Connie Slocum is a fifth year Molecular Medicine student in Dr. Caroline Genco’s laboratory in the Department of Medicine, Section of Infectious Diseases. Currently, she is working with P. gingivalis lipid A mutant strains that differentially activate TLR4 in order to understand the role of TLR4 in chronic inflammation and atherosclerosis mediated by a bacterial infection. Connie determined that P. gingivalis evasion of TLR4 signaling prevents inflammasome activation thereby attenuating the inflammatory response to this bacterium. She has determined the intracellular survival of wild-type P. gingivalis and the lipid A mutants in BMDM. This work is described in a manuscript “Distinct Lipid A Moieties Contribute to Pathogen-Induced Site-Specific Vascular Inflammation” which is currently under revision at PLOS Pathogens. Utilizing a series of lipid A mutants, this manuscript demonstrated that P. gingivalis expression of antagonistic lipid A dampens induction of inflammatory mediators, increases bacterial survival, and promotes chronic inflammation in an ApoE-/- mouse model of atherosclerosis. Autophagy may provide the mechanistic link for how P. gingivalis survives intracellularly in the macrophage to evade host immune detection. Connie has generated preliminary data demonstrating that P. gingivalis manipulates autophagy. Future studies will include utilizing the P. gingivalis lipid A mutants to determine if the presence of distinct lipid A structures dictates the ability of this pathogen to enter the autophagic pathway to promote its intracellular survival.
Caitlin Leibowitz is a third year student in the laboratory of Dr. Deborah Stearns Kurosawa in the Department of Pathology. Caitlin’s thesis project is focused on defining how the Citrobacter rodentium Stx-2 toxin leads to kidney injury. The Citrobacter rodentium model of Stx2-induced AKI in a bacterial setting was established and Caitlin demonstrated that animals challenged with the Stx2 producing strain of C. rodentium develop kidney injury, with increasing plasma BUN (p<0.005 by day 7), and renal NGAL and KIM1 mRNA (p<0.005 for both markers), with tubular injury visualized by histopathology. She also identified elevated kidney expression of AKI markers NGAL and KIM1 as robust markers of kidney injury. These markers were elevated by day 3 post challenge (p<0.05 for NGAL, n.s. for KIM1), and expression was further increased by euthanasia endpoint (p<0.01 for NGAL, p<0.005 for KIM1). Furthermore, she demonstrated that renal tubular injury was present by day 3-post challenge and continued through euthanasia endpoint. Her studies also revealed leukopenia did not influence plasma BUN or kidney NGAL and KIM1 mRNA levels after Stx2 challenge. When Stx2 was incubated with mouse whole blood ex vivo, toxin binding was not detectable by FACS to any white blood cell subset, and similar results were found when Stx2 was incubated with mouse bone marrow cells. Caitlin is currently working on two manuscripts describing these studies.
Melissa Ghulam-Smith is an MD PHD student in the laboratory of Dr. Manish Sagar in the Department of Medicine, Section of Infectious Diseases. Her project is focused on defining the role of neutralizing and non-neutralizing antibodies in protecting infants exposed to HIV-1 (subtype C) through breastfeeding. Human Immunodeficiency Virus Type I (HIV-1) can be transferred from mother-to-infant in utero, intrapartum, or post-partum through breastfeeding. In the absence of antiretrovirals, breastfeeding introduces a 15-20% risk of transmission. The relative inefficiency of this mode of transmission may be due to the presence of neutralizing and/or non-neutralizing antibodies against specific epitopes on autologous HIV-1 strains that are passed from the mother to infant. Her project proposes to study the ability of antibodies found in infant plasma against replication competent recombinant viruses, which incorporate HIV-1 envelopes of the viruses circulating in the corresponding mother’s breast milk. She has obtained samples from the control arm of the Breastfeeding, Antiretroviral, and Nutrition Study, in which mother-infant pairs were only exposed to antiretrovirals immediately after birth and not during the breastfeeding period eliminating selective pressures on the virus and ensuring transmission occurred through breastfeeding. Neutralization assays will be carried out in the presence of cell-free and cell-associated virus and contribution of antibodies against the gp120 and gp41 viral epitopes will be compared between infant groups. She hypothesizes that infants exposed to HIV-1 though breastfeeding who did not acquire infection may have higher titers of neutralizing and/or non-neutralizing antibodies. Additionally, differences in this neutralizing activity may be driven by differences in target epitopes of antibodies present in the infant plasma. This study will aid in understanding the characteristics and specificities of antibodies that prevent transmission, which may have important implications for future vaccine development efforts. Over the past year her worked has focused on producing virus from breast milk samples acquired from Malawi. She successfully produced replication competent recombinant virus and performed neutralization studies.
