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W Harnett

Publications and source records attributed to W Harnett.

At least 19 recordsLinked to original sources
PubMed2006

Notch is constitutively active in Theileria-transformed B cells and can be further stimulated by the filarial nematode-secreted product, ES-62.

Theileria parva-infected B cells express Jagged-1 and activate Notch signalling in a parasite-dependent manner. ES-62, a filarial nematode-secreted phosphorylcholine-containing glycoprotein, is able to further stimulate Notch-mediated signalling in parasitized cells. Notch is also activated to a similar extent by addition of exogenous IL-10, and this occurs prior to any increase in proliferation in T. parva-infected B cells.

Animals↗
PubMed2006

Dissecting Ascaris glycosphingolipids for immunomodulatory moieties--the use of synthetic structural glycosphingolipid analogues.

We have previously shown glycosphingolipids of Ascaris suum to have phosphorylcholine (PC) and non-PC immunomodulatory moieties. In the present study we further investigated the nature of the immunomodulatory moieties by employing three synthetic glycosphingolipids each possessing features of the original molecule to examine effects on macrophage and dendritic cell (DC) cytokine production and surface co-stimulatory molecule expression. Compound 2, which lacked PC but contained ceramide, had no effect on either macrophages or DCs. Surprisingly however, Compound 1, which contained PC and hence arguably most resembled the native material, had, with the exception of a small increase in surface antigen expression, no immunomodulatory properties. Conversely, Compound 3, which contained PC but was otherwise least like the native molecule, demonstrated a number of effects on both macrophages and DCs, including induction of Th-1/pro-inflammatory cytokines, inhibition of such cytokines induced by IFN-gamma/LPS and increased expression of co-stimulatory molecules. Taken together these results indicate: (i) that although PC is an immunomodulatory component of the native molecule other structural feature are necessary to allow it to act; (ii) that carbohydrate rather than ceramide is likely to represent a non-PC immunomodulatory moiety; and (iii) that synthetic PC-containing molecules have the potential to act as immunomodulatory drugs.

Animals↗
PubMed2006

Molecular basis of worm-induced immunomodulation.

Long-term infection with parasitic worms is generally associated with an immunological phenotype that is Th2-like and anti-inflammatory. This phenotype is probably an unintentional consequence of molecular characteristics of worms (as free-living worms also express polarising molecules) in combination with deliberate attempts by the parasites, via molecular secretions, to modulate the phenotype. This review is concerned with the identity of immunomodulatory worm products, the receptors that they interact with and the signal transduction pathways that they activate. It hopes to indicate how knowledge of these factors can explain the changes in gene expression that result in the characteristic worm-induced immunological phenotype.

Animals↗
PubMed2006

Investigation of strategies with potential for producing a phosphorylcholine-free version of the filarial nematode immunomodulator, ES-62.

Phosphorylcholine (PC) is found attached to N-type glycans of proteins secreted by filarial nematodes, where it appears to act as an immunomodulator. Based on information on the structure and biosynthesis of the PC-glycan of a major secreted protein, ES-62, strategies were designed with potential for preparing PC-free material to better understand the importance of PC in filarial nematode immunomodulation. The strategies involve either enzymatic removal of PC or inhibition of its attachment during ES-62 synthesis. No method tested was found to be 100% effective although approximately 70% removal was obtained by culturing worms in Et18OCH3. Reasons for failure to obtain complete absence of PC moieties are discussed in relation to the structure and synthesis of PC-glycans and in addition PC-glycan biosynthesis is briefly commented on as a target for chemotherapy.

Animals↗
PubMed2005

Lack of immunological cross-reactivity between parasite-derived and recombinant forms of ES-62, a secreted protein of Acanthocheilonema viteae.

The longevity of filarial nematodes is dependent on secreted immunomodulatory products. Previous investigation of one such product, ES-62, has suggested a critical role for post-translationally attached phosphorylcholine (PC) moieties. In order to further investigate this, ES-62 lacking PC was produced, using the Pichia pastoris recombinant gene expression system. Unlike parasite-derived ES-62, which is tetrameric the recombinant material was found to consist of a mixture of apparently stable tetramers, dimers and monomers. Nevertheless, the recombinant protein was considered to be an adequate PC-free ES-62 as it was recognized by existing antisera against the parasite-derived protein. However, subsequent to this, recognition of parasite-derived ES-62 by antibodies produced against the recombinant protein was found to be absent. In an attempt to explain this, recombinant ES-62 was subjected to structural analysis and was found to (i) contain 3 changes in amino acid composition; (ii) demonstrate significant alterations in glycosylation; (iii) show major differences in protein secondary structure. The effects of these alterations in relation to the observed change in immunogenicity were investigated and are discussed. The data presented clearly show that recognition by existing antibodies is insufficient proof that recombinant proteins can be used to mimic parasite-derived material in studies on nematode immunology and vaccination.

