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Background: There are many hypotheses about the neuropathological origin of sporadic Alzheimer’s disease (AD). The amyloid cascade hypothesis remains the most widely accepted by the scientific community, although the role of amyloid-β (Aβ) as the sole cause of neuropathological features of the non-inherited form of AD is questionable. Methods: The 5xFAD mouse model shows amyloid plaques formation and cognitive impairment from 4 months of age. However, no attention is paid to the effect of genetic load, heterozygous (HTZ) versus homozygous (HZ) condition, on the behaviour, histology, metabolic physiology, and composition of gut microbiota. We studied the phenotype of HTZ and HZ mice of both sexes at 4-6 months (early disease onset) and 10-12 months of age (late disease onset). Results: We described that from 4-6 months old, both HTZ and HZ mice exhibited hippocampal impairment caused by accumulation of Aβ40 and Aβ42, increased neuroinflammatory markers and altered PI3K-Akt pathway. However, only HZ mice showed cognitive impairment in the Y-maze test and the Morris water maze. In addition, dysregulation of insulin-linked metabolic pathways was detected at the peripheral level between HTZ and HZ mice. This striking difference between behavioural, immunopathological and physiological phenotypes extends to the microbiota in terms of Firmicutes/Bacteroidetes ratio, being most pronounced in HZ mice at 10-12 months old. Conclusions: Taken together, our results suggest that Aβ aggregation may not be the sole cause of worsening cognitive impairment, but that other factors such as PI3K-Akt, insulin-linked metabolic pathways, and microbiota composition could be influencing disease progression, allowing for alternative disease-modifying interventions.

The present study characterizes the oral pharmacokinetics of D-Pinitol, a natural insulin mimetic inositol, in human healthy volunteers (14 males and 11 females). D-Pinitol absorption was studied in (a) subjects receiving a single oral dose of 15 mg/kg (n = 10), or (b) 5 mg/kg pure D-Pinitol (n = 6), and (c) subjects receiving D-Pinitol as part of carbohydrate-containing carob pods-derived syrup with a 3.2% D-Pinitol (Dose of 1600 mg/subject, n = 9). The volunteers received a randomly assigned single dose of either D-Pinitol or carob pod-derived syrup. Blood samples were collected at 0, 15, 30, 45, 60, 90, 120, 180, 240, 360 and 1440 min after intake. Plasma concentration of D-Pinitol was measured and pharmacokinetic parameters obtained. The data indicate that when given alone, the oral absorption of D-Pinitol is dose-dependent and of extended duration, with a Tmax reached after almost 4 h, and a half-life greater than 5 h. When the source of D-Pinitol was a carob pods-derived syrup, Cmax was reduced to 40% of the expected based on the data of D-Pinitol alone, suggesting a reduced absorption probably because of competition with monosaccharide transport. In this group, Tmax was reached before that of D-Pinitol alone, but the estimated half-life remained the same. In the D-Pinitol groups, plasma concentrations of glucose, insulin, glucagon, ghrelin, free fatty acids, and pituitary hormones were additionally measured. A dose of 15 mg/kg of D-Pinitol did not affect glucose levels in healthy volunteers, but reduced insulin and increased glucagon and ghrelin concentrations. D-Pinitol did not increase other hormones known to enhance plasma glucose, such as cortisol or GH, which were surprisingly reduced after the ingestion of this inositol. Other pituitary hormones (gonadotropins, prolactin, and thyroid-stimulating hormone) were not affected after D-Pinitol ingestion. In a conclusion, D-Pinitol is absorbed through the oral route, having an extended half-life and displaying the pharmacological profile of an endocrine pancreas protector, a pharmacological activity of potential interest for the treatment or prevention of insulin resistance-associated conditions.

18 Pág. Departamento de Reproducción animal., Recent evidence links brain insulin resistance with neurodegenerative diseases, where hyperphosphorylated tau protein contributes to neuronal cell death. In the present study, we aimed to evaluate if d-pinitol inositol, which acts as an insulin sensitizer, affects the phosphorylation status of tau protein., This research was funded by the European Regional Development Funds-European Union (ERDF-EU), FATZHEIMER project (EU-LAC HEALTH 2020, 16/T010131), “Una manera de hacer Europa”; Ministerio de Economía, Industria y Competitividad, Gobierno de España, Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad (RTC-2016-4983-1); ERDF-EU-Instituto de Salud Carlos III (ISCIII), Proyectos de investigación en salud (PI19/01577); Consejería de Salud y Familias, Junta de Andalucía, Proyecto de Investigación en Salud (PI-0139-2018); Consejería de Economía, Conocimiento y Universidad, Junta de Andalucía, Plan Andaluz de Investigación, Desarrollo e Innovación (P18-TP-5194); Delegación del Gobierno para el Plan Nacional sobre Drogas, Ministerio de Salud, Gobierno de España (PND2020/048). D.M-V. (FI20/00227) holds a “PFIS” predoctoral contract from the National System of Health, ERDF-EU-Instituto de Salud Carlos III. A.J.L.-G. (IFI18/00042) holds an “iPFIS” predoctoral contract from the National System of Health, ERDF-EU-ISCIII. P.R. (CP19/00068), F.J.P. (CPII19/00022) and J.D. (CP21/00021) hold a “Miguel Servet” research contract from the National System of Health, ISCIII co-funded by European Social Fund, “Investing in your future,” Gobierno de España.

Peptidic agonists of the glucagon-like peptide-1 receptor (GLP-1R) have gained a prominent role in the therapy of type-2 diabetes and are being considered for reducing food intake in obesity. Potential advantages of small molecules acting as positive allosteric modulators (PAMs) of GLP-1R, including oral administration and reduced unwanted effects, could improve the utility of this class of drugs. Here, we describe the discovery of compound 9 (4- {[1-({3-[4-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5-yl}methyl)- piperidin-3-yl]methyl}morpholine, V-0219) that exhibits enhanced efficacy of GLP-1R stimulation, subnanomolar potency in the potentiation of insulin secretion, and no significant off-target activities. The identified GLP-1R PAM shows a remarkable in vivo activity, reducing food intake and improving glucose handling in normal and diabetic rodents. Enantioselective synthesis revealed oral efficacy for (S)-9 in animal models. Compound 9 behavior bolsters the interest of a small-molecule PAM of GLP-1R as a promising therapeutic approach for the increasingly prevalent obesity-associated diabetes.