Jennifer Smith is a postdoctoral trainee in the laboratory of Dr. Ellen Weinberg in the Department of Medicine, Section of Infectious Diseases. Jennifer’s project is to examine the role of IL-33 in P. gingivalis induced oral bone loss and inflammatory atherosclerosis using a newly developed IL-33-/- mouse model. Because IL-33 has been shown to have a protective role in atherosclerosis, she hypothesizes that IL-33-/- mice will show signs of greater inflammation compared to WT and that P. gingivalis will induce inflammatory bone loss and atherosclerosis development that is even greater in the IL-33-/- mouse. Her aims are to define the role of IL-33 in P. gingivalis -induced chronic inflammation, plaque accumulation, and oral inflammatory bone loss. She has made significant progress toward the aims of her research proposal. She finished all experiments toward a manuscript that is now in preparation titled, “The role of IL-33 in the resolution of cockroach-antigen induced allergic airway inflammation.” Using a novel IL-33-/- mouse treated with cockroach antigen one time per week over the course of three weeks, she measured airway resistance, examined cytokine expression and secretion, looked at whole blood hematology profiles, did a histological analysis of whole lung sections, and a cytological analysis of the Broncho alveolar lavage. The results of this study indicate that IL-33 plays a role in the resolution of lung inflammation that may offer protection for severe asthmatics that are allergic to this clinically relevant allergen. IL-33 has previously been shown to also offer cardiac protection by reducing inflammation of adipose tissue. Future studies will use the IL-33-/- mouse to look at its effect on atherosclerosis and determine if P. gingivalis is able to induce atherosclerosis more readily in the IL-33-/- mouse as compared to the wild type strain.
Julia Payne is a postdoctoral trainee in the laboratory of Andrew Wilson in the Department of Medicine, Section of Pulmonary, Allergy, Sleep, and Critical Care. Julia is a Pulmonary and Critical Care Medical fellow. Her project consists of the application of a novel adeno-associated virus (AVV) vector for alpha-1 antitrypsin gene therapy. Alpha-1 antitrypsin deficiency (AATD) is an inherited disease caused by a single amino acid substitution in the glycoprotein alpha-1 antitrypsin (AAT). AAT, predominantly produced by hepatocytes, is an essential serine protease inhibitor, which functions to counteract the injurious effects of neutrophil elastase in the lung. Deficient circulating levels of AAT can lead to destruction of lung elastin and development of pan-acinar emphysema at an early age. Standard treatment for patients with AATD, weekly infusion of pooled human AAT protein, is both costly and invasive, providing a rationale to seek alternative treatment approaches. Recent advances in AAV vector design might allow for higher-level transgene production than was previously possible, paving the way for a potentially therapeutic gene delivery system. Julie has been working to characterize and test an adeno-associated virus (serotype 8) as a candidate vector for pulmonary gene transfer. In the past, she has tested the capacity of this vector to deliver reporter genes (luciferase, eGFP) to cells of the murine lungs following intratracheal and intramuscular administration. She observed that high levels of bioluminescence were detectible in the thoracic regions of C57BL6J mice up to a year following intratracheal administration of AAV8-CASI-luc. To phenotype the cells transduced by the vector, the GFP transgene was delivered intratracheally. Histologic examination revealed ciliated airway epithelium, club cells, alveolar macrophages, and type 2 alveolar epithelial cells were transduced. These observations suggested that the optimized AAV8 vector may be suitable for therapeutic gene transfer. Alpha1- antitrypsin deficiency (AATD) has long been a target for gene therapists. Human clinical trials evaluating intramuscular (IM) gene delivery for AATD have demonstrated the safety and feasibility of viral gene transfer, but have had difficulty reaching the high serum concentration of alpha-1 antitrypsin (AAT) protein required to protect the lung from neutrophil elastase-induced damage. Within the lung epithelial lining fluid (ELF), however, a much lower AAT concentration is thought to be protective. Direct lung-targeted vector delivery might therefore circumvent the requirement for high AAT transgene expression requirements. Over the past year, she has investigated the utility of lung-targeted AAT gene delivery using an AAV2/8 vector in a murine elastase-injury model of emphysema. Results suggest that this vector is capable of achieving durable gene delivery of hAAT within the epithelial lining fluid of the lung, and ameliorates elastase-induced emphysema. Currently, she is writing up these results in a first-author manuscript to be submitted to the American Journal of Respiratory and Critical Care Medicine.