Amino Acid Sequence↗
PubMed2005

Subversion of immune cell signal transduction pathways by the secreted filarial nematode product, ES-62.

Filarial nematodes achieve longevity within the infected host by suppressing and modulating the host immune response. To do this, the worms actively secrete products that have been demonstrated to possess immunomodulatory properties. In this article we discuss the immunomodulatory effects of the phosphorylcholine-containing filarial nematode secreted glycoprotein ES-62. In particular we describe how it modulates intracellular signal transduction pathways in a number of different cells of the immune system, in particular B-lymphocytes, T-lymphocytes, macrophages and dendritic cells.

Animals↗
PubMed2005

ES-62 is unable to modulate Toxoplasma gondii-driven Th1 responses and pathology.

ES-62, a filarial nematode-derived anti-inflammatory immunomodulator, was administered to mice in an attempt to prevent pathology associated with Toxoplasma gondii infection. The nematode product was shown to elevate mitogen and T. gondii-specific IL-10 production but was unable to inhibit Th1 responses. Consequently ES-62 could not prevent Th1 generated immunopathology.

Animals↗
PubMed2004

Onchocerca ochengi acquisition in zebu Gudali cattle exposed to natural transmission: parasite population dynamics and IgG antibody subclass responses to Ov10/Ov11 recombinant antigens.

Ngaoundere Gudali zebu cattle naturally exposed to Simulium damnosum s.l. and Culicoides spp. bites were examined during 4 years for O. ochengi adult worm acquisition, Onchocerca ochengi and Onchocerca gutturosa skin microfilaria dynamics, and IgG1 and IgG2 antibody subclass responses. Eleven animals acquired a total of 465 O. ochengi nodules (average of 17 per female and 72 per male). The O. ochengi nodule load was highly variable in individual animals and exacerbated in mature male cattle. Three patterns of acquisition of O. ochengi (resistant to new infestation, early susceptibility and late susceptibility), not associated with Simulium biting intensity (P > 0.05), were distinguished. The minimum prepatent periods for O. ochengi nodules, O. ochengi microfilariae and O. gutturosa microfilariae were 10, 20 and 21 months, respectively. The O. ochengi microfilaria density significantly (P < 0.001) increased with age, was higher in young mature bulls than female animals (P < 0.001) and finally reached highest levels (P < 0.005) during the dry season. Antibody responses to Ov10/Ov11 recombinant O. volvulus antigens were predominantly of the IgG1 subclass. High levels of this subclass (not IgG2) observed in new born calves declined to almost zero levels at the age of 5-8 months but IgG1 levels significantly increased (P < 0.05) with age subsequently during patency. Put together the acquisition and accumulation of O. ochengi parasites in zebu cattle, apart from being season, sex (gender) and host age associated, may also suggest a density-dependent regulation of parasite establishment in a proportion of the exposed population.

Age Factors↗
PubMed2004

Stage-specific and species-specific differences in the production of the mRNA and protein for the filarial nematode secreted product, ES-62.

Previous studies have shown that the secreted phosphorylcholine-containing glycoprotein of filarial nematodes, ES-62, is only present in the post-infective life-cycle stages, but that the mRNA is transcribed throughout the worm's life-cycle. The aim of this current study was to investigate whether the presence or absence of protein expression simply reflects differences in mRNA abundance. To this end, we investigated the relative abundance of ES-62 using TaqMan real time RT-PCR, in different life-cycle stages of 2 model filarial nematode parasites, Acanthocheilonema viteae and Brugia pahangi. For B. pahangi, microfilariae, infective larvae and adult worms were each found to have approximately similar levels of ES-62 mRNA. However, the corresponding stages of A. viteae differed greatly from each other with a pattern of increased mRNA production with maturation. As a rule A. viteae had higher levels of ES-62 mRNA than B. pahangi, and this was particularly noticeable in the adult stage where the difference was approximately 3500-fold higher. However, this significant difference in mRNA abundance was not reflected in the quantity of ES-62 protein secreted by the adult worms of each species, as A. viteae only secreted approximately 3 times as much ES-62 as B. pahangi. Thus, overall, the results obtained from this study indicate that ES-62 protein production does not solely reflect mRNA levels, and also suggest that the 2 nematodes may employ different mechanisms for regulating protein production.

