Isolation and partial structural characterization of new Kunitz-type trypsin inhibitors from the pike cestode Triaenophorus nodulosus.

The inhibitors produced by the parasitic worms successfully protect them from the host's proteases and are supposed to underlie the host-parasite specificity. Our previous study has shown that the extracts from the pike tapeworm Triaenophorus nodulosus inhibit host proteinases and commercial trypsin. We aimed to isolate and identify the components responsible for trypsin inactivation. After a two-step separation the molecular masses were measured by SE-HPLC. The sample proved to contain four fractions represented by polypeptides (1-45 kDa) and low-molecular hydrophobic compounds. According to SDS-PAGE analysis, the major polypeptides in the fractions displaying the highest inhibition had masses of 14.4 kDa. The study culminated in partial N-terminal amino acid sequence analysis with a further search for homology. The research revealed two novel Kunitz-type proteins potentially responsible for the inhibitory capacity of the tapeworms against trypsin. Our findings extend the list of cestodes relying on Kunitz-type proteins in the host-parasite molecular cross-talk.

Effects of tapeworm infection on absorption and excretion of zinc and cadmium by experimental rats.

The main objective of this study was to determine how rat tapeworms affect the excretion of zinc and cadmium through rat feces. Male rats (Rattus norvegicus var. alba) were divided into four groups, and the experiment was conducted over a 6-week period. The control groups (00; 0T) were provided with a standard ST-1 rodent mixture and received 10.5 mg of Zn/week. Groups P0 and PT were fed a mixture supplemented with the hyperaccumulating plant Arabidopsis halleri at a dosage of 123 mg Zn/week and 2.46 mg Cd/week. Groups 0T and PT were infected with the rat tapeworm (Hymenolepis diminuta). Fecal samples were collected 24 h post exposure. Zinc and cadmium concentrations in rat feces were analyzed using inductively coupled plasma optical emission spectrometry. Tapeworm presence decreased the amount of metals excreted through the feces of the host throughout the entire experiment, with the exception of 1 week (control group). No statistically significant differences between zinc excretion rates in the control groups (00 and 0T) were detected at any time throughout the experiment. A statistically significant difference between zinc excretion rates (p < 0.05) in the exposed groups (P0 and PT) was detected in 2 of the 6 monitored weeks. Group PT excreted significantly less cadmium (p < 0.01) than group P0 did in three of the 6 weeks. Overall, our results indicate that tapeworms are able to influence the excretion of metals by their host. Tapeworms accumulate metals from intestinal contents. It is not clear whether tapeworms carry out this process before the host tissues absorb the metals from the intestines or the tapeworms accumulate metals excreted from the body of the host back to the intestines. Most likely, it is a combination of both phenomena.

Studies on some metacestodes immunohistochemical response in mice as a model for human cysticercosis: I. Role of Th1 and Th2 cytokines in experimental liver granuloma of Mesocestoides corti infected mice.

Intraperitoneal infection of female BALB/C mice with the Mesocestoides corti larvae leading to an intense inflammatory response associated with symptoms started to appear between 4-5 weeks post-infection. The hepatic changes in the process of granuloma formation after intraperitoneal infection with the tetrathiredia of M. corti were analyzed. Histopathological changes were observed after five days of infection. As a result of this parasitic infection, an extensive inflammatory response took place with infiltrating cells first tracking the migratory pathway surrounding the parasites. The pathology associated with these processes was very destructive for the liver parenchyma. As the infection progressed, neutrophils, macrophages, fibroblasts, mast cells and lymphocytes were recruited in the tissue. These immune cells started to surround the parasites, leading to the formation of granuloma around them. Both Th1 and Th2 cytokines interact with each other to regulate and modulate the hepatic granuloma formation in infected mice.

Schistocephalus solidus infections increase gonadotropins and gonadotropin releasing hormone (GnRH3) mRNA levels in the three-spined stickleback, Gasterosteus aculeatus.

