M. McConville

Adult triclabendazole-resistant Fasciola hepatica : surface and subsurface tegumental responses to in vitro treatment with the sulphoxide metabolite of the experimental fasciolicide compound alpha

Mature Fasciola hepatica of the triclabendazole-resistant Sligo isolate were incubated in vitro with 10 microg/ml of the sulphoxide metabolite of compound alpha [5-chloro-2-methylthio-6-(1-naphthyloxy)-H-benzimidazole]; the metabolite will be referred to as alpha.SO. Changes resulting from drug treatment were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and tubulin immunocytochemistry (ICC). SEM revealed that disruption to the tegumental surface mainly took the form of swelling and blebbing. Extensive spine loss occurred on the ventral surface of the oral cone, and sloughing of the tegument was observed along the lateral margins of the fluke. Examination of sections from the anterior mid-body region at the TEM level revealed that treatment with alpha.SO led to swelling of the basal infolds and mitochondria within the tegumental syncytium; also, accumulations of secretory bodies beneath the apical plasma membrane. The tegumental cell bodies contained swollen mitochondria and cisternae of granular endoplasmic reticulum, but few Golgi complexes were observed. An increase in T2 secretory bodies was observed, whilst in the T1 tegumental cells, the T1 secretory bodies had decreased in number. Immunocytochemical (ICC) studies showed that incubation with alpha.SO, ABZ.SO and TCBZ.SO did not cause significant changes to the distribution of tubulin within the tegumental syncytium of the Sligo isolate. In contrast, alpha.SO, ABZ.SO and TCBZ.SO caused severe disruption to tubulin organization within the syncytial layer of the TCBZ-susceptible Cullompton isolate. The EM results confirm that compound alpha is a fasciolicide capable of disrupting the tegument of mature TCBZ-resistant F. hepatica; however, this was not accompanied by any change in tubulin immunoreactivity.

An evaluation of the efficacy of compound alpha and triclabendazole against two isolates of Fasciola hepatica.

Seventy indoor-reared sheep were divided into 10 groups to test the efficacy of the experimental fasciolicide, compound alpha (15mg/kg) against triclabendazole (TCBZ)-resistant and TCBZ-susceptible F. hepatica infections. Activity against the Sligo TCBZ-resistant isolate was tested at three time points post-infection (p.i.): 3 days, 4 weeks and 12 weeks (Groups 1-3, respectively). A parallel trial was carried out using TCBZ (10mg/kg) (Groups 5-7): this provided a direct comparison between the efficacies of the two drugs. Group 4 served as an untreated Sligo control. Groups 8 and 9 were setup to test the efficacy of TCBZ and compound alpha against 12-week-old and 4-week-old TCBZ-susceptible, Cullompton infections, respectively. Group 10 served as an untreated Cullompton control. Sheep were sacrificed at 16 weeks p.i. and efficacies were determined. All remaining flukes were collected and measured, before being processed for whole-mount staining to assess the condition of their reproductive structures (testis, vitellaria, ovary and uterus). A second study was carried out to test the activity of compound alpha (15mg/kg) against mature 12-week-old TCBZ-susceptible F. hepatica infections in sheep. Eighteen sheep were divided into two groups, A and B. Group A was treated and Group B served as an untreated control group. Efficacy was determined by reduction in faecal egg counts. The results showed that, whilst compound alpha was very active against adult TCBZ-susceptible flukes, producing a 100% reduction in faecal egg counts, it only caused a 62.5% reduction in fluke burden against juvenile flukes. Moreover, compound alpha was not effective against any stage of infection with TCBZ-resistant F. hepatica in sheep. Data from the trial also revealed biological differences between the two isolates. Thus, Sligo flukes were smaller in size and produced fewer eggs than the Cullompton flukes and their cysts were less infective to sheep. However, they reached the bile ducts more quickly and their eggs appeared in the faeces >2 weeks earlier.

