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Tapeworms alter the physiological environment of the host's small intestinal lumen by contracting the intestinal smooth muscle, thereby slowing the transit of intestinal contents. We hypothesize that parasite-to-host molecular signaling is responsible for the specific patterns of small intestinal smooth muscle contraction observed both during tapeworm infection and after the infusion of tapeworm-secreted molecules into the intestinal lumen of unanesthetized rats. Of the tapeworm-secreted compounds tested, only lumenal infusion of guanosine 3',5'-cyclic monophosphate (cGMP) induced contractile patterns that mimic those observed during tapeworm infection. The response to cGMP occurred in a concentration-dependent fashion. Our study clearly demonstrates that cGMP can serve as an extracellular signal molecule regulating small intestinal motility mechanisms in vivo.

Abstract Luminal infection by the noninvasive tapeworm, H. diminuta, alters rat small intestinal myoelectric activity. The significance of continuity between small intestinal enteric nervous system (ENS) and that of both the stomach/pylorus and colon/caecum regarding the induction of tapeworm-altered myoelectric patterns was evaluated. A total of 32 rats were implanted with four serosal electrodes placed at sites in the duodenum through the mid-jejunum. Sixteen of the 32 rats underwent intestinal transections and anastomoses at both the duodenum and ileum. After recording myoelectrical activity of both normal and transected intestines, eight rats from each group (normal and transected) were infected with H.diminuta. Phase III frequency, duration of the migrating myoelectric complex (MMC), slow wave frequency, percentage of slow waves associated with spike potentials and the occurrence of the the two tapeworm-initiated myoelectric patterns, repetitive bursts of action potentials (RBAP) and sustained spike potentials (SSP), were measured. In infected rats, the frequency of the RBAP and SSP electric patterns were significantly reduced by the double transection. Intestinal transection did not affect the other changes caused by infection, such as decreased MMC phase III frequency and percentage of slow waves associated with spike potentials. In conclusion, a small intestinal ENS in continuity with other segments of the GI tract is required to generate maximal numbers of tapeworm-induced SSP and RBAP myoelectric activity in the small intestine of the rat.

The rat tapeworm Hymenolepis diminuta alters the myoelectric activity of the small intestine. To determine if secreted factors from the tapeworm are responsible for these alterations of intestinal smooth muscle activity, tapeworm-conditioned medium (TCM) obtained from in vitro culture was infused via an indwelling cannula into the duodenum of an uninfected rat. Myoelectric recordings were analyzed for sustained spike potentials (SSP) and repetitive bursts of action potentials (RBAP), the previously characterized tapeworm modifications of the normal interdigestive myoelectric pattern. Results indicated that TCM initiated SSP, but not RBAP in the intestine of the uninfected rat. The SSP-inducing signal factor activity, present in TCM, was retained after boiling, prolonged freezing, proteinase treatment, and passage through a 10-kDa exclusion filter. The signal factor was soluble in the aqueous phase on lipid extraction. It was concluded that the SSP-inducing signal factor is a nonproteinaceous, heat-resistant, low-molecular weight, water soluble molecule.

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 host's 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 host's intestine.

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 tapeworm's 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 tapeworm's 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.

The effects of tunicamycin (TM) on the uptake and incorporation of tritiated galactose into the tegumental membrane and carcass from adult Hymenolepis diminuta were examined to assess the potential usefulness of this inhibitor for studying the function of the tapeworm surface glycocalyx. Hymenolepis diminuta adults (11 days old) were preincubated for 1 hr, pulsed for 30 min with [3H]galactose and [14C]leucine, and chased for 2 hr; replicate experiments were conducted in which all media contained no TM or TM at 10 micrograms/ml. Tunicamycin significantly inhibited the incorporation of tritiated galactose into the tapeworm's carcass and 30,000-g tegumental membrane fraction. Incorporation of tritiated galactose into the tapeworm's tegumental surface membrane also was inhibited significantly when expressed relative to the incorporation of [14C]leucine. Tunicamycin did not affect the amounts of free, i.e., soluble, [3H]galactose or [14C]leucine recovered from the tapeworms not did it affect the short-term (2 min) uptake of [3H]galactose by tapeworms. Thus, the inhibitory effect of TM appears to be at the level of protein glycosylation rather than carbohydrate (galactose) transport. The data indicate that TM might be useful for producing tapeworm surface membranes with diminished carbohydrate moieties.

