Ghrelin-induced orexigenic effect in rats depends on the metabolic status and is counteracted by peripheral CB1 receptor antagonism.

Ghrelin is an endogenous regulator of energy homeostasis synthesized by the stomach to stimulate appetite and positive energy balance. Similarly, the endocannabinoid system is part of our internal machinery controlling food intake and energy expenditure. Both peripheral and central mechanisms regulate CB1-mediated control of food intake and a functional relationship between hypothalamic ghrelin and cannabinoid CB1 receptor has been proposed. First of all, we investigated brain ghrelin actions on food intake in rats with different metabolic status (negative or equilibrate energy balance). Secondly, we tested a sub-anxiogenic ultra-low dose of the CB1 antagonist SR141716A (Rimonabant) and the peripheral-acting CB1 antagonist LH-21 on ghrelin orexigenic actions. We found that: 1) central administration of ghrelin promotes food intake in free feeding animals but not in 24 h food-deprived or chronically food-restricted animals; 2) an ultra-low dose of SR141716A (a subthreshold dose 75 folds lower than the EC50 for induction of anxiety) completely counteracts the orexigenic actions of central ghrelin in free feeding animals; 3) the peripheral-restricted CB1 antagonist LH-21 blocks ghrelin-induced hyperphagia in free feeding animals. Our study highlights the importance of the animaĺs metabolic status for the effectiveness of ghrelin in promoting feeding, and suggests that the peripheral endocannabinoid system may interact with ghrelińs signal in the control of food intake under equilibrate energy balance conditions.

Cell vacuolation induced by Haemophilus influenzae supernatants in HEp-2 cells

Haemophilus influenzae belongs to respiratory tract microbiota. We observed vacuoles formation in previous studies with H. influenzae culture supernatants, so in this work we characterised that cytotoxic effect. We observed an abundant production of acidic cytoplasmic vacuoles due to the presence of a “vacuolating factor” in H. influenzae supernatants which was characterised as thermolabile. Greatest vacuolating activity was observed when utilizing the fraction > 50 kDa. The presence of a large number of vacuoles in HEp-2 cells was verified by transmission electron microscopy and some vacuoles were identified with a double membrane and/or being surrounded by ribosomes. These results suggest similar behaviour to that of vacuolating effects described by autotransporter proteins an undescribed cytotoxic effect induced by H. influenzae .

Intérêt des marqueurs sanguins dans la prédiction de la toxicité radio-induite [Interest of blood markers in predicting radiation-induced toxicity].

The oncologic outcome and the total dose are highly correlated with the treatment by ionizing radiation. The dose increase (total or per fraction) may provoke late-side effects that are potentially irreversible. The radiation-induced CD8 lymphocyte apoptotic value and the molecular modifications within the lymphocyte are capable of predicting the level of risk of developing late-side effects after curative intent radiotherapy. In this review, we present the different blood assays in this setting and discuss the current possibilities of researches, namely those involving the proteomic process.

PARP-1 regulates metastatic melanoma through modulation of vimentin-induced malignant transformation.

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.

Blood-brain barrier disruption associated with topiramate-induced angle-closure glaucoma of acute onset.

BACKGROUND: Topiramate (Topamax(R)) is an anti-epileptic drug of the sulfamate group used secondarily for bipolar disease. HISTORY AND SIGNS: One week after initiation of topiramate treatment for a bipolar disorder, a 57-year-old man presented with blurred vision. Clinical examination revealed a bilateral conjunctivitis, areflexic mydriasis, severe anterior chamber shallowing, with a myopic shift and vitritis. THERAPY AND OUTCOME: A spinal tap revealed an increased protein content of 1581 mg/L on cerebrospinal fluid (CSF) analysis, being compatible with a rupture of the blood-brain barrier (BBB). UBM exposed bilateral ciliochoroidal effusions with secondary angle-closure. Topiramate was promptly discontinued, whereas visual acuity, intraocular pressure (IOP), and anterior and posterior segments anatomy normalized within 1 week. One month later, bilateral iris atrophy was present. CONCLUSION: The presence of BBB disruption with increased protein content in CSF with simultaneous blood ocular barrier breakdown may suggest a common inflammatory mechanism.

Detection of neuronal activity and metabolism in a model of dehydration-induced anorexia in rats at 14.1 T using manganese-enhanced MRI and 1H MRS.

