Microbiology and Immunology

Conventional Dendritic cells (cDCs), a specialized group of immunological sentinels with tree-like or dendritic shapes, are critical for recognition of danger signals, presentation of antigens and control of a spectrum of innate and adaptive immune responses. Type I interferons (IFNs), as important danger signals, activate cDCs through the canonical type I IFN receptor signaling. Type I IFNs are the first line of host defense against viral infection by up-regulating IFN-stimulated genes (ISGs). However, there are circumstances in which the silencing of excessive type I IFNs could be beneficial to the host, such as IFN-dependent autoimmune diseases, gene therapy that uses viral vectors and transplantation. The role of type I IFNs in DC development, activation and antigen presentation function remains to be completely investigated. In this dissertation, we studied the regulation of Type I IFNs in murine DCs, both cDCs and plasmacytoid DCs (pDCs), and specifically we investigated the role of two molecules, Signal Transducer and Activator of Transcription 2 (STAT2) and Three prime Repair EXonuclease 1 (Trex1), in DC biology. Our research furthers our understanding of DC development, activation and function, and provides important data for the therapeutic application of modified DCs to induce immunological tolerance in gene therapy, IFN-dependent autoimmune diseases and transplantation. STAT2 is a nuclear transcription factor downstream of type I IFN receptor-mediated signaling, the role of which has been mostly explored in antiviral responses mediated by type I IFNs. However, the involvement of STAT2 in cDC activation and function such as cross-presentation remains hitherto unclear. We report that STAT2 is essential for murine cDC activation upon TLR3, -4, -7 and -9 stimulation. In the absence of STAT2, cDCs displayed impaired up-regulation of type I IFN response (costimulatory molecules and type I IFN-stimulated genes), and reduced inflammatory cytokine production when stimulated with TLR ligands. STAT2 was required in all of the DC responses to exogenous IFNα, suggesting that the canonical STAT1-STAT2 heterodimers are the major signaling transducers downstream of type I IFNs in DCs. Of interest, LPS-induced TNFα and IL6 production were reduced in STAT2-/- DCs but not in IFNAR1-/- DCs, suggesting a novel STAT2-dependent pathway mediated by LPS, bypassing type I IFN-receptor signaling. STAT2-deficient cDCs showed impaired cross-presentation leading to decreased CD8+ T cell response both in vitro and CTL killing in vivo, indicating that STAT2 is essential for TLR-induced cross-presentation. These results demonstrate that STAT2 is critical in the regulation of TLR-induced DC activation and cross-presentation, suggesting an essential role for STAT2 in anti-viral and anti-tumor immune responses. We also propose a novel regulatory function of STAT2 in the LPS response independent of type I IFN receptor signaling. Trex1 mutations are associated with a spectrum of type I IFN-dependent…

Microbiology and Immunology

The Adeno-Associated Virus(AAV) is a small, single stranded DNA virus that has been developed as a gene transfer vector. Early clinical trials using recombinant AAV vectors (rAAV) have identified the following concerns that need to be addressed in order to increase efficiency of these vectors. It has since been determined that AAV vector efficiency decreases due to the following mechanisms: ineffective endocytosis, endosomal degradation, inefficient trafficking, and the need to convert from a single-stranded AAV genome to a transcriptional active double-stranded form. The purpose of this study is to elucidate mechanisms that help stabilize the AAV genome in order to make it a more efficient vector for gene therapy. Previously, there have been studies using fluorescent labeling to track the movement of AAV into the nucleus. An integral part of AAV genomic stability may be the obstacles it encounters in the cytoplasm prior to entering the nucleus. Previous studies on improving AAV transduction have focused primarily on the nucleus. The study will hopefully shed light on the hurdles AAV encounters as it moves through the cytoplasm. Thus, this project has designed and utilized a new system for specifically tracking the status of AAV genomes in the cytoplasm as well as in the nucleus. This project utilizes a novel dual luciferase reporter system to track the movement of AAV particles from the cytoplasm into the nucleus to elucidate mechanisms that could contribute to the stabilization of the AAV genome. The novel dual reporter system is comprised of a single-stranded vector containing two different types of secreted luciferases: Cypridina Luciferase and Gaussia Luciferase. Cypridina Luciferase is placed under the control of a nuclear promoter and therefore it is expressed only in the nucleus. The second, Gaussia Luciferase, is under the control of a cytoplasmic promoter that will only be expressed in the cytoplasm upon the presence of T7 RNA polymerase. Using this dual reporter luciferase system along with RT qPCR quantification in a Hek293 cell line expressing the T7 RNA polymerase, demonstrated that genomes are present in the cytoplasm at 18 and 24 hours post infection. The second part of this study is using the dual reporter system to understand the trafficking patterns of rAAV and looking at ways to enhance transduction. One method could be administering rAAV vectors in conjunction with a drug; this approach may help overcome some of these cellular barriers encountered during infection as well as help stabilize the genome. Cidofovir (CDV) is a monophosphate nucleotide analogue that competitively inhibits the incorporation of deoxycytidine triphosphate into viral DNA via viral DNA polymerase. In vitro, CDV actively inhibits a number of DNA viruses including herpes viruses, adenovirus, polyomavirus, papillomavirus, and poxviruses. Cidofovir has already been approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus (CMV) retinitis in patients with acquired…

