liposomes, part c

Preface The origins of liposome research can be traced to the contributions by Alec Bangham and colleagues in the mid 1960s. The description of lecithin disper- sions as containing ‘‘spherulites composed of concentric lamellae’’ (A.D. Bangham and R.W. Horne, J. Mol. Biol. 8, 660, 1964) was followed by the observation that ‘‘the diffusion of univalent cations and anions out of spontan- eously formed liquid crystals of lecithin is remarkably similar to the diffusion of such ions across biological membranes (A.D. Bangham, M.M. Standish and J.C. Watkins, J. Mol. Biol. 13, 238, 1965). Following early studies on the biophysical characterization of multilamellar and unilamellar liposomes, inves- tigators began to utilize liposomes as a well-defined model to understand the structure and function of biological membranes. It was also recognized by pioneers including Gregory Gregoriadis and Demetrios Papahadjopoulos that liposomes could be used as drug delivery vehicles. It is gratifying that their efforts and the work of those inspired by them have lead to the development of liposomal formulations of doxorubicin, daunorubicin and amphotericin B now utilized in the clinic. Other medical applications of liposomes include their use as vaccine adjuvants and gene delivery vehicles, which are being explored in the laboratory as well as in clinical trials. The field has progressed enormously in the 38 years since 1965. This volume includes applications of liposomes in immunology, diagnostics, and gene delivery and gene therapy. I hope that these chapters will facilitate the work of graduate students, post-doctoral fellows, and established scientists entering liposome research. Other volumes in this series cover additional subdisciplines in liposomology. The areas represented in this volume are by no means exhaustive. I have tried to identify the experts in each area of liposome research, particularly those who have contributed to the field over some time. It is unfortunate that I was unable to convince some prominent investigators to contribute to the volume. Some invited contributors were not able to prepare their chapters, despite generous extensions of time. In some cases I may have inadvertently overlooked some experts in a particular area, and to these individuals I extend my apologies. Their primary contributions to the field will, nevertheless, not go unnoticed, in the citations in these volumes and in the hearts and minds of the many investigators in liposome research. xv I would like to express my gratitude to all the colleagues who graciously contributed to these volumes. I would like to thank Shirley Light of Academic Press for her encouragement for this project, and Noelle Gracy of Elsevier Science for her help at the later stages of the project. I am especially thankful to my wife Diana Flasher for her understanding, support and love during the seemingly never-ending editing process, and my children Avery and Maxine for their unique curiosity, creativity, cheer, and love. Finally, I wish to dedicate this volume to two other members of my family who have been influential in my life, with their love and support since my childhood days, my aunt Sevim Uygurer and my brother Dr. Arda Du ¨ zgu ¨ nes, Nejat Du ¨ zgu ¨ nes, xvi preface METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Contributors to Volume 373 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Salvador F. Alin ˜ o (26), Departamento de Famacologia, Facultad de Medicina, Uni- versidad de Valencia, Avda Blasco Ibanez 15, 46010 Valencia, Spain Carl R. Alving (2, 3, 10), Department of Membrane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 M. A. Arangoa (22), Department of Pharmacology and Pharmaceutical Technology, School of Pharmacy, Univer- sity of Navarra, 31080 Pamplona, Spain Udo Bakowsky (18), Department of Pharmaceutical Technology and Biophar- macy, University of Saarbruecken, Germany Richard R. Bankert (33), Department of Microbiology, SUNY at Buffalo, 138 Farber Hall, 3435 Main Street, Buffalo, New York 14214 Lajos Baranyi (10), Department of Mem- brane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Marta Benet (26), Departamento de Fa- macologia, Facultad de Medicina, Univer- sidad de Valencia, Avda Blasco Ibanez 15, 46010 Valencia, Spain Michael Bodo (10), Department of Mem- brane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Otto