John A. Oaks

ISOLATION AND PARTIAL BIOCHEMICAL CHARACTERIZATION OF THE BRUSH BORDER PLASMA MEMBRANE FROM THE CESTODE, HYMENOLEPIS DIMINUTA*

A procedure was devised to remove the tegument from the cestode, Hymenolepis diminuta, using 0.2% Triton X-100. The brush border fragments were collected separately from cytoplasmic vesicles by differential centrifugation, and the brush border plasma membrane was further enriched by disruption in high osmolarity Tris-HC1 buffer. The distribution of plasma membrane was quantified by comparing the specific activity of 3H-concanavalin A in all fractions following incubation of intact organisms with the radioactive lectin. SDS-polyacrylamide gels stained with Coomassie Blue reveal no qualitative differences between plasma membranes and vesicles. However, when gels are stained with Stains-all, 7 proteins of the plasma membrane stain blue, whereas the vesicular counterparts stain red. Treatment of plasma mem- brane with calf intestinal alkaline phosphatase results in a decrease in the intensity of 5 of the blue- staining bands indicating that these proteins are phosphoproteins. Membrane-bound alkaline phosphatase is resistant to dodecyl sulfate plus dithiothreitol and was detected histochemically in a region correspond- ing to 104,000 daltons following SDS polyacrylamide gel electrophoresis. Lactoperoxidase catalyzes the iodination of 7 externally oriented plasma membrane macromolecules, 6 of which stain as glycoproteins with PAS. The incorporation of 125I-iodide increases approximately 10-fold when the organisms are prein- cubated in buffer for 30 min. This suggests that either absorbed proteins are being removed or that, in the absence of host proteases, these external macromolecules are accumulating in the plasma membrane with time.

IMMUNOCYTOCHEMICAL LOCALIZATION OF THE MAJOR GLUTATHIONE S-TRANSFERASES IN ADULT SCHISTOSOMA MANSONI

Indirect immunofluorescence was used to investigate the tissue distribution of the major isoenzymes of Schistosoma mansoni glutathione S-transferase (GSH S-transferase). When polyclonal rabbit antisera against GSH S-transferase isoenzymes SmGST-1, -02, and -3 were applied to cryostat or plastic-embedded sections of fixed adult worms, a punctate pattern of enzyme distribution was observed that was restricted to the parenchyma. Labeling was much more pronounced in males than females, consistent with the biochemically determined distribution of these enzymes between the sexes. Intense immunolabeling was noted within the subectocytoplasmic core tissue of the tubercles of the male that appeared to be connected to deep parenchymal cells by immunoreactive cell processes. Immunofluorescence could be blocked completely by prior incubation of antisera with affinity-purified enzyme. Although schistosome GSH S-transferases have been reported to be protective antigens, no immunoreactivity was detected within or on the tegument, including the dorsal spines of the male. The lack of tegumental immunoreactivity was confirmed by immunoblotting of tegumental membrane preparations following SDS-PAGE. Muscle fibers, vitelline cells, and cecal epithelium also failed to react. The fact that the GSH S-transferases were not uniformly distributed among all parenchymal cells suggests the existence of subpopulations of parenchymal cells that are preferentially involved in the conjugation of electrophiles with glutathione.

Disruption and removal of the tegument from Schistosoma mansoni with triton X-100.

Tegumental membranes of Schistosoma mansoni were disrupted by 0.2% Triton X-100 in Tris-maleate buffered/Kreb-Ringers solution. Subsequent differential centrifugation of the disruption solution at 2,500 g and 30,000 g produced two pellets which contained membrane components. Examination of the carcass by scanning electron microscopy revealed that most of the exposed tegument of both male and female worms was removed, while surface membrane protected by close apposition of another surface (i.e., in the gynecophoral canal) remained intact. The parenchymal tissue (e.g., subtegumental muscle and tegumental perikarya), excretory and gut epithelia, and the teguments basement membrane also remained intact. The selectivity of the disruption suggests that membrane in both pellets originated almost exclusively from the tegument. Although larger morphological features (i.e., surface crypts) present in the intact tegument did not maintain their form in the 2,500 g pellet, the high specific activity of 3H-concanavalin A retained by this fraction, and the presence of numerous spines and large pieces of membrane, suggest that the 2,500 g pellet contained most of the worms disrupted surface membrane. Transmission electron microscopy demonstrated the presence of dense spinelike material and vesicles of various sizes and densities, as well as some mitochondria in the 30,000 g pellet. Low specific activity of 3H-concanavalin A in the post-30,000 g supernatant suggests that relatively few externally oriented, saccharide-containing molecules were solubilized from tegumental membranes by Triton X-100.

