Hymenolepis diminuta Infection Affects Apoptosis in the Small and Large Intestine.

The rat tapeworm Hymenolepis diminuta has been shown to cause alterations in gastrointestinal tissues. Since hymenolepiasis induces a number of reactions in the host, it is reasonable to assume that it may also be involved in the mechanisms of apoptosis in the intestines. Individual research tasks included an examination of the effect of H. diminuta infection on; (i) the cellular localization of the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2, as well as caspase-3 and caspase-9, and (ii) the effects of the infection on the expression of Bcl-2, Bax, Cas-3 and Cas-9, at the mRNA and protein levels. Molecular tests (including mRNA (qRT PCR) and the protein (Western blot) expression of Bax, Bcl-2, and caspases-3, -9) and immunohistochemical tests were performed during the experiment. They showed that H. diminuta infection activates the intrinsic apoptosis pathway in the small and large intestine of the host. H. diminuta infection triggered the apoptosis via the activation of the caspase cascade, including Cas-3 and Cas-9. Hymenolepiasis enhanced apoptosis in the small and large intestine of the host by increasing the expression of the pro-apoptotic gene and protein Bax and by decreasing the expression of the anti-apoptotic gene and protein Bcl-2.

Immunoproteomics and Surfaceomics of the Adult Tapeworm Hymenolepis diminuta.

In cestodiasis, mechanical and molecular contact between the parasite and the host activates the immune response of the host and may result in inflammatory processes, leading to ulceration and intestinal dysfunctions. The aim of the present study was to identify antigenic proteins of the adult cestode Hymenolepis diminuta by subjecting the total protein extracts from adult tapeworms to 2DE immunoblotting (two-dimensional electrophoresis combined with immunoblotting) using sera collected from experimentally infected rats. A total of 36 protein spots cross-reacting with the rat sera were identified using LC-MS/MS. As a result, 68 proteins, including certain structural muscle proteins (actin, myosin, and paramyosin) and moonlighters (heat shock proteins, kinases, phosphatases, and glycolytic enzymes) were identified; most of these were predicted to possess binding and/or catalytic activity required in various metabolic and cellular processes, and reported here as potential antigens of the adult cestode for the first time. As several of these antigens can also be found at the cell surface, the surface-associated proteins were extracted and subjected to in-solution digestion for LC-MS/MS identification (surfaceomics). As a result, a total of 76 proteins were identified, from which 31 proteins, based on 2DE immunoblotting, were predicted to be immunogenic. These included structural proteins actin, myosin and tubulin as well as certain moonlighting proteins (heat-shock chaperones) while enzymes with diverse catalytic activities were found as the most dominating group of proteins. In conclusion, the present study shed new light into the complexity of the enteric cestodiasis by showing that the H. diminuta somatic proteins exposed to the host possess immunomodulatory functions, and that the immune response of the host could be stimulated by diverse mechanisms, involving also those triggering protein export via yet unknown pathways.

How tapeworm infection and consumption of a Cd and Zn hyperaccumulating plant may affect Cu, Fe, and Mn concentrations in an animal-a plant consumer and tapeworm host.

This study evaluated the effects of a hyperaccumulator plant (Arabidopsis halleri), containing surplus of cadmium (Cd) and zinc (Zn) and being an admixture to the rat feed, on concentrations of copper (Cu), iron (Fe), and manganese (Mn) in the tissues of experimental rats infected/uninfected with the tapeworm (Hymenolepis diminuta). Male Wistar rats were divided into three groups (00, P0, and PT); the P0 and PT animals were fed a standard mixture for rats (ST-1) supplemented with the plant A. halleri at a weekly Zn and Cd dosage of 123 and 1 mg, respectively. Moreover, rats from the group PT were infected with the tapeworm. The group 00 served as control animals fed only ST-1 having no tapeworm infection. Rats were euthanized after 6 weeks, and Cu, Fe, and Mn levels were determined in rat and tapeworm tissues. The results indicated that both the consumption of hyperaccumulator plant and/or presence of tapeworms did have significant effect on Cu, Fe, and Mn concentrations in the host tissues. Concentrations of all the elements were higher in the rat liver and partially kidneys than in the tapeworms, and the concentrations of Cu, Fe, and Mn were affected by the consumption of Cd/Zn hyperaccumulator plants. Particularly, Fe concentrations in all rat tissues were significantly increased by consumption of A. halleri while decreased by the presence of tapeworms. Overall, the consumption of a Cd/Zn hyperaccumulator plant and tapeworm infection cause an imbalance in Cu, Fe, and Mn concentrations in the tissues of a consumer (experimental rats).

