Microbiology and Immunology Department Theses and Dissertations

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THE ROLE OF PAK1 IN THE CELLULAR AND MOLECULAR COMPONENTS OF PLEXIFORM NEUROFIBROMAS
(2008-10-10T18:12:42Z) McDaniel, Andrew S.; Clapp, D. Wade; Broxmeyer, Hal E.; Ingram, David A.; Srour, Edward F.
Neurofibromatosis type I (NF1) is a common genetic disease that affects over 200,000 patients in North America, Europe, and Japan. Individuals with NF1 display a wide variety of pathologies; importantly, 15-40% of NF1 patients are affected by plexiform neurofibromas. Neurofibromas are complex tumors consisting of tumorgenic Schwann cells surrounded by endothelial cells, fibroblasts, and inflammatory mast cells. These peripheral nerve sheath tumors contribute significantly to the morbidity and mortality associated with NF1. Currently, no medical therapies exist for treating neurofibromas. Recent evidence indicates that the hematopoietic tumor microenvironment carries out a crucial function in the formation of plexiform neurofibromas. Neurofibromatosis is the result of mutations at the NF1 locus, which encodes the GTPase activating protein neurofibromin. Neurofibromin is a negative regulator of the proto-oncogene Ras. Ras hyperactivation is the molecular basis of NF1 associated phenotypes, and it has been demonstrated that restoration of Ras signaling to wild type levels can correct NF1 associated phenotypes in vitro and in vivo. In keeping with the long term goal of detecting potential molecular targets for medical therapies to treat human plexiform neurofibromas, we have identified the kinase Pak1 as a possible downstream intermediary of Ras signaling in NF1 deficient cells. Studies described here utilized murine genetic models to study the effects of genetic inactivation of Pak1 on molecular signaling and cellular functions related to neurofibromas. We demonstrate that inactivation of Pak1 leads to correction of SCF mediated gain-in-function phenotypes seen in Nf1 haploinsufficient mast cells, in vivo and in vitro. However, by using a conditional Nf1 knockout mouse that is a reliable model of plexiform neurofibroma formation, we shown that loss of Pak1 alone in the hematopoeitic compartement is not sufficient to prevent neurofibroma formation. Additionally, we describe a key role for Pak1 in regulating PDGF and TGF-β mediated fibroblast functions, in vitro and in vivo. These studies provide insight into the causes of debilitating tumors related to a common genetic disease, and this research could potentially lead to the development of medical therapies for these tumors, increasing the quality of life for tens of thousands of affected individuals each year.
IN VIVO HEMATOPOIETIC CELL ENGRAFTMENT IS MODULATED BY DPPIV/CD26 INHIBITION AND RHEB2 OVEREXPRESSION
(2009-03-18T18:36:08Z) Campbell, Timothy Brandon; Broxmeyer, Hal E.; Clapp, D. Wade; Quilliam, Lawrence; Srour, Edward
Hematopoietic cell transplantation (HCT) is an important modality used to treat patients with hematologic diseases and malignancies. A better understanding of the biological processes controlling hematopoietic cell functions such as migration/homing, proliferation and self-renewal is required for improving HCT therapies. This study focused on the role of two biologically relevant proteins, dipeptidylpeptidase IV (DPPIV/CD26) and Ras homologue enriched in brain 2 (Rheb2), in modulating hematopoietic cell engraftment. The first goal of this study was to determine the role of the protein DPPIV/CD26 in modulating the engraftment of human umbilical cord blood (hUCB) CD34+ stem/progenitor cells using a NOD/SCID mouse xenograft model, and based upon previous work demonstrating a role for this enzyme in Stromal-Derived Factor-1/CXCL12 mediated migration and homing. Related to this first goal, pretreatment with an inhibitor of DPPIV/CD26 peptidase activity increased