Effects of 5-hydroxytryptamine, cyclic AMP, AMP, and fructose 2,6-bisphosphate on phosphofructokinase activity in Hymenolepis diminuta

1. 5-HT (10(-4) M) had no effect on the activity of phosphofructokinase in Hymenolepis diminuta. Concentrations of ATP above 33 microM inhibited PFK activity; AMP and cyclic AMP relieved this inhibition. 2. Local levels of cyclic AMP may be indirectly modulated by NaF, guanylyl imidophosphate, or 5-HT in the presence of GTP, which stimulates adenylyl cyclase activity x2 in H. diminuta homogenates. 3. Fructose 2,6-bisphosphate (F2BP), a physiological regulator of PFK activity in rat liver, also relieved ATP-induced inhibition of PFK. F2BP was present in supernatants from the worms at about 20 mumol/g wet wt. 4. 5-HT may cause an increase in the rate of glycolysis in H. diminuta by elevating either cyclic AMP and/or AMP levels; these nucleotides can in turn increase PFK activity.     

Specific cross-immunity between Hymenolepis nana and H. diminuta: immunization with heterologous and homologous light infections

The consequences of previous and concurrent infection with two related species of cestodes, Hymenolepis nana and H. diminuta, were studied in CD1 mice. A H. diminuta infection strongly affected the establishment and the survival of a secondary H. nana egg or cyst infection administered 30 days later. An infection of 20 H. nana eggs strongly protected against a 5-cyst H. diminuta challenge, whereas an infection of 10 H. nana cysts was ineffective; 20 H. nana eggs also protected against a challenge with 5 cysts of H. diminuta administered 5 days later. No effects were observed in either parasite during a concurrent infection established by administration of cysts. An H. nana egg-infection was unable to affect the establishment of a secondary H. nana cyst-infection given 1 month later; however a significant decrease in growth was found. Similar results were found when a primary H. nana egg-infection was followed 5 days later by the homologous cyst-infection. But an infection with 5 H. nana cysts was unable to protect against a homologous challenge of 5 cysts or 200 eggs. The reciprocal cross immunity between the heterologous parasites and the failure of protection of homologous challenges are discussed in relation to light infections.     

Hymenolepis diminuta and H. nana: cross immunity against the lumen phase in BALB/c mice

When BALB/c mice initially given cysticercoids of Hymenolepis diminuta orally (Day 0) were challenged with eggs or cysticercoids of H. nana, almost all the mice became completely resistant to H. nana challenges from Day 30 onward, and no luminal adults of H. nana were established. There was a tendency for the number of tissue cysticercoids recovered 4 days after egg challenge in immunized mice to be much less than that in control mice (P less than 0.001, Student's t test). However, when these cysticercoids recovered from immune group mice were inoculated into uninfected mice, they matured in the lumen. Thus, the cross immunity to H. nana challenge evoked by an initial prepatent infection with H. diminuta appeared to be directed not against the tissue phase but against the lumen phase of H. nana. When BALB/c mice initially given eggs of H. nana were challenged with H. diminuta, they became resistant to H. diminuta from Day 15 onward. When the mice given eggs of H. nana were treated with a cestocide, praziquantel, at the beginning of the expected luminal development of H. nana and experienced a tissue phase only before challenge with H. diminuta, they showed no resistance to H. diminuta. Thus, the cross immunity to H. diminuta challenge evoked by an initial patent infection with H. nana appeared to be due to the immunogens of the lumen phase of H. nana but not those of the tissue phase. The cross immunity may be, therefore, essentially evoked by the lumen phase of these two phylogenetically closely related species and not by or against the tissue phase of H. nana.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 5-hydroxytryptamine (serotonin) on the incorporation of 32P-inorganic phosphate into phospholipids in Hymenolepis diminuta (Cestoda)

5-Hydroxytryptamine (5-HT) (1,2 and 5 mM) significantly stimulated the incorporation of radioactive inorganic phosphate (32Pi) into phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid and also total phospholipid fraction of Hymenolepis diminuta after one hour of incubation. Such effect was both time and concentration dependent. In the presence of 5-HT early labelling of phosphatidylinositol was observed. Also, the percentage stimulation by 5-HT was the highest in this fraction under all experimental conditions. The inorganic, organic, total and phosphatidylcholine-bound phosphate of H. diminuta incubated with 5-HT were not significantly different from those of the control under all incubation conditions. Results reported herein suggest that messenger molecules that are derived from phosphoinositides may be involved in the stimulatory mechanism of 5-HT in H. diminuta.     

