Faculty

Human Cytomegalovirus (HCMV)

Human Cytomegalovirus (HCMV) is a significant cause of disease in the immunocompromised and is the leading cause of infection-related congenital birth defects. Disease associated with HCMV infections is an increasing problem due to the emergence of drug-resistant viruses. Proteins contained within the HCMV tegument are important for both establishment of virus infection and assembly of new viral particles and thus make attractive therapeutic targets. Our research is focused on understanding the mechanisms that regulate the subcellular trafficking of the tegument proteins, focusing on the phosphoprotein pp71. This protein initially travels to the nucleus, where it plays an important role in switching on viral gene expression. At the late stage of infection, pp71 is found in the cytoplasm in viral assembly compartments associated with trans-Golgi Network (TGN)-derived membranes. Our laboratory has identified a domain of pp71 that is important for both nuclear and TGN-trafficking. Together with our collaborators in the George L. Wright Center for Biomedical Proteomics, we have shown that nuclear localization is regulated by phosphorylation of a specific threonine residue within pp71. Further, our studies have identified a specific sequence that dictates pp71 trafficking to the mitochondria, suggesting additional functions for this key viral tegument protein. We are currently determining the mechanisms of pp71 trafficking and assessing the biological functions of cytoplasmic pp71 during infection.

Faculty

Dr. Campbell's laboratory pursues studies on the pathogenesis of the herpesvirus, cytomegalovirus (CMV). This virus causes severe complications in infected newborns, AIDS patients and immunosuppressed bone marrow or organ transplant recipients. Acute and latent infections occur in a variety of organs and cell types within the host.

One prominent cell type infected during both acute and latent stages of infection is the macrophage. This laboratory has identified a gene region of murine CMV (MCMV) that is required for efficient infection of macrophages and hence infectivity in vivo. Deletion of three genes within this region results in a mutant virus that replicates poorly in macrophages in vitro and in mice. The product of these three genes has been characterized, and they form a stable complex in infected cells. By using a combination of genetic manipulations, molecular biology, proteomics, and in vivo studies, the laboratory pursues studies to characterize the structure of this complex and to decipher the function of these viral proteins in regulating cell tropism.

A second project aims to identify the immune response to MCMV in the salivary gland, a site of viral persistence. Assessment of the phenotype and cytokine/chemokine profiles of cells infiltrating in response to MCMV infection has identified populations of cells unique to this mucosal site. Continued studies aim to identify the function of the site-specific cells in controlling infection.

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Dr. Ciavarra and his team are currently focused on analysis of the cellular interactions essential for responses against infectious agents (viruses) or tumor cells. With respect to host resistance to viral pathogens, current studies focus on how innate and adaptive antiviral immune responses are regulated in the central nervous system (CNS).

The brain and spinal cord represent special challenges for the immune system to achieve viral clearance without destroying sensitive and irreplaceable nerve cells. Within this focus, his laboratory is investigating how CNS resident cells such as microglia (brain macrophages) and astrocytes contribute to anti-viral immunity and how these cells interact with other blood cells that infiltrate the brain during viral encephalitis to achieve viral clearance from this organ.

A second area of interest in this laboratory is the production of novel cellular vaccines for the treatment of prostate cancer. Tumor cells have been modified to contain specific genes that should boost the anti-tumor immune response. These genes are activated within the tumor microenvironment at distinct times during tumor progression and the development of metastatic disease. His laboratory has developed several animal models to assess the efficacy of these novel cellular vaccines.

Faculty

Applications accepted from committed researchers.

Our research is an exploration of molecular mechanisms underlying eukaryotic DNA replication and how these mechanisms are targeted in disease. Discovery at this level is aimed at translation into clinical studies for the prevention and treatment of disease and the bioengineering of novel protein applications. We use both cell culture and human pathology specimens to perform studies that compliment each other. These studies involve various cellular, molecular, biochemical and biophysical approaches. We study two proteins about which little is currently known. These projects are described below.

