principal investigator/program directorphys.columbia.edu/~tosti/icidr/archive/nih_proposal.pdf ·...

Principal Investigator/Program Director (Last, first, middle): Brittenham, Gary M., M.D.

PHS 398 (Rev. 5/01) Form Page 2

DESCRIPTION. State the application’s broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This description is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED.

The proposed research will use quantitative magnetic resonance methods to determine the role of red blood cell sequestration in the pathogenesis of cerebral malaria. Cerebral malaria is a major life-threatening complication of infection with Plasmodium falciparum, diagnosed clinically as unrousable coma in the absence of other attributable causes. We propose non-invasive high-field (3.0 Tesla) magnetic resonance (MR) studies to determine the extent of microvascular red blood cell sequestration in conjunction with assays of plasma cytokines in patients with cerebral malaria, in patients with other forms of severe and uncomplicated malaria, and in uninfected controls in Bangkok, Thailand. This research program will take advantage of a unique convergence of resources and expertise at Mahidol University in Bangkok: (i) the Bangkok Hospital for Tropical Disease, Faculty of Tropical Medicine, a world-renowned malarial research facility, (ii) the Ramathibodi Hospital, Faculty of Medicine, Department of Radiology, fully equipped with a Phillips Intera 3.0 Tesla MR system, and (iii) an established, decade-long collaborative relationship with investigators at Columbia University, now enhanced by a scientific linkage with the Hatch Magnetic Resonance Research Center. The proposed research has three specific aims:

(1) to test the hypothesis that the extent of red blood cell sequestration in the cerebral vasculature is greater in cerebral malaria than in other forms of severe malaria, as determined by time of flight MR angiography and cerebral perfusion studies using arterial spin labeling techniques;

(2) to test the hypothesis that cerebral metabolic dysfunction is greater in cerebral malaria than in other forms of severe malaria, as determined by proton (1H) MR spectroscopic measurements of ventricular lactate and of an indicator of axonal injury, N-acetylaspartate, in brain tissue; and

(3) to test the hypothesis that cerebral hemozoin deposition is greater in cerebral malaria than in other forms of severe malaria, as determined by a novel MR method, magnetic field correlation imaging.

By determining definitively the role of cerebral sequestration of P. falciparum-infected red blood cells, the proposed studies would help guide development of effective treatment for cerebral malaria.

PERFORMANCE SITE(S) (organization, city, state) Columbia University College of Physicians and Surgeons, New York, New York, U.S.A. Mahidol University, Hospital of Tropical Diseases and Ramathibodi Hospital, Bangkok, Thailand New York University School of Medicine New York, New York, , U.S.A.

KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Role on Project

Brittenham, Gary M., M.D. Columbia University College of P&S Principal Investigator Looareesuwan, Sornchai, M.D. Mahidol Univ., Faculty Tropical Med. Major Foreign Collaborator Boongird, Prasert, M.D. Mahidol Univ., Faculty Tropical Med. Co-Investigator Brown, Truman R., Ph.D. Columbia University College of P&S Co-Investigator DeLaPaz, Robert L., M.D. Columbia University College of P&S Co-Investigator Jensen, Jens H., Ph.D. New York Univ. School of Medicine Co-Investigator Kitayaporn, Dwip, M.D. Mahidol Univ., Faculty Tropical Med. Co-Investigator Laothamatas, Jiraporn, M.D. Mahidol Univ., Faculty Tropical Med. Co-Investigator Swaminathan, Srirama, Ph.D. Philips Med. System/Columbia Univ. Co-Investigator Wilairatana, P., M.D., Ph.D. Mahidol Univ., Faculty Tropical Med. Co-Investigator

Disclosure Permission Statement. Applicable to SBIR/STTR Only. See instructions. Yes No

Principal Investigator/Program Director (Last, first, middle):

PHS 398 (Rev. 05/01) Page _______ Form

The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page.

RESEARCH GRANT

TABLE OF CONTENTS Page Numbers

Face Page.................................................................................................................................................. 1 Description, Performance Sites, and Personnel ................................................................................... 2- Table of Contents ..................................................................................................................................... Detailed Budget for Initial Budget Period (or Modular Budget)........................................................... Budget for Entire Proposed Period of Support (not applicable with Modular Budget)........................... Budgets Pertaining to Consortium/Contractual Arrangements (not applicable with Modular Budget) Biographical Sketch—Principal Investigator/Program Director (Not to exceed four pages) .................. Other Biographical Sketches (Not to exceed four pages for each – See instructions)) ........................ Resources ................................................................................................................................................. Research Plan Introduction to Revised Application (Not to exceed 3 pages)......................................................................................................... Introduction to Supplemental Application (Not to exceed one page).............................................................................................. A. Specific Aims ..................................................................................................................................................................... B. Background and Significance............................................................................................................................................. C. Preliminary Studies/Progress Report/ (Items A-D: not to exceed 25 pages*) Phase I Progress Report (SBIR/STTR Phase II ONLY) * SBIR/STTR Phase I: Items A-D limited to 15 pages. D. Research Design and Methods .......................................................................................................................................... E. Human Subjects................................................................................................................................................................. Protection of Human Subjects (Required if Item 4 on the Face Page is marked “Yes”) Inclusion of Women (Required if Item 4 on the Face Page is marked “Yes”) ................................................................. Inclusion of Minorities (Required if Item 4 on the Face Page is marked “Yes”) ............................................................... Inclusion of Children (Required if Item 4 on the Face Page is marked “Yes”) ................................................................. Data and Safety Monitoring Plan (Required if Item 4 on the Face Page is marked “Yes” and a Phase I, II, or III clinical

