Principal Investigator

Kara Bernstein, PhD
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Location

2.35 University of Pittsburgh Cancer Institute
5117 Centre Avenue
Pittsburgh, PA 15213

Research Description

Repair of DNA damage is crucial to prevent accumulation of mutations that can cause human disease, such as cancer. Our lab studies how double-strand breaks in the DNA, one of the most lethal types of DNA lesions, are repaired. Many proteins are important for DNA repair including the Shu complex and the Rad51 paralogs, whose mutation is associated with cancer predisposition and Fanconi anemia. Our lab uses cell biological, molecular, and genetic approaches to study the role of double-strand break repair proteins, such as the Shu complex and the Rad51 paralogs, in response to DNA damage. By understanding the mechanism of double-strand break repair and the role of DNA repair proteins in this process, we will uncover mechanisms of tumorigenesis and cancer progression. We will then use this knowledge to aid in diagnosis/prognosis of different types of cancers and to find novel therapeutic targets. 

Dr. Bernstein conducts her research at the University of Pittsburgh Cancer Institute. 

Members 

Braulio Bonilla - Graduate Student

Thong Luong - Graduate Student

Sarah Hengel - Post Doctoral Associate



Principal Investigator

William B. Klimstra, PhD
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Location

8037 Biomedical Science Tower 3
3501 Fifth Avenue
Pittsburgh, PA 15213

Research Description

The goal of my laboratory has been to define the host and viral factors that determine the success or failure of the innate immune response to infection with arthropod-borne viruses. The specific approach is to examine at the single cell level, the molecular mechanisms that determine host cell permissivity to the alphaviruses (e.g., Sindbis virus [SB], Venezuelan equine encephalitis virus [VEEV], eastern equine encephalitis virus [EEEV], western equine encephalitis virus [WEEV], Chikungunya virus [CHIKV] and Ross River virus [RRV]) and the contribution of replication in specific cells to the pathogenesis of viral disease. Upon introduction into a susceptible host, alphaviruses initially replicate within cells of the dendritic cell (DC) and macrophage lineages. In young animals, this replication is unrestrained and leads to induction of a toxic proinflammatory cytokine response. However, in adults virus replication and cytokine induction are restricted by one or more as yet uncharacterized mechanisms. These mechanisms likely involve changes in host cell permissivity to virus infection. Ongoing studies include: determination of the relationship between infection of DC/macrophage and induction of the systemic inflammatory response, identification and characterization of cellular receptors that promote virus infection and identification of host innate immune mechanisms that control virus replication within individual cells. Furthermore, since the extent of virus replication, viral cellular tropism and the host response to infection are critical factors in stimulation of robust and appropriate immune responses to immunogens, we also strive to translate information gained from pathogenesis studies into strategies for improvement of alphavirus-based gene delivery systems.

Dr. Klimstra conducts his research through the Center for Vaccine Research. Learn more>



Principal Investigator

Gutian Xiao, PhD
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and

Zhaoxia (Julia) Qu, PhD
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Location

1.18 Hillman Cancer Center
5117 Centre Avenue
Pittsburgh, PA 15213

Research Description

NF-κB is a family of transcription factors that control expression of numerous genes involved in diverse biological processes, including inflammation, immune response, and cell growth.  NF-κB activity is normally inhibited by IκBs (Inhibitors of NF-κB) or IκB-like protein p100.  Accordingly, NF-κB activation requires IκB degradation or p100 processing to selectively degrade its C-terminal IκB-like domain.  The IκB degradation depends on an IκB kinase (IKK) complex, which consists of two catalytic subunits IKKα (IKK1) and IKKβ (IKK2), and one regulatory subunit IKKγ (NEMO), while the p100 processing is specifically mediated by IKKα and its upstream kinase NIK, NF-κB-inducing kinase.  These two major mechanisms leading to NF-κB activation are now termed canonical and non-canonical NF-κB signaling pathways, respectively.  



 

 

 

 

 

 

 

 

 

 

 

Activation of the NF-κB signaling pathways, particularly the canonical signaling pathway, is tightly regulated and rapidly curtailed following the initial activating stimulus.  Transient activation of NF-κB is physiologically important because persistent activation of either pathway can result in deleterious or even fatal conditions, such as septic shock, inflammatory diseases, autoimmune diseases, and cancers.  Although the molecular mechanisms by which NF-κB is induced under physiological conditions have been well defined, how NF-κB is aberrantly activated for human pathogenesis remains largely unclear. Given the fact that it is unfeasible to block NF-κB activation for disease therapy using ‘classical’ NF-κB inhibitors because of the physiological importance of NF-κB in humans, it is of interest and importance to address the difference in the pathogenic and physiological activation of NF-κB, which is important for the development of novel approaches to specifically target pathogenic NF-κB activation for disease prevention and treatment.  Currently, we are energetically studying how NF-κB is constitutively activated for the pathogenesis and therapy resistance of the following diseases and translating this knowledge into clinical settings.

