Rajagopal N. Aravalli

Molecular mechanisms of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) typically has poor prognosis, because it is often diagnosed at an advanced stage. Heterogeneous phenotypic and genetic traits of affected individuals and a wide range of risk factors have classified it a complex disease. HCC is not amenable to standard chemotherapy and is resistant to radiotherapy. In most cases, surgical resection and liver transplantation remain the only curative treatment options. Therefore, development of novel, effective therapies is of prime importance. Extensive research over the past decade has identified a number of molecular biomarkers as well as cellular networks and signaling pathways affected in liver cancer. Recent studies using a combination of “omics” technologies, microRNA studies, combinatorial chemistry, and bioinformatics are providing new insights into the gene expression and protein profiles during various stages of the disease. In this review, we discuss the contribution of these newer approaches toward an understanding of molecular mechanisms of HCC and for the development of novel cancer therapeutics. (HEPATOLOGY 2008;48:2047‐2063.)

Cutting Edge: TLR2-Mediated Proinflammatory Cytokine and Chemokine Production by Microglial Cells in Response to Herpes Simplex Virus

Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2−/− as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-α, IL-1β, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and CXCL10 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2−/− mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.

Shuttle vectors for hyperthermophilic archaea

Abstract Progress in understanding the basic molecular, biochemical, and physiological characteristics of archaeal hyperthermophiles has been limited by the lack of suitable expression vectors. Here, we report the construction of versatile shuttle vectors that can be maintained, and selected for, in both archaea and bacteria. The primary construct, pAG1, was produced by ligating portions of the archaeal cryptic plasmid pGT5 and the bacterial plasmid pUC19, both of which exhibit high copy numbers. A second vector construct, pAG2, was generated, with a reduced copy number in Escherichia coli, by introducing the Rom/Rop gene from pBR322 into pAG1. After transformation, both pAG1 and pAG2 were stably maintained and propagated in the euryarchaeote Pyrococcus furiosus, the crenarchaeote Sulfolobus acidocaldarius, and in Escherichia coli. An archaeal selective marker, the alcohol dehydrogenase gene from Sulfolobus solfataricus, was isolated by polymerase chain reaction (PCR) amplification and cloned into the two constructs. They were stably maintained and expressed in the two archaea and conferred resistance to butanol and benzyl alcohol. However, the vector pAG21, deriving from pAG2, proved the more stable in E. coli probably due to its lower copy number in the bacterium. Conditions are presented for the use of the vectors which, potentially, can be used for other hyperthermophilic archaea.

Toll-like receptor 2 signaling is a mediator of apoptosis in herpes simplex virus-infected microglia

BackgroundInformation regarding the response of brain cells to infection with herpes simplex virus (HSV)-1 is needed for a complete understanding of viral neuropathogenesis. We have recently demonstrated that microglial cells respond to HSV infection by producing a number of proinflammatory cytokines and chemokines through a mechanism involving Toll-like receptor 2 (TLR2). Following this cytokine burst, microglial cells rapidly undergo cell death by apoptosis. We hypothesized that TLR2 signaling might mediate the cell death process as well.MethodsTo test this hypothesis, we investigated HSV-induced cell death of microglia obtained from both wild-type and TLR2-/- mice. Cell death was studied by oligonucleosomal ELISA and TUNEL staining, and the mechanisms of apoptosis were further analyzed using murine apoptosis-specific microarrays. The data obtained from microarray analysis were then validated using quantitative real-time PCR assays.ResultsHSV infection induced apoptotic cell death in microglial cells from wild-type as well as TLR2 cells. However, the cell death at 24 h p.i. was markedly lower in knockout cells. Furthermore, microarray analyses clearly showed that the expression of pro-apoptotic genes was down-regulated at the time when wild-type cells were actively undergoing apoptosis, indicating a differential response to HSV in cells with or without TLR2.ConclusionWe demonstrate here that HSV induces an apoptotic response in microglial cells which is mediated through TLR2 signaling.

Potentiation of HIV-1 Expression in Microglial Cells by Nicotine: Involvement of Transforming Growth Factor-β1

HIV-1 infection and nicotine addiction are global public health crises. In the central nervous system, HIV-1 causes a devastating neurodegenerative disease. It is well recognized that microglial cells play a pivotal role in the neuropathogenesis of HIV-1 and that drugs of abuse not only contribute to the spread of this agent but may facilitate viral expression in these brain macrophages. Nicotine has been shown to stimulate the production of HIV-1 by in vitro-infected alveolar macrophages, and the HIV-1 protein gp120 binds to nicotinic receptors. In this study, we demonstrated the constitutive expression of nicotinic acetylcholine receptor mRNA in primary human microglial cells and showed that the pretreatment of microglia with nicotine increased HIV-1 expression in a concentration-dependent manner, as measured by p24 antigen levels in culture supernatants. We also found that nicotine robustly altered the gene expression profile of HIV-1-infected microglia and that the transforming growth factor-β1 is involved in the enhanced expression of HIV-1 by nicotine.