Parasites often impair the reproduction of their hosts, one well known case being the cestode Schistocephalus solidus which is a common parasite in three-spined sticklebacks, Gasterosteus aculeatus. One of the possible ways that this could be exerted is by suppression on the brain-pituitary-gonadal (BPG) axis. In this study, mRNA levels of FSH-ß and LH-ß and of GnRH2 (cGnRH II) and GnRH3 (sGnRH) were measured via Q-PCR in infected and uninfected fish sampled from the field a few weeks before the onset of breeding. The pituitary mRNA levels of both FSH-ß and LH-ß were higher in infected males than in uninfected males. Also in females, FSH-ß mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-ß expression. Brain mRNA levels of GnRH3 were higher in infected fish than in uninfected fish in both sexes, but no difference was found in GnRH2 mRNA levels. Thus, infection by S. solidus was able to alter the expressions not only of gonadotropins (GtHs), but also of GnRH which has not been observed previously. However, the effects are opposite to what should be expected if the parasite suppressed reproduction via actions on the brain-pituitary level. The gonads are perhaps more likely to be impaired by the parasites in other ways, and changed feedbacks on the BPG axis could then lead to the increases in GtHs and GnRH.

Dynamics of hepatic stellate cells, collagen types I and III synthesis and gene expression of selected cytokines during hepatic fibrogenesis following Mesocestoides vogae (Cestoda) infection in mice.

In the present study, the relationship between progression of Mesocestoides vogae infection in the liver of mice, the accumulation rate of collagen types I and III, gene expression of fibrogenic factors and cytokines was examined within 6weeks p.i. Due to asexual multiplication, the total number of larvae in the liver increased considerably and 63.4% were found in collagen capsules on day 42 p.i. Intense staining for both collagens was recorded in the activated hepatic stellate cells (HSCs) throughout the period of this study in the inflammatory lesions. With progressing infection, cellular expression of both collagens was confined to the flat cells, myofibroblasts, which were scattered among collagen fibres in parenchymal lesions and capsules. Collagen-positive areas mirrored immunostaining of alpha-smooth muscle actin (alpha-SMA) in HSCs and myofibroblasts. Gene expression of both collagens increased rapidly within 14days p.i. and their expression pattern resembled that for pro-fibrotic cytokine transforming growth factor (TGF)-beta1 and alpha-SMA protein. IL-10 cytokine expression was up-regulated following day 14 p.i. and that of IL-13 was up-regulated early p.i., then transcription elevated gradually mirroring the activity of other pro-fibrotic markers. In contrast, transcription activity of TNF-alpha and IFN-gamma was elevated shortly after infection, followed by the partial down-regulation of gene expression, indicating the lack of larval killing, enhanced granulomatous inflammation and the perpetuation of hepatic fibrosis. Histomorphometric analysis of the parenchymal fibrous lesions, surface areas of larvae surrounded with the inflammatory infiltrates and surface areas of developing or mature larva-containing granulomas, correlated with the proportion of free and encapsulated larvae, immunostaining and gene expression patterns of collagens and pro-fibrotic markers. At a later stage of infection (day 28 p.i. onwards) collagen I-positive areas occupied a greater surface area and formed mature larval capsules and scars in the liver. In contrast, collagen III was less abundant and was localised mainly in the fibrous lesions in damaged parenchyma, suggesting their specific up-regulation as the part of host-protecting and tissue-healing responses.

IL-4(-/-) mice with lethal Mesocestoides corti infections--reduced Th2 cytokines and alternatively activated macrophages.