D-Pinitol (DPIN) is a natural occurring inositol capable of activating the insulin pathway in peripheral tissues, whereas this has not been thoroughly studied in the central nervous system. The present study assessed the potential regulatory effects of DPIN on the hypothalamic insulin signaling pathway. To this end we investigated the Phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (Akt) signaling cascade in a rat model following oral administration of DPIN. The PI3K/Akt-associated proteins were quantified by Western blot in terms of phosphorylation and total expression. Results indicate that the acute administration of DPIN induced time-dependent phosphorylation of PI3K/Akt and its related substrates within the hypothalamus, indicating an activation of the insulin signaling pathway. This profile is consistent with DPIN as an insulin sensitizer since we also found a decrease in the circulating concentration of this hormone. Overall, the present study shows the pharmacological action of DPIN in the hypothalamus through the PI3K/Akt pathway when giving in fasted animals. These findings suggest that DPIN might be a candidate to treat brain insulin-resistance associated disorders by activating insulin response beyond the insulin receptor., This work was supported by grants from the following institutions: Ministerio de Economía y Competitividad, Gobierno de España (Grant RTC-2016-4983-1), EU-ERDF-Instituto de Salud Carlos III (Grant PI19/01577), and Consejería de Economía, Conocimiento y Universidad, Junta de Andalucía (Grant P18-TP-5194), Proyecto de Investigación en Salud, Consejería de Salud y Familias (PI-0139-2018). D.M-V. (FI20/00227) holds a “PFIS” predoctoral contract from the National System of Health, EU-ERDF-Instituto de Salud Carlos III

This work was supported by grants from the following institutions: Grant for international postdoctoral stay. Jose Castillejo Program (Grant CAS15/00257), Ministerio de Educación Cultura y Deporte. Gobierno de España. Proyecto de Investigación en Salud, Consejería de Salud y Familias (Grant PI-0139-2018). EU-ERDF-Instituto de Salud Carlos III (Grant PI19/01577). Proyectos dedesarrollo tecnológico, Ministerio de Ciencia e Innovación (Grant DTS19/00125). Ministerio de Economía, Industria y Competitividad (Grant RTC-2019-007329-1).

Polycystic ovarian syndrome (PCOS) is the main cause of female infertility. It is a multifactorial disorder with varying clinical manifestations including metabolic/endocrine abnormalities, hyperandrogenism, and ovarian cysts, among other conditions. D-Chiro-inositol (DCI) is the main treatment available for PCOS in humans. To address some of the mechanisms of this complex disorder and its treatment, this study examines the effect of DCI on reproduction during the development of different PCOS-associated phenotypes in aged females and two mouse models of PCOS. Aged females (8 months old) were treated or not (control) with DCI for 2 months. PCOS models were generated by treatment with dihydrotestosterone (DHT) on Days 16, 17, and 18 of gestation, or by testosterone propionate (TP) treatment on the first day of life. At two months of age, PCOS mice were treated with DCI for 2 months and their reproductive parameters analyzed. No effects of DCI treatment were produced on body weight or ovary/body weight ratio. However, treatment reduced the number of follicles with an atretic cyst-like appearance and improved embryo development in the PCOS models, and also increased implantation rates in both aged and PCOS mice. DCI modified the expression of genes related to oocyte quality, oxidative stress, and luteal sufficiency in cumulus-oocyte complexes (COCs) obtained from the aged and PCOS models. Further, the phosphorylation of AKT, a main metabolic sensor activated by insulin in the liver, was enhanced only in the DHT group, which was the only PCOS model showing glucose intolerance and AKT dephosphorylation. The effect of DCI in the TP model seemed mediated by its influence on oxidative stress and follicle insufficiency. Our results indicate that DCI works in preclinical models of PCOS and offer insight into its mechanism of action when used to treat this infertility-associated syndrome.

To characterize the metabolic actions of D-Pinitol, a dietary inositol, in male Wistar rats, we analyzed its oral pharmacokinetics and its effects on (a) the secretion of hormones regulating metabolism (insulin, glucagon, IGF-1, ghrelin, leptin and adiponectin), (b) insulin signaling in the liver and (c) the expression of glycolytic and neoglucogenesis enzymes. Oral D-Pinitol administration (100 or 500 mg/Kg) resulted in its rapid absorption and distribution to plasma and liver compartments. Its administration reduced insulinemia and HOMA-IR, while maintaining glycaemia thanks to increased glucagon activity. In the liver, D-Pinitol reduced the key glycolytic enzyme pyruvate kinase and decreased the phosphorylation of the enzymes AKT and GSK-3. These observations were associated with an increase in ghrelin concentrations, a known inhibitor of insulin secretion. The profile of D-Pinitol suggests its potential use as a pancreatic protector decreasing insulin secretion through ghrelin upregulation, while sustaining glycaemia through the liver-based mechanisms of glycolysis control.

The peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that promote ligand-dependent transcription of target genes that regulate energy production, lipid metabolism, and inflammation. The PPAR superfamily comprises three subtypes, PPARα, PPARγ, and PPARβ/δ, with differential tissue distributions. In addition to their different roles in the regulation of energy balance and carbohydrate and lipid metabolism, an emerging function of PPARs includes normal homeostasis of intestinal tissue. PPARα activation represses NF-κB signaling, which decreases the inflammatory cytokine production by different cell types, while PPARγ ligands can inhibit activation of macrophages and the production of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and Il-1β. In this regard, the anti-inflammatory responses induced by PPAR activation might restore physiopathological imbalances associated with inflammatory bowel diseases (IBD). Thus, PPARs and their ligands have important therapeutic potential. This review briefly discusses the roles of PPARs in the physiopathology and therapies of the most important IBDs, ulcerative colitis (UC), and Crohn's disease (CD), as well some new experimental compounds with PPAR activity as promising drugs for IBD treatment.

