Silencing of essential genes by RNA interference in Haemonchus contortus.

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.

The flightless I protein and the gelsolin family in nuclear hormone receptor-mediated signalling.

The Drosophila melanogaster flightless I protein and its homologues in higher eukaryotes (FliI) are conserved members of the gelsolin family of actin-binding proteins. Members of the gelsolin family generally contain three or six copies of a 125-amino-acid residue gelsolin-related repeating unit, and may contain additional domains including the C-terminal villin-related 'headpiece' or N-terminal extensions such as the leucine-rich repeat of the FliI protein. Numerous studies including work done with mouse knockouts for gelsolin, villin and CapG support a role for the family in cytoskeletal actin dynamics. In both fruitfly and mouse, the FliI protein is also essential for early development. Recent studies indicate that supervillin, gelsolin and FliI are involved in intracellular signalling via nuclear hormone receptors including the androgen, oestrogen and thyroid hormone receptors. This unexpected role in signalling has opened a new area in research on the gelsolin family and is providing important new insights into the mechanisms of gene regulation via nuclear receptors.

Trehalose metabolism genes in Caenorhabditis elegans and filarial nematodes.

The sugar trehalose is claimed to be important in the physiology of nematodes where it may function in sugar transport, energy storage and protection against environmental stresses. In this study we investigated the role of trehalose metabolism in nematodes, using Caenorhabditis elegans as a model, and also identified complementary DNA clones putatively encoding genes involved in trehalose pathways in filarial nematodes. In C. elegans two putative trehalose-6-phosphate synthase (tps) genes encode the enzymes that catalyse trehalose synthesis and five putative trehalase (tre) genes encode enzymes catalysing hydrolysis of the sugar. We showed by RT-PCR or Northern analysis that each of these genes is expressed as mRNA at all stages of the C. elegans life cycle. Database searches and sequencing of expressed sequence tag clones revealed that at least one tps gene and two tre genes are expressed in the filarial nematode Brugia malayi, while one tps gene and at least one tre gene were identified for Onchocerca volvulus. We used the feeding method of RNA interference in C. elegans to knock down temporarily the expression of each of the tps and tre genes. Semiquantitative RT-PCR analysis confirmed that expression of each gene was silenced by RNA interference. We did not observe an obvious phenotype for any of the genes silenced individually but gas-chromatographic analysis showed >90% decline in trehalose levels when both tps genes were targeted simultaneously. This decline in trehalose content did not affect viability or development of the nematodes.

The role of eosinophils in parasitic helminth infections: insights from genetically modified mice.

Eosinophilia - an increase in the number of eosinophils in the blood or tissues - has historically been recognized as a distinctive feature of helminth infections in mammals. Yet the precise functions of these cells are still poorly understood. Many scientists consider that their primary function is protection against parasites, although there is little unequivocal in vivo evidence to prove this. Eosinophils are also responsible for considerable pathology in mammals because they are inevitably present in large numbers in inflammatory lesions associated with helminth infections or allergic conditions. In this review, Carolyn Behm and Karen Ovington outline some of the cellular and biological properties of eosinophils and evaluate the evidence for their role(s) in parasitic infections.

Involvement of the compatible solutes trehalose and sucrose in the response to salt stress of a cyanobacterial Scytonema species isolated from desert soils.

The response to moderate salt stress of a Scytonema species isolated from a soil crust in the arid region of central Australia was studied. An increase in intracellular trehalose and sucrose concentrations was detected by NMR and HPLC analysis following salt stress, maximal amounts being produced by exposure to 150 mM NaCl after 48 h. When the organism was subsequently returned to normal growth conditions, the cellular concentrations of these solutes decreased. The biosynthesis of trehalose and sucrose was studied and found, in both cases, to involve both sugar phosphate synthase and phosphatase enzymes. The combined synthase activities and the individual phosphatase activities in cell extracts were increased by salt stress. Trehalose phosphorylase was the only catabolic enzyme detected for trehalose; neither trehalase nor phosphotrehalase activities could be detected. This is the first report of trehalose phosphorylase activity in cyanobacteria. Both trehalose and sucrose phosphorylase activities increased in salt-stressed cells, whereas the activity of invertase did not change.