trial is proposed...................................................................................................................................................... F. Vertebrate Animals ............................................................................................................................................................ G. Literature Cited .................................................................................................................................................................. H. Consortium/Contractual Arrangements .............................................................................................................................. I. Letters of Support (e.g., Consultants)................................................................................................................................. J. Product Development Plan (SBIR/STTR Phase II and Fast-Track ONLY) ......................................................................... Checklist.................................................................................................................................................... Appendix (Five collated sets. No page numbering necessary for Appendix.)

Appendices NOT PERMITTED for Phase I SBIR/STTR unless specifically solicited.

Number of publications and manuscripts accepted for publication (not to exceed 10)

Check if Appendix is Included

Other items (list):

http://grants.nih.gov/grants/funding/phs398/section_1.html#6_bios

http://grants.nih.gov/grants/funding/phs398/section_1.html#6_bios

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JUSTIFICATION. Follow the budget justification instructions exactly. Use continuation pages as needed.

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COLUMBIA UNIVERSITY COLLEGE OF PHYSICIANS AND SURGEONS: YEAR 1:

Specific functions of personnel:

Gary M. Brittenham, M.D., Professor of Pediatrics and Medicine, will serve as Principal Investigator for the project with overall scientific responsibility for the direction, conduct and completion of the proposed studies, devoting 25% of his professional effort to this purpose.

Truman R. Brown, Ph.D., Professor of Radiology and Biomedical Engineering, will serve as a co-Investigator for the project with scientific responsibility for the radiological aspects of the research and for the design and interpretation of the MR examinations, devoting 10% of his professional effort to this purpose.

Robert L. DeLaPaz, M.D., Professor of Radiology will serve as a co-Investigator for the project, with responsibility for design and modification of the MR protocols and experiments, and for directing quantitative analysis and interpretation of the findings, devoting 25% of his professional effort to this purpose.

A Research Associate, to be named, will assist in testing and evaluation of protocols and in quantitative analysis of the data.

Supplies: The cost of reagents, materials and labware for preparation of phantoms to examine resonance effects of hemozoin (beta-hematin) has been estmated at $5,000.

Travel: $25,180 has been allocated for travel by Investigators to Bangkok, Thailand, and to attend the annual ICTDR meeting in Bethesda, MD. Travel expenses to Thailand using U.S. government per diem rates for Bangkok were calculated as $182/day (= $125 lodging/day + $57 meals and incidental expenses/d) x 80 days (2 x 30 day visits for the Principal Investigator + 2 x 10 d visits for co-Investigators) + 4 x $1910 (coach round-trip airfare, New York to Bangkok) = $22,200. Travel expenses to Bethesda to attend the annual ICTDR meeting were estimated using U.S. government per diem rates for the Principal Investigator and a co-Investigator and calculated as 2 x 5 d x $201/d (= $150 lodging/day + $51 meals and incidental expenses/d) + 2 x $485 (coach [shuttle] round-trip airfare, New York to Washington) = $2,980.

Other expenses: Telephone and facsimile expenses, photocopying, mailing and shipping costs have been estimated at $4,000. YEARS 2 TO 5: No changes are planned in the specific functions or effort of the personnel in the project or in other categories.

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MAHIDOL UNIVERSITY FACULTY OF TROPICAL MEDICINE: YEAR 1:

Specific functions of personnel:

Sornchai Looareesuwan, M.D., Dean and Professor of Clinical Tropical Medicine, Major Foreign Collaborator and Principal Investigator for the Mahidol University subcontract (40% effort), will have overall responsibility for the conduct of the ICIDR program at the Hospital for Tropical Diseases. Polrat Wilairatana, M.D, Ph.D., Professor of Clinical Tropical Medicine and Co-Investigator (20% effort), will be responsible for carrying out day-to-day study activities, enrolling patients, obtaining informed consent and overseeing blood sampling and other project procedures. Srivicha Krudsood, M.D., Associate Professor of Clinical Tropical Medicine and Co-Investigator (50% effort), will assist in day-to-day study procedures and in enrolling patients, obtaining informed consent and in carrying out study procedures. Weerapong Phumratanaprapin, M.D., Assistant Professor of Clinical Tropical Medicine and Co-Investigator (20% effort), will assist in study procedures and accompany patients to Ramathibodi Hospital for MRI studies. Udomsak Silachamroon, M.D., Professor of Clinical Tropical Medicine and Co-Investigator (15% effort), will assist in study procedures and accompany patients to Ramathibodi Hospital for MRI studies. Prasert Boongird, M.D., Professor of Neurology and Co-Investigator (15% effort), will oversee neurological evaluation of patients at Ramathibodi Hospital at the time of MRI examinations. Jiraporn Laothamatas, M.D., Professor of Radiology and Co-Investigator (25% effort), will be responsibl for MRI examinations of patients at Ramathibodi Hospital. Ratana Kunnatiranont, M.D., Radiologist and Co-Investigator (25% effort), will assist in review and interpretations of the MRI examinations. Dwip Kitayaporn, M.D., Ph.D., Professor of Epidemiology (10% effort), will serve as the designated biostatistician for the ICIDR program. Jaranit Kaewkungwal, Ph.D., Assistant Professor of Epidemiology and Research Associate (100% effort), will serve as the designated Data Manager for the project, with responsibility for the collection of all study data, its entry into the study data base, and initial tabulations and summaries. Teerachai Panumaporn, M.Sc, Research Associate (20% effort), will serve as Data Clerk, assisting the Data Manager in data collection, entry and database maintenance.

Supplies: The cost of reagents and labware for cytokine measurements have been estimated at $2,000.

Travel: $6,000 has been allocated for travel by the Major Foreign Collaborator and a co-Investigator to Bethesda, MD, to attend the annual ICTDR and calculated as 2 x 10 d x $201/d (= $150 lodging/day + $51 meals and incidental expenses/d) + 2 x $1910 (coach round-trip airfare, Bangkok to Washington) + $170 (hotel or home to airport transportation in Thailand and the U.S.) = $6,000.

Patient care costs (inpatient): Anticipating admission of as many as 45 patients with cerebral, severe or uncomplicated malaria for study purposes, the study charge for the entire 28 day hospitalization is $1,000 per patient or a total of $45,000.

Other expenses: Ramathibodi Hospital Department of Radiology charges for 125 MRI examinations, at $280/study, are a total of $35,000. In accordance with RFA instructions, $15,000 are budgeted for Pathogen Study Group expenses. Charges for import of kits for the cytokine assays and for telephone and facsimile expenses, photocopying, mailing and shipping costs have been estimated at $10,000.

YEARS 2 TO 5: No changes are planned in the specific functions or effort of the personnel in the project or in other categories.

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PHS 398/2590 (Rev. 05/01) Page __13__ Biographical Sketch Format Page Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.

BIOGRAPHICAL SKETCH Provide the following information for the key personnel in the order listed for Form Page 2.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME POSITION TITLE

Gary M. Brittenham, M.D. Professor of Pediatrics and Medicine EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE (if applicable) YEAR(s) FIELD OF STUDY

Johns Hopkins University, Baltimore, Maryland B.A.(Honors) 1961-1966 Philosophy École Pratique des Hautes Études, Paris, France 1966-1967 Anthropology Western Reserve University School of Medicine, M.D. 1967-1971 Medicine Cleveland, Ohio

A. Positions and Honors. 1. Professional Appointments and Experience: 1971-77 Intern, Junior Assistant, Senior Assistant and Chief Resident, NIH Hematology Trainee and

Teaching Fellow, Department of Medicine, Cleveland Metropolitan General Hospital, Case Western Reserve University School of Medicine.

1977-90 Assistant (1977-82) and Associate (1982-90) Professor, Department of Medicine and Department of Anthropology, Case Western Reserve University.

1987-90 Associate Professor of General Medical Sciences (Oncology), Division of General Medical Sciences, Case Western Reserve University School of Medicine.

1990-98 Professor of Medicine, Professor of Anthropology, Professor of General Medical Sciences (Oncology) and Professor of International Health, Case Western Reserve University.

1990-98 Director, Research Hematology Training Program, Case Western Reserve University. 1998- Professor of Pediatrics and Medicine, Departments of Pediatrics and Medicine, Columbia

University College of Physicians and Surgeons. 2001- Director, Pediatric Research Hematology Training Program, Columbia University College of

Physicians and Surgeons. 2. Honors: 1976-79 Teaching and Research Scholar in Medical Nutrition, American College of Physicians. 1977-79 Research Scholar, Indo-American Fellowship Program, Indo-U.S. Subcommission on Education

and Culture. 1991 University Medal, Osaka City University School of Medicine. 1993 US Public Health Service Award for Exceptional Achievement in Orphan Products Development 1995 International Association for the Study of Disorders of Iron Metabolism: Award for

