Schistosoma mansoni: production of cercarial eicosanoids as correlates of penetration and transformation

A method was developed, using a 0.25% agar matrix, to incorporate varying concentrations of linoleate and correlate cercarial transformation and eicosanoid production in vitro. Schistosoma mansoni cercariae were stimulated to penetrate over a wide range of linoleate concentrations; however, the transformation process occurred over a narrow range. Approximately 25% of cercariae penetrated the agar matrix in controls (no linoleate) and 0.003 mM linoleate. Penetration rates rose gradually until, at linoleate concentrations of 0.3 mM or greater, penetration approached 100%. The transformation process did not begin until the linoleate concentration in agar reached 2.0 mM (3.8%), and achieved maximum (91%) at 3.0 mM. A concentration of 9.0 mM linoleate gave 100% penetration and transformation rates, but penetration was superficial and cercariae were not viable. Cercarial eicosanoid production was concentration-related. Various eicosanoid classes were associated with cercarial penetration and transformation. Penetration rates were correlated with increasing leukotriene (LT, R = 0.9541) and hydroxyeicosatetraenoic acid (HETE, R = 0.8363) levels, while transformation rates correlated with increasing prostaglandin levels (R = 0.9225). Correlating eicosanoid production with penetration and transformation rates strengthened the hypothesis that successful cercarial penetration and transformation are dependent on both skin essential fatty acid levels and resulting cercarial eicosanoid production.     

Schistosoma mansoni: cercarial eicosanoid production and penetration response inhibited by esculetin and ibuprofen

Cercariae of Schistosoma mansoni produce a wide variety of eicosanoids when stimulated by 3.3 mM linoleate. High-performance liquid chromatography indicated that 10(-5) M esculetin dramatically decreased eicosanoid production by cercariae. Ibuprofen (10(-4) M) also decreased eicosanoid production, but to a lesser extent. These results were confirmed by radioimmunoassay using time-dose curves for esculetin and time curves for ibuprofen. The results reported here indicated that, for cercariae, ibuprofen was neither a specific cyclo-oxygenase inhibitor, as has been reported for platelet and endothelial cells, nor was esculetin a specific inhibitor of lipoxygenase, as has been reported for platelets and mastocytoma cells. Rather, both drugs inhibited cyclo-oxygenase and lipoxygenase enzyme systems. Further, the data indicated that two forms of cyclo-oxygenase exist in cercariae (isozymes?), one initiating the conversion of gamma-dihomolinolenate into the 1-series prostaglandins and another acting on arachidonate forming the 2-series prostaglandins. The cyclo-oxygenase acting on arachidonate has a greater sensitivity to both ibuprofen and esculetin than the enzyme acting on gamma-dihomolinolenate. Cercarial lipoxygenases also varied greatly in their sensitivity to ibuprofen.     

Schistosoma mansoni: mechanism of cercarial tail loss and its significance to host penetration

The mechanism of tail loss from cercariae of Schistosoma mansoni was investigated under experimental conditions. Tail loss from 90% or more of cercariae occurred in less than 10 min when packed organisms were incubated at 30 C in a minimal volume of water. Proteolytic secretions from the acetabular glands did not play a significant part in this process since the addition of known protease inhibitors nor the addition of secretions collected from other cercariae influenced the rate of the process. Tail loss was inhibited when the packed cercariae were incubated in saline at concentrations of 0.05 M or above though glandular secretion occurred at an equal rate in both water and saline. Tail loss was also inhibited by the chelating agents ethylenediaminotetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA). A marked feature of cercariae packed in water was their clumping and adhesion to the walls of the containing vessel by secretions from the postacetabular glands. Such clumping did not occur in saline or chelant solutions. It is suggested that the most probable mechanism for tail loss is a simple mechanical trauma effected by the movement of the tail acting against the resistance of the secretion-fixed body.During incubation under conditions which remove the “coat” from the body of the cercaria, the “coat” of the tail remains intact.     

