Alessandro G. Salerno

Cholesteryl ester transfer protein (CETP) increases postprandial triglyceridaemia and delays triacylglycerol plasma clearance in transgenic mice.

The CETP (cholesteryl ester transfer protein) is a plasma protein synthesized in several tissues, mainly in the liver; CETP reduces plasma HDL (high-density lipoprotein) cholesterol and increases the risk of atherosclerosis. The effect of CETP levels on postprandial intravascular metabolism of TAGs (triacylglycerols) is an often-overlooked aspect of the relationship between CETP and lipoprotein metabolism. Here, we tested the hypothesis that CETP delays the plasma clearance of TAG-rich lipoprotein by comparing human CETP expressing Tg (transgenic) and non-Tg mice. After an oral fat load, the postprandial triglyceridaemia curve was markedly increased in CETP-Tg compared with non-Tg mice (280+/-30 versus 190+/-20 mg/dl per 6 h respectively, P<0.02). No differences in intestinal fat absorption and VLDL (very-low-density lipoprotein) secretion rates were observed. Kinetic studies of double-labelled chylomicron-like EMs (emulsions) showed that both [(3)H]triolein and [(14)C]cholesteryl oleate FCRs (fractional clearance rates) were significantly reduced ( approximately 20%) in CETP-Tg mice. Furthermore, TAG from lipid EM pre-incubated with CETP-Tg plasma had plasma clearance and liver uptake significantly lower than the non-Tg plasma-treated lipid EM. In addition, reductions in post-heparin plasma LPL (lipoprotein lipase) activity (50%) and adipose tissue mRNA abundance (39%) were verified in CETP-Tg mice. Therefore we conclude that CETP expression in Tg mice delays plasma clearance and liver uptake of TAG-rich lipoproteins by two mechanisms: (i) transferring TAG to HDLs and increasing CE content of the remnant particles and (ii) by diminishing LPL expression. These findings show that the level of CETP expression can influence the responsiveness to dietary fat and may lead to fat intolerance.

Effects of diabetes and CETP expression on diet-induced atherosclerosis in LDL receptor-deficient mice.

The role of CETP expression and diabetes in atherogenesis was investigated in mice with heterozygous disruption of the LDL receptor gene (LDLR1). LDLR1 mice with and without CETP expression were treated with streptozotocin (STZ) and maintained on a standard diet for one month before switching to an atherogenic diet for an additional month. STZ‐sensitive mice had ∼2.5‐fold higher glycemia and 7.5‐ to 8.0‐fold higher cholesterolemia. Factorial analysis of variance showed no significant effect of diabetes, CETP or diabetes‐CETP interaction on the size of the atherosclerotic lesions. CETP expression in non‐diabetic mice resulted in a 50% reduction in the area of the atherosclerotic lesions. Multiple regression analysis showed a positive and independent atherogenic effect of triglyceridemia in LDLR1 mice and of cholesterolemia in diabetic mice. Logistic analysis showed that elevated plasma cholesterol level significantly increased the risk of developing large lesion size (>75th percentile). In conclusion, CETP expression did not alter the lesion formation in response to diabetes, although it may be protective in the euglycemic state; the triglyceride level was an independent risk factor for LDL receptor‐deficient mice but not for CETP‐expressing mice; and elevated plasma cholesterol levels increased the risk of developing large atherosclerotic lesions, independently of CETP and diabetes.

Chronic use of pravastatin reduces insulin exocytosis and increases β-cell death in hypercholesterolemic mice.

We have previously demonstrated that hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice secrete less insulin than wild-type mice. Removing cholesterol from isolated islets using methyl-beta-cyclodextrin reversed this defect. In this study, we hypothesized that in vivo treatment of LDLr(-/-) mice with the HMGCoA reductase inhibitor pravastatin would improve glucose-stimulated insulin secretion. Female LDLr(-/-) mice were treated with pravastatin (400mg/L) for 1-3 months. Isolated pancreatic islets were assayed for insulin secretion rates, intracellular calcium oscillations, cholesterol levels, NAD(P)H and SNARE protein levels, apoptosis indicators and lipidomic profile. Two months pravastatin treatment reduced cholesterol levels in plasma, liver and islets by 35%, 25% and 50%, respectively. Contrary to our hypothesis, pravastatin treatment increased fasting and fed plasma levels of glucose and decreased markedly (40%) fed plasma levels of insulin. In addition, ex vivo glucose stimulated insulin secretion was significantly reduced after two and three months (36-48%, p<0.05) of pravastatin treatment. Although reducing insulin secretion and insulinemia, two months pravastatin treatment did not affect glucose tolerance because it improved global insulin sensitivity. Pravastatin induced islet dysfunction was associated with marked reductions of exocytosis-related SNARE proteins (SNAP25, Syntaxin 1A, VAMP2) and increased apoptosis markers (Bax/Bcl2 protein ratio, cleaved caspase-3 and lower NAD(P)H production rates) observed in pancreatic islets from treated mice. In addition, several oxidized phospholipids, tri- and diacylglycerols and the proapoptotic lipid molecule ceramide were identified as markers of pravastatin-treated islets. Cell death and oxidative stress (H2O2 production) were confirmed in insulin secreting INS-1E cells treated with pravastatin. These results indicate that chronic treatment with pravastatin impairs the insulin exocytosis machinery and increases β-cell death. These findings suggest that prolonged use of statins may have a diabetogenic effect.