Hymenolepis diminuta: analysis of the expression of Toll-like receptor genes and protein (TLR3 and TLR9) in the small and large intestines of rats.

Toll-like receptors (TLRs) play a fundamental role in the rapid activation of innate immune responses to a variety of pathogen-associated molecular patterns (PAMPs). In a previous study we observed an increase in the level of expression of TLR2 and TLR4 mRNA in the jejunum and colon during experimental hymenolepidosis in rats. In this study, we performed a quantitative real-time polymerase chain reaction (qRT-PCR), Western blot analysis and immunohistochemical staining of TLR3 and TLR9 receptors during experimental hymenolepidosis in rats. The levels of mRNA and protein expression of TLR3 and TLR9 in the jejunum had increased at 16 days post Hymenolepis diminuta infection (dpi) in the case of TLR3 and at 16 and 25 dpi in the case of TLR9. In the colon the expression of TLR3 and TLR9 had increased at 16, 25 and 40 dpi. The results of the immunohistochemical reactions showed that H. diminuta infected rats (16, 25, 40 and 60 dpi) exhibited changes in TLR3 and TLR9 localization and intensity in the epithelial cells of the jejunum and colon. The changes in the level of TLR3 and TLR9 expression may confirm involvement of the innate immune system in the pathomechanism of hymenolepidosis.

Is the tapeworm able to affect tissue Pb-concentrations in white rat?

The effect of gastrointestinal helminths on Pb accumulation in the host body is ambiguous. A laboratory experiment with Rattus norvegicus/Hymenolepis diminuta model was conducted to determine Pb toxicokinetics in a terrestrial host-parasite system. The ET-AAS or ICP-OES techniques were used to determine Pb concentrations (CPb) in both tapeworms and host tissues (kidney, liver, bone, testes, muscle and intestinal wall). Concerning the entire host-parasite system, the highest CPb were detected in H. diminuta. Rat kidneys and bone were the only two tissues whose mean Pb levels were lower in parasitized animals than they were in non-infected subjects after both levels of exposure. At low Pb exposure, parasitization slightly changed the Pb toxicokinetics in the host body. However, with respect to tissue at the same exposure level, no significant differences were detected between the parasitized and non-parasitized animals and no significant correlations were found between CPb in tapeworms and those of host tissues. The results of this study indicate that H. diminuta does not protect rat from elevated Pb exposure even if tapeworm accumulates a higher portion of ingested Pb dose compared with that of the most Pb-loaded host soft tissue. The portion of Pb dose accumulated in H. diminuta correlates positively with parasite biomass.

Hymenolepis diminuta: analysis of the expression of Toll-like receptor genes (TLR2 and TLR4) in the small and large intestines of rats.

Toll receptors play a critical role in the rapid activation of innate immune responses to a variety of pathogens. In mammals, Toll-like receptors (TLR) have been found in both immune related cells and other cells. At present little is known about the participation of TLR in host defense mechanisms during parasitic infections. The aim of this study was to determine the expression of TLR2 and TLR4 genes in rat intestines during experimental hymenolepidosis. There is difference in expression of TLR2 and TLR4 genes in the colon and jejunum in uninfected rats: in the colon, mRNA of the examined TLR is present in much higher amounts than the jejunum, while the protein of the TLR also had a segmented specific distribution. In the jejunum isolated rats infected with Hymeolepis diminuta 6 and 8 days post infection (dpi), mRNA for TLR4 and TLR2 were significantly more strongly expressed in comparison with the uninfected controls. In the colon, a statistically significantly increased expression of TLR4 gene was observed only at 6 dpi, and at 8 dpi for the TLR2 gene. Moreover, we observed that during inflammation, the immunopositive cell number and the intensity of immunohistochemical staining (indicating the presence of TLR within intestinal epithelial cells), increased together with the duration of the infection period.

