Basic Investigators Research
Antibiotic Action
Antibiotic Research Laboratory
In this laboratory, research of Drs. Robert C. Moellering, Jr., George M. Eliopoulos, Howard S. Gold, Claudie Thauvin-Eliopoulos and Satish K. Pillai is directed towards understanding the mechanisms of action of new antimicrobials, exploring their in vitro activities as single agents and in combination, and investigating mechanisms by which bacteria develop resistance to these agents.
The major focus of the laboratory has been on multiply drug-resistant gram-positive pathogens (staphylococci and enterococci). This laboratory reported the mechanism of resistance to linezolid in S. aureus and continues to explore the basis of resistance to linezolid and to daptomycin. In addition, members of the laboratory have investigated antimicrobial resistance mechanisms in Enterobacteriaceae, including carbapenemases, inhibitor-resistant beta-lactamases and other extended-spectrum enzymes.
The laboratory has provided molecular epidemiologic support to studies of clinical outbreaks of vancomycin-resistant enterococci and carbapenem-resistant Enterobacteriaceae. Other projects have explored how mutations in global regulator genes affect bacterial virulence in S. aureus.
Experimental models are used to evaluate therapeutic regimens in vivo against highly antibiotic-resistant pathogens. These studies have utilized infective endocarditis models to study enterococcal and MRSA infections and intra-abdominal abscess models to investigate polymicrobial infections or those due to extended-spectrum beta-lactamase-producing Enterobacteriaceae.
Recent work has involved nematode and insect models for the study of virulence in antibiotic-resistant S. aureus and in Acinetobacter spp. We have used the greater wax moth, Galleria mellonella, to investigate the virulence of vancomycin-intermediate S. aureus and of S. aureus with mutations associated with decreased susceptibility to daptomycin. Prokaryote-eukaryote interactions, specifically between C. albicans and A. baumannii were explored in the nematode, Caenorhabditis elegans.
Viral Pathogenesis
This laboratory, under the direction of Dr. Norman Letvin, studies the immune response to HIV infection in non-human primate models and in man. Dr. Letvin and his co-workers have defined an AIDS-like disease of monkeys, demonstrated that it is induced by the simian immunodeficiency virus (SIV), a close relative of the human AIDS virus, and have used this disease as an animal model for the study of AIDS. They are employing this model to explore vaccination strategies for protection against infection with the AIDS virus. In particular, they are assessing the use of live vector systems, peptide formulations and the novel vaccine modality of naked DNA to elicit AIDS virus-specific cytotoxic T lymphocytes (CTL) in monkeys.
The investigators in this laboratory are also utilizing SIV-induced disease of monkeys as a model for studying mechanisms by which cells of the immune system contain the spread of the AIDS virus in an infected individual. These studies have demonstrated the crucial role played by the CD8+ lymphocyte in controlling AIDS virus replication. Studies of immune containment of HIV-1 in infected humans, including the mechanisms by which CD8+ lymphocytes inhibit HIV-1 replication in CD4+ cells, are ongoing in the laboratory. Moreover, vaccine strategies for eliciting HIV-1 specific cellular immunity in humans are under investigation. The laboratory also uses SIV-induced disease in monkeys as a model system for examining novel therapeutic approaches to the treatment of AIDS.
The Cellular and Molecular Bases of Inflammation
Dr. Peter Weller has many active areas of basic laboratory research centered around understanding basic mechanisms of leukocyte functioning in forms of inflammation. The two principal areas of investigation are:
1) the immunobiology of eosinophilic leukocytes and
2) the intracellular regulation and compartmentalization of inducible mediators of inflammation in neutrophils and other leukocytes.
Studies of human eosinophils are aimed at defining mechanisms whereby eosinophils may collaboratively interact with other cellular elements of the immune system. These studies include investigations of the mechanisms whereby eosinophils may function as antigen-presenting cells in governing T-lymphocyte dependent immune responses, and include investigations of the in vivo migration and function of eosinophils and of the regulated expression of cell surface proteins involved in collaborative interactions between eosinophils and other cell types.
Additional studies are focused on defining the molecular mechanisms governing the synthesis, granule storage and release mechanisms of eosinophil derived cytokines. The roles of eosinophils in wound healing and fibrosis and the activities of chemokines and cytokines released by eosinophils that contribute to tissue remodeling are being studied. The second area of research involves the molecular and cellular biologic bases of inducible responses of leukocytes participating in host defense and other forms of inflammation. These are centered on a unique intracellular compartment, termed the lipid body, whose formation is rapidly inducible in leukocytes. The intracellular signaling mechanisms responsible for lipid body induction and especially the roles of lipid bodies as distinct sites of cytokine and eicosanoid mediator formation are being studied.