Cellular, molecular and biochemical analysis of MCM8 function:

The role of the minichromosome maintenance (MCM) proteins in initiation of DNA replication has been extensively studied, and the MCM2-7 heterohexamer is proposed to be a replicative helicase unwinding DNA ahead of the replication fork during S phase. Additional functions are currently being ascribed to these family members. In 2003, we discovered the gene for MCM8, a new family member that is not found in yeast, but which has seemly evolved to adapt to the more complex nuclear structure of higher eukaryotes. We, and others, have shown the MCM8 gene to be either mutated or variantly spliced in a choriocarcinoma and virally interrupted in a hepatocarcinoma, respectively. The archael MCM proteins, MCM2-7 and MCM8 proteins are AAA+ (ATPases associated with a variety of cellular activities) enzymes. MCM8 is unique among the family members in that it has intrinsic in vitro helicase activity without the presence of family members MCM2-7. MCM8 interacts with proteins involved in DNA replication, including Cdc6, RPA, Cdk2 and other MCM family members. We are studying the extent to which MCM8 is involved in human pathology.

 Analysis of Pur-gamma and WRN interaction in cancer, aging and in AIDS-associated neurological disorders:

We are studying the protein Pur-gamma in regard to cancer, aging and neurological disorders associated with AIDS. The Pur-gamma gene is located head to head with the WRN gene and we are investigating the concurrent regulation of these genes and the effect of each protein on the activity of the other. Very little is currently known about Pur-gamma, which at first was thought to be an embryonic form in mice. Our data has revealed expression of this protein under certain pathological conditions in the adult. Our work with this protein is leading to the expansion of our research into DNA repair mechanisms.

Faculty

Atherosclerosis

Our laboratory is interested in the involvement of the immune system in the development and progression of atherosclerosis. This is a disease of large vessels that is characterized by a formation of atherosclerotic plaques consisting of necrotic core, calcified regions, accumulated modified lipids, inflamed smooth muscle cells, endothelial cells, leukocytes and foam cells.

Atherosclerosis is the most common pathological process that leads to cardiovascular diseases. In the past decade, a growing body of data shows that B and T cells, macrophages, NKT cells and dendritic cells participate in the development of atherosclerosis. This strongly suggests that the innate and adaptive immune systems are deeply involved in the initiation and progression of atherosclerosis. However, the exact mechanisms of the recruitment, activation, proliferation and retention of different types of immune cells within the aortic wall remain unclear.

Our research focuses on the identification of the immune cell composition of the aortic wall under normal/non-inflamed conditions and during atherosclerosis, and the mechanisms of migration and retention of lymphocytes within the aortas. Recently we showed that L-selectin is at least partially responsible for the recruitment of lymphocytes into the aortic wall. We also demonstrated that the chemokine receptor CXCR6 regulates the homing of T cell subset into the atherosclerosis-prone aortic wall.

Our laboratory is also interested in the mechanisms that lead to the formation of lymphoid-like structures within the aortas and local immune response during the development and progression of atherosclerosis. Understanding of immune reactions that participate in atherosclerosis and functions of aortic tertiary lymphoid structures will help to design new approaches towards the prevention and treatment of this disease.

In addition to our work on the immune response during atherosclerosis, our laboratory has also become involved in the studies devoted understanding of mechanisms of diabetes-accelerated atherosclerosis. Insulin resistance and type 2 diabetes are associated with accelerated atherosclerosis in patients, but the availability of mouse models to study connections between these two diseases has been limited. We develop a mouse model of insulin-signaling dependent accelerated atherosclerosis, and clearly demonstrated that the pre-diabetic state already accelerates the development of atherosclerosis. It will be important to further investigate roles of hyperinsulinemia and hyperlipidemia in atherosclerosis and dissect a role of the immune system, particularly T and B cells, in this model.

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Faculty

 AIDS, DNA replication, neurovirology, virology, environmental carcinogenesis, nucleic acids, cancer, cell cycle, tumor suppressors, lung cancer, chromatin,  gene, transcription, brain pathology, HIV.

Work in the laboratory of Dr. Edward M. Johnson is concerned with control of DNA replication in cancer and AIDS. An important current project involves an opportunistic infection of the brain, caused by the virus JCV, in AIDS patients. We have hypothesized that activation of JCV in glial cells of the brain is influenced by HIV-1 infection. We have found that JCV late gene transcription is stimulated by the HIV-1 Tat protein through action at sequence elements bound by the cellular protein, Pur-alpha. A complex between Tat and Pur-alpha acts to stimulate transcription at both the HIV-1 TAR RNA element and the JCV late promoter. The complex also interacts with T-antigen to enhance JCV DNA replication. Pur-alpha is a frequent partner of Cyclin/Cdk complexes, as is another Tat-binding protein, Cyclin T1.

