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Research Plan

The Mucosal Immunity Program is organized into four project groups, supported by the administrative and proteomic cores. The research projects address the hypothesis that dysregulation of key innate immune molecules of the oral epithelium predispose oral tissue to disease following chronic HIV infection. Projects #1 and #2 of this program characterize the crosstalk between beta defensins and cells of the adaptive immune system by investigating the interactions of hBD-3 with chemokine and Toll-like receptors. Project #3 utilizes proteomic analyses to characterize the protein expression of beta defensins and other innate defense molecules in HIV-exposed oral epithelial cells, and Project #4 investigates the role of beta defensin gene copy number in susceptibility to HIV oral complications. Thus, all four projects examine basic mechanisms of innate immune function, as well as assessment of the genetic and immunologic determinants of oral diseases prevalent in HIV+ individuals. The projects are:

Project 1: Epithelial immunity and oral complications of HIV infection
Project 2: HBD-3-CXCR4: Molecular mechanisms in innate immunity
Project 3: Proteomic response of epithelial cell interactions with HIV
Project 4: The genetic variation of innate immune genes with oral manifestations of HIV
Supplemental Grant: EBV and innate immunity in HIV+ and normal oral epithelium
Proteomics and Biostatistics Core
Administrative Core

Project 1. Epithelial immunity and oral complications of HIV infection

Principal Investigator: Dr. Scott Sieg

Investigators: Drs. Michael Lederman, Aaron Weinberg, Zhimin Feng, Tom McCormick

This project (1) examines the effects of hBD-3 on antigen presenting cells and T cell maturation. HBD-3 activates and promotes APC maturation: how does hBD-3-induced activation and maturation influence APC function, including APC ability to induce T cell maturation and activation? Although most of the analyses are centered on the direct effects of hBD-3 on APC to induce naïve CD4 helper cell activation and differentiation, the study addresses the complexities introduced by APC/oral epithelial interactions since these will be important to consider for the development of hBD-3 molecules as mucosal adjuvants. The project will also (2) determine the structural requirements for the TLR1/2 agonist activity of hBD-3. The novel finding that hBD-3 interacts with TLR 1/2 to promote APC maturation requires identifying the functional domains of hBD-3 that contribute to this activity. These studies will help to ascertain if hBD-3 molecules can be designed to mediate selective biological functions; translationally important in developing hBD-3 based therapeutics, microbicides and vaccine adjuvants. Finally, Project 1 will (3) investigate hBD-3 responsiveness in APCs from HIV-infected persons. This is a natural outcome of the group's preliminary studies showing that monocytes from HIV-infected persons respond poorly to stimulation with hBD-3, and that TLR1 expression in these cells is lower than in HIV negative individuals.

Project 2. HBD-3-CXCR4: Molecular Mechanisms in Innate Immunity

Principal Investigator: Dr. Aaron Weinberg

Investigators: Drs. Zhimin Feng, George Dubyak, Michael Lederman, David McDonald

This project includes (1) structure-function studies of hBD-3 and CXCR4 to identify critical sites of interaction on each molecule. The group's preliminary studies have identified candidate functional sites on both molecules. Therefore, continued site directed mutagenesis will reveal important functional domains, at the amino acid level, that dictate the interaction between both molecules. The project will also (2) determine the mechanism(s) underlying hBD-3 induced CXCR4 antagonism. Successful completion of this aim will allow definition of the mechanism whereby hBD-3 induces CXCR4 internalization without apparent activation of downstream signaling pathways. The project will conduct signaling studies to test whether hBD-3 blocks the accelerated GTP/GDP exchange and GTPase activity of Gi/Gq proteins that comprise most proximal actions of activated CXCR4. Internalization studies will define the possible roles of CXCR4 phosphorylation, arrestin, myosin 2A, dynamin, and clathrin coated-pit dependent endocytosis. Finally, the project will (3) study intracellular trafficking of hBD-3 and CXCR4 to ascertain why hBD-3 induces a slow recycling of the receptor when compared to the natural ligand for CXCR4, SDF-1. These studies collectively are vitally important to ascertain how an endogenous innate immune molecule of the mucosa protects the microenvironment, and how hBD-3 and its derivatives could be exploited to alter CXCR4 dependent biological outcomes.

Project 3. Proteomic response of epithelial cell interactions with HIV

Principal Investigator: Dr. Tom McCormick

Investigators: Drs. Ge Jin, Alison Quayle, Scott Sieg, Aaron Weinberg

his project (1) compares proteomic profiles of human oral epithelial cells (HOECs), female genital track epithelial cells (FGTECs) and skin-derived epithelial cells (SDECs). The group's preliminary studies show that HIV challenged HOECs respond by generating hBDs, while FGTECs do not. This suggests that different mucosal compartments respond differentially to HIV. Successful completion of the first aim in this project will define the proteome of various epithelial cells and determine the alteration(s) induced by HIV infection. Moreover, the preliminary proteomics data suggest that chronic HIV infection and/or highly active antiretroviral therapy (HAART) predisposes the oral mucosae to both cellular and innate immune impairment. The project will also (2) compare innate immune response molecules and altered target proteins among HOECs, FGTECs and SDECs in wart tissue from oral and skin sources, since our preliminary data demonstrated different innate immune responses in these tissues. Wart tissue from HIV and non HIV sources will be studied proteomically in order to define innate and other altered protein response molecule differences. Finally, the project will (3) examine the potential "cross-talk" between epithelial cells and dendritic cells that will have been exposed previously to hBD3 to determine if protein response of epithelial cells is altered by co-incubation with undifferentiated and/or differentiated monocytes. Findings from this study will help elucidate the role innate immune molecules play in mucosal protection during health and disease.

