Anthelmintic drugs and nematicides: studies in Caenorhabditis elegans.

Parasitic nematodes infect many species of animals throughout the phyla, including humans. Moreover, nematodes that parasitise plants are a global problem for agriculture. As such, these nematodes place a major burden on human health, on livestock production, on the welfare of companion animals and on crop production. In the 21st century there are two major challenges posed by the wide-spread prevalence of parasitic nematodes. First, many anthelmintic drugs are losing their effectiveness because nematode strains with resistance are emerging. Second, serious concerns regarding the environmental impact of the nematicides used for crop protection have prompted legislation to remove them from use, leaving agriculture at increased risk from nematode pests. There is clearly a need for a concerted effort to address these challenges. Over the last few decades the free-living nematode Caenorhabditis elegans has provided the opportunity to use molecular genetic techniques for mode of action studies for anthelmintics and nematicides. These approaches continue to be of considerable value. Less fruitful so far, but nonetheless potentially very useful, has been the direct use of C. elegans for anthelmintic and nematicide discovery programmes. Here we provide an introduction to the use of C. elegans as a 'model' parasitic nematode, briefly review the study of nematode control using C. elegans and highlight approaches that have been of particular value with a view to facilitating wider-use of C. elegans as a platform for anthelmintic and nematicide discovery and development.

Pioglitazone normalizes insulin signaling in the diabetic rat retina through reduction in tumor necrosis factor α and suppressor of cytokine signaling 3.

Dysfunctional insulin signaling is a key component of type 2 diabetes. Little is understood of the effects of systemic diabetes on retinal insulin signaling. A number of agents are used to treat patients with type 2 diabetes to normalize glucose levels and improve insulin signaling; however, little has been done to investigate the effects of these agents on retinal insulin signal transduction. We hypothesized that pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, would normalize retinal insulin signal transduction through reduced tumor necrosis factor α (TNFα) and suppressor of cytokine signaling 3 (SOCS3) activities in whole retina and retinal endothelial cells (REC) and Müller cells. To test this hypothesis, we used the BBZDR/Wor type 2 diabetic rat model, as well as REC and Müller cells cultured in normoglycemia and hyperglycemic conditions, to investigate the effects of pioglitazone on TNFα, SOCS3, and downstream insulin signal transduction proteins. We also evaluated pioglitazone's effects on retinal function using electroretinogram and markers of apoptosis. Data demonstrate that 2 months of pioglitazone significantly increased electroretinogram amplitudes in type 2 diabetic obese rats, which was associated with improved insulin receptor activation. These changes occurred in both REC and Müller cells treated with pioglitazone, suggesting that these two cell types are key to insulin resistance in the retina. Taken together, these data provide evidence of impaired insulin signaling in type 2 diabetes rats, which was improved by increasing PPARγ activity. Further investigations of PPARγ actions in the retina may provide improved treatment options.

Red blood cell survival in long-term dialysis patients.

BACKGROUND: Shortening of red blood cell (RBC) survival contributes to the anemia of chronic kidney disease. The toxic uremic environment accounts for the decreased RBC life span. The contribution of mechanical damage caused by hemodialysis to the shortened life span is unclear. Reductions up to 70% in RBC survival have been reported in uremic patients. To date, no accurate well-controlled RBC survival data exist in dialysis patients treated using different dialysis modalities and receiving erythropoiesis-stimulating agent (ESA) therapy. The aim of this study was to determine RBC survival in hemodialysis (HD) and peritoneal dialysis (PD) patients compared with healthy persons. STUDY DESIGN: Observational study. SETTING & PARTICIPANTS: 14 HD patients and 5 PD patients were recruited from the dialysis unit. Healthy volunteers (n = 14) age- and sex-matched to HD participants were included. All dialysis patients received either ESA therapy or regular iron supplementation. PREDICTOR: Dialysis patients versus age- and sex-matched healthy controls. OUTCOMES: RBC survival. MEASUREMENTS: RBC survival was determined using radioactive chromium labeling. RESULTS: More than 85% of dialysis patients were anemic (hemoglobin, 12.0 ± 1.1 g/dL); hemoglobin concentrations were not significantly different between HD and PD patients. Median RBC survival was significantly decreased by 20% in HD patients compared with healthy controls: 58.1 (25th-75th percentile, 54.6-71.2) versus 72.9 (25th-75th percentile, 63.4-87.8) days (P = 0.02). No difference was shown between the PD and HD groups: 55.3 (25th-75th percentile, 49.0-60.2) versus 58.1 (25th-75th percentile, 54.6-71.2) days (P = 0.2). LIMITATIONS: Label loss from RBCs associated with the chromium 51 labeling technique needs to be accounted for in the interpretation of RBC survival data. CONCLUSIONS: Despite current ESA therapy, decreased RBC survival contributes to chronic kidney disease-related anemia, although the reduction is less than previously reported. There does not appear to be net mechanical damage associated with HD therapy resulting in decreased RBC life span.