C. Boerman (15), Department of Nu- clear Medicine (565), University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Elena Bogdanenko (28), V N Orekhovich Institute of Biomedical Chemistry, Rus- sian Academy of Medical Sciences, 10, Pogodinska ya Street, 119832 Moscow, Russia Jeff W.M. Bulte (12), Department of Radiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Laura Bungener (5), Department of Med- ical Microbiology, Molecular Virology Section, University of Groningen, 9713 AV Groningen, The Netherlands Rolf Bunger (10), Department of Membrane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Gerardo Byk (23), Laboratory of Peptido- mimetics and Genetic Chemistry, Bar Ilan University, Department of Chemistry, 52900 Ramat Gan, Israel Jin-Soo Chang (9), Morgan Biotechnology Research Institute, 341 Pojung-Ri, Koon- sung-Myon, Youngin City, Kyonggi-Do 449-910, South Korea Myeong-Jun Choi (9), Charmzone Co.,Ltd., Bioimaterial Research Center, 301 Hankang Building, 184-11 Kwang- jang-dong, Kwangjin-ju, Seoul, Korea Jaime Crespo (26), Departamento de Fa- macologia, Facultad de Medicina, Univer- sidad de Valencia, Avda Blasco Ibanez 15, 46010 Valencia, Spain Toos Daemen (5), Department of Medical Microbiology, Molecular Virology Section, University of Groningen, 9713 AV Groningen, The Netherlands ix Sumeet Dagar (13), Departments of Pharmaceutics and Pharmacodynamics, University of Illinois at Chicago, 833 Wood Street, Chicago, Illinois 60612 Francisco Dası ´ (26), Departamento de Fa- macologia, Facultad de Medicina, Univer- sidad de Valencia, Avda Blasco Ibanez 15, 46010 Valencia, Spain Robert J. Debs (34), Geraldine Brush Cancer Research Institute, 2330 Clay Street, San Francisco, California 94115 Marcel De Cuyper (12), Interdisciplinary Research Center, Katholieke Universiteit Leuven, Campus Kortrijk, B-8500 Kortrijk, Belgium C. Tros De Ilarduya (22), Department of Pharmacology and Pharmaceutical Tech- nology, School of Pharmacy, University of Navarra, 31080 Pamplona, Spain Nejat Du ¨ zgu ¨ nes, (19, 22, 24, 28), Depart- ment of Microbiology, University of the Pacific School of Dentistry, 2155 Webster Street, San Francisco, California 94115 Nejat K. Eg ˇ ilmez (33), Department of Microbiology, SUNY at Buffalo, 138 Farber Hall, 3435 Main Street, Buffalo, New York 14214 Abdelatif Elouahabi (20), Center for Structural Biology and Bioinformatics, CP 206/2, Campus Plaine-ULB, Blv du Triomphe, 1050 Brussels, Belgium Henrique Faneca (19), Department of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, 3000 Coimbra, Portugal Sylvia Fong (34), Geraldine Brush Cancer Research Institute, 2330 Clay Street, San Francisco, California 94115 Benoı ˆ t Frisch (4), Laboratoire de Chimie Bioorganique, UMR 7514 CNRS-ULP, Faculte de Pharmacie, 74 Route du Rhin, Illkirch 67400, France Stephen J. Frost (16), Department of Clin- ical Biochemistry, The Princess Royal Hospital, Lewes Rd. Haywards Heath, West Suxxes RH16 3LU, England M. Teresa Gira ˜ o Da Cruz (24), Depart- ment of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, 3000 Coimbra, Portugal Laurent Giraudo (7), Centre d’Immuno- logie de Marseille-Luminy, Campus de Luminy, Case 906, 13288 Marsielle Cedex 09, France Mitsuru Hashida (25), Graduate School of Pharmaceutical Sciences, Kyoto Uni- versity, Sakyo-ku, Kyoto 606-850, Japan Kazuya Hiraoka (30), Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, 2-2 Yama- da-oka, Suita City, Osaka 565-0871, Japan Dick Hoekstra (18), Department of Mem- brane Cell Biology, University of Gron- ingen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands Leaf Huang (21), Center for Pharmacoge- netics, School of Pharmacy, University of Pittsburgh, 633 Salk Hall, Pittsburgh, Pennsylvania 15213 Anke Huckreide (5), Department of Med- ical Microbiology, Molecular Virology Section, University of Groningen, 9713 AV Groningen, The Netherlands Yasufumi Kaneda (30), Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, 2-2 Yamada- oka, Suita City, Osaka 565-0871, Japan Shigeru Kawakami (25), Faculty of Pharmaceutical Sciences, Nagasaki University, Magaski 852-8521, Japan Chong-Kook Kim (17), College of Phar- macy, Seoul National University, San 56-1, Shinlim-Doug, Kwanak-Gu, Seoul, South Korea x contributors to volume 373 Kenji Kono (27), Department of Applied Materials Science, Graduate School of Engineering, Osaka Prefecture Univer- sity, 