News and Publications
DEPARTMENT OF IMMUNOLOGY
|Richard J. Ulevitch, Ph.D. ||Member and Chairman |
|Carole L. Banka, Ph.D. ||Assistant Member |
|Richard Boismenu, Ph.D. ||Assistant Member |
|Gary M. Bokoch, Ph.D. ||Associate Member |
|Linda M. Bradley, Ph.D. ||Assistant Member |
|Dennis R. Burton, Ph.D.* ||Member |
|David A. Cheresh, Ph.D. ||Associate Member |
|Robert W. Chesnut, Ph.D. ||Adjunct Associate Member |
|Charles G. Cochrane, M.D. ||Member |
|Neil R. Cooper, M.D. ||Member |
|Linda K. Curtiss, Ph.D. ||Associate Member |
|Frank J. Dixon, M.D. ||Member, Director Emeritus, TSRI |
|Thomas S. Edgington, M.D. ||Member |
|Sao-Tah Fan, Ph.D. ||Assistant Member |
|Ann J. Feeney, Ph.D. ||Associate Member |
|Lily Feng, M.D. ||Assistant Member |
|Robert I. Fox, M.D., Ph.D. ||Adjunct Assistant Member |
|Nicholas R.J. Gascoigne, Ph.D. ||Associate Member |
|Elizabeth D. Getzoff, Ph.D.* ||Associate Member |
|Peter Ghazal, Ph.D.** ||Associate Member |
|Howard Gray, Ph.D. ||Adjunct Member |
|Jiahuai Han, Ph.D. ||Associate Member |
|Wendy L. Havran, Ph.D. ||Associate Member |
|Bessie Pei-Hsi Huang, Ph.D.* ||Associate Member |
|Tony E. Hugli, Ph.D. ||Member |
|Janis H. Jackson, M.D. ||Assistant Member |
|Jonathan G. Kaye, Ph.D. ||Associate Member |
|Norman R. Klinman, M.D., Ph.D. ||Member |
|Ulla Gissi Knaus, Ph.D. ||Assistant Member |
|Dwight H. Kono, M.D. ||Assistant Member |
|Vladimir V. Kravchenko, Ph.D. ||Assistant Member |
|Jiing-Dwan Lee, Ph.D. ||Assistant Member |
|David D. Lo, M.D., Ph.D. ||Associate Member |
|Nigel Mackman, Ph.D. ||Associate Member |
|David L. McElligott, Ph.D. ||Assistant Member |
|Anthony M. Montgomery, Ph.D. ||Assistant Member |
|Edward L. Morgan, Ph.D.*** ||Associate Member, Sidney Kimmel Cancer Center, La Jolla, CA |
|Donald E. Mosier, M.D., Ph.D. ||Member |
|Barbara M. Mueller, Ph.D. ||Assistant Member |
|Robert M. Nakamura, M.D.**** ||Member |
|Glen R. Nemerow, Ph.D. ||Associate Member |
|Anne M. O'Rourke, Ph.D. ||Assistant Member |
|Paul Parren, Ph.D. ||Assistant Member |
|Per A. Peterson, M.D., Ph.D. ||Adjunct Member |
|Mary Laurie Phillips, Ph.D. ||Adjunct Assistant Member |
|Eric R. Prossnitz, Ph.D. ||Assistant Member |
|Ralph A. Reisfeld, Ph.D. ||Member |
|Wolfram Ruf, M.D. ||Associate Member |
|Daniel R. Salomon, M.D. ||Adjunct Assistant Member |
|Nora Sarvetnick, Ph.D. ||Associate Member |
|David D. Schlaepfer, Ph.D. || Assistant Member |
|Ingrid U. Schraufstatter, M.D. ||Assistant Member |
|Alessandro Sette, Ph.D. ||Adjunct Assistant Member |
|Linda A. Sherman, Ph.D. ||Associate Member |
|Jonathan Sprent, M.D. ||Member |
|Charles D. Surh, Ph.D. ||Assistant Member |
|Luc Teyton, M.D., Ph.D. ||Associate Member |
|Argyrios N. Theofilopoulos, M.D. ||Member |
|Marilyn Thoman, Ph.D.*** ||Assistant Member, Sidney Kimmel Cancer Center, La Jolla, CA |
|Peter S. Tobias, Ph.D. ||Associate Member |
|Bruce E. Torbett, Ph.D. ||Assistant Member |
|Ernest S. Tucker III, M.D.***** ||Member |
|Susan R. Webb, Ph.D. ||Associate Member |
|William O. Weigle, Ph.D. ||Member |
|Curtis B. Wilson, M.D. ||Member |
|Richard D. Ye, M.D., Ph.D. ||Associate Member |
SENIOR RESEARCH ASSOCIATES
|Syed Munir Alam, Ph.D. |
|Ana Angulo-Aguado, Ph.D. |
|William A. Boisvert, Ph.D. |
|Peter C. Brooks, Ph.D.*** ||University of Southern California, Los Angeles, CA |
|Mary Ann Campbell, Ph.D.*** ||La Jolla Pharmaceuticals, La Jolla, CA |
|Tsung-Hsien Chuang, Ph.D. |
|Surganghanie Dharmawardhane, Ph.D. |
|Pam Daffern, Ph.D. |
|Joanna Davies, Ph.D. |
|Craig Dickenson, Ph.D. |
|Henrik Ditzel, Ph.D. |
|Steven R. Duncan, M.D. |
|Julia A. Ember, Ph.D. |
|Colleen Fearns, Ph.D. |
|Shuang Huang, Ph.D. |
|Richard Klemke, Ph.D. |
|Tania Massamiri, Ph.D.+ |
|Sally Orr, Ph.D. |
|Graham C. Parry, Ph.D. |
|Gaston Picchio, Ph.D. |
|M. Germana Sanna, Ph.D. |
|Bee-Cheng Sim, Ph.D. |
|David Tough, Ph.D.*** ||Edward Jenner Institute, Compton, England |
|R. Anthony Williamson, Ph.D. |
|Isabelle Atencio, Ph.D.+ |
|Balaji Balasa, Ph.D. |
|Dimitrios Balomenos, Ph.D.+ |
|Valerie Benard, Ph.D. |
|Jan Bohuslav, Ph.D. |
|Nancy Boudreau, Ph.D.*** ||Virginia Commonwealth University, Richmond, VA |
|Ingo Brink, M.D.*** ||University of Freiburg, Freiburg, Germany |
|Darren Browning, Ph.D. |
|Zeling Cai, M.D., Ph.D. |
|Ta-Hsiang Chao, Ph.D. |
|Shi Zhong Chen, Ph.D. |
|Weiping Chen, M.D., Ph.D. |
|Yaping Chen, Ph.D. |
|Efthalia Chronopoulou, Ph.D. |
|Mary Crowley, Ph.D. |
|Robert H. Daniels, Ph.D. |
|Fernando Donate, Ph.D. |
|Torsten Dreier, Ph.D. |
|Brian P. Eliceiri, Ph.D. |
|Jonathan H. Erlich, Ph.D. |
|Marika Falcone, Ph.D. |
|Wolfgang Fischer, Ph.D.+ |
|Paola Fisicaro, Ph.D. |
|Scott Gallichan, Ph.D. |
|Gabriella Garcia, Ph.D. |
|Julie Ann Gegner, Ph.D. |
|Jacqueline Glynn, Ph.D.*** ||Chiron, La Jolla, CA |
|Lothar Goretzki, Ph.D. |
|Carl Haarstad, Ph.D. |
|Javier Hernandez-Gil, Ph.D. |
|Robert C. Hoch, Ph.D. |
|Matthew H. Hsu, Ph.D. |
|Huang Huang, Ph.D. |
|Hui-Y Huang, Ph.D. |
|Inkyu Hwang, Ph.D. |
|Mark A. Jagels, Ph.D. |
|Yong Jiang, M.D. |
|Michael K. Jones, Ph.D.*** ||University of California, Irvine, CA |
|Archana Kapoor-Munchi, Ph.D.+ |
|Yutaka Kato, M.D., Ph.D. |
|Curtis R. Kelly, Ph.D. |
|Cecelia M. King, Ph.D. |
|Charles King, Ph.D. |
|Hidehiro Kishimoto, M.D., Ph.D. |
|Gregory H. Kline, Ph.D. |
|Nobuhiko Kubo, Ph.D. |
|Lisa Dimolfotto Landon, Ph.D. |
|James Le Blanc, Ph.D. |
|Dong Sup Lee, M.D. |
|Jie Leng, Ph.D. |
|Erguang Li, Ph.D. |
|Li Li, Ph.D. |
|Cheng Liu, M.D., Ph.D. |
|Holger Lode, M.D. |
|Joseph Lustgarten, Ph.D. |
|Marion Marsh, Ph.D. |
|Toshiaki Maruyama, M.D., Ph.D. |
|Marielena McGuire, Ph.D. |
|Denise McKinney, Ph.D. |
|David John Morgan, Ph.D. |
|Yohei Miyagi, M.D., Ph.D.*** ||Yokohama University School of Medicine, Yokohama, Japan |
|Thomas M. Mohler, M.D. |
|Regular Mueller, Ph.D.+ |
|Bertrand Nadel, Ph.D. |
|Naushaba Nayeem, Ph.D. |
|C. Thomas Nugent, Ph.D. |
|Ilka Ott, M.D., Ph.D. |
|Zhinxing Pan, M.D., Ph.D.*** ||La Jolla Institute for Allergy and Immunology, La Jolla, CA |
|James Pancook, Ph.D.+ |
|Mary Pauza, Ph.D. |
|Eric Petitclerc, Ph.D.*** ||University of Southern California, Los Angeles CA |
|Philippe H. Pfeifer, Ph.D.*** ||BMA Biomedicals, Augst, Switzerland |
|Pascal R. Poignard, Ph.D. |
|Stella Redpath, Ph.D. |
|Helen Sabzevari, Ph.D. |
|Luraynne Sanders, Ph.D. |
|Urban Scheuring, M.D. |
|Deborah E. Schiff, M.D. |
|Jan Schimke, M.D. |
|Li Karine Schiotz, Ph.D. |
|Hui Shao, Ph.D. |
|Steven A. Silletti, Ph.D. |
|Mark Slifka, Ph.D. |
|Staffan Stromblad, Ph.D+ |
|Dwayne G. Stupack, Ph.D. |
|Naoyuki Sugano, Ph.D.+ |
|Siquan Sun, Ph.D. |
|Hiroshi Takamori, M.D., Ph.D. |
|Richard Tapping, Ph.D. |
|Sharon Tracy, Ph.D.+ |
|Kimberly Victor, Ph.D. |
|Silvia Vidal, Ph.D. |
|Julia Voice, Ph.D. |
|Daniel Von Seggern, Ph.D. |
|Vincent R. Walker, Ph.D. |
|Kena Wang, Ph.D. |
|Mei-Ying Wang, Ph.D. |
|Meng Wang, M.D. |
|Catherine Werts-Larzilliere, Ph.D. |
|Karen Whitmer, Ph.D.*** ||Stanford University, Palo Alto CA |
|Yiyang Xia, M.D. |
|Rong Xiang, M.D., Ph.D. |
|Li Xu, Ph.D. |
|Jin Yao, M.D. |
|Mayra Yebra, Ph.D. |
|Tomasz Zal, Ph.D. |
|Frank T. Zenke, Ph.D. |
|Ming Zhao, M.D. |
|Qi Zhong, Ph.D. |
|Kemin Zhou, Ph.D. |
|Bonnie M. Bradt |
|John C. Mathison, Ph.D. |
|Jennifer Price |
|Susan D. Revak |
|Carole G. Romball |
|Martin J. Glennie, Ph.D. ||University of Southampton, Southampton, England |
|Tadhashi Yamamoto, M.D. ||Niigata University School of Medicine, Niigata, Japan |
| * Joint appointment in Department of Molecular Biology |
| ** Joint appointment in Department of Neuropharmacology |
| *** Appointment completed; new location shown |
| **** Joint appointment in Department of Molecular and Experimental Medicine |
|***** Joint appointment in Scripps Reference Laboratory |
| + Appointment completed |
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Richard J. Ulevitch, Ph.D.
