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Seshadri S.,University of Liverpool | Bates M.,Molecular and Biochemical Parasitology Group | Vince G.,Lancaster University | Lewis Jones D.I.,Liverpool Womens Hospital
Andrologia | Year: 2011

Cytokines are released by various immunocompetent cell subsets in the male urogenital tract and are thought to affect sperm cell function and reproductive process. The aim of the study was to evaluate the levels and a possible role of seven seminal plasma cytokines with fertilisation rates in men attending an in vitro fertilisation (IVF) programme. A total of 36 men of couples who were undergoing traditional IVF in a regional reproductive medicine unit were recruited into this prospective study. Cytokines such as interleukin (IL)-6, IL-8, IL-10, IL-11, IL-12, tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) in the seminal plasma were determined using enzyme linked immunosorbent assay. IL-6, IL-8, IL-10, IL-11 and IFN-γ were detected in all samples. IL-12, and TNF-α were detected in most samples. Levels of IL-11 were significantly higher in the good fertiliser group (P≤0.05). Positive correlation between cytokines such as IL-6 and IL-8 (P<0.03), IL-10 and IL-11 (P<0.001) and IFN-γ and IL-10 and IL-11 (P<0.04 and P<0.0001 respectively) were found. Our study confirms that the six cytokines other than IL-11 do not affect spermatozoon-oocyte interaction and fertilisation rates in IVF. IL-11 could have a role in the fertilising capacity of the spermatozoa. Significant correlation exists among these cytokines which shows that cytokines rarely act in isolation but rather in a network. © 2011 Blackwell Verlag GmbH.


Lynd A.,Vector Group | Weetman D.,Vector Group | Barbosa S.,Molecular and Biochemical Parasitology Group | Egyir Yawson A.,Ghana Atomic Energy Commission | And 4 more authors.
Molecular Biology and Evolution | Year: 2010

Alleles subject to strong, recent positive selection will be swept toward fixation together with contiguous sections of the genome. Whether the genomic signatures of such selection will be readily detectable in outbred wild populations is unclear. In this study, we employ haplotype diversity analysis to examine evidence for selective sweeps around knockdown resistance (kdr) mutations associated with resistance to dichlorodiphenyltrichloroethane and pyrethroid insecticides in the mosquito Anopheles gambiae. Both kdr mutations have significantly lower haplotype diversity than the wild-type (nonresistant) allele, with kdr L1014F showing the most pronounced footprint of selection. We complement these data with a time series of collections showing that the L1014F allele has increased in frequency from 0.05 to 0.54 in 5 years, consistent with a maximum likelihood-fitted selection coefficient of 0.16 and a dominance coefficient of 0.25. Our data show that strong, recent positive selective events, such as those caused by insecticide resistance, can be identified in wild insect populations. © 2010 The Author. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.


LaCourse E.J.,Aberystwyth University | LaCourse E.J.,Molecular and Biochemical Parasitology Group | Perally S.,Aberystwyth University | Morphew R.M.,Aberystwyth University | And 11 more authors.
PLoS Neglected Tropical Diseases | Year: 2012

Background: Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised. Methodology/Principal Findings: In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection. Conclusions/Significance: We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity. © 2012 LaCourse et al.


Patzewitz E.-M.,University of Glasgow | Patzewitz E.-M.,University of Nottingham | Salcedo-Sora J.E.,Molecular and Biochemical Parasitology Group | Wong E.H.,University of Glasgow | And 8 more authors.
Antioxidants and Redox Signaling | Year: 2013

Aims: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. Results: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. Innovation: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. Conclusion: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV. Antioxid. Redox Signal. 19, 683-695. © Copyright 2013, Mary Ann Liebert, Inc. 2013.