Regulation of primary Strongyloides ratti infections in mice: a role for interleukin-5.

C57BL/6 mice genetically deficient in interleukin-5 (IL-5-/-) and normal C57BL/6 (IL-5+/+) mice were infected with larvae of a homogonic strain of the nematode Strongyloides ratti. In primary infections both male and female IL-5-/- mice released two to four times more eggs and larvae than IL-5+/+ mice. IL-5-/- mice harboured about 60% more intestinal worms, which were more fecund, than IL-5+/+ mice. The duration of the infection was similar in normal and IL-5-deficient mice. Both IL-5-/- and IL-5+/+ mice resisted a secondary infection. IL5-/- mice lost more weight during the infection than normal mice and took longer to regain their initial weight after expelling the worms. The number of eosinophils increased in the bone marrow, peritoneal cavity and small intestine of IL-5+/+ mice, but not IL-5-/- mice, following infection. No significant differences between infected IL-5+/+ and IL-5-/- mice in mast cells or other leucocytes were observed in the peritoneal cavity. Thus, IL-5 functions to protect the host in a primary infection of S. ratti by limiting the number and fecundity of worms establishing in the small intestine. This protection is correlated with elevated blood and tissue eosinophilia which occurs in normal mice but not in IL-5-/- mice.

Factors influencing the development and carbohydrate metabolism of Echinococcus granulosus in dogs.

Echinococcus granulosus adult worms, 35 days postinfection, were measured for dispersion in the intestines of 10 dogs, a range of morphological characters, and the excreted end products of carbohydrate catabolism following 4 hr incubation in vitro. Most worms were found in the proximal sections of the small intestine, but the pattern of dispersion differed between dogs. Worm development varied both between dogs and between different regions of the small intestine of individual dogs. Overall there was a high level of variability with no simple patterns. Worm metabolism was related to worm development and, also independently, to local population density within the intestine. Larger, more mature worms produced less lactate and, at higher densities, worms tended to produce more acetate and succinate (pathways with a higher energy yield than lactate) and less ethanol. Thus, both more developed worms and high population density are associated with a shift from cytosolic to mitochondrial metabolism. The variation between worm populations along the small intestine along with the observed variation between worm populations from sibling dogs infected with genetically identical parasites suggests that the local host environment has a significant effect on parasite development.

Eosinophilia, parasite burden and lung damage in Toxocara canis infection in C57Bl/6 mice genetically deficient in IL-5.

C57Bl/6 mice genetically deficient in interleukin (IL)-5 (IL-5-/-) and mice with the normal IL-5 gene (IL-5+/+) were infected with embryonated eggs of Toxocara canis. IL-5+/+ mice developed a marked eosinophilia in their peripheral bloods and bone marrows after infection. In contrast, the number of eosinophils at these sites actually decreased during the acute phase of infection in IL-5-/- mice. A smaller number of eosinophils infiltrated the lung, liver, heart and skeletal muscle of infected IL-5-/- mice than those of infected IL-5+/+ mice. Eosinophils were not produced in cultures of bone marrow cells from either IL-5+/+ or IL-5-/- mice which were stimulated with excretory secretory antigen of T. canis larvae. The capacity of cells from the bone marrow to differentiate into eosinophils when stimulated in vitro with recombinant murine IL-5 was the same whether the cells were from IL-5+/+ or IL-5-/- mice. Taken together, these results show that an IL-5-like molecule is not produced by the T. canis larvae and that IL-5 produced by host cells is solely responsible for the eosinophilia in mice infected with this nematode. The number and location of T. canis larvae were not altered in the absence of IL-5. In contrast, lung damage in infected IL-5-/- mice was less extensive than that in infected IL-5+/+ mice, although structures resembling Charcot-Leyden crystals were seen in the lungs of both IL-5+/+ and IL-5-/- mice. These results suggest that eosinophils play a role in the pathology in mice infected with T. canis.