Claire Filone is a postdoctoral fellow in the laboratory of Dr. John Connors in the Department of Microbiology. Her postdoctoral work has focused on identifying host-pathogen interactions and novel antiviral through unique screening strategies. Along with supporting work on several projects that have led to publication, she has been the primary lead on 3 research projects. She identified a novel indoline alkaloid compound as an antiviral for nonsegmented negative strand RNA virus replication, including VSV and Ebola (published March 2013). She initiated, led and managed two collaborations with the Broad Institute Genomic Perturbation Platform to design and execute whole genome-scale pooled RNAi screens to identify host factors necessary for orthopoxvirus or filovirus infection. In this role, she designed novel pooled shRNA screens for host factors necessary for viral infection working at Boston University as well as traveling to complete high containment work at United States Army Medical Research Institute for Infectious Diseases (USAMRIID). She confirmed the necessity of the master regulator for the heat shock response, HSF1, for orthopoxvirus infection (published February 2014) and she is currently validating the candidate genes necessary for filovirus infection. Her work describing a novel antiviral against nonsegmented negative strand viruses, published in March 2013, was named on of the top 100 discoveries of 2013 by Discover Magazine (#42). She was awarded a Career Development Grant for postdoctoral women from the American Society of Microbiology.
Ariana Darcy just completed her thesis studies (4/2014). Her mentor is Dr. Monty Montano, in the Section of Infectious Diseases and a Junior Faculty Member on this training program. Her studies have focused on studying the observation that patients with HIV experience normal aging co-morbidities at an accelerated rate compared to uninfected age matched controls. More specifically, the potential role chronic inflammation and immunosenescence may play in the increased incidence of osteoporosis in these patients. In the past year she has continued to work closely with Dr. Louis Gerstenfeld, who serves as a Co- Mentor. Her thesis project focuses on the potential role of T cell activation inhibiting osteoblast differentiation in chronically infected HIV patients. HIV patients tend to have lower bone density compared to their age-matched uninfected counterparts and the mechanisms behind this have yet to be fully understood. HIV patients also have higher immune activation levels compared to uninfected individuals and therefore it is possible that this immune activation is detrimental to healthy bone homeostasis. Ariana has recently found that supernatants from activated T cells, both infected or uninfected with HIV, similarly inhibit osteoblast differentiation. Co-culturing osteoblasts with activated T cells also prevents osteoblast differentiation and it appears to kill the osteoblasts completely. HIV infected T cells that are unactivated are able to prevent osteoblast differentiation whereas uninfected T cells do not do this. In the current year Ariana established that activated CD4+ T cells inhibit osteoblast differentiation and this was attenuated using the immunosuppressants, rapamycin and JQ1. Furthermore, she found that HIV infected CD4+ T cells behaved differently and did not respond to rapamycin and JQ1 treatment. The plan is to establish a mechanism for inhibition of osteoblast differentiation mediated by T cell activation and publish a manuscript on these findings.
Tuan Pham is a fifth year student in the Department of Biomedical Engineering. His research mentor is Dr. James Hamilton in the Department of Biophysics. The goal of his proposed research project is to use a rabbit model of atherosclerosis and determine the effects of acute therapeutic protocols on the vulnerability of formed plaques using serial Magnetic Resonance Imaging. During the span of this year, he completed collecting data for this serial MRI of atherosclerotic plaque progression project. Results from this experiment will help design intervention type experiments in future studies. A paper has been drafted and will be submitted shortly for review. Additionally, he has collected a majority of data for a second series of studies examining therapeutic treatments modalities for atherosclerosis (RvD1). In these studies Tuan observed that resolving treated rabbits exhibited marked decrease in the number of plaque ruptures present after pharmacological triggering. These promising results have laid the groundwork for continuing studies to develop resolvins for plaque regression therapies.