Barbosa S.,Molecular and Biochemical Parasitology Group | Black IV W.C.,Colorado State University | Hastings I.,Molecular and Biochemical Parasitology Group
PLoS Neglected Tropical Diseases | Year: 2011

Insecticide resistance has the potential to compromise the enormous effort put into the control of dengue and malaria vector populations. It is therefore important to quantify the amount of selection acting on resistance alleles, their contributions to fitness in heterozygotes (dominance) and their initial frequencies, as a means to predict the rate of spread of resistance in natural populations. We investigate practical problems of obtaining such estimates, with particular emphasis on Mexican populations of the dengue vector Aedes aegypti. Selection and dominance coefficients can be estimated by fitting genetic models to field data using maximum likelihood (ML) methodology. This methodology, although widely used, makes many assumptions so we investigated how well such models perform when data are sparse or when spatial and temporal heterogeneity occur. As expected, ML methodologies reliably estimated selection and dominance coefficients under idealised conditions but it was difficult to recover the true values when datasets were sparse during the time that resistance alleles increased in frequency, or when spatial and temporal heterogeneity occurred. We analysed published data on pyrethroid resistance in Mexico that consists of the frequency of a Ile1,016 mutation. The estimates for selection coefficient and initial allele frequency on the field dataset were in the expected range, dominance coefficient points to incomplete dominance as observed in the laboratory, although these estimates are accompanied by strong caveats about possible impact of spatial and temporal heterogeneity in selection. © 2011 Barbosa et al.


Janneh O.,Coventry University | Janneh O.,University of Liverpool | Bray P.G.,Molecular and Biochemical Parasitology Group | Jones E.,University of Liverpool | And 4 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2010

Background: The intracellular and plasma concentrations of HIV protease inhibitors (HPIs) vary widely in vivo. It is unclear whether there is a concentration-dependent effect of HPIs such that at increasing concentration they may either block their own efflux (leading to 'autoboosting') or influx (leading to saturability/decreased intracellular accumulation). Method: The effects of various concentrations (0-30 μM) of lopinavir, saquinavir, ritonavir and atazanavir on the accumulation of [14C]lopinavir, [3H]saquinavir, [3H]ritonavir and [3H]atazanavir, respectively, were investigated in CEMparental, CEMVBL [P-glycoprotein (ABCB1) overexpressing], CEME1000 (MRP1 overexpressing) and in peripheral blood mononuclear cells (PBMCs). We also investigated the effects of inhibitors of ABCB1/ABCG2 (tariquidar), ABCC (MK571) and ABCC1/2 (frusemide), singly and in combination with HPIs, on cellular accumulation. Results: In all the cell lines, with increasing concentration of lopinavir, saquinavir and ritonavir, there was a significant increase in the cellular accumulation of [14C]lopinavir, [3H]saquinavir and [3H]ritonavir. Tariquidar, MK571 and frusemide (alone and in combination with lopinavir, saquinavir and ritonavir) significantly increased the accumulation of [14C]lopinavir, [3H]saquinavir and [3H]ritonavir. Ritonavir (alone or in combination with tariquidar) decreased the intracellular accumulation of [3H]ritonavir in PBMCs. Atazanavir decreased the accumulation of [3H]atazanavir in a concentration-dependent manner in all of the cells tested. Conclusions: There are complex and variable drug-specific rather than class-specific effects of the HPIs on their own accumulation. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Moss D.M.,Molecular and Biochemical Parasitology Group | Siccardi M.,University of Liverpool | Back D.J.,University of Liverpool | Owen A.,University of Liverpool
Journal of Antimicrobial Chemotherapy | Year: 2013

Objectives: Raltegravir pharmacokinetics (PK) show high intra- and inter-patient variability and are also influenced by co-administered substances that alter the gastrointestinal tract environment, such as pH-altering or metal-containing agents. The aim of this investigation was to develop a population-based absorption, distribution, metabolism and excretion (ADME) model to investigate the effects of gastrointestinal pH and ingested magnesium on raltegravir PK. Methods: In vitro data describing the disposition of raltegravir were obtained from literature sources or generated by standard methods. Raltegravir (400 mg single dose) PK were simulated in healthy volunteers (50 subjects per group, 20-50 years old, 0.5 proportion female subjects) over a 12 h period. Results: Simulated raltegravir PK correlated well with data from clinical trials, with a mean deviation in Cmax, AUC0-12 and Ctrough of ,20%. Solubility of raltegravir in the gastrointestinal tract was increased at higher luminal pH. Increased intestinal pH and transit time both correlated with higher raltegravir absorption (P<0.001). Magnesium ingestion reduced raltegravir exposure in simulated subjects, with mean Ctrough reduced by 32% (P<0.001). Conclusions: The in vitro-in vivo extrapolation model developed in this study predicted raltegravir PK in virtual individuals with different gastrointestinal pH profiles. The main PK variables were predicted with good accuracy compared with reference data, and both luminal pH and magnesium were able to influence drug absorption. This modelling system provides a tool for investigating the absorption of other drugs, including HIV integrase inhibitors currently in development, which have also shown interactions with food and metal-containing products. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Bell D.J.,University of Liverpool | Bell D.J.,Wellcome Trust Clinical Research Programme | Nyirongo S.K.,Wellcome Trust Clinical Research Programme | Mukaka M.,Wellcome Trust Clinical Research Programme | And 3 more authors.
Clinical Pharmacology and Therapeutics | Year: 2011