The role of trehalose in the physiology of nematodes.

The sugar trehalose, an alpha-1-linked non-reducing disaccharide of glucose, is important in the physiology of many micro-organisms as well as in some groups of metazoan organisms, including insects and nematodes. Trehalose is a stress protectant in biological systems as it interacts with and directly protects lipid membranes and proteins from the damage caused by environmental stresses such as desiccation and freezing. Trehalose is present in many nematode species where its concentration often exceeds that of glucose but is usually lower than that of glycogen. In Ascaris suum it is found in all tissues, with highest concentrations in muscle, haemolymph and the female and male reproductive organs. Trehalose acts as an energy reserve in some nematodes and their eggs, and may be important in uptake of glucose; it appears to function as the major circulating blood sugar. Trehalose accumulates in nematodes that can withstand dehydration and may be important in supercooling of nematodes or eggs that can withstand freezing. In many nematodes trehalose is also important in the process of egg hatching. The combined action of 2 enzymes, trehalose 6-phosphate (T6P) synthase and T6P phosphatase, catalyses the synthesis of trehalose in most organisms. Hydrolysis of trehalose glucose is catalysed by trehalase. These enzymes to have been detected in nematodes but the processes regulating their activity are unknown. Trehalose metabolism may provide new molecular targets for attack in nematodes parasitic in mammals.

The enigmatic eosinophil: investigation of the biological role of eosinophils in parasitic helminth infection.

In many helminth infected hosts the number of eosinophils increases dramatically, often without any concurrent increases in the number of other leukocytes, so that eosinophils become the dominant cell type. Many experimental investigations have shown that the eosinophilia is induced by interleukin-5 (IL-5) but its functional significance remains unclear. Mice genetically deficient in IL-5 (IL-5-/-) have been used to evaluate the functional consequences of the IL-5 dependent eosinophilia in helminth infected hosts. Host pathology and level of infection were determined in IL-5-/- and wild type mice infected with a range of species representative of each major group of helminths. The effects of IL-5 deficiency were very heterogeneous. Of the six species of helminth examined, IL-5 dependent immune responses had no detectable effect in infections with three species, namely the cestodes Mesocestoides corti and Hymenolepis diminuta and the trematode Fasciola hepatica. In contrast, IL-5 dependent immune responses were functionally important in mice infected with three species, notably all nematodes. Damage to the lungs caused by migrating larvae of Toxocara canis was reduced in IL-5-/- mice. Infections of the intestine by adult stages of either Strongyloides ratti or Heligmosomoides polygyrus were more severe in IL-5-/- mice. Adult intestinal nematodes were clearly deleteriously affected by IL- 5 dependent processes since in its presence there were fewer worms which had reduced fecundity and longevity. The implications of these results for the viability of using inhibitors of IL-5 as a therapy for asthma are considered.

Fasciola hepatica infection in sheep: changes in liver metabolism.

Several aspects of liver function during infection with Fasciola hepatica were examined in sheep four weeks after infection and compared with the changes observed in infected rats. Previously reported respiratory abnormalities in mitochondria isolated from the left lobe of the liver of infected sheep were characterised further. Evidence is presented that the respiratory lesion is located in the mitochondrial electron transport chain and that the aberrant respiratory behaviour is not associated with an increase in nonesterified fatty acids and the depletion of mitochondrial phospholipids, as is the case in the rat. Microsomal membranes, which have also been shown to be depleted of phospholipids in the fluke-infected rat liver, showed no such changes in the sheep. However, in common with the rat, a substantial loss of cytochrome P450 was recorded in microsomes prepared from the left lobe, and the glycogen content of the left lobe was found to be less than 50 per cent of control values. No change was observed in glucose 6-phosphatase activity. All these changes were localised effects, confined to areas of fluke infiltration.

IL-5-deficient mice have a developmental defect in CD5+ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses.

