Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

Increased expression of the SNARE accessory protein Munc18c in lipid-mediated insulin resistance

Fatty acids inhibit insulin-mediated glucose metabolism in skeletal muscle, an effect largely attributed to defects in insulin-mediated glucose transport. Insulin-resistant mice transgenic for the overexpression of lipoprotein lipase (LPL) in skeletal muscle were used to examine the molecular mechanism(s) in more detail. Using DNA gene chip array technology, and confirmation by RT-PCR and Western analysis, increases in the yeast Sec1p homolog Munc18c mRNA and protein were found in the gastrocnemius muscle of transgenic mice, but not other tissues. Munc18c has been previously demonstrated to impair insulin-mediated glucose transport in mammalian cells in vitro. Of interest, stably transfected C2C12 cells overexpressing LPL not only demonstrated increases in Munc18c mRNA and protein but also in transcription rates of the Munc18c gene. jlr To confirm the relevance of fatty acid metabolism and insulin resistance to the expression of Munc18c in vivo, a 2-fold increase in Munc18c protein was demonstrated in mice fed a high-fat diet for 4 weeks. Together, these data are the first to implicate in vivo increases in Munc18c as a potential contributing mechanism to fatty acid-induced insulin resistance.

A randomized trial of aggressive lipid reduction for improvement of myocardial ischemia, symptom status, and vascular function in patients with coronary artery disease not amenable to intervention

PURPOSE: To determine the effects of aggressive lipid lowering on markers of ischemia, resistance vessel function, atherosclerotic burden, and Symptom status in patients with symptomatic coronary artery disease. METHODS: Sixty consecutive patients with coronary artery disease that was unsuitable for revascularization were assigned randomly to either usual therapy of lipids for patients with a low-density lipoprotein (LDL) cholesterol target level <116 mg/dL, or to a, more aggressive lipid-lowering strategy involving up to 80 mg/d of atorvastatin, with a target LDL cholesterol level <77 mg/dL. The extent and severity of inducible ischemia (by dobutamine echocardiography), vascular function.(brachial artery reactivity), atheroma burden (carotid intima-media thickness), and symptom status were evaluated blindly at baseline and after 12 weeks of treatment. RESULTS: After 12 weeks of treatment, patients in the aggressive therapy group had a significantly greater decrease in mean (+/- SD) LDL cholesterol level than those in the usual care group (29 +/- 38 mg/dL vs. 7 +/- 24 mg/dL, P = 0.03). Patients in the aggressive therapy group had a reduction in the number of ischemic wall segments (mean between-group difference of 1.3; 95% confidence interval: 0.1 to 2.0; P = 0.04), flow-mediated dilatation (mean between-group difference of 5.9%; 95% confidence interval: 2.5% to 9.4%; P = 0.001), and angina score after 12 weeks. There were no significant changes in atherosclerotic burden in either group. CONCLUSION: Patients with symptomatic coronary artery disease who are treated with aggressive lipid lowering have improvement of symptom status and ischemia that appears to reflect improved vascular function but not atheroma burden. Am J Med. 2003;114:445-453. (C) 2003 by Excerpta Medica Inc.

Are thiazide diuretics preferred as first-line therapy for hypertension? An appraisal of The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT)

No Abstract

Lipid rafts and caveolae as portals for endocytosis: New insights and common mechanisms

Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane of mammalian cells. While our understanding of the machinery regulating and driving clathrin-coated pit-mediated endocytosis has progressed dramatically, including the elucidation of the structure of individual components and partial in vitro reconstitution, the role of caveolae as alternative endocytic carriers still remains elusive 50 years after their discovery. However, recent work has started to provide new insights into endocytosis by caveolae and into apparently related pathways involving lipid raft domains. These pathways, distinguished by their exquisite sensitivity to cholesterol-sequestering agents, can involve caveolae but also exist in cells devoid of caveolins and caveolae. This review examines the current evidence for the involvement of rafts and caveolae in endocytosis and the molecular players involved in their regulation.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyltransferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARgamma/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