In addition to parasite resistance, inadequate levels of exposure to antimalarial drugs may contribute to treatment failure. We developed population pharmacokinetic (PK) models to describe the distribution of sulfadoxine (SDX) and pyrimethamine (PYM) in children with uncomplicated malaria in Malawi. The concentration levels of antimalarial drugs in whole blood were determined using high-performance liquid chromatography. We found no evidence of underdosing in children as compared with adults; the children had drug exposure levels similar to those described in adults. Treatment failure was more likely in children with lower PYM concentrations on day 14 (P = 0.024), and there was a trend for lower SDX concentrations on day 14 (P = 0.061). SDX and PYM concentrations at levels predictive of treatment failure have been identified at day 14. Less than one-third of the children displayed drug concentration levels above these thresholds after receiving the recommended SDX-pyrimethamine (SP) dose. Our findings suggest that PK factors contributed to the observed high rate of treatment failure, and we therefore recommend a higher SP dose for children under the age of 5 years. © 2011 american Society for Clinical Pharmacology and Therapeutics.


Currier R.B.,Alistair Reid Venom Research Unit | Harrison R.A.,Alistair Reid Venom Research Unit | Rowley P.D.,Alistair Reid Venom Research Unit | Laing G.D.,Molecular and Biochemical Parasitology Group | Wagstaff S.C.,Alistair Reid Venom Research Unit
Toxicon | Year: 2010

Bitis arietans is considered one of the most medically significant snakes in Africa, primarily due to a combination of its extensive geographical distribution, common occurrence and highly potent haemorrhagic and cytotoxic venom. Our investigation has revealed a remarkable degree of intra-species variation between pooled venom samples from different geographical origins across sub-Saharan Africa and Arabia, and within a group of individual specimens from the same origin in Nigeria as determined by a combination of immunological, biochemical and proteomic assays. We demonstrate significant quantitative and qualitative differences between B. arietans venom in terms of protein expression, immunogenicity and activity of snake venom metalloproteinases (SVMPs); toxins with a primary role in the haemorrhagic and tissue-necrotic pathologies suffered by envenomed victims. Specifically, we have identified a processed PII SVMP that exhibits striking inter-specimen variability. © 2009 Elsevier Ltd.


PubMed | Molecular and Biochemical Parasitology Group
Type: Journal Article | Journal: The Journal of antimicrobial chemotherapy | Year: 2013

Raltegravir pharmacokinetics (PK) show high intra- and inter-patient variability and are also influenced by co-administered substances that alter the gastrointestinal tract environment, such as pH-altering or metal-containing agents. The aim of this investigation was to develop a population-based absorption, distribution, metabolism and excretion (ADME) model to investigate the effects of gastrointestinal pH and ingested magnesium on raltegravir PK.In vitro data describing the disposition of raltegravir were obtained from literature sources or generated by standard methods. Raltegravir (400 mg single dose) PK were simulated in healthy volunteers (50 subjects per group, 20-50 years old, 0.5 proportion female subjects) over a 12 h period.Simulated raltegravir PK correlated well with data from clinical trials, with a mean deviation in C(max), AUC(0-12) and C(trough) of <20%. Solubility of raltegravir in the gastrointestinal tract was increased at higher luminal pH. Increased intestinal pH and transit time both correlated with higher raltegravir absorption (P<0.001). Magnesium ingestion reduced raltegravir exposure in simulated subjects, with mean C(trough) reduced by 32% (P<0.001).The in vitro-in vivo extrapolation model developed in this study predicted raltegravir PK in virtual individuals with different gastrointestinal pH profiles. The main PK variables were predicted with good accuracy compared with reference data, and both luminal pH and magnesium were able to influence drug absorption. This modelling system provides a tool for investigating the absorption of other drugs, including HIV integrase inhibitors currently in development, which have also shown interactions with food and metal-containing products.

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