1. Cancers

A. Viral Oncogenesis. We are focusing on two NF-κB-dependent oncogenic viruses: human T-cell leukemia virus type I (HTLV-I) and Kaposi's sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV8).  While HTLV-I is the etiologic agent of adult T-cell leukemia/lymphoma (ATL), KSHV causes Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD).  Interestingly, both HTLV-I and KSHV are often detected in individuals infected with HIV. In fact, KS is the most prevalent malignancy among patients with AIDS.  Currently, no effective therapies exist for these fatal diseases.

B. Cellular Oncogenesis. We study the top three deadliest tumors-lung, breast and colon, which account for approximately 241,000 deaths every year in the United States alone.  We are investigating how NF-κB is deregulated in tumor cells and tumor microenvironment, particularly tumor-associated inflammatory cells, as well as how deregulated NF-κB promotes the formation, progression, metastasis and chemoradioresistance of these solid tumors.  We are also actively investigating human leukemia/lymphomas associated with somatic mutations of the nf-κb2 gene and one hematologic malignancy termed early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), a newly identified type of highly lethal pediatric leukemia with no known cause or cure.  Recently, we have established the first reproducible and the only available in vivo model for studying this aggressive childhood leukemia.

2. Inflammatory and Autoimmune Diseases.  In addition to tumors, we are also interested in chronic obstructive pulmonary disease (COPD), colitis, multiple sclerosis (MS) and rheumatoid arthritis (RA).  In particular, we are investigating the molecular and cellular mechanisms by which NF-κB is able to promote COPD and colitis for the development of lung and colon cancers, but NF-κB-driven MS and RA are not associated with tumorigenesis.

3. Graft-Versus-Host Disease (GVHD).  Hematopoietic stem cell transplantation (HSCT) is an effective therapy for patients with a broad range of hematologic malignancies, and is the only potential option for several types of leukemia that no effective therapy exists, such as ATL and EPT-ALL.  However, allogeneic HSCT (alloSCT) causes GVHD in approximately 30-70% of patients, increasing morbidity and mortality as well as the cost of care.  Given the role of NF-κB in inflammation and inflammation-associated diseases, it is interesting to investigate whether and how NF-κB contributes to the development of GVHD and more importantly to target NF-κB for the prevention of GVHD.

Lab Members:

Lei Han - Postdoctoral Associate



Principal Investigator

Saumendra N. Sarkar, PhD
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Location

1.8 Hillman Cancer Center
5117 Centre Avenue
Pittsburgh, PA 15213

Research Description

Innate immunity of an organism is the inborn protection against invading pathogens. Because it is inborn, and entrusted with the protection of host from a vast array of previously unknown invaders, the innate immune system generates a generalized alert response upon pathogen detection. This alert is chemically mediated by a class of molecules called Cytokines. A critical task for this host protection system is to distinguish foreign or non-self, from self, and initiate their destruction or containment. The sensors or the receptors of the innate immune system accomplish this by recognizing specific molecular patterns, which are common to pathogens or pathogen associated molecules, but absent in the host. We focus on a particular subset of these sensors/receptors, which are involved in sensing virus infection.

In order to protect the host from viral invasion, the innate immune system has evolved sensors to detect the viral nucleic acids. Several unique features of virally produced DNA or RNA are exploited to distinguish viral nucleic acids from that of the host. One such unique nucleic acid is double-stranded RNA (dsRNA)—a common byproduct or intermediate in viral genome replication. In mammals, receptors like Toll-like Receptor 3 (TLR3), Retinoic acid-Inducible Gene I (RIG-I) and Melanoma Differentiation-Associated gene-5 (MDA5) are the three known sensors of dsRNA. Single-stranded viral RNA is sensed by Toll-like Receptor 7 and 8 (TLR7 and TLR8), while viral DNA is detected by Toll-like Receptor 9 (TLR9), and other less well characterized receptors.