Hymenolepis diminuta: experimental studies on the antioxidant system with short and long term infection periods in the rats.

Many helminths cause long-lasting infections, living for several years in mammalian hosts reflecting a well balanced coexistence between host and parasite. There are many possible explanations as to how they can survive for lengthy periods. One possibility is their antioxidant systems, which can serve as defence mechanisms against host-generated oxygen radicals. Therefore, the aim of this experimental study was to examine the antioxidant system in Hymenolepisdiminuta during short (1.5 months young tapeworms) and long (1.5 years old tapeworms) term infection in the rat small intestine. The strobilae of H. diminuta tapeworms (14 young and three old) were divided into three pieces: the anterior part, containing the genital primordiae in the immature segments; the medial part, containing the early uterus in the mature, hermaphroditic proglottids and the terminal part with the mature gravid uterus in the gravid segments. Supernatants of these fragments were used for determination of markers of oxidative stress: concentration of thiobarbiturate reactive substances (TBARS) and of reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (GSHPxs), glutathione transferase (GST) and glutathione reductase (GSHR). The results indicated changes in levels of oxidative stress markers and antioxidant enzyme activity in both the young and old forms of H. diminuta. Relatively high activity of SOD (particularly in the anterior part of young tapeworms) was observed, as was increased activity of total GSHPx and a relatively high concentration of GSH in all parts of the tapeworms. These are caused by exposure to increased amount of ROS, which are produced during the inflammatory state. Due to the high activity of antioxidant enzymes, the anterior section of young and old tapeworms is equipped with a very effective antioxidant system. Old organisms also effectively resist oxidative stress due to reduced levels of lipid peroxidation and the high activity of GST, all of which suggest good adaptation to the hostile environment in the host's intestine.

Hymenolepis diminuta: Activity of anti-oxidant enzymes in different parts of rat gastrointestinal tract.

The aim of this study was to assess the intensity of oxidative stress by measuring levels of lipid peroxidation products in the duodenum, jejunum and colon of rats infected with Hymenolepis diminuta and evaluate the effectiveness of protection against oxidative stress by measuring the glutathione levels and activity of anti-oxidant enzymes: superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. In exposed rats we observed a significant increase of lipid peroxidation products in the duodenum and jejunum. A significant decrease in superoxide dismutase activity in all the examined parts of the digestive tract was observed. Additionally, rats from 16 to 40 days post H. diminuta infection (dpi) had a decreased catalase activity in the colon, while at 60dpi it increased. The glutathione peroxidase activity increased significantly in the colon at 60dpi. The increase in glutathione reductase activity was observed in the colon in rats 60dpi. There was a lack of changes in the levels of glutathione in the duodenum and a significant increase in its concentration in the jejunum and colon from 40 to 60dpi and from 16 to 40dpi, respectively. In this study we observed altered activity of anti-oxidant enzymes and glutathione level in experimental hymenolepidosis, as a consequence of oxidative stress. It may indicate a decrease in the efficiency of intestinal protection against oxidative stress induced by the presence of the parasite. The imbalance between oxidant and anti-oxidant processes may play a major role in pathology associated with hymenolepidosis.

Hymenolepis diminuta: the effects of infection on transepithelial ion transport and tight junctions in rat intestines.

In this study, we examine the effect of Hymenolepis diminuta on ion transport in the ileum and on tight junctions in the ileum and colon of rats. We also evaluate the effect of H. diminuta on C-fiber endings in the ileum, the direct habitat of H. diminuta, before and after mechanical stimulation and pharmacological modification by capsaicin (C-fiber irritant). Wistar rats were orally infected with five cysticercoids of H. diminuta. Using a modified Ussing chamber, electrophysiological parameters of the ileum were measured (transepithelial electrical potential difference and transepithelial electrical resistance) as well as the deposition of occludin (a tight junction protein) in the ileum and colon of the rats 8, 16, 25, 35, 40 and 60 days post infection. We observed a significant reduction in transepithelial electrical potential difference in the ileum of rats infected with H. diminuta. In both the ileum and colon of rats infected with H. diminuta we also observed a decrease in occludin deposition, which indicates leakage of tight junctions, correlating with the decrease in transepithelial electrical resistance of these tissues. The application of capsaicin confirmed the hypothesis that H. diminuta in rats affects the C-fiber sensory receptors, causing changes in ion transport in the ileum. The results of the performed electrophysiological and immunohistochemical examinations indicate hymenolepidosis-related changes in the active transport of ions and the passive movement of ions.

