Burkholderia pseudomallei (Bp) is a major public health concern in the endemic regions of southeast Asia and northern Australia, yet the organism has a worldwide distribution and cases are likely under-reported. In northeast Thailand the mortality rate associated with Bp infection is over 40%. The inherent resistance of Bp to multiple antibiotics impairs treatment, and relapse is seen in more than 25% of survivors. Beyond its public health significance, Bp is considered a potential biological warfare agent by the U.S. DHHS and was recently listed as a Tier 1 select agent. Despite enhanced research and vaccine efforts, traditional vaccine strategies employing attenuated bacterial strains, recombinant proteins, or purified polysaccharides have failed to elicit complete protection against aerosol challenge with Bp. We have previously shown that immunization with outer membrane vesicles (OMVs) derived from Bp can protect mice from lethal melioidosis. In this work we characterize the interactions of OMVs with antigen presenting cells in order to elucidate innate immune responses to the OMV vaccine. Vaccine-mediated antibody responses and protective efficacy were characterized in BALB/c mice. We also tested the safety and immunogenicity of the OMV vaccine in non-human primates (NHP). We show that Bp OMVs interact with dendritic cells and macrophages and are internalized by these antigen presenting cells (APCs).Internalization is dependent on actin polymerization and cholesterol present in APC membranes. OMVs also upregulate MHC class I and II on APCs, as well as promote the production of pro-inflammatory cytokines in a TLR2/4 dependent manner. Immunization of mice with Bp OMVs by the s.c. and i.m. routes induced the production of OMV-specific IgM and IgG and significantly protected mice against aerosol challenge. Addition of alum and MPL did not significantly change the antibody profiles of immunized mice and did not significantly enhance vaccine mediated protection. OMVs were well tolerated in a large animal NHP model. There were no adverse clinical reactions, and NHPs mounted significantly increased levels of OMV-specific IgG and OMV specific CD4+ T cell responses. These results suggest that Bp OMVs can stimulate innate and adaptive immune responses and may represent a safe and efficacious vaccine against melioidosis / acase@tulane.edu

Rupture of atherosclerotic plaque can cause acute life-threatening events such as myocardial infarction and ischemic stroke; therefore, there is much interest in developing therapies aimed at increasing plaque stability. More stable lesions are characterized as having high collagen content and containing a large number of vascular smooth muscle cells (SMCs) of contractile/differentiated phenotype. In our previous studies using an apolipoprotein E-deficient (Apoe-/-) mouse model of atherosclerosis, we found that insulin-like growth factor-1 (IGF-1)-infusion not only reduced total plaque burden, but also increased collagen expression and the number of alpha-smooth muscle actin (αSMA)-positive cells in plaque. In this study, we identify cellular mechanisms responsible for these observations. We found that in human aortic smooth muscle cells (HASMCs) grown in culture, IGF-1 post-transcriptionally upregulated expression of the procollagen type I alpha-1 subunit (pro-α1(I)) as well as contractile proteins, αSMA and smooth muscle 22-alpha (SM22α), via a PI3K-dependent but Erk1/2- and mTOR-independent signaling mechanism. Furthermore, experiments using an inhibitor of collagen synthesis or a blocking antibody against the alpha2beta1-integrin (α2β1) suggested that interaction with collagen type I promotes HASMC contractile phenotype. To elucidate mechanisms underlying IGF-1 upregulation of collagen synthesis we investigated the effect of IGF-1 on the mRNA-binding protein, la ribonucleoprotein domain family member 6 (LARP6), which had been shown to bind a conserved stem-loop secondary motif in the 5’UTR of COL1a1 and COL1a2 mRNA. IGF-1 rapidly increased LARP6 expression in HASMCs leading to increased COL1a1 and COL1a2 mRNA bound LARP6 and increased synthesis of collagen type I. Mutation of the 5’stem-loop of Col1a1 mRNA (that inhibited binding by LARP6) or overexpression of a 5’stem-loop RNA molecular decoy (that sequesters LARP6) both prevented the ability of IGF-1 to increase pro-α1(I) synthesis as well as mature α1(I) expression in cultured medium. Furthermore, IGF-1-infusion in Apoe-/- mice increased LARP6 and pro-α1(I) expression in aortic lysates, and SMC-specific IGF-1-overexpression in transgenic mice robustly increased collagen fibrillogenesis in atherosclerotic plaque. In conclusion, this work identifies LARP6 as a critical mediator by which IGF-1 augments synthesis of collagen type I in vascular smooth muscle, and uncovers key mechanisms whereby IGF-1 promotes atherosclerotic plaque stability. / acase@tulane.edu