Cocaine induces neuroinflammatory response and interleukin-1 beta (IL1β) is suggested a final effector for many cocaine-induced inflammatory signals. Recently, the chemokine fractalkine (CX

Background and purpose Lipogenesis is intimately controlled by hormones and cytokines as well as nutritional conditions. IL-6 participates in the regulation of fatty acid metabolism in the liver. We investigated the role of IL-6 in mediating fasting/re-feeding changes in the expression of hepatic lipogenic enzymes. Experimental approach Gene and protein expression of lipogenic enzymes were examined in livers of wild-type (WT) and IL-6-deficient (IL-6(-/-) ) mice during fasting and re-feeding conditions. Effects of exogenous IL-6 administration on gene expression of these enzymes were evaluated in vivo. The involvement of STAT3 in mediating these IL-6 responses was investigated by using siRNA in human HepG2 cells. Key results During feeding, the up-regulation in the hepatic expression of lipogenic genes presented similar time kinetics in WT and IL-6(-/-) mice. During fasting, expression of lipogenic genes decreased gradually over time in both strains, although the initial drop was more marked in IL-6(-/-) mice. Protein levels of hepatic lipogenic enzymes were lower in IL-6(-/-) than in WT mice at the end of the fasting period. In WT, circulating IL-6 levels paralleled gene expression of hepatic lipogenic enzymes. IL-6 administration in vivo and in vitro showed that IL-6-mediated signalling was associated with the up-regulation of hepatic lipogenic enzyme genes. Moreover, silencing STAT3 in HepG2 cells attenuated IL-6 mediated up-regulation of lipogenic gene transcription levels. Conclusions and implications IL-6 sustains levels of hepatic lipogenic enzymes during fasting through activation of STAT3. Our findings indicate that clinical use of STAT3-associated signalling cytokines, particularly against steatosis, should be undertaken with caution.

14 p.-8 fig., Chronic NP-1 administration reduces body weight and hepatic steatosis despite induction of tolerance in adiponectin gene transcription with respect to the acute actions of this drug. This study explored the hypothesis that NP-1 could exert these effects through mechanisms independent of adiponectin. To this aim, we took advantage of the Zucker (fa/fa) rat model, which exhibits obesity, fatty liver and elevated leptin and adiponectin levels. Body weight and food intake were reduced after chronic NP-1 treatment. Plasma TNF alpha concentrations were elevated but no increase in adiponectin was found. Even so, NP-1 ameliorated fatty liver and corrected dyslipidemia by mechanisms probably associated with reduced feeding, transcription of Cpt1 and down-regulation of Hmgcr-CoA expression. In brown fat tissue NP-1 increased Dnmt1 (inhibitor of Adipoq) while it reduced Ucp1 expression and heat production, which excludes thermogenesis as a mechanism of the NP-1 slimming effect. The anti-obesity action of chronic NP-1 administration might be mediated by TNF alpha, which is known to have anorectic actions in the hypothalamus and to regulate both Dmnt1 and Ucp1 expression in adipose tissues. This finding opens up the possibility of using NP-1-mediated TNF alpha-induced weight loss as an innovative treatment of complicated obesity under strict pharmacologic control., This work was supported by Instituto de Salud Carlos III (ISCIII), Ministerio de Economía y Competitividad (MINECO) co-funded by UE-ERDF program (JS: CP12/03109, PI16/01374; FRF: PI16/01698); Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, UE-ERDF (FRF: CTS-8221); Consejería de Salud, Junta de Andalucía, UE-ERDF (FRF: SAS111224). JS (CPII17/00024), AS (CP14/00173) and FJP (CP14/00212) hold “Miguel Servet” research contracts from the Instituto de Salud Carlos III (ISCIII). PR holds a “Sara Borrel” research contract from the Instituto de Salud Carlos III (ISCIII) (CD16/00067).

Interleukin-6 (IL-6) has emerged as an important mediator of fatty acid metabolism with paradoxical effects in the liver. Administration of IL-6 has been reported to confer protection against steatosis, but plasma and tissue IL-6 concentrations are elevated in chronic liver diseases, including fatty liver diseases associated with obesity and alcoholic ingestion. In this study, we further investigated the role of IL-6 on steatosis induced through a high-fat diet (HFD) in wild-type (WT) and IL-6-deficient (IL-6−/−) mice. Additionally, HFD-fed IL-6−/− mice were also chronically treated with recombinant IL-6 (rIL-6). Obesity in WT mice fed a HFD associated with elevated serum IL-6 levels, fatty liver, upregulation of carnitine palmitoyltransferase 1 (CPT1) and signal transducer and activator of transcription-3 (STAT3), increased AMP kinase phosphorylation (p-AMPK), and downregulation of the hepatic lipogenic enzymes fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). The HFD-fed IL-6−/− mice showed severe steatosis, no changes in CPT1 levels or AMPK activity, no increase in STAT3 amounts, inactivated STAT3, and marked downregulation of the expression of acetyl-CoA carboxylase (ACCα/β), FAS and SCD1. The IL-6 chronic replacement in HFD-fed IL-6−/− mice restored hepatic STAT3 and AMPK activation but also increased the expression of the lipogenic enzymes ACCα/β, FAS and SCD1. Furthermore, rIL-6 administration was associated with aggravated steatosis and elevated fat content in the liver. We conclude that, in the context of HFD-induced obesity, the administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis., Summary: The administration of rIL-6 might contribute to the aggravation of fatty liver disease through increasing lipogenesis in HFD-induced obesity.

Cocaine induces neuroinflammatory response and interleukin-1 beta (IL1β) is suggested a final effector for many cocaine-induced inflammatory signals. Recently, the chemokine fractalkine (CX3CL1) has been reported to regulate hippocampus-dependent neuroinflammation and synaptic plasticity via CX3C-receptor 1 (CX3CR1), but little is known about the impact of cocaine. This study is mainly focused on the characterization of CX3CL1, IL1β and relevant inflammatory signal transduction pathways in the hippocampus in acute and repeated cocaine-treated male mice. Complementarily, the rewarding properties of cocaine were also assessed in Cx3cr1-knockout (KO) mice using a conditioned place preference (CPP). We observed significant increases in CX3CL1 and IL1β concentrations after cocaine, although repeated cocaine produced an enhancement of CX3CL1 concentrations. CX3CL1 and IL1β concentrations were positively correlated in acute (r = +0.61) and repeated (r = +0.82) cocaine-treated mice. Inflammatory signal transduction pathways were assessed. Whereas acute cocaine-treated mice showed transient increases in p-ERK1/2/ERK1/2 and p-p65/p65 NFκB ratios after cocaine injection, repeated cocaine-treated mice showed transient increases in p-ERK1/2/ERK1/2, p-p38/p38 MAPK, p-NFκB p65/NF-κB p65 and p-CREB/CREB ratios. Baseline p-p38/p38 MAPK and p-CREB/CREB ratios were downregulated in repeated cocaine-treated mice. Regarding the cocaine-induced CPP, Cx3cr1-KO mice showed a notably impaired extinction but no differences during acquisition and reinstatement. These results indicate that cocaine induces alterations in CX3CL1 concentrations, which are associated with IL1β concentrations, and activates convergent inflammatory pathways in the hippocampus. Furthermore, the CX3CL1/CX3CR1 signaling could mediate the processes involved in the extinction of cocaine-induced CPP.