In this study, hypothalamic activation was performed by dehydration-induced anorexia (DIA) and overnight food suppression (OFS) in female rats. The assessment of the hypothalamic response to these challenges by manganese-enhanced MRI showed increased neuronal activity in the paraventricular nuclei (PVN) and lateral hypothalamus (LH), both known to be areas involved in the regulation of food intake. The effects of DIA and OFS were compared by generating T-score maps. Increased neuronal activation was detected in the PVN and LH of DIA rats relative to OFS rats. In addition, the neurochemical profile of the PVN and LH were measured by (1) H MRS at 14.1T. Significant increases in metabolite levels were measured in DIA and OFS relative to control rats. Statistically significant increases in γ-aminobutyric acid were found in DIA (p=0.0007) and OFS (p<0.001) relative to control rats. Lactate increased significantly in DIA (p=0.03), but not in OFS, rats. This work shows that manganese-enhanced MRI coupled to (1) H MRS at high field is a promising noninvasive method for the investigation of the neural pathways and mechanisms involved in the control of food intake, in the autonomic and endocrine control of energy metabolism and in the regulation of body weight.

Effects of a lifestyle intervention on adiposity and fitness in overweight or low fit preschoolers (Ballabeina).

OBJECTIVE: Overweight (OW) and low fit children represent cardiovascular high-risk groups. A multidimensional school-based lifestyle intervention performed in 652 preschoolers reduced skinfold thickness and waist circumference, and improved fitness, but did not affect BMI. The objective of this study is to examine whether the intervention was equally effective in OW (≥90th national percentile) and/or low fit (lowest sex- and age-adjusted quartile of aerobic fitness) children compared to their normal weight and normal fit counterparts. DESIGN AND METHODS: Cluster randomized controlled single blinded trial, conducted in 2008/09 in 40 randomly selected preschool classes in Switzerland. The intervention included a playful physical activity program and lessons on nutrition, media use and sleeps. Primary outcomes were BMI and aerobic fitness; secondary outcomes included sum of four skinfolds, waist circumference and motor agility. Modification of intervention effects by BMI-group and fitness-group was tested by interaction terms. RESULTS: Compared to their counterparts, OW children (n = 130) had more beneficial effects on waist circumference (p for interaction = 0.001) and low fit children (n = 154) more beneficial effects on all adiposity outcomes (p for interaction ≤0.03). The intervention effects on both fitness outcomes were not modified by BMI- or fitness-group (all p for interaction ≥0.2). Average intervention effect sizes for BMI were -0.12, -0.05, -0.26 and -0.02 kg/m(2) and for aerobic fitness were 0.40, 0.30, 0.12 and 0.36 stages for OW, normal weight, low fit and normal fit children, respectively. Conclusions: This multidimensional intervention was equally and for some adiposity measures even more effective in high-risk preschoolers and represents a promising option for these children.

Water-induced modulation of Helicobacter pylori virulence properties

While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium’s ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.

Pharmacological blockade of either cannabinoid CB1 or CB2 receptors prevents both cocaine-induced conditioned locomotion and cocaine-induced reduction of cell proliferation in the hippocampus of adult male rat.

Addiction to major drugs of abuse, such as cocaine, has recently been linked to alterations in adult neurogenesis in the hippocampus. The endogenous cannabinoid system modulates this proliferative response as demonstrated by the finding that pharmacological activation/blockade of cannabinoid CB1 and CB2 receptors not only modulates neurogenesis but also modulates cell death in the brain. In the present study, we evaluated whether the endogenous cannabinoid system affects cocaine-induced alterations in cell proliferation. To this end, we examined whether pharmacological blockade of either CB1 (Rimonabant, 3 mg/kg) or CB2 receptors (AM630, 3 mg/kg) would affect cell proliferation [the cells were labeled with 5-bromo-2'-deoxyuridine (BrdU)] in the subventricular zone (SVZ) of the lateral ventricle and the dentate subgranular zone (SGZ). Additionally, we measured cell apoptosis (as monitored by the expression of cleaved caspase-3) and glial activation [by analyzing the expression of glial fibrillary acidic protein (GFAP) and Iba-1] in the striatum and hippocampus during acute and repeated (4 days) cocaine administration (20 mg/kg). The results showed that acute cocaine exposure decreased the number of BrdU-immunoreactive (ir) cells in the SVZ and SGZ. In contrast, repeated cocaine exposure reduced the number of BrdU-ir cells only in the SVZ. Both acute and repeated cocaine exposure increased the number of cleaved caspase-3-, GFAP- and Iba1-ir cells in the hippocampus, and this effect was counteracted by AM630 or Rimonabant, which increased the number of BrdU-, GFAP-, and Iba1-ir cells in the hippocampus. These results indicate that the changes in neurogenic, apoptotic and gliotic processes that were produced by repeated cocaine administration were normalized by pharmacological blockade of CB1 and CB2. The restorative effects of cannabinoid receptor blockade on hippocampal cell proliferation were associated with the prevention of the induction of conditioned locomotion but not with the prevention of cocaine-induced sensitization.