Blood lipid associations in 18 year-old men

The association of cigarette smoking, physical activity at work, and social class with total cholesterol and with high and low density lipoprotein cholesterol were examined in a random sample of 238 males, of 18 years of age, of Rosario, Argerntina. The mean (mg/dl) total serum cholesterol of the whole sample was 174.7, the high density lipoprotein cholesterol 52.8, and the low density lipoprotein cholesterol 121.5. Black tobacco consumers, evenly distributed by social class, had higher levels of total and low density lipoprotein cholesterol. Total cholesterol was higher in the high social class, differently from what smokers' distribution by social class, would lead one to expect. While a highly negative association was found between social class and physical activity at work, there were no significant diferences in lipoprotein levels between manual and non-manual workers. It is possible that the nutritional differences by social class still prevail over the smoking habit in their influence on the lipoprotein levels in these subjects.

Lipid content of frozen fish: comparison of different extraction methods and variability during freezing storage

In this work, a microwave-assisted extraction (MAE) methodology was compared with several conventional extraction methods (Soxhlet, Bligh & Dyer, modified Bligh & Dyer, Folch, modified Folch, Hara & Radin, Roese-Gottlieb) for quantification of total lipid content of three fish species: horse mackerel (Trachurus trachurus), chub mackerel (Scomber japonicus), and sardine (Sardina pilchardus). The influence of species, extraction method and frozen storage time (varying from fresh to 9 months of freezing) on total lipid content was analysed in detail. The efficiencies of methods MAE, Bligh & Dyer, Folch, modified Folch and Hara & Radin were the highest and although they were not statistically different, differences existed in terms of variability, with MAE showing the highest repeatability (CV = 0.034). Roese-Gottlieb, Soxhlet, and modified Bligh & Dyer methods were very poor in terms of efficiency as well as repeatability (CV between 0.13 and 0.18).

Lipid content and fatty acid profile of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles as affected by feed containing different amounts of plant protein sources

A growth trial with Senegalese Sole (Solea senegalensis Kaup, 1858) juveniles fed with diets containing increasing replacement levels of fishmeal by mixtures of plant protein sources was conducted over 12 weeks. Total fat contents of muscle, liver, viscera, skin, fins and head tissues were determined, as well as fatty acid profiles of muscle and liver (GC-FID analysis). Liver was the preferential local for fat deposition (5.5–10.8% of fat) followed by fins (3.4–6.7% fat). Increasing levels of plant protein in the diets seems to be related to increased levels of total lipids in the liver. Sole muscle is lean (2.4–4.0% fat), with total lipids being similar among treatments. Liver fatty acid profile varied significantly among treatments. Plant protein diets induced increased levels of C16:1 and C18:2 n -6 and a decrease in ARA and EPA levels. Muscle fatty acid profile also evidenced increasing levels of C18:2 n 6, while ARA and DHA remained similar among treatments. Substitution of fishmeal by plant protein is hence possible without major differences on the lipid content and fatty acid profile of the main edible portion of the fish – the muscle.

Lipid extractions from marine deep-sea invertebrates : modifications to the Bligh & Dyer method to increase lipid yields

The deep-sea environment is difficult to sample, and often only small quantities of samples can be obtained when using less destructive methods than dredging. When working with marine animals that are difficult to sample and with limited quantities of tissue to extract lipids, it is essential to ensure that the used method extracts the maximum possible quantity of lipids. This study evaluates the efficiency of introducing modifications to the method originally described by Bligh & Dyer (1959). This lipid extraction method is broadly used with modifications, although these usually lack proper description and evaluation of increment in lipids. In this study we consider the improvement in terms of amount of lipids extracted by changing the method. Lipid content was determined by gravimetric measurements in eight invertebrates from the deep-sea, including deep-sea hydrothermal vents animals, using three different approaches. Results show increases of 14% to 30% in lipid contents obtained from hydrothermal vent invertebrate tissues and whole animals by placing the samples in methanol for 24 hours before applying the Bligh & Dyer mixture. Efficiency of the extractions using frozen and freeze-dried samples was also compared. For large sponges, the use of lyophilized materials resulted in increases of 3 to 7 times more lipids extracted when compared with extractions using frozen samples.