Monitoring of florfenicol residues in fish muscle by HPLC-UV with confirmation of suspect results by LC-MS/MS.

Florfenicol, a derivative of thiamphenicol in which the hydroxyl group at C-3 has been replaced with fluorine, is listed by the World Health Organization as an antibacterial agent for human medicine that is critically important in risk management strategies for non-human use. AOAC International has also identified it as an important molecule for the development of effective methods for the seafood sector. Following inspection missions from the European Union and United States of America, it was introduced in the Brazilian Residues Control Program to fulfill export and internal national requirements with a Maximum Residue Limit of 800 µg/kg. A high performance liquid chromatography method with ultraviolet detection at a wavelength of 230 nm (λ = 230 nm) for the detection of florfenicol in fish muscle was developed and validated according to the Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). Fish samples were extracted with ethyl acetate and hexane followed by C18 solid phase clean-up and chromatographic separation on a reversed-phase C18 LC column with acetonitrile:water as mobile phase. The method results were also was compared with those obtained using liquid chromatography-tandem quadrupole mass spectrometry. The method meets the Brazilian regulatory requirements with a decision limit (CCα) of 840 µg/kg and detection capability (CCβ) of 879 µg/kg. This method is easy to use and has been implemented into Brazils residue control program, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation of any suspect samples using the same method.

Laboratory validation of an LC-MS/MS method for the detection of ractopamine, clenbuterol and salbutamol in bovine and swine muscle at sub-μg kg–1 regulatory limits

ABSTRACT Ractopamine (RAC), is a β-adrenergic agonist increasingly used in the swine and cattle industry. This compound redirects nutrients to favour leanness rather than fat deposition, improves growth and carcass traits gaining higher economic benefit to producers. Countries around the world are split over whether to allow the use of RAC in meat production. Clenbuterol (CLB) and salbutamol (SLB) are anillinic and phenolic β-agonists, respectively, with the same capacity of producing economic benefits for the meat sector. However, they are prohibited because of the potentially adverse reactions they can cause in consumers. The three β-agonist compounds have been included in the Brazilian National Regulatory Survey and consequentially there is an eminent need for reliable methods capable of detecting those substances at the same time and reduce analytical costs. Therefore, an LC-MS/MS method for the simultaneous determination of residual RAC, CLB and SAL in swine and cattle muscle was developed and validated with quantification levels respecting the action levels established for Brazil which are 0.1, 0.2 and 5 µg kg–1 for RAC, CLB and SAL, respectively. Samples were quantified using RAC-d5, CLB-d9 and SLB-d6 as internal standards. The validation was performed according to European Union Decision 2002/657, which includes criteria (CCα, CCβ, recovery, repeatability, reproducibility and calibration curve). The method meets the Brazilian regulatory requirement that establishes criteria and procedures for the determination of parameters such as CCα, CCβ, precision and recovery. CCα values were 0.02, 0.21 and 5.42 µg kg–1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples; CCβ values were 0.03, 0.22 and 5.8 µg kg–1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples. Average recoveries fortified with 0.05–7.5 µg kg–1 of the studied β-agonist leads around 95%. The method was demonstrated to be suitable for the determination of RAC, CLB and SLB in swine and cattle muscle samples.

Soy protein containing isoflavones favorably influences macrophage lipoprotein metabolism but not the development of atherosclerosis in CETP transgenic mice

The possibility that soy protein containing isoflavones influences the development of experimental atherosclerosis has been investigated in ovariectomized mice heterozygous for the human CETP transgene and for the LDL-receptor null allele (LDLr+/− CETP+/−). After ovariectomy at 8 wk of age they were fed a fat/cholesterol-rich diet for 19 wk and divided into three experimental groups: dietary unmodified soy protein containing isoflavones (mg/g of diet), either at low-dose (Iso Low, 0.272, n=25), or at high-dose (Iso High, 0.535, n=28); and the atherogenic diet containing an isoflavone-depleted alcohol-washed soy protein as a control group (n=28). Aortic root lipid-stained lesion area (mean μm2×103±SD) did not differ among Iso Low (12.3±9.9), Iso High (7.4±6.4), and controls (10.7±12.8). Autoantibody titers against plasma oxidized LDL did not differ among the experimental groups. Using the control mice as the reference value (100%), in vitro mouse peritoneal macrophage uptake of labeled acetylated LDL-cholesterol was lower in the Iso High (68%) than in the Iso Low (85%) group. The in vitro percent removal by exogenous HDL of labeled unesterified cholesterol from macrophages previously enriched with human [4-14C]-cholesteryl oleate acetylated LDL was enhanced in the Iso High group (50%). In spite of these in vitro potentially antiatherogenic actions, soy protein containing isoflavones did not modify the average size of lipid-stained area in the aortic root.

