Chemical analyses and anthelmintic effects of Artemisia campestris essential oil.

The present study aimed at analyzing the chemical composition and evaluating the in vitro and in vivo anthelmintic activity of Artemisia campestris essential oil aerial parts. The chemical composition was analysed by gaz chromatography/mass chromatography (GC/MS). Fifty compounds were identified representing 99.98% of the total oil. A. campestris essential oil was dominated by beta-pinene (36.40%) and 2-undecanone (14.7%). The in vitro anthelmintic activity tests of A. campestris essential oil were performed on Haemonchus contortus using egg hatch assay (EHA) and adult worm's motility assay (AWMA) compared with a reference drug albendazole. In the EHA 100% inhibition was observed at 2 mg/ml after 48 h incubation (IC50 = 0.93 mg/ml). In the AWMA, essential oil induced 66.6% inhibition at 0.5 mg/ml after 8 h post exposure. The nematicidal effect of essential oil was evaluated on Heligmosomoides polygyrus. It was monitored through faecal egg count reduction (FECR) and total worm count reduction (TWCR). Three doses (2000, 4000 and 5000 mg/kg) were studied using a bioassay. The dose of 5000 mg/kg showed a high nematicidal activity (72.1% FECR and 72% TWCR), 7 days post-treatment. The results of the present study suggest that A. campestris essential oil has a potential anthelmintic activity and further studies are required in order to establish its mechanisms of action.

In Vivo Nematicidal Potential of Camel Milk on Heligmosomoides Polygyrus Gastro-intestinal Nematode of Rodents.

Following our previous findings on the in vitro anthelmintic effect of camel milk on Haemonchus contortus, the current study aimed at investigating its in vivo effect. Investigations were carried out using mice infected with Heligmosomoides polygyrus which is a parasite commonly used to test the efficacy of anthelmintics. Thirty six Swiss white mice of both sexes aged 5 - 6 weeks old, and weighing between 20 and 25 g were orally infected with 0.5 ml dose of 100, 1-week-old H. polygyrus infective larvae (L3). After the pre-patent period, infected animals were randomly divided into 6 groups of 6 animals each. The nematicidal efficacy of camel milk was monitored through faecal egg count reduction (FECR) and total worm count reduction (TWCR). Four doses (8.25; 16.5; 33.0; 66.0 ml/kg body weight (bw)) for fresh camel milk and 22 mg/kg bw for albendazole were studied using a bioassay. Albendazole and 4 % dimethylsulfoxide were included in the protocol as reference drug and placebo, respectively. For all tested doses except 8.25 ml/kg bw, camel milk was effective in vivo against H. polygyrus reducing both faecal egg count and worm count (p < 0.05). The dose 66 ml/kg bw showed the highest nematicidal activity causing a 76.75 % FECR and a 69.62 % TWCR 7 day after initiating the treatment. These results support the possible use of camel milk in the control of gastro-intestinal helminthiasis.

Parasitic helminth infections and the control of human allergic and autoimmune disorders.

The profile of global health today presents a striking reciprocal distribution between parasitic diseases in many of the world's lower-income countries, and ever-increasing levels of inflammatory disorders such as allergy, autoimmunity and inflammatory bowel diseases in the more affluent societies. Attention is particularly focused on helminth worm parasites, which are associated with protection from allergy and inflammation in both epidemiologic and laboratory settings. One mechanistic explanation of this is that helminths drive the regulatory arm of the immune system, abrogating the ability of the host to expel the parasites, while also dampening reactivity to many bystander specificities. Interest has therefore heightened into whether helminth parasites, or their products, hold therapeutic potential for immunologic disorders of the developed world. In this narrative review, progress across a range of trials is discussed, together with prospects for isolating individual molecular mediators from helminths that may offer defined new therapies for inflammatory conditions.

Low-level regulatory T-cell activity is essential for functional type-2 effector immunity to expel gastrointestinal helminths.