One strength of the faculty of the Department of Immunology is being able to recognize great opportunities disguised as what others perceive as insoluble scientific problems. This approach to research is reflected by the accomplishments of the scientific staff of the department. We can look to many important achievements as shown by peer recognition. Publication of seminal findings, remarkable success in obtaining research funding, invitations to organize and speak at outstanding scientific meetings, and recruitment of the best postdoctoral fellows characterize the past year.
Our postdoctoral fellows deserve a special mention. They are the largest group of scientists--currently more than 100--within the department. The group includes trainees from the United States and from more than a dozen European and Asian countries. The fellows not only contribute to the scientific productivity and environment but also provide a unique opportunity for all of us to have colleagues with diverse cultural and ethnic backgrounds.
Among the notable achievements of this past year are publications documenting the research of our members that promises to provide new approaches to treat human disease. Research performed in the laboratory of Glen Nemerow has provided insights into how adenoviruses, important viral vectors for gene therapy, interact with cell membranes. The Nemerow group, together with members of the Cheresh laboratory, first showed how specific members of the integrin family promote viral uptake. Using information gained from these pioneering studies, this group has now shown how delivery of an antiviral ribozyme can inhibit processes essential for the proliferation of Epstein-Barr virus. This virus is thought to be responsible for several human malignant neoplasms and lymphoproliferative disease in immunocompromised patients. The results of these efforts, including demonstration of the in vivo efficacy of this approach, are described in a 1997 article in Proceedings of the National Academy of Sciences of the United States of America.
Dennis Burton and his colleagues have recently addressed issues related to understanding the structure of the prion protein. This work, done in collaboration with S.B. Prusiner, University of California, San Francisco, has been led by R.A. Williamson, with contributions from other members of the Burton laboratory. Prion diseases produce degenerative changes in the CNS by a unique mechanism that involves infectious prion particles. Although the exact nature of this process is not understood, apparently the fundamental lesion involves conversion of the cellular prion protein to an isoform that produces the disease. Antibodies directed to a range of conformational and linear epitopes are being used as probes to develop new structure-function relationships for prions. Results of these pioneering studies are described in another 1997 article in the Proceedings of the National Academy of Sciences of the United States of America.
Luc Teyton, a recent recruit, provides new strength to the department in structure-function relationships of essential receptors on immune system cells. In the past year, he has provided us with new information that markedly broadens our understanding of the function of T-cell receptors. The description in the journal Science of the crystal structure of a T-cell receptor heterodimer was the result of a collaborative effort between the Teyton group and the laboratory of Ian Wilson, Department of Molecular Biology. This seminal work provides a molecular description that will aid in understanding how the T-cell receptor functions in regulating various aspects of the immune response.
Appearing in the journal Nature are data from the Teyton laboratory that provide new insights into how another cell-surface protein, CD8, enhances formation of a stable complex consisting of the T-cell receptor and MHC class I molecules. This work reflects the ongoing commitment in this department to investigate various aspects of the immune system to acquire a full understanding of functional immune responses. In addition to providing new scientific information, this research on CD8, performed in collaboration with the Wilson laboratory, is an example of how the unique scientific environment of TSRI facilitates collaborations that would otherwise be difficult to put into practice.
I also wish to highlight several accomplishments that reflect the prominence that scientists in this department have achieved in the field of intracellular signaling. Work from the laboratory of Gary Bokoch continues to provide new information about small GTP-binding proteins and intracellular kinase cascades. Dr. Bokoch, together with Sandy Schmid, Department of Cell Biology, and other members of the Schmid laboratory, described an important role for small GTP-binding proteins in receptor-mediated apoptosis. In other work, reported in Science, the Bokoch group discovered an important role for a member of the p21-activated kinase family in apoptosis. Jiahuia Han, Shuang Huang, and their colleagues provided evidence in an article published in Immunity for an important role for the intracellular kinase MKK6b in apoptosis. Both of these papers provide seminal findings that will help us understand how programmed cell death is regulated.
Finally, in studies described in Nature, J. Han and R.J. Ulevitch found that a member of the myocyte-enhancing-factor family of transcription factors is an important downstream target of the MAP kinase known as p38. These data offer new insights on regulation of gene expression during inflammation.
Just as scientific progress is marked by transitions from one state of knowledge to another, the makeup of any research department also undergoes change. During the past several years, we have added many new staff members. The most recent addition is Philippe Gallay, who will implement a research program focused on the molecular mechanisms of HIV infection of mammalian cells. Finally, I want to note one additional change in the Department of Immunology and in TSRI: William Weigle, who has been a member of the staff of TSRI since 1961, announced his retirement. Bill Weigle, as he is known to his colleagues, leaves a permanent legacy in the many dozens of trainees who benefited from his mentoring, in the hundreds of publications in the scientific literature, and in his continuous efforts to ensure that the Department of Immunology and TSRI remain at the forefront of science. In his new role as professor emeritus, Bill will still be available to provide the benefit of his experience to all of us.
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R. Boismenu, Y. Chen, K. Chou, N. Chen
The intestinal mucosa shields the body from the external environment but still allows intake of essential nutrients. This epithelial barrier is continuously subjected to potentially toxic, infectious, and otherwise harmful agents. Distinct immune mechanisms have evolved to maintain the integrity of this compartment. In particular, T cells belonging to both the and the ß cell lineages are in close physical association with the mucosa. Previously, we showed that activated intestinal cells but not ß cells produce the epithelial growth factor FGF-7. Interestingly, FGF-7 appears to be the only cytokine expressed by intestinal cells that can support the survival of epithelial cell lines in tissue culture. In addition, we have shown that intestinal cells synthesize several chemokines whose function is to recruit inflammatory cells. These findings suggested to us that intestinal cells and the soluble factors they produce play important roles in maintaining intestinal homeostasis in normal and disease conditions.
ROLE OF CELLS IN INTESTINAL HOMEOSTASIS
We are evaluating the role of intestinal cells in a murine model of colitis induced by feeding the animals dextran sulfate sodium. In this model, focal areas of disease appear within the first 3 days of treatment with dextran sulfate sodium. Large numbers of activated cells accumulate in the affected intestinal segments. In contrast, marked inflammation occurs only after 5 days of treatment. We are pursuing the hypothesis that local production of chemokines by resident cells participate in the initiation of this inflammatory response. Using cellular and molecular approaches, we have shown that in this model system intestinal cells are a major source of FGF-7 and of several cytokines known to modulate immune responses. In ongoing projects, we will characterize the colitis induced by dextran sulfate sodium by using a variety of gene-knockout and transgenic mice that have perturbations in cytokine production and T-cell populations. In addition, we have begun studies to determine antigens recognized by intestinal cells.
ROLE OF CD81 IN THYMOCYTE DEVELOPMENT
In collaboration with W.L. Havran, Department of Immunology, we are continuing our investigation of the mechanisms that control the early stages of T-cell development. We previously showed that CD81, a surface protein normally expressed by thymic epithelial cells in the fetal thymus, is involved in the phenotypic conversion of immature CD4-CD8- thymocytes to more mature CD4+CD8+ cells. Currently, we are defining further the phenotype of immature thymocytes capable of interaction with CD81+ epithelial cells. This project includes analysis of defined cell-surface molecules, proteins associated with the cell cycle, and rearrangements of the genes for the T-cell receptor in immature thymocytes. In a related study, we are using biochemical and molecular techniques to isolate cell-surface proteins that interact with CD81.
Boismenu, R., Havran, W.L. T cells in host defense and epithelial cell biology. Clin. Immunol. Immunopathol., in press.
Boismenu, R., Havran, W.L. An innate view of T cells. Curr. Opin. Immunol. 9:57, 1997.
Boismenu, R., Hobbs, M.V., Boullier, S., Havran, W.L. Molecular and cellular biology of dendritic epidermal T cells. Semin. Immunol. 8:323, 1996.
Boismenu, R., Semeniuk, D., Murgita, R.A. Purification and characterization of human and mouse recombinant -fetoproteins expressed in Escherichia coli. Protein Expr. Purif. 10:10, 1997.
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Regulation of Cell Function by Rho GTPases
G.M. Bokoch, V. Benard, T.H. Chuang, R.H. Daniels, S. Dharmawardhane, C.C. King, T. Rudel, L.C. Sanders, F.T. Zenke, B.P. Bohl, P. Hall, Y. Wang
The Rho family of GTP-binding proteins (Rho, Rac, and Cdc42) has been a focus of research in our group. Rho GTPases control the assembly and activity of the actin cytoskeleton, the production of oxidants by leukocytes, the activity of regulatory kinase and phospholipase cascades, and signaling pathways that control cell growth. This spectrum of regulatory activities places the Rho GTPases as key components of such physiologic and pathologic processes as tumor growth and metastasis, wound healing, inflammatory responses, and even development. We have taken a variety of cellular, molecular, and biochemical approaches to understand how the activities of Rho GTPases are regulated, to determine the effector targets these proteins interact with to control cell function, and to determine how these regulatory processes are abnormal in various disease states.
