Philip Furmanski - US grants

Studies in our laboratory have demonstrated that macrophages can directly affect erythropoiesis, both in vitro and in vivo. Using a unique model system developed in our laboratory, we have found that this macrophage regulation of erythropoiesis is impaired in leukemia. Regulation can be restored and the leukemia reversed by the treatment of fully leukemic animals with normal macrophages. When macrophages are transferred to normal or leukemic animals, erythroid colony formation (CFU-E) is significantly suppressed. The data suggest that macrophages do not suppress erythropoiesis in vivo through regulation of EPO levels but by an influence on novel erythropoietic stimulatory factor. This factor is produced in response to anemic stress and is present in impure but not purified preparations of EPO. The factor can be measured by its ability to reverse macrophage suppression of CFU-E and has no activity in the traditional in vivo standard plasma EPO assay. Preliminary characterization reveals that the activity is a glycoprotein of approximately 40,000 daltons. No suppressive activity has been detected by normal macrophages in vitro. The production in vitro of a CFU-E stimulatory factor by virus-infected macrophages correlates with the failure of leukemic animals to experience regression. Our goals for the coming year are to: (1) extend our present studies to include isolation and characterization of the factor related to EPO that appears to be regulated either directly or indirectly by normal macrophages; (2) determine the factor's role in vivo in regulation of erythropoiesis; and (3) characterize the in vitro stimulatory factor produced by virus-infected macrophages. (MB)

Vesicular stomatitis virus (VSV) will be used to study regulation of macromolecular synthesis with particular emphasis on (1) the role of interference caused by defective interfering (DI) particles in vivo and on (2) the functional interactions of the viral glycoprotein G with antibody and with the host cell. These studies will relate to the possibility of using DI particles or DI-specific nucleic acid sequences to regulate viral infections. Studies on the glycoprotein will define functional domains and elucidate the basic mechanisms by which the host defends against viral infections and how viruses overcome these defenses. These studies also provide a novel way to examine human tumor antigens. The experimental approach with DI particles will be to initiate infections in mice with known quantities of DI particles and standard VSV. The detection of synthesis of DI particles will be by Northern blotting with DI-specific cloned 32P-DNA sequences and correlating the presence of DI RNA with the disease process. Naturally virulent New Jersey isolates of VSV will be examined for their ability to generate DI particles and mapped physically against laboratory strains to determine if virulence markers are associated with certain regions of the genome. G protein will be approached via monoclonal and anti-peptide antibodies. The parameters to be measured will be neutralization, precipitation of whole virions, precipitation of G protein, and antibody-induced degradation of G protein. Mechanisms of these interactions will be elucidated. Non-VSV proteins acquired by VSV during the budding-out process from HeLA cells will be characterized in respect to their phosphate and carbohydrate content and to their synthesis and association with tumorigenesis.

No Abstract Provided

No Abstract Provided

Immunotherapy offers an attractive approach to the control of cancer. Passive immunotherapy with specifically sensitized cells holds particular promise, but requires the ability to consistently develop large numbers of lymphoid cells capable of eliminating the tumor in vivo and an understanding of T cell functions and interactions. Newly developed methods for isolation, characterization, expansion, long-term growth and cloning of lymphoid cells in culture, make it possible to achieve these goals. Reliable experimental models are now required to test and refine this potentially very valuable mode of treatment and determine its applicability to naturally occurring, established cancers. In an unique model system developed in our laboratory, acute leukemia in mice undergoes predictable, immunologically-mediated, spontaneous regression. Normal T cell and macrophage functions are essential for regression to occur. In a consistent fraction of clinically regressed animals, the leukemia spontaneously recurs. Under support of this grant, we have shown that leukemia regression can be efficiently induced in progressor leukemia mice by transfer of in vitro cultured T cells that are specifically reactive to virus/leukemia cell antigens. This immunotherapy is effective even in fully leukemic animals and requires no concurrent or prior adjunctive treatment such as irradiation or cytotoxic drugs. Helper Lyt 1+ cells are implicated in causing permanent disease cures, while cytotoxic Lyt 2+ cells cause temporary leukemia remissions. The aims of the present study include: complete characterization of the effector cells, their antigenic specificity and mechanism of action in vivo; determination of the involvement of lymphokines in successful immunotherapy; and development and characterization of effective immunotherapeutic protocols for prevention and treatment of leukemic recurrence, the outstanding problem in cancer management.