engraftment of hUCB CD34+ cells in vivo in recipient Non Obese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) mice while not disturbing their differentiation potential following transplantation. These results support using DPPIV/CD26 inhibition as a strategy for enhancing the efficacy of cord blood transplantation. The second goal was to determine, by overexpression, the role of the Rheb2 in affecting the balance between proliferation and in vivo repopulating activity of mouse hematopoietic cells. Rheb2 is known to activate the mammalian target of rapamycin (mTOR) pathway, a pathway important in hematopoiesis. Rheb2 overexpression increased the proliferation and mTOR signaling of two hematopoietic cell lines, 32D and BaF3, in response to delayed IL-3 addition. In primary mouse hematopoietic cells, Rheb2 overexpression enhanced the proliferation and expansion of hematopoietic progenitor cells (HPCs) and phenotypic hematopoietic stem cells (HSCs) in vitro. In addition, HPC survival was enhanced by Rheb2 overexpression. Using in vivo competitive repopulation assays, Rheb2 overexpression transiently expanded immature HPC/HSC populations shortly after transplantation, but reduced the engraftment of total transduced cells. These findings support previous work showing that signaling proteins able to enhance the proliferative status of hematopoietic stem cells often cause exhaustion of self-renewal and repopulating ability. These studies of hematopoietic engraftment modulated by both of these molecules provide information which may be important to future work on HCT.
Obstacles and Circumvention Strategies for Hematopoietic Stem Cell Transduction by Recombinant Adeno-associated Virus Vectors
(2009-03-18T18:55:15Z) Maina, Caroline Njeri; Srivastava, Arun; Clapp, D. Wade; Yoder, Mervin C.; He, Johnny J.
High-efficiency transduction of hematopoietic stem cells (HSCs) by recombinant adeno-associated virus serotype 2 (AAV2) vectors is limited by (i) inadequate expression of cellular receptor/co-receptors for AAV2; (ii) impaired intracellular trafficking and uncoating in the nucleus; (iii) failure of the genome to undergo second-strand DNA synthesis; and (iv) use of sub-optimal promoters. Systematic studies were undertaken to develop alternative strategies to achieve high-efficiency transduction of primary murine HSCs and lineage-restricted transgene expression in a bone marrow transplant model in vivo. These included the use of: (i) additional AAV serotype (AAV1, AAV7, AAV8, AAV10) vectors; (ii) self-complementary AAV (scAAV) vectors; and (iii) erythroid cell-specific promoters. scAAV1 and scAAV7 vectors containing an enhanced green-fluorescent protein (EGFP) reporter gene under the control of hematopoietic cell-specific enhancers/promoters allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. Self complementary AAV vectors containing an anti-sickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer, or the human parvovirus B19 promoter at map-unit 6 (B19p6) were tested for their efficacy in a human erythroid cell line (K562), and in primary murine hematopoietic progenitor cells (c-kit+, lin-). These studies revealed that (i) scAAV2-beta-globin vectors containing only the HS2 enhancer are more efficient than ssAAV2-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (ii) scAAV-beta-globin vectors containing only the B19p6 promoter are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (iii) scAAV2-B19p6-beta-globin vectors in K562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit+, lin- cells, yield efficient expression of the beta-globin protein. These studies suggest that the combined use of scAAV serotype vectors and the B19p6 promoter may lead to expression of therapeutic levels of beta-globin gene in human erythroid cells, which has implications in the potential gene therapy of beta-thalassemia and sickle cell disease.