CELLULAR REGULATION BY P21-ACTIVATED KINASES
During the course of studies on Rho GTPase regulation of human phagocyte function, we discovered that a family of serine/threonine kinases were targets for the active forms of Rac and Cdc42. Functional and structural analysis of the kinases, termed PAKs for p21-activated kinases, revealed their bifunctional nature. The C-terminal kinase domain phosphorylates substrates involved in regulating NADPH oxidase, stress responses, and the cellular actinomyosin system. The N terminus interacts with SH3 domain--containing proteins to modulate the actin cytoskeleton and direct polarized cell morphologies (Fig. 1).
PAKs thus appear to be potentially important mediators of cell motility, wound healing, tumor metastasis, neurite outgrowth, and many other processes that depend on the dynamics of the actin cytoskeleton. Our current studies, in collaboration with U.G. Knaus, Department of Immunology, and others at TSRI, are directed at using molecular approaches to understand the function, regulation, and biology of PAKs.
RHO GTPASES AS COMPONENTS OF APOPTOSIS
Cells involved in formation of tissues and organs, development of the immune system, inflammatory responses, and even abnormally growing precancerous cells are normally removed from the body by a highly regulated death response termed apoptosis. Although initiated by diverse stimuli, the death response occurs via a well-defined program involving dramatic biochemical and morphologic events that have been investigated at only a basic level. Thus, the cytoplasm of the dying cells condenses toward the nucleus as the edges of the cell retract. Membrane blebbing occurs during and at the completion of the contraction phase. Finally, the cell breaks into fragments that can be effectively removed by noninflammatory phagocytic mechanisms. How this complicated series of cytoskeletal events is controlled is unknown.
We have found that Rho GTPases participate in apoptosis at both biochemical and morphologic levels. Cdc42 can induce apoptotic death through its ability to stimulate the Jun/p38 kinase cascades. In collaborative studies with K. Hahn, Department of Cell Biology, we have generated Rho GTPases tagged with a fluorescent marker. These tagged enzymes will be used to determine the role of GTPases in regulating apoptotic cytoskeletal dynamics. Preliminary findings suggest critical roles for Rho GTPases and their effectors in controlling the cell death response.
We have established that PAK2 is a particularly important target for apoptotic proteases. PAK2 is cleaved into active N- and C-terminal pieces that can regulate aspects of the apoptotic program, including morphologic and membrane changes (Fig. 2).
Continuing studies will involve determining the importance of PAK2 cleavage during apoptosis of breast cancer cells and elucidating the molecular mechanisms of PAK action in the cell death program.
Bokoch, G.M., Wang, Y., Bohl, B.P., Sells, M.A., Quilliam, L.A., Knaus, U.G. Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J. Biol. Chem. 271: 25746, 1996.
Brzeska, H., Knaus, U.G., Wang, Z.-Y., Bokoch, G.M., Korn, E.D. p21-Activated kinase (PAK) has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I. Proc. Natl. Acad. Sci. U.S.A. 94:1092, 1997.
Chuang, T.H., Hahn, K., Lee, J.-D., Danley, D.E., Bokoch, G.M. The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes. Mol. Biol. Cell, in press.
Danley, D.E., Chuang, T.-H., Bokoch, G.M. Defective Rho GTPase regulation by IL-1b converting enzyme-mediated cleavage of D4 GDP dissociation inhibitor. J. Immunol. 157:500, 1996.
Dharmawardhane, S., Sanders, L.C., Daniels, R.H., Martin, S.S., Bokoch, G.M. Localization of p21-activated kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in stimulated cells. J. Cell Biol., in press.
Ding, J., Knaus, U.G., Bokoch, G.M., Badwey, J.A. The renaturable 69 and 63 kDa protein kinases that undergo rapid activation in chemoattractant-stimulated neutrophils are p21-activated kinases (Paks). J. Biol. Chem. 271:24869, 1996.
Ptasznik, A., Prossnitz, E.R., Yoshikawa, D., Smrcka, A., Traynor-Kaplan, A.E., Bokoch, G.M. A tyrosine kinase signaling pathway accounts for the majority of PIP3 formation in chemoattractant-stimulated human neutrophils. J. Biol. Chem. 271: 25204, 1996.
Rudel, T., Bokoch, G.M. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276:1571, 1997.
Sells, M.A., Knaus, U.G., Bagrodia, S., Ambrose, D., Bokoch, G.M., Chernoff, J. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7:202, 1997.
Thomas, J.E., Venugopalan, M., Galvin, R., Wang, Y., Bokoch, G.M., Vlahos, C.J. Inhibition of MG-63 cell proliferation and PGDF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase. J. Cell. Biochem. 64:182, 1997.
Xu, X., Wang, Y., Barry, D.C., Chanock, S., Bokoch, G.M. Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of responsiveness to guanine nucleotide dissociation stimulator. Biochemistry 36:626, 1997.
Zhou, K., Gorski, J.L., Chan, A., Kazusa cDNA group, Collard, J., Bokoch, G.M. Guanine nucleotide exchange factors dictate downstream signaling from Rac and Cdc42. J. Biol. Chem., in press.
Zigmond, S.H., Joyce, M., Borleis, J., Bokoch, G.M., Devreotes, P.N. Regulation of actin polymerization in cell-free systems by GTPgS and Cdc42. J. Cell Biol., in press.
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A key feature of the immune system is its ability to respond quickly and specifically to previously encountered pathogens by mobilizing memory T and B lymphocytes generated in response to initial exposure to antigens. Despite the fundamental nature of immunologic memory, little is known about mechanisms that control its development, particularly within the T-cell population. CD4 cells regulate immune responses by producing cytokines that affect the differentiation and functions of T cells, B cells, and antigen-presenting cells.
Studies in this laboratory have identified four stages of differentiation of CD4 cells in vivo that are distinguished by a combination of functional, phenotypic, and temporal criteria: naive precursors, primary effectors, resting memory cells, and memory effector cells. These stages mark the progressive differentiation of peripheral CD4 cells during an immune response and show that development of memory is a multistep process that begins with the response of naive CD4 cells to antigen. Once activated, naive cells differentiate into transient effector cells that secrete cytokines. Although some effector cells are terminal cells that undergo apoptosis as the primary response subsides, many return to rest, becoming memory cells. Our current studies focus on the role of antigens, antigen-presenting cells, and cytokines in the development of memory CD4 subsets, as shown by secretion of cytokines associated with type 1 (IFN-, TNF-ß, and IL-2) and with type 2 (IL-4, IL-5, IL-6, and IL-10) T-helper cells in responses elicited by reexposure to antigen. Our studies indicate that memory CD4 cells influence their own development more so than do naive cells through autocrine use of IFN- and IL-4, cytokines that regulate differentiation of type 1 and type 2 T-helper cells, respectively.
Adhesion molecules such as CD44 and ß1, ß2, and ß7 integrins are upregulated after activation of naive CD4 cells, and on memory cells, the concentrations remain elevated, resulting in fundamental alterations in the biology and migratory behavior of antigen-primed CD4 lymphocytes. Greater expression of adhesion molecule contributes to decreased requirements for costimulation of memory cells compared with naive cells and to the capacity for activation with a broad range of antigen-presenting cells by lower doses of antigen.
Differences in adhesion molecules are also thought to underlie distinct recirculation pathways of memory and naive CD4 cells. Our studies of murine CD4 cells have shown that unlike naive cells, most memory cells lack L-selectin and do not home to lymphoid tissues where entry is controlled through high endothelial venules. Recirculation of naive cells in these sites can be abolished by a blockade of L-selectin and 4 integrins. In contrast, memory cells remain in the blood and tissues, where no adhesion requirements for entry of cells have been found. Many memory CD4 cells are found in the spleen, where they are ideally situated to encounter systemic antigens, and are recruited to sites of antigen exposure. We are using transgenic mouse models to study the regulation of infiltration of CD4 subsets into sites of inflammation such as the pancreas in autoimmune diabetes, lung allografts, and inflamed skin by cytokines, chemokines, and antigen.
Balasa, B., Deng, C., Lee, J., Bradley, L.M., Dalton, D.K., Christadoss, P., Sarvetnick, N. IFN-g is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice. J. Exp. Med., in press.
Bradley, L.M., Dalton, D.K., Croft. M. A direct role for IFN-g in regulation of Th1 development. J. Immunol. 157:1350, 1996.
Bradley, L.M., Malo, M.E, Tonkonogy, S., Watson, S.R. L-selectin is not essential for naive CD4 trafficking or development of primary responses in Peyer's patches. Eur. J. Immunol. 27:1140, 1997.
Bradley, L.M., Watson, S.R. Lymphocyte migration into tissue: The paradigm derived from CD4 subsets. Curr. Opin. Immunol. 8:312, 1996.
Dutton, R.W., Bradley, L.M., Swain, S.L. T-cell memory. Adv. Immunol., in press.
Swain, S.L., Croft, M., Dubey, C., Haynes, L. Rogers, P., Zhang, X., Bradley, L.M. From naive to memory T cells. Immunol. Rev. 150:143, 1996.
Yoshimoto, K., Swain, S.L., Bradley, L.M. Enhanced development of Th2-like primary CD4 effectors in response to sustained exposure to limited rIL-4 in vivo. J. Immunol. 156: 3267, 1996.
Zhang, X., Brunner, T., Carter, L., Dutton, R.W., Rogers, P., Bradley, L.M., Sato, T., Reed, J., Green, D., Swain, S.L. Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185:1837, 1997.
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Human Antibodies and Viral Infection
D.R. Burton, P.W.H.I. Parren, R.B. Bastidas, H.J. Ditzel, A. Hessell, P. Fisicaro, E. E. Karrer, T. Maruyama, P. Poignard, H. Sakurai, M. Wang, R.A. Williamson, C.F. Barbas III,* R.M. Chanock,** C.J. Peters,*** P.P. Sanna****
* Department of Molecular Biology, TSRI
** National Institute of Allergy and Infectious Diseases, Bethesda, MD
*** Centers for Disease Control and Prevention, Atlanta, GA
**** Department of Neuropharmacology, TSRI
The perception of humans as easy victors over microbes has changed dramatically in the last decade. Vaccination has offered protection against a number of viral pathogens, but it is increasingly recognized that the strategies used in the past will not be successful against all viruses. More understanding of viral pathogenesis and of the interaction of viruses with the immune system is required. We focus on the interplay of antibodies and viruses in humans. We make particular use of combinatorial antibody technology, which gives ready access to human antibodies. The viruses we are studying are HIV type 1 (HIV-1), respiratory syncytial virus (RSV), herpes simplex viruses 1 and 2 (HSV-1, HSV-2), and Ebola virus.
