Matilde Bustos

Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas

Stimulation of the antitumor immune response by dendritic cells (DC) is critically dependent on their tightly regulated ability to produce interleukin-12 (IL-12). To enhance this effect artificially, bone marrow (BM)-derived DC were genetically engineered to produce high levels of functional IL-12 by ex vivo infection with a recombinant defective adenovirus (AdCMVIL-12). DC-expressing IL-12 injected into the malignant tissue eradicated 50–100% well established malignant nodules derived from the injection of two murine colon adenocarcinoma cell lines. Successful therapy was dependent on IL-12 transfection and was mediated only by syngeneic, but not allogeneic BM-derived DC, indicating that compatible antigen-presenting molecules were required. The antitumor effect was inhibited by in vivo depletion of CD8+ T cells and completely abrogated by simultaneous depletion with anti-CD4 and anti-CD8 mAbs. Mice which had undergone tumor regression remained immune to a rechallenge with tumor cells, showing the achievement of long-lasting systemic immunity that also was able to reject simultaneously induced concomitant untreated tumors. Tumor regression was associated with a detectable CTL response directed against tumor-specific antigens probably captured by DC artificially released inside tumor nodules. Our results open the possibility of similarly treating the corresponding human malignancies.

Role of obesity-associated dysfunctional adipose tissue in cancer: A molecular nutrition approach

Obesity is a complex disease caused by the interaction of a myriad of genetic, dietary, lifestyle and environmental factors, which favors a chronic positive energy balance, leading to increased body fat mass. There is emerging evidence of a strong association between obesity and an increased risk of cancer. However, the mechanisms linking both diseases are not fully understood. Here, we analyze the current knowledge about the potential contribution that expanding adipose tissue in obesity could make to the development of cancer via dysregulated secretion of pro-inflammatory cytokines, chemokines and adipokines such as TNF-α, IL-6, leptin, adiponectin, visfatin and PAI-1. Dietary factors play an important role in the risk of suffering obesity and cancer. The identification of bioactive dietary factors or substances that affect some of the components of energy balance to prevent/reduce weight gain as well as cancer is a promising avenue of research. This article reviews the beneficial effects of some bioactive food molecules (n-3 PUFA, CLA, resveratrol and lipoic acid) in energy metabolism and cancer, focusing on the molecular mechanisms involved, which may provide new therapeutic targets in obesity and cancer.

A synthetic peptide from transforming growth factor β type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury

Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine, which displays potent profibrogenic effects and is highly expressed in fibrotic livers. For this reason, development of TGF-B1 inhibitors might be of great importance to control liver fibrogenesis as well as other undesired side effects due to this cytokine. Potential peptide inhibitors of TGF-beta1 (derived from TGF-beta1 and from its type III receptor) were tested in vitro and in vivo using different assays. Peptides P11 and P12, derived from TGF-beta1, and P54 and P144, derived from its type III receptor, prevented TGF-beta1-dependent inhibition of MV1Lu proliferation in vitro and markedly reduced binding of TGF-beta1 to its receptors. P144 blocked TGF-beta1-dependent stimulation of a reporter gene under the control of human alpha2(I) collagen promoter. Intraperitoneal administration of P144 also showed potent antifibrogenic activity in vivo in the liver of rats receiving CCl4. These rats also showed a significant decrease in the number of activated hepatic stellate cells as compared with those treated with saline only. These results suggest that short synthetic peptides derived from TGF-beta1 type III receptor may be of value in reducing liver fibrosis in chronic liver injury.

Protection against liver damage by cardiotrophin-1: a hepatocyte survival factor up-regulated in the regenerating liver in rats

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 beta (TGF-beta) 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 microg 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.

INDUCTION OF TIMP-1 EXPRESSION IN RAT HEPATIC STELLATE CELLS AND HEPATOCYTES : A NEW ROLE FOR HOMOCYSTEINE IN LIVER FIBROSIS