Lyme disease is the most commonly reported vector-borne disease in the United States. The causative agent of Lyme disease, can alter gene expression to enable survival in a diverse set of conditions, including the tick midgut and the mammalian host. External environmental changes can trigger gene expression in B. burgdorferi, and the data demonstrate that B. burgdorferi can similarly alter gene expression as a stress-response when it is treated with the antibiotic doxycycine. After treatment with the minimum bactericidal concentration (MBC) of doxycycline, a subpopulation can alter its phenotype to survive antibiotic treatment, and to host adapt and successfully infect a mammalian host. Furthermore, our data demonstrate that if a population is treated with the MBC of doxycycline, a subpopulation may alter its phenotype to adopt a state of dormancy until the removal of the antibiotic, whereupon the subpopulation can regrow. We demonstrate that the chance of regrowth occurring increases as a population reaches stationary phase, and present a mathematical model for predicting the probability of a persister subpopulation within a larger population, and ascertain the quantity of a persister subpopulation. To determine which genes are expressed as stress-response genes, RNA Sequencing analysis, or RNASeq, was performed on treated, untreated, and treated and regrown B. burgdorferi samples. The results suggest several genes were significantly different in the treated group, compared to the untreated group, and in the untreated and regrown group compared to the untreated group, including a 50S ribosomal stress-response protein, coded from BB_0786. The appendices discuss the theory and methods that were used in RNA Sequencing (RNASeq) analysis, and provide an overview of the database that was created for the B. burgdorferi transcriptome. Additional studies may demonstrate further how persister subpopulations form, and which genes can trigger a persister state in B. burgdorferi. / acase@tulane.edu

Profound vascular leakage in conjunction with elevated viremia is the hallmark of Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS). Antibody (Ab)-dependent enhancement (ADE), in which pre-existing, cross-reactive Abs enhance virus infectivity, is thought to be responsible for increased viremia, while loss of endothelial cell (EC) barrier integrity is the precursor to plasma leakage. However, the relationship between viremia and vascular leak has not been established. The objective of this dissertation project was to determine the involvement of antibodies in the pathogenesis of vascular leak syndrome associated with DHF/DSS by establishing a relationship between Ab-mediated increase in viremia and changes in vascular permeability, the hallmark of DHF/DSS. Our approach focused on characterization of human monoclonal antibodies (hMAbs) from a previously dengue virus (DENV)-infected patient for their ability to both neutralize and enhance infection and increase vascular permeability in vitro. Our results revealed that the human antibody response to DENV E protein elicited by natural infection is predominantly comprised of broadly cross-reactive antibodies targeting domain II epitopes. Using a multiplex cytokine immunoassay, qRT-PCR, and plaque assay, we demonstrated an association between viral load and cytokine production in DENV-infected FcγR-bearing K562 cells, and determined that DENV infection of K562 cells in the presence of hMAb resulted in a modulated inflammatory cytokine response with an overall pro-inflammatory profile. Using human microvascular ECs (HMEC-1), we further demonstrated an association between viral load, cytokine production, and the onset of permeability changes via an indirect mechanism in which inflammatory mediators released by DENV-infected K562 cells altered HMEC-1 barrier function and observed a synergistic effect between active DENV infection and release of inflammatory mediators by both K562 and HMEC-1 that increased permeability. Collectively, our results support the multifactorial nature of the pathogenesis underlying vascular leak, involving a complex interaction between ECs and FcγR-bearing cells, and a synergistic relationship between enhanced viremia and inflammatory mediators leading to increased permeability. Our use of hMAbs provided a novel approach to understanding how Abs impact the vasculature during DENV infection and enable identification of Ab characteristics that may trigger vascular leak, a crucial concern for DENV vaccine design. / acase@tulane.edu

Influenza virus is an enveloped virus with a negative sense single strand RNA. The viral surface is characterized by two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA)(Chen et al., 2007; Samuel, 2010). The HA subunit is responsible for the attachment of the virus to the host cell by binding to the sialic acid receptors. Influenza virus infection, occurring in the endosome of the host cell is a fusion dependent process (Daniels et al., 1983). Low pH inside the endosome facilitates the fusion process by triggering a major conformational change of HA. This conformational change exposes the fusion initiation region of the protein subsequently releasing a hydrophobic fusion peptide (which is otherwise buried inside the protein core). This hydrophobic peptide slips into the host cell membrane resulting in HA transiently being a part of the viral and cell membrane. The HA pulls the two membranes together, thus completing the fusion process and forming a clear passage for the release of the viral genetic material into the cytoplasm (Stevens et al., 2004; Ramalho-Santos and Pedroso De Lima, 1999; Carr and Kim, 1993). A 16 amino acid peptide sequence (Flufirvitide-3) derived from the fusion initiation region of the HA protein has shown effective inhibition of influenza virus infection. Plaque inhibition assays and animal studies show high efficacy of the peptide against the virus. However, the mechanism of action of this peptide is still unclear. We have extensively studied the ability of FF-3 to interact with and affect purified HA, pure lipid bilayers and whole viruses. Taken together, the results suggest a novel mechanism of action. / acase@tulane.edu