Endocrinology CETP expression prolongs mice survival rate in sepsis by increasing leukocyte migration and reducing the concentrations of plasma IL-6 and of hepatic TLR4

D due to cardiovascular disease is predominantly a result of atherosclerosis, a condition where lesions form on the artery wall and restrict blood flow. A major outcome of atherosclerosis is an increase in vessel stiffness due to arterial remodeling. While atherosclerosis normally develops with age, accelerated progression of this condition occurs in diabetes, chronic kidney disease and rheumatoid arthritis. However, while some medications can treat the symptoms of atherosclerosis, no pharmaceutical intervention is currently capable of stopping arterial remodeling. However, scientific evidence suggesting certain phytochemicals present in legumes may be capable of lessening arterial stiffness led us to examine whether consumption of legumes could be used to treat atherosclerotic diseases. A human study that evaluated the effect of eating 1⁄2 cup per day of non-soy legumes (beans, peas, chickpeas, lentils) over an 8 week period on vascular function in persons with peripheral artery disease (PAD), a manifestation of atherosclerosis in which blood flow to the legs is reduced was therefore conducted. This study revealed that eating non-soy legumes could improve the ankle-brachial index, the primary diagnostic tool used to identify PAD, and increase blood flow to the legs. In a subsequent animal study designed to investigate mechanism of action, lentils were found to reverse arterial remodeling. Our results are the first to show that eating a specific food can reverse the main cause of atherosclerotic disease, and that the amount of food required obtaining this benefit can be reasonably incorporated into our regular diet.M is a neurohormone mainly synthesized by the pineal gland at night. It is the main endocrine output of the circadian master clock in the way that it relays the succession of nights and days to central and peripheral organs. Melatonin acts through 3 main reported proteins: The seven trans-membrane domain G-coupled receptors, MT1 and MT2; and the MT3 binding site, identical to the cytosolic enzyme quinone reductase 2. The exact characteristics of the binding of the ligands at MT1 and MT2 receptors is poorly documented, as often in the GPCR area, because purification of a functional receptor and its crystallization are difficult, at best. We succeeded in assessing a protocol leading to an active MT1 receptor in milligram amounts, with binding characteristics similar to those of the membrane MT1 receptor. We are using this material to reconstitute a functional MT1 in nanodiscs. Depending on the nature of the signaling pathways measured, more and more is reported concerning the biased nature of receptor ligands, particularly the agonists. We report here the molecular pharmacology of both recombinant human MT1 and MT2 receptors with a set of 24 molecules and 5 signalization pathways (GTPγS, cAMP, cellular dielectric spectroscopy, receptor internalisation, β-arrestin) as well as their binding affinities. These studies clearly show that depending on the chemical nature of the ligands, the pathway by which the signal is transmitted is different and the nature of the GPCR/ligand might vary from very partial agonist (almost antagonist) to pure agonist. These data will help the scientists in the field to build better compounds purer in their actions, and probably more specific.O is a frequent metabolic disorder but an effective therapy is still scarce. Anorexigenic neuropeptides seem to be potential specific tools for obesity treatment but their peptide character complicates their delivery to the brain, necessary for their central effect, and makes them subjects of fast degradation. In order to overcome the unfavorable facts mentioned, lipidization of anorexigenic neuropeptide prolactin-releasing peptide (PrRP) whose strong anorexigenic effect was demonstrated with the fact that both PrRP and its receptor knock-outs resulted in obese phenotypes, was applied. Analogs of PrRP with attached fatty acids showed unabated biological activities compared to the native PrRP regarding binding affinity and signaling in cells expressing PrRP receptor. Most interestingly, these analogs revealed a strong and long-lasting anorexigenic effects after their peripheral administrations to fasted mice and rats and neuronal activation in brain areas involved in food intake regulation. Moreover, repeated subcutaneous administration of palmitoylated PrRP lowered food intake and body weight and improved metabolic parameters in diet-induced obese mice. For the anorexigenic effect of native PrRP, central administration was necessary. Lipidization of PrRP enabled its peripheral administration for its anorexigenic effect in the brain, most probably by enhancing its stability in the blood and mediating the peptide to cross the blood brain barrier.G Wide Association Studies (GWAS) are extraordinary opportunities to find culprit genes in complex conditions such as diabetes, obesity, metabolic syndrome (MetS) or polycystic ovarian syndrome (PCOS). Despite enormous progress (e.g. 831 MetS genes register in GAD) associated genes displayed relative low odds ratio and hardly explain heritability in populations. Although there may be multiple explanations, the European MEDIGENE program (FP7-279171) launched the hypothesis that dissecting anthropological lineages would be able to better stratify