Maria A. Gonzalez-Carmona

Angiotensin‐II type 1 receptor‐mediated Janus kinase 2 activation induces liver fibrosis

Activation of the renin angiotensin system resulting in stimulation of angiotensin‐II (AngII) type I receptor (AT1R) is an important factor in the development of liver fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real‐time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human‐derived LX2 cells. JAK2 expression and activity were increased in experimental rodent and human liver fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild‐type animals led to activation of HSCs and fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho‐kinase activation. These effects were prevented in AT1R−/− mice. Pharmacological inhibition of JAK2 attenuated liver fibrosis in rodent fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII‐induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490. Conclusion: Our study substantiates the important cell‐intrinsic role of JAK2 in HSCs for development of liver fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver fibrosis. (Hepatology 2014;60:334–348)

CD40ligand‐expressing dendritic cells induce regression of hepatocellular carcinoma by activating innate and acquired immunity in vivo

Dendritic cells (DCs) are professional antigen‐presenting cells able to prime T‐cells against tumor‐associated antigens (TAA), but their potential to induce hepatocellular carcinoma (HCC) regression is still limited. CD40/CD40L interaction is essential for DC activation and induction of antigen‐specific T‐cells. In this study, transduction of TAA‐pulsed DC with a CD40L‐encoding adenovirus (Ad‐CD40L) was used to improve the immune response induced by DC toward HCC. Bone marrow–derived DC from C3H/HeNcrl mice were cultured with granulocyte‐macrophage colony‐stimulating factor and interleukin‐4. On day 6, tumor‐lysate pulsed DCs were infected with adenoviruses. HCCs were induced by inoculation of mice with Hepa129‐cells subcutaneously. When tumor‐volume was 100 to 400 mm3, DCs were injected intratumorally, subcutaneously, or intravenously. Ad‐CD40L transduction exerted CD40/CD40L interactions between DCs, increasing DC immunostimulation with up‐regulation of CD80/CD86‐ and interleukin‐12 (IL‐12) expression. Intratumoral injection of CD40L‐DC was superior to intravenous or subcutaneous treatments, yielding tumor elimination in almost 70% of mice. Moreover, all tumor‐free animals were protected against hepatic tumor cell rechallenge. In a preventive setting, subcutaneous injection of CD40L‐expressing DCs protected 50% of mice for more than 3 months toward tumor cell challenge. The induced immune response seemed to be dependent on cross‐priming with Th1‐lymphocytes in the lymph nodes, because transduced DCs were redetected in lymphoid tissues. In addition, immunohistochemistry of tumors indicated a significant tumor infiltration with CD4+, CD8+ T cells and natural killer (NK) cells. Tumor‐infiltrating lymphocytes were tumor‐specific, as shown in interferon‐gamma (IFN‐γ) enzyme‐linked immunosorbent spot and T‐cell proliferation assays. Conclusion: Transduction of DCs with Ad‐CD40L increases significantly the stimulatory capacity of DCs. Intratumoral injection of DCs activates both acquired and innate immunity, inducing complete regression of established tumors and long‐term immunity against tumor recurrence. This approach improves the antitumoral potential of DCs. (HEPATOLOGY 2008.)

Patient-derived dendritic cells transduced with an a-fetoprotein-encoding adenovirus and co-cultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against hepatocellular carcinoma cells

Abstract: Background/Aims: Breaking immunologic tolerance towards the hepatocellular carcinoma (HCC)‐associated α‐fetoprotein (AFP) antigen is possible. The use of this potential for the treatment of immunocompromised HCC patients is limited. In this study, we analyzed whether dendritic cells (DCs) from HCC patients transduced with a human AFP (hAFP)‐expressing adenovirus and co‐cultured with cytokine‐induced killer (CIK) cells can induce a strong specific immune response against HCC‐cells.

Clinical Studies Applying Cytokine-Induced Killer Cells for the Treatment of Gastrointestinal Tumors

Tumors of the gastrointestinal system represent a significant share of solid tumors worldwide. Despite the advances in diagnosis and treatment, the prognosis of gastrointestinal tumors is still very poor and improved therapies are indispensable. Cytokine-induced killer (CIK) cells are feasible for an immunotherapeutic approach as they are easily available and have an advantageous biologic profile; they are rapidly proliferating and their high cytotoxicity is non-MHC-restricted. We summarize and discuss twenty recent clinical studies applying CIK cells for the treatment of gastric, pancreatic, hepatocellular, and colorectal cancer. Autologous CIK cells were transfused intravenously, intraperitoneally, or via the common hepatic artery. In all studies side effects and toxicity of CIK cell therapy were mild and easily controllable. The combination of CIK cell therapy with conventional adjuvant or palliative therapies was superior to the standard therapy alone, indicating the benefit of CIK cell therapy for cancer patients. Thus, CIK cells represent a promising immunotherapy for the treatment of gastrointestinal tumors. The optimal treatment schedule and ideal combination with conventional therapies should be evaluated in further clinical studies.

