Blood-clotting response tests for resistance to diphacinone and chlorophacinone in the Norway rat (Rattus norvegicus Berk.)

Resistance baselines were obtained for the first generation anticoagulant rodenticides chlorophacinone and diphacinone using laboratory, caesarian-derived Norway rats (Rattus norvegicus) as the susceptible strain and the blood clotting response test method. The ED99 estimates for a quantal response were: chlorophacinone, males 0.86 mg kg−1, females 1.03 mg kg−1; diphacinone, males 1.26 mg kg−1, females 1.60 mg kg−1. The dose-response data also showed that chlorophacinone was significantly (p<0.0001) more potent than diphacinone for both male and female rats, and that male rats were more susceptible than females to both compounds (p<0.002). The ED99 doses were then given to groups of five male and five female rats of the Welsh and Hampshire warfarin-resistant strains. Twenty-four hours later, prothrombin times were slightly elevated in both strains but all the animals were classified as resistant to the two compounds, indicating cross-resistance from warfarin to diphacinone and chlorophacinone. When rats of the two resistant strains were fed for six consecutive days on baits containing either diphacinone or chlorophacinone, many animals survived, indicating that their resistance might enable them to survive treatments with these compounds in the field.

A reappraisal of blood clotting response tests for anticoagulant resistance and a proposal for a standardised BCR test methodology

Blood clotting response (BCR) resistance tests are available for a number of anticoagulant rodenticides. However, during the development of these tests many of the test parameters have been changed, making meaningful comparisons between results difficult. It was recognised that a standard methodology was urgently required for future BCR resistance tests and, accordingly, this document presents a reappraisal of published tests, and proposes a standard protocol for future use (see Appendix). The protocol can be used to provide information on the incidence and degree of resistance in a particular rodent population; to provide a simple comparison of resistance factors between active ingredients, thus giving clear information about cross-resistance for any given strain; and to provide comparisons of susceptibility or resistance between different populations. The methodology has a sound statistical basis in being based on the ED50 response, and requires many fewer animals than the resistance tests in current use. Most importantly, tests can be used to give a clear indication of the likely practical impact of the resistance on field efficacy. The present study was commissioned and funded by the Rodenticide Resistance Action Committee (RRAC) of CropLife International.

The effect of ram replacement and sex ratio on the sexual response of anoestrous ewes

It is accepted that an important source of variation in the response of anoestrous ewes, to the introduction of rams, is the intensity of male stimulation. The aim of this study was to investigate strategies capable of increasing the impact and transmission of the ram stimuli. In Experiment 1, two groups of seven ewes (Bluefaced Leicester male x Swaledale female) were individually penned with one ram and for the next 6 h the rams either remained in the pen or were replaced hourly. Blood samples revealed no difference in the pattern of plasma LH secretion. In Experiment 2, three groups of 16 ewes were either introduced to one ram, individually (H) or in groups of 8 (L), or remained isolated. Ram introduction increased the plasma LH pulsatility (P < 0.001). H ewes displayed more (nine versus six) male-induced LH pulses (pulses occurring within the first 45 min) and more pulses per 8 h intervals than the L group of ewes (1.9 +/- 0.3 versus 1.3 +/- 0.3), but these differences were not significant. It was concluded that (i) frequent replacement of rams within a few hours following ram introduction to ewes does not further improve the response of ewes, especially if the ram:ewe ratio is high; (ii) the characterization of the plasma LH secretion parameters during a period of 6-8 h does not seem to be an effective method to detect small differences in the intensity of stimulation received by the ewes when exposed to rams; (iii) North Country Mule ewes (Bluefaced Leicester male x Swaledale female) in the UK respond to the presence of rams in spring (late oestrous/early anoestrous season) with an elevation in plasma LH secretion. (c) 2005 Elsevier B.V. All rights reserved.