ANTI-TUMOR AND RADIO-SENSITIZING PROPERTIES OF AD-IU2, A PROSTATE-SPECIFIC REPLICATION-COMPETENT ADENOVIRUS ARMED WITH TRAIL
(2009-03-18T18:58:00Z) Jimenez, Juan Antonio; Gardner, Thomas A.; Kao, Chinghai; Crabb, David W.; Harrington, Maureen A.; Roman, Ann
In this thesis, I investigated the preclinical utility and antitumor efficacy of TRAIL delivered by Ad-IU2, a prostate-specific replication-competent adenovirus (PSRCA), against androgen-independent prostate cancer. Through transcriptional control of adenoviral early genes E1a, E1b and E4, as well as TRAIL by two bidirectional prostate-specific enhancing sequences (PSES), expression of TRAIL as well as adenoviral replication was limited to prostate-specific antigen and prostate-specific membrane antigen (PSA/PSMA)-expressing cells. Ad-IU2 replicated efficiently in and was restricted to PSA/PSMA-positive prostate cancer cells and induced 5-fold greater apoptosis in androgen-independent CWR22rv and C4-2 prostate cancer cells than the PSRCA control not expressing TRAIL. Ad-IU2 exhibited superior killing efficiency in PSA/PSMA-positive prostate cancer cells at doses 5 to 8-fold lower than that required by a non-TRAIL expressing PSRCA to produce a similar effect. This enhanced cytotoxic effect was not observed in non-prostatic cells, however. As an enhancement of its therapeutic efficacy, Ad-IU2 exerted a bystander effect through either direct cell-to-cell contact or soluble factors present in conditioned media from Ad-IU2-infected cells. In vivo, Ad-IU2, as compared to a control PSRCA, markedly suppressed the growth of subcutaneous CWR22rv xenografts at six weeks post-treatment (3.1 vs. 17.1-fold growth of tumor). The treatment of androgen-independent prostate cancer with Ad-IU2 prior to external beam radiation therapy (EBRT) significantly reduced clonogenic survival with dose reduction factors of 4.91 and 2.43 for CWR22rv and C4-2 cells, respectively. Radio-sensitization by Ad-IU2 was restricted to PSA/PSMA-positive cells. Combinatorial radio-gene therapy resulted in accumulation of cells in G1 phase and a perturbation of the radiation-induced G2 phase arrest. This multi-modal approach combining viral lysis, apoptosis-inducing gene therapy, and radiation therapy could have great impact in achieving complete local tumor control while reducing radiation dose and associated treatment morbidities. This would result in improvement of the clinical outcome of patients with high risk prostate cancer.
STAT PROTEIN REGULATION OF FOXP3 EXPRESSION AND INFLAMMATORY CYTOKINE PRODUCTION IN T HELPER CELL SUBSETS
(2009-03-19T19:00:06Z) O'Malley, John Thomas; Kaplan, Mark H.; Blum, Janice S.; Clapp, D. Wade; Travers, Jeffrey B.
The differentiation of naïve CD4+ T cells into subsets of T helper cells (Th) is an essential process that impacts host defense and the pathogenesis of immunemediated diseases. Signal transducers and activators of transcription (STAT) proteins, activated downstream of instructive cytokines, dictate and perpetuate the lineage decision of Th cells through both positive and negative effects. This is accomplished by regulating transcription factors, surface receptors and promoting epigenetic changes in gene expression through chromatin remodeling. Transforming growth factor-β1 (TGF-β1) can induce Foxp3 in developing Th cells and these Foxp3-expressing adaptive T regulatory cells (aTregs) are able to suppress inflammation in vitro and in vivo. To define the mechanism by which STAT proteins regulate Th cell pro- and anti-inflammatory phenotypes, we examined T cells deficient in Stat3, Stat4, and Stat6 as well as T cells expressing two STAT4 isoforms after being cultured in the presence or absence of TGF-β1 and cytokines known to be instructive in Th cell development. The negative effects of STAT proteins are demonstrated by our results indicating STAT3, STAT4 and STAT6 proteins activated downstream of the instructive cytokines IL- 6, IL-12 and IL-4, respectively, negatively regulate the development of TGF-β induced Foxp3 and aTreg development. STAT3, STAT4, and STAT6 utilize a vi Mark H. Kaplan, Ph.D., Chair common mechanism to inhibit aTreg generation by inhibiting STAT5, a positive regulator of Foxp3 expression, from binding to the Foxp3 gene. STAT proteins positively effecting inflammatory immunity are demonstrated by our analysis of STAT4 isoforms and their ability to regulate the production of proinflammatory cytokines downstream of IL-12. STAT4β, a STAT4 splice isoform that lacks a Cterminal domain, and STAT4α, a full-length isoform are both capable of mediating inflammatory cell development. However, STAT4β promotes greater inflammation in vivo than STAT4α independent of its ability to repress Foxp3. Instead, the inflammation correlates with STAT4 isoform-dependent expression of inflammatory cytokines. Thus, cytokine-stimulated STAT proteins orchestrate T helper cell pro- and anti-inflammatory cell phenotypes.