Helminth infection is frequently associated with the expansion of regulatory T cells (Tregs) and suppression of immune responses to bystander antigens. We show that infection of mice with the chronic gastrointestinal helminth Heligmosomoides polygyrus drives rapid polyclonal expansion of Foxp3(+)Helios(+)CD4(+) thymic (t)Tregs in the lamina propria and mesenteric lymph nodes while Foxp3(+)Helios(-)CD4(+) peripheral (p)Treg expand more slowly. Notably, in partially resistant BALB/c mice parasite survival positively correlates with Foxp3(+)Helios(+)CD4(+) tTreg numbers. Boosting of Foxp3(+)Helios(+)CD4(+) tTreg populations by administration of recombinant interleukin-2 (rIL-2):anti-IL-2 (IL-2C) complex increased worm persistence by diminishing type-2 responsiveness in vivo, including suppression of alternatively activated macrophage and granulomatous responses at the sites of infection. IL-2C also increased innate lymphoid cell (ILC) numbers, indicating that Treg functions dominate over ILC effects in this setting. Surprisingly, complete removal of Tregs in transgenic Foxp3-DTR mice also resulted in increased worm burdens, with "immunological chaos" evident in high levels of the pro-inflammatory cytokines IL-6 and interferon-γ. In contrast, worm clearance could be induced by anti-CD25 antibody-mediated partial depletion of early Treg, alongside increased T helper type 2 responses and without incurring pathology. These findings highlight the overarching importance of the early Treg response to infection and the non-linear association between inflammation and the prevailing Treg frequency.

Helminths in the hygiene hypothesis: sooner or later?

There is increasing recognition that exposures to infectious agents evoke fundamental effects on the development and behaviour of the immune system. Moreover, where infections (especially parasitic infections) have declined, immune responses appear to be increasingly prone to hyperactivity. For example, epidemiological studies of parasite-endemic areas indicate that prenatal or early-life experience of infections can imprint an individual's immunological reactivity. However, the ability of helminths to dampen pathology in established inflammatory diseases implies that they can have therapeutic effects even if the immune system has developed in a low-infection setting. With recent investigations of how parasites are able to modulate host immune pathology at the level of individual parasite molecules and host cell populations, we are now able to dissect the nature of the host-parasite interaction at both the initiation and recall phases of the immune response. Thus the question remains - is the influence of parasites on immunity one that acts primarily in early life, and at initiation of the immune response, or in adulthood and when recall responses occur? In short, parasite immunosuppression - sooner or later?

Blockade of IL-33 release and suppression of type 2 innate lymphoid cell responses by helminth secreted products in airway allergy.

Helminth parasites such as the nematode Heligmosomoides polygyrus strongly inhibit T helper type 2 (Th2) allergy, as well as colitis and autoimmunity. Here, we show that the soluble excretory/secretory products of H. polygyrus (HES) potently suppress inflammation induced by allergens from the common fungus Alternaria alternata. Alternaria extract, when administered to mice intranasally with ovalbumin (OVA) protein, induces a rapid (1-48 h) innate response while also priming an OVA-specific Th2 response that can be evoked 14 days later by intranasal administration of OVA alone. In this model, HES coadministration with Alternaria/OVA suppressed early IL-33 release, innate lymphoid cell (ILC) production of IL-4, IL-5, and IL-13, and localized eosinophilia. Upon OVA challenge, type 2 ILC (ILC2)/Th2 cytokine production and eosinophilia were diminished in HES-treated mice. HES administration 6 h before Alternaria blocked the allergic response, and its suppressive activity was abolished by heat treatment. Administration of recombinant IL-33 at sensitization with Alternaria/OVA/HES abrogated HES suppression of OVA-specific responses at challenge, indicating that suppression of early Alternaria-induced IL-33 release could be central to the anti-allergic effects of HES. Thus, this helminth parasite targets IL-33 production as part of its armory of suppressive effects, forestalling the development of the type 2 immune response to infection and allergic sensitization.

Differential recognition patterns of Schistosoma haematobium adult worm antigens by the human antibodies IgA, IgE, IgG1 and IgG4.