The free-surface of the tapeworm's tegument was examined for morphological evidence of secretion after fixation by rapid freezing-freeze substitution and alternatively by immersion in low concentrations of glutaraldehyde maintained at room temperature. After low-aldehyde fixation, omega profiles were at the bases of tegumental microvilli, arguing for the participation of some of the ectocytoplasm's vesicles in secretion of their contents to the intestinal lumen. The almost instantaneous fixation provided by the rapid freezing-freeze substitution technique documents the constitutive production of 0.03 to 0.075-micron microvesicles from outpocketings from the plasma membrane of the tegumental brush border. Observed in secretory epithelial cells of other species, microvesicles are recognized as a secretory pathway for constituent molecules of surface membranes. We conclude that in addition to the primary route of merocrine exocytotic secretion provided by the fusion of the Golgi-derived, ectocytoplasmic vesicles at the bases of the brush border microvilli, tegumental microvesicles provide a second secretory pathway for endogenous macromolecules across the tegumental free surface.

Dimethyl sulfoxide (DMSO), commonly used for cryoprotection or for the solvation of cytoskeleton-modifying drugs, causes changes in the topology of the plasma membrane of the tegumental brush border in the tapeworm Hymenolepis diminuta; however, relatively long exposures of high concentrations are required. In tapeworms treated with DMSO concentrations of greater than or equal to 1% in the present study, the interaction of the tegumental surface membrane with the underlying cytoskeleton may have been disrupted at focal points in the brush border, resulting in a partial loss of the membrane anchoring required for the structural integrity of the brush border. Blebbing of the tegumental surface was prominent only after exposure to 1% DMSO for 20 h in in vitro culture with RPMI 1640, and vesiculation of the membrane along the microvillar (microtriche) shafts, which may have been related to the in vitro conditions, was amplified by the presence of concentrations of greater than or equal to 1% DMSO in the incubation medium. The tegumental response to DMSO was not uniform but regional, consistently appearing to be more prevalent on the distal aspects of each proglottid rather than on the edge proximal to the scolex. Blebbing and vesiculation were not seen on the basal aspect of the tegument, including the basal ectocytoplasmic plasma membrane, the perikarya, and the internuncial processes. Microvillar core bundles of actin microfilaments persisted following 8 h in vitro exposure to all three concentrations of DMSO tested (0.1%, 1%, 5%); however, only in tapeworms that were treated in vitro with 5% DMSO for greater than or equal to 8 h did core microfilament bundles appear to lose the rigidly straight and parallel organization characteristic of control tapeworms that were incubated either in the absence of DMSO or with 0.1% DMSO. Other components of the brush border cytoskeleton (i.e., microvillar caps, junctional tubules, and tunics) appeared unaffected by DMSO except at foci where blebbing occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

The epidermis of the tapeworm Hymenolepis 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.

Polysaccharides and other complex carbohydrates were released by proteolysis of the chloroform-methanol insoluble residue of 10 day-old worms and eggs of Hymenolepis diminuta. Gas-liquid chromatographic analysis of alditol acetate derivatives of monosaccharides released from the polysaccharides by hydrolysis revealed that in the 10 day-old worm, glucose was the most abundant sugar, followed by galactose, glucosamine, galactosamine, fucose and possibly rhamnose. Mannose was least abundant and xylose was absent. In the egg, glucose and galactose were equally abundant, followed by the same sugars found in 10 day-old worms, and xylose was present. Uronic acid was detected in both fractions by specific chemical tests. None of the saccharide material from eggs and worms was susceptible to degradation by Streptomyces hyaluronidase, chondroitinase AC, and slightly susceptible to chondroitinase ABC, as shown by electrophoretic analysis on composite 2.2% acrylamide-agarose slab gels and 4.5/12.5% polyacrylamide gels before and after enzymatic treatment. One of the gel-separable bands, however, was degradable by both nitrous acid and Flavobacterium heparinase. Both bands from eggs were degradable by nitrous acid. These results suggest that eggs contain heparin and/or heparan sulfate and perhaps dermatan sulfate and that 10 day-old worms also have these polyglycans but possibly not chondroitin sulfate or hyaluronic acid.