 A major project involving lung cancer aims to elucidate the mechanism by which cells distinguish newly-replicated DNA, and its associated proteins, in S and G2 phases of the cell cycle and prevent that DNA from reinitiating replication in the same cycle. A primary hypothesis is that alterations in activity of an MCM4, 6, 7 helicase complex, necessary for initiation or reinitiation, are mediated by Cdk-dependent phosphorylation.  We are pursuing findings that Cyclin A/Cdk2 and sequence-specific single-stranded DNA-binding protein, Pur-alpha, are colocalized with once-replicated DNA in S and G2, that Pur-alpha modulates activity of Cdk2 and that Pur-alpha colocalizes with MCM7 on chromatin. We shall ascertain using chromatin immunoprecipitation (ChIP) and re-ChIP with successive antibodies the location and timing of assembly and dissociation of MCM4, 6 and 7 helicase components and the helicase inhibitor, MCM2, upstream of the c-MYC gene in the cell cycle of small-cell lung carcinoma cells and normal controls. We are examining whether Pur-alpha modulates the MCM4, 6, 7 helicase, either by association with Cyclin A/Cdk2 or by DNA unwinding. Results allow identification of links between control of initiation of replication and imposition of checkpoint controls that are critical in preventing progression to cancer.

 We are exploring the dynamics of the interactions between Tat, T-antigen and Pur-alpha during the course of JCV infection of oligodendrocytes and are determining whether transcriptional activation involves activity of Cyclin T1/Cdk9. We are detailing the mechanism by which Tat enhances replication initiated at the JCV origin in human oligodendrocytes both in vivo and using a new in vitro system. We are also examining the ability of TAT and Pur-alpha to interact with Smad effectors of the TGF-beta1 pathway. Results will help elucidate pathways of activation of HIV-1 and JCV in the brain and will help target particular molecular interactions for therapy.

 Our laboratory has generated five patents.

Faculty

Cancer is marked by uncontrolled proliferation and inappropriate survival of damaged cells in the body. Many processes used to direct the proper growth, differentiation and cell death of tissues in the developing embryo are identical to the genetic pathways that are perturbed in the cancerous state. Recently, an abundant class of non-coding RNAs, microRNAs (miRNAs), has been implicated to function as tumor suppressor genes and oncogenes and is often dysregulated in human cancers. Little is known regarding how these molecules contribute to cellular transformation and tumor formation. MiRNAs are small ~22 nucleotide single-stranded RNAs that negatively regulate expression of their gene targets. Animal miRNAs bind to complementary sequences located in the 3’ untranslated region (3’ UTR) of their target protein-coding messenger RNAs (mRNAs), resulting in translational inhibition and/or mRNA degradation.

Dr. Kerscher is very interested in studying the role miRNAs play in controlling developmental events and how this relates to cancer progression. The lab employs the simple roundworm, Caenorhabditis elegans, an organism easily grown and studied in the laboratory and amenable to genetic manipulation, to characterize the biological function of novel miRNA genes. Specifically, the lab focuses on the lin-4 and let-7 miRNA families, which they have found to direct important developmental processes such as cell-fate specification and gonad formation. The lin-4 and let-7 miRNAs are highly conserved across animal phylae and provide a unique opportunity to apply knowledge gained in the nematode to elucidate the mechanisms of human disease.

Faculty

The focus of my research is on AIDS pathogenesis, neuroAIDS and the study of monocyte maturation, infection and traffic. The goals of these studies are to define the role of macrophages in the central nervous system, and monocytes and CD8+ T lymphocytes outside the brain, contributing to pathogenesis of disease. Much of this work is done in non-human primate models.
We have described target cells infected in the CNS, their turnover and replacement by cells from the blood and bone marrow, and emerging subpopulations of monocytes that expand with disease. While investigating a pathogenic role of activated/infected monocytes in the induction of brain infection and inflammation, we are currently working on immunologic agents that selectively target SIV and HIV infected, activated monocyte/macrophages. We use the CD8 lymphocyte depletion and rapid AIDS model to study the consequences of monocyte/macrophage activation and traffic, the role of viral sequences within SIV that may drive CNS disease.

