John P. Caulfield

Schistosoma mansoni: synthesis and release of phospholipids, lysophospholipids, and neutral lipids by schistosomula.

Lipids in the two surface membranes of Schistosoma mansoni may play an important role in the parasites defense against host immunity. In particular, lysophosphatidylcholine lyses erythrocytes attached to the parasite and alters the lateral mobilities of their membrane proteins and lipids (Golan et al. 1986). Here, we have studied the incorporation of radiolabeled precursors into the major lipid classes of schistosomula as well as into lipids released by schistosomula into the medium. Radiolabeled polar head groups (choline and ethanolamine) and fatty acid precursors (palmitate and oleate) were linearly incorporated into parasite phospholipids. Fatty acids were differentially incorporated into the various phospholipid classes, principally into phosphatidylcholine and, to a lesser extent, into phosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine. The major neutral lipid class labeled, triglycerides, had a decrease in specific activity with time after pulse labeling and the specific activity of the phospholipids increased with time. Thus, triglycerides may provide acyl chains for phospholipid synthesis. Choline was incorporated into phosphatidylcholine and lysophosphatidylcholine, and ethanolamine into phosphatidylethanolamine and lysophosphatidylethanolamine. No evidence was found for phospholipid methylation or demethylation in schistosomula. Labeled lipids were linearly and selectively released into the medium. Triglycerides were released at the highest rate with measurable quantities of phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine also observed. Monopalmitoylphosphatidylcholine was the only lysophosphatidylcholine present in the medium as demonstrated by reverse-phase chromatography of released choline-labeled lysophosphatidylcholine. These studies demonstrate that schistosomula synthesize phospholipids and neutral lipids and release some of them into the culture medium. In particular, they release a single molecular species of a potent biologically active molecule, monopalmitoylphosphatidylcholine, that may play a role in the parasites evasion of the immune response.

ULTRASTRUCTURE, CARBOHYDRATE, AND AMINO ACID ANALYSIS OF TWO PREPARATIONS OF THE CERCARIAL GLYCOCALYX OF SCHISTOSOMA MANSONI

This study determined the amino acid and carbohydrate composition of 2 cercarial glycocalyx preparations obtained after phenol-water extraction and subsequent gel chromatography. Labeled cercariae were subjected to 85% phenol, thereby dissociating the glycocalyx into the aqueous phase, which was dialyzed and chromatographed on Sepharose CL-2B or -4B in either 4 M guanidine hydrochloride (GuHCl) or 0.1% sodium dodecyl sulfate (SDS). The label eluted primarily in the void volume and was antigenic as tested with rabbit polyclonal antibodies by immunoblotting. Approximately 6 micrograms of protein and 28 micrograms of carbohydrate were obtained from 10(5) cercariae in the antigenic void volume fraction after SDS chromatography. Threonine, serine, and glutamic acid comprised 44% of the amino acid residues of the protein. The predominant sugar was fucose. Galactosamine, glucosamine, galactose, and mannose were also detected. After GuHCl chromatography, free amino acids, predominantly glycine and serine, comprised 17% of the total protein. The carbohydrate composition was similar to that of SDS-chromatographed extracts. Phenol-water extracts eluting in the void volume of Sepharose CL-2B were compared by negative staining and scanning electron microscopy with material obtained from medium in which cercariae shed glycocalyx during transformation to schistosomula. Both the isolated material and the transformation medium contained particles 20-50 nm in diameter, with subunits of 15-20 nm. These studies show that the cercarial glycocalyx is particulate, contains mainly carbohydrate and some protein, and is solubilized by phenol-water extraction.

Schistosoma mansoni: Sterol and phospholipid composition of cercariae, schistosomula, and adults

The sterol and phospholipid composition of cercariae, schistosomula, and adult Schistosoma mansoni was analyzed by gas-liquid chromatography and high-performance liquid chromatography (HPLC). Cercariae and schistosomula contained cholesterol, desmosterol, campesterol, stigmasterol, and beta-sitosterol while adults contained only cholesterol. In all stages cholesterol comprised greater than 50% of the total sterols, and in cercariae and schistosomula desmosterol comprised 38 and 21% of the total sterols, respectively. The other three sterols, campesterol, stigmasterol, and beta-sitosterol, made up approximately 10% of the total. The same five sterols found in cercariae and schistosomula were present in the hepatopancreas of uninfected snails but with a much higher desmosterol concentration in the parasite, 38%, than in the snail, 2%. As in cercariae and schistosomula the three minor sterols comprised approximately 10%. Thus, the sterol composition of cercariae and schistosomula was similar but not identical to that of the snail host. Phosphatidylcholine was the major phospholipid of all three stages (50%) as determined by two HPLC procedures. The remaining phospholipids consisted of phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. In addition, in adults there were small quantities of sphingomyelin and lysophosphatidylcholine. The percentage of each phospholipid was similar among stages with the exception of a slight increase in phosphatidylserine in adults compared to cercariae and schistosomula. These results show that a characteristic lipid composition is found in cercariae, schistosomula, and adults.

