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Terry W Spithill

Publications and source records attributed to Terry W Spithill.

10 recordsLinked to original sources
PubMed2007

Peritoneal lavage cells of Indonesian thin-tail sheep mediate antibody-dependent superoxide radical cytotoxicity in vitro against newly excysted juvenile Fasciola gigantica but not juvenile Fasciola hepatica.

Indonesian thin-tail (ITT) sheep resist infection by Fasciola gigantica by an immunological mechanism within 2 to 4 weeks of infection yet are susceptible to F. hepatica infection. Studies of ITT sheep show that little liver damage occurs following F. gigantica infection, suggesting that the invading parasites are killed within the peritoneum or shortly after reaching the liver. We investigated whether cells isolated from the peritoneums of ITT sheep could kill newly excysted juvenile F. gigantica in vitro and act as a potential mechanism of resistance against F. gigantica infection. Peritoneal cells from F. gigantica-infected sheep, rich in macrophages and eosinophils, mediated antibody-dependent cytotoxicity against juvenile F. gigantica in vitro. Cytotoxicity was dependent on contact between the parasite and effector cells. Isolated mammary gland eosinophils of F. gigantica-infected sheep, or resident peritoneal monocytes/macrophages from uninfected sheep, also killed the juvenile parasites in vitro. By using inhibitors, we show that the molecular mechanism of killing in these assays was dependent on the production of superoxide radicals by macrophages and eosinophils. In contrast, this cytotoxic mechanism was ineffective against juvenile F. hepatica parasites in vitro. Analysis of superoxide dismutase activity and mRNA levels showed that activity and gene expression were higher in F. hepatica than in F. gigantica, suggesting a possible role for this enzyme in the resistance of F. hepatica to superoxide-mediated killing. We suggest that ovine macrophages and eosinophils, acting in concert with a specific antibody, may be important effector cells involved in the resistance of ITT sheep to F. gigantica.

Animals↗
PubMed2006

The A/T-specific DNA alkylating agent adozelesin inhibits Plasmodium falciparum growth in vitro and protects mice against Plasmodium chabaudi adami infection.

There is an urgent need for new anti-malarial drugs to combat the resurgence of resistance to current therapies. To exploit the A/T richness of malaria DNA as a potential target for anti-malarial drugs we tested an A/T-specific DNA synthesis inhibitor, adozelesin, for activity against Plasmodium falciparum in vitro and Plasmodium chabaudi adami in mice. Adozelesin is a DNA alkylating agent that exhibits specificity for the motif A/T, A/T and A. In P. falciparum 3D7 cultures, adozelesin acts as a powerful inhibitor of parasite growth (IC(50) of 70 pM) and is equally potent at killing the drug-resistant strains FCR3 and 7G8. Using a real-time PCR assay, we show that treatment with adozelesin in vitro results in damage of P. falciparum genomic DNA. In synchronized cultures, adozelesin exhibits a concentration-dependent effect on parasitemia and on the development of parasites through the asexual cycle. In asynchronous cultures, parasites arrest at all stages of the asexual cycle suggesting that adozelesin exerts other anti-parasitic effects in addition to inhibiting DNA replication. These anti-parasite effects are irreversible since cultures exposed to adozelesin for more than 6h fail to recover upon removal of the drug. Furthermore, adozelesin is very effective at suppressing malaria infection in vivo; growth of P. c. adami DK in mice was highly impaired by a single injection of adozelesin (25 microg/kg) at 4 days post-infection. These results demonstrate that adozelesin irreversibly blocks parasite growth in vitro and suppresses parasite infection in vivo, suggesting that A/T-specific DNA damaging agents represent a new class of compounds with potential as anti-malarials.

Adenine↗
PubMed2006

Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite.

The liver fluke, Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed in Pichia pastoris and purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion by F. hepatica.

Animals↗
PubMed2005

DNA vaccines in sheep: CTLA-4 mediated targeting and CpG motifs enhance immunogenicity in a DNA prime/protein boost strategy.