populations for GWAS. This is based on the ability to geo-localized individual DNA in Europe based on SNP (single nucleotide polymorphism), more or less concordant to mitochondrial (mt)DNA or Chr Y lineages. Focusing on Mediterranean populations, the project is studying native and immigrant populations (Albanians, Turkish, Maghrebin and Romanian) in Europe affected by MetS or PCOS. Among 30 prioritized genes screened by their leader SNP or proxies, we discovered in French and Romanians one of the most influential gene in PCOS acting as dual phosphatase. Other genes were more specific to male/female susceptibility for MetS. More than 4621 DNA samples with clinical and anthropological information were collected in the Mediterranean area or from more genetically-distant populations in Eastern Europe, Lithuania, Lebanon and Russia. We also obtained 400 samples of ancient (a) DNA from antique Romans from Tarragona Necropolis. While efforts are spent to detail haplotype structure with a customized Affymetrix chip, locus refining using Roche 454 technology is currently used for identification of rare SNP with major effect and that may be used to develop significant genetic markers at a clinical scale.G hormone (GH) receptor activation rapidly activates the JAK2-STAT5 pathway, which is a core cancer pathway that can drive other essential core cancer pathways such as survival and cell cycle progression. STAT5 is a weak transcription factor that needs glucocorticoid (GC) receptor (GR) cofactor function for potent gene regulation. GH signalling can also promote differentiation and it is an important cytokine for metabolic function throughout the whole body. The liver has several ways to communicate with neuroendocrine control and if hepatic GH and GC signalling is impaired it can mobilize fuel from muscle or adipose tissue to convert it to glucose. Overall, these processes are important to understand development of type II diabetes, obesity or metabolic liver cancer development. Imbalanced GH levels are involved in diseases such as dwarfism or chronic inflammation with polycystic kidney disease and liver cancer formation. Thus, we conditionally deleted the STAT5a/b and/or GR transcription factors or JAK2 tyrosine kinase in liver epithelial cells to understand these diseases in a physiologic context better. We compared their phenotypes for normal liver function or upon inflammatory hyper GHinduced hepatocellular carcinoma development. We found that STAT5 is a more prominent tumour suppressor than JAK2 and we undertook a careful genetic, biochemical and key protein analysis to explain differences in liver disease. The key role of STAT5 as an oncogene in hematopoietic cancers is well established, other cancer types like carcinomas lack behind. We found that STAT5 and GR function in adipocytes were crucial for development of metabolic liver cancer. Therefore, we followed a conditional approach do genetically deplete STAT5 or GR proteins in white and brown adipose tissue. We dissected their key function for lipolysis or other metabolic processes controlled by adipocytes. Surprising defects were manifested in the beta-oxidation pathway and catecholamine signalling. Despite that we know today from the cancer genome that ~90% of the mutations happen in tumour suppressor genes, we still have an incomplete understanding if JAK2-STAT5 activity is good or bad. Our data for loss of STAT5 and GR proteins pinpoint to tight regulation of many metabolic pathways and loss or disturbance of the STAT5-GR axis caused severe metabolic disturbances with rapid development of liver cancers.C disease (CVD) is the leading cause of morbidity and mortality in the United States as well as globally. Although the consumption of green leafy vegetables (GLVs) has been demonstrated to reduce the risks associated with cardiovascular and other diseases, very little attention has been devoted to the traditional and relatively novel GLVs available to individuals most at risk. Our research examined the influence of collard greens, purslane and sweet potato greens, supplemented into the diet of spontaneously hypertensive rats over a 6 week period, on the hepatic fatty acid profile. This research revealed that consumption of diets containing these GLVs resulted in improved fatty acid profiles, namely decreased saturated fatty acid percentages and increased polyunsaturated and omega-3 fatty acid percentages. These findings suggest the ability of GLVs to modulate the hepatic fatty acid composition, thus attenuating elevations in atherogenic fatty acids, which may be implicated in CVD pathogenesis. Other research studies with these GLVs have yielded similar observations, with the cardioprotective benefits of these vegetables increasing with decreasing dietary omega-6/omega-3 fatty acid ratios. Future research will focus on extensively investigating the influence of these diets on the cardiovascular system and other biological systems. As CVD is both exacerbated by and exacerbates other co-morbities and endocrine disorders, which may adversely affect the cardiovascular system, efforts to mitigate CVD should involve an integrative approach, with practical and accessible intervention strategies particularly among those at highest risk.P of diabetic nephropathy is strongly associated with irreversible loss of renal function, which remains undiagnosed till the appearance of microalbuminuria, decrease in creatinine clearance and/or increase in serum creatinine, observed at the late stage 2 