A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

Computational and Biological Evaluation of N-octadecyl-N'-propylsulfamide, a Selective PPARα Agonist Structurally Related to N-acylethanolamines.

To further understand the pharmacological properties of N-oleoylethanolamine (OEA), a naturally occurring lipid that activates peroxisome proliferator-activated receptor alpha (PPARα), we designed sulfamoyl analogs based on its structure. Among the compounds tested, N-octadecyl-N'-propylsulfamide (CC7) was selected for functional comparison with OEA. The performed studies include the following computational and biological approaches: 1) molecular docking analyses; 2) molecular biology studies with PPARα; 3) pharmacological studies on feeding behavior and visceral analgesia. For the docking studies, we compared OEA and CC7 data with crystallization data obtained with the reference PPARα agonist GW409544. OEA and CC7 interacted with the ligand-binding domain of PPARα in a similar manner to GW409544. Both compounds produced similar transcriptional activation by in vitro assays, including the GST pull-down assay and reporter gene analysis. In addition, CC7 and OEA induced the mRNA expression of CPT1a in HpeG2 cells through PPARα and the induction was avoided with PPARα-specific siRNA. In vivo studies in rats showed that OEA and CC7 had anorectic and antiobesity activity and induced both lipopenia and decreases in hepatic fat content. However, different effects were observed when measuring visceral pain; OEA produced visceral analgesia whereas CC7 showed no effects. These results suggest that OEA activity on the PPARα receptor (e.g., lipid metabolism and feeding behavior) may be dissociated from other actions at alternative targets (e.g., pain) because other non cannabimimetic ligands that interact with PPARα, such as CC7, do not reproduce the full spectrum of the pharmacological activity of OEA. These results provide new opportunities for the development of specific PPARα-activating drugs focused on sulfamide derivatives with a long alkyl chain for the treatment of metabolic dysfunction.

Tuberculosis in migrant populations. A systematic review of the qualitative literature.

BACKGROUND The re-emergence of tuberculosis (TB) in low-incidence countries and its disproportionate burden on immigrants is a public health concern posing specific social and ethical challenges. This review explores perceptions, knowledge, attitudes and treatment adherence behaviour relating to TB and their social implications as reported in the qualitative literature. METHODS Systematic review in four electronic databases. Findings from thirty selected studies extracted, tabulated, compared and synthesized. FINDINGS TB was attributed to many non-exclusive causes including air-born transmission of bacteria, genetics, malnutrition, excessive work, irresponsible lifestyles, casual contact with infected persons or objects; and exposure to low temperatures, dirt, stress and witchcraft. Perceived as curable but potentially lethal and highly contagious, there was confusion around a condition surrounded by fears. A range of economic, legislative, cultural, social and health system barriers could delay treatment seeking. Fears of deportation and having contacts traced could prevent individuals from seeking medical assistance. Once on treatment, family support and "the personal touch" of health providers emerged as key factors facilitating adherence. The concept of latent infection was difficult to comprehend and while TB screening was often seen as a socially responsible act, it could be perceived as discriminatory. Immigration and the infectiousness of TB mutually reinforced each another exacerbating stigma. This was further aggravated by indirect costs such as losing a job, being evicted by a landlord or not being able to attend school. CONCLUSIONS Understanding immigrants' views of TB and the obstacles that they face when accessing the health system and adhering to a treatment programme-taking into consideration their previous experiences at countries of origin as well as the social, economic and legislative context in which they live at host countries- has an important role and should be considered in the design, evaluation and adaptation of programmes.

Effect of a specific supplement enriched with n-3 polyunsaturated fatty acids on markers of inflammation, oxidative stress and metabolic status of ear, nose and throat cancer patients.