The role of essential fatty acids and prostaglandins in cercarial penetration (Schistosoma mansoni)

We used rat skin membranes to test the putative role of prostaglandins (PG) and essential fatty acids (EFA) in the penetration response of Schistosoma mansoni cercariae. To examine the effects of EFA on cercarial penetration an EFA-deficient rat model was used. Dams were fed an EFA-deficient diet during lactation and the pups were weaned to this diet. Cercarial penetration of EFA-deficient rat skin membranes was not reduced from control levels until 12 wk on the diet. At this time a decrease of 64.3% was observed. This decrease remained constant up to 16 wk, after which the study was terminated. Other normal rats were treated with 20 mg/kg ibuprofen, a PG inhibitor, to examine the role of PG in the penetration response. Treated rat skin contained a mean of 2 micrograms ibuprofen per 30 mm3 of skin (25-mm skin disc) at 1.5 hours post-injection. Skin from treated rats inhibited penetration by over 81%. These studies indicate that skin EFA and PG may have a critical role in the completeness of penetration by cercariae through the skin, although it is not clear whether cercarial or host PG are involved in the penetration response.     

Schistosoma mansoni: inhibition of cercarial "penetration" proteases by components of mammalian blood.

1. Normal human sera and plasma were fractionated in order to identify inhibitors of the "penetration" proteases of Schistosoma mansoni cercariae. 2. The main inhibitor, accounting for 90% of the total activity of serum, appears to be alpha 1-antitrypsin (alpha 1-AT) as identified by separation on DEAE-cellulose and Sephadex, by immunoelectrophoresis and by anticercarial protease activity of purified alpha 1-AT preparations. 3. The inhibition profiles of purified preparations of the 6 known serum antiproteases suggest that the parasite protease is similar to vertebrate chymotrypsin. 4. On a molar basis, the order of inhibitory activity against the cercarial protease is: alpha 1-AT = alpha 2-macroglobulin; C'-1-inactivator; alpha 1-antichymotrypsin. No inhibition was obtained with inter-alpha-inhibitor or antithrombin III.     

Schistosoma mansoni: analysis of cercarial transformation methods

Four methods of transforming cercariae to schistosomulae in vitro in ELAC buffer (pH 7.2, 37 C, 0-6 hr incubation) were compared in relation to biochemical and ultrastructural characteristics. The transformation methods used were chemical (3 mM linoleate), mechanical (centrifuge/vortex), mechanical/chemical, and heat (incubation at 37 C). Ultrastructural characteristics examined were based on the presence or absence of glycocalyx, heptalaminate membrane, cyton granules, and nuclear condition. Two EM fixation methods were used. Biochemical parameters assayed were loss of water tolerance (uptake of trypan blue dye), eicosanoid biosynthesis (PGE, LTB4, and 5-HETE), protein synthesis (leucine uptake), RNA synthesis (uracil and orotic acid uptake), and DNA synthesis (thymidine uptake). EM characteristics were remarkably similar for all transformation methods except heat incubation, with transformed cercariae evidencing the characteristics of schistosomulae (cyton granule migration, absence of glycocalyx and heptalaminate membrane); however, euchromatic nuclei could not be demonstrated using in vivo or in vitro transformation methods. Despite the ultrastructural similarities between transformation methods, biochemical data demonstrated that the resultant organisms were quite different. The chemical transformation method gave the highest rate of loss of water tolerance and eicosanoid production. RNA and protein synthesis were not correlated to ultrastructural changes and were highest in those organisms undergoing mechanical transformation methods, significantly higher than in those cercariae transformed by the chemical method. DNA synthesis was not demonstrated using any transformation method, although thymidine uptake did occur. Our data indicate substantial biochemical differences exist between morphologically similar organisms. Thus, experiments using any type of artificially transformed schistosomule must be interpreted with caution until additional biochemical and physiological studies on cercarial transformation are undertaken.     