of diabetic nephropathy. Proximal tubular damage plays a central role in pathogenesis of Type 2 diabetic nephropathy (T2DM) and the site specific enzymes located in renal tubules may be the useful prognostic markers. Thus, this study was aimed to evaluate the prognostic accuracy of urinary excretion of N-acetyl β-D-glucosaminidase (NAG), which is localized in the lysosomes of renal tubules for the onset of T2DM. The study was conducted on the total of 491 eligible participants including 76 healthy controls, 194 T2DM patients with duration 0-5 yrs, 5-10 yrs, 10-15 yrs and 15-20 yrs of diabetes, 71 microalbuminuric patients, 100 diabetic nephropathy patients and 50 non-diabetic nephropathy patients. Fasting glucose, serum fructosamine, HbA1C, urinary microalbumin, serum creatinine, estimated glomerular filtration rate (eGFR), serum-NAG and urinary-NAG were estimated. The urinary excretion of NAG was compared with other well established markers like microalbuminuria, eGFR, serum creatinine, which are routinely used for assessment of diabetic nephropathy. UrinaryNAG excretion was increased by and 8 and 12 folds in Type 2 diabetic patients of 10-15 yrs and 15-20 yrs of diabetes duration respectively without showing microalbuminuria. The 16 and 18 fold increased urinary-NAG was observed in microalbuminuric and diabetic nephropathy patients respectively, which has not shown any alteration in non-diabetic nephropathy patients. A cutoff value of 3 U/L of urinary NAG has demonstrated a sensitivity of 96.1% and a specificity of 100% to discriminating healthy controls from patients with diabetes duration 10-15 yrs (AUC 1.000), 15-20 yrs of diabetes (AUC 0.999), microalbuminuria (AUC 0.999) and diabetic nephropathy (AUC 1.000). Excretion of urinary-NAG was gradually increased with the duration of diabetes and appeared much before the microalbuminuria and increased serum creatinine. Thus, urinary-NAG may be used as a potential site specific early tubular damage marker leading to diabetic nephropathy.O the last few decades, diabetes has evolved as the most common medical complication of pregnancy. The increasing prevalence of Type 2 diabetes in general, and in younger people in particular, has led to an increasing number of pregnancies with this complication. Gestational diabetes is such a disease that puts two generations at risk of developing future diabetes mellitus. It is a disease which does not discriminate based on race, religion or community. Indications and methods of screening, the diagnostic criteria and management has been the subject of considerable controversy. After the reports of HAPO study, many medical associations including ADA are now in favour of universal screening. Compliance with diet and insulin, close glycemic monitoring, antenatal fetal surveillance for sudden intrauterine demise, management of macrosomia and shoulder dystocia, are only a handful of the numerous tough challenges which obstetricians face. Since complications are directly related to the glycemic status, so a timely diagnosis and optimal management is prudent. Strict glycemic targets, home glucose monitoring and meticulous fetal monitoring are the cornerstones for successful feto-maternal outcomes. It is high time now that all clinicians should unite to fight against this giant enemy.T MRL mouse strain has long been studied for its autoimmune phenotypes, its super healing abilities and now we are investigating the mouse strain because it proves to be resistant to high fat diet-induced type 2 diabetes (T2D) pathology. Amongst the T2D characteristics that the control C57Bl/6J mice display, after HFD feeding are; hyperglycemia, insulin resistance, and cardiac hypertrophy. The HFD-fed MRL mice display none of these phenotypes. They remain sensitive to insulin and glucose and their cardiac function remains normal. In the preliminary steps of identifying the causative molecular mechanisms behind this HFD resistance we have identified increased skeletal muscle pAMPK and glycolysis. Both of these features are well known to be indicative of T2D resistance. We also assessed the levels of various hormones implicated in the pathogenesis of T2D. Surprisingly, we identified decreases in adiponectin levels in both HFD MRL and control diet (CD) MRL mice compared to their diet matched controls. As expected, insulin levels were increased in the control C57Bl6/J strain with HFD feeding. However, in the MRL mice insulin was decreased in the HFD mice. We also assessed food intake and grams gained for the four mouse groups. Both of the MRL mouse groups consumed significantly fewer food calories but gained significantly more weight. We are now attempting to combine the data for a model which explains the metabolic resilience of the MRL mice.article i nfo Background: Obesity in adulthood is associated with increased risk for diabetes mellitus (DM). It is uncertain whether this risk is attenuated in adulthood who are overweight or obese initially but not obese subsequently. Methods and results: The data were collected in 1992 and then again in 2007 from the same group of 687 participants (male: 58.1%, age: 48.1±6.2 years).The participants were categorized into four groups on the basis of adiposity status in 1992 and 2007: group I included subjects with a normal BMI in 1992 and 2007; group II, those with a normal BMI in 1992 who were overweight or obese in 2007; group III, those who were overweight or obese in 1992 but normal BMI in 2007; and group IV, those who were overweight