Mécanismes moléculaires de la fonction anti-apoptotique des HDLs sur la cellule béta pancréatique : 4E-BP1 comme potentielle cible thérapeutique. [Molecular mechanisms of anti-apoptotic function of HDLs on pancreatic beta cells : 4E-BP1 as a potential therapeutic target]

Introduction : Les particules de HDL (High Density Lipoprotein) ont des fonctions diverses notamment en raison de leur structure très hétérogène. Tout d'abord, les HDLs assurent le transport du cholestérol de la périphérie vers le foie mais sont également dotées de nombreuses vertus protectrices. Un grand nombre d'études démontre les mécanismes de protection des HDL sur les cellules endothéliales. Sachant que les patients diabétiques ont ses niveaux bas de HDL, le but de cette étude est d'investiguer les mécanismes moléculaires de protection sur la cellule beta pancréatique. Résultats : Une étude « microarray » nous a permis d'obtenir une liste de gènes régulés par le stress, comme la privation de sérum, en présence ou en absence de HDL. Parmi ces gènes, nous nous sommes particulièrement intéressés à un répresseur de la synthèse protéique « cap » -dépendante, 4EBP1. Dans notre étude transcriptomique, les niveaux d'ARNm de 4E-BP1 augmentaient de 30þ% dans des conditions sans sérum alors que les HDLs bloquaient cette élévation. Au niveau protéique, les niveaux totaux de 4EBP1 étaient augmentés dans les conditions de stress et cette élévation était contrée par les HDLs. D'autres expériences de transfection ou d'infection de 4E-BP1 ont montrés que cette protéine était capable d'induire l'apoptose dans les cellules beta, imitant ainsi l'effet de la privation de sérum. Afin de déterminer le rôle direct de 4E-BP1 dans la mort cellulaire, ses niveaux ont été réduits par interférence ARN. Le niveau de mort cellulaire induit par l'absence de sérum était moins élevé dans des cellules à taux réduits de 4EBP1 par RNAi que dans des cellules contrôle. Conclusion : Ces données montrent que les HDL protègent les cellules beta suite à différents stress et que 4E-BP1 est une des protéines pro-apoptotiques inhibées par les HDL. 4E-BP1 est capable d'induire la mort cellulaire dans les cellules bêta et cette réponse peut-être réduite en diminuant l'expression de cette protéine. Nos données suggèrent que 4E-BP1 est une cible potentielle pour le traitement du diabète.

Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target?

Tumor angiogenesis is an essential step in tumor progression and metastasis formation. Suppression of tumor angiogenesis results in the inhibition of tumor growth. Recent evidence indicates that vascular integrins, in particular alpha V beta 3, are important regulators of angiogenesis, including tumor angiogenesis. Integrin alpha V beta 3 antagonists, such as blocking antibodies or peptides, suppress tumor angiogenesis and tumor progression in many preclinical tumor models. The potential therapeutic efficacy of extracellular integrin antagonists in human cancer is currently being tested in clinical trials. Selective disruption of the tumor vasculature by high doses of tumor necrosis factor (TNF) and interferon gamma (IFN-gamma), and the antiangiogenic activity of nonsteroidal anti-inflammatory drugs are associated with the suppression of integrin alpha V beta 3 function and signaling in endothelial cells. Furthermore, expression of isolated integrin cytoplasmic domains disrupts integrin-dependent adhesion, resulting in endothelial cell detachment and apoptosis. These results confirm the critical role of vascular integrins in promoting endothelial cell survival and angiogenesis and suggest that intracellular targeting of integrin function and signaling may be an alternative strategy to extracellular integrin antagonists for the therapeutic inhibition of tumor angiogenesis.

