Development of a human liver microphysiological coculture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.

Primary Human Cell-Derived Extracellular Matrix from Decellularized Fibroblast Microtissues with Tissue-Dependent Composition and Microstructure.

Human extracellular matrix (ECM) exhibits complex protein composition and architecture depending on tissue and disease state, which remains challenging to reverse engineer. One promising approach is based on cell-secreted ECM from human fibroblasts, which can then be decellularized into an acellular biomaterial. However, fibroblasts initially seeded on rigid tissue culture plastic or biomaterial scaffolds experience aberrant mechanical cues that influence ECM deposition. Here, we show that engineered microtissues of primary human fibroblasts seeded in low-adhesion microwells can be decellularized to produce human, tissue-specific ECM. We investigate: 1) cardiac fibroblasts, as well as 2) lung fibroblasts from healthy, idiopathic fibrosis and chronic obstructive pulmonary disease donors. We demonstrate optimized culture and decellularization conditions, then characterize gene expression and protein composition. We further characterize ECM microstructure and mechanical properties. We envision that this method could be utilized for biomanufacturing of patient and tissue-specific ECM for organoid drug screening as well as implantable scaffolds. Impact: In this study, we demonstrate a method for preparing decellularized matrix using primary human fibroblasts with tissue and disease-specific features. We aggregate single cell dispersions into engineered tissues using low adhesion microwells and show culture conditions that promote ECM deposition. We demonstrate this approach for cardiac fibroblasts as well as lung fibroblasts (both normal and diseased). We systematically investigate tissue morphology, matrix architecture, and mechanical properties, along with transcriptomic and proteomic analysis. This approach should be widely applicable for generating personalized ECM with features of patient tissues and disease state, relevant for culturing patient cells ex vivo as well as implantation for therapeutic treatments.

3D Microtissues Mimic the Architecture, Estradiol Synthesis, and Gap Junction Intercellular Communication of the Avascular Granulosa.

Humans are consistently exposed to thousands of untested chemicals that have been detected in the follicular fluid of the ovaries, and can disrupt reproductive health. Human granulosa cells (GCs) are the functional unit of the ovarian follicle with steroidogenic and signaling activities, and play a pivotal role in oocyte development. During follicle progression, GCs multiply to form a 3D avascular structure, and establish gap junction intercellular communication (GJIC) that is critical to maintaining optimal viability and function. We developed a high-throughput in vitro platform of human GCs for the screening of chemicals that can impact GJIC and estradiol (E2) production of human granulosa. Our granulosa 3D microtissues fabricated with human ovarian granulosa-like tumor KGN cells are multicell-layered structures that mimic the avascular granulosa layers surrounding the oocyte. These microtissues robustly expressed the steroidogenic CYP19 aromatase enzyme and GJIC intercellular membrane channel, connexin 43. Granulosa microtissues produced E2 at rates comparable to primary human GCs as previously reported. E2 production was suppressed by the CYP19 inhibitor, letrozole, and induced by CYP19 activators, bisphenol A at 100 µM, and genistein at 100 µM. Granulosa microtissues displayed active GJIC function, as demonstrated by the connexin 43-dependent diffusion of calcein fluorescent dye from microtissue surface to the core using high-throughput confocal microscopy in conjunction with our open-sourced automated image analysis tool. Overall, our 3D human granulosa screening platform is highly promising for predictive and efficient in vitro toxicity testing to screen for chemicals that contaminate follicular fluid and may affect fertility.

Does fetal leptin and adiponectin influence children's lung function and risk of wheeze?