or obese in 1992 and 2007. With group I as reference, the HR is 0.818 for group II (95% CI: 0.341-1.962, p=0.653), 2.231 for group III (95% CI: 1.087-4.579, p=0.029) and 1.855 for group IV (95% CI: 1.049-3.279, p=0.034) after adjustment for confounders. It was not significantly different between groups I and II, as well as between groups III and IV. Conclusion: In adulthood, becoming nonobese could not reverse the adverse effects of obesity on DM, as com- pared with the subjects who persist being overweight or obese. Keeping weight in the normal BMI range should be emphasized in the public for preventing DM.P cancer (PCa) ranks first in incidence among cancers in American males and second as a cause of death by cancer. Beside age, etiologic and progression factors are still not fully identified. Accumulating evidence point to changes in signalling molecules and pathways as means to confer survival and/or growth advantages to tumor cells as they evolve with time and become resistant to androgen deprivationand chemo-therapies. Our studies on the non-receptor Fak and Fer tyrosine kinases (TKs) indicate that beside their up-regulation in PCa, these kinases are key elements controlling PCa cell motility and survival/growth, respectively. Notably, Fak signaling complexes with Src, paxillin and integrins are involved in PCa cell response to neuroendocrine (NE) products, eliciting selective patterns based on androgen receptor (AR) expression in PCa cells. The Fer TK integrates signals emanating from interleukin (IL)-6 and to a lesser extent growth factors (EGF, IGF-1) and also androgens. This occurs through STAT3 and AR, both identified as Fer substrates and binding partners and accumulating into the nucleus where they regulate transcription. Altogether, these mechanisms favor the adaptation of androgen-sensitive luminal-like tumor cells and the concomitant selection of androgen-independent tumor cell subsets (stem, NE). They concur to cell heterogeneity of tumors and metastases as observed during PCa progression.T 2 diabetes in developing countries has reached epidemic proportions especially in the Persian Gulf where most of these countries have recently gone through a very rapid socioeconomic transition. Coupled with this rise in the incidence of type 2 diabetes is a sharp increase in the incidence of diabetic foot ulcerations. Most of the patients with diabetic foot ulcers (DFU) present late at primary care level and a lot of patients refuse to go to secondary care fearing amputations. In order to overcome this problem and provide a patient centered care we have developed a diabetic foot clinic at primary care level led by a senior consultant family physician with special interest in diabetic foot management. During the five year period we have utilised natural honey in the management of chronic diabetic foot ulcers which proved to be an efficacious and cost effective alternative to advanced wound products. The budget for wound products has dropped by 70% after the inclusion of natural honey in the management of (DFU). During this oral presentation we will provide clinical data demonstrating the feasibility of treating (DFU) at primary care level utilising natural honey which is evidence based, aesthetically acceptable, efficacious and cost effective.T 2 diabetes is a complex metabolic disorder characterized by hyperglycemia arising from a combination of insufficient insulin secretion together with resistance to insulin action. The incretin effect describes the observation that oral glucose has a greater stimulatory effect on insulin secretion than the intravenous glucose at the same circulating glucose concentration. In humans, this effect seems to be primarily mediated by GLP1 and GIP. GLP1 is produced from the proglucagon gene in intestinal L cells and is secreted in response to nutrients. GLP1 stimulates insulin secretion in a glucose-dependent fashion, inhibits inappropriate hyperglucagonemia, slows gastric emptying, reduces appetite and improves satiety, and has beta-cell proliferative, antiapoptotic, and differentiation effects at least in vitro and in preclinical models. GLP1 has a very short halflife in plasma (1 to 2 minutes) due to amino terminal degradation by the enzyme dipeptidyl peptidase IV (DPP4). A variety of pharmacologic techniques have been developed to harness the potential of GLP1 signaling to treat diabetes, including GLP1 receptor agonists, which are peptides that produce increases of 10-fold or higher inGLP1 activity, and DPP4 inhibitors, which are small molecule inhibitors of the degradation of GLP1 and GIP as well as other hormones. Exenatide is synthetic exendin-4 and was the first GLP1-based therapeutic agent to be approved for human use. Liraglutide, Albiglutide, Taspoglutide & Lixisenitide are other GLP1 receptor agonists under various trials for efficacy and cardiovascular safety. Sitagliptin was the 1st DPP4 inhibitors available for therapy. Many new drugs Saxagliptin, Vildagliptin, Aloegliptin are under various stages of clinical trial for approval. They have advantage over GLP1 analogues as they are oral and better side effect profile. Availability of these new drugs has made treatment of type 2 diabetes easier. Long term effects of on cardiovascular safety of these drugs is being studies in long term RCTs. Role of incretin based therapy in type 1 diabetes is scope for further research.A asthma is a disease of the airways that affects over 25 million people in the United States incurring over