The role of PU.1 and IRF4 interaction in the biology and function of T helper 2 cells
(2009-05-19T18:01:29Z) Ahyi, Ayele-Nati; Kaplan, Mark H.; Blum, Janice S.; Klemsz, Michael J.; Yoder, Mervin C.
Adaptive and innate immune responses play a critical role in the protection against extracellular or intracellular pathogens. The function of these two types of immune responses is coordinated by CD4+ T-helper (Th) cells. Depending on the cytokine environment, Th progenitor (Thp) cells differentiate into three functionally different effector subsets. T-helper-1 (Th1) cells which mediate cell-mediated immunity, T-helper-2 (Th2) which orchestrates humoral immunity and T-helper-17 (Th17) cells key players in autoimmunity response. Cytokine induced transcription factors that are differentially expressed in Th cells are required for the development and commitment to a specific Th lineage. The population of Th2 cells can be subdivided in subpopulations depending on the level of a cytokine and the subsets of cytokines they produce. Very limited information is available about the regulation of cytokine production in this array of Th2 cells. We have recently identified the ETS family transcription factor PU.1 as regulating heterogeneity in Th2 populations. To define additional factors that might contribute to Th2 heterogeneity, we examined the PU.1 interacting protein IFN-regulatory factor (IRF)-4, a transcription factor expressed in lymphocytes and macrophages. When Th2 cells are separated based on levels of IL-10 secretion, IRF4 expression segregates into the subset of Th2 cells expressing high levels of IL-10. To investigate the role of IRF4 in cytokine heterogeneity, Th2 cells were infected with retrovirus expressing IRF4. The cells overexpressing IRF4 secreted significantly higher levels of IL-10 and IL-4 compared to cells infected with a control vector at the same time the level of IL-9 decreases. To understand the mechanism by which IRF4 regulates IL-10 expression in various Th2 cell subpopulations we used co-immunoprecipitation assays to determine transcription factors that interact with IRF4. Our data shows that PU.1, IRF4 and NFATc2 form a complex in Th2 nuclear extract. We also demonstrated by ChIP assay that IRF4 directly binds the Il10 and Il4 loci in a time dependent manner. The role of these protein-protein and protein-DNA complexes and their contribution towards Th2 heterogeneity will be further defined. Understanding the regulation of the anti-inflammatory cytokine IL-10 in Th2 cells may give us a tool to control inflammation.
DISTINCT LOCALIZATION OF NADPH OXIDASE FLAVOCYTOCHROME B IN RESTING AND INTERFERON GAMMA ACTIVATED MACROPHAGES
(2009-06-22T19:52:25Z) Casbon, Amy Jo; Dinauer, Mary C; Kaplan, Mark H.; Bauer, Margaret E.; Pavalko, Fredrick M.