Malnutrition affects 40-50% of patients with ear, nose and throat (ENT) cancer. The aim of this study was to assess changes induced by a specific nutritional supplement enriched with n-3 polyunsaturated fatty acids, fiber and greater amounts of proteins and electrolytes, as compared with a standard nutritional supplement, on markers of inflammation, oxidative stress and metabolic status of ENT cancer patients undergoing radiotherapy (RT). Fourteen days after starting RT, 26 patients were randomly allocated to one of two groups, 13 supplemented with Prosure, an oncologic formula enriched with n-3 polyunsaturated fatty acids, fiber and greater amounts of proteins and electrolytes (specific supplement), and 13 supplemented with Standard-Isosource (standard supplement). Patients were evaluated before RT, and 14, 28 and 90 days after starting RT. The results showed that there were no significant differences between the groups, but greater changes were observed in the standard supplement group, such as a decline in body mass index (BMI), reductions in hematocrit, erythrocyte, eosinophil and albumin levels, and a rise in creatinine and urea levels. We concluded that metabolic, inflammatory and oxidative stress parameters were altered during RT, and began to normalize at the end of the study. Patients supplemented with Prosure showed an earlier normalization of these parameters, with more favorable changes in oxidative stress markers and a more balanced evolution, although the difference was not significant.

Long-term effects of varying consumption of ω3 fatty acids in ear, nose and throat cancer patients: assessment 1 year after radiotherapy.

Abstract A prospective 1-year follow-up study in ear, nose, and throat (ENT) cancer patients was carried out one year after radiotherapy to assess the effect of varying consumption of ω3 fatty acid according to whether they consumed more or less than the 50th percentile of ω3 fatty acids. Clinical, analytical, inflammatory (CRP and IL-6), and oxidative variables (TAC, GPx, GST, and SOD) were evaluated. The study comprised 31 patients (87.1% men), with a mean age of 61.3 ± 9.1 years. Hematological variables showed significant differences in the patients with a lower consumption of ω3 fatty acids. A lower mortality and longer survival were found in the group with ω3 fatty acid consumption ≥50th percentile but the differences were not significant. No significant difference was reached in toxicity, inflammation, and oxidative stress markers. The group with ω3 fatty acid consumption <50th percentile significantly experienced more hematological and immune changes.

The administration of atomoxetine during alcohol deprivation induces a time-limited increase in alcohol consumption after relapse.

The administration of selective serotonin reuptake inhibitors (SSRIs) typically used as antidepressants increases alcohol consumption after an alcohol deprivation period in rats. However, the appearance of this effect after the treatment with selective noradrenaline reuptake inhibitors (SNRIs) has not been studied. In the present work we examined the effects of a 15-d treatment with the SNRI atomoxetine (1, 3 and 10 mg/kg, i.p.) in male rats trained to drink alcohol solutions in a 4-bottle choice test. The treatment with atomoxetine (10 mg/kg, i.p.) during an alcohol deprivation period increased alcohol consumption after relapse. This effect only lasted one week, disappearing thereafter. Treatment with atomoxetine did not cause a behavioral sensitized response to a challenge dose of amphetamine (1.5 mg/kg, i.p.), indicating the absence of a supersensitive dopaminergic transmission. This effect is markedly different from that of SSRI antidepressants that produced both long-lasting increases in alcohol consumption and behavioral sensitization. Clinical implications are discussed.

CB1 blockade potentiates down-regulation of lipogenic gene expression in perirenal adipose tissue in high carbohydrate diet-induced obesity.

De novo lipogenesis and hypercaloric diets are thought to contribute to increased fat mass, particularly in abdominal fat depots. CB1 is highly expressed in adipose tissue, and CB1-mediated signalling is associated with stimulation of lipogenesis and diet-induced obesity, though its contribution to increasing fat deposition in adipose tissue is controversial. Lipogenesis is regulated by transcription factors such as liver X receptor (LXR), sterol-response element binding protein (SREBP) and carbohydrate-responsive-element-binding protein (ChREBP). We evaluated the role of CB1 in the gene expression of these factors and their target genes in relation to lipogenesis in the perirenal adipose tissue (PrAT) of rats fed a high-carbohydrate diet (HCHD) or a high-fat diet (HFD). Both obesity models showed an up-regulated gene expression of CB1 and Lxrα in this adipose pad. The Srebf-1 and ChREBP gene expressions were down-regulated in HFD but not in HCHD. The expression of their target genes encoding for lipogenic enzymes showed a decrease in diet-induced obesity and was particularly dramatic in HFD. In HCHD, CB1 blockade by AM251 reduced the Srebf-1 and ChREBP expression and totally abrogated the remnant gene expression of their target lipogenic enzymes. The phosphorylated form of the extracellular signal-regulated kinase (ERK-p), which participates in the CB1-mediated signalling pathway, was markedly present in the PrAT of obese rats. ERK-p was drastically repressed by AM251 indicating that CB1 is actually functional in PrAT of obese animals, though its activation loses the ability to stimulate lipogenesis in PrAT of obese rats. Even so, the remnant expression levels of lipogenic transcription factors found in HCHD-fed rats are still dependent on CB1 activity. Hence, in HCHD-induced obesity, CB1 blockade may help to further potentiate the reduction of lipogenesis in PrAT by means of inducing down-regulation of the ChREBP and Srebf-1 gene expression, and consequently in the expression of lipogenic enzymes.