The treatment for cocaine use constitutes a clinical challenge because of the lack of appropriate therapies and the high rate of relapse. Recent evidence indicates that the immune system might be involved in the pathogenesis of cocaine addiction and its co-morbid psychiatric disorders. This work examined the plasma pro-inflammatory cytokine and chemokine profile in abstinent cocaine users (n = 82) who sought outpatient cocaine treatment and age/sex/body mass-matched controls (n = 65). Participants were assessed with the diagnostic interview Psychiatric Research Interview for Substance and Mental Diseases according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR). Tumor necrosis factor-alpha, chemokine (C-C motif) ligand 2/monocyte chemotactic protein-1 and chemokine (C-X-C motif) ligand 12 (CXCL12)/stromal cell-derived factor-1 (SDF-1) were decreased in cocaine users, although all cytokines were identified as predictors of a lifetime pathological use of cocaine. Interleukin-1 beta (IL-1β), chemokine (C-X3-C motif) ligand 1 (CX3CL1)/fractalkine and CXCL12/SDF-1 positively correlated with the cocaine symptom severity when using the DSM-IV-TR criteria for cocaine abuse/dependence. These cytokines allowed the categorization of the outpatients into subgroups according to severity, identifying a subgroup of severe cocaine users (9-11 criteria) with increased prevalence of co-morbid psychiatric disorders [mood (54%), anxiety (32%), psychotic (30%) and personality (60%) disorders]. IL-1β was observed to be increased in users with such psychiatric disorders relative to those users with no diagnosis. In addition to these clinical data, studies in mice demonstrated that plasma IL-1β, CX3CL1 and CXCL12 were also affected after acute and chronic cocaine administration, providing a preclinical model for further research. In conclusion, cocaine exposure modifies the circulating levels of pro-inflammatory mediators. Plasma cytokine/chemokine monitoring could improve the stratification of cocaine consumers seeking treatment and thus facilitate the application of appropriate interventions, including management of heightened risk of psychiatric co-morbidity. Further research is necessary to elucidate the role of the immune system in the etiology of cocaine addiction.

The current epidemic of obesity in western countries is being worsened by the lack of effective pharmacotherapies. The apparent success of a central nervous systemacting cannabinoid CB1 receptor antagonist-based treatment for obesity was hampered by the appearance of psychiatric side effects in certain patients. These adverse effects forced its withdrawal from the market. However, the discovery that the main beneficial metabolic effects of cannabinoid CB1 receptor antagonists were derived of its activity in peripheral tissues, including the adipose tissue, opened the possibility of rescuing this type of therapy. This goal might be achieved by differential medicinal chemistry approaches. The present review examines these options that include peripheral-restricted cannabinoid CB1 receptor antagonists, dual ligands and combinatorial therapies using sub-effective doses of CB1 receptor antagonists that might be devoid of side effects.

Enhancement of adiponectin level has been shown to have beneficial effects, including antiobesity, antidiabetic, and hepatoprotective effects. This evidence supports the therapeutic utility of adiponectin in complicated obesity. The present study characterized the in vivo effects of sustained adiponectin release by NP-1, a new class of thiazol derivative that increases adiponectin levels. Acute administration of NP-1 reduced feeding, increased plasma adiponectin, and improved insulin sensitivity without inducing malaise, as revealed by conditioned taste aversion studies. Short-term (7 days) treatment with NP-1 also reduced feeding and body weight gain and increased phosphorylation of AMPK in muscle, a main intracellular effector of adiponectin. NP-1 was also evaluated in diet-induced obesity, and adult male Wistar rats were fed two different types of diet: a standard high-carbohydrate/low-fat diet (SD) and a high-fat diet (HFD). Once obesity was established, animals were treated daily with NP-1 (5 mg/kg) for 14 consecutive days. Chronic NP-1 induced body weight loss and reduction of food intake and resulted in both a marked decrease in liver steatosis and an improvement of biochemical indexes of liver damage in HFD-fed rats. However, a marked induction of tolerance in adiponectin gene transcription and release was observed after chronic NP-1 with respect to the acute actions of this drug. The present results support the role of adiponectin signaling in diet-induced obesity and set in place a potential use of compounds able to induce adiponectin release for the treatment of obesity and nonalcoholic fatty liver, with the limits imposed by the induction of pharmacological tolerance.

Journal Article; High-fat diet-induced obesity (DIO) is associated with fatty liver and elevated IL-6 circulating levels. IL-6 administration in rodents has yielded contradictory results regarding its effects on steatosis progression. In some models of fatty liver disease, high doses of human IL-6 ameliorate the liver steatosis, whereas restoration of IL-6 in DIO IL-6-/- mice up-regulates hepatic lipogenic enzymes and aggravates steatosis. We further examined the effects of chronic low doses of murine IL-6 on hepatic lipid metabolism in WT mice in DIO. IL-6 was delivered twice daily in C57BL/6J DIO mice for 15 days. The status and expression of IL-6-signalling mediators and targets were investigated in relation to the steatosis and lipid content in blood and in liver. IL-6 administration in DIO mice markedly raised circulating levels of lipids, glucose and leptin, elevated fat liver content and aggravated steatosis. Under IL-6 treatment there was hepatic Stat3 activation and increased gene expression of Socs3 and Tnf-alpha whereas the gene expression of endogenous IL-6, IL-6-receptor, Stat3, Cpt1 and the enzymes involved in lipogenesis was suppressed. These data further implicate IL-6 in fatty liver disease modulation in the context of DIO, and indicate that continuous stimulation with IL-6 attenuates the IL-6-receptor response, which is associated with high serum levels of leptin, glucose and lipids, the lowering levels of lipogenic and Cpt1 hepatic enzymes and with increased Tnf-alpha hepatic expression, a scenario evoking that observed in IL-6-/- mice exposed to DIO and in obese Zucker rats. Yes

