Institute for Immunity, Transplantation and Infection

Seed Grant Programs

Institutes of Medicine Seed Grant Fund in Translational Research

The Institutes of Medicine Seed Grant Fund in Translational Research  has been established at the School of Medicine to support important translational research in each of the five Institutes of Medicine.  Each year for five years, a $25,000 seed grant will support one research project in one of the five Institutes, with the award rotating each year to a new Institute until all five Institutes have received a year of seed funding.  The Dean of the School of Medicine determined that this year (2008-2009) the seed grant will be given to the Institute for Immunity, Transplantation and Infection (ITI).  ITI received 34 proposals which were reviewed by the Director of ITI, Mark Davis, and the ITI Steering Committee.   They have decided to award the seed grant to Shirit Einav, Medical Fellow in Jeffrey Glenn’s lab. Her project is entitled: “Novel Mechanisms for HCV Induced Cellular Transformation.”

There were many good proposals that were submitted and two runners-up were selected, the funding of which will come from ITI and other sources.  They are:

Below are abstracts of the three projects starting with the Seed Grant winner:

Novel mechanisms for HCV induced cellular transformation
Shirit Einav, MD
Chronic infection with the hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC). HCC is the sixth most common cancer, but the third leading cause of cancer death in the world. The incidence of HCC and the mortality rate associated with it are increasing dramatically. While chronic inflammation, fibrosis and liver cell proliferation are considered the major factors contributing to the development of HCC, there is accumulating data supporting direct viral effects. Despite advances in the understanding of HCV's biology, mechanisms whereby the virus promotes cellular transformation remain poorly understood. Current therapies are inadequate for the majority of HCV patients with or without HCC. HCC is the most common indication for liver transplantation. Our long-term goals are to better understand the HCV life cycle and the viral factors that mediate development of HCC and to translate this knowledge into novel antiviral strategies that would potentially inhibit not only viral replication but also viral-mediated cellular transformation. In this proposal we seek to study the role of viral factors in mediating development of HCV induced HCC – the leading indication for liver transplantation, by studying their effect on host cellular factors (cytokines, other host cell proteins and microRNA). Thus, this proposal integrates the three disciplines of the ITI: Infection, Transplantation and Immunology.
While previously characterized by a complete lack of known function, the HCV non-structural (NS) protein NS4B has emerged as a key player in viral replication and in cellular transformation. NS4B was previously shown by others to transform NIH3T3 cells when co-expressed with the Ha-ras gene. While the mechanism(s) for NS4B’s transformation activity remains unknown, disrupting this function may help inhibiting development of HCC. We have previously identified a nucleotide binding motif (NBM) within NS4B. This motif consists of a set of conserved amino acids found in both the GTP-binding members of the G-protein family, as well as in the superfamily of viral proteins with nucleotide binding domains. The most highly-conserved element within these nucleotide-binding domains of GTP-binding proteins is the A motif. Other conserved elements known from crystal structures to participate in nucleotide binding include the G, PM2 and B motifs. We have shown that NS4B’s NBM mediates both binding and hydrolysis of GTP and HCV RNA replication. Furthermore, we have recently shown that NS4B can mediate cellular transformation and tumor formation in mice independently of the Ha-ras gene. Similar to the NBM of human oncogenes, such as ras, known to mediate malignant transformation, the NBM was found to mediate NS4B’s role in cellular transformation.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting messenger RNAs through translational repression or RNA degradation. Data is accumulating in regard to the role miRNA play in the settings of infection and cancer. miRNAs interact with classic oncogenes and tumor suppressor networks and contribute to the initiation and progression of many human malignancies. Recently, Randall, et al. showed that HCV replication modulates the expression of a limited set of host miRNAs in replicon cells. Nevertheless, the temporal effects of HCV replication on miRNAs profile haven’t been studied yet. Furthermore, the involvement of various miRNA in HCV induced oncogenesis hasn’t been studied either. We, in collaboration with Dr. Andrew Fire’s laboratory, have recently profiled the miRNA expression following HCV infection. A significant increase in the frequency of two miRNAs was detected several days post infection. One of these miRNAs is known to be involved in development of tumors. We have shown that NS4B but not the other non-structural HCV proteins known to transform cells (NS3 and NS5A), is capable of inducing this miRNA levels. Furthermore, the levels of this miRNA were found to be elevated in NS4B transformed cell lines in correlation with NS4B’s levels.
Our overall hypothesis is that nucleotide binding and hydrolysis leading to induction of miRNA are essential for mediating NS4B’s role in cellular transformation and oncogenesis. Thus to the extent viral factors contribute to the increased risk of HCC, approaches designed to disrupt these functions may be potentially used to inhibit HCV associated HCC. Pharmacologic inhibition of the NBM may also inhibit HCV replication.
These results shed light on the mechanism by which NS4B mediates cellular transformation and have exciting implications. The NBM is conserved across natural HCV isolates suggesting that it is essential for infection in vivo. The involvement of miRNAs in HCV induced transformation represents a novel mechanism of viral-mediated carcinogenesis. Finally, these results may also lead to development of novel strategies for prevention and treatment of HCC in HCV patients.

