Obesity dependent metabolic signatures associated with nonalcoholic fatty liver disease progression

Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultra-performance liquid chromatography coupled to mass spectrometry (UPLCMS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual’s level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values = 0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention

³Neural stimulation and recording electrodes´

HE use of diamond for neural stimulation has been of interest, as the mechanical, chemical and biological stability of the material are advantageous All coatings were applied to a semi-spherical monopolar Ti6Al4V substrate (surface area: 0.06 cm 2 ). The TiN coatings were deposited using magnetron sputtering and had a thickness of ~4 µm. B-NCD was grown using a pulsed microwave plasma enhanced chemical vapor deposition apparatus with linear antenna delivery system [3] operating at low pressures with a CH 4 -H 2 -CO 2 chemistry with trimethylboron (TMB) as a boron dopant. Electrochemical measurements on three B-NCD electrodes were compared to measurements on a smooth and a porous TiN electrode. Measurements were performed in phosphate buffered saline (PBS) at room temperature. EIS was performed using Solartron, Model 1294 in conjunction with 1260 Impedance/gain-phase Analyzer (Solartron Analytical, UK). The impedance spectrum was measured from 0.1 Hz ± 100 kHz using a current of 5.0 µA. The electrolyte resistance was subtracted from the measured impedance. Voltage transient measurements (VTM) and cyclic voltammetry (CV) were performed using a VersaSTAT 3 potentio-galvanostat (Princeton applied research, USA). The water window potentials were established for the B-NCD electrodes, after which CV measurements were made at sweep rates of 0.05, 0.1, 0.5 and 1.0 V/s. For TiN -0.6 and 0.9 V vs. Ag|AgCl were used as water window potentials. The CSC c was computed at 0.05 V/

Diagnòstic de dèficit de producció d'anticossos específics : eficàcia del tractament amb immunoglobulines en el control de l'afectació pulmonar /