Transcriptional profiling of the LPS induced NF-kappaB response in macrophages

BACKGROUND: Exposure of macrophages to bacterial products such as lipopolysaccharide (LPS) results in activation of the NF-kappaB transcription factor, which orchestrates a gene expression programme that underpins the macrophage-dependent immune response. These changes include the induction or repression of a wide range of genes that regulate inflammation, cell proliferation, migration and cell survival. This process is tightly regulated and loss of control is associated with conditions such as septic shock, inflammatory diseases and cancer. To study this response, it is important to have in vitro model systems that reflect the behaviour of cells in vivo. In addition, it is necessary to understand the natural differences that can occur between individuals. In this report, we have investigated and compared the LPS response in macrophage derived cell lines and peripheral blood mononuclear cell (PBMC) derived macrophages. RESULTS: Gene expression profiles were determined following LPS treatment of THP-1 cells for 1 and 4 hours. LPS significantly induced or repressed 72 out of 465 genes selected as being known or putative NF-kappaB target genes, which exhibited 4 temporal patterns of expression. Results for 34 of these genes, including several genes not previously identified as LPS target genes, were validated using real time PCR. A high correlation between microarray and real time PCR data was found. Significantly, the LPS induced expression profile of THP-1 cells, as determined using real time PCR, was found to be very similar to that of human PBMC derived macrophages. Interestingly, some differences were observed in the LPS response between the two donor PBMC macrophage populations. Surprisingly, we found that the LPS response in U937 cells was dramatically different to both THP-1 and PBMC derived macrophages. CONCLUSION: This study revealed a dynamic and diverse transcriptional response to LPS in macrophages, involving both the induction and repression of gene expression in a time dependent manner. Moreover, we demonstrated that the LPS induced transcriptional response in the THP-1 cell line is very similar to primary PBMC derived macrophages. Therefore, THP-1 cells represent a good model system for studying the mechanisms of LPS and NF-kappaB dependent gene expression.

Rate-limiting steps in the development of atherosclerosis: the response-to-influx theory

A large number of processes are involved in the pathogenesis of atherosclerosis but it is unclear which of them play a rate-limiting role. One way of resolving this problem is to investigate the highly non-uniform distribution of disease within the arterial system; critical steps in lesion development should be revealed by identifying arterial properties that differ between susceptible and protected sites. Although the localisation of atherosclerotic lesions has been investigated intensively over much of the 20th century, this review argues that the factor determining the distribution of human disease has only recently been identified. Recognition that the distribution changes with age has, for the first time, allowed it to be explained by variation in transport properties of the arterial wall; hitherto, this view could only be applied to experimental atherosclerosis in animals. The newly discovered transport variations which appear to play a critical role in the development of adult disease have underlying mechanisms that differ from those elucidated for the transport variations relevant to experimental atherosclerosis: they depend on endogenous NO synthesis and on blood flow. Manipulation of transport properties might have therapeutic potential. Copyright (C) 2004 S. Karger AG, Basel.

The reproductive response of rams to thyroidectomy: mediation by impaired inhibin feedback rather than a change in LH pulsatility

A series of experiments was conducted to examine the mechanism by which removal of the thyroid glands in seasonally suppressed rams brings about rapid testicular growth. In the first experiment, thyroidectomy at the nadir of the testicular cycle (late winter) initiated testis growth without any detectable change in the extent of spermatogenesis compared with sham-operated controls. The serum concentration of FSH, but not LH, was also markedly increased by thyroidectomy. In the second experiment, serum FSH concentration was again increased by thyroidectomy in late winter but there was no effect of thyroidectomy on LH concentration, LH pulses (measured in frequent blood samples) or testosterone concentration. Furthermore, there was no evidence of a change in central dopaminergic inhibition of GnRH, as measured by the pulsatile LH response to an i.m. injection of the dopaminergic D-2 agonist bromocriptine or antagonist sulpiride. The rapid increase in FSH concentration occurred despite a markedly increased serum inhibin A concentration in thyroidectomized rams. Therefore, the efficacy of inhibin feedback was examined by testing the FSH-suppressive effect of an inhibin preparation (5 ml charcoal-stripped bovine follicular fluid i.v.) in long-term thyroidectomized and thyroid intact castrated rams. Bovine follicular fluid suppressed FSH concentrations in control rams as expected but in marked contrast, was completely without effect in thyroidectomized animals. In castrated rams, the FSH concentration was only marginally increased by thyroidectomy, indicating that there is a major component of the mediation of the effects of thyroidectomy that is testicular in origin. It was concluded that a reduction in the ability of endogenous inhibin to inhibit FSH release at the pituitary, rather than a hypothalamic mechanism, is the primary cause of the stimulation of testis growth by thyroidectomy.