The generation of diversity is a hallmark of the malignant process. This diversity results in the observed heterogeneity in tumor cell properties, the acquisition of metastatic capacity and the development of resistance to treatment. Diversity could be the result of accumulated, random point mutations and cell selection. An alternative, which has recently gained in experimental support, is that tumor diversity is promoted through cell-cell fusion and chromosome selection. The goal of our study is to extend these findings and critically assess the contribution of cell fusion to tumor development and progression using sensitive, unambiguous and nonselective markers for fusion events. This will be accomplished by analysis of the formation of heteropolymers of autosomal, multimeric alloisozymes in tumor-bearing allophenic (tetraparental, chimeric) mice. The results obtained will be independent of host cell contamination, can be analyzed on a tissue or individual cell basis, and will be free of histocompatibility effects or selection. Appropriate marker enzymes are available representative of different chromosomes, including glucose phosphate isomerase, malate dehydrogenase, phosphoglucomutase, isocitrate dehydrogenase and autosomal glucose-6-phosphate dehydrogenase. The primary tumor system to be employed is the methylcholanthrene-induced sarcoma. Using this system, the aims of our study are to determine the contribution of cell fusion to tumor development, to metastatic progression and to the acquisition of resistance to therapy, and to identify the cell partner for tumor fusion events. The results of these studies could provide greater insight into the pathogenesis of malignancy and the mechanisms by which tumors respond to their environment.

ultraviolet spectrometry; biomedical equipment resource;

biomedical equipment resource; biomedical equipment purchase;

Breast cancer is second only to lung cancer as the most common neoplasm in women and a leading cause of cancer death in women. Progress in gaining an understanding of breast cancer is proceeding at an extremely rapid pace. Major technological developments have been made that promise to greatly improve our ability to detect and manage the disease. Laboratories and clinical units throughout the world are addressing all aspects of the disease, from molecular biologic studies of its genetic origins to new strategies for therapy in the patient with advanced breast cancer. Knowledge gained from such studies is beginning to impact the burden of breast cancer in our society, although considerably more cancer control research is needed. To assure continued progress against the disease, and to foster the translation of research findings to the clinical problems in breast cancer, periodic meetings are required of those scientists and clinicians interested in breast cancer to: (1) assess the latest findings related to the origins, biology, diagnosis and treatment of breast cancer; 2) disseminate this knowledge to scientists and clinicians interested in the disease; 3) foster new initiatives and collaborations in the study of breast cancer, particularly at the interface between the laboratory and clinic; and, 4) explore new concepts and technologies from the diverse fields of modern biomedical research that could be brought to bear on the problems of breast cancer biology and management. This proposal requests funding for a Breast Cancer Research Conference to be held under the auspices of the International Association for Breast Cancer Research in Tel Aviv, Israel, March 5-9, 1989. The Meeting will consist of state- of-the-art plenary sessions, poster sessions, panel discussions and a workshop. This meeting will be a timely and significant stimulus to research on breast cancer, and an opportunity for the development of new insights and approaches to the study of the disease.