Schistosoma mansoni: development of the cercarial glycocalyx

The development of the cercarial glycocalyx of Schistosoma mansoni was studied by transmission electron microscopy and immunofluorescence light microscopy employing antibodies raised against extracted and chromatographed glycocalyx. By electron microscopy, cercariae present in the brood chamber of daughter sporocysts were surrounded by an electron-dense granular and fibrillar matrix. This material appeared structurally distinct from the glycocalyx which was coarsely fibrillar and located only on the surface of organisms that had developed a final tegument. The thickness of the glycocalyx apparently increased with the maturation of the tegument, since teguments that had many spines also had the thickest glycocalyx. Immunofluorescent staining of frozen sections of infected snail hepatopancreas showed that glycocalyx antigens were present on the surface of the cercariae and not in the matrix within the brood chamber or in snail tissues. Immunofluorescent staining of isolated larval cercariae showed staining of some but not all parasites with partially elongated tails. These studies suggest that the glycocalyx develops late in cercarial development (late in Stage 6 or in Stage 7 of Cheng and Bier), is made by the cercariae themselves, and is not a product of either the sporocyst wall cells or snail hepatopancreas.     

Schistosoma mansoni: ultrastructure of cercarial escape glands

While in the sporocyst, the cercaria of Schistosoma mansoni has a pair of unicellular escape glands whose funduses are located together on one side of the body. The funduses taper into microtubule lined ducts that open on the anterior surface of the oral sucker through desmosome supported pores in the immediate vicinity of the ducts of the acetabular glands. The glands contain membrane-bound secretory granules which have a fine medium-dense, homogeneously granular material in their matrices. A large membrane-bound reservoir of granular material, whose texture is similar to that of the secretory granules, is often seen in the cytoplasm of the funduses. In free-swimming cercariae, the ducts in the oral sucker are the only obvious remains of the escape gland.     

Schistosoma mansoni: correlations between mouse strain, skin eicosanoid production, and cercarial skin penetration

We have previously reported that cercarial penetration is highly correlated with cercarial production of leukotrienes (LT's) and hydroxyeicosatetraenoic acids (HETE's). Because skin also produces various eicosanoids, we undertook an investigation of skin eicosanoids in various strains of mice and 1 strain of rat in order to ascertain if skin eicosanoids could be correlated to cercarial penetration. SENCAR, ICR, NMRI, A/J, C3H/HeJ, C57Bl/6, ASEBIC, and BALB/c mouse strains were used in this study as well as the SD-Rat strain. The ability of cercariae to penetrate skin was strain specific. A/J and SENCAR mice had the highest penetration rates (approximately 98%), whereas the SD-Rat strain had the lowest (43%). These penetration rates we linearly correlated with tail skin HETE production at 10 min (R = 0.826), whereas HETE production at 60 min had a parabola-shaped relationship (R = 0.793). The primary infection of mice with Schistosoma mansoni cercariae may therefore be directly correlated with both the skin's innate ability to synthesize HETE, as well as with cercarial eicosanoid production, especially HETE levels. However, we believe that skin eicosanoid production is just one of many factors affecting cercarial skin penetration. Other factors discussed are: skin surface fatty acid levels, cercarial eicosanoid production, epidermal vs. dermal eicosanoid production, and the immunocompetence of the host.     

Schistosoma mansoni: fine structure of cercarial acetabular glands

Emerged cercariae of Schistosoma mansoni were studied with the electron microscope for the purpose of describing the acetabular (penetration) glands and their cellular investment. The two pre- and three postacetabular unicellular glands consist of enlarged aboral areas (funduses) and their oral extensions as ducts. The glands were morphologically similar except for their shape and secretory globules. In the funduses of the postacetabular glands the globules were of a single type, spheroidal to irregular in shape, with numerous electron-dense areas. Preacetabular secretory globules appeared to be of several types, varying in size, shape, and homogeneity. Some were of uniform density; others showed electron-lucid areas. The fine structure of both pre- and postacetabular globules changed as they were compared in the funduses, in progressively oral areas of the ducts and after extrusion. These changes were thought to be transitional. Microtubules and close cellular investiture of the glands by muscle, nerve, and unclassified cells with extended interwoven processes appeared to provide structural support.     