Hymenolepis diminuta: Effect of infection on ion transport in colon and blood picture of rats.

The aim of this study was to examine the effect of an infection with Hymenolepis diminuta on ion transport in an isolated colon and blood picture of rats. Fifty rats were orally infected with five cysticercoids of H. diminuta. The experimental groups of rats were assigned to four groups: group I - 8 days post-infection (dpi), group II - 16 dpi, group III - 40 dpi and group IV- 60 dpi. The control group comprised non-infected rats. The experiments consisted of measuring the transepithelial electrical potential difference (PD) and the transepithelial electrical resistance (R) of the rat colon under controlled conditions as well as during mechanical stimulation (MS) using a modified Ussing chamber. Ion transport was modified using inhibitors of the epithelial sodium channel (amiloride - AMI) and the epithelial chloride channel (bumetanide - BUME), and also using capsaicin (CAPSA), a substance which activates C-fibres. The experimental data presented in this study indicates that experimental hymenolepidosis inhibits sodium and chloride ion transport in the epithelium of the rat colon, with preserved tight junction continuity (except at 40 dpi) and a decreased mechanical sensitivity. The effect of capsaicin on ion transport in the rat colon was varied. In control rats it increased ionic current, and in H. diminuta-infected rats it did not cause any changes in PD. Blood picture in this study showed a statistically significantly lower red blood cells (RBC) count and haemoglobin (HGB) concentration in infected rats in comparison to non-infected. Red cell distribution width (RDW) values and platelet (PLT) count were negatively correlated with the duration of infection, whereas mean corpuscular volume (MCV) value was positively correlated. We did not observe leukocytosis during infection, and amongst the differential leukocyte counts eosinophils and basophils showed statistically significant lower values in infected rats in comparison to non-infected. Our results indicate that hymenolepidosis is associated with the activation of inflammatory mediators and stimulation of nervous fibres, which significantly affects the function of ion channels in the epithelium of the colon in the host. At the same time, a significant decrease in eosinophil count during infection suggests that such an infection did not trigger a strong immunological reaction in rats.

Cytokine production in Hymenolepis nana infection.

BACKGROUND: Although many experimental studies provide convincing evidence that type II immunity is protective against helminths, recent data in mice demonstrate that Th1 is also important in some cestodes like Hymenolepis nana. OBJECTIVES: To identify the role of Th1 and Th2 lymphocytes in immunity against H. nana, the levels of IL-12, IFNgamma, IL-5, and IL-13 were determined in serum of humans infected with this cestode. METHODS: A total of 31 patients (case) with H. nana infection and 30 clinically healthy individuals (control) was included in this study. Measurements of IL-12, IFNgamma, IL-13 and IL-5 in serum samples were performed by solid-phase sandwich enzyme linked immunosorbant assay. Differential leukocyte count was also done. T test, Mann Whitney U test and Wilcoxan W test were used for data analysis. RESULTS: The mean concentrations of IFNgamma, IL-12 and IL-5 in the sera of patients with H. nana infection were higher than the control group, but only the differences between the concentrations of IFNgamma (P<0.001) and IL-13 (P<0.05) in the two groups were significant. There was an increase in the percentage of monocytes, eosinophils and lymphocytes in patients when compared to the controls, but this increase was not significant. CONCLUSIONS: Results from the present study in humans are in agreement with experimental studies in animals in which both Th1 and Th2 responses occur in H. nana infection.

Helminth infection enhances disease in a murine TH2 model of colitis.