Cost-effectiveness of an exercise intervention program in perimenopausal women: the Fitness League Against MENopause COst (FLAMENCO) randomized controlled trial.

BACKGROUND The high prevalence of women that do not reach the recommended level of physical activity is worrisome. A sedentary lifestyle has negative consequences on health status and increases health care costs. The main objective of this project is to assess the cost-effectiveness of a primary care-based exercise intervention in perimenopausal women. METHODS/DESIGN The present study is a Randomized Controlled Trial. A total of 150 eligible women will be recruited and randomly assigned to either a 16-week exercise intervention (3 sessions/week), or to usual care (control) group. The primary outcome measure is the incremental cost-effectiveness ratio. The secondary outcome measures are: i) socio-demographic and clinical information; ii) body composition; iii) dietary patterns; iv) glycaemic and lipid profile; v) physical fitness; vi) physical activity and sedentary behaviour; vii) sleep quality; viii) quality of life, mental health and positive health; ix) menopause symptoms. All outcomes will be assessed at baseline and post intervention. The data will be analysed on an intention-to-treat basis and per protocol. In addition, we will conduct a cost effectiveness analysis from a health system perspective. DISCUSSION The intervention designed is feasible and if it proves to be clinically and cost effective, it can be easily transferred to other similar contexts. Consequently, the findings of this project might help the Health Systems to identify strategies for primary prevention and health promotion as well as to reduce health care requirements and costs. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT02358109 . Date of registration: 05/02/2015.

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells.

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.

Disruption of GIP/GIPR axis in human adipose tissue is linked to obesity and insulin resistance.

CONTEXT Glucose-dependent insulinotropic peptide (GIP) has a central role in glucose homeostasis through its amplification of insulin secretion; however, its physiological role in adipose tissue is unclear. OBJECTIVE Our objective was to define the function of GIP in human adipose tissue in relation to obesity and insulin resistance. DESIGN GIP receptor (GIPR) expression was analyzed in human sc adipose tissue (SAT) and visceral adipose (VAT) from lean and obese subjects in 3 independent cohorts. GIPR expression was associated with anthropometric and biochemical variables. GIP responsiveness on insulin sensitivity was analyzed in human adipocyte cell lines in normoxic and hypoxic environments as well as in adipose-derived stem cells obtained from lean and obese patients. RESULTS GIPR expression was downregulated in SAT from obese patients and correlated negatively with body mass index, waist circumference, systolic blood pressure, and glucose and triglyceride levels. Furthermore, homeostasis model assessment of insulin resistance, glucose, and G protein-coupled receptor kinase 2 (GRK2) emerged as variables strongly associated with GIPR expression in SAT. Glucose uptake studies and insulin signaling in human adipocytes revealed GIP as an insulin-sensitizer incretin. Immunoprecipitation experiments suggested that GIP promotes the interaction of GRK2 with GIPR and decreases the association of GRK2 to insulin receptor substrate 1. These effects of GIP observed under normoxia were lost in human fat cells cultured in hypoxia. In support of this, GIP increased insulin sensitivity in human adipose-derived stem cells from lean patients. GIP also induced GIPR expression, which was concomitant with a downregulation of the incretin-degrading enzyme dipeptidyl peptidase 4. None of the physiological effects of GIP were detected in human fat cells obtained from an obese environment with reduced levels of GIPR. CONCLUSIONS GIP/GIPR signaling is disrupted in insulin-resistant states, such as obesity, and normalizing this function might represent a potential therapy in the treatment of obesity-associated metabolic disorders.

Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase.

OBJECTIVE Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes. METHODS ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR. RESULTS ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG. CONCLUSIONS ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.

Modulation of Higher-Order Olfaction Components on Executive Functions in Humans.