Schistosoma mansoni: immunogenic glycoproteins of the cercarial glycocalyx

Immunochemical studies at the level of the light and electron microscope showed that a monoclonal antibody, 128C3/3, was directed to an epitope in the glycocalyx of Schistosoma mansoni cercariae. Immunoprecipitation of surface labeled cercarial extracts with this monoclonal antibody demonstrated that the glycocalyx is composed of at least five components, including a very large molecular size polypeptide and polypeptides of 220, 180, 170, and 15 kDa. After transformation of cercariae to schistosomula, these polypeptides were shed from the surface and were therefore no longer accessible to surface labeling. Monoclonal antibody 128C3/3 was also reactive with a 38 kDa polypeptide from schistosomula; this polypeptide was weakly expressed on the surface of cercariae. Analysis of immunoprecipitates of radioiodinated protein extracts of cercariae, newly transformed schistosomula, and 36 hr in vitro cultured schistosomula showed that the 180 and 170 kDa polypeptides continued to be expressed within the organism following transformation, but were not accessible to surface labeling. Lectin binding studies revealed differences in the oligosaccharide composition of the six polypeptides. With the exception of the 15 kDa antigen, all the polypeptides reactive with 128C3/3 were highly immunogenic in infected mice and humans.     

Schistosoma mansoni: a comparative study of artificially transformed schistosomula and schistosomula recovered after cercarial penetration of isolated skin.

A comparison was made of the ultrastructure, development and antigenic nature of the surfaces and of the viability of three types of Schistosoma mansoni: schistosomula formed after cercariae had penetrated isolated skin (SS) schistosomula produced after mechanical separation of cercarial tails from bodies (MS), and schistosomula transformed from cercariae after incubation in fresh rat serum (RS). Within 2h of transformation, the surface membranes of all three types of schistosomula had changed from trilaminate to heptalaminate structures and SS and MS had lost their cercarial glycocalyx. Initially a dense amorphous material was demonstrated on the surfaces of RS, which was thought to be the result of an interaction between a factor in rat serum and the glycocalyx; this material was greatly reduced within 2 h of transformation. The pre-acetabular glands of SS were emptied while those of MS and RS retained their contents. Immunofluorescent studies showed that all schistosomula bound serum from mice immune to S. mansoni, but the binding was stronger with MS and RS. The mixed agglutination reaction demonstrated the presence of human A and B blood group-like antigenic determinants on approximately 30% of 3h old SS; these determinants were not detected on MS or RS. In vitro, the development of MS and RS was similar to SS; the first schistosomula reached the "gut-closed" stage by day 10; 50-70% of SS reached this stage by day 12, in contrast to only 25-50% of MS and RS. Between 28 and 45% of all schistosomula developed to maturity when injected intravenously into mice. It was concluded that the two types of artificially prepared schistosomula fulfil the main criteria of transformation from cercaria to schistosomulum. Further, it is suggested that MS are the most appropriate source of material for immunochemical and physiological studies.     

Schistosoma mansoni: complement and cercarial tail loss during in vitro transformation.

When Schistosoma mansoni cercariae are incubated at 37 C in media containing serum, the organisms lose their tails and change into viable, infective schistosomula. Tail loss does not occur in the absence of serum, or when the serum is heat inactivated. In the present studies, tail loss during in vitro conversion was shown to be complement dependent. The capacity of fresh serum to promote tail loss was markedly suppressed or abolished by cobra venom factor, zymosan, Sepharose CL-4B AND anti-C3 antibody. The alternative rather than the classic complement pathway appeared to be responsible since (1) binding of anti-C3 to cercariae required magnesium, but not calcium; (2) both C4-deficient serum and C2-deficient serum supported tail loss; but (3) human serum heated to 50 C for 20 min to inactivate Factor B did not support tail loss. Cercarial tail loss also required the terminal complement components C5 through C8. The extent and rate of tail loss was normal in agammaglobulinemic sera indicating that the complement effect was not antibody dependent.     

Schistosoma mansoni: relationship between cercarial production levels and snail host susceptibility

Two populations of Biomphalaria glabrata snails differing slightly in their susceptibility to Schistosoma mansoni infection showed dramatic differences in cercarial output per snail. Exposed to five or more miracidia, snails from a group with a 90-100% susceptibility rate (Group A) produced nearly twice the number of cercariae as those from a group with a 70-80% susceptibility rate (Group B). Exposure of individual snails to known numbers of miracidia resulted in higher numbers of primary (mother) sporocysts in Group A snails than in Group B snails. However, monomiracidial exposure of snails from both groups resulted in equivalent numbers of cercariae produced per positive snail, indicating that, once established, all primary sporocysts possess a similar reproductive potential. Morphometric analysis of serially sectioned 9-day-old primary sporocysts supported this conclusion; the size of the primary sporocysts and the size and numbers of secondary (daughter) sporocysts within each primary sporocyst were comparable in snails from both groups. The data indicate cercarial production in this system is regulated prior to, and/or during, early development of the primary sporocyst.     