Relationship of imatinib-free plasma levels and target genotype with efficacy and tolerability

Imatinib has revolutionised the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours (GIST). Using a nonlinear mixed effects population model, individual estimates of pharmacokinetic parameters were derived and used to estimate imatinib exposure (area under the curve, AUC) in 58 patients. Plasma-free concentration was deduced from a model incorporating plasma levels of alpha(1)-acid glycoprotein. Associations between AUC (or clearance) and response or incidence of side effects were explored by logistic regression analysis. Influence of KIT genotype was also assessed in GIST patients. Both total (in GIST) and free drug exposure (in CML and GIST) correlated with the occurrence and number of side effects (e.g. odds ratio 2.7+/-0.6 for a two-fold free AUC increase in GIST; P<0.001). Higher free AUC also predicted a higher probability of therapeutic response in GIST (odds ratio 2.6+/-1.1; P=0.026) when taking into account tumour KIT genotype (strongest association in patients harbouring exon 9 mutation or wild-type KIT, known to decrease tumour sensitivity towards imatinib). In CML, no straightforward concentration-response relationships were obtained. Our findings represent additional arguments to further evaluate the usefulness of individualizing imatinib prescription based on a therapeutic drug monitoring programme, possibly associated with target genotype profiling of patients.

Substrate metabolism, nutrient balance and obesity development in children and adolescents: a target for intervention?

Obesity results from the organism's inability to maintain energy balance over a long term. Childhood obesity and its related factors and pathological consequences tend to persist into adulthood. A cluster of factors, including high energy density in the diet (high fat intake), low energy expenditure, and disturbed substrate oxidation, favour the increase in fat mass. Oxidation of three major macronutrients and their roles in the regulation of energy balance, particularly in children and adolescents, are discussed. Total glucose oxidation is not different between obese and lean children; exogenous glucose utilization is higher whereas endogenous glucose utilization is lower in obese compared with lean children. Carbohydrate composition of the diet determines carbohydrate oxidation regardless of fat content of the diet. Both exogenous and endogenous fat oxidation are higher in obese than in lean subjects. The influence of high fat intake on accumulation of fat mass is operative rather over a long term. Several future directions are addressed, such that a combination of increased physical activity and modification in diet composition, in terms of energy density and glycemic index, is recommended for children and adolescents.

CD44 attenuates activation of the hippo signaling pathway and is a prime therapeutic target for glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor that, by virtue of its resistance to chemotherapy and radiotherapy, is currently incurable. Identification of molecules whose targeting may eliminate GBM cells and/or sensitize glioblastoma cells to cytotoxic drugs is therefore urgently needed. CD44 is a major cell surface hyaluronan receptor and cancer stem cell marker that has been implicated in the progression of a variety of cancer types. However, the major downstream signaling pathways that mediate its protumor effects and the role of CD44 in the progression and chemoresponse of GBM have not been established. Here we show that CD44 is upregulated in GBM and that its depletion blocks GBM growth and sensitizes GBM cells to cytotoxic drugs in vivo. Consistent with this observation, CD44 antagonists potently inhibit glioma growth in preclinical mouse models. We provide the first evidence that CD44 functions upstream of the mammalian Hippo signaling pathway and that CD44 promotes tumor cell resistance to reactive oxygen species-induced and cytotoxic agent-induced stress by attenuating activation of the Hippo signaling pathway. Together, our results identify CD44 as a prime therapeutic target for GBM, establish potent antiglioma efficacy of CD44 antagonists, uncover a novel CD44 signaling pathway, and provide a first mechanistic explanation as to how upregulation of CD44 may constitute a key event in leading to cancer cell resistance to stresses of different origins. Finally, our results provide a rational explanation for the observation that functional inhibition of CD44 augments the efficacy of chemotherapy and radiation therapy.

Target organ damage: how to detect it and how to treat it?