Characterization of a serotonin transporter and an adenylate cyclase-linked serotonin receptor in the cestode Hymenolepis diminuta

[3H]5-HT exhibited specific binding in membrane preparations of Hymenolepis diminuta. The specific binding was saturable, reversible and temperature dependent. A non-linear Scatchard plot was obtained in a concentration range of 11 nM - 1000 nM [3H]5-HT, which could be resolved into sites having apparent dissociation constants (KD) of 0.10 microM and 6.25 microM for the high-affinity and low-affinity components, respectively. The latter could be selectively eliminated by binding [3H]5-HT to H. diminuta membranes in the presence of 10(-3) M nitroimipramine. Drug displacement studies, using 0.20 microM and 2.0 microM [3H]5-HT, revealed that while low-affinity [3H]5-HT binding was displaced by unlabelled 5-HT and inhibitors of 5-HT uptake, high affinity [3H]5-HT binding was affected only by tryptamine derivatives and, to a lesser extent, methysergide. In addition, high-affinity binding was stimulated by MgCl2 while low-affinity binding showed sodium-dependency. The data implicate the low-affinity site as a putative 5-HT transporter and the high-affinity site as a putative 5-HT 1 receptor. Exposure of H. diminuta membranes to 5-HT resulted in a 3-4 fold stimulation of cAMP levels. The EC 50 for the 5-HT-induced activation of adenylate cyclase (0.76 microM) was of the same order of magnitude as the apparent KD for high-affinity binding. Furthermore, the order of drug potency for the elevation of cAMP levels by 5-HT agonists and reversal by 5-HT antagonists was identical to the order of drug potency for the inhibition of high-affinity binding, suggesting linkage of the putative 5-HT 1 receptor to adenylate cyclase in H. diminuta.     

The occurrence, synthesis and metabolism of 5-hydroxytryptamine and 5-hydroxytryptophan in the cestode Hymenolepis diminuta: a high performance liquid chromatographic study

The biosynthesis and metabolism of 5-hydroxytryptamine (serotonin; 5-HT) in the cestode Hymenolepis diminuta was investigated by High Performance Liquid Chromatography (HPLC). Incubation of intact H. diminuta in [3H]tryptophan resulted in substantial radioactivity recovered in 5-HT, 5-hydroxytryptophan (5-HTP), and 5-hydroxyindoleacetic acid (5-HIAA). Furthermore, the tissue levels of 5-HT and 5-HTP, as determined by HPLC with electrochemical detection, were significantly depressed when the animals were deprived of tryptophan. On the other hand, the tissue levels of 5-HTP were significantly increased following incubation with the 5-HTP decarboxylase inhibitor m-hydroxybenzylhydrazine. The synthesis and metabolism of 5-HT are discussed in the light of 5-HT as a physiological transmitter in H. diminuta.     

Effect of mebendazole on the larval development of three Hymenolepidid cestodes.

Mebendazole or Telmin (which contains 16.7% mebendazole) markedly retarded the development of Hymenolepis diminuta and H. nana when their intermediate hosts Tribolium confusum were fed on flour mixed with it from day 1 to day 10 p.i. Though retardation also occurred with H. microstoma, the effect of the drug on this parasite was noticeably less pronounced than with the other 2 species of this genus.     

Hymenolepis nana: immunity against oncosphere challenge in mice previously given viable or non-viable oncospheres of H. nana, H. diminuta, H. microstoma and Taenia taeniaeformis

When mice, previously given oral inoculation with viable oncospheres of the heterologous cestode species (Hymenolepis diminuta, H. microstoma, Taenia taeniaeformis) and the homologous one (H. nana), were challenged with oncospheres of H. nana 4 days after the primary inoculation, they showed strong and complete resistance to H. nana challenge, respectively. However, the resistance was not evoked in mice given either infective eggs of Toxocara canis or non-viable oncospheres of all cestode species examined. Congenitally athymic nude mice given viable oncospheres did not show any resistance to H. nana either. Eosinophil infiltration around cysticercoids of H. nana in the intestinal villi appeared to be more prominent in mice previously given viable oncospheres of H. diminuta than in mice given non-viable oncospheres or PBS only. Some of the eosinophils in the villus harboring cysticercoid(s) of H. nana invaded the epithelia in the former, whereas all eosinophils remained in the lamina propria in the latter. There was almost no eosinophil infiltration in nude mice. Microscopic observations revealed that oncospheres of H. diminuta, which require beetles as the intermediate host like H. microstoma, could invade the mouse intestinal tissue. Therefore, it is strongly suggested that the strong cross resistance to H. nana in mice, induced by oncospheres of all heterologous cestode species, is thymus-dependent and due to oncospheral invasion into the intestinal tissue of mice.     