CETP expression enhances liver HDL-cholesteryl ester uptake but does not alter VLDL and biliary lipid secretion

50 billion in direct and indirect costs per year. Although we understand the symptoms associated with allergic asthma, the exact causes and sequence of events that follow allergen exposure warrants further investigation. Animal models that effectively recapitulate the hallmarks of human disease are important to delineate the pathways that lead to the immunological, architectural and physiological changes that occur. We have identified that the neuroimmunological axis plays a role in the development of allergic asthma using a novel murine model induced by Aspergillus fumigatus conidia. Vasoactive intestinal peptide (VIP) is a neuropeptide with cytokine properties which has been demonstrated to have an anti-inflammatory role in the lungs when acting through its receptor VPAC2. We showed that VIP localization in the columnar epithelium of the airways was dynamically regulated following allergen provocation with levels decreasing early during the allergic cascade and increasing after the influx of lymphocytes into the airways. The VIP/VPAC2 axis, previously shown to promote TH2 immunity, was demonstrated herein not to play a role in the development or maintenance of allergic asthma. VPAC2 null mice have elevated levels of IgG2a and IgA in response to allergen challenge, indicating its novel role in regulating the humoral immune response.C ester transfer protein (CETP) transfers neutral lipids among plasma lipoproteins; its inhibition raises plasma HDL. There has been considerable debate on the role of CETP in human atherogenesis. This may in part be explained by the involvement of CETP on the protection against against microbial infection and human sepsis. In order to evaluate the role of CETP in polymicrobial sepsis induced by caecum ligation and puncture (CLP), mice expressing human CETP, and wild-type mice (WT) underwent CLP sepsis. Sham-operated mice were utilized as controls. After CLP, mice survival rates were evaluated over five days. Also, mice were sacrificed at 24 or 48 hours after CLP, and blood, peritoneal cells and liver were collected. After CLP, as compared to wild type mice, CETP mice survived longer, had increased leukocyte migration into the peritoneal cavity, lower plasma IL-6 and TLR4 and acyloxyacyl hydrolase (AOAH) expressions in their liver. CETP mice had reduced liver inflammation and plasma inflammatory factors, and increased leukocyte recruitment to the infectious focus. Thus, CETP is involved in the first line of defense against an exacerbated production of proinflammatory mediators. These results indicate that the regulation of TLR4 in the liver plays a role in the proinflammatory response and pathophysiology of polymicrobial sepsis that helps explaining why CETP pharmacological inhibition has consistently failed to provide protection against atherosclerosis in human investigations.A D (apoD) is a member of the lipocalin super family. It transports small hydrophobic compounds such as arachidonic acid, progesterone and pregnenolone. In human, apoD is found in the plasma fraction, associated with high-density lipoprotein (HDL). It is highly expressed in the brain, adrenal glands, kidneys, pancreas and placenta but poorly expressed in intestine and liver. In contrast, the murine expression of the apoD gene is almost exclusively expressed in the central nervous system (CNS). Short overexpression of apoD in liver of obese mice using adenovirus improved triglyceride profiles, correlating with increased plasma LPL activity and enhanced postprandial fat tolerance. However, transgenic mice (Tg) with increased expression of human apoD (H-apoD) in the liver develop metabolic defects at one year of age. H-apoDTg mice are not obese and have normal lipid concentration in circulation. They are glucose intolerant, insulin resistant, and develop hepatic steatosis. In these mice, hepatic PPARγ expression is increased and consequently Plin2 and Cide A/C leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36 is also increased associated with elevated fatty acid uptake. Despite modulation of hepatic lipogenic gene expression, de novo lipogenesis is not altered. Elevated ApoD expression activates PPARγ transcriptional activity by increasing the transport of one of PPAR’s natural ligand arachidonic acid into the cell. It is now clear that apoD not only plays a role in the protection of the central nervous system but is also associated with lipid metabolism and associated metabolic syndrome.T incidence of thyroid cancer is rising faster than many other cancers, with the highest overall rate of increase in cancer deaths and it is associated with a higher number of advanced diseases characterized by the loss of cancer differentiation and metastatic spread. The development of new therapeutic drugs able to blockade the oncogenic kinases (BRAF V600E, RET/ PTC) or signalling kinases [vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptors (PDGFR)] involved in cellular growth and proliferation is now possible thanks to the knowledge of the molecular pathways involved in the pathogenesis of thyroid cancer. Some