Thiols stabilize cobblestone morphology of cultured mesothelial cells.

Cellular thiols including GSH (glutathione) and L-Cys (L-cysteine) are essential for cell signalling, growth and differentiation. L-Cys is derived from the extracellular thiol pool and is the rate-limiting compound for intracellular GSH biosynthesis. The present study investigated the effect of thiol-supplemented medium on cell growth, phenotype and total GSH of cultured hPMCs (human peritoneal mesothelial cells). Cells were cultured in medium M199 supplemented with 2% serum, with 'plus' or without 'minus' L-Cys and compared with medium supplemented with either ß-ME (ß-mercaptoethanol) (0.25 mmol/l) or the receptor tyrosine kinase ligand EGF (epidermal growth factor, 100 ng/ml). ß-ME produced a disproportionate increase in total GSH compared with L-Cys and other thiols tested [(procysteine (2-oxothiazolidine-4-carboxylic acid) or NAC (N-acetyl-L-cysteine)], while growth and morphology were identical. Cell behaviour of primary hPMCs is characterized by the transition of fibroblastoid to cobblestone morphology during early passage. L-Cys and ß-ME promoted a rapid MET (mesenchymal-to-epithelial transition) within 3 days of culture, confirmed by the presence of cobblestone cells, intact organelles, abundant microvilli, primary cilia and cortical actin. In contrast, EGF produced a biphasic response consisting of delayed growth and retention of a fibroblastoid morphology. During a rapid log phase of growth, MET was accompanied by rapid catch-up growth. Thiols may stabilize the epithelial phenotype by engaging redox-sensitive receptors and transcription factors that modulate differentiation. These data may benefit researchers working on thiol-mediated cell differentiation and strategies to regenerate damage to serosal membranes.

Postprandial cytokine concentrations and meal composition in obese and lean women.

The aim of this study was to compare the acute effect of (i) meals rich in saturated fat, oleic acid, and alpha-linolenic acid and (ii) meals rich in starch and fiber on markers of inflammation and oxidative stress in obese and lean women. In a crossover study, 15 abdominally obese women (age, 54 +/- 9 years; BMI, 37.3 +/- 5.5 kg/m2) and 14 lean women (age, 53 +/- 10 years; BMI, 22.9 +/- 1.9 kg/m2) consumed meals rich in cream (CR), olive oil (OL), canola oil (CAN), potato (POT), and All-Bran (BRAN) in random order. Blood samples were collected before and up to 6 h after the meals and plasma interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha (TNF-alpha), lipid peroxides (LPOs), free-fatty acids (FFAs), insulin, glucose, and cortisol were measured. Plasma IL-6 decreased significantly 1 h after the meals then increased significantly above baseline at 4h and 6h in obese women and at 6h in lean women. The incremental area under the curve (iAUC) for IL-6 was significantly (P = 0.02) higher in obese compared with lean women and was significantly lower following the high fiber BRAN meal compared with a POT meal (P = 0.003). Waist circumference (R = 0.491, P = 0.007) and cortisol AUC (R = -0.415, P = 0.03) were significant determinants of the magnitude of 6h changes in plasma IL-6 after the meals. These findings suggest that the postprandial response of plasma IL-6 concentrations may be influenced by the type of carbohydrate in the meal, central adiposity, and circulating cortisol concentrations in women.

Vitamin E supplementation and plasma 8-isoprostane and adiponectin in overweight subjects.