Schistosoma haematobium antigen recognition profiles of the human isotypes IgA, IgE, IgG1 and IgG4 were compared by image analysis of western blots. Adult worm antigens separated by two-dimensional gel electrophoresis were probed with pooled sera from Zimbabweans resident in a S. haematobium endemic area, followed by the identification of individual antigenic parasite proteins using mass spectrometry. Overall, IgG1 reacted with the largest number of antigens, followed by IgE and IgA which detected the same number, while IgG4 detected the fewest antigens. IgE recognized all antigens reactive with IgG4 as well as an additional four antigens, an isoform of 28-kDa GST, phosphoglycerate kinase, actin 1 and calreticulin. IgG1 additionally recognized fatty acid-binding protein, triose-phosphate isomerase and heat shock protein 70, which were not recognized by IgA. Recognition patterns varied between some isoforms, e.g. the two fructose 1-6-bis-phosphate aldolase isoforms were differentially recognized by IgA and IgG1. Although the majority of S. haematobium adult worm antigens are recognized by all of the four isotypes, there are clear restrictions in antibody recognition for some antigens. This may partly explain differences observed in isotype dynamics at a population level. Differential recognition patterns for some isoforms indicated in the study have potential importance for vaccine development.

Acquired immune heterogeneity and its sources in human helminth infection.

Similarities in the immunobiology of different parasitic worm infections indicate that co-evolution of humans and helminths has shaped a common anti-helminth immune response. However, recent in vitro and immuno-epidemiological studies highlight fundamental differences and plasticity within host-helminth interactions. The 'trade-off' between immunity and immunopathology inherent in host immune responses occurs on a background of genetic polymorphism, variable exposure patterns and infection history. For the parasite, variation in life-cycle and antigen expression can influence the effector responses directed against them. This is particularly apparent when comparing gastrointestinal and tissue-dwelling helminths. Furthermore, insights into the impact of anti-helminthic treatment and co-infection on acquired immunity suggest that immune heterogeneity arises not from hosts and parasites in isolation, but also from the environment in which immune responses develop. Large-scale differences observed in the epidemiology of human helminthiases are a product of complex host-parasite-environment interactions which, given potential for exposure to parasite antigens in utero, can arise even before a parasite interacts with its human host. This review summarizes key differences identified in human acquired immune responses to nematode and trematode infections of public health importance and explores the factors contributing to these variations.

A macrophage migration inhibitory factor-like tautomerase from Teladorsagia circumcincta (Nematoda: Strongylida).

A macrophage migration inhibitory factor (MIF)-like molecule, Tci-MIF-1, was isolated from Teladorsagia circumcincta and subjected to detailed characterization. A cDNA representing Tci-mif-1 was isolated following its identification in third-stage larvae (L3)-enriched cDNA population. Sequencing of the cDNA indicated a 348-bp open reading frame (ORF) with the closest orthologue being a MIF derived from the human hookworm Ancylostoma ceylanicum. Messenger RNA (mRNA) representing the Tci-MIF-1 transcript was detected in eggs, L3 and adult stages of T. circumcincta. The transcript was also present, but to a lesser extent in fourth-stage larvae (L4). Detection of Tci-MIF-1 protein in T. circumcincta developmental stages reflected the transcript levels identified by reverse transcriptase-PCR. Using immunohistochemistry, the Tci-MIF-1 protein was shown to have a diffuse distribution in L3 tissue, and in L4 and adult stages, the protein was localized to the nematode gut. A recombinant version of Tci-MIF-1 was produced, and enzymic assays indicated that this recombinant protein and a somatic extract of L3 possessed dopachrome tautomerase activity as has been observed previously in other MIF-like molecules. Neither native, purified Tci-MIF nor recombinant Tci-MIF-1 dramatically influenced the in vitro migration of sheep monocytes.

99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: induction and control of regulatory T cells in the gastrointestinal tract: consequences for local and peripheral immune responses.

Regulatory T cells play a crucial role in normal gut homeostasis, as well as during infection with microbial or parasitic pathogens. Prior to infection, interactions with the commensal microflora are essential to differentiation of a healthy steady-state level of immunoregulation, mediated through both Toll-like receptor-dependent and -independent pathways. The ingress of pathogenic organisms may, according to the context, promote or reverse the regulatory environment, with onward consequences for inflammation in both the intestinal and extra-intestinal settings. Appropriate regulation of gut immunity thus depends upon a complex three-way interplay between host cells, commensals and pathogens, and can exert a major impact on systemic responses including allergy and autoimmunity.

daf-7-related TGF-beta homologues from Trichostrongyloid nematodes show contrasting life-cycle expression patterns.