Our overall research goal is focused on the development of a malaria transmission-blocking vaccine (TBV). The antigenic target, Pfs48/45 protein, is expressed on Plasmodium gametocytes, which are stages responsible for establishing parasite infection in the mosquito vector. The epitopes recognized by functional antibodies targeting Pfs48/45 are disulfide-bond (S-S) constrained, conformational epitopes. As Pfs48/45 protein has not been crystallized, precise location of the S-S bonds and the topology of epitopes are unknown. It has been shown previously that the ability to reduce S-S in antigens can greatly influence the epitopes presented by antigen-presenting cells (APCs) and thus influence induction of effective immune responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is an enzyme expressed in APCs that mediates reduction of S-S bonds contained within antigens, for subsequent display of peptides on MHC molecules. Using non-reduced (NR) and reduced/alkylated (RA) Pfs48/45 antigens, we sought to investigate the role of GILT on induction of protective immunity. We hypothesized that the ability to reduce S-S bonds in Pfs48/45 will impact the generation of T cell epitopes, and thus influence helper T cell responses required for B cell stimulation and production of protective antibody. We conducted immunogenicity studies in wild type (WT) and GILT-/- (KO) mice using the two structural forms of Pfs48/45 and analyzed immune responses to full length Pfs48/45, five overlapping fragments and 39 overlapping peptides. Results indicated that generation of Pfs48/45 antibodies is not significantly impacted by the availability of GILT, however there was uniquely Th2-biased T and B cell responses in the KO mice, and a contrasting Th1 bias in WT mice. Results also revealed possible effects of GILT on induction of long-lived plasma cells and memory B cells responsible for resting and antigen-recall responses to Pfs48/45. Data presented also shows reduced immunogenicity of the RA Pfs48/45 antigen and immune responses differed in magnitude and specificity between male and female animals. Overall, we aimed to gain a better understanding of the immunological mechanisms critical to generate protective and lasting immunity against Pfs48/45. These and future studies will contribute significantly to our understanding of antigenic features of Pfs48/45 important for use as a TBV. / acase@tulane.edu

As a novel therapy for neurodegenerative diseases, transplantation of multipotent mesenchymal stromal cells (MSCs) requires extensive optimization in animal models before being implemented in clinical trials. It is a goal of our laboratory to understand the mechanism of action of these cells and to improve their therapeutic efficacy. To address these goals, this study aims to optimize the cell dosage, cell type, administration route and timing, and/or donor age for stem cell therapy in two mouse models of demyelinating diseases: globoid cell leukodystrophy (GLD; Krabbe’s disease) and experimental autoimmune encephalomyelitis (EAE). GLD is a neurodegenerative lysosomal storage disease caused by the deficiency of galactocerebrosidase (GALC). Accumulation of toxic byproducts in myelin producing oligodendrocytes leads to the demyelination of neurons and increase in brain inflammation. The twitcher mouse model of GLD was used to test the therapeutic effects of MSCs after injection through intracerebroventricular (ICV) or intraperitoneal (IP) routes. Weekly MSC IP injections and single IP GALC-transduced MSC injections were performed. Other twitcher mouse cohorts received temporal vein (TV) or intracerebral (IC) injections of GALC-containing adeno-associated virus serotype 9 (AAV9-GALC) with or without IP MSC injections. All GLD affected mice treated with peripheral MSC and/or vector therapy had extended lifespans with improved motor function. The ameliorating effects of MSCs were related to their potent anti-apoptotic and anti-inflammatory effects on the peripheral and central nervous systems. These results indicate a promising future for peripheral administration of MSCs and vectors as non-invasive, adjunct therapies for patients affected with GLD. A similar study was performed using the EAE mouse model of multiple sclerosis (MS), which is a demyelinating disease due to an autoimmune reaction to myelin. The results demonstrated that biological age of the donor reduces the ability of MSCs to alleviate symptoms and improve pathology in the EAE mouse model. Upon transplantation, the young, but not old, MSCs provided neuroprotective effects through immunomodulation and remyelination in the central nervous system (CNS). The age-related therapeutic differences corroborate recent findings that biologic aging occurs in stem cells and highlight the potential need for allogeneic transplantation of MSCs in older MS patients. / acase@tulane.edu