1-1, Gakuencho, Sakai, Osaka 599-8531, Japan Krystyna Konopka (31), Department of Microbiology, University of the Pacific School of Dentistry, 2155 Webster Street, San Francisco, California 94115 Lakshmi Krishnan (11), Institute for Bio- logical Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada Peter E. Jensen (8), Department of Path- ology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322 Lawrence B. Lachman (6), Department of Bioimmunotherapy, Box 422, The Univer- sity of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas, 77030 Olivier Lambert (29), Institut Curie, Section de Recherche, UMR-CNRS 168 et LRC-CEA 8, 11 rue Pierre et Marie Curie, 75231 Paris, France Peter Laverman (15), Department of Nu- clear Medicine (565), University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Paul J. Lee (14), Vitreoretinal Surgical Fellow, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112 Lee Leserman (7), Centre d’Immunologie de Marseille-Luminy, Campus de Lu- miny, Case 906, 13288 Marsielle Cedex 09, France Song Li (21), Center for Pharmacogenetics, School of Pharmacy, University of Pitts- burgh, 633 Salk Hall, Pittsburgh, Penn- sylvania 15213 Soo-Jeong Lim (17), College of Pharmacy, Seoul National University, San 56-1, Shinlim-Doug, Kwanak-Gu, Seoul, South Korea Yong Liu (34), Geraldine Brush Cancer Research Institute, 2330 Clay Street, San Francisco, California 94115 Patrick Machy (7), Centre d’Immunologie de Marseille-Luminy, Campus de Lu- miny, Case 906, 13288 Marsielle Cedex 09, France Miguel Mano (19), Department of Bio- chemistry, Faculty of Sciences and Tech- nology, University of Coimbra, 3000 Coimbra, Portugal Gary R. Matyas (3), Department of Mem- brane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Nathalie Mignet (23), UMR 7001, Labor- atoire de Chimie Bioorganiquet et de Bio- technologie Moleculaire et Cellulaire, Ecole National Superieure de Chimie de Paris, 13 Quai Jules Guesde, BP 14, 94403 Vitry sur Siene, France Janos Milosevits (10), Department of Membrane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Alexey Moskovtsev (28), V N Orekhovich Institute of Biomedical Chemistry, Rus- sian Academy of Medical Sciences, 10, Pogodinska ya Street, 119832 Moscow, Russia Jean M. Muderhwa (3), Department of Membrane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Makiya Nshikawa (25), Graduate School of Pharmaceutical Sciences, Kyoto Uni- versity, Sakyo-ku, Kyoto 606-850, Japan contributors to volume 373 xi Volker Oberle (18), Department of Mem- brane Cell Biology, University of Gron- ingen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands Hayat O ¨ nkyu ¨ ksel (13), Departments of Pharmaceutics and Pharmacodynamics, University of Illinois at Chicago, 833 Wood Street, Chicago, Illinois 60612 Bu ¨ lent O ¨ zpolat (6), Department of Bioim- munotherapy, Box 422, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas, 77030 William M. Pardridge (32), University of California-Los Angeles, Warren Hall, 13- 164, 900 Veteran Avenue, Los Angeles, California 90024 Girishchandra B. Patel (11), Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada Ve ´ ronique Pector (20), Center for Struc- tural Biology and Bioinformatics, CP 206/2, Campus Plaine-ULB, Blv du Triomphe, 1050 Brussels, Belgium Maria C. Pedroso De Lima (19, 24), De- partment of Biochemistry, Faculty of Sci- ences and Technology, University of Coimbra, 3000 Coimbra, Portugal Nuno Penacho (19), Department of Bio- chemistry, Faculty of Sciences and Tech- nology, University of Coimbra, 300 Coimbra, Portugal Gholam A. Peyman (14), Ophthalmology Department (SL 69), Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112 Pedro Pires (24), Center for Neuroscience and Cell Biology, University of Coimbra, 3000 Coimbra, Portugal Olga Podobed (28), V N Orekhovich Insti- tute of Biomedical Chemistry, Russian Academy of Medical Sciences, 10, Pogo- dinska ya Street, 119832 Moscow, Russia Mangala Rao (2), Department of Mem- brane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Jean-Louis Rigaud (29), Institut Curie, Section de Recherche, UMR-CNRS 168 et LRC-CEA 8, 11 rue Pierre et Marie Curie, 75231 Paris, France Audrey Roth (4), Laboratoire de Chimie Bioorganique, UMR 7514 CNRS-ULP, Faculte de Pharmacie, 74 Route du Rhin, Illkirch 67400, France Stephen W. Rothwell (2), Department of Membrane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Israel