Immune regulation by helminth parasites: cellular and molecular mechanisms

Immunology was founded by studying the body's response to infectious microorganisms, and yet microbial prokaryotes only tell half the story of the immune system. Eukaryotic pathogens--protozoa, helminths, fungi and ectoparasites--have all been powerful selective forces for immune evolution. Often, as with lethal protozoal parasites, the focus has been on acute infections and the inflammatory responses they evoke. Long-lived parasites such as the helminths, however, are more remarkable for their ability to downregulate host immunity, protecting themselves from elimination and minimizing severe pathology in the host.     

Drug transport mechanisms in helminth parasites: passive diffusion of benzimidazole anthelmintics

Anthelmintic molecules must reach their receptors inside target parasites to exert the pharmacological effect. Available data suggest that the main route of entry of antiparasitic drugs into helminth parasites would be through their external surface. However, it is unclear if trans-tegumental/cuticular penetration is the most important way of entry of benzimidazole (BZD) anthelmintics into their target parasites compared to oral ingestion. The relative involvement of active and passive transport mechanisms has not been defined. The goal of the work reported here was to determine the main processes involved in the entry of BZD anthelmintic molecules into the three main classes of helminth parasites. Adult specimens of Moniezia benedeni (cestode), Fasciola hepatica (trematode) and Ascaris suum (nematode) were incubated in Kreb's Ringer Tris buffer (pH 7.4, 37 degrees C) (1g parasite/10 ml incubation medium) for 15, 45, and 90 min, respectively, in the presence of a concentration gradient of either fenbendazole (FBZ), oxfendazole or triclabendazole sulphoxide (TCBZSO) (1-30 mol/ml, n=4). Dead helminth specimens were also incubated with the same drug concentration gradient. Specimens of F. hepatica with the oral route closed off by ligation were incubated with TCBZSO in the presence or absence of bovine serum albumin. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography analysis to measure drug/metabolite concentrations. Equivalent drug concentrations were measured within ligated and non-ligated liver flukes, demonstrating that BZD do mainly penetrate by trans-tegumental diffusion. The higher the concentration of BZD molecules in the incubation medium, the greater their concentration recovered within the helminth parasites. High correlation coefficients (>0.98) were obtained between initial drug concentration in the incubation medium and those measured inside the nematode, cestode, and trematode parasites. FBZ concentrations recovered from tissues of dead cestodes/nematodes over time were significantly greater compared to those measured in living parasites. These differences in drug diffusion may be related to the morphological/functional properties of the parasite's external surfaces. The outcome of the work reported here indicates that passive drug transfer through the external helminth surface is the main transport mechanism accounting for BZD accumulation into target parasites.     

G-quadruplexes in helminth parasites

Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.     

Replicating helminth parasites of man

Most helminth parasites of man are unable to replicate within the human host. Thus, the worm burden of an infected person (on which the pathology largely depends - see Box 1) is a function of the number of infective forms to which the person is exposed. But for some species of helminths, the ability to replicate in man has a marked effect on the course and duration of infection, and for the pathogenesis of disease. In this review, David Grove discusses the mechanisms by which such replication may occur, and considers how this ability affects our approach to therapy and control.     

Molecules released by helminth parasites involved in host colonization

Parasites are designed by evolution to invade the host and survive in its organism until they are ready to reproduce. Parasites release a variety of molecules that help them to penetrate the defensive barriers and avoid the immune attack of the host. In this respect, particularly interesting are enzymes and their inhibitors secreted by the parasites. Serine-, aspartic-, cysteine-, and metalloproteinases are involved in tissue invasion and extracellular protein digestion. Helminths secrete inhibitors of these enzymes (serpins, aspins, and cystatins) to inhibit proteinases, both of the host and their own. Proteinases and their inhibitors, as well as helminth homologues of cytokines and molecules containing phosphorylcholine, influence the immune response of the host biasing it towards the anti-inflammatory Th2 type. Nucleotide-metabolizing enzymes and cholinesterase are secreted by worms to reduce inflammation and expel the parasites from the gastrointestinal tract. An intracellular metazoan parasite, Trichinella spiralis, secretes, among others, protein kinases and phosphatases, endonucleases, and DNA-binding proteins, which are all thought to interfere with the host cellular signals for muscle cell differentiation. Secretion of antioxidant enzymes is believed to protect the parasite from reactive oxygen species which arise from the infection-stimulated host phagocytes. Aside from superoxide dismutase, catalase (rarely found in helminths), and glutathione peroxidase (selenium-independent, thus having a poor activity with H(2)O(2)), peroxiredoxins are probably the major H(2)O(2)-detoxifying enzymes in helminths. Secretion of antioxidant enzymes is stage-specific and there are examples of regulation of their expression by the concentration of reactive oxygen species surrounding the parasite. The majority of parasite-secreted molecules are commonly found in free-living organisms, thus parasites have only adapted them to use in their way of life.     

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.     