Human Immunodeficiency Virus -1 infects CD4+ cells, and the subsequent loss of these cells cause Acquired Immune Deficiency Syndrome. Highly active antiretroviral therapy (HAART) is crucial to control viremia in the clinical management of AIDS/HIV infection; however, drug regimens are complex, expensive, and require life-long intervention with potential side effects. Current conventional anti-HIV drugs target different phases of the HIV life cycle and can be categorized as nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, entry inhibitors (co-receptor antagonists and fusion inhibitors), and integrase inhibitors(II). Enfuvirtide (Fuzeon, or T-20) is the first fusion inhibitor approved by the FDA and has substantial side effects and drug delivery issues with most patients developing some local injection site reaction. The subcutaneous application of enfuvirtide and its short half-life, which requires twice daily administration, has disadvantages in patients who are already burdened by complex oral therapy. To overcome these drug issues, we propose an alternative method to administer the HIV-1 peptide fusion inhibitor C46. Stem cells can be a vehicle for delivering genes to specific tissues in the body and their therapeutic delivery systems are extensively used in cancer research. For many years, restoration of blood and immune system function has been used as a component in the care of cancer patients who have been treated with chemotherapeutic agents. Mesenchymal Stem Cells (MSCs) have been demonstrated as a delivery vehicle for gene therapy applications based on their ability to engraft and home to inflamed tissues. MSCs are multi-potent and have immunological function in several human diseases. To investigate MSCs immune suppressive ability in HIV infection system, we will evaluate the crosstalk between MSCs and HIV infection immune-modulatory network. / acase@tulane.edu

Triple-negative breast cancer (TNBC) accounts for 15% of all diagnosed breast cancers nationally and affects African-American women 3 times more likely than any other ethnic group. Locally, African-American women in the New Orleans area see higher incidence of TNBC cases versus African-American women from the rest of the state of Louisiana, which represents an area of heightened public health interest for the metropolitan area. TNBC is a highly metastatic disease, and targeted therapies such as tamoxifen and herceptin are ineffective due to the lack of estrogen receptor (ER) and HER2/neu target expression in TNBC tumors. Chemotherapy remains the only effective drug therapy in TNBC cases. Evaluating new classes of drugs for clinical use against TNBC as well as furthering our understanding of underlying regulatory mechanisms in TNBC is a priority. Pan-deacetylase inhibitors (DACi), like panobinostat, have shown promise in clinical trials as therapies in other cancers. Pre-clinical data of panobinostat treatment in TNBC cell lines published by this lab has been positive so far, exhibiting a reduction in TNBC metastatic potential. DACi can alter multiple signaling pathways and are known to restore dysregulated microRNA (miRNA) expression patterns (miRnome) in cancers. MiRNA are a relatively new class of non-protein coding regulatory biomolecules that exhibit a variety of cancer-related properties, many still unknown in TNBC. Pan-DACi treatment combined with miRnome analysis in TNBC cell lines can be used to identify previously unknown miRNA cancer-related properties in TNBC. The specific aim of this project consists of using DACi treatment on TNBC cell lines in conjunction with miRnome analysis to identify previously undescribed anti-cancer miRNAs and elucidate their cancer-related properties in TNBC while uncovering affected cancer pathways, detecting miRNA targets, and revealing affected downstream components. Initial miRNA expression analysis of MDA-MB-231 TNBC cells treated with panobinostat or trichostatin A versus controls produced a list of potential anti-cancer miRNA candidates for further study. Among them, investigations into miR-203 and miR-335 produced unclear results as these were theorized to have anti-metastatic properties in TNBC yet enhanced cancer properties in our models and assays. Overexpression of mir-215 (a tumor suppressor in other cancers) unexpectedly enhanced tumor growth five fold in SCID mice xenografted with lentivirally-transduced MDA-MB-231 breast cancer cells stably overexpressing miR-215 (231/215+). Further qPCR analysis of 231/215+ cells uncovered upregulation of the breast cancer-associated lncRNA, HOTAIR; the breast cancer-associated miRNA, miR-196a; as well as the entire HOXC cluster in which they reside. This represents a previously unidentified regulatory mechanism of the HOXC cluster in humans. Additionally, miR-200b overexpression in MDA-MB-231 cells induced a change in cell morphology to an epithelial-like phenotype, reduced migration by 50%, and re-expressed the epithelial marker CDH1. This demonstrates a partial reversal of epithelial-mesenchymal transition (EMT), which indicates a reduction in metastatic potential by miR-200b overexpression in MDA-MB-231 cells. Additionally, these cells exhibited increased estrogen receptor alpha and related signaling pathways while also being susceptible to reduced proliferation with the anti-estrogen drug fulvestrant at high doses. Using pan-DACi treatment of TNBC cells to analyze changes in the miRnome for unknown cancer-related miRNA candidates suitable for further investigation in TNBC, we identified miR-215 overexpression in MDA-MB-231 cells as an oncogenic event that enhances tumor growth, cell proliferation, and HOXC cluster transcription while miR-200b is an anti-metastatic miRNA that partially reverses EMT and reduces fulvestrant resistance through re-expression of estrogen receptor signaling. / acase@tulane.edu

acase@tulane.edu