Selected ABCB1, ABCB4 and ABCC2 Polymorphisms Do Not Enhance the Risk of Drug-Induced Hepatotoxicity in a Spanish Cohort.

BACKGROUND AND AIMS Flawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort. METHODS A total of ten polymorphisms in ABCB1 (1236T>C, 2677G>T,A, 3435T>C), ABCB4 (1954A>G) and ABCC2 (-1774G>del, -1549A>G, -24C>T, 1249G>A, 3972C>T and 4544G>A) were genotyped using Taqman 5' allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed. RESULTS None of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCC2 -1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 -1774G/-1549A/-24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI. CONCLUSIONS Our findings do not support a role for the analysed polymorphisms in the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 -1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.

Molecular characterization of signalling complexes involved in G-protein coupled receptor-induced cardiac hypertrophy

In response to pathological stresses, the heart undergoes a remodelling process associated with cardiac hypertrophy. Since sustained hypertrophy can progress to heart failure, there is an intense investigation about the intracellular signalling pathways that control cardiomyocyte growth. Accumulating evidence has demonstrated that most stimuli known to initiate pathological changes associated with the development of cardiac hypertrophy activate G protein-coupled receptors (GPCRs) including the αl-adrenergic- (αl-AR), Angiotensin II- (AT-R) and endothelin-1- (ET-R) receptors. In this context, we have previously identified a cardiac scaffolding protein, called AKAP-Lbc (Α-kinase anchoring protein), with an intrinsic Rho specific guanine nucleotide exchange factor activity, that plays a key role in integrating and transducing hypertrophic signals initiated by these GPCRs (Appert-Collin, Cotecchia et al. 2007). Activated RhoA controls the transcriptional activation of genes involved in cardiomyocyte hypertrophy through signalling pathways that remain to be characterized. Here, we identified the nuclear factor-Kappa Β (NF-κΒ) activating kinase ΙΚΚβ as a novel AKAP-Lbc interacting protein. This raises the hypothesis that AKAP-Lbc might promote cardiomyocyte growth by maintaining a signalling complex that promotes the activation of the pro-hypertrophic transcription factor NF-κΒ. In fact, the activation of NF- κΒ-dependent transcription has been detected in numerous disease contexts, including hypertrophy, ischemia/reperfusion injury, myocardial infarction, allograft rejection, myocarditis, apoptosis, and more (Hall, Hasday et al. 2006). While it is known by more than a decade that NF-κΒ is a critical mediator of cardiac hypertrophy, it is currently poorly understood how pro-hypertrophic signals controlling NF-κΒ transcriptional activity are integrated and coordinated within cardiomyocytes. In this study, we show that AKAP-Lbc and ΙΚΚβ form a transduction complex in cardiomyocytes that couples activation of αl-ARs to NF-κB-mediated transcriptional reprogramming events associated with cardiomyocyte hypertrophy. In particular, we can show that activation of ΙΚΚβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukine-6 (IL-6), which, in turn, enhances foetal gene expression. These findings indicate that the AKAP-Lbc/ΙΚΚβ complex is critical for selectively directing catecholamine signals to the induction of cardiomyocyte hypertrophy.

The use of liver biopsy evaluation in discrimination of idiopathic autoimmune hepatitis versus drug-induced liver injury.

Distinguishing drug-induced liver injury (DILI) from idiopathic autoimmune hepatitis (AIH) can be challenging. We performed a standardized histologic evaluation to explore potential hallmarks to differentiate AIH versus DILI. Biopsies from patients with clinically well-characterized DILI [n = 35, including 19 hepatocellular injury (HC) and 16 cholestatic/mixed injury (CS)] and AIH (n = 28) were evaluated for Ishak scores, prominent inflammatory cell types in portal and intra-acinar areas, the presence or absence of emperipolesis, rosette formation, and cholestasis in a blinded fashion by four experienced hepatopathologists. Histologic diagnosis was concordant with clinical diagnosis in 65% of cases; but agreement on final diagnosis among the four pathologists was complete in only 46% of cases. Interface hepatitis, focal necrosis, and portal inflammation were present in all evaluated cases, but were more severe in AIH (P < 0.05) than DILI (HC). Portal and intra-acinar plasma cells, rosette formation, and emperiopolesis were features that favored AIH (P < 0.02). A model combining portal inflammation, portal plasma cells, intra-acinar lymphocytes and eosinophils, rosette formation, and canalicular cholestasis yielded an area under the receiver operating characteristic curve (AUROC) of 0.90 in predicting DILI (HC) versus AIH. All Ishak inflammation scores were more severe in AIH than DILI (CS) (P ≤ 0.05). The four AIH-favoring features listed above were consistently more prevalent in AIH, whereas portal neutrophils and intracellular (hepatocellular) cholestasis were more prevalent in DILI (CS) (P < 0.02). The combination of portal inflammation, fibrosis, portal neutrophils and plasma cells, and intracellular (hepatocellular) cholestasis yielded an AUC of 0.91 in predicting DILI (CS) versus AIH. Conclusion: Although an overlap of histologic findings exists for AIH and DILI, sufficient differences exist so that pathologists can use the pattern of injury to suggest the correct diagnosis.