Protection against Mesocestoides corti, a cestode that invades vital organs, is dependent on the production of IL-4, as IL-4(-/-) mice were found to have higher parasite burdens when compared with wild-type mice. The goal of this study was to investigate the role of IL-4 in immunity to M. corti, focusing on the immunological profile and on potential mediators of pathology. IL-4(-/-) mice infected with M. corti showed 100% mortality by 32 days, whereas wild-type mice survived for approximately 1 year. Parasite burdens were significantly increased in the liver, peritoneal, and thoracic cavities of IL-4(-/-) mice, associated with impaired recruitment of inflammatory cells and a reduction in monocytes and macrophages. IL-5 production by splenocytes and expression in liver tissue was decreased in infected IL-4(-/-) mice compared with wild-type mice. In contrast, IL-4(-/-) mice produced increased amounts of IFNgamma and TNFalpha. Alternatively activated macrophages were a major feature of liver granulomas in wild-type mice evidenced by Arginase I expression, while livers from infected IL-4(-/-) mice showed impaired alternative macrophage activation without increased classical macrophage activation. Thus, lethality during M. corti infection of IL-4(-/-) mice is associated with decreased Th2 cytokines, increased Th1 cytokines and impairment of alternatively activated macrophages.

Increased glial-derived neurotrophic factor in the small intestine of rats infected with the tapeworm, Hymenolepis diminuta.

The neurotrophin, glial-derived neurotrophic factor (GDNF), is essential for the development of the enteric nervous system (ENS) in both the embryo and neonate and may be important for maintenance and plasticity of ENS. The tapeworm, Hymenolepis diminuta, altered the number of cells containing GNDF in the host's jejunum and ileum. Numbers and locations of GDNF-containing cells were determined by applying monoclonal anti-GDNF antibody to intestinal segments collected from infected and uninfected age-matched rats during the initial 34 days post-infection (dpi). Most cells staining positive for GDNF were present in the lamina propria of the jejunum and ileum from both infected and uninfected rats. The co-localization of staining by the antibodies, anti-GDNF and anti-ED2 (a nuclear specific antibody for resident macrophages) indicated that at least 74% of the cells staining for GDNF were macrophages. Mast cells did not stain with the anti-GDNF antibody. The increased number of GDNF+ cells in the infected rat intestine suggests that this neurotrophin may play a role in the neural and mucosal responses to lumenal tapeworm infection.

Mucin-type O-glycosylation in Mesocestoides vogae (syn. corti).

Protein glycosylation is an important post-translational modification underlying host-parasite interactions, which may determine the outcome of infection. Although Mesocestoides vogae represents an important model for investigating the various aspects of cestode biology, virtually no information is available about the structure and synthesis of glycans in this parasite. In this work, focused on the initiation pathway of mucin-type O-glycosylation in M. vogae, we characterized O-glycoproteins bearing the simple mucin-type cancer-associated Tn and sialyl-Tn antigens, and the expression and activity of ppGalNAc-T, the key enzyme responsible for the first step of mucin-type O-glycosylation. Using immunohistochemistry, Tn and sialyl-Tn antigens were detected mainly in the tegument (microtriches) and in parenchymal cells. Tn expression was also observed in lateral nerve cords. Both Tn and sialyl-Tn antigens were detected in in vitro cultured parasites. Based on their electrophoretic mobility, Tn- and sialyl-Tn-bearing glycoproteins from M. vogae were separated into several components of 22 to 60 kDa. The observation that Tn and sialyl-Tn glycoproteins remained in the 0.6N perchloric acid-soluble fraction suggested that they could be good candidates for characterizing mucin-type glycosylation in this parasite. O-glycoproteins were purified and initially characterized using a proteomic approach. Immunohistochemical analysis of the tissue distribution of ppGalNAc-T revealed that this enzyme is expressed in the sub-tegumental region and in the parenchyma of the parasite. In M. vogae cultured in vitro, ppGalNAc-T was mainly detected in the suckers. Using a panel of 8 acceptor substrate synthetic peptides, we found that M. vogae ppGalNAc-T preferentially glycosylate threonine residues, the best substrates being peptides derived from human mucin MUC1 and from Trypanosoma cruzi mucin. These results suggest that M. vogae might represent a useful model to study O-glycosylation, and provide new research avenues for future studies on the glycopathobiology of helminth parasites.

Expression and distribution of Toll-like receptors in the brain during murine neurocysticercosis.