clinical trials have been conducted showing the ability of targeted therapies (sorafenib, sunitinib, axitinib, imanitib, vandetanib, pazopanib, gefitinib, cabozantinib) in stabilizing the course of the disease. Until now, no consensus guidelines have been established for patient selection. Thereby the effects on survival are unclear, because of lack of complete responses and because of the discrepancies between the radiographic tumor responses and the effective improvement of survival. To reach the goal to extend life duration assuring a good quality of life, we need to have more data on toxicities of single agent and of combination of different drugs, in order to identify specific biomarkers able to predict the treatment efficacy, the clinical outcome and to guarantee a tailored dosage of the drugs. Moreover, the possibility to test in vitro (in thyroid cancer cells in primary cultures) these novel drugs may help to improve the personalization of the treatment.P are used clinically in the treatment of many diseases. One major issue regarding the clinical use of many peptides is their short life span in the body, due to the rapid clearance of those proteins from the circulation. The low stability of peptides has thus often posed a difficulty to researchers and hindered their adoption in potential medical applications. At the clinical level, there is a need for a regime of frequent injections of the peptides into the patients to overcome this low stability factor.T African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. We previously indicated that a W-linked gene Dm-W could direct female development as a sex-determining gene. However, the mechanisms during early sex differentiation remain unclear. Therefore we screened genes showing sexually dimorphic expression by microarray using the ZZ and ZW gonads at early sex differentiation in X. laevis. Several genes for TGFβ members and steroidogenic enzymes displayed sexual dimorphism on the array. Importantly, steroid 17α-hydroxylase gene Cyp17a1 and estrogen synthesizing enzyme (aromatase) gene Cyp19a1 showed ZZ and ZW gonad-enriched expression just after sex determination, respectively. In both ZZ and ZW gonads at the stage, we discovered that Cyp17a1 and/or Cyp19a1 were expressed in very unique ‘massin-line’ structure; several masses sectioned by basement membranes in line along the anteroposterior axis of the gonads. In fact, ovarian cavities formed inside each mass consisting of Cyp17a1and Cyp19a1-positive cells in the ZW ovaries during differentiation. However, the ‘mass-in-line’ structure disappeared in the ZZ testes during testicular development. These results suggested that the ‘mass-in-line’ structure in both ZZ and ZW gonads just after sex determination might be formed in advance for ovarian cavities followed by oocyte production in X. laevis.C psychological stress is associated with behavioral and somatic disorders in modern society. Mediators of the stress response include the sympathetic nervous system and the hypothalamic-pituitary-adrenocortical (HPA) axis and their final mediators, adrenaline/noradrenaline, and glucocorticoids (i.e. cortisol in humans and corticosterone in rodents). These systems are modulated by brain neurotransmitters with serotonin (5-HT) playing an important role; indeed, drugs acting on this system (e.g. 5-HT uptake inhibitors) are prescribed clinically. HPA axis dysregulation involving hypercortisolemia is a common pathophysiological feature of stress-related disorders (SRD), such as major depression. Evidence suggests that chronic stress may lead to endocrine disruption and hypercortisolemia though the mechanisms involved remain unclear. It has been shown that relatively long periods of chronic stress may promote endocrine disruption, which translates into magnified corticosterone responses to acute stress in rodents. Interestingly, pharmacological blockade of 5-HT7 receptors normalized increased acute stress-induced corticosterone secretion in chronically stressed animals. Furthermore, disruption of the HPA axis by chronic stress was found to parallel increased expression of 5-HT7 receptors and increased 5-HT immunoreactivity in the adrenal cortex along with higher 5-HT levels and turnover in whole adrenals. Interestingly, ectopic expression of 5-HT7 receptors and increased potency of 5-HT to induce cortisol secretion via 5-HT7 receptors have been observed in human cortisol-producing adrenocortical adenomas. Remarkably, 5-HT7 receptor antagonists have been demonstrated to produce fast antidepressant-like effects and accelerate the antidepressant-like effects of several classes of antidepressant drugs. Then, blockade of 5-HT7 receptors, possibly at the adrenal level, might represent a novel therapeutic strategy in SRD.Methods: HepG2 cells and hamsters fed a high-fat diet were used to test the effects of IMM-H007 on lipid metabolism. Western blots, chemical intervention, HPLC, SAMS peptide assay, 14C-labelled acetate and palmitate assays, molecular docking assay and siRNA targeting the AMPK γ1 subunit were used to investigate the effect of IMM-H007 on AMPK activation as well as the underlying mechanism involved in this activation.