Effects of duration of exposure, co-supplementation with iron and Nippostrongylus brasiliensis infection on accumulation of trichloroacetic acid-insoluble copper in rats given molybdate in drinking water.

Thiolation can convert molybdate (MoO4) into a series of thiomolybdates (MoSxO4-x) in the rumen, terminating in tetrathiomolybdate (MoS4), a potent antagonist of copper absorption and, if absorbed, donor of reactive sulphide in tissues. Systemic exposure to MoS4 increases trichloroacetic acid-insoluble copper (TCAI Cu) concentrations in the plasma of ruminants and induction of TCAI Cu in rats given MoO4 in drinking water would support the hypothesis that rats, like ruminants, can thiolate MoO4. Data on TCAI Cu are presented from two experiments involving MoO4 supplementation that had broader objectives. In experiment 1, plasma Cu concentrations (P Cu) tripled in female rats infected with Nippostrongylus brasiliensis after only 5 days exposure to drinking water containing 70 mg Mo L-1, due largely to an increase in TCAI Cu; activities of erythrocyte superoxide dismutase and plasma caeruloplasmin oxidase (CpOA) were unaffected. Exposure for 45-51 days did not raise P Cu further but TCA-soluble (TCAS) Cu concentrations increased temporarily 5 days post infection (dpi) and weakened the linear relationship between CpOA and TCAS Cu. In experiment 2, infected rats were given less MoO4 (10 mg Mo L-1), with or without iron (Fe, 300 mg L-1), for 67 days and killed 7 or 9 dpi. P Cu was again tripled by MoO4 but co-supplementation with Fe reduced TCAI Cu from 65 ± 8.9 to 36 ± 3.8 µmol L-l. Alone, Fe and MoO4 each reduced TCAS Cu in females and males when values were higher (7 and 9 dpi, respectively). Thiolation probably occurred in the large intestine but was inhibited by precipitation of sulphide as ferrous sulphide. Fe alone may have inhibited caeruloplasmin synthesis during the acute phase response to infection, which impacts thiomolybdate metabolism.

Intestinal epithelial tuft cell induction is negated by a murine helminth and its secreted products.

Helminth parasites are adept manipulators of the immune system, using multiple strategies to evade the host type 2 response. In the intestinal niche, the epithelium is crucial for initiating type 2 immunity via tuft cells, which together with goblet cells expand dramatically in response to the type 2 cytokines IL-4 and IL-13. However, it is not known whether helminths modulate these epithelial cell populations. In vitro, using small intestinal organoids, we found that excretory/secretory products (HpES) from Heligmosomoides polygyrus blocked the effects of IL-4/13, inhibiting tuft and goblet cell gene expression and expansion, and inducing spheroid growth characteristic of fetal epithelium and homeostatic repair. Similar outcomes were seen in organoids exposed to parasite larvae. In vivo, H. polygyrus infection inhibited tuft cell responses to heterologous Nippostrongylus brasiliensis infection or succinate, and HpES also reduced succinate-stimulated tuft cell expansion. Our results demonstrate that helminth parasites reshape their intestinal environment in a novel strategy for undermining the host protective response.

Th9 Cells and Parasitic Inflammation: Use of Nippostrongylus and Schistosoma Models.

Th9 cells are a new subpopulation of CD4+ T helper cells, characterized by the expression of IL-9 that have been involved in type 2 immune responses, antitumor responses and autoimmune diseases. Here, we describe two different parasitic models frequently maintained in the laboratory where Th9 cells or IL-9 (the cytokine produced by Th9 cells) has been shown to play critical roles in pathogen clearance and immune response activation: the nematode Nippostrongylus brasiliensis and the trematode Schistosoma mansoni.

Activation of Nippostrongylus brasiliensis infective larvae is regulated by a pathway distinct from the hookworm Ancylostoma caninum.