The primary targets for HIV infection are CD4+ T lymphocytes and cells of the macrophage-monocyte lineage. Infection of T cells leads to their destruction and profound immunosuppression, while infection of macrophage- monocytes does not generally cause cytopathology, but may represent a sanctuary for the virus, serving as a reservoir for HIV persistence, spread and continual infection of T cells in the face of a significant, early immune response against viral antigens. Macrophages are also the likely routes of HIV infection of the central nervous system. The mechanisms by which virus-infected macrophages evade the host immune response are unknown. Unless they are elucidated, the pathogenesis of AIDS will not be fully understood and the prevention or therapy of HIV infections by immunologic means may not be achieved. The studies proposed are designed to systematically determine the mechanisms by which retroviral infection of macrophage lineage cells evades host immune responses and to identify the circumstances in which host immunity can be made capable of eliminating the infected cells. This will be approached using a novel experimental system developed in our laboratory involving Friend virus-induced erythroleukemias in mice. We have demonstrated that virus infection of macrophages occurs and is a critical factor in the pathogenesis of this disease. The conditions, the host is able to mount an immune response that eradicates the virus infected macrophages and this leads to leukemia regression. We have also found that CNS macrophages are productively infected with virus in this system, and that these infected cells are absent in regressor animals. This system thus permits identification of the factors involved in retroviral escape from host immune responses by establishment of a sanctuary infection in macrophages, as occurs in progressor animals. Moreover, it uniquely permits identification of the mechanisms by which the immune system can successfully eradicate infected macrophages and bring the disease under control, as occurs in regressor animals. This will be accomplished by: 1) determining if the viral and MHC antigens expressed on infected cells of the macrophage lineage differ from those on infected erythroid, lymphoid or fibroblastic cells; 2) determining whether expression of viral and MHC antigens on infected macrophages is modulated by cytokines, including GM-CSF, TNF-alpha, IFN's; and, 3) determining whether infected macrophages differ from other infected lineages in susceptibility to cell-mediated immune effectors.

This proposal is offered by New York University's College of Arts and Science. A new, integrated series of introductory level science courses will be developed for 1,600 non-science majors annually. The new curriculum takes into account the level of preparation and the extent of alienation of students from science and mathematics. The proposed curriculum aims to teach quantitative reasoning and emphasize the open-ended questioning aspect of scientific thinking by means of lectures, inquiry-based laboratory experiences and recitations, and cooperative study teams. The intention is to develop, pilot, and refine an experimental version of such a multicourse curriculum in 1993-95, and in 1995 to replace the existing elective science and mathematics requirements at NYU with this program. All non-science majors will eventually have equal exposure to a corpus of interesting material in a Science Core Program. Design features will permit a certain measure of individualization in learning, as well as encourage the development of small, cooperative problem-solving study groups, breaking students out of isolation and replicating the research model of scientific inquiry.

Lactoferrin (Lf), the principal and most avid Fe-binding protein in milk and other epithelial secretions and the major constituent of the specific granules of neutrophils, has been implicated in a wide variety of functions. Its chief role appears to be defense against infectious agents, through strong chelation of the Fe required for microbial growth, but functions as diverse as Fe absorption in the gut, feedback suppression of myelopoiesis and potent stimulation of macrophage and NK toxicity, among many others, have been attributed to the molecule. Some of these functions appear independent of Fe binding, and some relate to the strong but unexplained binding affinity of Lf for nucleic acids. Defects in Lf expression are observed in some cancers, and may contribute to the pathogenesis or clinical manifestations of the disease. By virtue of its profound affinity for Fe and multitude of related and unrelated effects, Lf offers interesting potential as a therapeutic in infectious diseases and neoplasms or as an immunomodulator. However, many of these proposed functions of Lf are controversial and virtually all are unproven at the physiological level. We have made the unique observation that there are multiple forms of Lf. At least one isoform binds no Fe and instead, expresses a potent ribonuclease activity. These isoforms have similar, if not identical, primary structures and are not interconvertible. It is our long-term objective to determine how the varied and powerful functions of Lf can be exploited therapeutically. Our working hypothesis is that the existence of multiple forms of Lf explains the broad and often inconsistent functions proposed for Lf. In order to test this hypothesis and ultimately to realize the therapeutic potential for the isoforms, we must gain an understanding of Lf pathophysiology and determine the precise in vivo role for each isoform. We also have the opportunity to determine how isoforms with such similar structures could express such disparate and nonoverlapping activities. This will be accomplished by: 1. determining the structural differences that account for the different activities of the isoforms; 2. determining the significance of the avid DNA binding activity of Lf isoforms; 3. cloning and expressing the isoforms; 4. examining structure-function relationships in the isoforms using site-directed mutagenesis; 5. analyzing the regulatory sequences that direct the unique tissue distribution of Lf; 6. constructing transgenic lines of mice in which the Lf gene has been disrupted and testing their phenotype and responsiveness to infection and neoplasia.