Adipocytokines, which are secreted during fetal development by both mothers and fetuses, may influence fetal lung development, but little human data are available. We used data from the HOME Study to investigate the associations of cord blood adipocytokine concentrations with children's lung forced expiratory volume (FEV1; N = 160) and their risk of wheeze (N = 281). We measured umbilical cord serum adipocytokine concentrations using enzyme-linked immunosorbent assays and FEV1 using a portable spirometer at ages 4 and 5 to calculate the percent predicted FEV1 (%FEV1). Parents completed standardized questionnaires of their child's wheeze symptoms every 6 months from birth to age 5, then again at ages 6 and 8. We used multivariable linear mixed models and modified Poisson regression with generalized estimating equations to estimate associations of adipocytokine concentrations (log2-transformed) with children's %FEV1 and the risk of wheeze, respectively, adjusting for sociodemographic, perinatal, and child factors. Cord serum leptin was not associated with children's %FEV1. Higher cord serum adiponectin concentrations were associated with higher %FEV1 in girls (ß = 3.1, 95% confidence interval [CI]: 0.6, 5.6), but not in boys (ß = -1.3, 95% CI: -5.9, 3.3) (sex × adiponectin p-value = 0.05). Higher leptin was associated with lower risk of wheeze in girls (RR = 0.74, 95% CI: 0.66, 0.84), but not boys (RR = 0.87, 95% CI: 0.69, 1.11) (sex × leptin p-value = 0.01). In contrast, higher adiponectin concentrations were associated with lower risk of wheeze (RR = 0.84, 95% CI: 0.73, 0.96) in both boys and girls. These data suggest that fetal adipocytokines may impact lung development and function in early childhood. Future studies are needed to confirm these findings and explore the mechanisms underlying these associations.

Breast Cancer Cells Transition from Mesenchymal to Amoeboid Migration in Tunable Three-Dimensional Silk-Collagen Hydrogels.

Invading cancer cells adapt their migration phenotype in response to mechanical and biochemical cues from the extracellular matrix. For instance, mesenchymal migration is associated with strong cell-matrix adhesions and an elongated morphology, while amoeboid migration is associated with minimal cell-matrix adhesions and a rounded morphology. However, it remains challenging to elucidate the role of matrix mechan-ics and biochemistry, since these are both dependent on ECM protein concentration. Here, we demonstrate a composite silk fibroin and collagen I hydrogel where stiffness and microstructure can be systematically tuned over a wide range. Using an overlay assay geometry, we show that the invasion of metastatic breast cancer cells exhibits a biphasic dependence on silk fibroin concentration at fixed collagen I concentration, first increasing as the hydrogel stiffness increases, then decreasing as the pore size of silk fibroin decreases. Indeed, mesenchymal morphology exhibits a similar biphasic depen-dence on silk fibroin concentration, while amoeboid morphologies were favored when cell-matrix adhesions were less effective. We used exogenous biochemical treatment to perturb cells towards increased contractility and a mesenchymal morphology, as well as to disrupt cytoskeletal function and promote an amoeboid morphology. Overall, we envision that this tunable biomaterial platform in a 96-well plate format will be widely applicable to screen cancer cell migration against combinations of designer biomaterials and targeted inhibitors.

Fatty Acid Metabolites Combine with Reduced ß Oxidation to Activate Th17 Inflammation in Human Type 2 Diabetes.

Mechanisms that regulate metabolites and downstream energy generation are key determinants of T cell cytokine production, but the processes underlying the Th17 profile that predicts the metabolic status of people with obesity are untested. Th17 function requires fatty acid uptake, and our new data show that blockade of CPT1A inhibits Th17-associated cytokine production by cells from people with type 2 diabetes (T2D). A low CACT:CPT1A ratio in immune cells from T2D subjects indicates altered mitochondrial function and coincides with the preference of these cells to generate ATP through glycolysis rather than fatty acid oxidation. However, glycolysis was not critical for Th17 cytokines. Instead, ß oxidation blockade or CACT knockdown in T cells from lean subjects to mimic characteristics of T2D causes cells to utilize 16C-fatty acylcarnitine to support Th17 cytokines. These data show long-chain acylcarnitine combines with compromised ß oxidation to promote disease-predictive inflammation in human T2D.

Hydrodynamics of the Bio-Gripper: A Fluid-Driven "Claw Machine" for Soft Microtissue Translocation.

Technological advances in solid organ tissue engineering that rely on the assembly of small tissue-building parts require a novel transport method suited for soft, deformable, living objects of submillimeter- to centimeter-length scale. We describe a technology that utilizes membrane flow through a gripper to generate optimized pressure differentials across the top and bottom surfaces of microtissue so that the part may be gripped and lifted. The flow and geometry parameters are developed for automation by analyzing the fluid mechanics framework by which a gripper can lift tissue parts off solid and porous surfaces. For the axisymmetric part and gripper geometries, we examine the lift force on the part as a function of various parameters related to the gripper design, its operation, and the tissue parts and environments with which it operates. We believe our bio-gripping model can be used in various applications in high-throughput tissue engineering.

Adaptive Immunity and Metabolic Health: Harmony Becomes Dissonant in Obesity and Aging.