Elevated plasma levels of homocysteine have been shown to interfere with normal cell function in a variety of tissues and organs, such as the vascular wall and the liver. However, the molecular mechanisms behind homocysteine effects are not completely understood. In order to better characterize the cellular effects of homocysteine, we have searched for changes in gene expression induced by this amino acid. Our results show that homocysteine is able to induce the expression and synthesis of the tissue inhibitor of metalloproteinases-1 (TIMP-1) in a variety of cell types ranging from vascular smooth muscle cells to hepatocytes, HepG2 cells and hepatic stellate cells. In this latter cell type, homocysteine also stimulated alpha 1(I) procollagen mRNA expression. TIMP-1 induction by homocysteine appears to be mediated by its thiol group. Additionally, we demonstrate that homocysteine is able to promote activating protein-1 (AP-1) binding activity, which has been shown to be critical for TIMP-1 induction. Our findings suggest that homocysteine may alter extracellular matrix homeostasis on diverse tissular backgrounds besides the vascular wall. The liver could be considered as another target for such action of homocysteine. Consequently, the elevated plasma levels of this amino acid found in different pathological or nutritional circumstances may cooperate with other agents, such as ethanol, in the onset of liver fibrosis.

A blood-tumor barrier limits gene transfer to experimental liver cancer: the effect of vasoactive compounds.

We have evaluated gene transfer efficiency to tumor nodules in diethylnitrosoamine (DENA)-induced hepatocellular carcinoma (HCC) in rats using adenoviral vectors administered by three different routes: intraportal, intra-arterial and intratumoral injection. Our results showed that intraportal infusion could not transduce tumor nodules greater than 1 mm in diameter while the intra-arterial route allowed transduction of nodules up to 2–5 mm in diameter. Tumors greater than this size were resistant to transduction by intravascular route, but could be transduced by direct intratumoral injection, indicating that the obstacle preventing gene transfer to tumor cells was mainly at the level of tumor vasculature and not at the level of neoplastic cells. We have studied the extracellular matrix in tumoral lesions to assess whether nodules with different size and histological pattern have different profiles in relation to transduction efficacy. Immunohistochemical detection showed a high expression of fibronectin (FN), laminin (LN) and α-smooth muscle actin (α-SMA) in those large HCC, which were resistant to adenoviral infection. Intra-arterial infusion of vasoactive compounds (histamine, angiotensin II or nitric oxide donor nitroglycerin) before vector administration enhanced gene transfer to tumor nodules that were poorly transduced without pre-treatment. Nitroglycerin was active to enhance transduction of large tumors with trabecular or pseudoglandular histological pattern, which were impermeable to adenoviral vectors even after histamine or angiotensin treatments. Our data indicate the presence of a physical barrier between blood and neoplastic cells, which prevents transduction of the tumor by vectors given by the intravascular route. The thickness and impermeability of the barrier increases as the tumor nodule grows. Vasoactive compounds may be of value in gene therapy of liver cancer by increasing transduction efficiency by intravascularly administered vectors.

Cardiotrophin-1 is a key regulator of glucose and lipid metabolism.

Cardiotrophin-1 (CT-1) is a member of the gp130 family of cytokines. We observed that ct-1(-/-) mice develop mature-onset obesity, insulin resistance, and hypercholesterolemia despite reduced calorie intake. Decreased energy expenditure preceded and accompanied the development of obesity. Acute treatment with rCT-1 decreased blood glucose in an insulin-independent manner and increased insulin-stimulated AKT phosphorylation in muscle. These changes were associated with stimulation of fatty acid oxidation, an effect that was absent in AMPKα2(-/-) mice. Chronic rCT-1 treatment reduced food intake, enhanced energy expenditure, and induced white adipose tissue remodeling characterized by upregulation of genes implicated in the control of lipolysis, fatty acid oxidation, and mitochondrial biogenesis and genes typifying brown fat phenotype. Moreover, rCT-1 reduced body weight and corrected insulin resistance in ob/ob and in high-fat-fed obese mice. We conclude that CT-1 is a master regulator of fat and glucose metabolism with potential applications for treatment of obesity and insulin resistance.