Contributions to the Understanding of Disorders of Iron Metabolism, 1995. 1997-04 Visiting Professor, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand 2003 John W. Harris, M.D., Visiting Professor, MHMC, Case Western Reserve University 3. Memberships:

Association of American Physicians, American Society for Clinical Investigation, Central Society for Clinical Research, American Federation for Medical Research, American Society of Hematology, International Society of Hematology, American Society of Tropical Medicine and Hygiene, American College of Physicians, Society for Experimental Biology and Medicine; Initial Review Group, FDA Orphan Products Program, 1984-94; Editorial Board, Journal of Laboratory and Clinical Medicine, 1989-94; FASEB, Expert Panel on Iron Nutrition, 1989-91; NIH NHLBI Cooley’s Anemia Progress Review Committee, 1994-5; NIH Hematology Study Section, 1987-91; 1993-97; NHLBI Research Training Review Special Emphasis Panel, 1996- ; ASH Scientific Subcommittee on Iron and Heme, 1999-2003; CDC Expert Panel on Hemochromatosis, 1999- ; NHLBI Observational Study Monitoring Board (OSMB) for the Hemochromatosis and Iron Overload Screening Study, 2000- ; Referee, Ministero dell'Istruzione, dell'Università e della Ricerca, Italia, 2003 - ; Technical Consultant, WHO, Dept. of Nutrition for Health and Development, 2004-


PHS 398/2590 (Rev. 05/01) Page __14__ Continuation Format Page Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.

B. Selected peer-reviewed publications: Brittenham GM. Genetic model for the observed distribution of Hb S in sickle cell trait. Nature 1977;

268:635-636. Brittenham GM, Farrell DE, Harris JW, Feldman ES, Danish EH, Muir WA, Tripp JH, Bellon EM. Magnetic

susceptibility measurements of human iron stores. New Engl J Med 1982; 307:1671-75. Atkinson CT, Bayne MT, Gordeuk VR, Brittenham GM, Aikawa M. Stage-specific ultrastructural effects of

desferrioxamine on Plasmodium falciparum in vitro. Am J Trop Med Hyg 1991;45:593-601. Gordeuk V, Thuma P, Brittenham G, McLaren C, Parry D, Backenstose A, Biemba G, Msiska R, Holmes L,

McKinley E, Vargas L, Gilkeson R, Poltera AA. Effect of iron chelation therapy on recovery from deep coma in children with cerebral malaria. New Engl J Med 1992;327:1473-1477.

Brittenham GM, Griffith PM, Nienhuis AW, McLaren CE, Young NS, Tucker EE, Allen CJ, Farrell DE, Harris JW. Efficacy of deferoxamine in preventing complications of iron overload in patients with thalassemia major. New Engl J Med 1994;331:567-573.

Looareesuwan S, Wilairatana P, Vannaphan S, Gordeuk VR, Taylor TE, Meshnick SR, Brittenham GM. Coadministration of desferrioxamine B with artesunate in malaria: an assessment of safety and tolerance. Trans R Soc Trop Med Hyg 1996;90:551-554.

Angelucci E, Giovagnoni A, Valeri GL, Paci R, Ripanti M, Muretto P, McLaren CE, Brittenham GM, Lucarelli G. Limitations of magnetic resonance imaging in measurement of hepatic iron. Blood 1997;90:4736-4742.

van der Torn M, Thuma PE, Mabeza GF, Biemba G, Moyo VM, McLaren CE, Brittenham GM, Gordeuk VR. Loading dose of quinine in cerebral malaria. Trans Roy Soc Trop Med Hyg 1998;92:325-331.

Looareesuwan S, Wilairatana P, Vannaphan S, Wananratana V, Wenisch C, Aikawa M, Brittenham G, Granninger W, Wernsdorfer WH. Pentoxifylline used as ancillary treatment for severe falciparum malaria in Thailand. Am J Trop Med Hyg 1998;58:348-353.

Camacho LH, Gordeuk VR, Wilairatana P, Pootrakul P, Brittenham GM, Looareesuwan S. The course of anaemia after the treatment of acute falciparum malaria. Ann Trop Med Parasitol 1998;92:525-37.

Hutagalung R, Wilairatana P, Looareesuwan S, Brittenham GM, Gordeuk VR. Influence of hemoglobin E trait on the severity of falciparum malaria. J Infect Disease 1999;179:283-286.

Wilairatana P, Westerlund EK, Aursudkij B, Vannaphan S, Krudsood S, Viriyavejakul P, Chokejindachai W, Treeprasertsuk S, Srisuriya P, Gordeuk VR, Brittenham GM, Neild G, Looareesuwan S. Treatment of malarial acute renal failure by hemodialysis. Am J Trop Med Hyg 1999;61:233-237.

