 0.625 microgram/mL. It is proposed that the mechanism of action of these compounds is based on their ability to interfere with the essential metal metabolism at intracellular sites of the epimastigote involving iron, copper, or zinc. The results also indicate that a certain degree of hydrophobicity may be necessary for the groups attached to the literal metal-bonding structure if the compounds are to successfully inhibit the epimastigotes of Trypanosoma cruzi. The development of antiprotozoal drugs which are chelating agents specifically designed to selectively disrupt the essential metal metabolism of Trypanosoma cruzi should furnish a new generation of drugs which can be used in the treatment of Chagas' disease.

The feeding of A type red blood cells in vitro and the ability of Schistosoma mansoni schistosomules to acquire A epitopes on their surfaces.

In vitro-raised Schistosoma mansoni schistosomules fed human A type red blood cells at day 7 postpenetration displayed A epitopes on their surfaces but not B epitopes when tested by the mixed agglutination procedure. Schistosomules treated with colchicine prior to exposure to red blood cells, exposed to plasma derived from human A type blood, or not exposed to host red blood cells did not display A epitopes on their surfaces. Under the conditions used in these experiments, it is likely that feeding of host red blood cells may be necessary for the tegument to become responsive to adsorption of host red blood cell epitopes.

Cytochemical localization of peroxidase activity in the miracidium of Schistosoma mansoni.

Distribution of peroxidase activity in the mitochondria of the miracidium of the blood fluke, Schistosoma mansoni, was investigated cytochemically using the diaminobenzidine (DAB) technique. Re-action product was localized in the mitochondria of this larvae stage at pH 7.4 and 9.7. The reaction was peroxide-dependent and insensitive to either potassium cyanide, sodium azide, or 3-amino-1,2,4-triazole at the concentrations used. The reaction was inactivated by heat and by pretreatment with methanol-nitro-ferricyanide, and inhibitor of peroxidase. A perioxide-independent reaction was also observed in the mitochondria. This latter reaction was sensitive to potassium cyanide and sodium azide. It is hypothesized that the peroxidase either may act where peroxide is an electron acceptor in a flavoprotein-linked system or may be a vestige of a more primitive pathway. No peroxidase activity was observed in the mitochondria of other stages of the life cycle of the worm.

Cytochemistry of gastrodermal autophagy following starvation in Schistosoma mansoni.

The gastrodermis of adult Schistosoma mansoni was examined by electron microscopy to determine the effects of starvation and the effects of hycanthone, administered in vitro. Special attention was focused on the relationship of the Golgi complexes with the process of autophagy. In general, autophagy was increased in the gastrodermis when it was exposed to stress conditions such as starvation and hycanthone. Acid phosphatase and thiamine pyrophosphatase activities were used as enzyme markers for the Golgi complexes and lysosomes. During the early stages of starvation, there was a 4-fold increase in the number of Golgi complexes per unit area in the gastrodermis. A progressive increase in the number of secondary lysosomes was evident as starvation time was increased. Hycanthone accelerated the effects of starvation. It was hypothesized that acid hydrolases are passed to the Golgi complexes via ER-derived vesicles. The enzymes are subsequently released as primary lysosomes from the Golgi complex to fuse with cytosegresomes and form secondary lysosomes (cytosomes).

In vitro effects of inhibitors on ingestion and digestion of hemoglobin by Schistosoma mansoni schistosomules.

Hemoglobin in solution was added to cultures of Schistosoma mansoni schistosomules grown in vitro. Colchicine-treated and Actinomycin D-treated 7-day parasites ingested the hemoglobin, but only the former group displayed ability to digest the hemeprotein. Parasites fed hemoglobin immediately following skin penetration displayed digestive abilities after 48 hr. Perhaps the granules secreted from the esophageal gland are not necessary for the digestion of hemoglobin but may be required for the lysis of intact membranes of host erythrocytes.

The in vitro effects of various lysosomotropic agents on the gut of Schistosoma mansoni schistosomula.