Drug-induced autoimmune liver disease: A diagnostic dilemma of an increasingly reported disease.

The aetiology of autoimmune hepatitis (AIH) is uncertain but the disease can be triggered in susceptible patients by external factors such as viruses or drugs. AIH usually develops in individuals with a genetic background mainly consisting of some risk alleles of the major histocompatibility complex (HLA). Many drugs have been linked to AIH phenotypes, which sometimes persist after drug discontinuation, suggesting that they awaken latent autoimmunity. At least three clinical scenarios have been proposed that refers to drug- induced autoimmune liver disease (DIAILD): AIH with drug-induced liver injury (DILI); drug induced-AIH (DI-AIH); and immune mediated DILI (IM-DILI). In addition, there are instances showing mixed features of DI-AIH and IM-DILI, as well as DILI cases with positive autoantibodies. Histologically distinguishing DILI from AIH remains a challenge. Even more challenging is the differentiation of AIH from DI-AIH mainly relying in histological features; however, a detailed standardised histologic evaluation of large cohorts of AIH and DI-AIH patients would probably render more subtle features that could be of help in the differential diagnosis between both entities. Growing information on the relationship of drugs and AIH is being available, being drugs like statins and biologic agents more frequently involved in cases of DIAILD. In addition, there is some evidence on the fact that patients diagnosed with DIAILD may have had a previous episode of hepatotoxicity. Further collaborative studies in DIAILD will strengthen the knowledge and understanding of this intriguing and complex disorder which might represent different phenotypes across the spectrum of disease.

The rise of soluble TWEAK levels in severely obese subjects after bariatric surgery may affect adipocyte-cytokine production induced by TNFα.

CONTEXT Soluble TNF-like weak inducer of apoptosis (sTWEAK) is generated by the intracellular proteolytic cleavage of full-length membrane-bound TNF-like weak inducer of apoptosis (mTWEAK). sTWEAK levels are reduced in diseases with an inflammatory component. Additionally, sTWEAK hampers TNFα activity in human cells. OBJECTIVES The objectives of the study were as follows: 1) to determine circulating sTWEAK in severe obesity and after bariatric surgery; 2) to study m/sTWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) protein expression in sc adipose tissue (SAT) of severely obese subjects, in SAT stromal vascular fraction (SVF), and isolated adipocytes and in human monocyte-derived macrophages; and 3) to explore, on human adipocytes, the sTWEAK effect on TNFα proinflammatory activity. DESIGN sTWEAK levels were measured in cohort 1: severely obese subjects (n = 23) and a control group (n = 35); and in cohort 2: (n = 23) severely obese subjects before and after surgery. The m/sTWEAK and Fn14 expressions were determined in SAT biopsies, SVF, and isolated adipocytes from severely obese and control subjects and in human monocyte-derived macrophages. In human primary cultured adipocytes, sTWEAK pretreated and TNFα challenged, IL-6, IL-8, and adiponectin protein and gene expressions were determined and nuclear factor-κ B and MAPK signaling analyzed. RESULTS sTWEAK levels were reduced in severely obese subjects. After surgery, sTWEAK levels rose in 69% of patients. mTWEAK protein expression was increased in SAT and SVF of severely obese subjects, whereas Fn14 was up-regulated in isolated adipocytes. M2 human monocyte-derived macrophages overexpress mTWEAK. In human adipocytes, sTWEAK down-regulates TNFα cytokine production by hampering TNFα intracellular signaling events. CONCLUSION The decrease of sTWEAK in severely obese patients may favor the proinflammatory activity elicited by TNFα.

Inconspicuous assessment of diet-induced thermogenesis using whole-body indirect calorimetry.

We report a novel technique for computing diet-induced thermogenesis using data from 24-h respiration chamber measurements of 76 subjects. Physical activity (PA) was determined using a radar system to assess its duration and an accelerometer to evaluate its intensity. The regression line relating PA and energy expenditure facilitated calculation of the integrated thermogenic response to the total energy ingested (11.4% ± 3.8%), which is consistent with the values classically reported in the literature (10%) at the group level.