Faculty

Human astroviruses; pre-clinical development of a novel inhibitor of the innate immune response in humans

Our laboratory studies the human astroviruses, a family of non-enveloped, icosahedral RNA viruses that cause gastroenteritis, predominantly in infants. We have demonstrated that astrovirus capsid protein suppresses the complement system, a fundamental component of the innate immune response against pathogens in vertebrates. We have recently defined the complement inhibiting region to a peptide of 15 amino acid residues. We are interested in determining the mechanism whereby these peptides inhibit complement activation.

A second area of interest for our laboratory is the development of these complement suppressing peptides as a therapeutic for complement-mediated disease. Whilst the human complement system represents a front-line defense against pathogens such as bacteria and viruses, its uncontrolled activation can lead to severe pathology in many different inflammatory and autoimmune disorders with an immune component such as systemic lupus erythematosis, rheumatoid arthritis, inflammatory bowel disease, ischemia-reperfusion injury (myocardial infarct, stroke), glomerulonephritis, adult respiratory distress syndrome, transplant rejection, graft versus host disease and burn injuries. Given the very potent inhibition of the astrovirus derived peptides on the complement system, we are currently interested in developing these peptides into a therapeutic compound as method for regulating aberrant complement activity.

Faculty

Herpes simplex virus 1 (HSV-1)

Dr. Lundberg's laboratory works on the immune response to the agent behind common cold sores, herpes simplex virus 1 (HSV-1). HSV-1 is a neurotropic pathogen that, as part of the normal course of infection, enters into a dormant state in the infected neuron. When disturbed through stress (sunburn, physical, mental), HSV-1 can reactivate. This is why patients almost always experience cold sores in the same location; that is, where the infected neuron connects to the skin surface. However, there are several areas of human HSV-1 related disease with the potential for much more dire consequences than an irritating blister.

When HSV-1 infects the cornea (and subsequently, the brain), the pathology that results is directly tied to the quality of the immune response during acute infection. In the case of sensitive tissues, such as the eye and the central nervous system (CNS), this means an immune response of appropriate strength under suitable control to avoid “collateral” damage.

Unfortunately, some patients still suffer the consequences of an overly exuberant inflammatory response during HSV-1 infection at these sites. Current focus is on the role of macrophages in the acute inflammation that develops within the CNS of susceptible individuals during HSV-1 infection. To do this, Dr. Lundberg's lab uses a mouse infection model to study very early changes in gene expression in the CNS and uses this information to understand why severe pathology develops in some strains of mice while others can control the infection without significant tissue destruction.

Faculty

Keywords:  MicroRNA biology, Cancer Research, Lung cancer and biology, Cancer genomics, Molecular study of oncogene mechanism, Oncogenic signaling, Cancer cell metabolism, and Finding new uses of old drugs.

I. MicroRNAs and mechanism of lung cancer genes

Oncogenes activated via gene amplification have a proven track record of being amenable to invention of new anti-cancer therapies. We and others discovered a recurrent amplified region in lung cancer genomes. This amplicon contains the TTF-1 gene (thyroid transcription factor 1 or known as NKX2-1) which is essential for lung development and morphogenesis. We are interested in mapping the interconnection between TTF-1 and microRNAs to afford novel entry points to investigate TTF-1-linked lung biology. Using a variety of experimental approaches, our laboratory is the first to discover the two types of TTF-1-linked microRNAs – an upstream microRNA that directly regulates TTF-1 expression and downstream microRNAs that are regulated by TTF-1. Currently, we are investigating the biology of these interactions between microRNAs and a lung cancer/development gene.

II. New uses of old drugs

In order to minimize the exorbitant costs and risks of de novo drug discovery and development, our strategy is to use off-patent marketed small molecule drugs as the starting point of drug discovery efforts, i.e. repurposing/repositioning an old drug for a new use. Medications that have come off patent are affordable for patients and have well documented biological, toxicological, and pharmacokinetic studies associated with them. Consequently, functional screening of such “old drugs” may readily yield chemicals for immediate clinical trials. Furthermore, “off-label” prescribing allows physicians to innovate with treatments based on emerging research data. Towards this end, we are conducting a multitude of cell-based screens to uncover new utilities of “old drugs” in fighting cancers. This line of research entails a highly translational goal in sight.  Although cancer is the focal point of our work, our approach is in principle transplantable and could be used to discover novel therapeutic strategies to treat other diseases. There are approximately 9990 drugs known to clinical medicine. Each drug should be considered an information-rich entity that merits exploration especially as treatment of orphan diseases.