Recent patents on experimental therapy for hepatitis C virus infection (1999 – 2002)

Despite encouraging progress, antiviral treatment is far from achieving eradication of hepatitis C virus (HCV) infection. Therefore, a large body of new patents concerning novel therapeutic concepts and agents is claimed every year. This review comprises information revealed about granted patents on experimental therapy for HCV infection from 1999 to 2002 but does not intend to be comprehensive. A great part of the inventions described represent peptidomimetic inhibitors of NS3/4–protease, but chemicals, nucleoside analogues or oligodeoxynucleotides are also discussed. The efficacy, specificity and toxicity for most of the claimed drugs are tested mainly in vitro and clinical trial data are rare. Most compounds are intended for combination therapy with well-known antivirals. Other claimed agents, such as recombinant adenoviruses or fusion proteins, are used to improve drug delivery into the cell. A new developing direction of research is the use of immunotherapeutic approaches for HCV therapy.

Beta‐glucosidase 2 knockout mice with increased glucosylceramide show impaired liver regeneration

Glycolipids have been shown to serve specialized functions in cell signalling, proliferation and differentiation processes, which are all important during liver regeneration. We previously generated beta‐glucosidase 2 (GBA2) knockout mice that accumulate the glycolipid glucosylceramide in various tissues, including the liver. The present study addressed the role of GBA2‐deficiency and subsequent glucosylceramide accumulation in liver regeneration.

Plasminogen fragment K1–5 improves survival in a murine hepatocellular carcinoma model

Background: The prognosis of patients with hepatocellular carcinoma (HCC) remains poor, and new alternative treatments are needed. Aims: To comparatively test the angiostatic and antitumour effects of adenoviral gene transfer of angiostatin (PlgK1–4, amino acids 1–440) and full kringles 1–5 (PlgK1–5, amino acids 1–546) in a model of subcutaneously transferred HCC in mice. Methods: PlgK1–4 and PlgK1–5 were generated from human WtPlg cDNA and used for adenovirus construction. Vector function and angiostatic effects were confirmed in vitro and in vivo. Antitumoral efficacies of intratumoral vector injections were studied in a model of subcutaneously transferred HCC model. Results: Cell supernatants containing PlgK1–4 and PlgK1–5 reduced endothelial tube formation in vitro by about 30%, whereas WtPlg exerted no inhibitory effect. Endothelial cell infiltration in vivo was decreased by about 60%, but not in AdWtPlg-treated animals. Intratumoral treatment of subcutaneous HCC tumours inhibited growth by 40% for AdPlgK1–4 and 63% for AdPlgK1–5 in surviving mice 12 days after initiation of treatment, whereas treatment with AdWtPlg even led to accelerated growth. Although PlgK1–4 and PlgK1–5 have similar inhibitory effects on intratumoral microvessels, PlgK1–5 markedly improved the survival time compared with PlgK1–4. Conclusion: PlgK1–5 and PlgK1–4 effectively inhibited HCC growth. As PlgK1–5 could also prolong the survival time, inducing complete tumour elimination in half of the AdPlgK1–5-treated mice, PlgK1–5 might be the most potential plasminogen fragment for treatment of experimental HCC.

Improving immunotherapy of hepatocellular carcinoma (HCC) using dendritic cells (DC) engineered to express IL-12 in vivo

Interleukin 12 (IL‐12), one of the most potent Th1‐cytokines, has been used to improve dendritic cells (DC)‐based immunotherapy of cancer. However, it failed to achieve clinical response in patients with hepatocellular carcinoma (HCC). In this study, improved conditions of immunotherapy with DC engineered to express IL‐12 were studied in murine subcutaneous HCC.

Increase of in vivo antitumoral activity by CD40L (CD154) gene transfer into pancreatic tumor cell-dendritic cell hybrids.

Objectives: Fusion of dendritic cells (DC) with tumor cells is an approach in immunotherapy combining antigenicity and capacity of antigen presentation to activate T cells for the induction of tumor-specific cytotoxic immunity. Although there have been reports of clinical benefit, response rates have been limited and further improvements are warranted. Methods: We used murine DC and a novel protocol for an effective fusion of those cells with the murine pancreatic cell line Panc02. Results: We observed 2 events: only moderate in vitro and in vivo cytotoxicity of tumor cell/DC hybrids and a down-regulation of costimulatory molecules on fused cells. Therefore, we transfected tumor cell/DC hybrids with an adenovirus expressing CD154 to improve DC activation and generating antitumor immune response without the need of CD4+ T cells. High CD154 expression could be obtained by transfection of DC and Panc02 cells prior fusion. Furthermore, vaccination with CD154-transfected tumor cell/DC hybrid led to a significantly increased induction of cytotoxic T cells in vitro and to an improved antitumoral effect in an orthotopic in vivo mouse model. Conclusions: CD154-transfected tumor cell/DC hybrids are a promising approach to increase the efficiency of antitumoral response.

Risk estimation for biliary tract cancer: Development and validation of a prognostic score

Biliary tract cancer is a rare tumour entity characterized by a poor prognosis. We aimed to identify prognostic factors and create a prognostic score to estimate survival.