Camacho LH, Wilairatana P, Weiss G, Mercader MA, Brittenham GM, Looareesuwan S, Gordeuk VR. The eosinophilic response and haematological recovery after treatment for Plasmodium falciparum malaria. Trop Med Int Health 1999;4:471-475.

Hutagalung R, Wilairatana P, Looareesuwan S, Brittenham GM, Gordeuk VR. Influence of hemoglobin E trait on the antimalarial effect of artemisinin derivatives. J Infect Disease 2000;181:1513-1516.

Kreil A, Wenisch C, Brittenham G, Looareesuwan S, Peck-Radosavljevic M. Thrombopoietin in plasmodium falciparum malaria. Br J Hæmatol 2000;109:534-6.

Perlmann P, Perlmann H, Looareesuwan S, Krudsood S, Kano S, Matsumoto Y, Brittenham G, Troye-Blomberg M, Aikawa M. Contrasting functions of IgG and IgE antimalarial antibodies in uncomplicated and severe Plasmodium falciparum malaria. Am J Trop Med Hyg 2000; 62:373-7.

Angelucci E, Brittenham GM, McLaren CE, Ripalti M, Baronciani D, Giardini C, Lucarelli G. Hepatic iron concentration and mobilizable body iron in thalassemia major. New Engl J Med 2000; 343:327-31.

Silachamroon U, Krudsood S, Treeprasertsuk S, Wilairatana P, Chalearmrult K, Mint HY, Maneekan P, White NJ, Gourdeuk VR, Brittenham GM, Looareesuwan S. Clinical trial of oral artesunate with or without high dose primaquine for the treatment of vivax malaria in Thailand. Am J Trop Med Hyg 2003;69:14-18.

Brittenham GM, Badman DG. Noninvasive measurement of iron: Report of an NIDDK Workshop. Blood 2003;101:15-19.

Sibmooh N, Yamanont P, Krudsod S, Leowattana W, Brittenham G, Looareesuwan S, Udomsangpetch R. Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules. Lipids Health Dis. 2004;3:15.

Ohashi J, Naka I, Patarapotikul J, Hananantachai H, Brittenham G, Looareesuwan S, Clark AG, Tokunaga K. Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection. Am J Hum Genet 2004;74:1198-1208.



Selected research projects ongoing or completed during the last three years:

Project: "Magnetic resonance measurement of heart and liver iron"

Principal Investigator: Gary M. Brittenham, M.D.

Agency: NIDDK

Type: R01 (DK66251)

Period: 30 Sept 2003 to 29 Sept 2007

This research project integrates radiological, clinical and laboratory efforts in the development and validation of quantitative magnetic resonance methods for the measurement of heart and liver iron. The overall goal of the research is to acquire a fundamental theoretical and physical understanding of the resonance effects of tissue iron as a guide in the development of novel MR techniques that will provide clinically applicable methods for the quantitative measurement of hepatic and cardiac iron.

Project: "Pathogenesis of severe malarial anemia in Thailand"


Agency: NIAID

Type: R01 (AI51310)

Period: 1 July 2002 to 30 June 2006

This research project examines the pathogenesis of severe malarial anemia in Thailand to define those elements attributable to infection with Plasmodium falciparum. This mutlidisciplinary collaborative investigation is designed to determine the relative contributions of accelerated destruction and impaired production of erythrocytes in the pathogenesis of the severe anemia of falciparum malaria, using a newly available, non-invasive method for estimating red cell survival, measurement of the alveolar carbon monoxide (CO) concentration, estimates of the mass of sequestered, parasitized erythrocytes, automated reticulocyte counts, determinations of serum transferrin receptor, phenotypic and genotypic studies of malarial susceptibility and measurements of pro- (Th-1) and anti- (Th-2) inflammatory cytokines.

Project: "International Malarial Anemia Research Training Program"


Agency: NIH, Fogarty International Center

Type: D43 (TW006240)

Period: 1 March 2003 to 28 Feb 2007

This international research training program is designed to prepare Thai physicians, scientists and other health professionals for careers in basic and clinical research on the pathogenesis of severe malarial anemia, utilizing the clinical and laboratory facilities of the Faculty of Tropical Medicine at Mahidol University and of the College of Physicians and Surgeons at Columbia University. Project: “High Tc susceptometer for magnetic measure of body iron.”


Agency: NIDDK

Type: R01 (DK57209)

Period: 30 Sept 2001 to 1 Oct 2006

This Bioengineering Research Partnership is developing a high-transition temperature (77°K; high-Tc) superconducting biosusceptometer for the direct, non-invasive magnetic measurement of hepatic iron stores in patients with iron overload from hereditary hemochromatosis, thalassemia major, sickle cell disease, aplastic anemia, myelodysplasia and other disorders.