Adipose tissue (AT) is the primary energy reservoir organ, and thereby plays a critical role in energy homeostasis and regulation of metabolism. AT expands in response to chronic overnutrition or aging and becomes a major source of inflammation that has marked influence on systemic metabolism. The chronic, sterile inflammation that occurs in the AT during the development of obesity or in aging contributes to onset of devastating diseases such as insulin resistance, diabetes, and cardiovascular pathologies. Numerous studies have shown that inflammation in the visceral AT of humans and animals is a critical trigger for the development of metabolic syndrome. This work underscores the well-supported conclusion that the inflammatory immune response and metabolic pathways in the AT are tightly interwoven by multiple layers of relatively conserved mechanisms. During the development of diet-induced obesity or age-associated adiposity, cells of the innate and the adaptive immune systems infiltrate and proliferate in the AT. Macrophages, which dominate AT-associated immune cells in mouse models of obesity, but are less dominant in obese people, have been studied extensively. However, cells of the adaptive immune system, including T cells and B cells, contribute significantly to AT inflammation, perhaps more in humans than in mice. Lymphocytes regulate recruitment of innate immune cells into AT, and produce cytokines that influence the helpful-to-harmful inflammatory balance that, in turn, regulates organismal metabolism. This review describes inflammation, or more precisely, metabolic inflammation (metaflammation) with an eye toward the AT and the roles lymphocytes play in regulation of systemic metabolism during obesity and aging. © 2017 American Physiological Society. Compr Physiol 7:1307-1337, 2017.

Advances in the quantification of mitochondrial function in primary human immune cells through extracellular flux analysis.

Numerous studies show that mitochondrial energy generation determines the effectiveness of immune responses. Furthermore, changes in mitochondrial function may regulate lymphocyte function in inflammatory diseases like type 2 diabetes. Analysis of lymphocyte mitochondrial function has been facilitated by introduction of 96-well format extracellular flux (XF96) analyzers, but the technology remains imperfect for analysis of human lymphocytes. Limitations in XF technology include the lack of practical protocols for analysis of archived human cells, and inadequate data analysis tools that require manual quality checks. Current analysis tools for XF outcomes are also unable to automatically assess data quality and delete untenable data from the relatively high number of biological replicates needed to power complex human cell studies. The objectives of work presented herein are to test the impact of common cellular manipulations on XF outcomes, and to develop and validate a new automated tool that objectively analyzes a virtually unlimited number of samples to quantitate mitochondrial function in immune cells. We present significant improvements on previous XF analyses of primary human cells that will be absolutely essential to test the prediction that changes in immune cell mitochondrial function and fuel sources support immune dysfunction in chronic inflammatory diseases like type 2 diabetes.

The bio-gripper: a fluid-driven micro-manipulator of living tissue constructs for additive bio-manufacturing.

We previously developed the Bio-Pick, Place, and Perfuse (Bio-P3) instrument to fabricate large perfusable tissue constructs by stacking and aligning scaffold-free living microtissues with integrated lumens. The Bio-P3 required an actuating mechanism to manipulate living microtissues of various sizes and shapes that are fragile, and must remain in an aqueous environment. The optical transparency of the Bio-P3 gripping device was essential to provide unobstructed visuals for accurate alignment of microtissues. We previously engineered a pilot fluid force-driven bio-gripper that can pick-and-place microtissue in planar position without causing cellular damage by pulling culture medium through track-etched membrane-integrated cell culture inserts. In this study, we invented a new flexible bio-gripper design that maximized the bio-gripper utilities. We utilized experimental approaches, multivariate analyzes, and theoretical modeling to elucidate how membrane characteristics (pore size, pore density, membrane thickness, membrane area, and surface chemistry) altered bio-gripper robustness and the flow rate (Q(c)) required for successful gripping. We devised two standardized tests and synthetic parts that mimicked microtissues, to systematically quantify bio-gripper performance. All thirteen syringe pump-driven bio-grippers except one successfully gripped and released synthetic parts with values of Q(c) that coincided with our mathematical simulation of the fluid mechanics of gripping. The bio-gripper could grip synthetic parts of various sizes, shapes and masses, demonstrating the robustness of the actuating mechanism. Multivariate analysis of experimental data indicated that both membrane porosity and thickness modulated Q(c), and in addition, revealed that membrane pore density determined membrane optical transparency. Fabricating large tissue constructs requires repeated stacking of microtissues. We showed that one bio-gripper could pick-and-place living microtissues thirty times with Q(c) corresponding to our simulation. Our bio-gripper was capable of stacking and aligning twenty microtissues. In summary, we successfully engineered a robust controllable fluid-driven bio-gripper to efficiently manipulate living microtissues and micro-objects in an aqueous environment.