In a mouse model of neurocysticercosis, the expression and distribution of Toll-like receptors (TLRs) was investigated by using both gene array analyses and in situ immunofluorescence microscopy (IF). In the normal uninfected brain, mRNA of all the TLRs are constitutively expressed albeit TLR5, TLR7, TLR8 and TLR9 to a lesser extent. In these animals, however, expression of TLR1, TLR3, TLR4 and TLR9 proteins was not detected. In contrast, parasite infection increased both gene and protein level expression of all the TLRs several fold except TLR5 where only the mRNA was upregulated. Importantly, TLRs were differentially distributed among various central nervous system (CNS) cell types and infiltrating leukocytes. TLR2 was almost exclusively localized to nervous tissue cells, particularly astrocytes, while TLR1 and TLR9 proteins were essentially limited to infiltrating leukocytes. All other TLRs tested were detected in both CNS and immune cell types. Interestingly, ependymal cells and neurofilaments of the cerebellar white matter of infected mice exhibited a substantial upregulation of TLR7 and TLR8 proteins respectively. These data provide a comprehensive analysis of TLR expression in the normal and parasite infected brain and suggest a role for TLRs in the interplay of immune cells and CNS cells during infection.

Parasitism in fish--an endocrine modulator of ecological relevance?

Wild bream (Abramis brama) were collected from the river Elbe, and the influences of parasitic infection by the tapeworm Ligula intestinalis on endocrine and related functions (vitellogenin [VTG]; plasma sex steroids: 17beta-estradiol [E2], 11-ketotestosterone [11-KT] and testosterone [T]; relative gonad [GSI] and liver [HSI] growth; maturation stages of germ cells [MS]; prominence of spawning tubercles [STI]) were investigated. Distinct regional differences in infection rates of bream with L. intestinalis were observed along the Elbe with the highest prevalences at the Czech border (up to 45%) and Magdeburg (up to 65%), areas that are heavily contaminated with complex mixtures of organic chemicals and metals. Parasitized fish of both sexes had significantly lower GSIs and poorly developed gonads (low MS). In males, a significant reduction in the prominence of spawning tubercles occurred. Infected females had significantly lower plasma VTG concentrations. A selective suppression of the sex steroids 11-KT and E2 was observed in male and female bream, respectively. Testosterone was not affected in the same manner in fish of both sexes. At sites with an elevated prevalence of L. intestinalis, the extent of the infection of an individual was significantly correlated with the suppression of the measured biomarkers. However, when applying a linear model to compare regional differences in infection prevalence with biological parameters, not all of the observed differences could be explained by parasitization by L. intestinalis. This indicates that other factors such as pollution may have contributed to the effects on reproductive and endocrine processes that occurred along the river. Given that sites with high prevalences of L. intestinalis were also characterized by elevated pollution, it is possible that there exists a combinatory effect of both pollution and parasitization that can have a serious impact on the reproductive capacity of a population, such as was observed at the sampling site at Magdeburg.

Distribution of [3H]cholesterol-labelled liposomes with or without praziquantel in mice infected with Mesocestoides corti (Cestoda) tetrathyridia.

The anthelmintic drug praziquantel (PZQ) has a short half-life in the circulation, necessitating repeated daily administration of PZQ for the therapy of larval stages of cestodes. The effect of incorporation of PZQ into multilamellar liposomes on their biodistribution in Mesocestoides corti (syn. M. vogae) infected mice has been examined using [3H]cholesterol as a liposomal marker. Incorporation of PZQ significantly increased the average size of liposomes with 70.3% of [3H]lip.PZQ particles up to 1.9 microm, whereas higher portion of [3H]liposomes (66.3% of total) were of smaller (up to 1.3 microm). Both liposome preparations were given intraperitoneally to avoid rapid sequestration in the liver. There were significant differences between [3H]liposomes and [3H]lip.PZQ-associated radioactivity in peritoneal adherent cells, liver- and peritoneal larvae, liver, spleen and lymph nodes within 16 days of examination. The highest uptake (about 2-fold more [3H]lip.PZQ than [3H]liposomes from the total dose) was found in peritoneal cells on day 1 post therapy (p.t.) followed by a rapid decline. The kinetic of decline in these cells recovered on day 1 p.t. was studied also in vitro. Disappearance of the marker due to the breakdown of liposomes and efflux of lipids and PZQ from cells was slower for [3H]lip.PZQ in comparison with drug-free liposomes and was not completed after 4 days-incubation. Significantly increased levels of radioactivity, more in [3H]liposomes treated groups, were recorded in the liver- and peritoneal larvae between days 8-16 p.t. indicating re-utilization of cholesterol by the larvae. The data suggest that incorporation of PZQ into liposomes contributes to the enlargement of liposome average size and slows down their degradation in phagocytosing cells. In this respect, these cells could serve as the secondary circulating depots for PZQ releasing it slowly to the circulation.