Project: “Cardiac disease in Cooley’s anemia: molecular and clinical studies”


Agency: NHLBI

Type: R01 (HL62882)


This research project has the goal of developing new non-invasive means of identifying those patients with thalassemia major and transfusional iron overload who are at the highest risk for iron-induced cardiac disease using clinically detectable alterations in the complexity of repolarization, as measured on special 24-hour ECG recordings. Project: “Oral iron chelators predicated on desferrithiocin”

Principal Investigator (CU subcontract): Gary M. Brittenham, M.D.

Agency: NIDDK

Type: R01 (DK49108)

Period: 1 Feb 1990 to 31 Jan 2010

The goal of this project is to examine desferrithiocin-based iron chelators developed by R. Bergeron, Ph.D., Principal Investigator of the parent project, in animal models and, ultimately, in patients with iron overload. This research uses our animal model of iron-induced cardiomyopathy with magnetic resonance methods to provide serial, non-invasive assessment of cardiac dimensions and function simultaneously with estimates of cardiac and hepatic iron concentrations. Project: "Pediatric Hematology Research Training Program"


Agency: NIDDK

Type: R01 (DK55462)

Period: 15 Aug 1999 to 31 Jul 2004

This research training grant supports the Pediatric Research Hematology Training Program in the Department of Pediatrics at the Columbia University College of Physicians and Surgeons. Project: “Non-transferrin-bound plasma iron and deferoxamine therapy”

Principal Investigator (CU subcontract): Gary M. Brittenham, M.D.

Agency: NIDDK

Type: R01 (DK55462)

Period: 15 Aug 1999 to 31 Jul 2004

This research project examines the effects of deferoxamine therapy on non-transferrin-bound iron in patients with thalassemia major. The overall goal of the Columbia component of this project is the non-invasive magnetic measurement of body iron with our superconducting quantum interference device (SQUID) susceptometer in patients with thalassemia studied by J. Porter, M.D., Principal Investigator of the parent project, to determine effects of the body iron load on non-transferrin-bound plasma iron.




NAME POSITION TITLE

Sornchai Looareesuwan, M.D. Professor, Clinical Tropical Medicine EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)


Mahidol University, Bangkok, Thailand B.Sc. 1972 Medicine Mahidol University, Bangkok, Thailand M.D. 1974 Medicine James Cook Univ. of North Queensland, Australia FACTM 1995 Tropical Medicine Royal College of Physicians, UK, Honorary Fellow FRCP 2002 Medicine

Research Training and Appointments

1974-75 Rotating Intern, Siriraj Hospital, Mahidol University 1976-78 Resident, General Medicine, Siriraj Hospital, Mahidol University 1979-81 Lecturer, Dept. of Clinical Tropical Medicine and Hospital for Tropical Diseases, Faculty of

Tropical Medicine, Mahidol University 1981-82 Wellcome Research Fellow, Nuffield Dept. of Clinical Medicine, University of Oxford, England 1982-85 Assistant Professor, Dept. of Clinical Tropical Medicine and Hospital for Tropical Diseases,

Faculty of Tropical Medicine, Mahidol University 1985-90 Associate Professor, Dept. of Clinical Tropical Medicine and Hospital for Tropical Diseases,

Faculty of Tropical Medicine, Mahidol University 1990-present Professor, Dept. of Clinical Tropical Medicine and Hospital for Tropical Diseases, Faculty of

Tropical Medicine, Mahidol University 1994-96 Deputy Director, Hospital for Tropical Diseases Head, Div. Critical Care in Tropical Diseases, Faculty of Tropical Medicine, Mahidol University 1994-present STC WHO Consultant 1995-00 USP Expert Advisory Panel on Parasitic and Tropical Disease, The United States Pharmacopeial

Convention, Inc., USA 1996-present Steering Board, Thailand-Tropical Disease Research Program 1996-present Director SEAMEO TROPMED Regional Centre 1996-present Dean, Faculty of Tropical Medicine, Mahidol University 1998-present Secretary General/Coordinator, SEAMEO TROPMED Network 2000-present Program manager, the Asian Centre of International Parasite Control (ACIPAC)

Awards The National Award for Best Research Work, National Research Council of Thailand-1987; 1994 Mepha Malaria Prize for Best Clinical Study on the Treatment of Plasmodium falciparum Malaria, Sociedade Brasileira de Medicina Tropical, Brazil; Mahidol University-B. Braun Price 1995 Medicine & Public Health of Thailand; Man of the Year for Scientific Achievement and Gold Record Achievement, International Society , American Biographical Institute, USA-1995; National Award 1995 for the Distinguished Researcher, National Research Council of Thailand; Who’s Who in Medicine and Healthcare, New Providence, NJ, USA-1996; The Most Productive Author and The Most Cited Author, Science and Technology of Thailand-1996; Vice-Chairman of the Joint Coordinating Board (JCB) of the Special Programme for Research and Training in Tropical Diseases (TDR), WHO Geneva (1998-1999); President, XVth International Congress of Tropical Medicine and Malaria, Cartagena, Colombia (2000)

Member Thai Medical Council; Medical Association of Thailand; Infectious Disease Association of Thailand; Parasitology and Tropical Medicine Association of Thailand; St. Cross College, Oxford University, 1981-82; American Society of Tropical Medicine and Hygiene



B. Peer-reviewed publications (selected from more than 400 peer-reviewed publications):

Looareesuwan S, Warrell DA, White NJ, Sutharasamai P, Chanthavanich P, Sundaravej K, Juel-Jensen BE, Bunnag D, Harinasuta T. Do patients with cerebral malaria have cerebral oedema? A computed tomography study. Lancet 1983; 1:434-437.