The early detection of cardiac organ damage in clinical practice is primordial for cardiovascular risk profiling of patients with hypertension. In this respect the determination of microalbuminuria is very appealing because it increasingly appears to be the most cost-effective means to identify cardiovascular and renal complications. Considering the treatment of patients with target organ damage, blockers of the renin-angiotensin system have a key position as they are very effective in regressing left ventricular hypertrophy, lowering urinary albumin excretion and delaying the progression of nephropathy. In high-risk patients with atherosclerosis, the use of a blocker of the renin-angiotensin system is also appealing, and it appears increasingly judicious to combine such a blocker with a calcium antagonist whenever required to control blood pressure.

Target Molecular Size and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Analysis of the ATP-and Pyrophosphate-Dependent Proton Pumps from Maize Root Tonoplast.

Tonoplast-enriched membranes were prepared from maize (Zea mays L. cv LG 11) primary roots, using sucrose nonlinear gradients. The functional molecular size of the tonoplast ATP-and PPi-dependent proton pumps were analyzed by radiation inactivation. Glucose-6-phosphate dehydrogenase (G6PDH) was added as an internal standard. Frozen samples (-196 degrees C) of the membranes were irradiated with (60)Co for different periods of time. After thawing the samples, the activities of G6PDH, ATPase, and PPase were tested. By applying target theory, the functional sizes of the ATPase and PPase in situ were found to be around 540 and 160 kilodaltons, respectively. The two activities were solubilized and separated by gel filtration chromatography. The different polypeptides copurifying with the two pumps were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands (around 59 and 65 kilodaltons) were associated with the ATPase activity, whereas a double band (around 40 kilodaltons) was recovered with the PPase activity.

Early investigational drugs that target epidermal growth factor receptors for the treatment of head and neck cancer.

INTRODUCTION: Squamous-cell carcinoma of the head and neck (SCCHN) remains a challenging clinical problem, due to the persistent high rate of local and distant failures and the occurrence of secondary primaries. For locally advanced SCCHN, a combination of chemotherapy (CT), radiation or surgery is often used, but there are limitations, which may reduce compliance. Molecular targeted therapies, namely anti-EGFR treatments, are in development with the aim of improving clinical outcomes and mitigating treatment-related toxicities. AREAS COVERED: This review provides an overview of early investigational drugs that target EGFR for the treatment of SCCHN and discusses the ongoing trials in this domain. EXPERT OPINION: Targeted therapies are increasingly used in oncology, especially in SCCHN. Cetuximab has demonstrated a significant improvement in the treatment outcome, both as a curative treatment in combination with radiation therapy and as a palliative treatment in combination with CT; however, it failed to show any benefit in combination with concomitant chemoradiotherapy. Presently, there are many new agents, including monoclonal antibodies and small-molecule tyrosine kinase inhibitors, which are either currently under investigation for or which warrant further investigation for treating SCCHN. The discovery of predictive factors that help to identify patients most likely to respond to EGFR inhibitors as well as patient-customized therapies would help to improve patient outcomes in the future.

Blood pressure target attainment in the background of guidelines: the very elderly in Swiss primary care.

Background There are only a few trials for the very elderly population (>79 years). No consensus, which blood pressure (BP) goals and substances should be applied, has been found yet. This survey was undertaken to investigate how octogenarians are treated and attain BP targets in the Swiss primary care. Methods Data from 4594 hypertensive patients were collected within 7 days. Eight hundred and seventy-seven patients met the requirement to be >79 years. We assessed substances/combinations and investigated pulse pressure and target blood pressure attainment (TBPA) using three different recommendations [Canadian Hypertension Education Program (CHEP), Swiss Society of Hypertension (SSH), European Society of Hypertension-European Society of Cardiology (ESH-ESC)]. Secondarily, we compared TBPA attained by angiotensin-converting enzyme inhibitor (ACEI)/diuretic (D), angiotensin receptor blocker (ARB)/D and calcium channel blocker (CCB)/D with any other dual therapy and investigated whether Ds/beta-blockers (BBs) or Ds/renin angiotensin-converting enzyme inhibitors (RAAS-Is) lead to higher TBPA. Finally, we assessed the impact of drug administration, practical work experience, location and specialization of GPs on TBPA. Results Octogenarians attained target blood pressure (TBP) between 44% (ESH-ESC) and 74% (SSH). Optimal/normal BP was reached in 22.8% of patients. Pulse pressure <65 mmHg was shown in 66.4% of patients. Monotherapy was most commonly applied followed by dual single-pill combination with ARB/D (46.5%) or ACEI/D (36.0%). No benefit in TBPA was found comparing a RAASI/D and CCB/D treatment with any other dual combination. There was also no difference between BB/D and RAAS-I/D combination therapy and between single-pill combination and dual free combinations. Conclusions GPs adhere to the use of substances proven in outcome trials and attain high TBP. No difference in meeting BP goals could be found using different drug classes. There is an unmet need to harmonize recommendations and to add additional information for the treatment of octogenarians.