Mice deficient in interleukin-5 (IL-5-/- mice) were generated by gene targeting in embryonal stem cells. Contrary to previous studies, no obligatory role for IL-5 was demonstrated in the regulation of conventional B (B-2) cells, in normal T cell-dependent antibody responses or in cytotoxic T cell development. However, CD5+ B cells (B-1 cells) in the peritoneal cavity were reduced by 50%-80% in 2-week-old IL-5-/- mice, returning to normal by 6-8 weeks of age. The IL-5-/- mice did not develop blood and tissue eosinophilia when infected with the helminth Mesocestoides corti, but basal levels of eosinophils with normal morphology were produced in the absence of IL-5. IL-5 deficiency did not affect the worm burden of infected mice, indicating that increased eosinophils do not play a significant role in the host defence in this parasite model.

Importance of T-cell-dependent inflammatory reactions in the decline of microsomal cytochrome P450 concentration in the livers of rats infected with Fasciola hepatica.

The concentration of cytochrome P450, measured spectrophotometrically in microsomal preparations from the livers of rats infected with 30 metacercariae of Fasciola hepatica, declined by approximately 50% at 3 weeks post-infection. Treatment of infected rats with the anti-inflammatory agent dexamethasone (2 mg/kg at 48 h intervals for 8 days prior to assay) abolished the decline in P450 content. Assay of P450 in infected congenitally athymic (nude) rats showed normal levels. These results demonstrate that the T-cell-dependent inflammatory response in the liver of the host is a necessary factor in the development of the decline in hepatic P450, which is known to compromise the metabolism of certain drugs in infected hosts.

A study of hepatic mitochondrial respiration and microsomal cytochrome P450 content in mice infected with the liver fluke Fasciola hepatica.

Previous studies of the effects of infection of Wistar rats with the common liver fluke, Fasciola hepatica, on liver bioenergetic and drug metabolism have demonstrated a loss of respiratory control in isolated mitochondria and reduced microsomal cytochrome P450 content, respectively, from 2 weeks post-infection throughout the acute phase of the infection. In the present study male Balb/c mice infected with F. hepatica showed a loss of respiratory control in isolated liver mitochondria only at 4 weeks post-infection. A similar time course was demonstrated for a reduction in hepatic microsomal cytochrome P450 content. Preparations from infected CBA mice showed similar changes to Balb/c mice but mitochondrial respiration in preparations from infected Swiss outbred mice was normal. A host difference between strains of mice and between mice and rats is therefore evident in the timing and extent of liver mitochondrial dysfunction and in the timing of the decrease in the cytochrome P450 content of hepatic microsomes. This difference between hosts may be related to the reported differences in cellular inflammatory responses to the migrating juvenile flukes in the livers of rats and mice.

Aberrant mitochondrial respiration in the livers of rats infected with Fasciola hepatica: the role of elevated non-esterified fatty acids and altered phospholipid composition.

The non-esterified fatty acid (NEFA) content and phospholipid composition of mitochondria isolated from the livers of Wistar rats infected with Fasciola hepatica were examined in relation to the aberrant mitochondrial respiration previously reported [Rule, Behm, and Bygrave (1989) Biochem. J. 260, 517-523]. At 2 weeks post-infection, elevated NEFA levels were associated with uncoupling of mitochondrial respiration that was reversible in vitro by the addition of BSA. State IV respiration rates showed a strong correlation with NEFA content. At 3 weeks post-infection, NEFA content had increased further and uncoupled mitochondria no longer showed any response to BSA. 31P-NMR analyses of cholate extracts of mitochondria from infected livers at 3 weeks post-infection revealed a marked loss of several major phospholipid species with a concomitant increase in catabolic products, particularly glycerophosphocholine and glycerophosphoethanolamine. Similar changes were observed in microsomal extracts. The NEFA content and phospholipid composition of mitochondria isolated from infected, athymic nude rats were not significantly different from uninfected, athymic rats. These findings suggest that uncoupling of liver mitochondria during infection with F. hepatica is the result of phospholipase activation mediated by the immune system of the host.

Characterization of the oligomycin-sensitivity properties of the F1F0-ATPase in mitochondria from rats infected with the liver fluke Fasciola hepatica.