For HIV-1, we have generated a large panel of antibodies against the proteins gp120 and gp41, which in trimeric form make up the envelope spike of the virus. This panel has enabled us to map the accessible epitopes on gp120 and gp41 in some detail. We have shown that the overwhelming majority of human antibodies to these proteins do not neutralize the virus and do not bind to native trimeric forms of the envelope. These findings have led us to suggest that the predominant antibody response in HIV-1 infection may be to forms of the envelope protein produced during lysis of infected cells ("viral debris") rather than to native virions. This conclusion was supported when live virus was used to select antibodies from libraries of human antibodies obtained from HIV-seropositive donors. The number of antibodies obtained was much less than the number obtained with isolated envelope proteins.
However, some antibodies capable of neutralizing laboratory strains and primary isolates of HIV-1 have been derived. In a trial by the National Institutes of Health, one particularly potent antibody was one of only three antibodies generated worldwide that were capable of broad, potent neutralization of primary isolates of HIV-1. The antibody protected against infection with primary isolates of HIV in an animal model in which mice populated with human cells were challenged with HIV-1. This result was the first time that an antibody had protected against infection by a primary isolate of HIV in an animal model. Furthermore, we have shown that the antibody provides protection even when given several hours after exposure to HIV. This finding greatly enhances the possible usefulness of the antibody in prophylactic therapy given after accidental exposure to HIV and in the interruption of mother-to-child transmission of the virus. It is also of considerable encouragement in attempts to generate an antibody-based vaccine to HIV.
RSV is the most important cause worldwide of severe viral infection of the lower respiratory tract in young children. Antibodies are known to mediate resistance to RSV infection. We have isolated a potent human monoclonal Fab fragment that neutralizes diverse RSV isolates and is highly effective therapeutically when administered intranasally to infected mice at the time of peak viral replication in the lungs. These results suggest that human recombinant Fabs, possibly as an aerosol, might be useful in the treatment of serious RSV disease and in other viral diseases in which replication of the virus in vivo is limited primarily to the lumenal lining of the respiratory tract.
HSV-1 and HSV-2 are associated with a number of conditions of variable severity, which are only partially responsive to current therapies. We have prepared panels of Fabs to HSV-1 and HSV-2 from antibody phage display libraries. One Fab effectively neutralizes HSV-1 and HSV-2, and a whole antibody version of this molecule is a potent antiviral in animal models.
Ebola virus attracted much attention in 1995 after an outbreak of an infection caused by this virus in Kikwit, Zaire, killed more than 300 people, with a mortality rate of 80%. We are interested in whether treatment with antibody can control such a rampant virus. We obtained bone marrow from survivors of the epidemic, constructed phage libraries, and isolated a panel of human antibodies. A number of these antibodies bind effectively to native viral envelope on infected cells, and we plan to investigate the ability of these antibodies to protect against viral challenge in animal models.
Barbas, C.F. III, Burton, D.R. Selection and evolution of high-affinity human anti-viral antibodies. Trends Biotechnol. 14:230, 1996.
Binley, J.M., Ditzel, H.J., Barbas, C.F. III, Sullivan, N., Sodroski, J., Parren, P.W.H.I., Burton, D.R. Human antibody responses to HIV-1 glycoprotein 41 cloned in phage display libraries suggest three major epitopes are recognized and give evidence for conserved antibody motifs in antigen binding. AIDS Res. Hum. Retroviruses 12:911, 1996.
Burton, D.R. Immunoglobulin, functions. In: Encyclopedia of Immunology. Roit, I.M., Delves, P.J. (Eds). Academic Press, San Diego, in press.
Burton, D.R. A vaccine for human immunodeficiency virus type 1: The antibody perspective. Proc. Natl. Acad. Sci. U.S.A., in press.
Burton, D.R., Montefiori., D.C. The antibody response in HIV-1 infection. AIDS 11(suppl A):S87, 1997.
Ditzel, H.J., Itoh, K., Burton, D.R. Determinants of polyreactivity in a large panel of recombinant human antibodies from HIV-1 infection. J. Immunol. 157:739, 1996.
Ditzel, H.J., Parren, P.W.H.I., Binley, J.M., Sodroski, J., Moore, J.P., Barbas, C.F. III, Burton, D.R. Mapping the protein surface of HIV-1 gp120 using human monoclonal antibodies from phage display libraries. J. Mol. Biol. 267:684, 1997.
D'Souza, M.P., Livnat, D., Bradac, J.A., Bridges, S., the AIDS Clinical Trials Group Antibody Selection Working Group, collaborating investigators. Evaluation of monoclonal antibodies to HIV-1 primary isolates by neutralization assays: Performance criteria for selecting candidate antibodies for clinical trials. J. Infect. Dis. 175:1056, 1997.
Graus, Y.F., de Baets, M.H., Parren, P.W.H.I., Berrih-Aknin, S., Wokke, J., van Breda Vriesman, P.J., Burton, D.R. Human anti-nicotinic acetylcholine receptor recombinant Fab fragments isolated from thymus-derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies. J. Immunol. 158:1919, 1997.
Kessler, J.A. II, McKenna, P.M., Emini, E.A., Chan, C.P., Patel, M.D., Gupta, S.K., Mark, G.E. III, Barbas, C.F. III, Burton, D.R., Conley, A.J. Recombinant human monoclonal antibody IgG1b12 neutralizes diverse human immunodeficiency virus type 1 primary isolates. AIDS Res. Hum. Retroviruses 13:575, 1997.
Li, A., Baba, T.W., Sodroski, J., Zolla-Pazner, S., Gorny, M.K., Robinson, J., Posner, M.R., Katinger, H., Barbas, C.F. III, Burton, D.R., Chou, T.-C., Ruprecht, R.M. Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins. AIDS Res. Hum. Retroviruses 13:647, 1997.
Maruyama, T., Parren, P.W.H.I., Sanchez, A., Rensink, I., Rodriguez, L.L., Khan, A.S., Peters, C.J., Burton, D.R. Recombinant human monoclonal antibodies to Ebola virus. J. Infect. Dis., in press.
Parren, P.W.H.I., Burton, D.R. Antibodies against HIV-1 from phage display libraries: Mapping of an immune response and progress towards anti-viral immunotherapy. Chem. Immunol. 65:18, 1997.
Parren, P.W.H.I., Fisicaro, P., Labrijn, A., Ditzel, H.J., Yang, W.-P., Barbas, C.F. III, Burton, D.R. In vitro antigen challenge of human antibody libraries for vaccine evaluation: The human immunodeficiency virus type 1 envelope. J. Virol. 70:9046, 1996.
Parren, P.W.H.I., Gauduin, M.-C., Koup, R.A., Poignard, P., Fisicaro, P., Burton, D.R. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol. Lett., in press.
Parren, P.W.H.I., Sattentau, Q.J., Burton, D.R. HIV-1 antibody: Debris or virion? Nature Med. 3:366, 1997.
Seligman, S.J., Binley, J.M., Gorny, M.K., Burton, D.R., Zolla-Pazner, S., Sokolowski, K.A. Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies. Mol. Immunol. 33:737, 1996.
Williamson, R.A., Lazzaroto, T., Sanna, P.P., Bastidas, R.B., Dalla Casa, B., Campisi, G., Burioni, R., Landini, M.P., Burton, D.R. Recombinant human antibody fragments for detection of cytomegalovirus antigenemia. J. Clin. Microbiol., in press.
Zeitlin, L., Whaley, K.J., Sanna, P.P., Moench, T.R., Bastidas, R., DeLogu, A., Williamson, R.A., Burton, D.R., Cone, R.A. Topically applied human recombinant monoclonal IgG1 antibody and its Fab and F(ab´)2 fragments protect mice from vaginal transmission of HSV-2. Virology 225:213, 1996.
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Antibody Recognition of DNA
D.R. Burton, R.A. Williamson, K.E. McLane,* J.F. LeBlanc, P. Ghazal
* Genentech, San Francisco, CA
A general method for the generation of molecules capable of recognizing double-stranded DNA with high affinity and specificity would have wide implications in chemistry, biology, and medicine. For instance, such reagents could be useful in genome mapping by blocking certain sites to enzyme action, in diagnostic procedures requiring identification of certain DNA sequences, and in therapeutic regimens requiring control of gene expression. Because they recognize a huge number of diverse molecular shapes, antibodies are promising candidates for high-affinity sequence-specific recognition of DNA. However, the generation of monoclonal antibodies to DNA by conventional immunization procedures has not been generally successful, because double-stranded DNA is ineffective in eliciting an immune response in normal animals.
To circumvent this problem, we have explored a number of strategies, including the affinity selection of high-affinity DNA binders from synthetic antibody libraries displayed on phage and the generation of binders from phage display libraries prepared from tissue from donors with the autoimmune disease systemic lupus erythematosus. In another approach, we have transplanted a 17 amino acid -helical domain from a transcription factor into a hypervariable part of the antibody molecule and have shown that the motif confers sequence-dependent DNA recognition on the mutant antibody molecule. Our most recent studies indicate that it is possible to generate high-affinity, specific DNA-binding antibodies. These molecules are being investigated for their ability to function as transcriptional repressors and activators.
In related studies, we are looking at the ability of antibodies to enter cells and target the nucleus when tagged with various motifs. The combination of sequence-specific recognition of DNA and nuclear targeting might provide useful reagents for control of gene expression.
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Probing the Structure of the Prion Protein
D.R. Burton, R.A. Williamson, R.B. Bastidas, D. Peretz,* S.B. Prusiner*
* University of California, San Francisco, CA
Prion diseases are degenerative illnesses of the CNS that affect animals and humans. These diseases may be inherited because of mutations in the chromosomal gene that encodes the prion protein (PrP), arise sporadically, or be acquired by transmission of infectious prion particles. The fundamental change underlying prion diseases is the conversion of cellular PrP into the scrapie isoform. Attempts to determine a chemical difference between the two forms have been unsuccessful. The scrapie isoform is formed from the cellular form after translation by a process that involves a profound conformational change, possibly the conversion of -helical structures to a ß-sheet.
For many years, investigators thought that scrapie and the related human prion diseases kuru and Creutzfeldt-Jakob disease were caused by slow viruses. Among the most puzzling features of these transmissible diseases was the lack of any detectable immune response to inoculated infectious particles. Neither scrapie-specific antibodies nor a cellular immune response has been detected.