Infants fed synthetic formulas suffer a higher incidence of iron-deficiency anemia and infection than do infants that are breast fed. As a result, there are plans to supplement formulas with natural constituents of milk that may be responsible for enhanced iron absorption and for anti-microbial activity. An extremely attractive candidate in this regard is lactoferrin(Lf), a molecule that has been implicated in both functions because of its extremely high avidity for iron. However, in spite of a compelling rationale, studies designed to test for the occurrence of these physiological activities of Lf in vivo have been inconsistent. Even when positive effects have been obtained, they have not necessarily pointed to a mechanism based on iron binding. The situation has been further complicated with the attribution to Lf of a wide array of additional, dissimilar functions, some of which appear to involve regulatory interactions with cells and also to be independent of iron-binding. It is the long-term objective of our laboratory to conclusively identify the in vivo physiological roles of Lf and to determine how they could be exploited for preventive or therapeutic purposes. our studies to date have revealed several novel aspects of Lf that could be germane to its in vivo activity: there exist multiple forms of Lf, at least one of which does not bind iron and, instead, exhibits a potent ribonuclease activity; Lf has antitumor activity which, importantly, occurs through activation of natural killer (NK) cells; and Lf can act directly to cause gene transcription in target cells. We believe that these findings offer a new perspective on Lf function an possibly an explanation for the disparate results obtained in prior studies. Our working hypothesis is that the broad functions proposed for Lf, and their inconsistent expression, are the result of the existence of these functionally distinct isoforms and the fact that they act, for some processes, through cells of the immune system. We propose to test this hypothesis directly in the context of Lf supplementation of infant formula for purposes of preventing infection. The studies will be carried out under 2 specific aims; 1. to determine the role of different isoforms in the anti-infective properties of Lf in milk; and 2. to determine the role of (gut associated and systemic) lymphoid cells in the anti-infective properties of Lf. We believe that the results of this work could provide an understanding of the effects and potential benefits of Lf supplementation of infant formula.

[unreadable] DESCRIPTION (provided by applicant): Lactoferrin (Lf), a multifunctional molecule first identified as a major constituent of human milk, has become the subject of intensive and widespread study. Although initially thought to be involved in iron transport and nutrition in the neonate, because of its strong avidity for Fe, it is now apparent that Lf is a key component of the body's first line of defense against infection and other invasive processes. We and others have shown that Lf also has antitumor activity and may be useful as a natural antibiotic. Yet, paradoxically, Lf is consistently re-expressed or ectopically expressed in a substantial proportion of many different cancer types. The significance of this finding has never been systematically examined. The goal of this study is to determine the functional consequences of Lf expression in human cancers. Our hypothesis, which is based on the known molecular properties of Lf and a striking new result obtained in our laboratory, is that Lf expression contributes to resistance of many cancers to oxidative damage that results from growth, local conditions and even treatment by certain chemotherapeutic agents and radiation. We will test this novel hypothesis by first determining the scope and characteristics of Lf-mediated resistance to cell damaging agents. We will then determine the mechanisms by which Lf mediates its effects, and determine whether the properties of Lf-expressing cells also involve activation of additional pathways of cellular resistance to oxidative damage. Finally, we will test whether Lf-mediated resistance in vitro to various chemotherapeutic agents that act at least in part by causing oxidative damage is manifest in resistance to therapy in vivo, using both syngeneic and xenotransplant model systems. The results of these studies could provide useful insights into the pathophysiology and natural history of some human cancers, mechanisms by which they develop resistance to treatment, and markers which may be applicable to predicting treatment response in patients with the disease.