The fatty acids of each lipid fraction and their use in providing energy source of the plerocercoid of Spirometra erinacei.

The fatty acid concentration of each lipid fraction of plerocercoids of Spirometra erinacei and the host snake serum was investigated. The major fatty acids of phospholipid of the plerocercoids were C18:1, C18:0 and C16:0, and those of the host snake serum were C16:0, C18:1 and C18:0, in order of amount in both cases. The changes of the fatty acid composition of phospholipid of the plerocercoids when they were incubated in physiological saline at 18 degrees C and at 37 degrees C for 24 h were investigated in both cases. Polyunsaturated fatty acids increased at 18 degrees C, and saturated fatty acids increased at 37 degrees C. Michaelis constants (Km) of beta-hydroxyacyl-CoA dehydrogenase (HAD), NADH: ubiquinone oxidoreductase (complex I) (NADH: ferricyanide reaction) and complex I (NADH: ubiquinone reaction) for NADH were 20.6, 50 and 13.3 microM, respectively. The ATP production in mitochondria of the plerocercoids was accelerated by adding ADP and inhibited by adding such electron transport system inhibitors as rotenone, antimycin A and sodium cyanide. These results suggested that the fatty acids in the plerocercoids played an important role in regulating the fluidity of membrane by changing the composition in membrane lipid corresponding with the change of temperature circumstance. The NADH reduced by HAD might be accepted by the complex I in the electron transport system, and thus the parasites were capable of ATP production in a classical pathway of the oxidative phosphorylation system.

Infection of IL5 transgenic mice with Mesocestoides corti induces very high levels of IL5 but depressed production of eosinophils.

Transgenic mice expressing interleukin 5 (IL5) have been demonstrated to show a lifelong high level eosinophilia. These mice were produced using a construct in which the dominant control region (DCR) of the human CD2 gene was ligated to a 10-kb fragment containing the mouse IL5 gene. The construct allows the expression of the IL5 gene under the control of its own promoter, but the DCR ensures constitutive expression by all T cells. Infection of these transgenic mice with Mesocestoides corti, which is itself a potent inducer of eosinophilia, increases serum IL5 to very high levels. This demonstrates that the transgenes retain inducibility, which is a feature of the endogenous gene. However, despite the high levels of IL5, the numbers of eosinophils in the blood, marrow, and spleen decrease during the period 1-4 weeks after infection. Furthermore, there is a decrease in eosinophil precursors, as assessed by the capacity of bone marrow to produce eosinophils in culture. After this decrease eosinophils return to their previous high levels, although the levels of IL5 remain high. These results suggest that a control mechanism is operating to limit the numbers of eosinophils produced. This control appears to act at the level of the precursor production and may not be directly related to the high levels of IL5.

A comparison of the metabolic effects of bovine growth hormone and growth factor from Spirometra mansonoides on rat liver in vivo.

The effects of bovine growth hormone(GH) and Spirometra growth factor (SGF) on the in vivo concentrationions of glycolytic and citricacid (TCA) cycle intermediates in rat liver were investigated. The effects of these agents on the mitochondrial and cytoplasmic redox states, liver phosphorylation states, and adenylate kinase activity were also evaluated. The effects of SGF were studied in both normal and hypophysectomized rats,wheras GH effects ere examined only in hypophysectomized rats. Growth homrone(0.25mg/rat) was injected dayily for 9 days...