A novel reciprocal and biphasic relationship between membrane cholesterol and β-secretase activity in SH-SY5Y cells and in human platelets

Research into the cause of Alzheimer’s disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters can modulate amyloid precursor protein (APP) processing, thus altering production of the Aβ peptides that deposit in cortical amyloid plaques. Processing depends on the encounter between APP and cellular secretases, and is thus subject to the influence of cholesterol‐dependent factors including protein trafficking, and distribution between membrane subdomains. We have directly investigated endogenous membrane β‐secretase activity in the presence of a range of membrane cholesterol levels in SH‐SY5Y human neuroblastoma cells and human platelets. Membrane cholesterol significantly influenced membrane β‐secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. Platelets from individuals with AD or mild cognitive impairment (n = 172) were significantly more likely to lie within the negative correlation zone than control platelets (n = 171). Pharmacological inhibition of SH‐SY5Y β‐secretase activity resulted in increased membrane cholesterol levels. Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane β‐secretase activity, and suggest that this regulatory mechanism is disrupted in platelets from individuals with cognitive impairment.

Ultrastructural changes to the tegumental system and the gastrodermal cells in adult Fasciola hepatica following in vivo treatment with the experimental fasciolicide, compound alpha

Sheep infected with the triclabendazole-susceptible, Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24, 48 and 72 h post-treatment (p.t.). Ultrastructural changes to the flukes were assessed using transmission electron microscopy (TEM), with a view to gathering information on the mechanism(s) of action for compound alpha and on the possible route of its entry into F. hepatica. The tegumental syncytium was more severely affected than the gut at all time-points p.t. with compound alpha, suggesting a predominantly trans-tegumental route of uptake. Disruption to the tegumental system became increasingly severe over time. A stress response was observed at 24 h p.t. and took the form of blebbing and increases in the production and transport of secretory bodies. By 72 h p.t., extensive tegumental loss and degeneration of the tegumental cell bodies had occurred. Degeneration of subtegumental tissues and internal flooding were also observed. Changes in the gastrodermal cells were slow to develop: reduced secretory activity was evident at 72 h p.t.. There was progressive disruption to the somatic muscle layers, with disorganization of the muscle blocks and loss of muscle fibres.

Platelet Redox Balance in Diabetic Patients With Hypertension Improved by n-3 Fatty Acids

OBJECTIVE Patients with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease, largely as a result of defective production of cardioprotective nitric oxide and a concomitant rise in oxidative stress. Dietary interventions that could reverse this trend would be extremely beneficial. Here we investigated whether dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation positively affected platelet nitroso-redox imbalance. RESEARCH DESIGN AND METHODS We randomized hypertensive T2DM patients (T2DM HT; n = 22) and age-and-sex matched hypertensive study participants without diabetes (HT alone; n = 23) in a double-blind, crossover fashion to receive 8 weeks of n-3 PUFAs (1.8 g eicosapentaenoic acid and 1.5 g docosahexaenoic acid) or identical olive oil capsules (placebo), with an intervening 8-week washout period. Platelet nitrite and superoxide were measured and compared before and after treatment; 8-isoprostane was determined by ELISA and subcellular compartmentalization of the NAD(P)H oxidase subunit p47-phox examined by Western blotting. RESULTS The n-3 PUFA supplementation reduced 8-isoprostane and superoxide levels in platelets from T2DM HT, but not HT alone, participants, without effect on nitrite production. This coincided with a significant decrease in p47-phox membrane localization and a similar reduction in superoxide to that achieved with apocynin. At baseline, a subcohort of T2DM HT and HT alone participants showed evidence of nitric oxide synthase (NOS)–derived superoxide production, indicating defective enzymatic activity. This was reversed significantly in T2DM HT participants after treatment, demonstrating improved NOS function. CONCLUSIONS Our finding that n-3 PUFAs diminish platelet superoxide production in T2DM HT patients in vivo suggests a therapeutic role for these agents in reducing the vascular-derived oxidative stress associated with diabetes.

P3-333: A novel reciprocal and biphasic relationship between membrane cholesterol and β-secretase activity in SH-SY5Y cells and human platelets

altered Notch processing. Therefore, it is essential to drug discovery efforts to have a high-throughput platform to quantitatively measure Notch cleavage by gamma-secretase. Methods: Here we present a novel in vitro ELISA, employing a biotin flag antibody, a cleaved Notch (V1744) specific antibody and a N100FLAG recombinant protein, to measure the generation of the Notch intracellular domain (NICD). Results: This assay compliments the in vitro APP C100FLAG abeta assay by allowing a direct comparison of compound efficacy on gamma secretase activity against the S3 Notch cleavage site compared to the -cleavage sites of APP. Conclusions: Together, these assays will aid in identifying a Notch-sparing mechanism of inhibiting gamma-secretase activity.