Animals↗
PubMed2004

Structure and synthesis of nematode phosphorylcholine-containing glycoconjugates.

Infection with filarial nematodes produces a chronic, long-lasting illness with adult worms able to survive within human hosts for up to 15 years. A contributor to the longevity of these parasites is the presence of phosphorylcholine (PC) on components of the worms' molecular secretions (ES). PC on ES modulates host immune responses towards an anti-inflammatory phenotype thereby generating an environment favourable for parasite survival. PC is attached to nematode ES via a covalent association with carbohydrate, which, although well-documented in bacteria and fungi, is absent from humans, making it an ideal target for the development of novel drugs. In order to produce such drugs it is first necessary to understand the structure and synthesis of nematode PC-glycans. ES-62 is the major PC-ES-product of Acanthocheilonema viteae and is a homologue of PC-ES found in human filarial nematodes. We have studied the structure and biosynthesis of PC-glycans of ES-62 by a combination of pulse-chase experiments, experiments involving the use of inhibitors of each of intracellular trafficking, oligosaccharide processing and phospholipid biosynthesis and various forms of mass spectrometry. Our indications indicate that PC is transferred in the lumen of the medial Golgi to an N-type glycan consisting of a trimannosyl core with or without core fucosylation bearing between 1 and 4 N-acetyl glucosamine residues. The structure of the PC-N-glycans found in ES-62 appears to be conserved amongst filarial nematodes in that it has additionally been identified in Onchocerca volvulus and O. gibsoni. Also, similar structures have been found in non-filarial parasitic nematodes and in the free-living nematode Caenorhabditis elegans. Finally, PC has also been recently found attached to the carbohydrate moieties of nematode glycosphingolipids and the structure of these will also be considered.

Animals↗
PubMed2003

Structural/functional aspects of ES-62--a secreted immunomodulatory phosphorylcholine-containing filarial nematode glycoprotein.

ES-62 is a major secreted glycoprotein of the rodent filarial nematode Acanthocheilonema viteae and homologue of molecules found in filarial nematodes which parasitise humans. The molecule consists of a tetramer of apparently identical monomers of ~62 kDa which we have shown by sedimentation equilibrium analytical ultracentrifugation to strongly associate. ES-62 is one of several filarial nematode proteins to contain the unusual post-translational modification of phosphorylcholine (PC) addition. Specifically, we have found that PC is attached to one of three distinct N-type glycans we have characterised on the molecule. The amino acid sequence of ES-62 shows 37-39% identity with a family of 6 other proteins, some of which have been predicted to be amino- or carboxy-peptidases. We have also found that ES-62 is able to interact with a number of cells of the immune system, specifically B- and T-lymphocytes, macrophages and dendritic cells. Lymphocytes exposed to ES-62 in vitro or in vivo are less able to proliferate in response to ligation via the antigen receptor. Peritoneal macrophages pre-exposed to the molecule are less able to produce the cytokines IL-12, IL-6 and TNF-alpha following subsequent incubation with the classical stimulators IFNgamma and LPS. Dendritic cells allowed to mature in the presence of ES-62 acquire a phenotype, which allows them to induce anti-inflammatory "TH2-type" responses. With respect to immunomodulation, the PC moiety of the parasite molecule appears to be predominantly responsible for the effects on lymphocyte proliferation at least and we have also found that its removal converts the murine IgG antibody response to ES-62 from solely IgG1 to mixed IgG1/IgG2a. ES-62 appears to interact with cells of the immune system in a PC-dependent manner and, at least in part, via a molecule of ~82 kDa. Studies of the interaction in lymphocytes show that it is associated with activation of certain signal transduction molecules including a number of protein tyrosine kinases and mitogen activated protein kinases (MAPkinases). Although such activation is insufficient to induce proliferation, it serves to almost completely desensitise the cells to antigen-receptor ligation-induced activation of the phosphoinositide 3-kinase (PI-3-kinase) and Ras/MAPkinase pathways, events critical for lymphocyte proliferation. Such desensitisation reflects ES-62-primed recruitment of a number of negative regulators of these pathways, such as the phosphatases SHP-1 and Pac-1.