The transforming growth factor-beta (TGF-beta) gene family regulates critical processes in animal development, and plays a crucial role in regulating the mammalian immune response. We aimed to identify TGF-beta homologues from 2 laboratory model nematodes (Heligmosomoides polygyrus and Nippostrongylus brasiliensis) and 2 major parasites of ruminant livestock (Haemonchus contortus and Teladorsagia circumcincta). Parasite cDNA was used as a template for gene-specific PCR and RACE. Homologues of the TGH-2 subfamily were isolated, and found to differ in length (301, 152, 349 and 305 amino acids respectively), with variably truncated N-terminal pre-proteins. All contained conserved C-terminal active domains (>85% identical over 115 amino acids) containing 9 cysteine residues, as in C. elegans DAF-7, Brugia malayi TGH-2 and mammalian TGF-beta. Surprisingly, only the H. contortus homologue retained a conventional signal sequence, absent from shorter proteins of other species. RT-PCR assays of transcription showed that in H. contortus and N. brasiliensis expression was maximal in the infective larval stage, and very low in adult worms. In contrast, in H. polygyrus and T. circumcincta, tgh-2 transcription is higher in adults than infective larvae. The molecular evolution of this gene family in parasitic nematodes has diversified the pre-protein and life-cycle expression patterns of TGF-beta homologues while conserving the structure of the active domain.

Exploring the immunology of parasitism--from surface antigens to the hygiene hypothesis.

Helminth immunology is a field which has changed beyond recognition in the past 30 years, transformed not only by new technologies from cDNA cloning to flow cytometry, but also conceptually as our definition of host immune pathways has matured. The molecular revolution defined key nematode surface and secreted antigens, and identified candidate immunomodulators that are likely to underpin parasites' success in eluding immune attack. The immunological advances in defining cytokine networks, lymphocyte subsets and innate cell recognition have also made a huge impact on our understanding of helminth infections. Most recently, the ideas of regulatory immune cells, in particular the regulatory T cell, have again overturned older thinking, but also may explain immune hyporesponsiveness observed in chronic helminth diseases, as well as the link to reduced allergic reactions observed in human and animal infections. The review concludes with a forward look to where we may make future advances towards the final eradication of helminth diseases.

Chitinases and chitinase-like proteins: potential therapeutic targets for the treatment of T-helper type 2 allergies.

Mammalian chitinase and chitinase-like proteins (CLPs) are a family of mediators increasingly associated with infection, T cell-mediated inflammation, wound healing, allergy and asthma. Although our current knowledge of the function of mammalian chitinases and CLPs is very limited, important information can be deduced from research carried out in lower organisms, and in different immunopathological conditions. Enzymatically active mammalian chitinase proteins may have evolved to degrade the copious amounts of chitin mammals are exposed to on a daily basis, and to form an innate barrier to chitin-containing organisms. CLPs are homologous to chitinases but lack the ability to degrade chitin. It is most striking that both chitinases and CLPs are up-regulated in T-helper type 2 (Th2)-driven conditions, and the first evidence is now emerging that these proteins may accentuate Th2 reactivity, and possibly contribute to the repair process that follows inflammation. Following studies demonstrating that chitinase inhibition leads to an attenuated allergic response, several strategies are being used to develop enzyme inhibitors for therapeutic use in human diseases. In this review, we will summarize recent insights into the effects of chitinases and CLPs in the context of Th2-dominated pathology with particular focus on allergy and asthma, discussing whether chitinase enzyme inhibitors may be of therapeutic value.

Expression of helminth genes in Leishmania: an experimental transfection system to test immunological function.

Functional analysis of genes from parasitic helminths requires, at the present time, heterologous expression. We have adapted the well-characterized system of transfection in Leishmania protozoal parasites, as a means of analysing the effect of single filarial genes on the mammalian immune system. For example, testing the function of the Brugia malayi abundant larval transcript (ALT) gene-transfected Leishmania mexicana were found to be significantly more virulent in macrophages in vitro. The course of infection in vivo is also aggravated by expression of the ALT gene. Examples are also given of transgenes which reduced in vitro growth within macrophages, as well as others which exert no effect on the protozoal parasitism. Thus, Leishmania transfection provides a tractable system to analyse helminth gene function within the context of the host immune system.

Persistent airway hyper-responsiveness and inflammation in Toxocara canis-infected BALB/c mice.