Drug transfer into target helminth parasites

The pharmacokinetics of an anthelmintic drug includes the time course of drug absorption, distribution, metabolism and elimination from the host and determines the concentration of the active drug that reaches the location of the parasite. However, the action of the anthelmintic also depends on the ability of the active drug to reach its specific receptor within the target parasite. Thus, drug entry and accumulation in target helminths are important issues when considering how best to achieve optimal efficacy. Passive drug transfer through the external helminth surface is the predominant entry mechanism for most widely used anthelmintics and is discussed in this article. Despite the structural differences between the external surface of nematodes (the cuticle) and the external surface of cestodes and trematodes (the tegument), the mechanism of drug entrance into both types of helminth depends on the lipophilicity of the anthelmintic and this is the major physicochemical determinant for the drug to reach a therapeutic concentration in the target parasite. Understanding the processes that regulate drug transfer into helminth parasites is an important aspect in improving the control of parasites in human and veterinary medicine.     

Density dependence and the control of helminth parasites

1. The transient dynamics and stability of a population are determined by the interplay between species density, its spatial distribution and the positive and negative density-dependent processes regulating population growth. 2. Using the human-helminth parasite system as an example, we propose that the life-stage upon which negative density dependence operates will influence the rate of host reinfection following anthelmintic chemotherapy, and the likely success of control programmes. 3. Simple deterministic models are developed which highlight how a parasite species whose population size is down-regulated by density-dependent establishment will reinfect a host population at a faster rate than a species with density-dependent parasite fecundity. 4. Different forms of density dependence can produce the same equilibrium behaviour but different transient dynamics. Under-representing the nature and magnitude of density-dependent mechanisms, and in particular those operating upon establishing life-stages, may cause the resilience of the parasite population to a control perturbation to be underestimated.     

Host fragmentation and helminth parasites: Hedging your bets against extinction

We consider the probability of parasite extinction due to anthropogenic fragmentation of host populations and in the absence of host extinction. We conclude that extinction at infrapopulation and infracommunity levels is both common and trivial. Extinction may occur in communities at higher levels but only if metapopulations or suprapopulations become extinct. Suprapopulations are highly complex and unlikely to become extinct in the face of simple host fragmentation. We acknowledge parasite metapopulations as being the most likely to become extinct, but only locally. Our reasoning for this is that, in the absence of complete host extinction, populations of the parasite in other fragments are likely to serve as sources for reinvasion (e.g. a rescue effect). We identify a number of features that may act as hedges against extinction for many parasites and conclude by attempting to identify what form an extinction might take.     

Growth factor receptors in helminth parasites: signalling and host-parasite relationships

Parasitic helminths remain major pathogens of both humans and animals throughout the world. The success of helminth infections depends on the capacity of the parasite to counteract host immune responses but also to exploit host-derived signal molecules for its development. Recent progress has been made in the characterization of growth factor receptors of various nematode and flatworm parasites with the demonstration that transforming growth factor beta (TGF-beta), epidermal growth factor (EGF) and insulin receptor signalling pathways are conserved in helminth parasites and potentially implicated in the host-parasite molecular dialogue and parasite development.     

Age-dependent epidemiological patterns and strain diversity in helminth parasites

Field studies of schistosomes and the major intestinal nematodes Trichuris trichiura and Ascaris lumbricoides repeatedly demonstrate that the intensity and prevalence of infection exhibit marked dependency on host age. Peak levels of infection typically occur in hosts aged between 10 and 14 yr in endemically infected communities. It has widely been assumed that the slow acquisition of resistance in adults is caused by repeated exposure to the same antigenic repertoire of a single parasite strain. Consequently, these empirical patterns have previously been taken to suggest that human immunity to helminth parasites confers poor protection against reinfection. Here, an alternative explanation is suggested on the basis of results from a simplified model of helminth transmission. It is proposed that the empirical observations can be attributed to the circulation of multiple helminth strains that each elicit highly protective immunity. If this hypothesis is correct, estimates of epidemiological parameters from field data and the potential for control of helminth diseases might require reevaluation.     

Population abundance and sex ratio in dioecious helminth parasites

Parasite populations are highly fragmented in space and time, and consist of aggregates of genetically similar individuals sharing the same host. To avoid inbreeding, theory predicts that female-biased sex ratios should be strongly favoured when either or both prevalence and intensity of infection are low. Other models indicate that if sex ratios are selected to increase the probability of mating, they should be less biased at a high intensity of infection in polygamous parasites, since at high intensities all females are mated. To test these predictions, the relationship between sex ratio and both the prevalence and intensity of infection was examined in comparative studies across 193 populations of nematode and acanthocephalan parasites. Sex ratios in these two dioecious, polygamous taxa are usually female biased. Among natural populations, no significant relationship was observed once the confounding effects of phylogeny had been removed. However, among experimental populations of nematodes, a negative relationship was found between intensity of infection and sex ratio, even after controlling for phylogeny. In other words, at high intensities, populations of nematodes are less female biased. This result must be treated with caution because of the unusually high numbers of worms per host in experimental infections. Nevertheless, combined with information on the proximate mechanisms regulating sex ratios in these parasites, it suggests a link between the characteristics of parasite populations and their sex ratio. Received: 21 November 1996 / Accepted: 26 March 1997     

Protozoan and helminth parasites of humans in mainland China

To date, 30 species of protozoa, 12 species of cestodes, 26 species of trematodes, 23 species of nematodes, two species of gordius and one acanthocephalan species hae been reported as parasites of man in mainland China.