The endogenous cannabinoid acylethanolamide AEA (arachidonoylethanolamide; also known as anandamide) participates in the neuroadaptations associated with chronic ethanol exposure. However, no studies have described the acute actions of ethanol on AEA production and degradation. In the present study, we investigated the time course of the effects of the intraperitoneal administration of ethanol (4 g/kg of body mass) on the endogenous levels of AEA in central and peripheral tissues. Acute ethanol administration decreased AEA in the cerebellum, the hippocampus and the nucleus accumbens of the ventral striatum, as well as in plasma and adipose tissue. Parallel decreases of a second acylethanolamide, PEA (palmitoylethanolamide), were observed in the brain. Effects were observed 45–90 min after ethanol administration. In vivo studies revealed that AEA decreases were associated with a remarkable inhibition of the release of both anandamide and glutamate in the nucleus accumbens. There were no changes in the expression and enzymatic activity of the main enzyme that degrades AEA, the fatty acid amidohydrolase. Acute ethanol administration did not change either the activity of N-acyltransferase, the enzyme that catalyses the synthesis of the AEA precursor, or the expression of NAPE-PLD (N-acylphosphatidylethanolamine-hydrolysing phospholipase D), the enzyme that releases AEA from membrane phospholipid precursors. These results suggest that receptor-mediated release of acylethanolamide is inhibited by the acute administration of ethanol, and that this effect is not derived from increased fatty acid ethanolamide degradation.

Combination therapy with interferon-alpha (IFNα) and ribavirin is the current treatment of choice for hepatitis C virus (HCV) infection. However, an important number of patients fail to respond to this therapeutic strategy. Factors determining IFN responsiveness are not well understood, and assessment of biomarkers that predict the response to IFN therapy in HCV patients is necessary. Several studies show that particular HCV proteins are able to block IFN function through interaction with important IFN-signal mediators, such as signal transducers and activators of transcription (STATs). We performed immunostaining analysis of STATs in liver tissue from IFN-responder vs. non-responder HCV patients in order to compare the expression profile of these proteins between both groups. Tissue arrays of liver biopsies were used to study the expression of STAT1, STAT2, STAT5 and PIAS1 (protein inhibitor of activated STAT1). Robust and higher expression levels of STAT1, STAT2 and STAT5 in liver tissue from HCV patients were found when compared with samples from healthy donors. However, no significant differences were observed between IFN-responder and -non-responder groups, but rather increasing levels of STAT1, STAT2 and STAT5 paralleled the degree of liver injury. Importantly, PIAS1 expression in the nucleus of most hepatocytes in HCV tissue biopsy sections, particularly of non-responder HCV patients, strongly indicated a regulatory effect on STAT1–DNA binding, likely affecting the IFN late signalling. In conclusion, our evidence indicates that intense PIAS1 nuclear staining, widely distributed in hepatocytes of infected livers, could be a good predictive factor of a defective response to IFN treatment, and a biomarker that is easily detectable by immunostaining during standard histopathological liver biopsy analysis. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background & Aims: Cardiotrophin-1 (CT-1) is a member of the interleukin 6 (IL-6) family of cytokines, which protect cardiac myocytes against thermal and ischemic insults. In this study, we investigated the expression of CT-1 by liver cells and its possible hepatoprotective properties. Methods: We analyzed the production, signaling, and antiapoptotic properties of CT-1 in hepatocytes and the expression of this cytokine during liver regeneration. We also investigated whether CT-1 might exert protective effects in animal models of liver damage. Results: We found that CT-1 is up-regulated during liver regeneration and exerts potent antiapoptotic effects on hepatocytic cells. Hepatocytes cultured under serum starvation or stimulated with the pro-apoptotic cytokine transforming growth factor β (TGF-β) produce CT-1, which behaves as an autocrine/paracrine survival factor. Treatment with an adenovirus encoding CT-1 efficiently protects rats against fulminant liver failure after subtotal hepatectomy, an intervention that causes 91% mortality in control animals whereas 54% of those receiving CT-1 gene therapy were long-term survivors. This protective effect was associated with reduced caspase-3 activity and activation of the antiapoptotic signaling cascades signal transducer and activator of transcription (Stat-3), extracellular regulated kinases (Erk) 1/2, and Akt in the remnant liver. Gene transfer of CT-1 to the liver also abrogated Concanavalin A (Con-A) liver injury and activated antiapoptotic pathways in the hepatic tissue. Similar protection was obtained by treating the animals with 5 μg of recombinant CT-1 given intravenously before Con-A administration. Conclusions: We show that CT-1 is a hepatocyte survival factor that efficiently reduces hepatocellular damage in animal models of acute liver injury. Our data point to CT-1 as a new promising hepatoprotective therapy.

© 2015 Society for the Study of Addiction. Oleoylethanolamide (OEA) is a satiety factor that controls motivational responses to dietary fat. Here we show that alcohol administration causes the release of OEA in rodents, which in turn reduces alcohol consumption by engaging peroxisome proliferator-activated receptor-alpha (PPAR-α). This effect appears to rely on peripheral signaling mechanisms as alcohol self-administration is unaltered by intracerebral PPAR-α agonist administration, and the lesion of sensory afferent fibers (by capsaicin) abrogates the effect of systemically administered OEA on alcohol intake. Additionally, OEA is shown to block cue-induced reinstatement of alcohol-seeking behavior (an animal model of relapse) and reduce the severity of somatic withdrawal symptoms in alcohol-dependent animals. Collectively, these findings demonstrate a homeostatic role for OEA signaling in the behavioral effects of alcohol exposure and highlight OEA as a novel therapeutic target for alcohol use disorders and alcoholism. Oleoylethanolamide (OEA) is a satiety factor that binds to the peroxisome proliferator-activated receptor-alpha (PPARα). This bioactive lipid mediator also acts as a homeostatic signal that controls multiple aspects of the physiological adaptations to alcohol exposure. OEA production is triggered by alcohol administration, contributes to the regulation of alcohol consumption and the acute motivational response to alcohol, and reduces the severity of somatic withdrawal symptoms.