Looareesuwan S, Ho M, Wattanagoon Y, White NJ, Warrell DA, Bunnag D, Harinasuta T & Wyler D. Dynamic alteration in splenic function during acute falciparum malaria. N Eng J Med 1987;317(11):675-679.

Looareesuwan S, Wilairatana P, Krishna S, Kendall B, Vannaphan S, Viravan C, White NJ. Magnetic resonance imaging of the brain in cerebral malaria. Clin Infect Dis 1995;21:300-309.

Looareesuwan S, Wilairatana P, Vannaphan S, Gordeuk VR, Taylor TE, Meshnick SR, Brittenham GM. Coadministration of desferrioxamine B with artesunate in malaria: an assessment of safety and tolerance. Trans R Soc Trop Med Hyg 1996;90:551-554.

Looareesuwan S, Wilairatana P, Vannaphan S, Wananratana V, Wenisch C, Aikawa M, Brittenham G, Granninger W, Wernsdorfer WH. Pentoxifylline used as ancillary treatment for severe falciparum malaria in Thailand. Am J Trop Med Hyg 1998;58:348-353.

Camacho LH, Gordeuk VR, Wilairatana P, Pootrakul P, Brittenham GM, Looareesuwan S. The course of anaemia after the treatment of acute falciparum malaria. Ann Trop Med Parasitol 1998;92:525-37.

Hutagalung R, Wilairatana P, Looareesuwan S, Brittenham GM, Gordeuk VR. Influence of hemoglobin E trait on the severity of falciparum malaria. J Infect Disease 1999;179:283-286.

Camacho LH, Wilairatana P, Weiss G, Mercader MA, Brittenham GM, Looareesuwan S, Gordeuk VR. The eosinophilic response and haematological recovery after treatment for Plasmodium falciparum malaria. Trop Med Int Health 1999;4:471-475.

Hutagalung R, Wilairatana P, Looareesuwan S, Brittenham GM, Gordeuk VR. Influence of hemoglobin E trait on the antimalarial effect of artemisinin derivatives. J Infect Disease 2000;181:1513-1516.

Kreil A, Wenisch C, Brittenham G, Looareesuwan S, Peck-Radosavljevic M. Thrombopoietin in plasmodium falciparum malaria. Br J Hæmatol 2000;109:534-6.

Perlmann P, Perlmann H, Looareesuwan S, Krudsood S, Kano S, Matsumoto Y, Brittenham G, Troye-Blomberg M, Aikawa M. Contrasting functions of IgG and IgE antimalarial antibodies in uncomplicated and severe Plasmodium falciparum malaria. Am J Trop Med Hyg 2000; 62:373-7.

Looareesuwan S, Oosterhuis B, Schilizzi BM, Sollie FA, Wilairatana P, Krudsood S, Lugt CB, Peeters PA, Peggins JO. Dose-finding and efficacy study for i.m. artemotil (beta-arteether) and comparison with i.m. artemether in acute uncomplicated P. falciparum malaria. Br J Clin Pharmacol 2002; 53, 492-500.

Yipp BG, Robbins SM, Resek ME, Baruch DI, Looareesuwan S, Ho M. Src-family kinase signaling modulates the adhesion of Plasmodium falciparum on human microvascular endothelium under flow. Blood 2003; 101, 2850-2857.

Treeprasertsuk S, Krudsood S, Tosukhowong T, Maek-A-Nantawat W, Vannaphan S, Saengnetswang T, Looareesuwan S, Kuhn WF, Brittenham G, Carroll J. N-acetylcysteine in severe falciparum malaria in Thailand. Southeast Asian J Trop Med Public Health 2003; 34, 37-42.

Silachamroon U, Krudsood S, Treeprasertsuk S, Wilairatana P, Chalearmrult K, Mint HY, Maneekan P, White NJ, Gourdeuk VR, Brittenham GM, Looareesuwan S. Clinical trial of oral artesunate with or without high dose primaquine for the treatment of vivax malaria in Thailand. Am J Trop Med Hyg 2003;69:14-18.

Looareesuwan S, Imwong M, Wilairatana P. Chlorproguanil-dapsone for malaria in Africa. Lancet 2004; 363, 1838-1839.