Blast cell activity in mice infected with Hymenolepis nana, H. diminuta and Trichinella spiralis: in vivo uptake of 125IUdR in lymphoid tissues and gut

The kinetics of the lymphoblast response in mice during the course of a primary infection with Hymenolepis nana was measured by the in vivo uptake of 125IUdR. The response was most marked in tissues local to the site of infection, involving the nodes draining the small intestine but not other areas, e.g., inguinal lymph nodes. A close correlation between these responses and the course of infection was observed. Uptake of 125IUdR was greatest in the mesenteric lymph node (MLN) but the peak reached in this organ was later than that in Peyer's patches (PP), small intestine (SI) and spleen (S). The increase in lymphoblast activity of the MLN was similar with Trichinella spiralis; no significant blast cell response to infection with H. diminuta was found till day 9 after injection, the results being similar to those obtained when H. nana infections were established using cysticercoids rather than eggs. It has been shown that the increase in lymphoblast activity was closely correlated with the presence of cells which are most effective in adoptive transfer immunity. A dose-dependent effect was detected in blast cell activity of MLN in different infection levels with T. spiralis and H. nana.     

Anthelmintic profile of methyl 5(6)-(4-methylpiperidin-1-yl) carbonylbenzimidazole-2-carbamate in experimental helminthiases

Biological evaluation of methyl 5(6)-(4-methylpiperidin-1-yl) carbonylbenzimidazole-2-carbamate against Ancylostoma ceylanicum, Nippostrongylus brasiliensis, Syphacia obvelata, Hymenolepis nana, H. diminuta and Cysticercus fasciolaris in experimental animals is reported. The compound (mg/kg) causes 100% elimination of A. ceylanicum (25 x 1), N. brasiliensis (100 x 1), S. obvelata (50 x 1), H. nana (250 x 3) and C. fasciolaris (50 x 10). It was also effective against the developing larvae (L3, L4 and L5) of A. ceylanicum at a single oral dose of 100 mg/kg. Another study indicated that the compound elicits 100% response within 32 hr of drug administration. The drug is well tolerated and LD50 is greater than 4500 mg/kg.     

Immunity to adult cestodes: basic knowledge and vaccination problems. A review.

Immunity in mammals to intestinal cestodes has been reviewed using the normal final host infected with the tapeworms Hymenolepis diminuta in rats and H. microstoma and H. nana in mice as a model. Primary infections up to a certain level continue to live as long the host, while most worms in infections with larger doses are destrobilated and expelled. It has been argued that concomitant immunity against a superimposed infection exists in rats and mice infected with H. diminuta and H. microstoma, respectively, and suggested that it also takes place in humans infected with Taenia spp. Immunity to secondary infections after expulsion of a primary infection occurs, but immunological memory is rather short-lived, although depression of worm growth occurs for at least two third of the rat's life. Serum antibodies have been shown to produce a direct precipitate on the surface of cestodes in vitro, but a direct effect of antibodies in vivo or the relationship with e.g. host effector cells, like mast cells and eosinophils, is unknown. It has been shown that peritoneal exudate cells from rats are able to kill H. diminuta in vitro. Very little is known about the mechanisms of tapeworms to counteract host immunological responses, but the tegumental glycoconjugates and discoidal secretory bodies are possible candidates. Passive transfer of immunity by mesenteric lymph node cells has only been successful using cells from H. nana egg-infected mice and has shown that only short-lived proliferating cells are responsible for transferring immunity. Vaccination procedures and problems are discussed with special reference to E. granulosus in dogs.     

Adsorption of bile salts by the cestodes, Hymenolepis diminuta and H. microstoma.