Differential apoptotic signaling in primary glial cells infected with herpes simplex virus 1

Microglial cells and astrocytes are glial cell types that perform distinct functions and generate innate immune responses to counter invading pathogens. Herpes simplex virus 1 (HSV-1) is a neurotropic virus that is capable of causing severe, necrotizing encephalitis. HSV-1 infects both of these glial cell types. Microglial cells undergo an abortive infection, yet respond to viral infection by inducing a burst of proinflammatory cytokine and chemokine production. Following this cytokine burst, they rapidly succumb to cell death. In contrast, astrocytes do permit productive viral replication, but do not generate these same innate immune mediators. Although apoptosis has been implicated in a number of acute and chronic neurological disorders, little is known about apoptosis during viral encephalitis. In the present study, the authors investigated the effect of HSV-1 infection on cell survival and studied the mechanisms of cell-death in virus-infected, primary murine glial cells. The authors report that although apoptosis occurred rapidly in microglia, it was delayed during productive infection of astrocytes. Furthermore, microarray studies revealed significant variations in the expression of apoptotic genes between these two types of glial cells, indicating crucial differences in signaling pathways. Intrinsic as well as extrinsic pathways of apoptosis were found to be activated in both glial cell types. Specifically, genes involved in the tumor necrosis factor (TNF) signaling pathway were predominantly up-regulated in microglia, whereas genes of the Fas pathway were induced during HSV infection of astrocytes.

Inhibition of Toll-like Receptor Signaling in Primary Murine Microglia

Microglial cells respond to the herpes simplex virus (HSV)-1 by producing proinflammatory cytokines and chemokines. After this inflammatory burst, these cells undergo apoptotic cell death. We have recently demonstrated that both virus-induced immune mediator production and apoptosis were induced through Toll-like receptor 2 (TLR2) signaling. Based upon these findings, we hypothesized that the inhibition of TLR2 signaling may serve as a means to alleviate excessive neuroinflammation. In the present study, we cloned four vaccinia virus (VV) proteins, which have been reported to disrupt either TLR signaling or NF-κB activation, and overexpressed them in HEK293T cells stably expressing murine TLR2 and in primary murine microglia. Using an NF-κB-driven luciferase reporter gene assay, we show that upon stimulation with HSV and Listeria monocytogenes, all four vaccinia proteins inhibited TLR2 signaling with different levels of inhibition in the TLR2-expressing cell line and primary microglia. We found similar results when microglial cells were stimulated with the TLR4 ligand LPS and the TLR9 ligand CpG ODN. Taken together, these data provide evidence that these VV proteins can function as inhibitors of TLR signaling in primary microglial cells.

Role of innate immunity in the development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. It is caused by a variety of risk factors, most common ones being infection with hepatitis viruses, alcohol, and obesity. HCC often develops in the background of underlying cirrhosis, and even though a number of interventional treatment methods are currently in use, recurrence is fairly common among patients who have had a resection. Therefore, whole liver transplantation remains the most practical treatment option for HCC. Due to the growing incidence of HCC, intense research efforts are being made to understand cellular and molecular mechanisms of the disease so that novel therapeutic strategies can be developed to combat liver cancer. In recent years, it has become clear that innate immunity plays a critical role in the development of a number of liver diseases, including HCC. In particular, the activation of Toll-like receptor signaling results in the generation of immune responses that often results in the production of pro-inflammatory cytokines and chemokines, and could cause acute inflammation in the liver. In this review, the current knowledge on the role of innate immune responses in the development and progression of HCC is examined, and emerging therapeutic strategies based on molecular mechanisms of HCC are discussed.

Hepatic differentiation of porcine induced pluripotent stem cells in vitro.

Porcine hepatocytes are potentially important in liver regeneration and in the treatment of humans with acute and chronic liver diseases. Induced pluripotent stem (iPS) cells are a valuable source of hepatocytes for these applications as they have unlimited potential to propagate in vitro. An efficient and robust differentiation of iPS cells generated from porcine fetal fibroblasts into functional hepatocyte-like cells in vitro is reported. The methodology followed a three-step differentiation protocol using several growth factors, namely, activin A, basic fibroblast growth factor, bone morphogenetic protein-4, and oncostatin M. Porcine iPS cell-derived hepatocyte-like (piPS-Hep) cells were characterized by morphological analysis and were tested for the expression of hepatocyte-specific genes using RT-PCR. Functional analyses for albumin production and glycogen storage were also carried out. These differentiated hepatocyte-like cells could represent a valuable source for studies of drug metabolism and for cell transplantation therapy for a variety of liver disorders.

Animal models of cancer in interventional radiology

Animal models will play an increasingly important role in oncology research, especially for solid tumours such as hepatocellular carcinoma that are resistant to chemotherapy. Many models have been used, but there is a need for increased awareness of the limitations of these models and also a need for guidance for future model development.