The Garden State-Louis Stokes Alliance for Minority Participation (GS-LSAMP) will bring together all of the major colleges and universities from the highly urbanized northeastern part of New Jersey to double the number of graduates from underrepresented minority groups in STEM disciplines in 5 years. The Alliance members will use the ?best practices? of other LSAMP?s as well as innovative techniques to best address the academic and social needs of the students and to change the attitude of these students towards STEM careers. Three well-documented causes of attrition in STEM fields at the university level will be targeted: academic support, financial need and social support. The GS-LSAMP will include Rutgers University-Newark (#1 nationally in diversity) in consortium with Rutgers-New Brunswick, Montclair State University, Kean University, William Paterson University, New Jersey City University (HSI), Fairleigh Dickinson University, Bloomfield College, and Essex County College using an innovative ?hub and spoke? model with northern, central, and southern clusters. Each cluster will develop independent and versatile sub-communities with critical masses of students and faculty within the larger LSAMP community. The GS-LSAMP will create a robust Collaborative Learning Community with outreach beyond the Alliance to impact local community colleges and urban school districts through the sharing of ?best practices? in teaching and learning in STEM fields. The GS-LSAMP will also impact local high school and community college communities through one-on-one interactions among GS-LSAMP Scholars and the students in those institutions. A ?cybercommunity? will be created using innovative and locally tested software and techniques. With the dense population and abundance of opportunities to interact with the public, service learning will be an important option for GS-LSAMP Scholars, as will internships/externships among the many industries with STEM needs in the area. There will also be both real and virtual international components.

Intellectual Merit: The ability of the project to create, through social and academic networking, a collegial inter- and intra-Alliance environment for the GS-LSAMP Scholars, professors and graduate students. Incoming students will acculturate to college life in a Summer Bridge experience that includes intensive academic workshops augmented by one-to-one tutoring, Academic Skills Seminars, Career Development workshops, and personal development advising. Academic support, including tutoring, advising and mentoring will continue throughout the academic year. The GS-LSAMP Scholars will receive support and encouragement to conduct research with STEM faculty and graduate students. They will share their research with their GS-LSAMP peers in periodic meetings and seminars, which will also provide face-to-face opportunities to build friendships and support networks, that supplement the cyber social networking community.

Broader Impact: The GARDEN-LSAMP has the potential to form a network of academic and social support for young scholars interested in pursuing STEM degrees and ultimately careers in STEM fields. The social networking software developed will be made available to all LSAMP alliances. This network will enhance the awareness of the opportunities for students who major in STEM disciplines and will lead to an overall increase in the numbers of underrepresented New Jersey students aspiring to STEM-related fields. The schools that are members of the GS-LSAMP alliance will gain a more diverse group of students with high graduation rates and successful post-graduate careers.

This project is combining the theoretical perspectives of "communities of practice" and "situated learning" with the innovative research, curriculum, and community best practices developed in four Integrative Graduate Research and Education Traineeship (IGERT) projects. This is done horizontally across the institution's STEM graduate programs and centers, and vertically within the undergraduate research experience programs. This extensive program of activities is improving the quality of undergraduate and graduate students completing STEM degrees. Additionally, the project is using proven methods to increase the participation of groups underrepresented in STEM fields, including first generation, economically disadvantaged, and disabled students. With attention to addressing critical educational junctures, the senior leadership team is creating the Graduate Innovation and Integration Center (GIIC) to enhance the integration of the institution's and research and educational efforts. GIIC center is working with five existing interdisciplinary campus centers to build collaborations and synergistic outcomes. A comprehensive plan is evaluating each aspect of the project including critical junctures as well as the impact of the project on promoting institutional collaboration and synergy. Longitudinal studies are being planned that follow students for three years post graduation. The broader impact of this project is the synergy between a wide array of undergraduate and graduate programs; the broadened participation of undergraduate students in research experiences, particularly students from populations underrepresented in STEM disciplines; the improved retention of a diverse body of graduate students; and a model for student success that can be used at other institutions.