Patients infected with hepatitis C virus (HCV) have an impaired response against HCV antigens while keeping immune competence for other antigens. We hypothesized that expression of HCV proteins in infected dendritic cells (DC) might impair their antigen-presenting function, leading to a defective anti-HCV T-cell immunity. To test this hypothesis, DC from normal donors were transduced with an adenovirus coding for HCV core and E1 proteins and these cells (DC-CE1) were used to stimulate T lymphocytes. DC-CE1 were poor stimulators of allogeneic reactions and of autologous primary and secondary proliferative responses. Autologous T cells stimulated with DC-CE1 exhibited a pattern of incomplete activation characterized by enhanced CD25 expression but reduced interleukin 2 production. The same pattern of incomplete lymphocyte activation was observed in CD4+T cells responding to HCV core in patients with chronic HCV infection. However, CD4+response to HCV core was normal in patients who cleared HCV after alpha interferon therapy. Moreover, a normal CD4+response to tetanus toxoid was found in both chronic HCV carriers and patients who had eliminated the infection. Our results suggest that expression of HCV structural antigens in infected DC disturbs their antigen-presenting function, leading to incomplete activation of anti-HCV-specific T cells and chronicity of infection. However, presentation of unrelated antigens by noninfected DC would allow normal T-cell immunity to other pathogens.

Dependence of murine pro-B Ba/F3 cells on interleukin-3 can be substituted by GH when cells are stably transfected with the GH receptor (GHR) complementary DNA. Recently, we demonstrated that Ba/F3 cells produce GH, which is responsible for the survival of cells expressing the GHR. This GH effect involves the activation of nuclear factor-kappaB (NF-kappaB). Here, we examined the signaling pathways mediating proliferation of growth factor-deprived Ba/F3 GHR cells. Exogenous GH stimulation of Ba/F3 GHR cells induced cyclins E and A and the cyclin-dependent kinase inhibitor p21(waf1/cip1) and repressed cyclin-dependent kinase inhibitor p27(kip1). The presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor Ly 294002 abolished proliferation induced by GH, arresting Ba/F3 GHR cells at the G(1)/S boundary, but did not promote apoptosis. Thus, the proliferative effect of GH is closely related to PI 3-kinase activation, whereas PI 3-kinase is not essential for GH-induced cell survival. Addition of Ly 294002 resulted in a moderate decrease in NF-kappaB activation by GH, suggesting a possible link between PI 3-kinase and NF-kappaB signaling by GH. Expression of c-myc was also induced by GH in Ba/F3 GHR cells, and inactivation of either PI 3-kinase or NF-kappaB reduced this induction. Overexpression of the dominant negative repressor mutant c-Myc-RX resulted in an inhibition of the GH proliferative effect, suggesting the involvement of c-myc in GH-induced proliferation. Taken together, these results suggest that the effects of GH on cell survival and proliferation are mediated through two different signaling pathways, NF-kappaB and PI 3-kinase, respectively; although cross-talk between them has not been excluded. NF-kappaB, which has been shown to be responsible for the antiapoptotic effect of GH, could also participate in GH-induced proliferation, as c-myc expression is promoted by PI 3-kinase, in an NF-kappaB-dependent and -independent manner.

Apoptosis is a form of physiological cell death which can be initiated in response to various stimuli including virus infections. We show that vaccinia virus (VV) infection induces apoptosis in an immature B lymphocyte line, WEHI-231. In these cells, several VV-specific proteins were synthesized during the infection, but neither virus production nor viral DNA synthesis were detected. The intracellular levels of the proto-oncogene Bcl-2, which effectively protects cells from programmed cell death, were found to be down-regulated by the VV infection, suggesting that this down-regulation might be involved in the viral induction of apoptosis in WEHI-231 cells. Stable transfectants overexpressing human Bcl-2 were shown to be resistant to the apoptosis produced by the infection, a finding consistent with the proposed role for the down-regulation of endogenous Bcl-2 in VV-induced apoptotic death.

Journal Article; Research Support, Non-U.S. Gov't; β-adrenergic receptor activation promotes brown adipose tissue (BAT) β-oxidation and thermogenesis by burning fatty acids during uncoupling respiration. Oleoylethanolamide (OEA) can inhibit feeding and stimulate lipolysis by activating peroxisome proliferator-activating receptor-α (PPARα) in white adipose tissue (WAT). Here we explore whether PPARα activation potentiates the effect of β3-adrenergic stimulation on energy balance mediated by the respective agonists OEA and CL316243. The effect of this pharmacological association on feeding, thermogenesis, β-oxidation, and lipid and cholesterol metabolism in epididymal (e)WAT was monitored. CL316243 (1 mg/kg) and OEA (5 mg/kg) co-administration over 6 days enhanced the reduction of both food intake and body weight gain, increased the energy expenditure and reduced the respiratory quotient (VCO2/VO2). This negative energy balance agreed with decreased fat mass and increased BAT weight and temperature, as well as with lowered plasma levels of triglycerides, cholesterol, nonessential fatty acids (NEFAs), and the adipokines leptin and TNF-α. Regarding eWAT, CL316243 and OEA treatment elevated levels of the thermogenic factors PPARα and UCP1, reduced p38-MAPK phosphorylation, and promoted brown-like features in the white adipocytes: the mitochondrial (Cox4i1, Cox4i2) and BAT (Fgf21, Prdm16) genes were overexpressed in eWAT. The enhancement of the fatty-acid β-oxidation factors Cpt1b and Acox1 in eWAT was accompanied by an upregulation of de novo lipogenesis and reduced expression of the unsaturated-fatty-acid-synthesis enzyme gene, Scd1. We propose that the combination of β-adrenergic and PPARα receptor agonists promotes therapeutic adipocyte remodelling in eWAT, and therefore has a potential clinical utility in the treatment of obesity. Yes

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 CB 1 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.