Flavocytochrome b558, the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91phox (NOX2) and a smaller subunit, p22phox. Localization of flavocytochrome b to the phagosome is essential for microbial killing, yet the subcellular distribution of flavocytochrome b in macrophages and how it is incorporated into macrophage phagosomes is not well characterized. In neutrophils, flavocytochrome b localizes primarily to specific granules that are rapidly mobilized to the phagosome upon stimulation. In contrast to neutrophils, macrophages do not contain specific granules, and trafficking of membrane proteins to the phagosome is more dynamic, involving fission and fusion events with endosomal compartments. We hypothesized that in macrophages, flavocytochrome b localizes to both plasma membrane and endosomal compartments that deliver flavocytochrome b to the phagosome. We generated fluorescently tagged versions of both p22phox and gp91phox, and rigorously verified their functionality in Chinese Hamster Ovary cells. Localization of flavocytochrome b was then examined in both RAW 264.7 murine macrophages and primary murine bone marrow derived macrophages (BMDM) in the presence and absence of interferon gamma (IFNg). We found that in “resting” macrophages, flavocytochrome b localizes primarily to the Rab11-positive endosome recycling compartment that recycles to the plasma membrane. In addition, phagocytosis assays showed flavocytochrome b is incorporated into the phagocytic cup and colocalized with Rab11 at the base of the cup, suggesting Rab11-positive endosomes may be involved in trafficking of flavocytochrome b between intracellular membranes and forming or nascent phagosomes. However, in IFNg activated macrophages, flavocytochrome b was localized predominantly in the plasma membrane, with little present in endosomal compartments. This shift in flavocytochrome b distribution occurred following sustained exposure to IFNg and correlated with increased flavocytochrome b protein expression and increased extracellular production of superoxide. Taken together, our results suggest the IFNg-induced redistribution of flavocytochrome b may be important for enhancing the production of superoxide at the cell surface and may be a potential new mechanism by which IFNg enhances antimicrobial activity in macrophages.
Sam68, Stress Granules, and translational control of HIV-1 nef mRNA
(2009-06-23T21:33:16Z) Henao-Mejia, Jorge Alejandro; He, Johnny J.; Blum, Janice S.; Roman, Ann; Wek, Ronald C.
More than 20 million people have died of AIDS since the early eighties, while nearly 34 millions are currently infected with the HIV. Anti-retroviral therapy (ART) directed at key viral enzymes has changed AIDS from uniformly fatal to a manageable chronic disease. However, ART-associated drug resistance and toxicity have posed a great challenge for long-term management of the disease and have called for development of new therapeutics. In this study, we focused on the viral factor Nef and the host factor Sam68. Nef is a major pathogenic viral determinant for HIV-1, and no therapeutics have been targeted to this factor. Sam68 is indispensible for HIV-1 propagation. We revealed that Sam68 variants were very potent in preventing Nef expression. We found that these effects were associated with their ability to form a macromolecular structure called stress granules (SG). In addition, we demonstrated that these variants bound to nef mRNA in a sequence-specific manner. Furthermore, we showed that these variants co-localized with nef mRNA in SG. Importantly, we validated these findings in the context of HIV-1 infection of its natural target cells and found significant loss of Nef function in these cells. Taken together, these results demonstrate that SG induction and nef mRNA sequestration account for translational suppression of Nef expression and offer a new strategy for development of anti-HIV therapeutics. Sam68 is implicated in a variety of other important cellular processes. Our findings that Sam68 variants were able to induce SG formation prompted us to investigate whether wild-type Sam68 was also recruited to SG. We found that Sam68 was increasingly recruited into SG under oxidative stress, and that its specific domains were involved. However, Sam68 knockdown had no effects on SG assembly, suggesting that Sam68 is not a constitutive component of SG assembly. Lastly, we demonstrated that Sam68 complexed with TIA-1, an essential SG component. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1, and suggest that SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions.
VIRAL MODULATION OF MHC CLASS II-MEDIATED ANTIGEN PRESENTATION
(2009-06-24T12:57:08Z) Wang, Nan; Blum, Janice Sherry, 1957-; He, Johnny J.; Kaplan, Mark H.; Gallagher, Patricia J.; Harrington, Maureen A.