Developmentally arrested infective larvae of strongylid nematodes are activated to resume growth by host-derived cues encountered during invasion of the mammalian host. Exposure of Nippostrongylus brasiliensis infective larvae to elevated temperature (37°C) is sufficient to activate signalling pathways which result in resumption of feeding and protein secretion. This occurs independently of exposure to serum or glutathione, in contrast to the hookworm Ancylostoma caninum, and is not initiated by chemical exsheathment. No qualitative differences in protein secretion were induced by host serum as visualised by two-dimensional SDS-PAGE, although exposure of larvae to an aqueous extract of rat skin did stimulate secretion of a small pre-synthesised bolus of proteins. Infective larvae began feeding after a lag period of 3-4 h at 37°C, reaching a maximum of 90% of the population feeding by 48 h. Neither a membrane permeant analogue of cyclic GMP nor muscarinic acetylcholine receptor agonists stimulated feeding at 20°C, and high concentrations of both compounds inhibited temperature-induced activation. LY294002, an inhibitor of phosphatidylinositol 3-kinase, Akt inhibitor IV, an inhibitor of Akt protein kinase, and ketoconazole, an inhibitor of cytochrome P450, all blocked resumption of feeding and protein secretion at 37°C. Serotonin increased the rate of feeding assessed by uptake of radiolabelled BSA, but could not initiate feeding independently of elevated temperature. Collectively, the data suggest that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge at pathways downstream of phosphatidylinositol 3-kinase involving steroid hormone synthesis.

Nippostrongylus brasiliensis: reversibility of reduced-energy status associated with the course of expulsion from the small intestine in rats.

Gastrointestinal nematodes require energy for active establishment in the gut against intestinal flow and peristaltic motion. In this study we employed CellTiter-Glo Luminescent Cell Viability Assay to measure the ATP value of individual adult Nippostrongylus brasiliensis during the course of immune-mediated expulsion from the small intestine in rats. The ATP values of adult worms taken from the lumen of the distal small intestine were lower than worms collected from the lumen of the proximal small intestine. Moreover, values from worms in the lumen of the proximal small intestine were lower than those from worms in the mucosa, the preferred site of adult N. brasiliensis. The reduction of ATP values in worms from each region was observed not only at expulsion phase, but also at established phases of the infection suggesting that energy metabolism of the parasites is independent of host immune response. When adult worms with low ATP values on day 12 post-infection were implanted surgically into the small intestine of naïve rats, the worms re-established in recipients and completely restored the ATP values. Short in vitro culture of adult worms under low oxygen tension resulted in low ATP value in the worms. These results suggested that adult worms were dislodged from their preferred site by intact energy metabolism activity.

Mucin-type O-glycosylation in helminth parasites from major taxonomic groups: evidence for widespread distribution of the Tn antigen (GalNAc-Ser/Thr) and identification of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity.

This article focuses on the initiation pathway of mucin-type O-glycosylation in helminth parasites. The presence of the GalNAc-O-Ser/Thr structure, also known as Tn antigen, a truncated determinant related to aberrant glycosylation in mammal cells, and the activity of the UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferase (ppGaNTase), the enzyme responsible for its synthesis, were studied in species from major taxonomic groups. Tn reactivity was determined in extracts from Taenia hydatigena, Mesocestoides corti, Fasciola hepatica, Nippostrongylus brasiliensis, and Toxocara canis using the monoclonal antibody 83D4. The Tn determinant was revealed in all preparations, and multiple patterns of Tn-bearing glycoproteins were observed by immunoblotting. Additionally, the first evidence that helminth parasites express ppGaNTase activity was obtained. This enzyme was studied in extracts from Echinococcus granulosus, F. hepatica, and T. canis by measuring the incorporation of UDP-(3H)GalNAc to both deglycosylated ovine syalomucin (dOSM) and synthetic peptide sequences derived from tandem repeats of human mucins. Whereas significant levels of ppGaNTase activity were detected in all the extracts when dOSM was used as a multisite acceptor, it was only observed in F. hepatica and E. granulosus extracts when mucin-derived peptides were used, suggesting that T. canis ppGaNTase enzyme(s) may represent a member of the gene family with a more restricted specificity for worm O-glycosylation motifs. The widespread expression of Tn antigen, capable of evoking both humoral and cellular immunity, strongly suggests that simple mucin-type O-glycosylation does not constitute an aberrant phenomenon in helminth parasites.