We study two related aspects of innate immunity: (A) the signaling process involved in cytokine production after virus infection and (B) develop modulators for these signaling pathways. To understand the details of the networks involved in transcriptional induction or ‘switching on’ of genes following the detection of viral nucleic acids, we use various immortalized human cell lines engineered to make TLR3 and TLR7, as model systems. These studies have led us to discover new roles of protein modifications (e.g. tyrosine phosphorylations) involved in TLR3 signaling. We have shown how these phosphorylations control subtle modifications of transcription machinery (transcription factors). Continuing this line of investigation we are now trying to understand how other signaling networks interact and modulate the innate immune signaling pathways in human cells.

In a separate but related project we are identifying new modifiers of innate immune signaling pathways using high throughput screening. Activation of innate immune receptors by their natural microbial agonists leads to up-regulation of a series of pro-inflammatory and cytokine genes to neutralize the infection and shape the subsequent acquired immune response. On the other hand, excess stimulation of the receptor system from invading pathogens or from internal tissue damage products, cause several major inflammatory diseases such as atherosclerosis, asthma, bacterial sepsis and rheumatoid arthritis. Besides chronic inflammatory diseases, innate immune receptors have also been linked to cancer. The most established one is the gastrointestinal malignancy. Epidemiological and genetic evidences have also established links between TLR response and ovarian, prostate, breast and several other cancers. Thus, modulation of innate immune receptor signaling pathways offers an attractive method to fight such diseases. Proofs of this principle have been already established in several cases. Among other TLRs, TLR3 has been shown to mediate inflammation and pathogenesis of viral infection. TLR3 deficient mice are more resistant to lethal infection by West Nile virus than wild type mice. Similarly, TLR3 increases disease morbidity and mortality from Vaccinia and Phlebovirus infection. Therefore, in specific viral infection models, TLR3 may contribute not only to host defense but also to pathogenesis.

We have established cell-based screening systems for dsRNA mediated gene induction. Currently we are screening chemical and genetic modifiers of TLR3 and RIG-I signaling pathway. Additionally, we are in the process of establishing cell-based screening system for TLR7 and TLR8 in order to identify modifiers of their signaling pathways.

Dr. Sarkar conducts his research at the University of Pittsburgh Cancer Institute.

Members

Lulu Shao - Post Doctoral Associate



Principal Investigator

Patrick S. Moore, MD, MPH
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Location

1.8 Hillman Cancer Center
5117 Centre Avenue
Pittsburgh, PA  15213

 

Research Description

We study 1) Kaposi’s sarcoma-associated herpesvirus (KSHV), the viral cause of Kaposi’s sarcoma, 2)  Merkel  cell  polyomavirus  (MCV), the viral cause of Merkel cell carcinoma and 3) methods to search for undiscovered human tumor viruses. Our recent studies on KSHV have shown that its major latency protein evades typical protein processing pathways to avoid provoking a cell-mediated immune response. We also have found that both KSHV and Epstein-Barr virus (EBV) undergo programmed frameshifting to generate novel latent proteins. This allows these viruses to increase their phenotypic diversity within tight biological constraints on their genome size. Surprisingly, these two distantly related viruses generate frameshifted proteins that are nearly identical to each other.

To search for new human tumor viruses, we have developed a technique called digital transcriptome subtraction (DTS) that allows us to sample tumor mRNA profiles for foreign transcripts. Unlike other new pathogen discovery techniques, DTS is quantitative so that we can essentially rule out infection if no foreign transcripts are found. We have successfully used DTS to measure KSHV transcription in infected cell lines, and we have demonstrated that an AIDS-related cancer, squamous cell conjunctival carcinoma, is unlikely to be caused by an exogenous infection.

Using DTS, we discovered Merkel cell polyomavirus (MCV), the likely cause of ~80% of Merkel cell carcinomas. MCV is the first polyomavirus to be found that is a likely cause of human cancer. We are characterizing the basic biology of this virus in host cell transformation, and we are searching for other diseases that may be related to MCV infection. We are developing monoclonal antibodies and serologic tests that will be useful diagnostic markers for infection. We have recently found a unique mutation in tumor-derived MCV that sheds light on tumor cell evolution and the fundamental mechanism for Merkel cell carcinogenesis. Recent studies by others have confirmed our initial findings for the role of MCV in MCC. Our laboratory has now discovered and characterized two of the seven known viral causes of human cancer. We are actively examining other cancers to search for additional human cancer viruses.

Dr. Moore conducts his research through the University of Pittsburgh Cancer Institute.