The function of the African swine fever virus (ASFV)bcl-2homologue, geneA179L,in the regulation of apoptosis was investigated using as a model system the human myeloid leukemia cell line K562 induced to die by apoptosis with inhibitors of macromolecular synthesis, a process that is prevented by overexpression of humanbcl-2.It is shown that transfection of K562 cells with the ASFVA179Lgene protects these cells from apoptotic cell death induced by a combination of cycloheximide and actinomycin D or by treatment with cytosine arabinoside. To test the functional role of the highly conserved BH1 domain present in the A179L protein, the Gly residue at position 85 was mutated to Ala, since it has been shown that substitution of the corresponding Gly in human Bcl-2 abrogates its death-repressor activity. It was found that the Gly-to-Ala mutation in the BH1 domain of the viral protein abolished its capacity to protect the K562 cells from apoptosis, indicating that this Gly is essential for A179L action. This finding stresses the functional similarity of the BH1 domains of the viral protein and cellular Bcl-2., This work was 29. Hockenberry, D. M., Oltvai, Z. N., Yin, X.-M., Milliman, C. L., and supported by grants from the Dirección General de Investigación Korsmeyer, S. J., Cell 75, 241 – 251 (1993). Científica y Técnica (PB93-0160-C02-02), the European Community 30. Nguyen, M., Branton, P. E., Walton, P. A., Oltvai, Z. N., Korsmeyer, (AIR-CT93-1332), and Comunidad de Madrid (AE00424/95), and by an S. J., and Shore, G. C., J. Biol. Chem. 269, 16521 – 16524 (1994). institutional grant from Fundacio´n Ramo´n Areces to the Centro de 31. Afonso, C. L., Neilan, J. G., Kutish, G. F., and Rock, D. L., J. Virol. 70, BiologıBa Molecular ‘‘Severo Ochoa.’’

et al., [Background]: LFABP plays a critical role in the uptake and intracellular transport of fatty acids (FA) and other peroxisome proliferator-activated receptor alpha (PPARα) ligands. PPARα activation by PPARα ligands bound to LFABP results in gene expression of FA oxidation enzymes and de novo LFABP. The cytokine IL-6 is involved in regulating liver lipid oxidation. [Aims]: To study the ability of IL-6 to modulate the expression of the LFABP in hepatocytes. Methods: HepG2 and mouse primary hepatocytes were used to test LFABP mRNA and protein expression after IL-6 and PPARα-ligand treatments. Mice lacking IL-6 and wild-type C57Bl/6 were subjected to a fasting/re-feeding cycle to monitor hepatic LFABP mRNA kinetics after food intake. [Results]: In hepatocyte cultures, IL-6 treatment stimulated a LFABP mRNA sustained expression. Combined treatment of IL-6 plus PPARα ligands further enhanced LFABP gene and protein expression. In contrast, pretreatment with the PPARα-antagonist GW-6471 prevented the up-regulation of LFABP mRNA induced by IL-6 in the late phase of LFABP kinetics. Furthermore, the up-regulation of LFABP mRNA observed in the liver of wild-type mice 8 h after re-feeding was absent in mice lacking IL-6. [Conclusions]: IL-6 induces LFABP kinetics in hepatocytes and is partially dependent on PPARα. The maximum increase in LFABP expression occurs when the stimulation with IL-6 and PPARα-ligands takes place simultaneously. The in vivo results indicate a postprandial regulation of LFABP that correlates with the presence of IL-6. These effects may have important implications in the postprandial increase in FA uptake and intracellular trafficking in the liver. © 2013 John Wiley & Sons A/S., This work was supported by Grants PI061109, from the Spanish Institute of Health ‘Carlos III’; TCMR0019, PI0552, PI0236/08, PI0608/10 from the Andalusian Ministry of Health; SAF2012-33283, SAF2010-22113, SAF2010-20521 and CTS-6747/2010; from the Spanish Ministry of Science and Innovation and ‘Centro de Investigacion Biomédica en Red de Diabetes y Enfermedades Metabolicas Asociadas’ (CIBER-DEM), and CIBERobn EU-ERDF(CB06/03/1008). Grants from the European Union’s 7th Framework Programme (Health-F2-2008-223713, REPROBESITY). Elena Baixeras is a tenured investigator through the I3SNS Program of the ‘Andalusian Progreso y Salud Foundation’, Spain.

Apoptosis of natural killer (NK) cells can be induced by non-specific physical damage (UV irradiation, heat shock) or by simultaneous ligation of the CD16 and the interleukin-2 receptor (IL-2R) molecules, but not with either anti-CD16 or IL-2 alone. Whereas blockade of GTP-binding protein (G protein)-mediated signal transduction using ADP-ribosylating bacterial toxins or the GTPase-resistant GTP analog guanosine 5'-0-(3-thiotriphosphate (GTP gamma S) does not affect non-specific induction of NK cell apoptosis, such interventions do inhibit induction of apoptosis by anti-CD16/IL-2. The G proteins involved in the regulation of activation-induced NK apoptosis are sensitive to pertussis toxin (PTX) and to the non-specific GTP analog GTP gamma S but not to cholera toxin, Pseudomonas exotoxin A or diphtheria toxin. A pertussis toxin mutant that lacks ADP-ribosylating activity, but conserves the membrane translocating and T cell-mitogenic effects of the native molecule, fails to inhibit NK apoptosis. To exert their apoptosis-inhibitory effect, PTX and GTP gamma S must be employed before cells are activated. Later addition has no effect, suggesting the implication of G proteins in the transmission of apoptosis-inducing signals, but not in the effector stage of apoptosis. Pre-incubation with PTX or GTP gamma S does not affect the activation of NK cells by CD16 cross-linking, IL-2 stimulation- or both, as assessed by the induction of CD69 expression, protein tyrosine phosphorylation and calcium mobilization. Moreover, neither PTX nor GTP gamma S compromise the effector function of NK cells or the susceptibility of target cells to NK-mediated lysis. These data suggest apoptosis as a novel mechanism by which NK responses may be controlled in vivo, as well as an experimental and therapeutical strategy to counteract endogenous down-regulation of NK responses.