Schistosoma mansoni: post-transformational surface changes in schistosomula grown in vitro and in mice.

Abstract The development of schistosomula during the first 4 days after transformation from cercariae has been examined in parasites isolated from the lungs of mice and in organisms cultured in lactalbumin and rabbit serum or in the defined serum-free medium, RPMI 1640. The development of organisms grown under all three conditions was the same. Schistosomula increased in length from 67 to 110 μm and decreased in width from 24 to 18 μm, so that the volume remained constant at approximately 2.7 × 104 μm3. The increase in length occurred mainly in the torso or posterior three-quarters of the worm which increased from 49 to 88 μm or 80%, whereas the head increased from 18 to 22 μm or 22%. The spines were lost from the surface that was most rapidly lengthening by gradual resorption into the tegument and were replaced by pits mainly during the first 3 days. These changes resulted in a 325% increase in the surface area of the schistosomula, from 1.2 × 104 to 3.9 × 104 μm2. In addition, the openings of the acetabular ducts, the ventral sucker, and the tail socket all became smaller and flatter over the four-day period. Internally, the major changes were the loss of the acetabular ducts in the pre- and post-acetabular glands and an increase in size of the caecum. In summary, these experiments show that the surface of the schistosomulum is extensively remodeled before intravascular migration occurs and demonstrate the efficacy of RPMI 1640 as a culture medium for schistosomula in the first 4 days after transformation.

Schistosoma mansoni: Immunization with cercarial glycocalyx preparation increases the adult worm burden

Cercariae are covered by a thick glycocalyx (GCX) that activates complement by the alternative pathway and is recognized by antibodies in the serum of infected animals (Standen 1952; Samuelson and Caulfield 1985). Further, it contains antigens reactive with protective monoclonal antibodies produced in rats and mice (Grzych et al., 1982; Ham et al. 1984). Recently, Veira et al. (1986) found that cercarial transformation fluid, which contained GCX and other cercarial components, suppressed the in vitro proliferative response of human peripheral blood lymphocytes to both schistosome antigens and mitogens. Despite this evidence that GCX is immunologically relevant in schistosome infections, its effect as a primary immunogen has not been studied. We have recently characterized GCX prepared with sodium dodecyl sulfate (SDS) or guanidine hydrochloride (GuHCl) as solubilizing agents (Caulfield et al. 1987). Both preparations were rich in carbohydrate, 40% for GuHCl-GCX and 80% for SDS-GCX, with fucose as the major sugar. Here we injected mice with pooled fractions of either preparation of GCX, using phosphate-buffered saline (PBS), Maalox, or Bacille Calmette-Guerin (BCG) as carriers and/or adjuvants. We assessed the effect of immunization with GCX by comparing the numbers of adult worms recovered (worm burden) be mesenteric vein perfusion (Stirewah et al. 1951; Warren and Peters 1967) in GCXand sham-immunized mice 6 weeks postcercarial challenge. The number of adult worms recovered from mice was dramatically different between GuHCl-GCX and sham-immunized groups. As shown in Table I, animals injected with GuHCl-GCX in either PBS or Maalox had increased adult worm burdens in ah four experiments. The data were compared statistically using a stratified Wilcoxon’s test (Lagakos 1982; Table I). The statistical significance of the difference between the GuHCl-GCX-immunized and sham-immunized controls was 0.02 < P < 0.01. Thus, GuHCl-GCX when administered with PBS or Maalox significantly increased the number of surviving adult worms at 6 weeks postinfection when compared to shamimmunized controls. When BCG was administered with GuHCI-GCX, we found that the adult worm burdens were the same as in mice which received BCG alone. Mice immunized with SDS-GCX gave very different results from mice immunized with GuHCl-GCX. In two separate experiments with SDS-GCX, the adult worm burdens of SDS-GCX-immunized and shamimmunized controls were not significantly different when PBS or Maalox were used as adjuvants. Thus, immunization with SDS-GCX did not affect the number of surviving adult worms when compared to control-immunized mice. Several immunological mechanisms could explain the increase in numbers of adult worms recovered in mice immunized with GuHCl-GCX. Immunization with GuHCl-GCX may have suppressed cellular immunity, as suggested by Veira et al. (1986). Other studies have also demonstrated that schistosome materials suppressed the proliferative response of peripheral blood lymphocytes in vitro (Dessaint et al. 1977; Colley et al. 1979; Camus et al. 1981). Significantly, egg antigens, some of which cross-react with the cercarial surface (Ham et al. 1984), have been implicated in the suppression of the cell-mediated immune response (Boros et al. 1975; Colley 1975; Naggar and Colley 1982). Alternatively, GuHCl-GCX-immunized mice may have produced blocking antibodies which would bind to and protect invading larval parasites. Blocking antibodies to cercarial tegumental epitopes have been demonstrated in the rat model, both in vitro and in vivo (Grzych et al. 1982; Khahfe et al. 1986). Lastly, immunization with GuHCl-GCX may have induced a state of tolerance with respect to the carbohydrate antigens in GCX, analogous to that induced