Consultable des del TDXTítol obtingut de la portada digitalitzadaLa resposta d'anticossos a la vacuna conjugada de l'H.influenzae tipus b (Hib) es va estudiar en 59 voluntaris sans (edat mitjana: 32 anys) i en 22 pacients amb immunodeficiència humoralknown (edat mitjana: 32 anys) per determinar la seva utilitat en el diagnòstic de dèficit de producció d'anticossos. Vint dels sans i 9 dels pacients van ser també immunitzats amb la vacuna del pneumococ. La mesura dels anticossos específics sèrics es va realitzar per tècnica d'ELISA. to Hib capsular polysaccharideLa resposta a les vacunes es va definir utilitzant el límit inferior de l'interval de probabilitat del 90%, dues cues, dels nivells d'anticossos específics post-immunització de la població sana. El valor d'anticossos específics mínim a incrementar per considerar un individu responedor va ser de 2,28 g/ml per la vacuna de l'Hib i 395 U/ml per la del pneumococ. Amb aquest criteri el 85% (50/59) dels sans van respondre amb la IgG específica contra l'Hib i el 75% (15/20) amb la IgG contra el pneumococ. Tots els sans que van rebre les dues vacunes van respondre al menys a una d'elles. Cap dels pacients amb immunodeficiències no va respondre a cap d'elles. L'avaluació de la resposta d'anticossos contra la vacuna del pneumococ i l'Hib pot facilitar el diagnòstic d'immunodeficiències primàries de tipus humoral i la indicació de tractament amb immunoglobulines. Per investigar si el dèficit d'anticossos amb nivells d'IgG normal s'associa a bronquièctasis, es va estudiar la resposta d'anticossos contra la vacuna polisacàrida del pneumococ i la conjugada de l'Hib en pacients amb bronquièctasis d'etiologia no coneguda, valorats ambulatoriament durant un període de 7 anys. El dèficit de producció d'anticossos es va definir com la manca de resposta a les dues vacunes. 107 pacients van ser inclosos en l'estudi (edat mitjana: 46.3 anys). El dèficit de producció d'anticossos es va diagnosticar en 12 pacients (11%); aquests pacients van presentar una major incidència d'otitis mitjana, nivells baixos d'IgG2 i nivells basals d'anticossos contra S.pneumoniae i Hib inferiors. El dèficit de producció d'anticossos amb IgG normal pot associar-se a bronquièctasis i hauria de valorar-se en pacients amb bronquièctasis d'etiologia no coneguda, especialment en pacients amb antecedents d'otitis mitjana, nivells baixos d'IgG2 i nivells baixos d'anticossos específics. La progressió de l'afectació pulmonar és l'alteració més freqüent en pacients amb immunodeficiència comuna variable (ICV). La dosi més eficaç i els paràmetres per avaluar-ne l'eficàcia no han estat ben establerts. Es va estudiar l'evolució de l'afectació pulmonar de 24 pacients amb ICV que van iniciar tractament amb immunoglobulines intravenoses (IVIG) amb dosis necessàries per mantenir uns nivells estables d'IgG superiors a 600 mg/dl. La dosi d'IVIG, els nivells sèrics d'IgG, la incidència d'infeccions bacterianes, proves de funció respiratòria (PFR) i TACAR de tòrax es van monitoritzar durant 2 anys. Es va determinar la variabilitat de la dosi d'IVIG (205 - 372 mg/kg/21 dies) per obtenir els nivells d'IgG establerts. Els pacients amb malaltia pulmonar crònica (MPC) van requerir dosis superiors d'immunoglobulines (p = 0.045). Durant el tractament es va observar una disminució significativa del número d'infeccions greus i lleus. No es van observar canvis en les PFR ni en la puntuació de la TACAR en pacients sense MPC, però van millorar en pacients amb MPC. Un increment de la puntuació de la TACAR es va detectar en dos pacients sense evidència d'alteració en l'estat clínic ni en les PFR. El mantenir nivells residuals d'IgG superiors a 600 mg/ml pot ajudar a evitar la progressió de l'afectació pulmonar en pacients amb ICV. Les manifestacions clíniques, les PFR i la TACAR de tòrax, al menys cada dos anys són aconsellables per detectar la progressió de l'afectació pulmonar.Antibody response to an H.influenzae type b (Hib) conjugated vaccine was studied in 59 healthy adults (mean age: 32 yr) and 22 patients with knownhumoral immunodeficiencies (mean age: 32 yr) to determine its usefulness in the diagnosis of defective antibody formation. Twenty of the healthy adults and 9 of the patients were also immunized withPNU-Immune 23 a pneumococcal vaccine. Serum specific antibodies were measured by ELISAto Hib capsular polysaccharide. Adequate response to both vaccines was defined using the lower limit of the two-tailed 90% probability interval of postimmunization specific IgG of the healthy adults. By using this cutoff, responders were considered to be those with an absolute increase in anti-Hib IgG titers higher than 2.28 g/ml, and in anti-S.pneumoniae IgG higher than 395 arbitrary units/ml. With these criteria, 85% (50/59) of the healthy adults responded with anti-Hib IgG and 75% (15/20) with anti-pneumococcal IgG. All healthy adults receiving both vaccines responded to at least one. None of the patients with humoral immunodeficiencies responded to either vaccine. Evaluation of the antibody response to both the Hib and pneumococcal vaccines may facilitate the diagnosis of humoral immunodeficiency and selection of patients to receive immunoglobulin therapy. To ascertain whether antibody production deficiency with normal IgG levels is associated with bronchiectasis, antibody response to a pneumococcal unconjugate vaccine and an Hib conjugate vaccine was studied in all adult patients with bronchiectasis of unknown etiology that were assessed in our chest outpatient clinic from January 1994 to October 2001. Antibody production deficiency was defined as a failure to respond to either vaccine. 107 patients were included in the study (mean age: 46.3 years). Antibody production deficiency was diagnosed in 12 patients (11%). A significantly higher incidence of otitis media, lower serum IgG2 subclass levels, and lower preimmunization antibody levels to S.pneumoniae and Hib were observed in patients with antibody production deficiency. Antibody production deficiency with normal IgG levels may be associated with bronchiectasis, making it advisable to evaluate the antibody response to both the H.influenzae and pneumococcal vaccines in patients with bronchiectasis of unknown etiology, particularly in those with a history of otitis media, low IgG2 subclass levels and low levels of baseline specific antibodies. Lung damage progression is the most frequent condition in patients with common variable immunodeficiency (CVID). Appropriate immunoglobulin dose adjustments and follow-up guidelines to evaluate this have not been well established. To assess the evolution of lung damage once stable residual serum levels of IgG over 600 mg/dl had been achieved, a prospective study was conducted in 24 adult patients with CVID, with no previous intravenous immunoglobulin (IVIG) treatment. IVIG dose, total serum IgG level, bacterial infection rate, pulmonary function tests (PFTs) and high resolution computed tomography (HRCT) of the thorax were monitored over two years. IVIG dose variability (205 - 372 mg/kg/21 days) to obtain the required serum IgG levels was determined. Patients with chronic pulmonary disease (CPD) needed higher doses than those without CPD (p = 0.045). A significant reduction in severe and mild infections/patient-year was observed during treatment. Overall, there were no changes in PFTs and HRCT scores in patients without CPD, but both improved in patients with CPD. An increase of over 15% in overall HRCT score was detected in two patients without evidence of impairment in either clinical status or PFT values. Residual levels of total IgG over 600 mg/dl may help prevent progression of lung damage in patients with CVID. Levels of IgG, clinical manifestations and PFTs seem sufficient for routine follow-up. HRCT of the thorax, at least biennially, may help to identify patients in whom lung injury is progressing even though they may remain symptom-free and with stable PFTs