Intravenous injection of a bacterial superantigen such as Staphylococcus aureus enterotoxin B (SEB) causes transient activation and expansion of SEB-reactive V beta 8+ T cells, as well as specific down-regulation of the immune response, through partial deletion of superantigen-reactive T cells. Here we demonstrate that co-administration of pertussis toxin (PTX) and SEB reduces the SEB-induced deletion of V beta 8+ T cells, although it does not affect T cell activation and proliferation. PTX abrogates the SEB-driven deletion of V beta 8+CD4+ (not V beta 8+CD8+) splenocytes that is observed early (12-24 h) after SEB injection. Moreover, it antagonizes the late (> or = 4 days) deletion of V beta 8+CD4+ and V beta 8+CD8+ peripheral T cells that follows transient expansion of such cells. This phenomenon is associated with significant reductions in apoptosis and endonucleolysis and is not caused by a compensatory increase in proliferation of SEB-reactive T cells, as we determined by using a combined fluorometric analysis of cell cycle and DNA alterations, which are associated with programmed cell death. These effects are also observed in thymectomized animals, thus excluding the possibility that PTX might act by enhancing the maturation and export of thymic T cells to the periphery. Moreover, the SEB-induced reduction of V beta 8+ splenocytes is antagonized by PTX in vitro. The capacity of PTX to reduce clonal deletion depends critically on its ADP-ribosyltransferase activity, inasmuch as a non-enzymatic PTX mutant fails to act in this biologic system. We conclude that PTX selectively antagonizes or impedes the delivery of negative signals to T cells, which are stimulated by superantigens, without interfering with the transmission of stimulatory signals.

The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3-expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.

7 páginas, 3 figuras, 2 tablas., Glucokinase is essential for glucose-stimulated insulin release from the pancreatic beta-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported, This work was supported by grants (to A.L.C.-M. and N.C.-V.) from Ministerio de Ciencia e Innovación, Dirección General de Investigación Científica y Técnica (SAF2005-08014; SAF2006-12863) and Junta de Andalucía (SAS/PI-024/2007; SAS/PI-0236/2009)

Interferon-alpha5 (IFN-alpha5) is the main IFN-alpha subtype expressed in the liver. Hepatitis C virus (HCV) infection is associated with low IFN-alpha5 mRNA levels, possibly reflecting an escape mechanism of the virus. In this work, we sought to compare IFN-alpha2 and IFN-alpha5 with respect to activation of early cell signaling cascades and induction of antiviral genes in the human hepatoma HepG2 and Huh7 cell lines. We found that the Tyr701 phosphorylation kinetics of Stat1 mediated by IFN stimulation was higher when cells were incubated with IFN-alpha5 than when using IFN-alpha2. Similarly, Tyr(1054/1055) phosphorylation kinetics of Tyk2 were more intense after exposure to IFN-alpha5 than when using IFN-alpha2. Concomitantly, Tyr705 phosphorylation of Stat3 was higher after stimulation with IFN-alpha5 than with IFN-alpha2. In parallel to these findings, the mRNA levels of the antiviral IFN-inducible gene 2',5'-oligoadenylate synthetase were higher in cell samples treated with IFN-alpha5 than with IFN-alpha2. These findings suggest that interaction of IFN-alpha5 and IFN-alpha2 subtypes with IFN type I receptor occurs differently, and this affects the intensity of expression of antiviral genes. In conclusion, our data show that in hepatocytic cells, IFN-alpha5 induces stronger signaling and higher expression of antiviral genes than IFN-alpha2. These data warrant clinical trials to evaluate the efficacy of IFN-alpha5 in chronic viral hepatitis.

Systemic treatment with an anti-ICAM-2 monoclonal antibody (mAb; EOL4G8) eradicates certain established mouse tumors through a mechanism dependent on the potentiation of a CTL-mediated response. However, well-established tumors derived from the MC38 colon carcinoma cell line were largely refractory to this treatment as well as to intratumor injection of a recombinant adenovirus encoding interleukin-12 (IL-12; AdCMVIL-12). We sought to design combined therapy strategies with AdCMVIL-12 plus anti-ICAM-2 mAbs and to identify their mechanism of action.Analysis of antitumor and toxic effects were performed with C57BL/6 mice bearing established MC38 tumors. Anti-ovalbumin T-cell receptor transgenic mice and tumors transfected with this antigen were used for in vitro and in vivo studies on activation-induced cell death (AICD) of CD8(+) T cells.Combined treatment with various systemic doses of EOL4G8 mAb plus intratumor injection of AdCMVIL-12 induced complete regression of MC38 tumors treated 7 days after implantation. Unfortunately, most of such mice succumbed to a systemic inflammatory syndrome that could be prevented if IFN-gamma activity were neutralized once tumors had been rejected. Importantly, dose reduction of EOL4G8 mAb opened a therapeutic window (complete cure of 9 of 18 cases without toxicity). We also show that ICAM-2 ligation by EOL4G8 mAb on activated CTLs prevents AICD, thus extending IFN-gamma production.Combination of intratumor gene transfer of IL-12and systemic anti-ICAM-2 mAb display synergistic therapeutic and toxic effects. CTL life extension resulting from AICD inhibition by anti-ICAM-2 mAbs is the plausible mechanism of action.

While growth hormone (GH) is classically defined as a peptide hormone, recent evidence supports a role for GH acting as a cytokine in the immune system under conditions of stress, counteracting immunosuppression by glucocorticoids. Lymphoid cells express the GH receptor, which belongs to the cytokine receptor superfamily, and GH can be produced by immune tissues, suggesting an autocrine/paracrine mode of action of GH. GH can act as a cytokine, promoting cell cycle progression of lymphoid cells and preventing apoptosis. These effects of GH were shown to be mainly mediated by the PI-3 kinase/Akt pathway and the transcription factor NF-κB. Expression of several cell cycle mediators, as well as Bcl-2, c-Myc and cyclin proteins were found to be regulated by GH. Survival of immune cells under conditions of stress was promoted by NF-κB. Thus, GH acts not only as a hormone but also as a cytokine, playing a potentially important role in immune system cells. Lastly, in this mini-review, we will discuss whether the discovery of these molecules in GH signaling pathways offers new insights into additional mechanisms of action whereby GH regulates apoptosis, proliferation and neoplastic transformation of cells of the immune system.