Tumor progression locus 2 ablation suppressed hepatocellular carcinoma development by inhibiting hepatic inflammation and steatosis in mice.

BACKGROUND: Tumor progression locus 2 (TPL2), a serine-threonine kinase, functions as a critical regulator of inflammatory pathways and mediates oncogenic events. The potential role of Tpl2 in nonalcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) development remains unknown. METHODS: Both wild-type and Tpl2 knockout male mice were initiated by a hepatic carcinogen (diethylnitrosamine, i.p. with a single dose of 25 mg.kg(-1))at 2 weeks of age, and then were given the high carbohydrate diet feeding to induce hepatic steatosis, inflammation, adenoma and HCC for 24 weeks. RESULTS: Tpl2 knockout mice had significantly lower incidences of liver tumor and developed hepatocellular adenoma only, which is contrast to wild-type mice where they all developed HCC. Tpl2 knockout mice had significantly down-regulated phosphorylation of JNK and ERK, and levels of mRNA expression of pro-inflammatory cytokines (Il-1ß, Il-18, Mcp-1 and Nalp3), which correlated with the reduced incidence and number of hepatic inflammatory foci. Furthermore, Tpl2 ablation resulted in decreased hepatic steatosis and expression of de novo lipogenesis related markers (ACC, SCD1, SREBP1C and AKT phosphorylation), as well as reduction of endoplasmic reticulum stress biomarkers PERK and eIF-2a. CONCLUSION: The study revealed for the first time that Tpl2 plays a significant role in promoting HCC development by its pro-inflammatory effect, which suggested that Tpl2 could be a molecular target for HCC prevention.

Lycopene and apo-10'-lycopenoic acid have differential mechanisms of protection against hepatic steatosis in ß-carotene-9',10'-oxygenase knockout male mice.

BACKGROUND: Nonalcoholic fatty liver disease is positively associated with obesity and cardiovascular disease risk. Apo-10'-lycopenoic acid (APO10LA), a potential oxidation product of apo-10'-lycopenal that is generated endogenously by ß-carotene-9',10'-oxygenase (BCO2) cleavage of lycopene, inhibited hepatic steatosis in BCO2-expressing mice. OBJECTIVE: The present study evaluated lycopene and APO10LA effects on hepatic steatosis in mice without BCO2 expression. METHODS: Male and female BCO2-knockout (BCO2-KO) mice were fed a high saturated fat diet (HSFD) with or without APO10LA (10 mg/kg diet) or lycopene (100 mg/kg diet) for 12 wk. RESULTS: Lycopene or APO10LA supplementation reduced hepatic steatosis incidence (78% and 72%, respectively) and severity in BCO2-KO male mice. Female mice did not develop steatosis, had greater hepatic total cholesterol (3.06 vs. 2.31 mg/g tissue) and cholesteryl ester (1.58 vs. 0.86 mg/g tissue), but had lower plasma triglyceride (TG) (229 vs. 282 mg/dL) and cholesterol (97.1 vs. 119 mg/dL) than male mice. APO10LA-mitigated steatosis in males was associated with reduced hepatic total cholesterol (18%) and activated sirtuin 1 signaling, which resulted in reduced fatty acids (FAs) and TG synthesis markers [stearoyl-coenzyme A (CoA) desaturase protein, 71%; acetyl-CoA carboxylase phosphorylation, 79%; AMP-activated protein kinase phosphorylation, 67%], and elevated cholesterol efflux genes (cytochrome P450 family 7A1, 65%; ATP-binding cassette transporter G5/8, 11%). These APO10LA-mediated effects were not mimicked by lycopene supplementation. Intriguingly, steatosis inhibition by lycopene induced peroxisome proliferator-activated receptor (PPAR)α- and PPARγ-related genes in mesenteric adipose tissue (MAT) that increases mitochondrial uncoupling [cell death-inducing DNA fragmentation factor, α subunit-like effector a, 55%; PR domain-containing 16, 47%; uncoupling protein 3 (Ucp3), 55%], FA ß-oxidation (PPARα, 53%; very long chain acyl-CoA dehydrogenase, 38%), and uptake (FA transport protein 4, 29%; lipoprotein lipase 43%). Expressions of 10 MAT PPAR-related genes were inversely correlated with steatosis score, suggesting that lycopene reduced steatosis by increasing MAT FA utilization. CONCLUSIONS: Our data suggest that lycopene and APO10LA inhibit HSFD-induced steatosis in BCO2-KO male mice through differential mechanisms. Sex disparity of BCO2-KO mice was observed in the outcomes of HSFD-induced liver steatosis and plasma lipids.