Ileal mucosal mast cell, eosinophil, and goblet cell populations during Hymenolepis diminuta infection of the rat.

The population dynamics in the enteric connective tissues of eosinophils, mucosal mast cells (MMC), and in the mucosal epithelium of goblet cells were examined morphometrically in fixed ileal tissue of outbred Sprague Dawley rats during the first 32 days of infection with the tapeworm Hymenolepis diminuta. MMC and eosinophils were present in the lamina propria and submucosa; however, only eosinophils were also present in the muscularis externa. Eosinophilic infiltrate was first observed in the lamina propria at 15 days postinfection (dpi) and the numbers of eosinophils remained elevated through 32 dpi. Initial mucosal mastocytosis was detected on 6 dpi and MMC numbers continued to rise over the study period without reaching a plateau. Goblet cell hyperplasia occurred only at 32 dpi. In contrast to some intestinal nematode infections where these same 3 cell types are associated with the hosts expulsion responses, H. diminuta is not lost by a rapid host response in the outbred Sprague Dawley rat strain used in these experiments. We suggest that either the induction of hyperplasia of these host effector cells in ileum tissue during H. diminuta infection is not capable of triggering parasite rejection mechanisms, or the function of the induced hyperplasia is necessary for some as yet unassociated physiological or tissue architecture change in the hosts intestine.

Dirofilaria immitis: molting process of third-stage larvae.

The objective of this study was to determine the molting process of Dirofilaria immitis third-stage larvae (L-3) to fourth-stage larvae (L-4), as it occurred in vitro. After 48 hr in vitro, the L-4 epicuticle was completely formed, and by 72 hr there was a clear separation between the L-3 and L-4 cuticles. The thickness of the newly formed L-4 cuticle was significantly less than that which has been described for larvae recovered from dogs after a similar incubation time period. If culture conditions were lacking in bovine albumin or proper temperature, larvae successfully developed the L-4 epicuticle but did not complete ecdysis. The molting process of D. immitis L-3 was thus shown to be multistepped with different factors required to induce the various developmental phases.

Ultrastructure of the tegumental microvilli (microtriches) of Hymenolepis diminuta

SummaryThe ultrastructure of microtriches of the rat tapeworm, Hymenolepis diminuta, was examined with a number of electron-microscopic techniques. Fixatives containing different buffers, non-ionic detergents, chelators, tannic acid and various concentrations of aldehydes were tested for ability to stabilize cytoskeletal components while extracting background material. These methods revealed features unique to these specialized microvilli, and permitted construction of a detailed model of microthrix architecture. The microtriches of H. diminuta are comprised of a microfilament-containing base, a dense cap and a complex junctional region between the base and cap. The microfilaments of the base are contiguous distally with a tubular structure (the junctional tubule) within the junctional region; proximally, the microfilaments end abruptly: a terminal web appears to be absent. A beveled bilayered cylinder of dense material (the core tunic) encircles the microfilamentous core. The core tunics and junctional tubules of the microtriches are specifically and uniformly aligned along the strobila. Microtriches therefore can be distinguished from other microvilli (e.g., those of enterocyte brush borders) by their complex ultrastructure and precise orientation upon the cytoplasmic surface.

A simple method of obtaining an enriched fraction of tegumental brush border from Hymenolepis diminuta.

A method for isolating an enriched preparation of tegumental brush border from the tapeworm, Hymenolepis diminuta, is described. Combining incubation of whole tapeworms in Krebs-Ringer/tris-maleate solution containing a hemolytic saponin, low shear-force agitation, and differential centrifugation, a pellet is obtained at 2,500 g which contains a significant concentration of surface brush border. The content of brush border in this fraction is identified by the presence of numerous microvilli, increased specific radioactivity after surface tagging with 3H-Concanavalin A, and relatively little mitochondrial contamination (succinic dehydrogenase). Based on morphological criteria, fractions sedimenting with greater force contain dense vesicles and mitochondria from the outer portion of the tegument.

Hymenolepis diminuta fractions but not previous tapeworm infection stimulate intestinal myoelectric alterations in vivo in the rat.