Body mass index for predicting hyperglycemia and serum lipid changes in Brazilian adolescents

OBJECTIVE: To determine the best cut-offs of body mass index for identifying alterations of blood lipids and glucose in adolescents. METHODS: A probabilistic sample including 577 adolescent students aged 12-19 years in 2003 (210 males and 367 females) from state public schools in the city of Niterói, Southeastern Brazil, was studied. The Receiver Operating Characteristic curve was used to identify the best age-adjusted BMI cut-off for predicting high levels of serum total cholesterol (>150mg/dL), LDL-C (>100mg/dL), serum triglycerides (>100mg/dL), plasma glucose (>100mg/dL) and low levels of HDL-C (< 45mg/dL). Four references were used to calculate sensitivity and specificity of BMI cut-offs: one Brazilian, one international and two American. RESULTS: The most prevalent metabolic alterations (>50%) were: high total cholesterol and low HDL-C. BMI predicted high levels of triglycerides in males, high LDL-C in females, and high total cholesterol and the occurrence of three or more metabolic alterations in both males and females (areas under the curve range: 0.59 to 0.67), with low sensitivity (57%-66%) and low specificity (58%-66%). The best BMI cut-offs for this sample (20.3 kg/m² to 21.0 kg/m²) were lower than those proposed in the references studied. CONCLUSIONS: Although BMI values lower than the International cut-offs were better predictor of some metabolic abnormalities in Brazilian adolescents, overall BMI is not a good predictor of these abnormalities in this population.

Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria

Background/Aims: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. Methods: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. Results: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P , 0:01), as well as disrupted protein mobility(P , 0:001). Consistent with increased permeability, cytochrome c was released from the intermembrane space(P , 0:01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P , 0:01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. Conclusions: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

Insulin resistance does not play a major role in lipid and apolipoprotein profile in obese women

Background: Obesity is associated with increased atherogenesis through alterations in lipids, among other potential factors. Some of those abnormalities might be mediated by insulin resistance (IR). Aims: To compare lipid and apolipoprotein profile between lean and obese women; to evaluate the influence of IR on lipid and apolipoprotein profile, in obese women. Methods: We studied 112 obese and 100 normal-weight premenopausal women without known cardiovascular disease. Both groups were characterized for anthropometrics and a fasting blood sample was collected for assessment of glucose, insulin, triglycerides, cholesterol (total, LDL and HDL), and apolipoproteins A-I, A-II, B, C-II, C-III, and E; IR was assessed by the homeostatic model assessment (HOMA-IR). We compared lipids between obese and lean women; we looked for correlation of those levels with anthropometrics and IR (independently from anthropometrics) in obese women. Results: Obese women were characterized by mean age=34.6±8.3 years, BMI=43.6±7.9 kg/m2, waist circumference (Wc)=117.5±15.1 cm, and HOMA-IR=4.28±3.5. Lean women (age=34.2±8.3 years, BMI=21.4±1.7 kg/m2, Wc=71.7±5.8 cm, and HOMA-IR=1.21±0.76) presented with significantly lower levels of total cholesterol (P=0.001), LDL-cholesterol (P<0.001), and triglycerides (P<0.001); they presented higher levels of HDL-cholesterol (P<0.001), Apo A-I (P<0.001) and Apo A-II (P=0.037). HOMA-IR showed no significant association with apolipoproteins. HOMA-IR was inversely associated with HDL-cholesterol (P=0.048; r=−0.187) but that association disappeared when we adjusted for waist circumference. Only triglycerides were directly associated with HOMA-IR (P<0.001; r=0.343) independently from anthropometrics. Conclusion: We confirm that obese women present worst lipid and apolipoprotein profile. However, with the exception for triglycerides, insulin resistance per se does not play a major role in lipid and apolipoprotein abnormalities observed in obese women.

Bisphenol A alters transcript levels of biomarker genes for major depressive disorder

Major depressive disorder is a moderately heritable disorder characterized by one or more major depressive episodes. Laboratory tests to suport MDD diagnosis are not available. Diagnosis and treatment are based on various signs and symptoms not always fitting into strict diagnostic categories. Research for biological markers of neuropsychiatric disorders has been a challenge.