Lab Members: 

Erdong Cheng - Post Doctoral Associate

Jennifer Alvarez Orellana - Post Doctoral Associate



Principal Investigator

Saleem A. Khan, PhD
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Location

545 Bridgeside Point II
450 Technology Dr.
Pittsburgh, PA 15219

Research Description

We are involved in three main areas of research. The first one deals with the role of microRNAs in human papillomavirus-associated cervical and oral cancers as well as role of miRNAs in aging. We are collaborating with several investigators in these studies, including Robert Ferris, Robert Edwards, Laura Niedernhofer and Patricia Opresko. The second area deals with the cellular functions and mechanism of action of the PcrA helicase which is specifically found in Gram-positive bacteria. In these studies, we are collaborating with Drs. Syam Anand and Sanford Leuba. The third area of our interest deals with a molecular analysis of the role of the RepX protein in the replication and segregation of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis. We have identified several cellular miRNAs that appear to be targeted by the HPV E6 oncogene. For example, we showed that the HPV-16 E6 oncogene downregulates miR-218, which in turn is involved in the regulation of the cellular LAMB3 gene. We also found that E6 upregulates miR-363 in HPV-positive oral cancer cell lines. Our ongoing studies deal with the identification of the domain(s) and mechanism by which E6 affects miR-218 expression, including whether or not this effect is mediated through p53. We are also investigating the role of HPVs in oral cancers. Our studies have shown that the presence of HPV-16 in SCCHN cell lines alters the miRNA profiles. In particular, miR-363 was found to be upregulated in HPV-positive SCCHN cell lines and tissues. Furthermore, the E6 oncogene of HPV-16 was required for the increased expression of miR-363. Our studies suggest that in addition to their known effects on tumor suppressor proteins such as p53 and Rb, the HPV oncogenes may also regulate cellular pathways by alteration of specific cellular miRNAs. We are using the XPF mouse and the human Werner Syndrome progeroid models to study the potential role of miRNAs in aging. Our initial studies have identified several miRNAs that are affected in XPF and WS cell lines as compared to normal controls. We are now investigating the role of such miRNAs in cellular senescence.

We are involved in the identification of functional domains of the PcrA helicase, using genetic, biochemical and single-molecule approaches. We have studied the substrate specificity of PcrA and have developed a model to explain how PcrA blocks recombination by displacing the RecA protein bound to the DNA. Using various PcrA mutants, we are studying its domains that are essential for cell survival.

We have shown that the pXO1-encoded RepX is a novel protein that may be involved in both the replication and segregation of this plasmid. We have shown that RepX belongs to the tubulin/ FtsZ family of GTPases and forms polymers both in vitro and in vivo. We have identified proteins that interact with RepX and are currently studying the role of such interactions in pXO1 replication, stability and segregation.



Principal Investigator

JoAnne L. Flynn, PhD
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Location

5065 Biomedical Science Tower 3
3501 Fifth Avenue
Pittsburgh, PA 15260

Research Description

Bone-marrow derived macrophages display heterogeneous phenotypesMy primary interest is in the interaction of pathogens with the host, with special emphasis on the immune mechanisms that protect against or exacerbate disease. Our focus is on Mycobacterium tuberculosis, the organism responsible for tuberculosis, which causes 2 million deaths per year worldwide. We are investigating the immune responses required for protection against tuberculosis, and the effect of these immune responses on both the host and the bacterium. We specifically study cytokine production, macrophage activation, and T cell subsets (CD4+ and CD8+ T cells) that are important in tuberculosis.

CD20... T cell- and B cell-mediated adaptive responses originate from cell-cell interactions that are initiated in lymph nodesFinally, we have a strong interest in the immune mechanisms responsible for maintaining a latent M. tuberculosis infection, and how deficiencies in the immune response can result in reactivation of disease. Our work is done in two model systems: mice and non-human primates. We have vaccine studies, drugs studies, and basic immunologic and pathogenesis studies ongoing. We also participate in projects involving mathematical modeling of the immune response to M. tuberculosis, and our plan is to incorporate nuclear imaging of live animals into our research.