A human in vitro granuloma model for the investigation of multinucleated giant cell and granuloma formation.

A method for the in vitro generation of granulomas and its use in the analysis of the human granulomatous response is summarized. As a target for the cellular response L3 larvae of Nippostrongylus brasiliensis are coincubated with human mononuclear blood cells, and within seven to fourteen days the development of blood monocytes to mature macrophages and to epithelioid cells and multinucleated giant cells (MGC) as typical constituents of granulomas clustered around the nematode is observed. The following review describes the uses and applications of this model for phenotyping, functional, formation and modulating studies of granulomas and MGCs, taking into account its unique features compared to other in vitro models. With respect to MGC formation, procedures are described and examples are given which allow the phenotyping of these cells using immunofluorescence and immunohistological techniques. In addition, the potential of this model for illuminating functional aspects of MGC is described applying an isolation protocol for MGC and a subsequent reverse-transcriptase polymerase chain reaction method for the analysis of single cells. Moreover, the significance and relevance of using this granuloma model is discussed in the follow up analysis of in vivo findings of interleukin-6 expression in MGC of granulomas of patients with sarcoidosis. These in vivo results implicated a role for interleukin-6 in granuloma and MGC development. The in vitro granuloma model was used to investigate potential modulatory effects of this cytokine by analysing the cell numbers and the number of MGC per in vitro granuloma, the size of the MGC formed, the fusion index and the morphology of the in vitro granuloma. The results demonstrated significant modulatory effects of interleukin-6 on the cell number per in vitro granuloma and on the morphology of the cells involved. Conceivably, elevated interleukin-6 levels may modulate granuloma formation with respect to the number of cells involved and in influencing distinct cell populations involved in granuloma formation.

Vasoactive intestinal polypeptide-like and peptide histidine isoleucine-like proteins excreted/secreted by Nippostrongylus brasiliensis, Nematodirus battus and Ascaridia galli.

Vasoactive intestinal polypeptide (VIP)-like protein was detected by dot blot analysis in the excretions/secretions (E/S) of Nematodirus battus and Ascaridia galli and was confirmed in the E/S of Nippostrongylus brasiliensis. ELISA analysis showed that N. brasiliensis E/S contained the highest proportion of VIP-like protein (28.04 pmoles/mg of total E/S protein) and A. galli E/S contained the lowest (10.89 pmoles/mg of total E/S protein). Peptide histidine isoleucine (PHI)-like protein was detected by dot blot analysis in the E/S products of N. brasiliensis, N. battus and A. galli. ELISA analysis suggested that A. galli E/S contained the highest proportion of PHI (20.77 nmoles/mg of total E/S protein) and N. battus E/S contained the lowest (0.67 nmoles/mg of total E/S protein). The possible significance of VIP-like and PHI-like substances in the E/S of gastrointestinal nematodes is discussed.

On the potential of amino acids to support survival and energy status of Nippostrongylus brasiliensis in vitro.

Aerobically but not anaerobically, amino acids can sustain motility as well as glycogen and ATP levels of N. brasiliensis as effectively as glucose can. Proline is the most active amino acid and in combination with lysine, cysteine and phenylalanine, can completely replace glucose.

Polyamine metabolism in some helminth parasites.

