Characterisation of antagonist binding sites on chemokine receptor CCR4

CCR4 is a chemokine receptor notably expressed on T helper 2 and regulatory T cells. CCR4 binds the chemokines CCL17 and CCL22. These are involved in T cell homeostasis and inflammatory diseases including asthma and atopic dermatitis, making CCR4 a potential therapeutic target. Previous studies suggested that CCL22 is dominant over CCL17 with respect to ligand-induced internalisation and desensitisation of CCR4. The biology of CCR4 was investigated in this project using point mutational studies. A C-terminal lysine within the conserved helix VIII region was determined to be dispensable for CCL22-induced chemotaxis but required for CCL17-induced chemotaxis, suggesting that the two chemokines stabilised distinct receptor conformations. The highly conserved GluVII:06 of helix VII was shown to be critical for chemokine binding and receptor function. Seven small molecule allosteric antagonists of CCR4, supplied by GlaxoSmithKline, were hypothesised to bind either a classical intrahelical site (site 1) within the receptor or a novel intracellular site (site 2). 22 amino acids were predicted to be involved in the binding of the antagonists. Antagonist binding was indirectly investigated by inhibiting either function or chemokine binding of the receptor mutants. Mutation of leucine 118 in transmembrane helix III significantly reduced CCR4 sensitivity to site 1 antagonism in chemotaxis and chemokine-binding assays. Mutants of phenylalanine 305 and leucine 307 at the end of transmembrane helix VII also showed a reduction in antagonist 2 sensitivity. Direct investigation of the effects of mutation on antagonist binding was performed using tritium-labelled antagonists. Mutation of GluVII:06 prevented site 1 antagonist binding to CCR4. Further investigation of site 1 antagonist binding was hindered by high non-specific binding of the compounds. A low-affinity site for the tritium-labelled site 2 antagonist was identified on untransfected cells, possibly within endogenously expressed chemokine receptors. This antagonist therefore may have potential as a broad-spectrum chemokine receptor inhibitor

Immunological Responses Elicited by Different Infection Regimes with Strongyloides ratti

Nematode infections are a ubiquitous feature of vertebrate life. In nature, such nematode infections are acquired by continued exposure to infective stages over a prolonged period of time. By contrast, experimental laboratory infections are typically induced by the administration of a single (and often large) dose of infective stages. Previous work has shown that the size of an infection dose can have significant effects on anti-nematode immune responses. Here we investigated the effect of different infection regimes of Strongyloides ratti, comparing single and repeated dose infections, on the host immune response that was elicited. We considered and compared infections of the same size, but administered in different ways. We considered infection size in two ways: the maximum dose of worms administered and the cumulative worm exposure time. We found that both infection regimes resulted in Th2-type immune response, characterised by IL4 and IL13 produced by S. ratti stimulated mesenteric lymph node cells, anti-S. ratti IgG1 and intestinal rat mast cell protease II (RMCPII) production. We observed some small quantitative immunological differences between different infection regimes, in which the concentration of IL4, IL13, anti-S. ratti IgG1 and IgG2a and RMCPII were affected. However, these differences were quantitatively relatively modest compared with the temporal dynamics of the anti-S. ratti immune response as a whole

The genetics of immune and infection phenotypes in wild mice, Mus musculus domesticus

Wild animals are under constant threat from a wide range of micro- and macroparasites in their environment. Animals make immune responses against parasites, and these are important in affecting the dynamics of parasite populations. Individual animals vary in their anti-parasite immune responses. Genetic polymorphism of immune-related loci contributes to inter-individual differences in immune responses, but most of what we know in this regard comes from studies of humans or laboratory animals; there are very few such studies of wild animals naturally infected with parasites. Here we have investigated the effect of single nucleotide polymorphisms (SNPs) in immune-related loci (the major histocompatibility complex [MHC], and loci coding for cytokines and Toll-like receptors) on a wide range of immune and infection phenotypes in UK wild house mice, Mus musculus domesticus. We found strong associations between SNPs in various MHC and cytokine-coding loci on both immune measures (antibody concentration and cytokine production) and on infection phenotypes (infection with mites, worms and viruses). Our study provides a comprehensive view of how polymorphism of immune-related loci affects immune and infection phenotypes in naturally infected wild rodent populations

Correction to: The population genetics of parasitic nematodes of wild animals.

Unfortunately, the original version of this article [1] contains an error. In the section entitled "Influence of anthropogenic disruption on parasitic nematode population genetics", the passage

<i>Strongyloides ratti</i> and <i>S. venezuelensis</i> – rodent models of <i>Strongyloides </i>infection

Strongyloides spp. are common parasites of vertebrates and two species, S. ratti and S. venezuelensis, parasitize rats; there are no known species that naturally infect mice. Strongyloides ratti and S. venezuelensis overlap in their geographical range and in these regions co-infections appear to be common. These species have been widely used as tractable laboratory systems in rats as well as mice. The core biology of these two species is similar, but there are clear differences in aspects of their within-host biology as well as in their free-living generation. Phylogenetic evidence suggests that S. ratti and S. venezuelensis are the result of two independent evolutionary transitions to parasitism of rats, which therefore presents an ideal opportunity to begin to investigate the basis of host specificity in Strongyloides spp

The genome of <i>Strongyloides </i>spp. gives insights into protein families with a putative role in nematode parasitism

SUMMARYParasitic nematodes are important and abundant parasites adapted to live a parasitic lifestyle, with these adaptations all aimed at facilitating their survival and reproduction in their hosts. The recently sequenced genomes of fourStrongyloidesspecies, gastrointestinal parasites of humans and other animals, alongside transcriptomic and proteomic analysis of free-living and parasitic stages of their life cycles have revealed a number of protein families with a putative role in their parasitism. Many of these protein families have also been associated with parasitism in other parasitic nematode species, suggesting that these proteins may play a fundamental role in nematode parasitism more generally. Here, we review key protein families that have a putative role inStrongyloides’ parasitism – acetylcholinesterases, astacins, aspartic proteases, prolyl oligopeptidases, proteinase inhibitors (trypsin inhibitors and cystatins), SCP/TAPS and transthyretin-like proteins – and the evidence for their key, yet diverse, roles in the parasitic lifestyle.</jats:p