OBJECTIVE: Isoprostanes are a marker of oxidant stress and atherosclerotic risk, and plasma concentrations are elevated in obesity. Adiponectin is a regulator of insulin sensitivity, and low circulating levels are associated with oxidant stress and obesity. The aim of this study was to determine the effect of vitamin E supplementation on plasma concentrations of 8-isoprostane and adiponectin in overweight/obese subjects. RESEARCH METHODS AND PROCEDURES: The study was a 6-month, randomized, double-blind, placebo-controlled trial in 80 overweight subjects (60 women and 20 men, BMI >27 kg/m(2)). Exclusion criteria were serious illness, smoking, or taking antioxidant supplements. Participants were randomized to receive 800 IU/d natural vitamin E (n = 39) or placebo (n = 41) for 3 months with an increase in the dose to 1200 IU/d for a further 3 months. Plasma 8-isoprostane and adiponectin concentrations were measured at baseline and 3 and 6 months. RESULTS: During 6 months of supplementation with vitamin E, plasma vitamin E concentration increased significantly (p < 0.001) by 76%, and plasma 8-isoprostane concentrations decreased significantly (-11%, p = 0.03), whereas plasma adiponectin concentrations did not change significantly. DISCUSSION: These findings suggest that supplementation with high-dose vitamin E decreases systemic oxidative stress and 8-isoprostane concentrations in overweight/obese individuals. A decrease in plasma 8-isoprostane has the potential to reduce risk of cardiovascular disease in obesity.

Effect of high-dose vitamin E on insulin resistance and associated parameters in overweight subjects.

OBJECTIVE: Markers of oxidative stress and plasma alanine transferase (ALT) levels are increased and circulating antioxidant concentrations are reduced in individuals with insulin resistance. Vitamin E improves glycemic control in people with diabetes. We tested the hypothesis that vitamin E would decrease markers of oxidative stress and plasma ALT levels and improve insulin sensitivity in overweight individuals. RESEARCH DESIGN AND METHODS: Eighty overweight individuals (BMI >27 kg/m(2)) were randomly allocated to receive either 800 IU vitamin E per day or a matching placebo for 3 months. The dose of vitamin E was increased to 1,200 IU per day for a further 3 months. RESULTS: Plasma peroxides decreased by 27% at 3 months and by 29% at 6 months in the group that received vitamin E and were positively correlated with plasma vitamin E concentrations at the 6-month time point. At 3 months, fasting plasma glucose and insulin concentrations were significantly reduced and homeostasis model assessment increased. These changes were not apparent at 6 months. Plasma ALT concentrations declined significantly throughout the study period. CONCLUSIONS: In conclusion, these findings indicate that vitamin E improves oxidative stress and hepatocellular function. Although insulin resistance also improves, this effect appears transient.

Formaldehyde scavenging from peritoneal dialysis solutions using reduced aminothiol compounds.

BACKGROUND: Aldehydes were identified in clinical solutions, including peritoneal dialysis (PD) and cryoprotection solutions, which were used to freeze cells, tissues and embryos. Aldehydes are associated with increased cellular injury and may contribute to peritoneal membrane damage that occurs in patients on peritoneal dialysis. Recently, it was demonstrated that aldehydes could be 'scavenged' from these solutions by using aminothiol compounds. Although aldehydes were removed during the scavenging process, the kinetics of scavenging and the products formed were not characterized. METHODS: Proton nuclear magnetic resonance (NMR) spectroscopy was used to investigate formaldehyde scavenging from an artificial PD solution supplemented with aminothiol compounds, cysteamine or l-cysteine. Artificial PD solutions were formulated on the basis of commercial PD solutions and consisted of 132 mmol/L NaCl, 0.25 mmol/L MgCl2, 1.25 mmol/L CaCl2, and buffered with lactate (4.0 mmol/L) and lacked d-glucose. Formaldehyde scavenging was a two-step process involving an intermediate step followed by the formation of stable thiazolidine compounds. These included the derivatives of cysteamine and l-cysteine; thiazolidine and thiazolidine-4-carboxylic acid, respectively. CONCLUSION: Scavenging with aminothiol compounds masked the destructive carbonyl group (C = O) of formaldehyde and formed a compound that has antioxidant properties. The addition of aminothiol compounds may improve the biocompatibility of commercial PD solutions.

Cultured peritoneal mesothelial cells exhibit apical primary cilia.