Project 4. The genetic variation of innate immune genes with oral manifestations of HIV

Principal Investigator: Dr. Peter Zimmerman

This project will (1) define the genetic determinants that predict the failure of host innate defenses during HIV-related immunosuppression. The group recently demonstrated that hBD-3 can promote the maturation of monocytes and myeloid dendritic cells through interaction with TLR1/2. Thus, a model of genetic control of hBD expression and genetic regulation of TLR-dependent responses to hBDs could determine the degree to which innate and adaptive defenses can be harnessed to resist oral complications of HIV-induced immunodeficiency. There is a specific SNP in the hBD-1 gene associated with low frequencies of oral candida carriage. Moreover, recent findings show an association between that SNP and high copy number polymorphisms in the hBD-3 gene. This is quite remarkable considering that copy number polymorphisms are rare in innate immune genes. It appears therefore that hBD expression levels likely vary in the human population. Since hBD-3 protects against the HIV X4 phenotype via internalization of the CXCR4 coreceptor, high hBD-3 expressors could be protected from X4 emergence. This project will (2) determine if genetic determinants of defensin expression (both gene duplications and nucleotide polymorphisms) have demonstrable impact on the course of HIV disease via restriction of the emergence of CXCR4 utilizing HIV strains.

Supplemental Grant: EBV and innate immunity in HIV+ and normal oral epithelium

Principal Investigator: Dr. Aaron Weinberg

Investigators: Drs. Richard Longnecker, Theodore Jardetzky

The central hypothesis of this Program Project is that alterations in innate defense mechanisms determine susceptibility to oral complications following HIV infection. The participating groups at Case Western Reserve University have shown that oral epithelial cell-derived beta defensins can (1) be induced by HIV, (2) inhibit the ability of the virus to infect immunocompetent cells and (3) interact with specific chemokine and toll-like receptors resulting in regulation of adaptive immune cells. Our proteomic studies suggest that cellular and innate immune mechanisms are impaired in HIV positive oral mucosa, when compared to HIV negative oral mucosa. We have proposed, therefore, that chronic HIV infection and/or highly active antiretroviral therapy (HAART) perturbs both cellular and innate defenses of the oral mucosa. We have an ongoing project examining the effects of HIV infection and HAART therapy on exacerbating human papilloma virus (HPV) associated pathology, In this project, we will examine the effects of HIV infection and HAART therapy on Epstein-Barr Virus (EBV), which is also a key contributor to oral pathologies, particularly in immunocompromised patients. The proposed studies with EBV will enable us to compare and contrast the effects of HAART on EBV and HPV, providing additional insights into oral health complications in HIV+ populations.

The Proteomics and Biostatistics Core

Under the direction of Dr. Mark Chance, The Case Center for Proteomics and Bioinformatics will perform quantitative analysis of changes in protein expression for Projects 1 and 3 using two dimensional differential in gel electrophoresis (2-D DIGE) coupled with mass spectrometry and label free proteomic approaches as well as genotyping, polymorphism analyses (SNPs and CNPs) and association studies in Project 4. The core will facilitate the analysis and interpretation of high throughput data with sophisticated statistical and functional tools such as DeCyder EDA, Pro Quant, R and network analysis tools such as Ingenuity. Finally, the core will integrate and correlate the results from different aims of the projects, whenever it applies, through appropriate statistical models, in order to validate results and address over-arching research questions.

The Administrative Core

Assigned to the Administrative Core is the (1) integration the various components and activities of the Program Project, (2) management of all fiscal operations, (3) coordination, development and facilitation of planning for the projects and cores and (4) implementation of an enrichment program to include research-in progress meetings, visiting scientist lectures, symposia and a training program. The Administrative Core will organize annual meetings among the Executive Committee and an External Science Steering Committee to conduct rigorous scientific scrutiny of information gathered through the Program Project.

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Career Openings

A postdoctoral position is available to work in the area of oral innate immunity, including studies to decipher how epithelial cell derived antimicrobial peptides (AMPs) induce G-protein coupled receptor antagonism in adaptive immune cells and the trafficking of AMPs within these cells. The qualified candidate must be trained in molecular biology techniques, PCR assays, transfections, transductions, RNA interference and with some knowledge of protein chemistry and tissue culture. Additional experience in immunology is desired.

Candidates must have earned their PhDs within the last 1-2 years. The opportunity for further advancement within the institution exists. Applicants should submit an updated CV, three references, and a cover letter describing their research goals and interests to: Dr. Aaron Weinberg, Chair, Department of Biological Sciences, Case Western Reserve University School of Dental Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4905 or by email: aaron.weinberg@case.edu.

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Research in the Case School of Dental Medicine

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