BACKGROUND & AIMS: There is convincing evidence from animal and human studies that infection with parasitic helminths can alleviate the histopathology and symptoms of colitis. Here the ability of the rat tapeworm Hymenolepis diminuta to affect the course of oxazolone-induced colitis (a TH2 model) was assessed. METHODS: Mice were infected with H diminuta and 8 days later they received oxazolone (3 mg in 50% EtOH, intrarectal). On autopsy (3 or 7 days postoxazolone), disease severity was assessed by macroscopic clinical scores, histologic damage scores, myeloperoxidase and eosinophil peroxidase activity, and cytokine synthesis. RESULTS: As gauged by all markers of gut function, infection with H diminuta caused a significant exacerbation of oxazolone-induced colitis. Indeed, while mice receiving oxazolone only began to recover approximately 3-4 days posttreatment, the cotreated group continued to deteriorate. Helminth infection, independent of oxazolone administration, enhanced IL-4, IL-5, IL-10, and IL-13 production from in vitro stimulated immune cells and evoked increases in colonic eosinophil peroxidase of cotreated mice. Finally, while knockout of natural killer (NK) and NK-T cells by administration of a neutralizing NK1.1 antibody reduced the inflammation in oxazolone and oxazolone + H diminuta-treated animals, mice in the latter group still displayed significant colitis. CONCLUSIONS: We have shown that H diminuta infection is beneficial in other models of colitis. The current data is presented as a caveat to the position that parasitic helminths in general can be considered as a therapy for heterogeneous inflammatory disorders without careful analysis of the immunologic basis of the condition.

Infection with the cestode Hymenolepis diminuta induces changes in acetylcholine metabolism and muscarinic receptor mRNA expression in the rat jejunum.

Total and neuron-specific uptake of [3H] choline into smooth muscle/myenteric plexus (SM/MP) preparations from the jejunum of rats infected with five Hymenolepis diminuta for 30 days compared to uninfected rats was significantly increased, as was choline acetyltransferase activity and acetylcholine biosynthesis. Although acetylcholinesterase and total cholinesterase activity levels in SM/MP preparations from infected rats were not significantly different from uninfected animals, pseudocholinesterase activity was significantly elevated in infected rats. Infection resulted in a significant elevation in the relative expression of muscarinic 2 (M2) receptor mRNA in jejunum compared to uninfected rats. Conversely, in rats infected with 50 worms for 30 days, the relative expression of muscarinic 1 (M1) receptor mRNA in the jejunum was significantly depressed, while the expression of M2 receptor mRNA was not significantly different from that in five worm infections. The relative expression of muscarinic 3 receptor mRNA was unaffected by infection. The present study shows that infection of rats with low numbers of an enteric cestode leads to a significant modulation of the cholinergic components of the myenteric plexus and M2 receptor mRNA, and that large number of worms result in suppression in the relative expression of M1 receptor mRNA.

Tapeworm infection reduces epithelial ion transport abnormalities in murine dextran sulfate sodium-induced colitis.

The rat tapeworm Hymenolepis diminuta was used to test the hypothesis that helminth infection could modulate murine colitis. Mice were infected with five H. diminuta cysticercoids, and colitis was evoked via free access to 4% (wt/vol) dextran sulfate sodium (DSS)-containing drinking water for 5 days. BALB/c mice were either infected with H. diminuta and 7 days later exposed to DSS (prophylactic strategy) or started on DSS and infected with H. diminuta 48 h later (treatment strategy). Naive and H. diminuta-only-infected mice served as controls. On autopsy, colonic segments were processed for histological examination and myeloperoxidase (MPO) measurement or mounted in Ussing chambers for assessment of epithelial ion transport. Cytokines (gamma interferon [IFN-gamma], interleukin 12 [IL-12], and IL-10) were measured in serum and colonic tissue homogenates. DSS treatment resulted in reduced ion responses (indicated by short-circuit current [Isc]) to electrical nerve stimulation, the cholinergic agonist carbachol, and the adenylate cyclase activator forskolin compared to controls. H. diminuta infection, either prophylactic or therapeutic, caused a significant (P < 0.05) amelioration of these DSS-induced irregularities in stimulated ion transport. In contrast, the histopathology (i.e., mixed immune cell infiltrate, edema, and ulcerative damage) and elevated MPO levels that accompany DSS colitis were unaffected by concomitant H. diminuta infection. Similarly, there were no significant differences in levels of IFN-gamma, IL-12, or IL-10 in serum or tissue from any of the treatment groups at the time of autopsy. We suggest that abolishment of colitis-induced epithelial ion transport abnormalities by H. diminuta infection provides proof-of-principle data and speculate that helminth therapy may provide relief of disease symptoms in colitis.