Lymphocyte roles in metabolic dysfunction: of men and mice.

Type 2 diabetes (T2D) is a metabolic disease associated with obesity-related insulin resistance (IR) and chronic inflammation. Animal studies indicate that IR can be caused and/or exacerbated by systemic and/or tissue-specific alterations in lymphocyte differentiation and function. Human studies also indicate that obesity-associated inflammation promotes IR. Nevertheless, clinical trials with anti-inflammatory therapies have yielded modest impacts on established T2D. Unlike mouse models, where obesity is predominantly associated with IR, 20-25% of obese humans are metabolically healthy with high insulin sensitivity. The uncoupling of obesity from IR in humans but not in animal models advocates for a more comprehensive understanding of mediators and mechanisms of human obesity-promoted IR, and better integration of knowledge from human studies into animal experiments to efficiently pursue T2D prevention and treatment.

Lycopene attenuated hepatic tumorigenesis via differential mechanisms depending on carotenoid cleavage enzyme in mice.

Obesity is associated with increased liver cancer risks and mortality. We recently showed that apo-10'-lycopenoic acid, a lycopene metabolite generated by beta-carotene-9',10'-oxygenase (BCO2), inhibited carcinogen-initiated, high-fat diet (HFD)-promoted liver inflammation, and hepatic tumorigenesis development. The present investigation examined the outstanding question of whether lycopene could suppress HFD-promoted hepatocellular carcinoma (HCC) progression, and if BCO2 expression is important using BCO2-knockout (BCO2-KO) and wild-type male mice. Results showed that lycopene supplementation (100 mg/kg diet) for 24 weeks resulted in comparable accumulation of hepatic lycopene (19.4 vs. 18.2 nmol/g) and had similar effects on suppressing HFD-promoted HCC incidence (19% vs. 20%) and multiplicity (58% vs. 62%) in wild-type and BCO2-KO mice, respectively. Intriguingly, lycopene chemopreventive effects in wild-type mice were associated with reduced hepatic proinflammatory signaling (phosphorylation of NK-κB p65 and STAT3; IL6 protein) and inflammatory foci. In contrast, the protective effects of lycopene in BCO2-KO but not in wild-type mice were associated with reduced hepatic endoplasmic reticulum stress-mediated unfolded protein response (ER(UPR)), through decreasing ER(UPR)-mediated protein kinase RNA-activated like kinase-eukaryotic initiation factor 2α activation, and inositol requiring 1α-X-box-binding protein 1 signaling. Lycopene supplementation in BCO2-KO mice suppressed oncogenic signals, including Met mRNA, ß-catenin protein, and mTOR complex 1 activation, which was associated with increased hepatic microRNA (miR)-199a/b and miR214 levels. These results provided novel experimental evidence that dietary lycopene can prevent HFD-promoted HCC incidence and multiplicity in mice, and may elicit different mechanisms depending on BCO2 expression.

High-refined-carbohydrate and high-fat diets induce comparable hepatic tumorigenesis in male mice.