Antitumoral properties of Sorafenib, Regorafenib, Cabozantinib and Lenvatinib in 3D tumor liver cell culture

Motivation: Most researchs to find effective therapies to treat cancer has been done in cell lines grown in monolayer that don´t take into account the three-dimensional structure of the tumor or the interactions between the tumor cells. In cell cultures in two dimensions, all cells are exposed in the same way to the therapeutic agent, so the results are not very precise.For this reason, it is important to develop cell culture techniques in which it is possible simulate in vivo conditions to predict more exactly the behavior and cellular interactions within the tumor. Like tumors, spheroids are three-dimensional aggregates of cancer cells that naturally form regions of hypoxia.Methods: To carry out this study we have used three different cell lines: HepG2, Hep3B and Huh7. First, the cells were cultured in 96-well plates coated with agarose. The spheroids were collected on day 5, 8 (when treated with the different drugs), 10, 12 and 15. The collected spheroids were fixed with paraformaldehyde, passed through a paraffination cycle, were introduced in paraffin blocks and cut with the microtome. Other spheroids were disintegrated by trypsinization to determine the number of cells and lysates to be able to determine caspase-3 activity. The parameters that we have measured have been apoptosis, cell proliferation, cell viability, cell death, hypoxia and cell growth. In addition, was carried out a study of the expression of  different growth factor receptors such as EGFR, VGFR, FGFR ans PDGFR by immunodetection.Results: Sorafenib and Regorafenib induced cell death and reduced cell proliferation both in 2D and 3D cultured HCC lines. These effects are higher than those observed with Cabozantinib and Lenvatinib in the same conditions. The expression of some receptors such as EGFR is reduced in the cells treated for 24 hours with sorafenib and regorafenib but no changes are observed in the cells treated with lenvatinib and cabozantinib with respect to the control.Conclusions: The observed resistance of Lenvatinib and Cabozantinib treatment to the induction of cell death and cell cycle arrest in comparison with that observed with Sorafenib and Regorafenib may be related to the induction of EGFR-dependent pathway

Nutritional regulation of gene expression: carbohydrate-, fat- and amino acid-dependent modulation of transcriptional activity