Rubinstein (13), Departments of Pharmaceutics and Pharmacodynamics, University of Illinois at Chicago, 833 Wood Street, Chicago, Illinois 60612 Jean-Marie Ruysschaert (20), Center for Structural Biology and Bioinformatics, CP 206/2, Campus Plaine-ULB, Blv du Triomphe, 1050 Brussels, Belgium Sandor Savay (10), Department of Mem- brane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Daniel Scherman (23), UMR 7001, La- boratoire de Chimie Bioorganiquet et de Biotechnologie Moleculaire et Cellulaire, Ecole National Superieure de Chimie de Paris, 13 Quai Jules Guesde, BP 14, 94403 Vitry sur Siene, France Francis Schuber (4), Laboratoire de Chi- mie Bioorganique, UMR 7514 CNRS- ULP, Faculte de Pharmacie, 74 Route du Rhin, Illkirch 67400, France Karine Serre (7), Centre d’Immunologie de Marseille-Luminy, Campus de Luminy, Case 906, 13288 Marsielle Cedex 09, France xii contributors to volume 373 Se ´ rgio Simo ˜ es (19, 24), Department of Bio- chemistry, Faculty of Sciences and Tech- nology, University of Coimbra, 3000 Coimbra, Portugal G. Dennis Sprott (11), Institute for Bio- logical Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada Gert Storm (15), Department of Pharma- ceutics, Utrecht Institute for Pharmaceut- ical Sciences (UIPS), Utrecht University, The Netherlands Janos Szebeni (10), Department of Mem- brane Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Toru Takagishi (27), Department of Ap- plied Materials Science, Graduate School of Engineering, Osaka Prefecture Univer- sity, 1-1, Gakuencho, Sakai, Osaka 599-8531, Japan Marc Thiry (20), Laboratory of Cell and Tissue Biology, University of Liege, Rue de Pitteurs, Liege, Belgium Michel Vandenbranden (20), Center for Structural Biology and Bioinformatics, CP 206/2, Campus Plaine-ULB, Blv du Triomphe, 1050 Brussels, Belgium Esther Van Kesteren-Hendrikx (1), De- partment of Cell and Immunology, Faculty of Medicine, Vrije Universiteit Medical Center, Van de Boechhorststraat 7, 1081 BT Amsterdam, The Netherlands Nico Van Rooijen (1), Department of Cell and Immunology, Faculty of Medicine, Vrije Universiteit Medical Center, Van de Boechhorststraat 7, 1081 BT Amster- dam, The Netherlands Larry E. Westerman (8), VGS/DVRD/ NCID, Centers for Disease Control and Prevention, Atlanta, Georgia 30322 Barbara Wetzer (23), UMR 7001, Laboratoire de Chimie Bioorganiquet et de Biotechnologie Moleculaire et Cellulaire, Ecole National Superieure de Chimie de Paris, 13 Quai Jules Guesde, BP 14, 94403 Vitry sur Siene, France Jan Wilschut (5), Department of Medical Microbiology, Molecular Virology Section, University of Groningen, 9713 AV Groningen, The Netherlands Seiji Yamamoto (30), Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, 2-2 Yama- da-oka, Suita City, Osaka 565-0871, Japan Fumiyoshi Yamashita (25), Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-850, Japan Jing-Shi Zhang (21), Center for Pharma- cogenetics, School of Pharmacy, Univer- sity of Pittsburgh, 633 Salk Hall, Pittsburgh, Pennsylvania 15213 Renat Zhdanov (28), V N Orekhovich In- stitute of Biomedical Chemistry, Russian Academy of Medical Sciences, 10, Pogo- dinska ya Street, 119832 Moscow, Russia contributors to volume 373 xiii [1] ‘‘In Vivo’’ Depletion of Macrophages by Liposome-Mediated ‘‘Suicide’’ By Nico van Rooijen and Esther van Kesteren-Hendrikx Introduction Macrophages are multifunctional cells. They play a key role in natural and acquired host defense reactions, in homeostasis, and in the regulation of numerous biological processes. The main tools they use to achieve these goals are phagocytosis followed by intracellular digestion, and production and release of soluble mediators such as cytokines, chemokines, and nitric oxide (NO). Macrophages can be found as resident cells in all organs of the body, and they can be recruited to sites of inflammation. Their immediate precursors are monocytes, which are released in the blood circulation from the bone marrow. After some time, monocytes leave the circulation, cross the barrier formed by the walls of blood vessels, and enter into one of the organs, where their final differentiation into mature macrophages will take place. Depletion of macrophages followed by functional studies in such macrophage-depleted animals forms a generally accepted approach to es- tablish their role in any particular biomedical phenomenon. Early methods for depletion of macrophages were based on the administration of silica, carrageenan, or by various other treatments. However, incompleteness of depletion, and even stimulation of macrophages, as well as unwanted effects on nonphagocytic cells, were obvious disadvantages. 