LIGHT AND ELECTRON MICROSCOPIC APPEARANCE OF RAT PERITONEAL MAST CELLS ADHERING TO SCHISTOSOMULA OF SCHISTOSOMA MANSONI BY MEANS OF COMPLEMENT OR ANTIBODY

Metrizamide-gradient purified rat peritoneal mast cells were allowed to adhere to schistosomula of S. mansoni that had been preincubated in either minimum essential medium with 5% fetal calf serum (MEM/FCS), heat-inactivated serum from rats 8 weeks after cercarial infection (IRS) fresh normal rat serum as a source of complement (NRS), or IRS and NRS. Adherence was evaluated in sections 0.3 micrometers thick. The preincubation conditions favoring cell adherence were IRS + NRS greater than NRS greater than IRS greater than MEM/FCS. Greater adherence was seen at 60 min than at 10 min. Discharge of mast cell granulates was seen infrequently, was not greater in adherent than nonadherent cells, was not increased by any preincubation condition, and did not occur against the parasites surface, as would be expected if antigen, antibody, and surface receptors were aggregated there. Electron microscopic examination showed that attachment to the parasite occurred through electron-dense material which was usually fibrillar in appearance. Membrane fusion, such as is seen between human neutrophils and schistosomula, did not occur.

Schistosoma mansoni: Ultrastructural demonstration of a miracidial glycocalyx that cross-reacts with antibodies raised against the cercarial glycocalyx

Cercariae are covered by a glycocalyx that is highly antigenic. Here, we have examined the surface of miracidia for a similar structure. The miracidia are covered by epithelial plates and syncytial ridges. By transmission electron microscopy, the plates and ridges were covered by a 0.5-micron-thick glycocalyx composed of a mesh of 9- to 10-nm fibrils that were stained by ruthenium red delivered in the aldehydes or ferrocyanide-reduced osmium tetroxide. Rabbit antibodies prepared against phenol extracted and chromatographed cercarial glycocalyx were detected by immunoelectron microscopy with secondary antibodies conjugated to horseradish peroxidase. Reaction product bound to both the miracidial and cercarial glycocalyx. In addition, the outer leaflets of the cercarial tegumental membrane and membranes of the miracidial surface structures, including plates, ridges, terebratorium, and sensory papillae, had reaction product. Controls incubated with nonspecific rabbit serum had no reaction product. By indirect immunofluorescence, antibodies against the cercarial glycocalyx stained both plates and ridges. As the miracidia transformed to sporocysts, the glycocalyx remained associated with the plates as they were sloughed. These studies demonstrate that miracidia possess a glycocalyx similar in structure and antigenicity to the cercarial glycocalyx.

Video Microscopy of Swimming and Secreting Cercariae of Schistosoma mansoni

termined with accuracy. However, E. monacis and E. perforoides oocysts sporulated first with about 85% sporulating after 5 days incubation while E. os took a slightly longer time (about 6.5 days). Eimeria tuscarorensis required about 20 days for a 50% sporulation. A short sporulation time may be a factor for the relative abundance of E. monacis and E. perforoides. Depending upon the time of year, between 77% and 90% of the oocysts were E. monacis and E. perforoides. In contrast, sporulation in E. tuscarorensis required at least 4 times longer than E. monacis and E. perforoides. The relatively long sporulation period would require more time for E. tuscarorensis to achieve sufficient levels of infective oocysts in the environment to produce an increase in oocyst numbers in the host. Furthermore, E. tuscarorensis oocysts were relatively more prevalent during the warmdry summer weather months (May-August) suggesting that the thick outer oocyst wall may resist desiccation and extend viability. Eimeria os were more prevalent in the Spring and Fall, suggesting less resistance to desiccation or heat. Indeed, E. os oocyst appeared to deteriorate much sooner than other species when incubated at 28-30 C in 2.5% potassium dichromate. The deterioration usually started at the region around the micropyle. In conclusion, coccidial parasites were abundant in the woodchuck population of southern Illinois. Although E. monacis and E. perforoides were the species which predominated through the non-hibernating months of the year, E. os and E. tuscarorensis appeared to show seasonal