The prefrontal (PFC) and orbitofrontal cortex (OFC) appear to be associated with both executive functions and olfaction. However, there is little data relating olfactory processing and executive functions in humans. The present study aimed at exploring the role of olfaction on executive functioning, making a distinction between primary and more cognitive aspects of olfaction. Three executive tasks of similar difficulty were used. One was used to assess hot executive functions (Iowa Gambling Task-IGT), and two as a measure of cold executive functioning (Stroop Colour and Word Test-SCWT and Wisconsin Card Sorting Test-WCST). Sixty two healthy participants were included: 31 with normosmia and 31 with hyposmia. Olfactory abilities were assessed using the ''Sniffin' Sticks'' test and the olfactory threshold, odour discrimination and odour identification measures were obtained. All participants were female, aged between 18 and 60. Results showed that participants with hyposmia displayed worse performance in decision making (IGT; Cohen's-d = 0.91) and cognitive flexibility (WCST; Cohen's-d between 0.54 and 0.68) compared to those with normosmia. Multiple regression adjusted by the covariates participants' age and education level showed a positive association between odour identification and the cognitive inhibition response (SCWT-interference; Beta = 0.29; p = .034). The odour discrimination capacity was not a predictor of the cognitive executive performance. Our results suggest that both hot and cold executive functions seem to be associated with higher-order olfactory functioning in humans. These results robustly support the hypothesis that olfaction and executive measures have a common neural substrate in PFC and OFC, and suggest that olfaction might be a reliable cognitive marker in psychiatric and neurologic disorders.

An in vitro iron superoxide dismutase inhibitor decreases the parasitemia levels of Trypanosoma cruzi in BALB/c mouse model during acute phase.

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), two hydroxyphthalazine derivative compounds were prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels, by (1)H NMR and TEM studies. Finally, as these compounds are potentially capable of causing oxidative damage in the parasites, the study was completed, by assessing their activity as potential iron superoxide dismutase (Fe-SOD) inhibitors. High-selectivity indices observed in vitro were the basis of promoting one of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Compound 2 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression. Compound 2 turned out to be a great inhibitor of Fe-SOD. The high antiparasitic activity and low toxicity together with the modest costs for the starting materials render this compound an appropriate molecule for the development of an affordable anti-Chagas agent.

Gut Microbiota Composition in Male Rat Models under Different Nutritional Status and Physical Activity and Its Association with Serum Leptin and Ghrelin Levels.

BACKGROUND Several evidences indicate that gut microbiota is involved in the control of host energy metabolism. OBJECTIVE To evaluate the differences in the composition of gut microbiota in rat models under different nutritional status and physical activity and to identify their associations with serum leptin and ghrelin levels. METHODS IN A CASE CONTROL STUDY, FORTY MALE RATS WERE RANDOMLY ASSIGNED TO ONE OF THESE FOUR EXPERIMENTAL GROUPS: ABA group with food restriction and free access to exercise; control ABA group with food restriction and no access to exercise; exercise group with free access to exercise and feed ad libitum and ad libitum group without access to exercise and feed ad libitum. The fecal bacteria composition was investigated by PCR-denaturing gradient gel electrophoresis and real-time qPCR. RESULTS In restricted eaters, we have found a significant increase in the number of Proteobacteria, Bacteroides, Clostridium, Enterococcus, Prevotella and M. smithii and a significant decrease in the quantities of Actinobacteria, Firmicutes, Bacteroidetes, B. coccoides-E. rectale group, Lactobacillus and Bifidobacterium with respect to unrestricted eaters. Moreover, a significant increase in the number of Lactobacillus, Bifidobacterium and B. coccoides-E. rectale group was observed in exercise group with respect to the rest of groups. We also found a significant positive correlation between the quantity of Bifidobacterium and Lactobacillus and serum leptin levels, and a significant and negative correlation among the number of Clostridium, Bacteroides and Prevotella and serum leptin levels in all experimental groups. Furthermore, serum ghrelin levels were negatively correlated with the quantity of Bifidobacterium, Lactobacillus and B. coccoides-Eubacterium rectale group and positively correlated with the number of Bacteroides and Prevotella. CONCLUSIONS Nutritional status and physical activity alter gut microbiota composition affecting the diversity and similarity. This study highlights the associations between gut microbiota and appetite-regulating hormones that may be important in terms of satiety and host metabolism.

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.