In addition to investigating previously undefined pathways of leukocyte responses to inflammation, these studies also offer the potential to identify novel anti-inflammatory therapeutic targets. Our research indicates that lipid bodies in leukocytes have roles as sites of regulated formation of eicosanoids and as distinctextranuclear sites of translation. The biology of these structures is intimately related to the roles of leukocytes in acute inflammation.
Study of Viruses Associated with Human Cancer
Dr. Joyce Fingeroth studies herpesviruses that are causally associated with human cancer. These studies include basic, translational and clinical research. In the laboratory Dr. Fingeroth inveestigates the Epstein-Barr virus (EBV) receptor. This 140 Mr glycoprotein is expressed on human B lymphocytes, some T lymphocytes and rarely other hematopoietic lineage, epithelial and smooth muscle cells. It is the cellular attachment protein for EBV and also for complement fragments. The spectrum of EBV-related neoplasms reflects the cellular distribution of this receptor. The function of this receptor is studied using the tools of molecular biology, protein biochemistry and cellular immunology to relate the regulation and structure of the molecule to its function as a virus receptor. HHV-8 or KSHV (Kaposi's sarcoma-associated herpesvirus), the most recently discovered member of the human herpesvirus family, appears to have a limited tropism similar to EBV. Preliminary experiments are underway to determine whether specific cellular attachment proteins can be identified. Translational studies in the laboratory have focused on characterizing the EBV and KSHV thymidine kinases as a targets for antiviral and antitumor therapy.
Study of the Complement System
Dr. Anne Nicholson-Weller directs this laboratory: We have been interested in how the complement system, which is part of the innate immune system, participates in host defense and in protection against autoimmunity. The first project is focused on understanding how the receptor for C1q and mannan binding lectin, which was identified in our laboratory, is able to mediate the phagocytosis of C1q-opsonized particles and transport C1q-opsonized immune complexes. C1q deficiency in humans is associated with a greater than 90% chance of developing an aggressive autoimmune, lupus-like illness. This might relate to clearance/removal of C1q-opsonized autoantigens, or to a unique C1q-mediated signaling pathway. With respect to the signaling hypothesis, we have studied calreticulin, a membrane receptor for immobilized C1q, and how its downstream signaling pathway intersects with that of beta 2 integrins. This work was done in collaboration with Dr. Lloyd B. Klickstein, MD, Ph.D. of the Brigham and Women's Hospital.
A second project involves the role of the human erythrocyte as a transporter of particles recognized by complement. A challenge for all organisms with an enclosed vascular space, is how to clear the circulation of noxious particles, be they viral or bacterial pathogens, apoptotic debris, or immune complexes. For humans and a few higher primates this is accomplished by tagging the particle with complement opsonic fragments (C1q, mannan binding lectin, C4b and C3b). The particle then adheres to an erythrocyte by binding its complement receptor 1 (CR1 or CC35), which has binding sites for these complement opsonins. During circulation though the liver and spleen the erythrocyte-adherent particles are removed by a process that spares the erythrocyte. In mice and most other vertebrates, intravascular clearance depends on complement activation and then the adherence of platelets, as opposed to erythrocytes. We are modeling the human erythrocyte-dependent clearance process in vitro. Our goals are to understand how the human erythrocyte both facilitates ingestion of its bound particle, and yet avoids its own ingestion. It is likely that the clustered nature of CR1 expression on the erythrocyte has a role in protecting it from being ingested. We have used fluorescent beads as well as Salmonella montevideo as test particles.
Laboratory and Clinical Studies of HIV Vaccines and Biodefense Vaccines
Dr. Raphael Dolin and colleagues have a major interest in the development and evaluation of HIV vaccines. As part of the NIH sponsored Harvard HIV Vaccine Trials Unit, studies of candidate HIV vaccines are carried out in normal volunteers for safety and immunogenicity. Recent vaccines which have been studied include DNA vaccines which code for HIV-1 envelope genes, and genes for gag, pol, and nef proteins. Studies of candidate vaccines based on adenovirus 5 vectors are also underway. Laboratory investigation of immune responses also has been undertaken, including studies of both humoral and cell-mediated immune responses. The latter employ techniques to measure T-cell responses by ELISPOT, intracellular cytokine staining, and tetramer assays. Establishment of highly sensitive, specific neutralization antibody assays is a particular interest of the group.
Dr. Dolin and colleagues are also interested in the development of safe and more tolerable smallpox vaccines, as part of the NIH sponsored Translational Immunology Center. Clinical studies of vaccinia and Modified Vaccinia Ankara (MVA) vaccines in human volunteers are being undertaken along with measurement of humoral and cell mediated immune responses in vaccines. The goal of the studies is to develop a new generation of genetically modified and defined vaccines for biodefense uses.