Cognitive issues in fingerprint analysis: Inter- and intra-expert consistency and the effect of a "target" comparison

Deciding whether two fingerprint marks originate from the same source requires examination and comparison of their features. Many cognitive factors play a major role in such information processing. In this paper we examined the consistency (both between- and within-experts) in the analysis of latent marks, and whether the presence of a 'target' comparison print affects this analysis. Our findings showed that the context of a comparison print affected analysis of the latent mark, possibly influencing allocation of attention, visual search, and threshold for determining a 'signal'. We also found that even without the context of the comparison print there was still a lack of consistency in analysing latent marks. Not only was this reflected by inconsistency between different experts, but the same experts at different times were inconsistent with their own analysis. However, the characterization of these inconsistencies depends on the standard and definition of what constitutes inconsistent. Furthermore, these effects were not uniform; the lack of consistency varied across fingerprints and experts. We propose solutions to mediate variability in the analysis of friction ridge skin.

Peroxisome proliferator-activated receptor beta/delta as a therapeutic target for metabolic diseases.

The peroxisome proliferator-activated receptor (PPAR) family comprises three distinct isotypes: PPARalpha, PPARbeta/delta and PPARgamma. PPARs are nuclear hormone receptors that mediate the effects of fatty acids and their derivatives at the transcriptional level. Until recently, the characterisation of the important role of PPARalpha in fatty acid oxidation and of PPARgamma in lipid storage contrasted with the sparse information concerning PPARbeta/delta. However, evidence is now emerging for a role of PPARbeta/delta in tissue repair and energy homeostasis. Experiments with tissue-specific overexpression of PPARbeta/delta or treatment of mice with selective PPARbeta/delta agonists demonstrated that activation of PPARbeta/delta in vivo increases lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in several animal models. PPARbeta/delta activation also prevents the development of obesity and improves cholesterol homeostasis in obesity-prone mouse models. These new insights into PPARbeta/delta functions suggest that targeting PPARbeta/delta may be helpful for treating disorders associated with the metabolic syndrome. Although these perspectives are promising, several independent and contradictory reports raise concerns about the safety of PPARbeta/delta ligands with respect to tumourigenic activity in the gut. Thus, it appears that further exploration of PPARbeta/delta functions is necessary to better define its potential as a therapeutic target.

Smoked cannabis and doping control: looking for the wrong target analyte?