This study examined primary cilia on cultured human and rabbit peritoneal mesothelial cells (PMC) and investigated factors that influence ciliary expression. Primary cilia were examined with indirect immunocytochemistry, laser scanning confocal microscopy and scanning electron microscopy. Ciliary expression was evaluated in cultures with or without l-cysteine (0.25 mM) or exposure to Ca(2+)-free Krebs-Ringer solution supplemented with EGTA, 0.5 mM. This treatment disrupted cell monolayer integrity. Cilia were counted and normalized to total cell counts using NIH image. Primary cilia were identified on both human and rabbit PMC. Cells treated with l-cysteine expressed significantly more cilia compared with monolayers deprived of l-cysteine. Exposure to Ca(2+)-free Krebs-Ringer solution significantly reduced cilia (5.9+/-1.0%, n=7). Although ciliary expression could be augmented with l-cysteine, approximately 60% of human PMC and 84% of rabbit PMC did not exhibit cilia. Together, these data show that monolayers of PMC express apical cilia that can be augmented with l-cysteine, independently of increased cell density.

Release of cholesterol from cell membranes to postprandial plasma from mildly hypercholesterolemic subjects: the effect of meals rich in olive and safflower oils.

Triglyceride-rich lipoproteins increase net transport of cell cholesterol to postprandial plasma from healthy subjects after a meal rich in fat and cholesterol. The aim of the present study was to determine the effect of meals rich in polyunsaturated fats (PUFA) and monounsaturated fats (MUFA) and low in cholesterol on net in vitro transport of cholesterol from red blood cells (RBCs) to postprandial plasma from 21 men with mild to moderate hypercholesterolemia in a randomized, crossover trial. Cholesterol concentration increased by 12% due to accumulation of cell cholesterol in fasted hypercholesterolemic plasma incubated with a 2/1 (vol/vol) excess of RBCs at 37 degrees C for 18 hours. The increase in cell cholesterol in plasma was mainly localized in the low-density lipoprotein (LDL) fraction (64%) and the remainder was approximately equally divided between the very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) fractions. Accumulation of cell cholesterol in the LDL fraction prevented the significant decrease in LDL cholesterol in plasma incubated alone. When RBCs were incubated with postprandial plasma isolated 4 hours and 6 hours after liquid meals rich in safflower and olive oils, the accumulation of cell cholesterol in plasma increased significantly (11%, P <.004) above values for fasted plasma and irrespective of the type of fat in the meal. Also, the content of cell cholesterol increased significantly (70%, P <.001) in triglyceride (TG)-rich lipoproteins and decreased significantly (P =.006) in the LDL fraction, which remained the main ultimate destination of cell cholesterol in postprandial plasma. The increased loss of cell cholesterol to fasted and postprandial plasma was closely correlated (r > 0.823, P <.001) with the concomitant increase in plasma cholesteryl esters (CE) generated by lecithin cholesterol acyltransferase (LCAT) activity. There was a small (5%), significant (P <.001) increase in plasma cholesterol esterification in postprandial plasma. These data suggest that high-fat meals rich in MUFA and PUFA and low in cholesterol may produce a small postprandial increase in the capacity of plasma to accept cell membrane cholesterol that is limited by a concomitant small increase in plasma cholesterol esterification, in hypercholesterolemic subjects. Thus, low-fat, lipid-lowering diets may have a minimal effect on this capacity but will reduce levels of atherogenic LDL cholesterol that appear to be maintained by diffusion of cell cholesterol to plasma.

Effect of meals rich in heated olive and safflower oils on oxidation of postprandial serum in healthy men.

The present randomised, crossover study sought to determine the effect of meals rich in safflower oil and olive oil (60 g) which had been heated for 8 h at 210 degrees C and the corresponding unheated oils on copper ion oxidation of dilute serum from 16 healthy men. Four hours after the meals rich in the heated oils, there were significant decreases of similar magnitude (-12%) in the lag time in conjugated diene formation during diluted serum oxidation. In the 12 subjects who consumed meals containing unheated oils, the lag time also decreased (-11%) significantly after the meal rich in unheated safflower oil (US) and did not change significantly after the unheated olive oil (UO) meal and these changes were different between the meals at a marginal level of significance (P=0.05). Our data suggest that susceptibility to oxidation of lipoproteins in low antioxidant environments similar to dilute serum may be increased in the postprandial period following meals rich in heat-modified vegetable oils and unheated oils rich in polyunsaturated fatty acids but not following meals rich in native olive oil. These findings may be relevant to the choice of fat to replace saturated fats in lipid-lowering diets and to low risk of coronary heart disease in communities which have a high consumption of olive oil.