Vaccinia virus (VV) has been used as a vaccine, yet safety concerns remain due to its viral immunoevasive properties. Among these, VV infection of antigen presentation cells (APC) perturbs MHC class II-mediated antigen (Ag) presentation. The goals of this project include: 1) to define mechanisms by which VV disrupts class II presentation; and 2) to examine whether disruption of the class II pathway by VV alters T cell responses in vitro and in vivo. A significant reduction in the expression of the class II chaperone, invariant chain (Ii), was observed during the late stage of VV infection. Yet surface expression of MHC class II molecules was maintained along with cell viability. To examine whether VV acts solely to disrupt host protein synthesis, B cells were treated with an inhibitor of translation-cycloheximide (CHX). Like VV, CHX negatively regulated Ii protein expression and class II presentation. Ii proteolysis also contributed in part to reduce Ii expression in VV infected and CHX treated APC. Yet only VV infection altered lysosomal protease expression, potentially influencing Ii degradation. Over-expression or ectopic-expression of Ii partially protected cells from VV-induced class II dysfunction. These studies suggest VV destabilizes class II molecules by disrupting Ii expression. To examine the presentation of viral Ags by class II, CD4 T cells from VV-primed mice were used. Viral proteins were presented by class II shortly after APC exposure to low concentrations of VV. The presentation of VV Ags correlated temporally with reductions in exogenous peptide presentation. At higher MOI (≥ 1), class II presentation of VV Ags was reduced. To examine the in vivo effects of VV on Ag presentation, a mouse model of ovalbumin-induced airway hypersensitivity was used. Th2 cytokine production was reduced, while a novel inflammatory cytokine Interleukin-17 (IL-17) production was enhanced in asthmatic VV-infected mice. In health mice, repeated VV infections lead to enhanced CD8 T cell production of Interferon-γ (IFN-γ) and IL-17. Finally, antibodies to a viral protein H3 were generated and shown to preserve class II presentation. Together these studies suggest VV disruption of the class II pathway may blunt T cell responses to VV.
The Haemophilus ducreyi SAP Transporter Contributes to Antimicrobial Peptide Resistance
(2009-09-30T19:56:19Z) Mount, Kristy Lee Beavers; Margaret E. Bauer, PhD
Haemophilus ducreyi is the causative agent of the genital ulcer disease chancroid, which has been shown to facilitate the transmission of HIV. H. ducreyi is likely exposed to multiple sources of antimicrobial peptides in vivo. APs are small, cationic molecules with both bactericidal and immunomodulatory functions. Because H. ducreyi is able to establish and maintain an infection in an environment rich with antimicrobial peptides, we hypothesized that the bacterium was resistant to the bactericidal effects of these peptides. Using a 96-well AP bactericidal assay, we examined H. ducreyi susceptibility to eight human APs likely to be encountered at the site of infection, including the α-defensins human neutrophil peptide-1, human neutrophil peptide-2, human neutrophil peptide-3, and human defensin 5, the β-defensins human β defensin-2, human beta defensin-3, and human beta defensin-4, and the human cathelicidin, LL-37. H. ducreyi survival was compared to the survival of Escherichia coli ML35, a strain known to be susceptible to several antimicrobial peptides. H. ducreyi was significantly more resistant than E. coli ML35 to the bactericidal effects of all peptides tested. Furthermore, we found that representative class I and class II strains of H. ducreyi were each resistant to APs of each functional category, indicating that resistance to antimicrobial peptides could represent a conserved method of pathogenesis for H. ducreyi as a species. The H. ducreyi genome contains a homolog for the Sap influx transporter. To study the role of the H. ducreyi Sap transporter in AP resistance, we generated an isogenic sapA mutant and used the 96-well AP bactericidal assay to compare the AP susceptibility profiles of wild-type H. ducreyi, the sapA mutant and the sapA trans-complement to α-defensins, β-defensins, and LL-37. We observed a 25% decrease in the survival of the sapA mutant when it was exposed to LL-37. These findings suggest that the H. ducreyi Sap transporter plays a role in H. ducreyi resistance to LL-37, but it is likely that other AP resistance mechanisms co-exist within the bacterium.

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