Electron microscopic examination of the epidermal basement membrane region of the rat tapeworm, Hymenolepis diminuta, has revealed specialized connective tissue structures that appear to anchor the epidermis, or tegument, to the parenchymal tissues of the helminth, as well as interconnect subtegumental muscle fibers, tegument, and parenchyma. Anchoring fibrils-cross-banded bundles of ca. 3 nm diameter filaments--were observed to directly interlink tegument and muscle, muscle and muscle, and tegument, muscle, and parenchymal connective tissue. Anchoring fibrils therefore appear to mechanically integrate epidermal tissue movements in response to subtegumental muscle contraction. A well-developed stratum of microfibrils, forming the lamina reticularis of the tegumental basement membrane, may also help anchor the tegument as well as to serve as a flexible, reinforcing sheath that protects parenchymal tissues from excessive radial displacement due to muscle contraction.

A hallmark feature of parasitic platyhelminths is a cytoarchitecturally unusual syncytial epidermis composed of a peripheral layer of continuous cytoplasm (the ectocytoplasm) connected to underlying nucleated cell bodies by small cytoplasmic bridges. The helminth epidermis, or tegument, plays important roles in protection and nutrient acquisition; cestodes, in fact, completely lack a gastrointestinal tract and absorb all nutritive material through the tegument. Perhaps not surprisingly, the cestode tegument bears certain resemblances to the mucosal epithelium of the vertebrate small intestine, including the possession of a microvillous brush border upon the surface of the ectocytoplasm. In contrast to the intestinal epithelial cell, however, very little is known concerning the nature and organization of the cytoskeleton within the helminth epidermis. Therefore, a number of different microscopical preparative techniques were used to examine the tegument of the tapeworm Hymenolepis diminuta for the presence and distribution of microfilaments, intermediate filaments, and microtubules. It was found that both actin-containing microfilaments and intermediate-sized filaments are present but are restricted to specific locations along the plasmalemmae of the ectocytoplasm. In contrast, microtubules are found throughout the tegument, and are concentrated in the supranuclear regions of the perikarya and in the cytoplasmic bridges interconnecting the perikarya and ectocytoplasm. Unlike brush borders of most other epithelia, the cestode epidermal brush border lacks a filamentous terminal web and is instead associated with microtubules. A network of fine filaments, 5-8 nm in diameter but distinct from actin-containing microfilaments, runs throughout the ectocytoplasm and appears to interlink tegumental vesicles. These fine filaments may represent the primary "skeletal" system responsible for maintaining the structure of the tegumental cytoplasm.

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.

Lipids comprised 37% and 22.1%, respectively, of the day weights of brush border- and vesicle-rich fractions separated by differential centrifugation of isolated H. diminuta tegument. Neutral lipids of both fractions were rich in cholesterol, but also contained small amounts of glycerides, sterol esters, and (in brush borders) free fatty acids. Phosphatidyl ethanolamine was the most prevalent polar lipid in both fractions, and was particularly abundant (63.4% of total polar lipids) in vesicles; sphingomyelin, not previously reported from H. diminuta, was also present. Polar lipids of both tegumental fractions resembled each other but differed from whole worm polar lipids in fatty acid composition. Tegumental polar lipids contained lower levels of long-chain, polyunsaturated fatty acids than reported for corresponding lipids of whole worms.