BACKGROUND: Infection with Toxocara canis, the roundworm of dogs, has been associated with asthmatic manifestations. Clinical symptoms such as wheezing, coughing and episodic airflow obstruction have been described for patients infected with this helminth. OBJECTIVE: In order to characterize the effect of T. canis infection on the lungs, we monitored immune responses, pulmonary pathology and lung function over a period of 60 days in BALB/c mice. METHODS: Infection was performed by a single oral administration of 1000 T. canis embryonated eggs. Airway responsiveness was measured in conscious, unrestrained mice at 7, 14, 30 and 60 days post-infection (p.i.). RESULTS: Infection of mice resulted in airway hyper-responsiveness (AHR) that persisted up to 30 days p.i. Pulmonary inflammation as well as increased levels of IgE and eosinophils in bronchoalveolar lavage (BAL) persisted up to 60 days p.i. Cytokine analysis in BAL indicated increased levels of IL-5 at day 7 and 14 p.i., whereas the levels of IL-2, IFN-gamma, IL-4 and IL-10 did not differ from those of uninfected controls. Toxocara-specific stimulation of spleen cells using recombinant TES-70 protein resulted in the induction of IL-5 at day 7 and 14 p.i. and IL-10 at day 14 p.i. Production of all other cytokines did not differ from that of uninfected controls. Evaluation of larval burden revealed that T. canis was still present in the lungs of infected mice at 60 days p.i. CONCLUSION: The presence of Toxocara larva in the lungs at 60 days p.i. following a single infection could explain the persistent pulmonary inflammation, airway hyper-reactivity, eosinophilia and increased IgE production observed in T. canis-infected BALB/c mice.

Multicentre laboratory evaluation of Brugia Rapid dipstick test for detection of brugian filariasis.

A multicentre evaluation of the Brugia Rapid dipstick test was performed using 1263 serum samples in four international laboratories, i.e. T.D. Medical College (TDMC, India), National Institutes of Health (NIH, USA), Swiss Tropical Institute (STI, Switzerland) and Leiden University Medical Centre (LUMC, Netherlands). In comparison with microscopy, the dipstick demonstrated sensitivities of 97.2% (70 of 72) at TDMC, 91.6% (175 of 191) at LUMC and 100% (six of six) at STI. Sera of chronic patients showed a positivity rate of 11.3% (19 of 168) and 61.2% (71of 116) at TDMC and LUMC, respectively. All 266 sera of non-endemic normals from STI, NIH and LUMC tested negative with the dipstick. At LUMC, sera of 'endemic normals' (amicrofilaraemics with no clinical disease) from an area with approximately 35% microfilaria positivity showed 60.8% positive results (31 of 51), thus demonstrating the likelihood of many cryptic infections occurring in this population. Specificities of the test with Onchocerca volvulus sera were 98.8% (80 of 81) and 100% (10 of 10) at the NIH and STI, respectively; while specificity with Loa loa sera at the NIH was 84.6% (44 of 52). At the STI, the dipstick test also demonstrated 100% specificity when tested with 75 sera from various protozoan and helminthic infections.

Variation and polymorphism in helminth parasites.

There are strong biological, evolutionary and immunological arguments for predicting extensive polymorphism among helminth parasites, but relatively little data and few instances from which the selective forces acting on parasite diversity can be discerned. The paucity of information on intraspecific variation stands in contrast to the fine detail with which helminth species have been delineated by morphological techniques, accentuating a trend towards considering laboratory strains as representative of a relatively invariant organism. However, in the fast-moving evolutionary race between host and parasite one would predict a monomorphic species would be driven to extinction. We review the arena of intraspecific variation for the major helminth parasites, ranging from biological properties such as host or vector preference, to biochemical and immunological characteristics, as well as molecular markers such as DNA sequence variants. These data are summarized, before focusing in more detail on polymorphisms within protein-coding genes of potential relevance to the host-parasite relationship, such as vaccine candidates. In particular, we discuss the available data on a number of major antigens from the filarial nematode Brugia malayi. Information is currently too sparse to answer the question of whether there is antigenic variation in filariasis, but the indications are that proteins from the blood-borne microfilarial stage show significant intraspecific variability. Future work will define whether polymorphisms in these antigens may be driven by exposure to the host immune response or reflect some other facet of parasite biology.