Amino Acid Sequence↗
PubMed2002

Expression of the filarial nematode phosphorylcholine-containing glycoprotein, ES62, is stage specific.

ES62, an immunomodulatory phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae, has previously been shown to be produced by L4 larvae and adult worms only. However, homologous sequences to ES62 have recently been found in L1 and L3 cDNA libraries of certain human filarial nematodes. Therefore, the various stages of A. viteae were re-examined and it was again found that only the post-L3 stages secreted ES62. Synthesis but not secretion by earlier stages was ruled out by examination of the protein content of whole worm extracts and by immunoelectron microscopy. However, examination by PCR of the mRNA for ES62 revealed that it was found in the L1 and L3 larvae. This may explain why homologous sequences to ES62 have been found in Brugia malayi and Onchocerca volvulus larval cDNA libraries. It also suggests that filarial nematodes, in general, may secrete ES62. To obtain evidence for this, we investigated production by Brugia pahangi, a close relation of B. malayi. We found that ES62 was indeed secreted but, as with A. viteae, only by the post-L3 stages, although again the mRNA for ES62 could be detected in the earlier stages. Overall our results suggest that production of ES62 is not species specific, that it is indeed stage specific, and that this may be due to post-transcriptional control of expression.

Amino Acid Sequence↗
PubMed2001

Modulation of macrophage cytokine production by ES-62, a secreted product of the filarial nematode Acanthocheilonema viteae.

Parasite survival and host health may depend on the ability of the parasite to modulate the host immune response by the release of immunomodulatory molecules. Excretory-secretory (ES)-62, one such well-defined molecule, is a major secreted protein of the rodent filarial nematode Acanthocheilonema viteae, and has homologues in human filarial nematodes. Previously we have shown that ES-62 is exclusively associated with a Th2 Ab response in mice. Here we provide a rationale for this polarized immune response by showing that the parasite molecule suppresses the IFN-gamma/LPS-induced production, by macrophages, of bioactive IL-12 (p70), a key cytokine in the development of Th1 responses. This suppression of the induction of a component of the host immune response extends to the production of the proinflammatory cytokines IL-6 and TNF-alpha, but not NO. The molecular mechanism underlying these findings awaits elucidation but, intriguingly, the initial response of macrophages to ES-62 is to demonstrate a low and transient release of these cytokines before becoming refractory to further release induced by IFN-gamma/LPS. The relevance of our observations is underscored by the finding that macrophages recovered from mice exposed to "physiological" levels of ES-62 by the novel approach of continuous release from implanted osmotic pumps in vivo were similarly refractory to release of IL-12, TNF-alpha, IL-6, but not NO, ex vivo. Therefore, our results suggest that exposure to ES-62 renders macrophages subsequently unable to produce Th1/proinflammatory cytokines. This likely contributes to the generation of immune responses with an anti-inflammatory Th2 phenotype, a well-documented feature of filarial nematode infection.

Adjuvants, Immunologic↗
PubMed2001

A filarial nematode-secreted phosphorylcholine-containing glycoprotein uncouples the B cell antigen receptor from extracellular signal-regulated kinase-mitogen-activated protein kinase by promoting the surface Ig-mediated recruitment of Src homology 2 domain-containing tyrosine phosphatase-1 and Pac-1 mitogen-activated kinase-phosphatase.

Unraveling the molecular mechanisms by which filarial nematodes, major human pathogens in the tropics, evade the host immune system remains an elusive goal. We have previously shown that excretory-secretory product-62 (ES-62), a homologue of phosphorylcholine-containing molecules that are secreted by human parasites and which is active in rodent models of filarial infection, is able to polyclonally activate certain protein tyrosine kinase and mitogen-activating protein kinase signal transduction elements in B lymphocytes. Such activation mediates desensitization of subsequent B cell Ag receptor (BCR) ligation-induced activation of extracellular signal-regulated kinase-mitogen-activated protein (ErkMAP) kinase and ultimately B cell proliferation. We now show that the desensitization is due to ES-62 targeting two major regulatory sites of B cell activation. Firstly, pre-exposure to ES-62 primes subsequent BCR-mediated recruitment of SHP-1 tyrosine phosphatase to abolish recruitment of the RasErkMAP kinase cascade via the Igalphabeta-ShcGrb2Sos adaptor complex interactions. Secondly, any ongoing ErkMAP kinase signaling in ES-62-primed B cells is terminated by the MAP kinase phosphatase, Pac-1 that is activated consequently to challenge via the BCR.