Modulation of caudal intestinal permeability in the rat during infection by the tapeworm Hymenolepis diminuta.

Bidirectional movement of solutes between the intestinal lumen and systemic circulation is restricted by tissue barriers that may be altered under conditions such as intestinal infection. In a study using an in vitro everted sac preparation to assess small intestinal permeability in a lumen-to-serosa direction, 51Cr-EDTA movement was compared regionally in the jejunum and ileum of rats infected and uninfected by tapeworms. Whereas jejunal segments showed no significant differences in permeability to 51Cr-EDTA at 6, 15, or 32 days postinfection (dpi), ileal segments displayed an increased permeability on 15 and 32 dpi, but not 6 dpi. The alterations in permeability were not reversed 1 wk after removal of the tapeworm from the intestine. In conclusion, the strictly lumen-dwelling tapeworm infection allows increased movement of molecules from the lumen into ileal, but not jejunal, tissues by 15 dpi.

The spatial and temporal concentrations of choline in the lumen contents of the small intestine of uninfected and Hymenolepis diminuta infected rats.

The spatial and temporal concentrations of free choline in the lumen of the small intestine of the uninfected and Hymenolepis diminuta-infected rat were investigated. In the unfed infected or uninfected rat, the choline concentrations ranged from approximately 500 microM in the duodenum to approximately 20 microM in the posterior ileum, with some segments in infected rats containing significantly higher choline levels than in uninfected rats. Following feeding, choline levels were significantly elevated to approximately 3 mM by 6 h, although these concentrations fell rapidly in transit down the intestine. By 12 h the choline levels were similar to those in the unfed rat. An initial small shift in the worm biomass toward the duodenum after feeding was followed by a redistribution of biomass along the length of the small intestine. The worm biomass, however, had little or no effect on the choline levels. The high concentrations of free choline observed in the anterior regions of the intestine are postulated to be predominantly determined by nutritional intake while the concentrations in the posterior region may in part be determined by blood choline levels. The high levels of choline indicate that choline is not limiting to support the growth of the worms.

A novel Na+/HCO3--codependent choline transporter in the syncytial epithelium of the cestode Hymenolepis diminuta.

Absorption of exogenous choline by the cestode Hymenolepis diminuta was found to be both Na+- and HCO3--dependent and, at pH 6 to 7, accounted for up to 65% of the total choline uptake. Na+/HCO3- dependent choline uptake was activated at approximately 6 mM HCO3- (EC50 approximately 9 mM), and, above 100 mM Na+, the rate of uptake was directly proportional to the Na+ concentration. Atempts to uncouple Na+-dependent uptake from HCO3--dependent uptake were not successful: K+-depolarization was without effect on HCO3--dependent choline uptake, and use of valinoomycin to hyperpolarize the brush-border membrane resulted in inhibition of uptake. Na-/HCO3--dependent choline uptake was not associated with solvent drag. The Na+/HCO3--dependent choline uptake displayed a Q10 of 6.4 (27 degrees to 37 degrees) and a relatively high activation energy of 126 kJ x mol(-1). At pH 6.0 and 7.0, Na-/HCO3--dependent choline uptake rates were similar, but Na+/HCO3--dependent choline uptake was reduced at pH 5.0. The Na+/HCO3--dependent choline uptake, at pH 7.0, displayed a Kt of approximately 500 microM and a Vmax of 4.01 pmol x mg wet weight(-1) x min(-1). The Na+/HCO3--dependent choline uptake was hemicholinium-3 sensitive, but not significantly inhibited by 200 microM bumetanide, 100 microM amiloride, benzamil, or EIPA or by 1 mM 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS) or 4-acetamido-4'-isothiocvanostilbene-2,2'-disulfonic acid (SITS). Although it remains to be shown that HCO3- uptake is coupled directly to both choline and Na+ uptake, the data suggest that choline up take occurs via choline/Na+/HCO3--co-trans porter.

Novel benzamil-sensitive sodium-independent choline transport in the cestode Hymenolepis diminuta: evidence for sodium channels uptake at low pH.