The ability to detect changes in nutrient levels and generate an adequate response to these changes is essential for the proper functioning of living organisms. Adaptation to the high degree of variability in nutrient intake requires precise control of metabolic pathways. Mammals have developed different mechanisms to detect the abundance of nutrients such as sugars, lipids and amino acids and provide an integrated response. These mechanisms include the control of gene expression (from transcription to translation). This review reports the main molecular mechanisms that connect nutrients' levels, gene expression and metabolism in health. The manuscript is focused on sugars' signaling through the carbohydrate-responsive element binding protein (ChREBP), the role of peroxisome proliferator-activated receptors (PPARs) in the response to fat and GCN2/activating transcription factor 4 (ATF4) and mTORC1 pathways that sense amino acid concentrations. Frequently, alterations in these pathways underlie the onset of several metabolic pathologies such as obesity, insulin resistance, type 2 diabetes, cardiovascular diseases or cancer. In this context, the complete understanding of these mechanisms may improve our knowledge of metabolic diseases and may offer new therapeutic approaches based on nutritional interventions and individual genetic makeup

Extracellular vesicles secretion by Lenvatinib and Sorafenib in HepG2 cells and their effect on cell death and proliferation

Motivation: Sorafenib, which acts on the RAF / MEK / ERK pathways through the inhibition of Raf kinase and different tyrosine kinases (VEGFR2, PDGFR, c-Kit receptors), is the drug currently used as a first-line treatment in hepatocellular carcinomas of advanced stage. It has recently been shown that Lenvatinib, another multi-kinase inhibitor, also improves mean progression-free survival and mean time to cancer progression. This finding motivated us to study the possible antiproliferative effects of Lenvatinib compared to Sorafenib, in addition to the secretion profile of extracellular vesicles in HepG2 cultures due to its recognized role in tumor progression and metastasis.Methods: To determine the percentage of proliferating cells in culture, the incorporation of bromodeoxyuridine (BrdU) was used as a marker, while the analysis of the apoptotic activity was done through a colorimetric test that allows detecting the amount of caspase 3/7 existing in culture. It is well known that there is a connection between apoptosis and autophagy, so we decided to study the changes that occurred in the latter process after treatment. For this, the level of expression of LC3-II was determined through an SDS-PAGE coupled to a Western-Blot analysis. The changes produced in the expression of VEGFR-2 and EGFR were also monitored and, finally, the secretion profile of extracellular vesicles was studied through the analysis of the expression of different markers (Lamp1, E-Selectin, CD63, TSG101, Grp78, GM130, Annexin V and Prohibitin) in fractions enriched in exosomes, extracellular vesicles and apoptotic bodies.Results and Conclusions: The results for the group treated with Sorafenib reproduced what has been described so far in the literature referring to hepatocellular carcinoma: decrease in cell proliferation caused by the downregulation of the expression of different growth factors (EGFR and VEGFR-2) and increase of cell death by apoptosis. However, Lenvatinib did not reproduce the pattern we expected for an antineoplastic drug, since it increased cell proliferation. With respect to the secretion profile of extracellular vesicles, no convincing results were obtained. We think that this could be due to the capacity of separation of the different fractions of the protocol used or to the difficulty of obtaining, from them, high amounts of proteins to proceed to its analysis by WB

Pig liver carnitine palmitoyltransferase. Chimera studies show that both the N- and C-terminal regions of the enzyme are important for the unusual high malonyl-CoA sensitivity

Pig and rat liver carnitine palmitoyltransferase I (L-CPTI) share common K(m) values for palmitoyl-CoA and carnitine. However, they differ widely in their sensitivity to malonyl-CoA inhibition. Thus, pig l-CPTI has an IC(50) for malonyl-CoA of 141 nm, while that of rat L-CPTI is 2 microm. Using chimeras between rat L-CPTI and pig L-CPTI, we show that the entire C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. The degree of malonyl-CoA sensitivity is determined by the structure adopted by this domain. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. We conclude that pig and rat L-CPTI have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acid residues interact differently with the C-terminal domain. This is the first study that describes how interactions between the C- and N-terminal regions can determine the malonyl-CoA sensitivity of L-CPTI enzymes using active C-terminal chimeras