Previous studies demonstrated that diet-induced obese mice fed a semi-purified high-fat diet (HFD) had greater liver tumorigenesis than mice fed a non-semi-purified diet. Because ingredients present in standard unpurified diets may elicit potential chemopreventive properties that are not present in semi-purified diets, the present study evaluated hepatic tumorigenic effects of dietary fat by replacing it with refined carbohydrates [digestible saccharides; high-carbohydrate diet (HCD)] in a semi-purified diet without altering other components. Two-wk-old C57Bl/6J male mice were randomly injected i.p. with either the liver-specific carcinogen diethylnitrosamine (25 mg/kg body weight) to induce liver cancer or saline as the nontumor control. At age 6 wk, mice with or without cancer initiation were further randomly assigned to an HFD (26% and 60% energy from carbohydrates and fat, respectively) or an HCD (66% and 12% energy from carbohydrates and fat, respectively) and consumed food ad libitum for 24 wk. Results showed that HCD-fed mice had a comparable degree of hepatic tumorigenesis (tumor number and volume) as HFD-fed mice, despite having significantly reduced body weights. HCD feeding induced greater hepatic endoplasmic reticulum (ER) stress-mediated protein kinase RNA-activated-like kinase (PERK) activation and oncogenic interleukin-6/signal transducer and activator of transcription 3 signaling than HFD feeding. HCD-stimulated PERK signaling was associated with elevated expression of prosurvival markers in tumors, including induced protein kinase B activation, increased extracellular signal-regulated kinases 1/2 phosphorylation, and elevated cyclin D1 protein expression. However, HCD-mediated PERK activation in tumors was also positively associated with markers of proapoptosis, which included elevated CCAAT/enhancer-binding protein homology protein expression and increased cleaved caspase-3. HCD-fed mice had greater severity in hepatic steatosis than HFD-fed mice. HCD-induced steatosis exacerbation was associated with increased expression in hepatic de novo lipogenic markers that can promote ER stress. Together, these data indicated that chronic HCD consumption by mice can produce comparable severity of hepatic tumorigenesis as HFD consumption, potentially through upregulating PERK-mediated ER stress.

Lycopene metabolite, apo-10'-lycopenoic acid, inhibits diethylnitrosamine-initiated, high fat diet-promoted hepatic inflammation and tumorigenesis in mice.

Obesity is associated with increased risk in hepatocellular carcinoma (HCC) development and mortality. An important disease control strategy is the prevention of obesity-related hepatic inflammation and tumorigenesis by dietary means. Here, we report that apo-10'-lycopenoic acid (APO10LA), a cleavage metabolite of lycopene at its 9',10'-double bond by carotene-9',10'-oxygenase, functions as an effective chemopreventative agent against hepatic tumorigenesis and inflammation. APO10LA treatment on human liver THLE-2 and HuH7 cells dose dependently inhibited cell growth and upregulated sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase that may suppress hepatic carcinogenesis. This observed SIRT1 induction was associated with decreased cyclin D1 protein, increased cyclin-dependent kinase inhibitor p21 protein expression, and induced apoptosis. APO10LA supplementation (10 mg/kg diet) for 24 weeks significantly reduced diethylnitrosamine-initiated, high fat diet (HFD)-promoted hepatic tumorigenesis (50% reduction in tumor multiplicity; 65% in volume) and lung tumor incidence (85% reduction) in C57Bl/6J mice. The chemopreventative effects of APO10LA were associated with increased hepatic SIRT1 protein and deacetylation of SIRT1 targets, as well as with decreased caspase-1 activation and SIRT1 protein cleavage. APO10LA supplementation in diet improved glucose intolerance and reduced hepatic inflammation [decreased inflammatory foci, TNFα, interleukin (IL)-6, NF-κB p65 protein expression, and STAT3 activation] in HFD-fed mice. Furthermore, APO10LA suppressed Akt activation, cyclin D1 gene, and protein expression and promoted PARP protein cleavage in transformed cells within liver tumors. Taken together, these data indicate that APO10LA can effectively inhibit HFD-promoted hepatic tumorigenesis by stimulating SIRT1 signaling while reducing hepatic inflammation.

[Adults with congenital heart diseases: a growing population, a multidisciplinary approach]. / Cardiopathies congénitates de l'âge adulte: nouvelle population de patients, prise en charge multidisciplinaire.

Untill recently, congenital heart disease was considered as a childhood's disease. With improvement in pediatric survival, adults with a congenital heart disease (ACHD) represent an emerging group of patients who need specialized medical care. In 2010, the ESC published newguidelines on global and specific management of adults with congenital heart disease. ACHD centers organize appropriate medical care for these patients, promote specialist training and national scientific research in collaboration with other national ACHD centers.

Aggravation of nonalcoholic steatohepatitis by moderate alcohol consumption is associated with decreased SIRT1 activity in rats.