1 For that reason, we have developed a more sophisticated approach, based on the liposome-mediated intracellular delivery of the bisphospho- nate clodronate. 2,3 In this approach, liposomes are used as a Trojan horse to get the small clodronate molecules into the macrophage. Once ingested by macrophages, the phospholipid bilayers of liposomes are disrupted under the influence of lysosomal phospholipases. The strongly hydrophilic clodro- nate molecules intracellularly released in this way do not escape from the cell, because they will not easily cross its cell membranes. As a result, the intracellular clodronate concentration increases as more liposomes are ingested and digested. At a certain clodronate concentration, irrevers- ible damage causes the macrophage to be killed by apoptosis. 4,5 Clodronate 1 N. van Rooijen and A. Sanders, J. Leuk. Biol. 62, 702 (1997). 2 N. van Rooijen and R. van Nieuwmegen, Cell Tiss. Res. 238, 355 (1984). 3 N. van Rooijen and A. Sanders, J. Immunol. Meth. 174, 83 (1994). [1] depletion of macrophages by liposomes 3 Copyright 2003, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 373 0076-6879/03 $35.00 molecules released in the circulation from dead macrophages will not enter cells again, because they are not able to cross cell membranes. Moreover, free clodronate molecules show an extremely short half-life in circulation and body fluids. They are removed by the renal system. The combination of low toxicity and short half-life of clodronate makes this drug the best choice for the liposome-mediated elimination of macrophages ‘‘in vivo’’. Clodronate in its free form is used widely as a drug for the treat- ment of malignant hypercalcemia 6 and painful bone metastasis caused by hormone-refractory prostate cancer, 7 emphasizing its nontoxic nature. Clodronate Liposomes in Research Clodronate Liposomes as a Tool to Investigate Macrophage Activities In Vivo With the liposome-mediated macrophage ‘‘suicide’’ approach, func- tional aspects of macrophages have been established in hundreds of studies up to now. Many of the resulting publications are listed in the website http://www.ClodronateLiposomes.com. Although liposomes can not cross vascular barriers such as the walls of capillaries, their meeting with particular macrophage populations can be achieved by choosing the right administration routes. Among the macro- phages that might become useful targets for manipulation by liposomes are Kupffer cells in the liver and splenic macrophages (to be reached by way of intravenous injection), 8 alveolar macrophages in the lungs (to be reached by way of intratracheal instillation or intranasal administration), 9 phagocytic synovial lining cells (by means of intra-articular injection in the synovial cavity), 10 peritoneal macrophages (by means of intraperito- neal injection), 11 macrophages in the testis (by means of local injection), 12 4 N. van Rooijen, A. Sanders, and T. van den Berg, J. Immunol. Meth. 193, 93 (1996). 5 M. Naito, H. Nagai, S. Kawanao, H. Umezu, H. Zhu, H. Moriyama, T. Yamamoto, H. Takatsuka, and Y. Takkei, J. Leuk. Biol. 60, 337 (1996). 6 A. List, Arch. Intern. Med. 151, 471 (1991). 7 A. Heidenreich, R. Hofmann, and U. H. Engelmann, J. Urol. 165, 136 (2001). 8 N. van Rooijen, N. Kors, M. van den Ende, and C. D. Dijkstra, Cell Tiss. Res. 260, 215 (1990). 9 T. Thepen, N. van Rooijen, and G. Kraal, J. Exp. Med. 170, 499 (1989). 10 P. L. E. M. van Lent, A. E. M. Holthuyzen, L. A. M. van den Bersselaar, N. van Rooijen, L. A. B. Joosten, F. A. J. van de Loo, L. B. A. van de Putte, and W. B. van de Berg. Arthritis Rheum. 39, 1545 (1996). 11 J. Biewenga, B. van de Ende, L. F. G. Krist, A. Borst, M. Chufron, and N. van Rooijen, Cell Tiss. Res. 280, 189 (1995). 12 A. Bergh, J. E. Damber, and N. van Rooijen, J. Endocrinol. 136, 407 (1993). 4 liposomes in immunology [1] [...]