Expression and immune recognition of Brugia malayi VAL-1, a homologue of vespid venom allergens and Ancylostoma secreted proteins.

Several important nematode parasites have been found to express members of a gene family variously termed as venom allergen antigen homologue (vah) or Ancylostoma secreted protein (asp). In some cases these products are secreted by infective larval stages and have been suggested to be effective vaccine immunogens. We isolated the corresponding gene from the human filarial nematode, Brugia malayi, by first searching the expressed sequence tag (EST) dataset generated by the Filarial Genome Project and then using gene-specific nondegenerate primers matching the selected gene for PCR, from B. malayi cDNA libraries. We report here the full-length gene sequence, which we have designated as Bm-val-1, for vah/asp-like. The corresponding protein (Bm-VAL-1) contains 232 amino acids in a single homology unit, unlike products from some other species in which there is a tandem repeat. A putative signal sequence is present at the 5' end and there are two potential N-glycosylation sites. Murine antibodies to recombinant Bm-VAL-1 react with a 28 kDa protein in L3 extracts and recombinant Bm-VAL-1 is recognised by murine T cells primed with soluble L3 proteins. Of 82 ESTs corresponding to Bm-val-1, 72 are recorded from the infective larval (L3) stage. However, PCR on the first-strand cDNA from later mammalian stages revealed some expression at most subsequent time points. Over 95% (20/21) of microfilaraemic human filariasis patients are seropositive for antibodies to Bm-VAL-1, with particularly high levels of IgG3 and IgG4 isotypes. The IgG4 subclass may indicate stimulation by adult and/or microfilarial-derived immunogens. The association of Bm-VAL-1 with the infective stage and its recognition by humans exposed to filariasis suggests that further evaluation of this antigen as a vaccine candidate should be performed.

A Brugia malayi homolog of macrophage migration inhibitory factor reveals an important link between macrophages and eosinophil recruitment during nematode infection.

Infections with the helminth parasite Brugia malayi share many key features with Th2-mediated allergic diseases, including recruitment of eosinophils. We have investigated the dynamics of inflammatory cell recruitment under type 2 cytokine conditions in mice infected with B. malayi. Among the cells recruited to the site of infection is a novel population of "alternatively activated" macrophages that ablate cell proliferation and enhance Th2 differentiation. By profiling gene expression in this macrophage population, we found a dramatic up-regulation of a recently described eosinophil chemotactic factor, eosinophil chemotactic factor-L/Ym1, representing over 9% of clones randomly selected from a cDNA library. Because B. malayi is known to secrete homologs (Bm macrophage migration inhibitory factor (MIF)-1 and -2) of the human cytokine MIF, we chose to investigate the role this cytokine mimic may play in the development of the novel macrophage phenotype observed during infection. Strikingly, administration of soluble recombinant Bm-MIF-1 was able to reproduce the effects of live parasites, leading both to the up-regulation of Ym1 by macrophages and a marked recruitment of eosinophils in vivo. Because activity of Bm-MIF-1 is dependent upon an amino-terminal proline, this residue was mutated to glycine; the resultant recombinant (Bm-MIF-1G) was unable to induce Ym1 transcription in macrophages or to mediate the recruitment of eosinophils. These data suggest that macrophages may provide a crucial link between helminth parasites, their active cytokine mimics, and the recruitment of eosinophils in infection.

Immunological genomics of Brugia malayi: filarial genes implicated in immune evasion and protective immunity.

Filarial nematodes are metazoan parasites with genome sizes of> 100 million base pairs, probably encoding 15 000-20 000 genes. Within this considerable gene complement, it seems likely that filariae have evolved a spectrum of immune evasion products which underpin their ability to live for many years within the human host. Moreover, no suitable vaccine currently exists for human filarial diseases, and few markers have yet been established for diagnostic use. In this review, we bring together biochemical and immunological data on prominent filarial proteins with the exciting new information provided by the Filarial Genome Project's expressed sequence tag (EST) database. In this discussion, we focus on those genes with the highest immunological profile, such as inhibitors of host enzymes, cytokine homologues and stage-specific surface proteins, as well as products associated with the mosquito-borne infective larva which offer the best opportunity for an anti-filarial vaccine. These gene products provide a fascinating glimpse of the molecular repertoire which helminth parasites have evolved to manipulate and evade the mammalian immune response.