Chronic alcohol intake decreases adiponectin and sirtuin 1 (SIRT1) expressions, both of which have been implicated in various biological processes including inflammation, apoptosis and metabolism. We have previously shown that moderate consumption of alcohol aggravates liver inflammation and apoptosis in rats with pre-existing nonalcoholic steatohepatitis (NASH). This study investigated whether moderate alcohol intake alters SIRT1 activity, adiponectin/Adiponectin receptor (AdipoR)-related signaling and lipid metabolism in a pre-existing NASH status. Sprague-Dawley rats were fed with a high-fat diet (71% energy from fat) for 6 weeks to induce NASH then subsequently divided into 2 sub-groups: fed either a modified high-fat diet (HFD, 55% energy from fat) or a modified high-fat alcoholic diet (HFA, 55% energy from fat and 16% energy from ethanol) for an additional 4 weeks. We observed in comparison to HFD group, HFA increased hepatic nuclear SIRT1 protein but decreased its deacetylase activity. SREBP-1c protein expression and FAS mRNA levels were significantly upregulated, while DGAT1/2 and CPT-I mRNA levels were downregulated in the livers of HFA compared to HFD. Although hepatic AdipoR1 decreased, HFA did not alter AdipoR2 and their downstream signaling. There were no significant changes in plasma adiponectin and free fatty acids (FFA), as well as adiponectin expression in adipose tissue between the two groups. The present study indicates that suppression in SIRT1 deacetylase activity contributes to alcohol-exacerbated hepatic inflammation and apoptosis in rats with pre-existing NASH. In addition, moderate alcohol intake did not modulate adiponectin/AdipoR signaling axis in this model.

Non-alcoholic steatohepatitis and hepatocellular carcinoma: implications for lycopene intervention.

Increased prevalence of non-alcoholic fatty liver disease (NAFLD) is one of the consequences of the current obesity epidemic. NAFLD is a major form of chronic liver disease that is highly prevalent in obese and overweight adults and children. Nonalcoholic steatohepatitis (NASH) is the severe form of NAFLD, and uncontrolled inflammation as displayed in NASH has been identified as one of the key events in enhancing hepatic carcinogenesis. Lycopene is a non-provitamin A carotenoid and the pigment principally responsible for the characteristic deep-red color of ripe tomato and tomato products, as well as some fruits and vegetables. Lycopene's innate antioxidant and anti-inflammatory properties have generated research interests on its capacity to protect against human diseases that are associated with oxidative stress and inflammation. In addition, differential mechanisms of lycopene metabolism including endogenous cleavage by carotenoid cleavage oxygenases (BCOs), generate lycopene metabolites that may also have significant impact on human disease development. However, it remains to be elucidated as to whether lycopene or its metabolites apolycopenoids have protective effects against obesity-related complications including inflammation and tumorigenesis. This article summarizes the in vivo experiments that elucidated molecular mechanisms associated with obesity-related hepatic inflammation and carcinogenesis. This review also provides an overview of lycopene metabolism, and the molecular pathways involved in the potential beneficial properties of lycopene and apolycopenoids. More research is clearly needed to fully unravel the importance of BCOs in tomato carotenoid metabolism and the consequence on human health and diseases.

Public response to an anthrax attack: reactions to mass prophylaxis in a scenario involving inhalation anthrax from an unidentified source.

An attack with Bacillus anthracis ("anthrax") is a known threat to the United States. When weaponized, it can cause inhalation anthrax, the deadliest form of the disease. Due to the rapid course of inhalation anthrax, delays in initiation of antibiotics may decrease survival chances. Because a rapid response would require cooperation from the public, there is a need to understand the public's response to possible mass dispensing programs. To examine the public's response to a mass prophylaxis program, this study used a nationally representative poll of 1,092 adults, supplemented by a targeted focus on 3 metropolitan areas where anthrax attacks occurred in 2001: New York City (n=517), Washington, DC (n=509), and Trenton/Mercer County, NJ (n=507). The poll was built around a "worst-case scenario" in which cases of inhalation anthrax are discovered without an identified source and the entire population of a city or town is asked to receive antibiotic prophylaxis within a 48-hour period. Findings from this poll provide important signs of public willingness to comply with public health recommendations for obtaining antibiotics from a dispensing site, although they also indicate that public health officials may face several challenges to compliance, including misinformation about the contagiousness of inhalation anthrax; fears about personal safety in crowds; distrust of government agencies to provide sufficient, safe, and effective medicine; and hesitation about ingesting antibiotic pills after receiving them. In general, people living in areas where anthrax attacks occurred in 2001 had responses similar to those of the nation as a whole.