Carolyn A. Behm

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.

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.

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.

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.

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.

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

Studies of regulatory metabolism in Moniezia expansa: General considerations

Abstract The activities of selected enzymes in the branched metabolic pathway to succinate or lactate were determined in cytosol and mitochondrial fractions. The enzymes of lowest activity in the cytosol, and thus possibly regulatory, are phosphofructokinase and pyruvate kinase. Malic enzyme activity could scarcely be detected in either compartment; phosphoenolpyruvate carboxykinase and malate dehydrogenase occur in both. The end products of metabolism are succinate and lactate; under anaerobic conditions lactate production increases whereas succinate production shows a small decrease. The presence of glucose in the medium does not influence the change, but causes an increase in total endproduct accumulation. Levels of metabolic intermediates in worms incubated aerobically and anaerobically are presented, and ‘cross-over’ plots and calculations of apparent equilibrium constants identify hexokinase, phosphofructokinase and pyruvate kinase as regulatory. Under aerobic conditions a large increase in the size of the malate pool is observed suggesting that the depression of lactate production is produced by its inhibitory effect on pyruvate kinase. Adenine nucleotide levels are maintained whether or not the worm is incubated under anaerobic conditions.

ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria

Abstract Kohler P. B. , Ryant C. and Behm Carolyn A. 1978. ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria. International Journal for Parasitology8: 399–404. Succinate decarboxylation was measured by the formation of 14CO2 from 1,4-14C-succinate in a particle free, dialysed mitochondrial extract from liver fluke. It has an absolute requirement for Mg2+ and CoA. ATP, ADP and inorganic phosphate are essential for optimal activity. Ap5A, an inhibitor of adenylate kinase, and glutathione are also necessary. GTP supports decarboxylation as well as ATP, provided ADP is also present. The formation of CO2 and propionate greatly exceeds the amount of ATP and CoA initially present in the reaction mixture. A net, substrate-level phosphorylation of ADP occurs, the amount of ATP formed being equivalent to the production of CO2 or propionate. This system is inhibited in flukes incubated in vitro with mebendazole. It is concluded that ATP is required to spark the fermentation system when succinate is the initial substrate and intermediate substrates are absent; that the terminal step in propionate formation is catalysed by a transferase which transfers CoA from propionyl CoA to succinate; and that ATP formation is coupled to the decarboxylation of methylmalonyl-CoA. A reaction scheme is presented.

Hosts use altered macronutrient intake to circumvent parasite-induced reduction in fecundity.

Explanations for the evolution of pathogen-induced fecundity reduction usually rely on a common principle: the trade-off between host longevity and reproduction. Recent advances in nutritional research have, however, challenged this assumption and shown that longevity and reproduction are not inextricably linked. In this study, we showed that beetles infected by cysticercoids of the tapeworm Hymenolepis diminuta increased their total food intake and, more particularly, their carbohydrate consumption compared with uninfected insects. This increased intake was only pronounced during the first 12 days p.i., when the parasite grows and develops into a mature metacestode. Despite consuming more nutrients, infected individuals sustained lower levels of body lipid and were less efficient at converting ingested protein to body protein. However they demonstrated a capacity to compose a diet that sustained high levels of reproductive output unless confined to foods that were nutritionally dilute. We did not find any indication that macronutrient intakes had an effect on host pro-phenoloxidase activity; however, phenoloxidase activity was significantly affected by protein intake. Our results showed that when offered nutritionally complementary diets, infected hosts do not systematically suffer a reduction in fecundity. Thus, in our view, the assumption that a reduction in host reproduction represents an adaptive response by the host or the parasite to divert resources away from reproduction toward other traits should be reassessed.

Differential gene expression at coral settlement and metamorphosis--a subtractive hybridization study.

Background A successful metamorphosis from a planktonic larva to a settled polyp, which under favorable conditions will establish a future colony, is critical for the survival of corals. However, in contrast to the situation in other animals, e.g., frogs and insects, little is known about the molecular basis of coral metamorphosis. We have begun to redress this situation with previous microarray studies, but there is still a great deal to learn. In the present paper we have utilized a different technology, subtractive hybridization, to characterize genes differentially expressed across this developmental transition and to compare the success of this method to microarray. Methodology/Principal Findings Suppressive subtractive hybridization (SSH) was used to identify two pools of transcripts from the coral, Acropora millepora. One is enriched for transcripts expressed at higher levels at the pre-settlement stage, and the other for transcripts expressed at higher levels at the post-settlement stage. Virtual northern blots were used to demonstrate the efficacy of the subtractive hybridization technique. Both pools contain transcripts coding for proteins in various functional classes but transcriptional regulatory proteins were represented more frequently in the post-settlement pool. Approximately 18% of the transcripts showed no significant similarity to any other sequence on the public databases. Transcripts of particular interest were further characterized by in situ hybridization, which showed that many are regulated spatially as well as temporally. Notably, many transcripts exhibit axially restricted expression patterns that correlate with the pool from which they were isolated. Several transcripts are expressed in patterns consistent with a role in calcification. Conclusions We have characterized over 200 transcripts that are differentially expressed between the planula larva and post-settlement polyp of the coral, Acropora millepora. Sequence, putative function, and in some cases temporal and spatial expression are reported.