The accumulation of purified sodium taurocholate (NaTC) and sodium glycocholate (NaGC) by Hymenolepis diminuta and Hymenolepis microstoma (Cestoda: Cyclophyllidea) was determined using radioactive bile salts. H. diminuta reached equilibrium levels of approximately 120 nmoles NaTC/g dry wt and 300 nmoles NaGC/g dry wt. Presentation of the bile salts in mixed micelles with 0.35 mM oleic acid did not alter these values. With H. microstoma, the maxima were 195 nmoles NaTC/g dry wt and 614 nmoles NaCG/g dry wt. These values were similarly unaffected by the addition of 0.35 mM oleic acid to the micelles. Equilibrium values of this magnitude, in media containing as much as 25 or 30 mM bile salt, and the maintenance of this level during incubations of 15 to 60 min eliminated the possibility that the accumulation was by diffusion or by any form of mediated transport into the worm. The accumulation on NaTC by H. diminuta was [Na+] independent, and insensitive to ouabain, DNP, and high [K+]. These observations, the maintenance of different levels of NaTC and NaGC, and the failure of the 2 bile salts to compete indicated that there was no active excretion mechanism operating in a fashion similar to the active transport of bile salts in the vertebrate small intestine. It was concluded that the accumulation of NaTC by H. diminuta was actually adsorption to the tegument. Comparable, although more limited, experiments extended this conclusion to the accumulation of NaGC by H. diminuta and of NaTC and NaGC by H. microstoma. It is suggested that bile salt monomers, rather than intact micelles, adsorb to specific loci on the tegument.     

Concurrent infections of the trematode Echinostoma caproni and the tapeworms Hymenolepis diminuta and Hymenolepis microstoma in mice

Superimposing the intestinal tapeworm Hymenolepis diminuta on an established infection with the trematode Echinostoma caproni or simultaneous infection of mice with H. diminuta and Hymenolepis microstoma caused destrobilation and expulsion of H. diminuta, whereas establishment and growth of H. microstoma under the same infection regimes were not affected. In contrast, simultaneous superimposition of H. diminuta and H. microstoma on an established E. caproni infection caused destrobilation and expulsion of both H. diminuta and H. microstoma.     

Differential establishment and survival of Hymenolepis diminuta in syngeneic and outbred rat strains.

Experimental Hymenolepis diminuta infection was carried out in inbred strains of rats (F344/N, JAR-2, LOU/M, TM, DA and DA-bg/bg) and outbred Wistar rats. All strains became infected with this cestode, but clear strain-dependent variation in the susceptibility to H. diminuta infection was observed. Marked differences in worm persistence and worm weight were found at 6 weeks post-infection in TM and DA rats. These strains would be useful to clarify the interactions between H. diminuta and its rat host.     

The uptake and metabolism of 5-hydroxytryptamine by tissue slices of the cestode Hymenolepis diminuta

The in vitro uptake of [3H]5HT was investigated in tissue slices of the cestode Hymenolepis diminuta. A concentrative, sodium sensitive, high affinity uptake mechanism (Km 1.43 X 10(-6) M; Vmax 222 fmoles/mg wet wt/min), together with a sodium insensitive component (linear up to 5 X 10(-6) M) were present. In the presence of 2-nitroimipramine the sodium sensitive component was significantly suppressed (Vmax 33 fmoles/mg/wet wt/min) although the Km (1.37 X 10(-6) M) was not affected. Nitroimipramine showed an IC50 of approximately 2 X 10(-6) M. The sodium insensitive component was not affected by nitroimipramine. Biogenic amines and related indoleamines were weak inhibitors of the sodium sensitive and sodium insensitive components of 5HT uptake. The tricyclic antidepressants and fluoxetine were effective inhibitors of the sodium sensitive component of 5-HT uptake; receptor ligands were weak inhibitors or without effect. The metabolism of [3H]5HT in tissue slices of H. diminuta was examined by HPLC. The role of the sodium sensitive uptake and metabolism of 5HT in terms of inactivation and recycling of neurally released 5HT and the possible importance of exogenous recruitment of 5HT are discussed.     

Biochemical, physiological and morphological variation in unarmed hymenolepids (Eucestoda: Cyclophyllidae)

The genus Hymenolepis contains a number of unarmed species. These frequently possess similar morphologies and are difficult to discriminate using the traditional method of comparative morphology. A parasite of the long-tailed field mouse, Apodemus sylvaticus in northeast Ireland, resembles the widespread H. diminuta which is usually a parasite of the rat. Analysis of general and specific proteins of the adults in A. sylvaticus , laboratory mice and rats suggests that the parasite found in the former host and H. diminuta are genetically distinct, though more closely allied than either is to H. nana, H. citelli and H. microstoma . Experimental analysis of the growth and expulsion of the Irish material and H. diminuta from SPF C57 laboratory mice, rats and wild caught A. sylvaticus suggests that there are behavioural and physiological differences in these taxa. Both are expelled from C57 mice though the hymenolepid from Irish A. sylvaticus persists for 3 days more than those of H. diminuta . The former prospers better in rats than H. diminuta in A. sylvaticus . Detailed comparison of the gross morphology of cysticercoid and adult H. diminuta and the Irish hymenolepid reveals differences in size rather than qualitative attributes. The occurrence of H. diminuta in A. sylvaticus is discussed. It is concluded that the hymenolepid recovered from Irish A. sylvaticus differs sufficiently from H. diminuta to warrant species status and that it has adapted to the alimentary canal of A. sylvaticus . This cestode material is described under the name of H. hibernia sp. nov.     