The uptake of choline by the tegument of Hymenolepis diminuta was investigated. The Q10 at pH 7.0 was 1.7, with an Ea of 90 kJ.mol-1. Choline transport was pH sensitive: At pH 5.0, a Na(+)-independent mechanism predominated, which was inhibited by 100 nM benzamil, 130 mM Na+, and 300 microM verapamil. At pH 7.0, the Na(+)-independent mechanism was inhibited by 130 mM Na+, amiloride, and EIPA with IC50's of 130 microM and 30 microM, respectively, and by benzamil with IC50's of 100 pM (high-potency Benzamil Sensitive Component; HBSC) and 70 microM (low-potency Benzamil Sensitive Component; LBSC). Calcium-free saline enhanced choline uptake non-specifically. Lanthanum3+, Gd3+, gramicidin, nigericin, and high-K+ did not affect choline uptake at pH 5.0 or pH 7.0, and 10 microM verapamil was without effect at pH 5.0, suggesting no significant role for the electrical potential difference across the brush-border membrane, a Na+/H+ antiporter, a Na+/Ca2+ antiporter, or Ca2+ channels in choline uptake. Under physiological conditions, the HBSC accounts for approximately 25% of the total choline taken up at pH 5.0, while the LBSC accounts for approximately 55% of the choline taken up at pH 7.0. The data suggest novel choline transporting mechanisms; an HBSC which displays properties in common with apical Na+ channels, and a unique LBSC of choline transport.

Reactive oxygen intermediates from eosinophils in mice infected with Hymenolepis nana.

A large number of eosinophils were recruited to the intestinal villi after infection with Hymenolepis nana. Eosinophil numbers were increased more rapidly in challenged mice than in primary infected mice. Local intestinal eosinophils from challenged mice showed more extracellular oxygen radical release, as assessed by histochemical methods using nitro blue tetrazolium, accompanied with tissue injury and larval degradation. Intestinal eosinophils isolated from the lamina propria induced specific oxygen radical generation in response to H. nana oncosphere extract as measured by luminol-dependent chemiluminescence. This response was stronger in challenged mice than in primary infected mice. Radical generation from uninfected mice was negligible. Lipid peroxidation in the small intestine, as measured by formation of malondialdehyde, was increased during H. nana challenge infection, the peak activity coinciding with the elimination of challenge larvae. Continuous administration of a NADPH oxidase inhibitor to sensitized mice interfered with the degeneration of challenge larvae. These results suggest that intestinal eosinophils may be the major contributor to oxygen radical production in response to H. nana and that reactive oxygen species may play a part of effector molecule in the resistance to reinfection with H. nana.

Hymenolepis diminuta-induced fecundity reduction may be caused by changes in hormone binding to Tenebrio molitor ovaries.

Aspects of vitellogenesis, known to be controlled by juvenile hormone, are adversely affected by Hymenolepis diminuta infection of Tenebrio molitor, in spite of circulating titres of the hormone remaining unchanged. It has therefore been proposed that juvenile hormone binding is disrupted at the tissue site level. Juvenile hormone III binding sites were located in the nuclear, microsomal and post-microsomal supernatant fractions of the follicle cells of Tenebrio molitor. When JH-III binding was quantified for both control and Hymenolepis diminuta-infected beetles, binding in the nucleus and cytosol were found to be largely unaffected. However, microsomal binding was severely disrupted; on days 3 and 6 post-infection, binding was greatly diminished, on day 9 post-infection, binding was slightly reduced and, by day 15, binding was 'restored' to that of control insects. Using follicle cell microsomes at day 3 post-infection, previous Scatchard analysis revealed the presence of at least two JH-III binding sites. The first is of higher affinity, Kd = 5.3 x 10(-8) M, Bmax = 1.5 x 10(-11) mol/mg protein and the second of lower affinity Kd = 7.7 x 10(-7) M, Bmax = 9.75 x 10(-11) mol/mg protein. A comparison with microsomal binding parameters of follicle cells from non-infected Tenebrio indicated that although the Bmax values were unchanged, the Kd value of the higher affinity site was increased by approximately 5-fold. These data are indicative of a parasite-induced competitive binding inhibitor.