Lab Members

Kara Kracinosky, Vet Tech  Joseph Zeppa, Postdoctoral Associate
Amy Fraser, Lab Manager Jake Borish, System Analyst
Alex White, Data Analyst
Jaime Tomko, Senior Research Specialist
Pauline Maiello, Data Analyst
Cassaundra Ameel, Research Technician
Lonnie Frye, Medical Imaging Technician
Sharie Ganchua, Graduate Student

Chelsea Causgrove, Senior Research Specialist

 

 Tonilynn Baranowski, Research Technician

Caylin Winchell, Post doctoral Associate

Erica Larson, Post doctoral Associate (Scanga Lab)




Principal Investigator

Neal A. DeLuca, PhD
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Location

547 Bridgeside Point II
450 Technology Dr.
Pittsburgh, PA 15219

Research Description

Repression and activation of persisting HSV genomes: Herpes simplex virus can undergo either a productive infection, where all the viral genes are expressed culminating in the production of progeny virus and cell death, or it can enter a latent state, which is characterized by the relative lack of viral gene expression, genome persistence, and cell survival. The latent state typically occurs only in neurons, and may involve the attenuation of immediate early (IE) gene expression, and thus the lack of later viral gene expression. The IE protein ICP0 has been shown to facilitate the transition from the latent to the lytic state, thus leading to reaction episodes that are typical of herpesviruses. We have been investigating the behavior of viruses that do not express any of the five IE proteins upon cellular infection. While the viral genomes are transcriptionally quiescent and cytopathic effects are not seen, they can be rendered transcriptionally active by supplying ICP0 in trans. Thus this system has some of the key features associated with HSV latency, but is also more amenable to biochemical and molecular study. We have found that quiescent genomes are largely repressed and exist in multiple states that differ with respect to nucleosome packaging, histone acetylation and methylation, and heterochromatin formation. The result is a stochastic mix of epigenetic states that differ with respect to the expressibility of the genome. We study the ability of viral activator proteins to overcome these repressive states and their mechanisms of action.

Virus-cell interactions affecting HSV gene expression:  In its productive life cycle, the genes of HSV are expressed in a sequential manner, with those controlling subsequent gene expression produced very early in infection. One of these, ICP4, is required for productive virus infection because it activates the expression of the remaining 80 or so early and late genes. ICP4 activates early and late genes by different mechanisms involving different interaction with TFIID, mediator, and other cellular transcription factors. To study these mechanisms, we examine the association of ICP4 and cellular transcription complexes on HSV promoters during infection, the regions of ICP4 that are important for these interactions are determined, and subsequently the effects of these interactions on the transcription and hence expression of different classes of HSV genes is determined. In addition, we have discovered that there are regions of ICP4 that are not required for productive viral growth in non-neuronal cells, but are absolutely required for function in neurons of the trigeminal ganglia, the site where HSV establishes latency. The basis for this requirement is current under study.

Members

Frances Sivrich, Research Technician



Principal Investigator

Cristian Apetrei, MD, PhD
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Location

9044 Biomedical Science Tower III
3501 Fifth Avenue
Pittsburgh, PA 15260

Research Description

My laboratory is interested in the study of the HIV/ SIV diversity and pathogenesis. The AIDS pandemic is produced by two different viruses, HIV-1 and HIV-2. These two viruses resulted from cross-species transmissions of SIVs, the viruses that naturally infect nonhuman primate species (NHPs) in Africa. HIV-1 resulted from 4 cross-species transmissions of SIVcpz/gor that infects chimpanzees and gorillas in West-Central Africa. HIV-2 emerged after 8 cross-species transmissions of SIVsmm that infects sooty mangabeys in West Africa. As SIVs naturally infect more than 40 species of non-human primates (NHPs) in Africa, our major concern is whether or not the remaining viruses infecting other species of African NHPs pose a major threat for humans. Our studies revealed that cross-species transmission of SIVs to humans are not the only requirement for the emergence on new virus strains and suggested that viral adaptation in the new host may play a decisive role for this event. Understanding the mechanisms of viral adaptation to new hosts upon cross-species transmission is of major interest for my laboratory. Using monkey models, we study the mechanisms of viral adaptation associated with viral emergence. Also, in order to better understand the AIDS pathogenesis, we are using various models of SIV infection in natural hosts. In African monkeys, SIV are not pathogenic in the vast majority of cases. My group is involved in the study of all currently available models (sooty mangabeys, African  green monkeys and mandrills) and generated significant results that challenged core paradigms of SIV pathogenesis. These studies may help us to control HIV infection in patients. Since no vaccine strategy currently developed seems to be effective, these alternative approaches may be essential in the control of AIDS pandemic.

Dr. Apetrei conducts research through the Center for Vaccine Research.  Learn more>

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