Characterization of innate immune pathways leading to inflammasome activation
Petr Broz, PhD
Bacterial infections still represent a major cause of death worldwide, especially in developing countries. In the developed world the emergence of antibiotic-resistant bacteria and the abandonment of vaccination has led to the re-appearance of infectious diseases. Indeed, the treatment of diseases caused by intracellular bacterial pathogens remains a major challenge for the biomedical community. For example, millions of human cases of Salmonellosis are reported worldwide every year, resulting in huge economic costs to society and thousands of deaths. Furthermore, there are 1.7 million deaths annually due to infections caused by Mycobacterium tuberculosis, the causative agent of Tuberculosis (WHO). Mammalian hosts have evolved mechanisms, collectively called the innate immune system, to detect and fight off many microbial infections quickly. In response, disease-causing bacteria have evolved specific mechanisms to survive in the host despite the presence of the mammalian immune system. I am interested in studying how the innate immune system recognizes bacterial pathogens, and how pathogens manipulate the host. To detect the presence of microbes, the host innate immune system relies on surveillance proteins, or receptors that recognize “foreign” microbial molecules. Some of these receptors are on the surface of host cells and detect extracellular microbes and their products, whereas other receptors are inside host cells and detect intracellular microbes and their products. Interestingly, the same receptors that are involved in the recognition of intracellular pathogens also recognize so-called “danger signals” resulting from cell injuries caused for example by the inhalation of airborne pollutants like asbestos and silica or from uric-acid crystals associated with gout. In addition mutations in these receptors have been shown to result in human disease. For example, mutations in Nod2 and NALP3, both intracellular receptors, are associated with Crohn’s disease, and the receptor NALP1 is associated with Vitiligo and other autoinflammatory diseases. Once these intracellular receptors recognize a microbial product or a danger signal, they trigger the assembly of an intracellular multiprotein complex termed the “inflammasome.” The inflammasome promotes the activation of caspase-1, an enzyme that is required for the processing and release of pro-inflammatory chemical messengers, called cytokines, such as interleukin (IL)-1b and IL-18, which alert other immune cells. Inflammasome activation can also lead to death of the infected cell, which is crucial in restricting intracellular replication of invasive bacterial pathogens such as Salmonella typhi, which causes typhoid fever, and Francisella tularensis, which causes tularemia, a highly infectious disease also known as “rabbit fever.” 
Despite the central role of the inflammasome complex in innate immunity, many aspects remain uncharacterized. Therefore, the aim of my project is to identify new inflammasome receptors and to characterize pathways that lead to inflammasome activation using in vitro and in vivo models of bacterial infections. In particular, I am interested in the molecular mechanisms that are used by macrophages (a crucial cell of the innate immune system) to recognize intracellular pathogens, such as Francisella tularensis and Salmonella typhi, and the subsequent activation of the inflammasome. The better understanding of the central inflammasome complex and the identification of receptors leading to its activation are of clinical relevance because the same receptors involved in detecting intracellular pathogens also recognize danger signals from cell injury and are involved in autoinflammatory diseases. These findings could lead to a better understanding of the pathogenesis of infectious diseases autoinflammatory diseases. Finally, this project could promote the development of novel immune modulators and/or anti-infectives.

Oral Bacteriophage Communities
David Pride, MD, PhD
Periodontitis is a state of the human oral cavity where there is inflammation of the supporting structures of the teeth. Unlike many other diseases, no specific bacterial cause has been attributed to periodontitis, and the prevailing belief is that the disease occurs as a result of changes in the oral bacterial community as a whole.  Periodontal disease often is associated with the presence of dental caries, which represents a disease condition affecting the structure of the teeth. Several different bacteria, including certain species of Streptococcus have been associated with dental caries, and promote the formation of dental caries through demineralization of the teeth through acid production. Bacteriophages are viruses that specifically infect bacteria and are not known to infect human cells. They represent the most abundant entities on the planet and have been found wherever bacteria exist. There are vast unexplored communities of bacteriophages that inhabit human bacterial communities, but because they do not directly infect human cells, they have not traditionally been thought to be involved in either promoting human health or disease. There have been numerous examples of changes in bacterial communities leading to human disease, such as antibiotic-associated diarrhea, where antibiotics eradicate beneficial gut bacteria, leading to an overgrowth of certain harmful bacteria. Bacteriophages, through their ability to enter human bacterial communities and eliminate certain bacteria, have the capacity to substantially alter bacterial communities, and thus, may play a role in human health and disease.
There have been no previous comprehensive studies of bacteriophage communities that inhabit humans.  We propose a multidisciplinary approach, which is the first of its kind, towards understanding whether bacteriophages play a role in human oral health and disease. We have recruited human subjects with both oral health and disease, and collected both saliva and dental plaque from each subject.  Our specific goals are: 1) To develop techniques to isolate and describe bacteriophages from the human oral cavity, 2) To identify bacteriophages that alter oral bacterial communities and play a role in oral health and disease, and 3) To understand the interplay between oral bacteria and bacteriophages through analysis of certain mechanisms of competition between the two communities. Through sampling of the human oral cavity in states of health and disease and isolation of bacterial and bacteriophage communities, we believe we can gain a more comprehensive view of the role of bacteriophage communities in the human oral cavity, and gain novel insight into the impact bacteriophages have on oral bacteria and oral health.

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