Microbiology and Immunology

Gene therapy for Hemophilia A (HA) using the recombinant Adeno-associated virus (rAAV) offers an alternative to classic treatment, which consists of FVIII protein infusions. However, due to limitations associated with rAAV and the FVIII protein itself, the end result is a transgene expression below therapeutic limits. One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications. Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances coagulation activity, and that it would be possible to improve FVIII activity through modifications of the light chain. Through the process of engineering, evaluation, and negative selection of kLC, a final construct was engineered. The hLC-K12 is a human Light Chain (hLC) construct containing 12 amino acid changes that work together to enhance coagulation activity. A comparison of the FVIII clotting activity to the amount of protein produced determined that hLC-K12 produced a 3.28 fold increase in specific activity over hLC in vitro. Similar in vitro results were observed when hLC-k12 was tested with the X5 heavy chain (X5HC), a heavy chain that has been genetically modified to enhance production. CD4KO/HA mice were injected with a rAAV vector carrying the hLC-K12 gene in conjugation with a rAAV vector carrying the X5HC gene. Replacing the hLC vector with the hLC-K12 vector produced an average 7.43 fold increase in FVIII clotting activity. An ELISA assay revealed no significant difference between productions of the heavy or light chains at any time point. By comparing the clotting activity to the amount of protein produced, it was determined that the increase in coagulation activity was due to an increase in specific activity. In fact, replacing the hLC vector with the hLC-K12 vector resulted in an average 5.8 fold increase in FVIII specific activity. The K12 modifications were evaluated using a single chain FVIII conformation. In vitro, the addition of the K12 mutations to the human heavy chain, hHCK12BDD, resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. There was however, a 3.3 fold increase in specific activity of the protein. Adding the K12 mutations to the X5 heavy chain, X5K12BDD, in vitro, resulted in a 2.7 fold increase in clotting activity and a 1.42 fold increase in specific activity of the protein. Single chain rAAV vectors were packaged and delivered to CD4KO/HA mice. Compared to mice injected with hFVIIIBDD, the hHCK12BDD produced an average 4.6 fold increase in clotting activity. An ELISA revealed no significant difference in production between these two groups. However, mice injected with hHCK12BDD produced FVIII with an average of 4.13 fold increase in specific activity. Similarly, when compared to mice injected with X5FVIIIBDD, the X5K12BDD produced an average 2.14 fold increase in clotting activity. An ELISA assay demonstrated no significant increase in protein production between these two…