Antibodies most likely would be valuable probes for exploring the conformation of prions, but because of tolerance, few monoclonal antibodies, and none to mouse prions, have been developed. We have solved this problem and have generated a large array of mouse monoclonal antibodies to mouse PrP. We immunized mice in which the gene for PrP had been ablated and then "rescued" antibodies by using phage display libraries.
Antibodies to a range of conformational and linear epitopes have been generated. The linear epitopes have been mapped by using overlapping PrP peptides and selection from PrP-fragment libraries expressed on the surface of phage. With these antibodies, we showed that a major difference in conformation between the cellular and the scrapie isoforms of PrP can be localized to the N-terminal part of the PrP molecule. In contrast, epitopes at the C-terminal part are conserved in the two forms of PrP. Nuclear magnetic resonance studies showed that the C-terminal part of cellular PrP has a well-defined -helical structure and that the N-terminal part has considerable flexibility. The antibody results suggest that a structural ordering in this region may be crucial in the acquisition of protein infectivity.
In another study, we have used the antibodies to show that recombinant cellular PrP undergoes a major conformational transition at low pH and only slowly returns to the native conformation. The possibility that this conformation is related to an intermediate form between the cellular and the scrapie isoforms of PrP is under study.
Peretz, D., Williamson, R.A., Matsunaga, Y., Serban, H., Pinilla, C., Bastidas, R.B., Rozenshteyn, R., James, T.L., Houghten, R.A., Cohen, F.E., Prusiner, S.B., Burton, D.R. A conformational transition at the N-terminus of the prion protein features in formation of the scrapie isoform. J. Mol. Biol., in press.
Williamson, R.A., Peretz, D., Smorodinsky, N., Bastidas, R., Blochberger, T., Serban, A., DeArmond, S., Prusiner, S.B., Burton, D.R. Circumventing tolerance in order to generate autologous monoclonal antibodies to the prion protein. Proc. Natl. Acad. Sci. U.S.A. 93:7279, 1996.
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Molecular and Biological Mechanisms Regulating Angiogenesis and Cellular Invasion
C. Andrews, P. Brooks, B. Eliceiri, M. Friedlander, R. Klemke, J. Leng, T. Möhler, R. Molander, G. Nemerow, E. Petitclerc, A. Reddy, M. Rosenfeld, L.C. Sanders, S. Silletti, S. Strömblad, C. Storgard, D. Stupack, T. von Schalscha, L. Yeh, D.A. Cheresh
BLOCKING OF ANGIOGENESIS BY ANTAGONISTS OF
Antibodies, synthetic peptides, and organic antagonists directed to integrin vß3 or vß5 block angiogenesis in chicken embryos, mice, and rabbits. These antagonists cause tumor regression in these animals by disrupting the tumors' blood supply. In a rabbit model of rheumatoid arthritis, a cyclic peptide antagonist of vß3 not only blocked angiogenesis in the knee but also prevented the inflammatory events leading to a decrease in the losses of cartilage and bone associated with this disease. These antagonists produce no apparent side effects because they do not affect preexisting blood vessels. On the basis of these findings, a humanized form of a monoclonal antibody to vß3 is being evaluated in a phase I clinical trial in patients with late-stage cancer. Thus far, the antibody appears to be safe and is providing clinical benefit to a number of these patients. Additional trials are being planned to test small synthetic peptide and nonpeptide antagonists in patients with cancer, arthritis, and ocular diseases.
ROLE OF MATRIX METALLOPROTEINASE 2 IN ANGIOGENESIS IN VIVO
The collagenase matrix metalloproteinase 2 (MMP-2) binds to the surface of invasive cells by virtue of its ability to interact with integrin vß3. This binding depends on the C-terminal hemopexin domain of the enzyme, because removal of the domain makes MMP-2 incapable of binding to cells bearing vß3. A recombinant fragment consisting of the hemopexin domain of MMP-2 blocked binding of vß3 to intact MMP-2. This recombinant fragment also blocked ongoing angiogenesis on chick chorioallantoic membranes in response to cytokines or human tumors. Systemic administration of this hemopexin domain blocked the activation of MMP-2 in tissues undergoing angiogenesis. Therefore, inhibition of MMP-2 binding to vß3 not only prevents enzyme activation and matrix degradation but also is a novel mechanism for disrupting angiogenesis in vivo.
MAP KINASE ACTIVITY DURING ANGIOGENESIS INDUCED BY BASIC FIBROBLAST GROWTH FACTOR
Angiogenesis depends on growth factors and vascular cell adhesion. Integrins and growth receptors have been colocalized on the cell surface and appear to cooperate in activating the ras/MAP kinase pathway. On initiation of angiogenesis by basic fibroblast growth factor, vascular cell MAP kinase was activated within 5 minutes, and activation was sustained for 20 hours. The initial MAP kinase activity (<2 hr) was refractory to integrin antagonists, whereas the sustained activity (>4 hr) depended on ligation of vß3 and was essential for angiogenesis. A MAP kinase kinase inhibitor administered during this sustained vß3-dependent signal also blocked MAP kinase activity and angiogenesis. Thus, angiogenesis depends on ligation of vß3, which promotes sustained MAP kinase activity in blood vessels stimulated with angiogenic growth factors.
REGULATION OF CELL MIGRATION
Interaction of cells with extracellular matrix proteins, cytokines, or growth factors promotes signaling events that regulate cell migration. Ligation of integrin or cytokine receptors induced MAP kinase activity and cell locomotion on the extracellular matrix. Activated MAP kinase influenced the cells' motility machinery by directly phosphorylating myosin light chain kinase, a step that led to increased phosphorylation of myosin light chains. In addition, cytokine- and integrin-mediated cell migration involved the adaptor proteins c-crk and CAS. Expression of c-crk and CAS in cells promoted interaction between the two proteins and the induction of cell movement. Moreover, activation of these signaling pathways is associated with induction of tumor cell invasion and pulmonary metastasis in vivo.
ROLE OF INTEGRINS AND SIGNALING MOLECULES IN CELL MIGRATION
Videomicroscopy can be used to measure cell movement in response to a growth factor gradient. By introducing genes coupled to green fluorescent protein, we are able to study how signaling molecules modulate the cell actin-myosin cytoskeleton and motility in living cells. A camera enables us to follow these molecules inside the migratory cells in real time or with time-lapse videography. These studies address how cell motility is regulated at the molecular and cellular level.
INTERACTION OF INTEGRIN CYTOPLASMIC DOMAINS WITH MYOSIN HEAVY CHAINS
Ligation of integrins promotes coordination of the microfilament cytoskeleton during cell motility, and although originally indicated as the molecular motor of muscle cells, myosin has been implicated in other functions, including contractility during mitosis and collagen contraction. We detected a region of the conventional type II myosin heavy chain that interacts directly with the cytoplasmic domain of the subunits of ß integrins. This domain comprises the C terminus of the nonmuscle B isoform of myosin, a variant originally characterized as embryonic. This finding implies a role for this interaction in migration during embryogenesis. Differential specificity for distinct subunits of ß integrins suggests discriminate regulation of myosin-integrin coupling, and because this region is highly divergent among myosin isoforms, another level of specificity may exist. Thus, we have defined a novel linkage between integrins and the cytoskeleton that is potentially important in cellular migration and cell contractility.
LIGAND VALENCY AND INTEGRIN ACTIVATION
The extracellular matrix undergoes dynamic reorganization during a variety of processes, including wound healing and neovascularization. We examined the influence of ligand multimerization on hematopoietic cells expressing integrin vß3 and found that multimeric ligands induce cellular adhesion. Adhesion requires local valency but is not influenced by gross ligand avidity. Signaling studies have revealed a role for pp72 syk in directing attachment, but interactions with either ligand form can activate alternative pathways. These results indicate a significant role for the local extracellular environment in directing adhesion of hematopoietic cells and provide an alternative to modulation of activation by chemokine and antigen receptors.
Boudreau, N., Andrews, C., Srebrow, A., Ravanpay, A., Cheresh, D.A. Regulation of the angiogenic phenotype by Hox D3. J. Cell Biol., in press.
Brooks, P.C., Klemke, R.L., Schön, S., Lewis, J.M., Schwartz, M.A., Cheresh, D.A. Insulin-like growth factor receptor cooperates with integrin vß5 to promote tumor cell dissemination in vivo. J. Clin. Invest. 99:1390, 1997.
Filardo, E.J., Deming, S.L., Cheresh, D.A. Regulation of cell migration by the integrin ß subunit ectodomain. J. Cell Sci. 109:1615, 1996.
Friedlander, M., Theesfeld, C.L., Sugita, M., Fruttiger, M., Thomas, M.A., Chang, S., Cheresh, D.A. Involvement of integrins vß3 and vß5 in ocular neovascular diseases. Proc. Natl. Acad. Sci. U.S.A. 93:9764, 1996.
Klemke, R.L., Schuang, C., Giannini, A.L., Gallagher, P.J., de Lanerolle, P., Cheresh, D.A. Regulation of cell motility by mitogen-activated protein kinase. J. Cell Biol. 137:481, 1997.
Mousa, S.A., Cheresh, D.A. Recent advances in cell adhesion molecules and extracellular matrix proteins: Potential clinical implications. Drug Discovery Today 2:9, 1997.
Okada, Y., Copeland, B.R., Hamann, G.F., Koziol, J.A., Cheresh, D.A., del Zoppo, G.J. Integrin vß3 is expressed in selected microvessels following focal cerebral ischemia. Am. J. Pathol. 149:37, 1996.
Strömblad, S., Becker, J.C., Yebra, M., Brooks, P.C., Cheresh, D.A. Suppression of p53 and p21WAF1/CIP1 expression by vascular cell integrin vß3 during angiogenesis in vivo. J. Clin. Invest. 98:426, 1996.
Strömblad, S., Cheresh, D.A. Cell adhesion and angiogenesis. Trends Cell Biol. 6:462, 1996.
Strömblad, S., Cheresh, D.A. Integrins, angiogenesis and vascular cell survival. Chem. Biol. 3:881, 1996.
Varner, J.A., Cheresh, D.A. Integrins and cancer. Curr. Opin. Cell Biol. 8:724, 1996.
Yebra, M., Parry, G.C.N., Strömblad, S., Mackman, N., Rosenberg, S., Mueller, B.M., Cheresh, D.A. Requirement of receptor-bound urokinase-type plasminogen activator for integrin vß3-directed cell migration. J. Biol. Chem. 271:29393, 1996.