Demonstration of specific high-affinity binding sites for [3H]5-hydroxytryptamine in the cestode Hymenolepis diminuta

[3H]5-HT (0.16-8.32 nM) exhibited saturable and specific binding in membrane preparations of Hymenolepis diminuta. The saturation data produced a non-linear Scatchard plot which could be resolved into sites having apparent dissociation constants (Kd) of 0.17 and 8.30 nM for the high-affinity and low-affinity components, respectively. Drug displacement studies, using radioligand concentrations of 0.6 and 6 nM, revealed that the two [3H]5-HT binding components are pharmacologically distinct and do not conform to any known class of 5-HT recognition site. The physiological significance of these putative 5-HT receptors and their potential usefulness for the selection of new antiparasitic agents are discussed.     

Molecular cloning and characterization of Hymenolepis diminuta alpha-tubulin gene.

To isolate a full-length alpha-tubulin cDNA from an eucestode, Hymenolepis diminuta, a lambda phage cDNA library was constructed. The alpha-tubulin gene was cloned, sequenced and characterized. The H. diminuta alpha-tubulin consisted of 450 amino acids. This protein contained putative sites for all posttranslational modifications as detyrosination/tyrosination at the carboxyl-terminal of protien, phosphorylation at residues R79 and K336, glycylation/glutamylation at residue G445 and acetylation at residue K40. Comparisons of H. diminuta alpha-tubulin with all full-length alpha-tubulin proteins revealed that H. diminuta alpha-tubulin possesses 10 distinctive residues, which are not found in any other alpha-tubulins. Phylogenetic analysis showed that H. diminuta alpha-tubulin has grouped in a separated branch adjacent eucestode and trematodes branch with 92% bootstrap value (1000 replicates). In conclusion, this is the first report of H. diminuta cDNA library construction, cloning and characterization of H. diminuta alpha-tubulin gene.     

Biochemical effects of thiabendazole and cambendazole on Hymenolepis diminuta (Cestoda) in vivo

An investigation of the chemotherapeutic and biochemical effects of two benzimidazole anthelmintics, thiabendazole (TBZ) and cambendazole (CBZ), on Hymenolepis diminuta in experimentally infected rats is reported. Thiabendazole was active against H. diminuta at a relatively high dosage. A single oral dose of TBZ at 250 mg/kg body weight on day 15 of infection eliminated 100% of the tapeworms as determined at necropsy 5 days after treatment. The chemotherapeutic actions of TBZ on H. diminuta were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of TBZ 24 hr earlier were significantly smaller and contained much less glycogen (as a percent of the wet weight) than worms from unmedicated controls. Protein concentrations increased in TBZ-treated worms and at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in TBZ-treated worms were significantly lower than the corresponding control values. Cambendazole proved to be five times more potent than TBZ against H. diminuta and produced the same basic changes in worm weight and chemical composition within 18 hr of treatment of the host. Administration of a single oral dose of TBZ or CBZ to the host produced in H. diminuta another change, the onset of which coincided with, or preceded, the gross alterations in worm weight and chemical composition. That change, observed in in vitro studies carried out 14 hr after treatment, revealed that tapeworms from drug-treated rats absorbed and metabolized much smaller quantities of exogenous glucose than did the controls, and the ability of the worm to accumulate glucose against a concentration difference was significantly depressed.     

Hymenolepis diminuta: lack of sulphate activation and sulphotransferase activity

There is no evidence that Hymenolepis diminuta can carry out sulphoconjugation reactions. Neither whole worms nor worm extracts were able to sulphate 4-methylumbelliferone. No sulphotransferase activity could be demonstrated in H. diminuta using a variety of substrates, nor was H. diminuta capable of synthesising the sulphate donor 3'-phosphoadenosine-5'-phosphosulphate from ATP and inorganic sulphate. Possible alternative sources of active sulphate in this parasite are discussed.