Microbiology and Immunology

The human immunodeficiency virus type-1 (HIV-1) is the major etiological agent of acquired immunodeficiency syndrome (AIDS), the cause of over 30 million deaths worldwide. Highly active antiretroviral therapy (HAART) has demonstrated great efficacy at suppressing viral load and is therefore the standard therapeutic treatment for HIV-1 infection. Noncompliance due to severe HAART-associated side effects significantly undermines therapeutic efficacy. Dronabinol, the synthetic form of the cannabinoid THC found in marijuana, is FDA-approved for countering some of these side effects. Studies have reported that cannabinoids restrict HIV-1 replication, although no mechanism has yet been proposed. Thus the purpose of this study was to characterize the effects of cannabinoids on HIV-1 infection and to determine the molecular basis of cannabinoid-induced viral suppression. By transcriptomic sequencing of T cells treated with cannabinoids, we have found that the expression of BAG6, a protein uncharacterized within the context of HIV-1 infection, was downregulated. To identify the role of this protein during infection, we knocked down BAG6 and were able to recapitulate the protective effects of cannabinoids by observing reduced severity of viral challenge. Moreover, we have also identified BAG6 to be a binding partner of two HIV-1 viral accessory proteins, Vif and Vpr. Importantly, we have discovered that Vpr mediates targeted degradation of BAG6 by leveraging the host proteasome during the early stages of the viral lifecycle, revealing a hitherto unknown function of this poorly-understood viral protein. We thus establish modulation of BAG6 expression as a novel mediator of the effects of cannaninoids on HIV-1 infection.

Temple University – Theses

Pharmacology

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease (CVD). We previously demonstrated that homocysteine (Hcy) suppresses endothelial cell (EC) proliferation, migration, and post-injury EC repair, but the molecular mechanism underlying Hcy-induced EC injury is unclear. In this study, we identified a novel gene, Carom, which mediates Hcy-induced suppression of EC migration and angiogenesis. We identified FCH and double SH3 domains 2 (FCHSD2), a novel gene, as an Hcy-responsive gene through Differential Display in Hcy (50µM)-treated human umbilical vein endothelial cells (HUVEC). FCHSD2 was initially named as Carom, based on the identification of this molecule as an interacting protein of calcium/calmodulin-dependent serine protein kinase (CASK) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1). In this thesis, we describe this gene as Carom. Carom belongs to the Fes/CIP4 homology and Bin/amphiphysin/Rvs (F-BAR) protein family, which is a group of multivalent adaptors linking plasma membrane and cytoskeleton, involved in endocytosis and cell migration. However, Carom's function is poorly characterized. Based on the findings that CASK and MAGI1 inhibit cell migration and growth, and the role of F-BAR proteins in cell migration, we hypothesize that Hcy up-regulates Carom to inhibit EC growth and/or migration, finally leading to CVD. We confirmed the significant induction of Carom mRNA expression in Hcy-treated HUVECs or human aortic endothelial cells (HAEC) by Northern blot and Real-time PCR. In addition, we found that Carom protein expressions were significantly increased both in Hcy-treated HAECs and lung ECs isolated from HHcy mice by Western blot using our homemade rabbit antibody against Carom. These data indicate that Hcy increases endothelial expression of Carom both in vitro and in vivo. Furthermore, in order to characterize Carom function in EC, we generated recombinant adenovirus Adv-Carom to transduce Carom for gain-of-function study and Adv-Carom-shRNA to express Carom shRNA for loss-of-function study. We found that neither adenovirus-transduced Carom expression nor adenoviral Carom shRNA had any impact on HUVEC proliferation by using [3H]-thymidine incorporation. Interestingly, we demonstrated that Adv-Carom inhibited HAEC migration, while Hcy-induced HEAC migration inhibition could be rescued by Adv-Carom-shRNA. These data suggest that Carom may inhibit angiogenesis via a cell proliferation-independent mechanism. Furthermore, we found that Hcy significantly increased the intracellular level of S-adenosyl homocysteine (SAH) but not S-adenosyl methionine (SAM), and decreased the SAM/SAH ratio, an indicator of cellular methylation, in HAECs, by using High-performance liquid chromatography/electrospray tandem mass spectrometry (HPLC-MS) to measure SAH and SAM levels. Meanwhile, Carom protein expression was significantly induced by azacytidine (AZC), a DNA methyltransferse inhibitor, in a dose-dependent manner in HAECs. Based on these…