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The Therapeutic Effect of Dilute KL4-Surfactant in Inflammatory Disease of the Lung
C.G. Cochrane, S.D. Revak, T.A. Merritt,* I.U. Schraufstatter, R.C. Hoch, C. Henderson,**
* St. Charles Medical Center, Bend, OR
** University of California, San Diego, CA
Pulmonary surfactant is a complex of phospholipids and proteins that forms a monolayer along the alveolar surface of pulmonary epithelial cells. Its main function is to prevent collapse of the alveoli caused by the recoil of elastic tissue in the alveolar septa. The surfactant protein with greatest activity for this function is surfactant protein B. When mixed with appropriate phospholipids, surfactant protein B induces the greatest surfactant function. Antibodies to surfactant protein B inhibit activity of the surfactant and induce atelectatic collapse in the lungs of rabbits. In addition, in human infants and mice deficient in surfactant protein B, inflation of the lungs does not occur despite normal levels of the other surfactant proteins and phospholipids.
Surfactant protein B is a peptide of 80 amino acids and consists of stretches of hydrophobic residues with intermittent hydrophilic residues; 9 of 12 of the hydrophilic residues are basic and have a positive charge. Surfactant protein B is soluble in lipids or organic solvents but not in aqueous media. Synthetic peptides that mimic the pattern of surfactant protein B function the same as the native peptide in aqueous dispersions of phospholipids. These mimic peptides consist of arginine or lysine (K) residues along with leucines (L), representing the hydrophobic residues. An example of these is KLLLLKLLLLKLLLLKLLLLK (KL4).
When tryptophan was incorporated in the leucine stretches, the fluorescence emission maximum indicated that the peptide lies in the acyl side chains, close to the polar-head groups. Removal of the charged arginine or lysine residues markedly decreased surfactant activity of the peptides, and magnetic resonance assays indicated an electrostatic interaction between lysine residues and negatively charged residues of the polar-head groups. Raman vibrational spectroscopy suggested, over a temperature range of 6--46°C, that the peptides increase lateral stability of the phospholipid molecules. These data provide the basis for a theory on the mechanism of surfactant protein B (or its peptide mimics) in providing the stability essential for surfactant function by a phospholipid layer.
The combination of KL4 peptide and the phospholipids dipalmitylphosphatidylcholine and palmityloleoylphosphatidylglycerol provided strong surfactant activity in fetal rabbits; macaques; and, in a phase I/II trial, in 39 human infants. In the human infants, treatment with KL4-Surfactant was begun within 4 hours of birth. Average weight of the infants was 1148 ± 279 g, and the average ratio of arterial to alveolar oxygen concentration was 0.14 ± 0.06. After treatment, the average ratio increased into the normal range within 12 hours. Fractions of inspired oxygen, mean airway pressures, and oxygen index decreased in inverse proportion to the arterial-to-alveolar ratio. No fatalities related to respiratory distress syndrome occurred. A single instillation of KL4-Surfactant was sufficient in all but a few of the infants. No safety issues were detected.
A new method has been devised to administer KL4-Surfactant to inflamed lungs of adult and infant animals with respiratory distress. Two models have been used in this regard: meconium aspiration in 2.5-kg rabbits and newborn macaques and lipopolysaccharide-induced injury of the lung after saline lavage to remove intrinsic surfactant in 2.5-kg rabbits. After injury developed in response to the lipopolysaccharide or meconium in 2.5-kg rabbits or, with meconium, in newborn macaques, the lungs of the animals were lavaged with a diluted form of KL4-Surfactant. With two to three lavages, inflammatory exudate or meconium was mostly removed from the alveolar spaces, and the alveoli rapidly expanded with air. In control animals, saline lavages removed exudate or meconium, but the alveolar spaces did not expand. In addition, when a bolus of KL4-Surfactant was administered to adult rabbits with lipopolysaccharide-injured and inflamed lungs or to rabbits with meconium injury, various zones of the lungs showed partial expansion, but others showed continued collapse, and no loss of inflammatory exudate occurred.
The partial pressure of arterial oxygen (Pao2) in rabbits receiving lavage with dilute KL4-Surfactant and 100% oxygen increased from less than 100 mm Hg to more than 400 mm Hg in less than 1 hour, whereas the Pao2 in rabbits lavaged with saline did not increase to more than 100 mm Hg. In rabbits given a bolus of KL4-Surfactant, the Pao2 increased moderately (to 200--300 mm Hg) but, as the inflammation progressed, decreased to 100 mm Hg. Of interest, inflammation in the lungs of rabbits lavaged with dilute KL4-Surfactant did not develop further, whereas rabbits treated with saline lavage or a bolus of surfactant showed marked inflammation at the end of the study, 2--4 hours after treatment.
In collaboration with R.M. Smith, University of California, San Diego, 20-kg pigs were injured by using saline lavage to remove intrinsic surfactant. Dilute KL4-Surfactant was administered through a fiber-optic bronchoscope after lung injury had developed and Pao2 levels had been less than 100 mm Hg for more than 1 hour. Segmental lavage with the dilute surfactant resulted in expansion of the treated segments and removal of inflammatory exudate. When more than 50% of the lung was lavaged, Pao2 levels increased to more than 500 mm Hg within minutes.
With these experimental data available, clinical trials have been initiated in centers throughout the United States for two indications: acute respiratory distress syndrome in adults and meconium aspiration syndrome in newborn infants. The results of these phase I/II clinical trials will be forthcoming.
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Analysis of the Early Stages of Infection of Human B Cells by Epstein-Barr Virus
N. Sugano,* W. Chen , M.L. Roberts,** N.R. Cooper
* Nihon University, Tokyo, Japan
** ISIS Pharmaceuticals, Carlsbad, CA
Our studies address the early stages of infection of human B lymphocytes by Epstein-Barr virus (EBV), a transforming human herpesvirus with oncogenic potential. Infection is initiated by binding of the virus to CD21, a membrane glycoprotein that also serves as the cellular receptor for the C3dg activation and processing fragment of the third component of complement. Unlike most viruses, EBV infects nonactivated, noncycling, resting cells, although expression of the viral and cellular genes that mediate the earliest stages of infection depends on the cellular transcription machinery. We evaluated the hypothesis that an intracellular signaling pathway triggered by EBV binding to CD21 enables EBV to infect resting B cells.
In exploring the signaling events initiated by EBV binding to CD21, we found that the ubiquitous transcription factor NF-B was rapidly activated and reached peak levels 30 minutes after binding of the virus to the membrane of purified resting human B cells. Activation of NF-B was triggered by ligand binding to the receptor, because the viral glycoprotein mediating EBV binding to CD21, C3dg, and a monoclonal antibody to CD21 all activated the transcription factor. Activation of NF-B was relevant for infection, because different inhibitors of NF-B blocked transcription of the earliest expressed viral gene and subsequent infection.
The target of NF-B activated by EBV binding to CD21 was the initial viral promoter, Wp, which contains an NF-B--like sequence. Transfection studies showed that NF-B activated the native Wp sequence but not a Wp construct with an altered NF-B--binding sequence, a finding that shows functional relevance. After binding to resting uninfected cells, transcription from Wp was initiated within the period of marked NF-B activation. Thus, EBV binding to its cell-surface receptor activates an NF-B--dependent signaling pathway that mediates activation of the initial viral promoter. This finding is the first example of a crucial role for an intracellular signaling pathway triggered by a virus-receptor interaction in permitting viral infection.
Latent membrane protein 1 (LMP1), one of the EBV latent genes, is the only EBV protein that has the properties of an oncogene in vitro and in vivo. In studies to evaluate the mechanisms involved in EBV-induced malignant transformation, LMP1 activated the ERK 1/2 mitogen-activated protein kinases. Cotransfection approaches showed that LMP1-mediated activation of the kinases proceeded via a ras-dependent pathway. Transfection studies also revealed that malignant transformation of fibroblasts initiated by LMP1 was ras dependent.
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Complement Activation by ß-Amyloid in Alzheimer's Disease
B.M. Bradt, N.R. Cooper
Alzheimer's disease is the most common cause of senile dementia, a symptom complex characterized by the age-related progressive loss of cognitive functions. The disease is characterized pathologically by the presence of large numbers of neuritic (senile) plaques in the hippocampus and neocortex, regions of the brain concerned with cognition and memory. The plaques are largely composed of a highly insoluble fibrillar form of a 40- to 43-residue ß-amyloid peptide (Aß). Damaged (dystrophic) neuronal processes, loss of neurons and synapses, increased numbers of activated (reactive) astrocytes and microglia, and components of the proinflammatory complement system are also associated with neuritic plaques.
Complement activation in the vicinity of neuritic plaques could generate the cytokine-like C5a peptide and trigger the influx and activation of astrocytes and microglia, because these cells have receptors for C5a and react to this peptide with activation and directed motion. The cell-signaling and cytotoxic properties of the complement membrane attack complex (C5b-7, C5b-8, and C5b-9) could potentially damage neurons and activate glial cells. For complement to be biologically relevant in this manner to the progression of Alzheimer's disease, the Aß peptide must be able to activate complement, because activation is a prerequisite for all the biological actions of the complement system.
We used a novel definitive approach to show that Aß directly and independently activates the classical and alternative complement pathways, leading to the formation of covalent complexes of Aß with the C3b and iC3b activation fragments of the third component of complement. We also found that Aß-mediated triggering of complement activates the terminal proinflammatory part of the complement reaction sequence and generates C5a and the C5b-9 complex. The C5b-9 membrane attack complex was functionally competent, because it could insert itself into neuronal precursor cell membranes and make the cells permeable to small molecules. These findings provide inflammation-based mechanisms to account for the presence of complement components in neuritic plaques, for the damage to neurons near neuritic plaques, and for the influx and activation of glial cells. They also have important potential implications for the therapy of Alzheimer's disease.
Cooper, N.R. Complement and viruses. In: The Human Complement System in Health and Disease. Volanakis, J., Frank, M. (Eds.). Marcel Dekker, New York, in press.
Cooper, N.R. Complement-dependent pro-inflammatory properties of the Alzheimer's disease ß-amyloid peptide. In: Controlling the Complement System for Novel Drug Development. Mazarakis, H., Swart, S.J. (Eds.). International Business Communications, Southborough, MA, 1997, p. 3.
Cooper, N.R. Complement-dependent virus neutralization. In: The Complement System. Rother, K., Till, G., Hänsch, M. (Eds.). Springer-Verlag, New York, in press.
Cooper, N.R. Evasion of complement mediated damage by microorganisms. In: The Complement System. Rother, K., Till, G., Hänsch, M. (Eds.). Springer-Verlag, New York, in press.