Cardiotrophin-1 defends the liver against ischemia-reperfusion injury and mediates the protective effect of ischemic preconditioning

Ischemia-reperfusion (I/R) liver injury occurs when blood flow is restored after prolonged ischemia. A short interruption of blood flow (ischemic preconditioning [IP]) induces tolerance to subsequent prolonged ischemia through ill-defined mechanisms. Cardiotrophin (CT)-1, a cytokine of the interleukin-6 family, exerts hepatoprotective effects and activates key survival pathways like JAK/STAT3. Here we show that administration of CT-1 to rats or mice protects against I/R liver injury and that CT-1–deficient mice are exceedingly sensitive to this type of damage. IP markedly reduced transaminase levels and abrogated caspase-3 and c-Jun–NH2-terminal kinase activation after I/R in normal mice but not in CT-1–null mice. Moreover, the protective effect afforded by IP was reduced by previous administration of neutralizing anti–CT-1 antibody. Prominent STAT3 phosphorylation in liver tissue was observed after IP plus I/R in normal mice but not in CT-1–null mice. Oxidative stress, a process involved in IP-induced hepatoprotection, was found to stimulate CT-1 release from isolated hepatocytes. Interestingly, brief ischemia followed by short reperfusion caused mild serum transaminase elevation and strong STAT3 activation in normal and IL-6–deficient mice, but failed to activate STAT3 and provoked marked hypertransaminasemia in CT-1–null animals. In conclusion, CT-1 is an essential endogenous defense of the liver against I/R and is a key mediator of the protective effect induced by IP.

Serotonin Skews Human Macrophage Polarization through HTR2B and HTR7

Besides its role as a neurotransmitter, serotonin (5-hydroxytryptamine, 5HT) regulates inflammation and tissue repair via a set of receptors (5HT1–7) whose pattern of expression varies among cell lineages. Considering the importance of macrophage polarization plasticity for inflammatory responses and tissue repair, we evaluated whether 5HT modulates human macrophage polarization. 5HT inhibited the LPS-induced release of proinflammatory cytokines without affecting IL-10 production, upregulated the expression of M2 polarization–associated genes (SERPINB2, THBS1, STAB1, COL23A1), and reduced the expression of M1-associated genes (INHBA, CCR2, MMP12, SERPINE1, CD1B, ALDH1A2). Whereas only 5HT7 mediated the inhibitory action of 5HT on the release of proinflammatory cytokines, both 5HT2B and 5HT7 receptors mediated the pro-M2 skewing effect of 5HT. In fact, blockade of both receptors during in vitro monocyte-to-macrophage differentiation preferentially modulated the acquisition of M2 polarization markers. 5HT2B was found to be preferentially expressed by anti-inflammatory M2(M-CSF) macrophages and was detected in vivo in liver Kupffer cells and in tumor-associated macrophages. Therefore, 5HT modulates macrophage polarization and contributes to the maintenance of an anti-inflammatory state via 5HT2B and 5HT7, whose identification as functionally relevant markers for anti-inflammatory/homeostatic human M2 macrophages suggests their potential therapeutic value in inflammatory pathologies.

Eicosapentaenoic acid stimulates AMP-activated protein kinase and increases visfatin secretion in cultured murine adipocytes

Visfatin is an adipokine highly expressed in visceral AT (adipose tissue) of humans and rodents, the production of which seems to be dysregulated in excessive fat accumulation and conditions of insulin resistance. EPA (eicosapentaenoic acid), an n-3 PUFA (polyunsaturated fatty acid), has been demonstrated to exert beneficial effects in obesity and insulin resistance conditions, which have been further linked to its reported ability to modulate adipokine production by adipocytes. TNF-alpha (tumour necrosis factor-alpha) is a pro-inflammatory cytokine whose production is increased in obesity and is involved in the development of insulin resistance. Control of adipokine production by some insulin-sensitizing compounds has been associated with the stimulation of AMPK (AMP-activated protein kinase). The aim of the present study was to examine in vitro the effects of EPA on visfatin production and the potential involvement of AMPK both in the absence or presence of TNF-alpha. Treatment with the pro-inflammatory cytokine TNF-alpha (1 ng/ml) did not modify visfatin gene expression and protein secretion in primary cultured rat adipocytes. However, treatment of these primary adipocytes with EPA (200 mumol/l) for 24 h significantly increased visfatin secretion (P<0.001) and mRNA gene expression (P<0.05). Moreover, the stimulatory effect of EPA on visfatin secretion was prevented by treatment with the AMPK inhibitor Compound C, but not with the PI3K (phosphoinositide 3-kinase) inhibitor LY294002. Similar results were observed in 3T3-L1 adipocytes. Moreover, EPA strongly stimulated AMPK phosphorylation alone or in combination with TNF-alpha in 3T3-L1 adipocytes and pre-adipocytes. The results of the present study suggest that the stimulatory action of EPA on visfatin production involves AMPK activation in adipocytes.