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Faculty

Proteomics, Malaria, Cancer

Dr. Nyalwidhe is interested in the application of functional, expressional and structural proteomics approaches to the study of human diseases with emphasis on infectious diseases and cancer. This includes research on human malaria caused by Plasmodium falciparum, a variety of other infectious agents, prostate cancer and viral-induced carcinomas, in addition to other forms of cancer.

The focus is on the mechanisms of interaction between infectious pathogens and their host cells, the mechanism of metastasis in cancer and the analysis of the proteome of human body fluids to identify clinically useful biomarkers for the diagnosis and prognosis of disease. Dr. Nyalwidhe is also using molecular biology and different mass spectrometry techniques in analyzing, monitoring and quantifying post-translational modifications in proteins to determine their influence in the progression and outcome of disease.

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Project I: Regulated Proteolysis in K-RAS-Mediated Tumorigenesis and Metastasis in human cancers

Dr. Tang's laboratory studies the RAS signal transduction pathway using multiple model organisms/systems including Drosophila, transgenic mice, human cancer cell lines and human cancer tissue specimens. As oncogenic RAS promotes the genesis of many human cancers, how best to contravene activated RAS signaling has been an intense area of investigation in the field of cancer biology for the past 30 years. Seven-In-Absentia (SINA), an E3 ubiquitin ligase, is an essential downstream component of the Drosophila RAS signal transduction pathway. The human homologue of SINA, SIAH, is a member of this evolutionarily highly conserved family of RING finger E3 ubiquitin ligases; however, the roles and regulation of SIAH-dependent proteolysis are not well understood in the context of RAS signal transduction in mammalian systems.

Dr. Tang's lab has accumulated evidence demonstrating the importance of proper SIAH function in mammalian K-RAS signaling. We show that by inhibiting the enzymatic activity of SIAH, and thus SIAH-mediated proteolysis, RAS-mediated neoplastic transformation and tumorigenesis can be effectively blocked in human cancer cells [Can Res 67(24):1798-810, 2007; JNCI 100(22):1606-29, 2008]. Furthermore, SIAH-deficient cells have reduced MAPK signaling, suggesting that SIAH might be involved in aberrant K-RAS signaling through a regulatory feedback loop mechanism. Thus, these studies provide an initial glimpse into the significance of the SIAH E3 ubiquitin ligase-regulated proteolysis in the K-RAS pathway during tumor initiation, progression and oncogenesis in human pancreatic cancer, lung cancer, invasive and metastatic breast cancer and hormonal-refractory prostate cancer.

Advancing understanding of the role of SIAH E3 ligases in K-RAS signaling and, more importantly, the potential to target SIAH as a novel new anti-K-RAS and anti-cancer target in the treatment of the most aggressive and the deadliest forms of human cancers represent exciting steps forward in the fields of K-RAS signaling, cancer biology and cancer therapy. Ultimately, we hope such SIAH-based anti-cancer therapies will lead to novel and efficacious treatments for human cancer patients, especially the ones with metastatic diseases.

Project II:   Innate Immunity and Cellular Defense

To understand how a host cell differentiates a pathogenic microbe from a nonpathogenic microorganism is a fundamental question in biology. Drosophila has an innate immune system that is similar to humans but is devoid of the complication of the adaptive immune system. We use the Drosophila as the model organism to study the molecular mechanism of how innate immunity is activated upon pathogen recognition. We found that the structural integrity of the sentinel receptors/innate sensors is modulated during infection and inflammation. We hypothesize that proteases release that is common during pathogen-host antagonism may provide an important cue for the host to distinguish a pathogenic versus a nonpathogenic microorganism. We are using transgenic fly models to demonstrate that protease release after pattern recognition provides a "tissue damage" signal that could alert host cells to the onset of endogenous tissue damage and exogenous pathogen invasion.

Project III:   Genetic Screens for Anti-Cancer Drug Resistance 

The development drug/chemical resistance is a recurring problem. There is an important need for us to understand the mechanisms by which drug/chemical resistance is acquired in multicellular organisms and cancers. We will carry out genetic screens in Drosophila for resistance to several key anticancer drugs that are prone to develop resistance. This effort, coupled with genomic and microarray analyses, should help to identify the alterations of key signaling pathways that could forecast and predict drug resistance development.

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