... the intracellular fate of liposomes and liposomal antigens in macrophages Protein antigens are processed and presented either by the major histocompatibility complex (MHC) class I or class II pathways.10,11 MHC class I molecules are expressed on the surface of all nucleated cells In contrast, MHC class II molecules are expressed only on the surface of antigen presenting cells (APCs), such as macrophages,... (Brinkman/Buchi, Dumstat, Germany) Fluorescence microscope (Leitz Orthoplan, Leica, Deerfield, IL) with color digital camera (DEI-470, Optronics Engineering, Goleta, CA) Adobe Photoshop software (Adobe Systems, Inc., San Jose, CA) 4–20% Polyacrylamide Sodium dodecyl sulfate (SDS) gels, SDSPAGE equipment (BioRad, Hercules, CA) CO2 incubator (Forma Scientific, Inc Marietta, OH) Refrigerated centrifuge... and then incubated with L(TR-CON), followed by staining the cells with C6 -NBD-ceramide Cells incubated with L(TR-CON) in the absence of the inhibitor transported the fluorescent peptides into the Golgi area (A), TR-CON (B), (NBDceramide staining) In cells incubated with 10 M lactacystin, the TR-CON remained widely distributed throughout the cells (C) , TR-CON (D), (NBD-ceramide staining) Scale bar:... into the endoplasmic reticulum and the Golgi complex by way of the transporter associated with antigen processing (TAP).28 In these compartments, the peptides bind to the MHC class I molecules Once bound, the antigenic peptides are transported to the cell surface to interact with receptors on T cells.29,30 The procedures that we use to study the intracellular trafficking of liposome-encapsulated proteins... macrophages play a role in graft rejection by promotion of T-cell infiltration.28 Interestingly, recent evidence supports the idea that T-cell–activated macrophages themselves are capable of recognizing and rejecting pancreatic islet xenografts.31 In the latter studies, it has been shown that CD4þT cells are required for macrophage activation in the presence of pancreatic islet xenografts However, once... Coverslips containing macrophages are incubated on ice with 2 nmoles/ml of C6 -NBD-ceramide for 30 min, then washed twice with HBSS, and trans ferred to 37 for 15 min After washing twice with HBSS, cells are mounted and viewed as described previously The localized liposomal conalbumin fluorescence (Fig 3A) can be superimposed on the Golgi fluorescence (Fig 3B) For localization of conalbumin by immunofluorescence... Because TAP1 knock-out is on a C5 7BL/6 background, the antigen of choice for trafficking 38 M J Androlewicz, P Cresswell, and K S Anderson, Proc Natl Acad Sci U S A 90, 9130 (1992) 39 S Li, K Paulsson, H Sjogren, and P Wang, J Biol Chem 274, 8649 (1999) [2] trafficking of liposomal antigens 29 Fig 4 Inhibition of processing of L(TR-CON) by lactacystin Macrophages were preincubated with 10 M lactacystin and... the MHC class II pathway Therefore, most soluble antigens are relatively ineffective for priming MHC class I–restricted cytotoxic T lymphocyte responses because of the inability of the antigen to gain access to the cytoplasmic compartment Several different methods have been used to channel antigens into the class I pathway.1,2, 13–23 Among these methods, liposomes have proven 6 ¨ R Gluck, Vaccine 17,... liposomes protected mice from PEA-, ConA-, or PEA/SEB–induced liver injury In the absence of Kupffer cells, liver damage was restricted to a few small necrotic areas These studies further indicated that Kupffer cells play an important role in T-cell activation-induced liver injury by contributing tumor necrosis factor.36 After administration of clodronate liposomes in nonobese diabetic (NOD) mice, it was... interference contrast objectives and a Leitz 63Â oil immersion lens designed for fluorescence microscopy Fluorescence signals are generated by use of fluorescence filters from Omega Optical that are optimized for Texas Red (excitation wavelength, 595; emission wavelength, 615) and fluorescein (excitation wavelength, 494; emission wavelength, 518) fluorochromes Images are collected with a color digital camera . Geraldine Brush Cancer Research Institute, 2330 Clay Street, San Francisco, California 94115 Marcel De Cuyper (12), Interdisciplinary Research Center, Katholieke Universiteit Leuven, Campus Kortrijk,. Curie, Section de Recherche, UMR-CNRS 168 et LRC-CEA 8, 11 rue Pierre et Marie Curie, 75231 Paris, France Peter Laverman (15), Department of Nu- clear Medicine (565), University Medical Center. doxorubicin, daunorubicin and amphotericin B now utilized in the clinic. Other medical applications of liposomes include their use as vaccine adjuvants and gene delivery vehicles, which are being