Cultured Schistosoma mansoni schistosomula of various ages were exposed to several lysosomotropic agents. Weak bases such as chloroquine, ammonium chloride, and acridine orange caused gut swelling upon protonation. The latter compound fluoresced a bright orange indicating the acidic nature of the gut contents. Hydrolysis of ingested L-amino acid methyl esters also resulted in gut swelling, indicating the nonpermeant nature of the resulting L-amino acids. Neither age nor feeding status influenced these swelling effects. Treatment of schistosomula with D-amino acid esters, free L-amino acids, or methanol had no effect. Thin-layer chromatographic analysis of worms treated with radiolabeled L-leucine methyl ester provided evidence that the ester was hydrolyzed. These results support the premise that the schistosome gut is an acidic compartment and is reminiscent of a secondary lysosome in its reaction to lysosomotropic agents.

Utilization of the heme moiety of hemoglobin by Schistosoma mansoni schistosomules in vitro.

The hemoglobin in mouse reticulocytes was labeled in vitro with either [3H], [14C] aminolevulinic acid (ALA), or [3H] leucine. Specific labeling of the globin moiety with labeled leucine, and the heme moiety with labeled ALA, was confirmed by carboxymethylcellulose chromatography and cyclohexanone extraction. Most of the leucine label recovered from reticulocytes that were incubated for 4 hr was incorporated in hemoglobin. However, 2 hr incubation of reticulocytes in the presence of labeled ALA followed by 4 hr in cell incubation medium in the absence of ALA was required for sufficient incorporation of the radionuclide into reticulocyte hemoglobin. In all reticulocyte labeling experiments, regardless of the radionuclide used, label was also observed in non-hemoglobin heme-containing molecules. Schistosoma mansoni schistosomules fed reticulocytes in vitro in which the heme moiety of hemoglobin was labeled displayed radioactivity in the protein fraction of the organisms, as determined by TCA precipitation, and in the ethanol-soluble component. In comparison, schistosomules fed reticulocytes containing globin-labeled hemoglobin displayed radioactivity only in the protein component. Pre-incubation of the schistosomules in puromycin prior to exposure to lyophilized, [14C] ALA-labeled hemoglobin partially inhibited incorporation of label. These results suggest that the organism utilizes not only the globin moiety of hemoglobin in its nutritional requirements, but the heme moiety as well.

Fluorescent histochemistry of acid proteases in adult Schistosoma mansoni and Schistosoma japonicum.

The acid proteases, cathepsin B, dipeptidyl aminopeptidase I and II were demonstrated by fluorescent histochemistry in adult Schistosoma mansoni and Schistosoma japonicum. Alanyl- and leucine-aminopeptidase activities were not observed. The substrates consisted of 4-methoxy-beta-naphthylamide derivatives coupled to the fluorescent molecule, 5-nitrosalicylaldehyde. Reaction product was observed as discrete granules in the gastrodermis; activity was also observed in the testes and vitelline cells of males and females, respectively. No activity was noted in the foregut or esophageal glands of either worm. In light of previous biochemical studies on "hemoglobinase" activity in the schistosomes, it appears likely that a group of acid proteases may be involved in the ultimate digestion of hemoglobin. The absence of activity in the esophageal glands lends further support to the hypothesis that the gastrodermis is the site of the proteases responsible for hemoglobin digestion.

Cytochemical localization of peroxidase activity in the mitochondria of Hymenolepis diminuta.

A cytochemical study of mitochondria of Hymenolepis diminuta indicates the presence of a mitochondrial peroxidase. Utilizing a 3,3'-diaminobenzidine (DAB) medium at pH 9.7, the reaction product is localized in the intracristal space, and between the inner and outer membranes of the mitochondria. No inhibitory effects are exerted on the peroxidase reaction by cyanide, azide, or aminotriazole. In addition, the mitochondria appear to have an enzyme which is cytochemically similar to vertebrate cytochrome c-oxidase. The possible physiological significance of the peroxidase is discussed.

Immunocytochemical studies on Schistosoma mansoni. II. Soluble cercarial antigens in cercarial and schistosomules.

The localization of soluble, cercarial antigen preparation (CAP) in cercariae and schistosomules of Schistosoma mansoni was performed at the light and electron microscope levels using the unlabeled antibody method. Reaction produce was observed associated with the contents of the pre- and postacetabular glands and with the filamentous coat of mature cercariae. No reaction product was observed associated with the glycoacalyx of schistosomules. However, several schistosomules did retain remnants of their filamentous coats and reaction product was observed associated with those remains. CAP components were also observed in the area surrounding the intrasporocyst cercariae.

