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

Despite the development of a vaccine and several antibiotics, tuberculosis continues to be one of the leading causes in mortality in the world. The pathogenesis of the main causative agent, Mycobacterium tuberculosis, has puzzled many researchers for over a century. Research on the origin of M. tuberculosis can provide new knowledge on how the organism has evolved into the dangerous pathogen it is today. This thesis reviews recent literature on how the evolution of tuberculosis has contributed to the genetic diversity and positive control of select genes in the tuberculosis genome and how this can impact future development of therapeutic agents. / 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

Mycobacterium tuberculosis (Mtb) remains a pathogen of significant importance with respect to global health. Although approximately one third of the world is infected with TB, only 5-10% develop clinical manifestations of active TB within 2 years post exposure. Infection with Mtb can cause active tuberculosis (ATB), inactive latent infection (LTBI) and be reactivated. The immune response is contained within the formation of a collection of immune cells, a granuloma, in the infected individual’s lungs, which is seen in all disease states. The granuloma functions both as an immune response to contain the bacterium from surrounding lung parenchyma as well as a site for the bacterium to remain in the individual; therefore, providing an environment in which the bacterium maintains the ability to reactivate. We currently lack a complete understanding of the physiology and the metabolic state of Mtb in this granulomatous environment during different states of infection. Leveraging a novel technique known as mesodisection, we microdissect TB granulomas from various infective stages as well as from different sections of the granuloma from non-human primate (NHP) derived formalin fixed paraffin embedded (FFPE) lung tissue. From these extracted tissue sections, RNA is extracted, amplified and subsequent microarray and nCounter analysis is performed; consequently, allowing us to uncover the Mtb specific transcriptomic profiles. First, our findings reveal statistically significant Mtb genes induced in ATB and LTBI in various granuloma types. Of the genes induced, many belong to the following categories: PE/ PPE, sigma factors, toxin-antitoxin complexes and the DosR regulon. In addition, our findings reveal a core group of genes commonly identified in both ATB and LTBI induced in ATB and LTBI in various granuloma types. Of the genes induced, many belong to the following categories: PE/ PPE, sigma factors, toxin-antitoxin complexes and the DosR regulon. In addition, our findings reveal a core group of genes commonly identified in both ATB and LTBI in all lesion types. Further, we propose that these findings improve our understanding of the physiology of the pathogen as well as its virulence which in turn can be used for the development of improved therapeutics, diagnostics and vaccines. / acase@tulane.edu

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

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic Mtb:ΔRv2745c mutant is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb, implicating a role for ClgR in the management of intraphagosomal redox stress. Our data indicates that ClgR plays a role in multiple regulatory networks in response to different stress conditions. Thus, redox stress leads to dysregulation of the σH/σE regulon in Mtb:ΔRv2745c. Induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions. Disruption of genes involved in sulfate assimilation also occurred in the knock out, implicating clgR as a possible regulator of downstream signaling cascades that facilitate Mtb survival. On the other hand, the expression of clgR during hypoxia is known to result in Clp protease induction. As such, the isogenic mutant has a significantly different growth profile upon hypoxia and reaeration. Transcriptomics reveal disruption of the dosR regulon, σH/σE regulon, and mycolic acid synthesis genes. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in vivo. Our in vivo findings in a low dose aerosolized model reveal deficiencies of the isogenic mutant when establishing an infection, leading to skewed immune responses throughout the course of infection. Thus, clgR plays a critical role in both establishing an infection that influence the immunogenic outcome. Additional studies investigating the role of clgR in a nonhuman primate model will further elucidate the contributions of clgR to the pathogenesis of Mtb in an animal model that is more representative of human TB disease. / acase@tulane.edu

No description available.

Salmonella spp. pose significant health risks to humans and animals. S. Typhi, the causative agent of typhoid fever, is responsible for 21 million new cases of enteric fever each year and an estimated 200,000 deaths worldwide. Approximately 5-8% of infected individuals will become lifelong bacterial carriers. It is currently unknown why bacteria persist within the host in the face of robust anti-bacterial immune responses. We hypothesize a stalemate between bacterial persistence and the host immune response is determined by anatomical location, and that this dictates CD4 T cell function and infection outcome. Using a mouse model of persistent S. Typhimurium infection, we show lymphoid Salmonella-specific Th1 cells are potent producers of IFN-γ and protect mice from challenge when adoptively transferred into naïve animals. Conversely, Salmonella-specific CD4 T cells from chronically infected livers exhibit a Tr1-like phenotype, produce large amounts of IL- 10, and increase mouse susceptibility to bacterial challenge. These differences in CD4 T cell phenotypes may inhibit macrophage ability to control intracellular bacterial replication; liver Tr1-like cells fail to activate bacterial killing, possibly through the production of IL-10, which reduces the expression of iNOS and nitric oxide production by macrophages. Additionally, we demonstrate liver macrophages from chronically infected mice exhibit an immunosuppressive, M2-like phenotype and are not classically primed to kill intracellular bacteria, unlike macrophages from lymphoid sites. Furthermore, we show that the liver may be responsible for inducing these Tr1-like cells, as liver macrophages are capable of activating and expanding Salmonella-specific CD4 T cells during infection. Thus, we believe the immunosuppressive environment in the liver affords a permissive niche for Salmonella persistence in vivo. However, we believe vaccination can protect mice from Salmonella infection, if potent Th1 cells are induced, as seen in lymphoid tissue during infection. Therefore, we developed a CD4 T cell peptide vaccine against a known Salmonella secreted epitope. We show vaccinated mice generate potent Th1 responses against Salmonella and mice are significantly protected from challenge. These studies provide new insight into the immunological mechanisms regulating bacterial persistence, as well as the role tissue microenvironments play in modulating pathogen-specific immune responses. / acase@tulane.edu

Indiana University-Purdue University Indianapolis (IUPUI) / Geographic technologies can be used to visualize and analyze data patterns that may go unnoticed from other approaches. The purpose of this project was to provide examples of how GIS and cartographic methods are being used to help facilitate communication and inform management processes for a complex statewide medical education system administered by the Indiana University School of Medicine, the largest medical school in the United States. The IU School of Medicine has nine regional campuses located around the state in addition to numerous partnering hospitals where medical students are trained. We illustrate geographic examples of various stages of medical student education from admissions, through campus assignments and clinical rotations, to residency training. These geographic processes are being used to inform reaccreditation processes as well as assisting administration with recruitment/retention strategies, statewide planning, and analysis in a complex medical education system.