Roberts, M.L., Luxembourg, A.T., Cooper, N.R. Epstein-Barr virus binding to CD21, the virus receptor, activates resting B cells via an intracellular pathway that is linked to B cell infection. J. Gen. Virol. 77:3077, 1996.
Sugano, N., Roberts, M.L., Cooper, N.R. EBV binding to CD21 activates the initial viral promoter via NF-B induction, J. Exp. Med., in press.
Webster, S., Bradt, B., Rogers, J., Cooper, N.R. Aggregation state-dependent activation of the classical complement pathway by the amyloid ß peptide (A ß). J. Neurochem. 69:388, 1997.
Webster, S., Lue, L.-F., Brachova, L., Tenner, A., McGeer, P., Walker, D., Bradt, B., Cooper, N.R, Rogers, J. Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease. Neurobiol. Aging, in press.
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Apolipoprotein E, Inflammation, and Atherosclerosis
W.A. Boisvert, I.R. Nikoulin,* R. Terkeltaub,** L.K. Curtiss
* Lidak Pharmaceuticals, La Jolla, CA
** Veterans Affairs Medical Center, San Diego, CA
Apolipoprotein E (apoE) regulates the clearance of plasma cholesterol by mediating binding of lipoproteins to their cellular receptors. Previously, we determined that a synthetic peptide, which represents a linear dimeric repeat of apoE amino acid residues 141--155, binds the receptor for low density lipoprotein (LDL). To prepare a synthetic peptide that had high affinity for both cholesterol-rich lipoproteins and cellular receptors for lipoprotein, we synthesized an N-terminal acetylated dimer peptide. This peptide was soluble at neutral pH, formed stable peptide-LDL complexes in vitro, and enhanced the binding of LDL to cellular receptors for lipoprotein in vitro. When peptide-LDL complexes were injected into C57BL/6J apoE-deficient mice, the rate of removal of the complexes from the blood was threefold faster than that of LDL alone. Administration of free peptide resulted in a 30--40% reduction in total plasma cholesterol within 10 minutes, a finding that was accounted for by a 40% and a 26% reduction in plasma levels of very low density and intermediate density lipoproteins, respectively. Therefore, a synthetic peptide analog of apoE can selectively associate with cholesterol-rich lipoproteins and facilitate their immediate clearance from plasma.
ApoE also promotes efflux of cholesterol from foam cells, which are a hallmark of early atherosclerotic lesions. However, the role of another apolipoprotein, apoAI, in this process in vivo is unknown. We used mice deficient in both apoE and apoAI to test the hypothesis that lipoproteins containing apoAI both bind secreted cholesterol and transport it out of the lesions within the vessel wall.
We lethally irradiated mice deficient in apoE alone or in both apoE and apoAI and reconstituted them with bone marrow cells from wild-type mice. The rationale for this treatment was to generate two sets of animals, one with lesions that contained both apoAI and apoE (AI + E) and another with lesions that contained apoE only. This experimental manipulation was possible because the apoE in lesions was derived from the transplanted wild-type macrophages, whereas the apoAI was not.
Macrophage-derived apoE decreased the hypercholesterolemia of both experimental groups; however, the plasma levels of cholesterol were twofold to threefold higher in the AI + E animals than in the animals with lesions containing apoE only. Despite this higher concentration of plasma cholesterol, total cholesterol deposited in the aortas was twofold lower in the AI + E group, and the mean atherosclerotic lesion area was 45% smaller. Therefore, locally available apoAI participates in the efflux of free cholesterol and retards the progression of lesions.
Inflammation mediated by mononuclear leukocytes occurs in atherogenesis, and expression of the monocyte chemotactic C-C chemokine JE/MCP-1 has been described. However, the C-X-C chemokines such as IL-8 also are expressed in atherosclerotic lesions, although these mediators are best recognized as neutrophil chemotaxins. To understand the role of C-X-C chemokines in atherogenesis, we studied diet-induced atherosclerosis in LDL-receptor knockout mice that were repopulated with bone marrow cells from mice deficient in the mouse receptor for IL-8 (IL-8R). Analysis confirmed that the peripheral blood leukocytes in these mice did not express receptors for IL-8. The mice had splenomegaly and a lack of germinal centers in their spleens, known characteristics of IL-8R knockout mice.
After being fed an atherogenic diet, all LDL-receptor knockout mice had dramatically increased plasma levels of cholesterol, but the levels in the mice with receptors for IL-8 were about 30% higher than the levels in the mice that lacked receptors for IL-8. Of importance, the area of the lesion was double that of the area in IL-8R--deficient mice, a finding that strongly suggests that IL-8R expression plays a key role in the early onset of atherogenesis.
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Estrogen and Atherosclerosis
M.M. Marsh, V.R. Walker, J.C. Rutledge,* L.K. Curtiss, C.L. Banka
* University of California, Davis, CA
The decreased risk of heart disease associated with endogenous or exogenous estrogen has traditionally been attributed to a decrease in circulating levels of low density lipoprotein (LDL) associated with an increase in hepatic receptors for LDL. To examine alternative mechanisms of estrogen protection, we established a mouse model in which LDL levels do not respond to estrogen.
Mice deficient in receptors for LDL (LDLR-/- mice) were ovariectomized and randomly assigned to treatment groups that received physiologic levels of 17ß-estradiol, 17-estradiol, or a control substance implanted in slow-release pellets. Control LDLR-/- males and females had sham operations. Animals were fed a high-fat, high-cholesterol diet for 12 weeks. All mice were hypercholesterolemic at baseline (cholesterol, 218 ± 5 mg/dl compared with an average of 70 mg/dl in wild-type mice).
Although fasting levels of plasma cholesterol increased approximately fourfold in all treatment groups on the high-cholesterol diet, neither the sex of the animal nor the hormone treatment had any effect on cholesterol levels. Despite nearly identical plasma levels of total cholesterol, estrogen-treated ovariectomized mice (both 17- and 17ß-estradiol) had smaller aortic atherosclerotic lesions than did placebo-treated ovariectomized females and male sham-operated mice. These data suggest that estrogen can protect LDLR-/- female mice from atherosclerosis in the absence of LDLR-mediated changes in plasma levels of cholesterol. Studies are ongoing to determine the alternative mechanisms of estrogen protection, including the possible action of estrogen as an antioxidant in the LDLR-/- mice.
To examine the primary effects of estrogen on the artery wall, we have collaborated with J. Rutledge at the University of California, Davis, to examine the effect of female sex hormones on lipid accumulation in a model system of isolated, perfused, rat carotid arteries. When oxidatively modified LDL or native LDL was perfused separately through isolated arteries, oxidatively modified LDL accumulated in the artery wall to a greater extent than did native LDL. Exposure of the artery to estradiol before perfusion with native or modified LDL had no effect on the rate of accumulation of normal LDL in the artery wall but significantly decreased the rate of accumulation of oxidatively modified LDL. Because perfusion with oxidatively modified LDL increased endothelial permeability, we are examining the possibility that estrogens may act as membrane antioxidants to protect the endothelium from damage that results in arterial accumulation of lipid, an early feature of atherosclerosis.
Banka, C.L. Antioxidant properties of estrogen: Selective protection of high density lipoprotein. In: Proceedings of the Workshop on Hormonal, Metabolic and Cellular Influences on Cardiovascular Disease. Forte, T.E. (Ed). Futura, New York, 1997, p. 193.
Banka, C.L. Non-genomic actions of estrogens: Estrogens as antioxidants. In: Estrogen and the Vessel Wall. Rubanyi, G. (Ed.). Harwood Academic, Reading, England, in press.
Boisvert, W.A., Spangenberg, J., Curtiss, L.K. Role of leukocyte-specific LDL receptors on plasma lipoprotein cholesterol distribution and atherosclerosis in mice. Arterioscler. Thromb. Vasc. Biol. 17:340, 1997.
Choi, S.Y., Pang, L., Kern, P.A., Kayden, H.J., Curtiss, L.K., Vanni-Reyes, T.M., Goldberg, I.J. Dissociation of LPL and LDL: Effects of lipoproteins and anti-apoB antibodies. J. Lipid Res. 38:77, 1997.
Exner, M., Susani, M., Witztum, J.L., Hovorka, A., Curtiss, L.K., Spitzauer, S., Kerjaschki, D. Lipoproteins accumulate in immune deposits and are modified by lipid peroxidation in passive Heymann nephritis. Am. J. Pathol. 149:1313, 1996.
Massamiri, T., Tobias, P.S., Curtiss, L.K. Structural determinants for the interaction of lipopolysaccharide binding protein with purified human high density lipoproteins: Role of apolipoprotein A-I. J. Lipid Res. 38:516, 1997.
Phillips, M.L., Pullinger, C., Kroes, I., Kroes, J., Hardman, D.A., Chen, G., Curtiss, L.K., Gutierrez, M.M., Kane, J.P., Schumaker, V.N. A single copy of apoprotein B-48 is present on the human chylomicron remnant. J. Lipd Res. 38:1170, 1997.
Rutledge, J.C., Woo, M.M., Rezai, A.A., Curtiss, L.K., Goldberg, I.J. Lipoprotein lipase increases lipoprotein binding to the artery wall and increases endothelial layer permeability by formation of lipolysis products. Circ. Res., 80:819, 1997.
Sorci-Thomas, M.G., Curtiss, L.K., Parks, J.S., Thomas, M.J., Kearns, M.W. Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity. J. Biol. Chem. 272:7278, 1997.
Spangenberg, J., Curtiss, L.K. Influence of macrophage-derived apoprotein E on plasma lipoprotein distribution of apolipoprotein A-I in apoprotein E-deficient mice. Biochem. Biophys. Acta, in press.
Spencer, T.A., Clark, D.S., Johnson, G.A., Erickson, S.K., Curtiss, L.K. Feasibility of an immunoassay for mevalonolactone. Bioorg. Med. Chem. 5:873, 1997.
Webb, N., deBeer, M.C., Kindy, M., van der Westhuyzen, D.R., Banka, C.L., Rader, D.J., deBeer, F.C. Adenoviral vector-mediated over-expression of SAA in apo-AI-deficient mice. J. Lipid Res. 39:45, 1997.