Schistosoma mansoni: ultrastructural studies on the esophageal secretory granules.

The posterior portion of the esophageal gland of Schistosoma mansoni produces a granule that is highly structured internally. Each granule consists of arrays of membrane-bound tubules enclosed by a membrane. Cytochemical tests indicate that the granules are not reactive for cytochrome c-oxidase but du react for macromolecular carbohydrates. It is believed that the granules are synthesized in the Golgi complex and are secreted at the base of the luminal amplifications of the esophagus. Colchicine treatment results in an accumulation of granules in the cyton region. Their physiological function is still undetermined, but it is hypothesized that they are involved with early stages of digestion of host red blood cells.

The effect of colchicine on translocation of incorporated [3H] proline in Hymenolepis diminuta.

Portions of adult Hymenolepis diminuta were exposed to a fixed concentration of colchicine (5 X 10(-4) M) in order to determine its effect upon incorporation of [3H] L-proline. Additional studies of the effect of colchicine upon tegumental morphology were performed. Autoradiographs showed a significant decrease in amount of incorporated label in the distal tegument of colchicine tissue and a heavy accumulation of label in the parenchyma. Radioassays indicated that the effect of colchicine on proline-incorporated protein was qualitative rather than quantitative suggesting that colchicine inhibits translocation in the tegument. It was hypothesized that microtubules within the internuncial processes facilitate movement of cell products from tegumentary cytons to the body surface.

In vitro effects of mannan and cytochalasin B on the uptake of horseradish peroxidase and [14C]sucrose by Trypanosoma cruzi epimastigotes.

Ultrastructural and quantitative studies were conducted to determine the in vitro effects of mannan and cytochalasin B (CB) on the transport of horseradish peroxidase (HRP) and [14C]sucrose by epimastigotes of Trypanosoma cruzi (strain Y). Time-dependent changes in HRP uptake were observed in cells incubated with the actin inhibitor CB. After 60 min incubation in CB, HRP and sucrose uptakes were inhibited by 48 +/- 15.4% and 16.5 +/- 3.96%, respectively. Morphological changed included HRP reaction product on the cell surface and a reduction in the number of HRP-positive reservosomes when compared to controls. After 120 min incubation, no inhibition was measured for either molecule. However, electron microscopy revealed HRP reaction product on the surface of the cells and in the cytosol. Also, perturbation of the plasma membrane was evident, suggesting that CB compromised the integrity of the plasma membrane, allowing HRP and sucrose to diffuse into the cytosol, giving misleading quantitative results. Mannan displayed a concentration-dependent inhibitory effect on HRP uptake but had little effect on sucrose uptake. Electron microscopic analysis revealed no change in the number of reservosomes per cell but reduction in the amount of HRP in reservosomes concomitant with mannan concentration. These results suggest that T. cruzi epimastigotes transport HRP by receptor-mediated and fluid-phase pinocytosis.

Detection of Schistosoma mansoni circulating cathodic antigen for evaluation of resistance induced by irradiated cercariae.

The appearance of serum levels of circulating cathodic antigen (CCA) detectable by a monoclonal antibody (mAb) (5H11) antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) system was evaluated during acute Schistosoma mansoni infections in female CF1 mice exposed to either 100 or 25 cercariae. Measurable CCA levels occurred in these groups at 5 and 7 wk after infection, respectively. The kinetics of appearance of CCA were thus related to the intensity of infection. The level of resistance developed by female C57BL/6 mice upon immunization with irradiated cercariae, as expressed by both worm burden and CCA levels after cercarial challenge was evaluated. Immunization conferred 44% protection against the challenge infection, and the level of CCA detected in the sera of the control group was significantly (P less than 0.02) higher than that found in the sera of the immunized group, 6 wk after challenge. These results demonstrate that CCA detection by the 5H11 mAb antigen-capture sandwich ELISA can reflect vaccine-induced resistance against S. mansoni. Localization studies showed that 5H11 reacts with a CCA epitope in the adult worm gut and to a lesser extent with the male tegument. Adaptations of this and other antigen detection systems may prove useful in monitoring the efficacy of developmental vaccines, an ability that may be essential for the extension of such studies to humans.