Post-haemorrhagic anaemia in sea bass, Dicentrarchus labrax (L.), caused by blood feeding of Ceratothoa oestroides (Isopoda: Cymothoidae)

The effects of the fish parasitic isopod, Ceratothoa oestroides (Risso), on haematological parameters of its cage-cultured sea bass host, Dicentrarchus labrax (L.), were studied. Analyses of blood parameters (cell counts, haemoglobin content and haematocrit) were carried out on parasitized and unparasitized sea bass from a fish farm in Turkey. Parasitized fish had significantly lowered erythrocyte counts, haematocrit and haemoglobin values and significantly increased leucocyte counts. Blood feeding by C. oestroides thus produces a post-haemorrhagic anaemia and the fish appear to mount an immune response to the presence of parasites.

Relation between the fatty acid composition of peripheral blood mononuclear cells and measures of immune cell function in health free-living subjects aged 25-72 y

Background: There is little information about the relation between the fatty acid composition of human immune cells and the function of those cells over the habitual range of fatty acid intakes. Objective: The objective of the study was to determine the relation between the fatty acid composition of human peripheral blood mononuclear cell (PBMC) phospholipids and the functions of human immune cells. Design: One hundred fifty healthy adult subjects provided a fasting blood sample. The phagocytic and oxidative burst activities of monocytes and neutrophils were measured in whole blood. PBMCs were isolated and used to measure lymphocyte proliferation in response to the T cell mitogen concanavalin A and the production of cytokines in response to concanavalin A or bacterial lipopolysaccharide. The fatty acid composition of plasma and PBMC phospholipids was determined. Results: Wide variations in fatty acid composition of PBMC phospholipids and immune cell functions were identified among the subjects. The proportions of total Polyunsaturated fatty acids (PUFAs), of total n-6 and n-3 PUFAs, and of several individual PUFAs in PBMC phospholipids were positively correlated with phagocytosis by neutrophils and monocytes, neutrophil oxidative burst, lymphocyte proliferation, and interferon gamma production. The ratios of saturated fatty acids to PUFAs and of n-6 to n-3 PUFAs were negatively correlated with these same immune functions. The relation of PBMC fatty acid composition to monocyte oxidative burst was the reverse of its relation to monocyte phagocytosis and neutrophil oxidative burst. Conclusion: Variations in the fatty acid composition of PBMC phospholipids account for some of the variability in immune cell functions among healthy adults.

The impact of age on the postprandial vascular response to a fish oil-enriched meal

Although chronic fish oil intervention had been shown to have a positive impact on vascular reactivity, very little is known about their acute effects during the postprandial phase. Our aim was to examine the impact of a fish oil-enriched test meal on postprandial vascular reactivity in healthy younger ( < 50 years) v. older ( ≥ 50 years) men. Vascular reactivity was measured at baseline (0 h), 2 and 4 h after the meal by laser Doppler iontophoresis and blood samples taken at 0 and 4 h for the measurement of plasma lipids, total nitrite, glucose and insulin. Acetylcholine- (ACh, endothelial-dependent vasodilator) and sodium nitroprusside (SNP, endothelial-independent vasodilator)-induced reactivities were greater at 4 h than at baseline or 2 h in the younger men (P < 0·04). These changes were not observed in the older men. Comparison of the male groups revealed significantly greater responses to ACh (P = 0·006) and SNP (P = 0·05) at 4 h in the younger compared with the older males. Postprandial NEFA concentrations were also greater at 4 h in the younger compared with the older men (P = 0·005), with no differences observed for any of the other analytes. Multiple regression analysis revealed age to be the most significant predictor of both ACh and SNP induced reactivity 4 h after the meal. In conclusion, the ingestion of a meal enriched in fish oil fatty acids was shown to improve postprandial vascular reactivity at 4 h in our younger men, with little benefit evident in our older men.

Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.

Watercress supplementation in diet reduces lymphocyte DNA damage and alters blood antioxidant status in healthy adults

Background: Cruciferous vegetable (CV) consumption is associated with a reduced risk of several cancers in epidemiologic studies. Objective: The aim of this study was to determine the effects of watercress (a CV) supplementation on biomarkers related to cancer risk in healthy adults. Design: A single-blind, randomized, crossover study was conducted in 30 men and 30 women (30 smokers and 30 nonsmokers) with a mean age of 33 y (range: 19-55 y). The subjects were fed 85 g raw watercress daily for 8 wk in addition to their habitual diet. The effect of supplementation was measured on a range of endpoints, including DNA damage in lymphocytes (with the comet assay), activity of detoxifying enzymes (glutathione peroxidase and superoxide dismutase) in erythrocytes, plasma antioxidants (retinol, ascorbic acid, a-tocopherol, lutein, and beta-carotene), plasma total antioxidant status with the use of the ferric reducing ability of plasma assay, and plasma lipid profile. Results: Watercress supplementation (active compared with control phase) was associated with reductions in basal DNA damage (by 17%; P = 0.03), in basal plus oxidative purine DNA damage (by 23.9%; P = 0.002), and in basal DNA damage in response to ex vivo hydrogen peroxide challenge (by 9.4%; P = 0.07). Beneficial changes seen after watercress intervention were greater and more significant in smokers than in nonsmokers. Plasma lutein and P-carotene increased significantly by 100% and 33% (P < 0.001), respectively, after watercress supplementation. Conclusion: The results support the theory that consumption of watercress can be linked to a reduced risk of cancer via decreased damage to DNA and possible modulation of antioxidant status by increasing carotenoid concentrations.

Effects of chronic and acute consumption of fruit- and vegetable-puree-based drinks on vasodilation, risk factors for CVD and the response as a result of the eNOS G298T polymorphism

The average UK adult consumes less than three portions of fruit and vegetables daily, despite evidence to suggest that consuming five portions daily could help prevent chronic diseases. It is recommended that fruit juice should only count as one of these portions, as juicing removes fibre and releases sugars. However, fruit juices contain beneficial compounds such as vitamin C and flavonoids and could be a useful source of dietary phytochemicals. Two randomised controlled cross-over intervention studies investigating the effects of chronic and acute consumption of commercially-available fruit- and vegetable-puree-based drinks (FVPD) on bioavailability, antioxidant status and CVD risk factors are described. Blood and urine samples were collected during both studies and vascular tone was measured using laser Doppler imaging. In the chronic intervention study FVPD consumption was found to significantly increase dietary carotenoids (P = 0.001) and vitamin C (P = 0.003). Plasma carotenoids were increased (P = 0.001), but the increase in plasma vitamin C was not significant. There were no significant effects on oxidative stress, antioxidant status and other CVD risk factors. In the acute intervention study FVPD were found to increase total plasma nitrate and nitrite (P = 0.001) and plasma vitamin C (P = 0.002). There was no effect on plasma lipids or uric acid, but there was a lower glucose and insulin peak concentration after consumption of the FVPD compared with the sugar-matched control. There was a trend towards increased vasodilation following both chronic and acute FVPD consumption. All volunteers were retrospectively genotyped for the eNOS G298T polymorphism and the effect of genotype on the measurements is discussed. Overall, there was a non-significant trend towards increased endothelium-dependent vasodilation following both acute and chronic FVPD consumption. However, there was a significant time x treatment effect (P < 0.05) of acute FVPD consumption in individuals with the GG variant of the eNOS gene.