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The Molecular Biology of Protease Receptors
T.S. Edgington, W. Ruf, C.D. Dickinson, P.E. Thorpe,* S. Dittmar,** P. Carmeliet,*** D. Collen,*** D.C. Altieri,**** A.C. Nicholson,***** R.L. Nachman,***** D.P. Hajjar,***** W. Risau+
* University of Texas Southwestern Medical Center, Dallas, TX
** Behringwerke AG--Hoechst Marion Roussel, Marburg, Germany
*** Center for Molecular and Vascular Biology, Leuven, Belgium
**** Yale University School of Medicine, New Haven, CT
***** Cornell University Medical College, New York, NY
+ Max-Planck Institute, Bad Nauheim, Germany
Tissue factor (TF), a structural member of the cytokine receptor superfamily, is the most extensively characterized protease receptor and a prototypic model for receptor-cofactor control of enzyme function. Apart from its role as the in vivo cell-surface initiator of the hemostatic and thrombogenic cascades and its role in the initiation of effector inflammatory responses associated with both innate immunity and antigen-driven cellular immune responses, recent evidence suggests that TF may have currently unknown ligands and resultant unanticipated biological functions. For example, TF expressed on the surface of tumor cells plays an important role in successful implantation and growth of hematogenous metastases, gene knockout studies indicate a role for TF in embryonic vascular development, and a role of TF as a recognitive molecule that can signal cellular responses has been advanced.
Furthermore, we have created soluble mutant forms of TF that have been fused to monoclonal antibodies, creating a "switchable" targetable thrombogen. In vivo, once it is switched on by docking on the vascular endothelium of a tumor, the thrombogen can eradicate solid tumors in mice by selective thrombosis of tumor vasculature and infarctive necrosis of tumor. Our studies of the three-dimensional structures of TF and of TF in complex with cognate ligands and specific monoclonal antibodies provide the knowledge needed to relate structure to function for this prototypic protease receptor and regulatory cofactor.
We have also studied other protease receptors, including effector protease receptor-1 (EPR-1), the receptor for coagulation factor Xa. The gene for EPR-1 is an activation-responsive gene that encodes expression of the receptor on endothelial cells and vascular smooth muscle cells after injury. Assembly of factor Xa on EPR-1 results in cellular responses important in the pathobiology of vascular injury and repair. We are determining the structures of the complex consisting of TF and factor VIIa and of the ternary complex consisting of TF, factor VIIa, and factor X. Elucidation of the structure of the ternary complex will provide insight to the cellular initiation of thrombosis and molecular detail. This information will facilitate more effective and safe molecular solutions to treatment of thrombotic diseases, the major cause of death in advanced societies.
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Transcriptional Regulation of the Gene for TNF-
S.-T. Fan, J. Yao, N. Mackman, T.S. Edgington
The cytokine TNF- has been implicated in a variety of inflammatory responses. The trimeric structure of the protein has been described, its cell-surface receptors have been determined, and the sometimes beneficial effects of antibody neutralization in vivo have been investigated. However, information is limited on the regulation of transcription of the gene for TNF- in cells of monocytic lineage, which are predominantly responsible for production of this cytokine in vivo.
We have now mapped and characterized various cis-acting regulatory elements in the promoter of the gene that are involved in initiation and control of transcription. Bacterial lipopolysaccharide was used as the agonist for induction of transcription of the gene for human TNF- in THP-1 cells of monoblastic leukemia origin. The 5´ aspect of the gene was mapped by transfecting the cells with plasmids containing various lengths of the putative promoter region and measuring the response of the reporter gene in the plasmid to stimulation with lipopolysaccharide.
The region from -182 to -37 had multiple regions that influenced the transcriptional response. Region I (-182 to -162) contained an overlapping Sp1/Egr-1 site. Region II (-119 to -88) contained CRE and NF-B sites; the NF-B site is the B3 site in the 5´ region of the gene. The results also showed constitutive occupancy of the CRE site by the proteins CREB and, to a lesser extent, c-Jun. Cell stimulation led to increased occupancy of the CRE site by c-Jun, with consequent displacement of CREB. The response to stimulation also resulted in occupancy of the EGR-1 site by EGR-1 protein and of the B3 site by the heterodimer p50/p65.
The CRE and B3 sites together conferred strong responsiveness to lipopolysaccharide to a heterologous promoter, although individually they were ineffective. The spacing in the DNA between CRE and B3 sites was critical, inferring cooperation between c-Jun and p50/p65 in the effective initiation and amplification of transcription of the gene for TNF- in these cells representative of cells of the monocytic lineage. Additional control elements have been suggested; however, the B3 and CRE elements appear to provide the major control of transcription in cells of monocytic lineage and to differ from control of this gene in cells of lymphocytic lineage.
Carmeliet, P., Mackman, N., Moons, L., Luther, T., Gressens, P., Van Vlaenderen, I., Demunck, H., Kasper, M., Breier, G., Evrard, P., Müller, M., Risau, W., Edgington, T., Collen, D. Role of tissue factor in blood vessel development. Nature 383:73, 1996.
Carmeliet, P., Moons, L., Dewerchin, M., Mackman, N., Luther, T., Breier, G., Ploplis, V., Müller, M., Nagy, A., Plow, E., Gerard, R., Edgington, T., Risau, W., Collen, D. Insights in vessel development and vascular disorders using targeted inactivation and transfer of vascular endothelial growth factor, the tissue factor receptor, and the plasminogen system. Ann. N. Y. Acad. Sci. 811:191, 1997.
Dittmar, S., Ruf, W., Edgington, T.S. Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation. Biochem. J. 321:787, 1997.
Edgington, T.S. More inflammatory effects of protease receptors. J. Clin. Invest. 99:2299, 1997.
Edgington, T.S., Dickinson, C.D., Ruf, W. The structural basis of function of the TF*VIIa complex in the cellular initiation of coagulation. Thromb. Haemost. 78:401, 1997.
Huang, X., Molema, G., King, S., Watkins, L., Edgington, T.S., Thorpe, P.E. Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature. Science 275:547, 1997.
Morrissey, J.H., Agis, H., Albrecht, S., Carson, S.D., Dignat-George, F., Edgington, T.S., Luther, T., Müller, M., Mutin, M., Nakamura, S., Valnet, P., Vercellotti, G.M. CD142 (tissue factor) workshop panel report. In: Leucocyte Typing VI. Garland Publishing, London, in press.
Nicholson, A.C., Nachman, R.L., Altieri, D.C., Summers, B.D., Ruf, W., Edgington, T.S., Hajjar, D.P. Effector cell protease receptor-1 is a vascular receptor for coagulation factor Xa. J. Biol. Chem. 271:28407, 1996.
Yao, J., Mackman, N., Edgington, T.S., Fan, S.-T. Lipopolysaccharide induction of the tumor necrosis factor- promoter in human monocytic cells: Regulation by Egr-1, c-Jun, and NF-B transcription factors. J. Biol. Chem., in press.
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Formation of B- and T-Cell Repertoires in Mice and Humans
A.J. Feeney, B. Nadel, K. Victor, G. Escuro, G. Lugo
The diversity of antibodies and T-cell receptors is due to both combinatorial and junctional diversity. The antigen-binding parts of each heterodimeric receptor are encoded by two V, one D, and two J segments, and many such germline segments are present at each receptor locus. These gene segments are far apart from each other in the genome, and in each lymphocyte precursor, a unique combination of V, D, and J gene segments recombines at the DNA level to form a continuous exon encoding the antigen-binding domain for that cell's receptor. In addition to this vast combinatorial diversity, significant diversity is created at the junctions of the V, D, and J segments by the removal of various numbers of nucleotides from the ends of the coding regions and the addition of nontemplated N nucleotides. The main focus of our laboratory is the analysis of factors that influence the composition of the primary repertoires of B and T cells. We are particularly interested in revealing biases imposed by the V(D)J recombination mechanism itself on nonrandom junctional diversity and gene utilization.
Although each receptor locus has many V, D, and J genes, the genes are not used equally. We are analyzing use of V genes in the human locus. We first analyzed the peripheral B-cell repertoire of newborn infants. Seventy-five percent of this repertoire was composed of only 9 V genes, and only 22 V genes of the 37 potentially functional V genes were detected at all. To determine if V genes recombine at different rates or whether this nonrandom use was due to antigenic selection, we are examining use of V genes in pre-B cells isolated from bone marrow. We also found nonrandom gene utilization in these pre-B cells, indicating that V genes do recombine at different rates. Strikingly, the V genes in the distal half of the locus were rarely used.
One factor likely to play a major role in this nonrandom recombination is the naturally occurring variation in the recombination signal sequences (RSSs) that flank each V, D, and J gene segment. The RSS is the site where the recombinase binds. Each change from the consensus reduces the efficiency of recombination of the adjacent gene segment to a variable degree. We have constructed plasmid-based competition recombination substrates to analyze the relative efficiency of recombination of various natural RSSs.
For example, the V gene A2 is the predominantly used V gene in the protective antibody response to the bacterium Haemophilus influenzae type b. Navajos have a 10-fold higher incidence of H. influenzae disease than other populations, and we previously found a new allele of A2, called A2b, in more than 50% of Navajos but not in control subjects. The A2b allele recombines much less frequently than the A2a allele does in vivo. Because one of the changes in the A2b allele is in the RSS, we tested the relative rate of recombination of the A2a and A2b alleles in these substrates. The A2b allele showed a fourfold to fivefold reduction in recombination in our in vitro system. Thus, the altered RSS of this gene is likely to be the reason for the gene's inability to recombine efficiently in vivo and thus may play a role in disease susceptibility.
During the past year, we have extended our analysis of junctional diversity through the use of these recombination substrates. Previously we observed that each V, D, and J coding end analyzed ex vivo had a unique pattern of nucleotide deletion. To further dissect the reasons for this nonrandom processing of coding ends, we put several of these natural coding ends, and also variants of them, into recombination substrates. The data derived from these studies directly show that the coding-end sequence dramatically influences the extent and pattern of nucleotide deletion. Because coding ends transiently undergo hairpin formation before being joined, we propose that the sequence of each coding end dictates a unique hairpin structure and that this structure determines how the hairpin is resolved. Thus, the sequence of each coding end directly influences the junctional diversity created from that gene segment.
Bain, G., Maandag, E.C.R., te Riele, H.P.J., Feeney, A.J., Sheehy, A., Schlissel, M., Shinton, S.A., Hardy, R.R., Murre, C. Both E12 and E47 allow commitment to the B cell lineage. Immunity 6:145, 1997.
Feeney, A.J., Lugo, G., Escuro, G. The human cord blood repertoire. J. Immunol. 158:3761, 1997.
McKercher, S.C., Torbett, B.E., Andersen, K.L., Henkel, G.W., Vestal, D.J., Baribault, H., Klemsz, M., Feeney, A.J., Wu, G., Paige, C., Maki, R.A. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15:5647, 1996.
Nadel, B., Feeney, A.J. Nucleotide deletion and P addition in V(D)J recombination: A determinant role of the coding end sequence. Mol. Cell. Biol. 17:3768, 1997.