Polyamine levels of some helminth parasites were analyzed by reverse phase HPLC of benzoyl derivatives. Setaria cervi, Acanthocheilonema viteae, Hymenolepis nana, H. diminuta, and Ascaridia galli contained higher levels of spermine than spermidine while in Ancylostoma ceylanicum and Nippostrongylus brasiliensis the spermidine levels were higher than spermine; putrescine was either absent or present in minor quantities. The enzymes of polyamine biosynthesis viz., ornithine decarboxylase, S-adenosyl methionine (SAM)-decarboxylase, and arginine decarboxylase were present in very low to negligible amounts in all the parasites examined. A. ceylanicum exhibited high activity of ornithine amino transferase (OAT) and catalyzed appreciable decarboxylation of ornithine. The ornithine decarboxylating activity of A. ceylanicum was localized in the particulate fraction containing mitochondria, not inhibited by alpha-difluoromethyl ornithine, the specific inhibitor of ornithine decarboxylase (ODC), but inhibited in the presence of glutamate, suggesting the involvement of mitochondrial OAT rather than a true ODC in ornithine decarboxylation in this parasite. Significant activity of polyamine oxidase was also detected in helminth parasites. The absence of polyamine biosynthesizing enzymes in helminth parasites suggests their dependence on hosts for uptake and interconversion of polyamines, providing a potential target for chemotherapy.

Polyamine metabolism in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Spermidine was detected as the major polyamine of Ancylostoma ceylanicum as well as Nippostrongylus brasiliensis. Spermine was present in lower amounts whereas the level of putrescine was even less. S-Adenosylmethionine decarboxylase, a rate-limiting enzyme in the biosynthetic pathway of polyamines, was demonstrated at low levels in both parasites. Decarboxylation of lysine and arginine was absent or negligible and that of ornithine questionable, as the enzyme activity was not inhibited by alpha-difluoromethylornithine while RMI 71,645, an irreversible inhibitor of ornithine aminotransferase, strongly inhibited the liberation of CO2 from ornithine. High activity of ornithine aminotransferase was observed in both the parasites and may interfere with the assay for ornithine decarboxylase. Adults of A. ceylanicum were found to rapidly take up spermidine and spermine from incubation medium while uptake of putrescine was very low. These results indicate that hookworms depend on uptake and interconversion rather than de novo synthesis for their polyamine requirement.

Steroid analogs inhibit hormone binding by an extract from Nippostrongylus brasiliensis (Nematoda).

1. An extract from the rodent nematode Nippostrongylus brasiliensis contained putative receptors that bound radiolabeled sex hormones, based on isoelectric focusing. 2. Binding of radiolabeled testosterone by receptors at pH 4.4 was highly inhibited by the androgen analogs, testosterone-3-oxime and 4-aza-5-androsten-3-on-17 beta-ol. 3. Binding of radiolabeled progesterone by receptors at pH 6.4 was highly inhibited by the progesterone analogs 3,5-seco-4-norpregnan-5-on-3-oic acid and 19-norethisterone or 21-deoxycorticosterone. 4. Binding of radiolabeled 17 beta-estradiol by receptors at pH 4.9 was highly inhibited by epiandrosterone. 5. In vivo development of N. brasiliensis to the adult was partially inhibited by selected steroid analogs.

Characterization of antigens of the nematode Nippostrongylus brasiliensis by monoclonal antibodies.

Hybridomas secreting monoclonal antibodies to Nippostrongylus brasiliensis antigens were generated by hybridization of IR983F myeloma cells with spleen cells from Lou/M/Wol rats infected with living third-stage larvae. Antibodies specific either for larval or worm antigens were identified by enzyme-linked immunosorbent assays with Nippostrongylus brasiliensis fragments, homogenates and secretions as antigens. The results demonstrate that all antibodies which recognized larval antigens (38 antibodies) also reacted with worm surfaces. Ten antibodies were specific only for worm antigens. Ten antibodies reacted with worm homogenate, three antibodies recognized components of worm secretion and 17 antibodies combined with acetylcholinesterase. The epitope specificity was investigated by the capacity of various glycosides, aminoacids, N-acetylneuraminic acid and phosphorylcholine to inhibit the binding to worm fragments. The analysis revealed that alpha-methylglucoside, alpha-methylmannoside, N-acetylglucosamine, N-acetylgalactosamine, fucose and the amino acids leucine, phenylalanine, tyrosine, serine, tryptophan did not combine with the antigen-binding sites of the antibodies. Proline, arginine and histidine, however, displayed inhibitory effects. With N-acetylneuraminic acid as inhibitor three groups of antibodies could be discriminated. At a concentration of 10-20 mM, phosphorylcholine was a potent inhibitor for all antibodies.