DNA vaccines have proven to be an efficient means of inducing immune responses in small laboratory animals; however, their efficacy in large out-bred animal models has been much less promising. In addressing this issue, we have investigated the ability of ovine cytotoxic lymphocyte antigen 4 (CTLA-4) mediated targeting and ruminant specific CpG optimised plasmids, both alone and in combination, to enhance immune responses in sheep to the pro cathepsin B (FhCatB) antigen from Fasciola hepatica. In this study, CTLA-4 mediated targeting enhanced the speed and magnitude of the primary antibody response and effectively primed for a potent memory response compared to conventional DNA vaccination alone, which failed to induce a detectable immune response. While the CpG-augmentation of the CTLA-4 targeted construct did not further enhance the magnitude or isotype profile of the CTLA-4 induced antibody titres, it did result in the induction of significant antigen-specific, lymphocyte-proliferative responses that were not observed in any other treatment group, showing for the first time that significant cellular responses can be induced in sheep following DNA vaccination. In contrast, CpG-augmentation in the absence of CTLA-4 mediated targeting failed to induce a detectable immune response. This is the first study to explore the potential adjuvant effects of ruminant specific CpG motifs on DNA vaccine induced immune responses in sheep. The ability of CpG-augmented CTLA-4 mediated targeting to induce both humoral and cellular immune responses in this study suggests that this may be an effective approach for enhancing the efficacy of DNA vaccines in large out-bred animal models.

Adjuvants, Immunologic↗
PubMed2004

DNA vaccines and their application against parasites--promise, limitations and potential solutions.

DNA or nucleic acid vaccines are being evaluated for efficacy against a range of parasitic diseases. Data from studies in rodent model systems have provided proof of principle that DNA vaccines are effective at inducing both humoral and T cell responses to a variety of candidate vaccine antigens. In particular, the induction of potent cellular responses often gives DNA vaccination an immunological advantage over subunit protein vaccination. Protection against parasite challenge has been demonstrated in a number of systems. However, application of parasite DNA vaccines in large animals including ruminants, primates and humans has been compromised by the relative lack of immune responsiveness to the vaccines, but the reasons for this hyporesponsiveness are not clear. Here, we review DNA vaccines against protozoan parasites, in particular vaccines for malaria, and the use of genomic approaches such as expression library immunization to generate novel vaccines. The application of DNA vaccines in ruminants is reviewed. We discuss some of the approaches being evaluated to improve responsiveness in large animals including the use of cytokines as adjuvants, targeting molecules as delivery ligands, electroporation and CpG oligonucleotides.

Animals↗
PubMed2003

The evolution of enzyme specificity in Fasciola spp.

Fasciola spp., commonly known as liver fluke, are significant trematode parasites of livestock and humans. They secrete several cathepsin L-like cysteine proteases, some of which differ in enzymatic properties and timing of expression in the parasite's life cycle. A detailed sequence and evolutionary analysis is presented, based on 18 cathepsin L-like enzymes isolated from Fasciola spp. (including a novel clone identified in this study). The enzymes form a monophyletic group which has experienced several gene duplication events over the last approximately 135 million years, giving rise to the present-day enzymatic repertoire of the parasite. This timing of these duplications appears to correlate with important points in the evolution of the mammalian hosts. Furthermore, the dates suggest that Fasciola hepatica and Fasciola gigantica diverged around 19 million years ago. A novel analysis, based on the pattern of amino acid diversity, was used to identify sites in the enzyme that are predicted to be subject to positive adaptive evolution. Many of these sites occur within the active site cleft of the enzymes, and hence would be expected to lead to differences in substrate specificity. Using homology modeling, with reference to previously obtained biochemical data, we are able to predict S2 subsite specificity for these enzymes: specifically those that can accommodate bulky hydrophobic residues in the P2 position and those that cannot. A number of other positions subject to evolutionary pressure and potentially significant for enzyme function are also identified, including sites anticipated to diminish cystatin binding affinity.

Amino Acid Sequence↗
PubMed2003

Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection.

The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.

Amino Acid Sequence↗
PubMed2002

Fasciola hepatica cathepsin L suppresses sheep lymphocyte proliferation in vitro and modulates surface CD4 expression on human and ovine T cells.

Fasciola hepatica infection has been shown to suppress sheep lymphocyte proliferation in vitro and this is at least partially attributable to excretory/secretory products (ES) released by F. hepatica parasites. We identified a suppressive component in ES by analysing the effect of ES fractions, separated by gel filtration, on the proliferation of sheep T cells in vitro. A major proportion of the suppressive activity in ES was shown to coelute with the cathepsin L proteases: E64, a cysteine protease inhibitor, blocked the suppressive activity of cathepsin L. In order to identify possible mechanisms by which cathepsin L could suppress T cell proliferation, the effect of ES and F. hepatica recombinant cathepsin L (rFhCatL) on the expression of 22 different sheep T cell surface markers was analysed by flow cytometry. Incubation of sheep T cells with ES or two rFhCatL significantly reduced surface CD4 expression and this effect was prevented in the presence of E64. In similar experiments with human T lymphocytes, ES and rFhCatL were shown to down regulate surface CD4 expression. These results show that F. hepatica cathepsin L both suppresses sheep T cell proliferation and reduces surface CD4 expression on both human and ovine T cells.

Animals↗