Proteomic biomarkers of peripheral blood mononuclear cells obtained from postmenopausal women undergoing an intervention with soy isoflavones

Background: The incidence of cardiovascular diseases increases after menopause, and soy consumption is suggested to inhibit disease development. Objective: The objective was to identify biomarkers of response to a dietary supplementation with an isoflavone extract in postmenopausal women by proteome analysis of peripheral blood mononuclear cells. Design: The study with healthy postmenopausal woman was performed in a placebo-controlled sequential design. Peripheral mononuclear blood cells were collected from 10 volunteers after 8 wk of receiving daily 2 placebo cereal bars and after a subsequent 8 wk of intervention with 2 cereal bars each providing 25 mg of isoflavones. The proteome of the cells was visualized after 2-dimensional gel electrophoresis, and peptide mass fingerprinting served to identify proteins that by the intervention displayed altered protein concentrations. Results: Twenty-nine proteins were identified that showed significantly altered expression in the mononuclear blood cells under the soy-isoflavone intervention, including a variety of proteins involved in an antiinflammatory response. Heat shock protein 70 or a lymphocyte-specific protein phosphatase and proteins that promote increased fibrinolysis, such as a-enolase, were found at increased intensities, whereas those that mediate adhesion, migration, and proliferation of vascular smooth muscle cells, such as galectin-1, were found at reduced intensities after soy extract consumption. Conclusion: Protcome analysis identified in vivo markers that respond to a dietary intervention with isoflavone-enriched soy extract in postmenopausal women. The nature of the proteins identified suggests that soy isoflavones may increase the anti inflammatory response in blood mononuclear cells that might contribute to the atherosclerosis-preventive activities of a soy-rich diet.

Effect of apoE genotype and dietary quercetin on blood lipids and TNF-alpha levels in apoE3 and apoE4 targeted gene replacement mice

The aim of this study was to determine the effect of dietary quercetin supplementation on blood lipids and TNF-alpha levels according to the apoE genotype in apoE3 and apoE4 targeted gene replacement mice. In a two-factorial design female apoE3 and apoE4 mice were fed semi-synthetic diets without (controls) and with quercetin (2 mg/g diet) for 6 weeks. Feeding the quercetin-supplemented diets significantly increased plasma levels of quercetin and isorhamnetin both in apoE3 and apoE4 mice. There was no significant effect of apoE genotype on plasma quercetin levels. ApoE3 and apoE4 transgenic mice exhibited similar plasma levels of apoE and cholesterol which were not significantly affected by dietary quercetin supplementation. In mice receiving the basal diet without quercetin supplementation, levels of TNF-alpha in whole blood stimulated ex vivo with lipopolysaccharide were higher in apoE3 as compared to apoE4 transgenic mice. Dietary quercetin significantly lowered levels of TNF-alpha by 44% in apoE3 mice relative to apoE3 mice receiving the unsupplemented diets. In apoE4 mice a moderate (20%) but not significant decrease in TNF-alpha levels in response to the quercetin supplementation was evident. Following quercetin supplementation TNF-alpha levels were similar between apoE3 and apoE4 transgenic mice. Current findings indicate that apoE3 mice are more responsive to the TNF-alpha lowering properties of dietary quercetin supplementation as compared to apoE4 animals.

The impact of EPA and DHA on blood lipids and lipoprotein metabolism: influence of apoE genotype

Fish and fish oil-rich sources of long-chain n-3 fatty acids have been shown to be cardio-protective, through a multitude of different pathways including effects on arrythymias, endothelial function, inflammation and thrombosis, as well as modulation of both the fasting and postprandial blood lipid profile. To date the majority of studies have examined the impact of EPA and DHA fed simultaneously as fish or fish oil supplements. However, a number of recent studies have compared the relative biopotency of EPA v. DHA in relation to their effect on blood lipid levels. Although many beneficial effects of fish oils have been demonstrated, concern exists about the potential deleterious impact of EPA and DHA on LDL-cholesterol, with a highly-heterogenous response of this lipid fraction reported in the literature. Recent evidence suggests that apoE genotype may be in part responsible. In the present review the impact of EPA and DHA on cardiovascular risk and the blood lipoprotein profile will be considered, with a focus on the apoE gene locus as a possible determinant of lipid responsiveness to fish oil intervention.