Infection of rats with the enteric, lumen-dwelling tapeworm Hymenolepis diminuta causes electric changes in host intestinal smooth muscle and decreased luminal transit. The mechanisms that stimulate host intestinal alterations during this nontissue invasive infection may include the tapeworms biomass, its diurnal migratory behavior, a host immune-mediated response, or direct parasite stimulation of host motor activity. In vivo intestinal myoelectric activity was monitored to evaluate the following: (1) that reinfection with H. diminuta is influenced by host immune regulation and (2) that administration of tapeworm fractions to never-before-infected rats initiates an alteration of enteric smooth muscle activity. To address the first hypothesis, we determined that altered intestinal myoelectric activity patterns were no different and did not occur earlier in a second infection with H. diminuta than in a primary infection. The lack of either a change in myoelectric pattern or an earlier onset of intestinal myoelectric changes indicates that tapeworm-induced myoelectric activity is not anamnestically stimulated by host immunomodulatory mechanisms. Consistent with the second hypothesis, administration of either H. diminuta carcass homogenate or tegument-enriched fractions directly into the intestinal lumen of tapeworm-naive rats initiated myoelectric patterns previously characteristic of chronic H. diminuta infection. Additionally, the appearance of characteristic nonmigrating myoelectric patterns in uninfected rats administered tapeworm fractions indicates that a substance from H. diminuta acts as the triggering signal molecule for intestinal myoelectric alterations. These findings also indicate that neither the tapeworms biomass nor its diurnal movement is required for initiation of H. diminuta-altered myoelectric patterns. We have shown that H. diminuta possess a signal molecule(s) that alters host enteric electric activity, and we suggest that these alterations may play an important role in the symbiotic rat-tapeworm interrelationship.

Praziquantel treatment normalizes intestinal myoelectric alterations associated with Hymenolepis diminuta-infected rats

Hymenolepis diminuta-associated alterations in rat intestinal myoelectric patterns are abolished following therapeutic administration of the anthelmintic praziquantel (PZQ). Host intestinal smooth muscle myoelectric patterns, reflecting smooth muscle contractility and intestinal phasic motility, were recorded using in vivo serosal electrodes, surgically implanted on the duodenum, jejunum, and ileum. Repeated electromyographic recording from unrestrained and unanesthetized rats began 5 days after electrode implantation surgery. Three initial control recordings from each rat confirmed the appearance of normal intestinal myoelectric patterns, characterized by the interdigestive migrating myoelectric complex (MMC). All animals were subsequently infected with H. diminuta and myoelectric recordings beginning after day 8 postinfection confirmed the appearance of diminished frequency of the MMC and 2 nonmigrating myoelectric patterns, i.e., repetitive bursts of action potentials and sustained spike potentials. PZQ was used to remove the tapeworms from rats 12 days after Hymenolepis diminuta infection, as intestinal myoelectric changes become maximal at this time in tapeworm-infected rats. PZQ administered to uninfected rats at either of 2 dose levels did not affect host interdigestive myoelectric activity. After removal of the parasite with PZQ, electromyographic recordings indicated a return to normal uninfected electrical patterns within 24 hr of drug treatment. We have demonstrated that the presence of Hymenolepis diminuta is necessary to induce and maintain abnormal intestinal myoelectric patterns. The altered motor properties of tapeworm-infected rat intestine and the rapid reconversion to preinfection myoelectric patterns provides a new and unique model to examine the regulatory mechanisms of intestinal motility and its control by luminal parasites.

Development and dynamics of regional specialization within the syncytial epidermis of the rat tapeworm,Hymenolepis diminuta

SummaryThe epidermis of the tapewormHymenolepis diminuta is a highly organized syncytium, composed of an outer layer of continuous cytoplasm, or ectocytoplasm, and an inner layer of nucleated cell bodies, or perikarya. The perikarya are in direct cytoplasmic continuity with the ectocytoplasm via narrow plasmalemma-bound bridges called internuncial process. Although distinct structural and functional differences are apparent between ectocytoplasm and perikarya, all of the perikarya along the body of the cestode are morphologically similar, as are all regions of ectocytoplasm. However, immunocytochemically distinct subpopulations of perikarya and regionally defined areas of ectocytoplasm were identified along the tapeworm strobila by the use of monoclonal antibodies raised against a preparation of isolated tegument. The different types of perikarya and the regionally specialized areas of ectocytoplasm were organized in a topographically precise manner along the body of the parasite. Examination of labeling patterns after colchicine treatment suggests that different types of perikarya are specialized for biosynthesis of specific tegumental molecules and for turnover or recycling of tegumental material. Furthermore, it appears that a 52 kDa polypeptide synthesized by one population of perikarya is transported through the syncytium and ultimately resorbed by a different population of tegumental perikarya. These data suggest that the syncytial epidermis of parasitic platyhelminthes exhibits a more complex organization of function than previously appreciated.