Introduction: Since 2004, cannabis is prohibited by the World Anti-Doping Agency (WADA) for all sports in competition. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In most cases, the athletes plausibly claim to have consumed cannabis several days or even weeks before competition and only for recreational purposes not related to competition. In doping analysis, the target analyte in urine samples is 11-nor-delta-9-tetrahydrocannabinol- 9-carboxylic acid (THC-COOH), the reporting threshold for laboratories is 15 ng/mL. However, the wide detection window of this long-term THC metabolite in urine does not allow a conclusion concerning the time of consumption or the impact on the physical performance. Aim: The purpose of the present pharmacokinetic study on volunteers was to evaluate target analytes with shorter urinary excretion time. Subsequently, urines from athletes tested positive for cannabis should be reanalyzed including these analytes. Methods: In an one-session clinical trial (approved by IRB, Swissmedic, and Federal Office of Public Health), 12 healthy, male volunteers (age 26 ± 3 yrs, BMI 24 ± 2 kg/m2) with cannabis experience (> once/month) smoked a Cannabis cigarette standardized to 70 mg THC/cigarette (Bedrobinol® 7%, Dutch Office for Medicinal Cannabis) following a paced-puffing procedure. Plasma and urine was collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-THC (THC-OH), and THC-COOH were determined after enzymatic hydrolyzation followed by SPE and GC/MS-SIM. The limit of quantitation (LOQ) for all analytes was 0.1 ng/mL. Visual analog scales (VAS) and vital functions were used for monitoring psychological and somatic side-effects at every timepoint of specimen collection (up to 480 min). Results: Eight puffs delivered a mean THC dose of 45 mg. Mean plasma levels of total THC, THC-OH and THC-COOH were measured in the range of 0.1-20.9, 0.1-1.8, and 1.8-7.5 ng/mL, respectively. Peak concentrations were observed at 5, 10, and 90 min. Mean urine levels were measured in the range of 0.1-0.7, 0.10-6.2, and 0.1-13.4 ng/mL, respectively. The detection windows were 2-8, 2-96, and 2-120 h. No or only mild effects were observed, such as dry mouth, sedation, and tachycardia. Besides high to very high THC-COOH levels (0-978 ng/mL), THC (0.1-24 ng/mL) and THC-OH (1-234 ng/mL) were found in 90 and 96% of the cannabis-positive urines from athletes. Conclusion: Instead of or in addition to THC-COOH, the pharmacologically active THC and THC-OH should be the target analytes for doping urine analysis. This would allow the estimation of more recent Cannabis consumption, probably influencing performance during competition. Keywords: cannabis, doping, clinical trial, plasma and urine levels, athlete's samples

Type I signal peptidase from Leishmania is a target of the immune response in human cutaneous and visceral leishmaniasis.

The gene encoding type I signal peptidase (Lmjsp) has been cloned from Leishmania major. Lmjsp encodes a protein of 180 amino residues with a predicted molecular mass of 20.5 kDa. Comparison of the protein sequence with those of known type I signal peptidases indicates homology in five conserved domains A-E which are known to be important, or essential, for catalytic activity. Southern blot hybridisation analysis indicates that there is a single copy of the Lmjsp gene. A recombinant SPase protein and a synthetic peptide of the L. major signal peptidase were used to examine the presence of specific antibodies in sera from either recovered or active individuals of both cutaneous and visceral leishmaniasis. This evaluation demonstrated that sera from cutaneous and visceral forms of leishmaniasis are highly reactive to both the recombinant and synthetic signal peptidase antigens. Therefore, the Leishmania signal peptidase, albeit localised intracellularly, is a significant target of the Leishmania specific immune response and highlights its potential use for serodiagnosis of cutaneous and visceral leishmaniasis.

MAF1: a new target of mTORC1.

Yeast and mammalian MAF1 are both regulated by the TOR (target of rapamycin) pathway. However, the exact mechanisms of regulation diverge at TOR, with yeast Maf1 phosphorylated mainly by the TORC1 (TOR complex 1) substrate Sch9 kinase and mammalian MAF1 by mTORC1 (mammalian target of rapamycin complex 1) itself. Sch9 phosphorylation of yeast Maf1 regulates Maf1 localization, but it is less clear whether phosphorylation of human MAF1 regulates its localization. Replacement of phosphosites with alanine decreases Pol III (RNA polymerase III) transcription, but the effect is much more pronounced for human MAF1 than for the yeast protein. In both cases, Pol III repression can be further increased by rapamycin treatment or, in mammalian cells, serum starvation, suggesting that the TOR pathway controls another aspect of Pol III transcription that is closely linked to MAF1, as it depends on the presence of MAF1.