Schistosoma mansoni: penetration apparatus and epidermis of the miracidium

Free swimming miracidia of Schistosoma mansoni were studied with the electron microscope for the purpose of describing the penetration apparatus and the epidermis. The penetration apparatus was composed of 3 unicellular glands which contain membrane-bound vesicles containing macromolecular diglycols. Each gland contained a nucleus, rough endoplasmic reticulum, Golgi complexes, numerous mitochondria, and glycogen stores. Each gland cell opened to the exterior through the apical papilla.The surface of the miracidium, with the exception of the apical papilla, was covered with ciliated epidermal cells containing numerous mitochondria, membranous bodies, and glycogen. No nuclei were detected within these epidermal cells. Intercellular ridges connecting with subepidermal cytons interrupted the epidermal cells at numerous points. The ridges were joined to the epidermal cells by septate desmosomes. Beneath the epidermal cells were found circular and longitudinal muscle bundles.Sensory structures of various types were associated with the outer covering. These consisted of (1) numerous “knob-like” cytoplasmic projections associated with epidermal cells, (2) bulbous, lamelloid structures with external cytoplasmic projections, and (3) ciliated nerve endings with posterior epidermal tiers and ciliated nerve pits associated with apical papilla.     

Schistosoma manonsi: cercarial penetration of host epidermis at the ultrastructural level

Invasion of the outer layers of the epidermis of mouse ear skin by cercariae of Schistosoma mansoni within 7 min of their application to it has been studied with the optical and the scanning and transmission electron microscopes.Entrance of cercariae was under the edges of the dead flattened keratinized cells of the horny layer (squames), and penetration through this layer was by disarticulation of the stacks of squames at their interdigitations. Mucus from the postacetabular glands was recognized with the light and electron microscopes on the skin surface, especially at squame edges and between layers of squames and along the keratogenous zone. The findings suggested that disarticulation of the squames was not effected solely by the muscular probing and pushing of the parasite, but that it might be aided by swelling of the mucus secretion deposited in the area from the postacetabular glands. Loosening of the interlocked edges of the squames by enzymatic action is also a possibility, but was not evaluated for this report.The migration path along the keratongenous zone was marked by extensive damage to the transitional cells of the granular layer subjacent to the squames. Packets of secretion from the cercarial preacetabular glands were identified below the horny layer in the cytoplasm of these cells. It was considered that the host tissue damage in this area was the result not only of tearing of the tissues by passage of the spiny schistosomules, but also of enzymatic activity, the enzyme source being the granules in the packets of preacetabular gland secretion.     

Schistosoma mansoni: the role of calcium in the stimulation of cercarial proteinase release.

We investigated the role of calcium mobilization in the induction of proteinase release from cercarial preacetabular glands. Proteinase release was measured by the ability of cercariae to break down a 3H-labeled proline extracellular fibroblast matrix and calcium influx was measured using 45Ca2+. The role of calcium in the activation of cercarial proteinase was examined by investigating the effects of calcium addition and removal on linoleate-induced matrix degradation, the ability of various calcium modulators (Verapamil, fendiline, nifedipine, SK-525A, BAY K-8644, Ryanodine, and SK-7171A) to stimulate or inhibit linoleate-induced proteinase release, the ability of calcium modulators directly to induce cercarial proteinase release, and the ability of various stimulants of proteinase release to induce calcium influx or efflux from cercariae. The results of these studies indicate that proteinase release is dependent on external calcium concentration, voltage-operated channels are either nonexistent in cercariae or have a minimal role in overall calcium influx, and that activation of Ca2+ influx can be caused by both free fatty acids and calcium modulators by a hypothesized receptor-operated channel. Although calcium uptake is important in cercarial proteinase release, it is not the only factor involved. Calcium uptake alone does not guarantee that proteinase will be secreted. On the other hand, if Ca2+ influx does not occur, proteinase will not be secreted.