The F1F0-ATPase activity of liver mitochondria isolated from rats infected with Fasciola hepatica at 3 and 4 weeks post-infection showed a marked loss of sensitivity to oligomycin and to N,N'-dicyclohexylcarbodiimide. A loss of sensitivity to diethylstilbestrol was also demonstrated at 4 weeks post-infection. Recovery was apparent in most cases by 6 weeks post-infection. No significant difference in latent ATPase activity was observed between mitochondria from control and infected livers at any stage of the infection. The mitochondria from infected livers were therefore considered to have a full complement of the F1 moiety of the F1F0-ATPase complex. Purification of the mitochondrial ATPase from 4-week infected livers resulted in a very low yield of an oligomycin-insensitive complex. This was due to a failure to enrich specific activity during purification. The evidence presented indicates that infection with Fasciola hepatica gives rise to alterations in the function of the host liver mitochondrial ATPase, namely loss of inhibitor sensitivity and apparent structural alterations of the ATPase complex.

Restoration of mitochondrial energy-linked reactions following dexamethasone treatment of rats infected with the liver fluke Fasciola hepatica.

Mitochondria isolated from male Wistar rats experimentally infected with the common liver fluke, Fasciola hepatica, exhibit loss of respiratory control from 2 weeks post-infection (Rule, et al. (1989) Biochem. J. 260, 517-523). We now report that subcutaneous injections of the anti-inflammatory drug, dexamethasone, during the final week of infection prevented the mitochondrial uncoupling and restored respiratory control almost to the levels of uninfected controls. Further investigations have shown that mitochondria from infected rat livers are unable to synthesize ATP and that abnormal respiration is also evident in hepatocytes isolated from infected rats. These abnormalities were absent when infected rats were treated with dexamethasone. In addition, liver mitochondrial function in infected, congenitally athymic, nude rats (CBH/R nu/nu) was not significantly different from that in uninfected nude or Wistar controls. These results provide evidence that the mitochondrial dysfunction in fascioliasis is host-mediated and that T lymphocytes in particular may be involved.

Beneficial effect of dexamethasone on attenuated hormone-induced uptake of calcium and glycogenolysis by perfused liver of rats infected with Fasciola hepatica.

Infection of rats with the liver fluke, Fasciola hepatica, impaired the responses of the perfused liver to calcium uptake and glucose release induced by the synergistic action of glucagon and vasopressin. Treatment of infected rats with dexamethasone prevented the impairment of each of these two responses.

Aberrant energy-linked reactions in mitochondria isolated from the livers of sheep infected with the liver fluke Fasciola hepatica.

Respiration by mitochondria isolated from the livers of sheep following infection up to 15 weeks with F. hepatica was measured with the respiratory substrates pyruvate (plus malate) and succinate in the absence and presence of ADP; the rates were compared with those obtained by mitochondria isolated from livers of uninfected sheep. It was found that respiration supported by both substrates in mitochondria isolated from the left lobe but not the middle lobe of 4-week infected sheep exhibited abnormalities such that the acceptor control ratios were only marginally above one. Some, but not total, recovery was seen in the later stages of infection. The aberrant respiratory behaviour is similar to that observed with infected rats.

The role of the host in the regulation of end-product formation in two strains of the rat tapeworm, Hymenolepis diminuta.

Individual worms from rats infected with different strains of Hymenolepis diminuta were incubated in vitro and the products lactate, succinate, acetate and ammonia assayed. Variability in excretion was not confined to differences between strains. Two metabolic types were identified. Where succinate was above 20 mumol g-1 h-1, lactate excretion was low. Where succinate was not detected, lactate excretion was high. Acetate excretion was variable. Lactate and ammonia excretion were positively correlated. All worms from one rat were of the same type but could be of either type from different rats. The host strain had no effect. A relationship was shown between lactate excretion and the number of worms from a standard inoculum present at 21 days of infection. The incidence of high lactate excretion was increased in worms from secondary infections. Components of the host immune response may thus exert effects on the metabolism of H. diminuta, manifest as shifts in emphasis on cytosolic and mitochondrial metabolism.