Animals↗
PubMed2001

Modulation of the host immune system by phosphorylcholine-containing glycoproteins secreted by parasitic filarial nematodes.

Phosphorylcholine (PC) is increasingly becoming recognised as a carbohydrate-associated component of a wide variety of procaryotic and eucaryotic pathogens. Studies employing nematode PC-containing molecules indicate that it possesses a plethora of immunomodulatory activities. ES-62 is a PC-containing glycoprotein, which is secreted by the rodent filarial nematode Acanthocheilonema viteae and which provides a model system for the dissection of the mechanisms of immune evasion induced by related PC-containing glycoproteins expressed by human filarial nematodes. At concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitised humans, ES-62 is able to inhibit antigen receptor-stimulated proliferation of B and T lymphocytes in vitro and in vivo. The active component of ES-62 appears to be PC, as PC conjugated to albumin or even PC alone broadly mimic the results obtained with ES-62. PC-induced impaired lymphocyte responsiveness appears to reflect uncoupling of the antigen receptors from key intracellular proliferative signalling events such as the phosphoinositide 3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways. Although PC-ES-62 can desensitise B and T cells, not all cells are affected, and in fact it is still possible to generate an antibody response to the molecule. Dissection of this response indicates that it is of the TH-2 type. This appears to reflect the ability of ES-62 to direct the polarity of the T cell response by suppressing the production of proinflammatory cytokines, inducing the induction of anti-inflammatory cytokines and by driving the maturation of dendritic cells that direct TH-2 T cell responses.

Adjuvants, Immunologic↗
PubMed2001

Antigen receptor signaling is subverted by an immunomodulatory product secreted by a filarial nematode.

ES-62 is a phosphorylcholine (PC)-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae which is able to inhibit antigen receptor-stimulated proliferation of B and T lymphocytes in vitro and in vivo. The active component of ES-62 appears to be PC, as the results obtained with ES-62 are broadly mimicked by PC conjugated to bovine serum albumin or PC alone. Such desensitization of lymphocyte responsiveness appears to reflect an uncoupling of the antigen receptors from key intracellular proliferative signaling events, such as the phosphoinositide-3-kinase, protein kinase C and Ras mitogen-activating protein kinase pathways. ES-62 mediates such immunomodulatory effects at concentrations equivalent to those found for PC-containing molecules in the bloodstream of parasitized humans and, thus, ES-62 provides a model system for dissecting the mechanisms of immune evasion induced by related PC-containing glycoproteins expressed by human filarial nematodes.

Adjuvants, Immunologic↗
PubMed2000

A filarial nematode-secreted product signals dendritic cells to acquire a phenotype that drives development of Th2 cells.

Although exogeneous "danger" signals such as LPS can activate APC to produce a Th1 response, the nature of events initiating a Th2 response is controversial. We now show that pathogen-derived products have the capacity to induce bone marrow-derived dendritic cell cultures to acquire a phenotype that promotes the differentiation of naive CD4+ T cells toward either a Th1 or Th2 phenotype. Thus, LPS-matured dendritic cells (DC1) promote a Th1 response (increased generation of IFN-gamma and reduced production of IL-4) by Ag-stimulated CD4+ T cells from the DO.11.10 transgenic mouse expressing a TCR specific for an OVA peptide (OVA323-339). In contrast, a phosphorylcholine-containing glycoprotein, ES-62, secreted by the filarial nematode, Acanthocheilonema viteae, which generates a Th2 Ab response in vivo, is found to induce the maturation of dendritic cells (DC2) with the capacity to induce Th2 responses (increased IL-4 and decreased IFN-gamma). In addition, we show that the switch to either Th1 or Th2 responses is not effected by differential regulation through CD80 or CD86 and that a Th2 response is achieved in the presence of IL-12.

Animals↗