Nematoda: aerobic respiratory pathways of adult parasitic species.

Aerobic respiratory pathways have been compared in adult parasitic nematodes, including Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ostertagia ostertagi, Cooperia oncophora, Haemonchus contortus, Oesophagostomum venulosum, Chabertia ovina, Dictyocaulus filaria, Dictyocaulus viviparus, and Ascaridia galli. Respiration was measured in both whole worm or tissue homogenates and isolated mitochondrial fractions, and delineated into the mammalian type or alternative respiratory pathways on the basis of their inhibition by antimycin A. The alternative, antimycin A-insensitive respiratory pathway was of comparable activity in all parasitic nematodes studied, irrespective of the body diameter or habitat of the worm. The mammalian-type, antimycin A-sensitive respiratory pathway showed variations; the extent of this pathway correlated with both the body diameter and habitat of the worm, being greater in thinner worms and those worms whose habitat is supposedly more aerobic.

The effect of anaerobiosis on adenosine triphosphate levels in larval Nippostrongylus brasiliensis and Haemonchus contortus.

The adenosine triphosphate (ATP) content of the pre-parasitic stages of Nippostrongylus brasiliensis, Haemonchus contortus (L1, L2 and L3) and the adults of the free-living nematode, Panagrellus redivivus, have been measured by bioluminescent photometry in aerated or near-anoxic conditions. The ATP content of the L1 and L2 stages of both parasitic species was unaltered by a lack of oxygen over a 90-min period. However, the L3 stage of both species and the adults of P. redivivus showed a significant fall in the level of ATP within 10 min of near-anoxia. This lower level of ATP was maintained during oxygen lack but the initial content was restored on return of the nematodes to aerobic conditions. The results suggest that measurement of ATP by bioluminescent photometry offers a readily measured and sensitive indicator of the capacity of a nematode to cope with transient changes in oxygen supply without undue metabolic stress.

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages.

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.

The effect of iron and protein deficiency on plasma levels and parasite uptake of [14C] fenbendazole in rats infected with Nippostrongylus brasiliensis.

The Nippostrongylus brasiliensis-rat model was used to determine whether iron and protein deficiency, which are commonly associated with parasitic infections, affected the pharmacokinetic behaviour of fenbendazole as measured by plasma concentrations and uptake by worms. Plasma 14C concentrations after [14C] fenbendazole administration were higher in iron and protein-deficient rats than in sufficient rats. However, the uptake of 14C by N. brasiliensis in iron and protein-deficient rats was significantly less than in worms from diet-sufficient rats. The reduced anthelmintic uptake by worms in protein and iron-deficient hosts may account, in part, for reduced anthelmintic efficacy under these circumstances. These findings are relevant to understanding variations in response to chemotherapy in populations of parasitised hosts containing malnourished individuals.

Changes in the adenylate energy charge of Nippostrongylus brasiliensis and Nematodirus battus during the development of immunity to these nematodes in their host.

Infection of rats with 2000 infective juveniles of Nippostrongylus brasiliensis and of lambs with 60 000 infective juveniles of Nematodirus battus results in a well-marked immunity to these nematodes in their respective host. There is a fall in the adenylate energy charge value of these nematodes during the course of these infections, reaching values of 0.37 in males and 0.27 in females of N. brasiliensis, and 0.31 in males and 0.23 in females of N. battus towards the end of the infections. In hosts given relatively small numbers of infective juveniles, the values for the nematodes removed from the hosts late in the infection remain at a relatively high level. These results indicate that the immune response of the host may affect the energy status of these nematodes, and this could help to explain their subsequent expulsion from the immune host.