Sibmooh N, Yamanont P, Krudsod S, Leowattana W, Brittenham G, Looareesuwan S, Udomsangpetch R. Increased fluidity and oxidation of malarial lipoproteins: relation with severity and induction of endothelial expression of adhesion molecules. Lipids Health Dis. 2004;3:15.

Ohashi J, Naka I, Patarapotikul J, Hananantachai H, Brittenham G, Looareesuwan S, Clark AG, Tokunaga K. Extended linkage disequilibrium surrounding the hemoglobin E variant due to malarial selection. Am J Hum Genet 2004;74:1198-1208.



Selected research projects ongoing or completed during the last three years:

1. Ongoing research support: Project: "Pathogenesis of severe malarial anemia in Thailand"


Agency: NIAID

Type: R01 (AI51310, Years 1-4)


The proposed research project is designed to examine the pathogenesis of severe malarial anemia in Thailand to define those elements attributable to infection with Plasmodium falciparum. Role: Principal Investigator, Mahidol University subcontract Project: " Malaria control in northwestern Thailand"

Principal Investigator: Sornchai Looareesuwan, M.D.

Agency: Gates Foundation

Type: Joint Grant

Period: 1 Dec 2000 to 30 Nov 2005

The Global Health Program of the Bill & Melinda Gates Foundation has provided a joint grant for the Mahidol University Faculty of Tropical Medicine, the Wellcome Mahidol Oxford Unit and the Communicable Disease Control Department (CDC) of the Thai Ministry of Public Health to support a trial of combined antimalarial therapy for control of falciparum malaria in northwestern Thailand. Role: Principal Investigator Project: “Effective antimalarial combination therapy”


Agency: Thai Ministry of University Affairs

Type: Grant


This project provides for research and development ofeffective antimalarial drugs in combination for widespead use in the treatment of malaria. Role: Principal Investigator

2. Completed research support: Project: “Cerebral malaria: role of iron in pathogenesis and therapy.”


Agency: NIAID

Type: U01 (AI35827, Years 1-6)

Period: 1 July 1994 to 30 Sept 2000

The overall goals of this project, based in Bangkok, Thailand, were to define the role of iron in the pathogenesis of cerebral malaria and to determine if the iron chelator deferoxamine could reduce morbidity and mortality in patients with this disorder. Role: Principal Investigator, Mahidol University subcontract





NAME POSITION TITLE

Prasert Boongird, M.D. Professor of Neurology EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)


Chulalongkorn University, Faculty of Science B.S. 1957 – 1958 Biology Chulalongkorn Medical School, Years I-VI 1966-1967 Medicine

A. Position, Appointments and Memberships 1. Professional Appointments and Experience:

Rotating intern, Chulalongkorn Hospital 1964 – 1965 Neurology resident, 1st year, Chulalongkorn Hospital 1965 – 1966 Rotating intern, City of Memphis Hospitals, Mephis, Tennessee, USA 1966 – 1967 Neurology resident, New York State University at Buffalo, USA 1967 – 1970 Research fellow, Clinical Neurophysiology, St. Michael Hospital, Universit of Toronto, Canada 1970 – 1971 Faculty staff, Neurology Division, Department of Medicine Ramathibodi Medical School, Mahidol University 1971 – 1975 Assistant Professor of Neurology, Mahidol University 1975 – 1979 Associate Professor of Neurology, Mahidol University 1979 – 1996 Professor of Neurology, Mahidol University 1996 – present

2. Professional Memberships Thai Royal College of Physicians The Medical Association of Thailand American Academy of Neurology, corresponding associate Vice-President, Epilepsy Society of Thailand 1998- present President, Thailand Section of Clinical Neurophysiology 1997- present President, ASEAN Neurological Association (ASNA) 1997- present

B. Selected peer-reviewed publications:

Boongird P, Smith BH. Focal seizures as a manifestation of hyperglycemia without ketoacidosis. A case report. J Med Assoc Thai 1971; 54, 858-862.

Amnueilaph R, Boongird P, Leechawengwongs M, Vejjajiva A. Heroin neuropathy. Lancet 1973; 1, 1517-1518. Boongird P, Vimolchalao M, Khantanaphar S, Bunyaratavej S. Acute spinal epidural abscess: a report of two

cases, one with autopsy findings. J Med Assoc Thai 1974; 57, 564-570. Phuapradit P, Roongwithu N, Limsukon P, Boongird P, Vejjajiva A. Radiculomyelitis complicating acute

haemorrhagic conjunctivitis. A clinical study. J Neurol Sci 1976; 27, 117-122. Boongird P, Phuapradit P, Siridej N, Chirachariyavej T, Chuahirun S, Vejjajiva A. Neurological manifestations

of gnathostomiasis. J Neurol Sci 1977; 31, 279-291.



Boongird P, Vejjajiva A. Electrophysiologic findings and prognosis in Bell's palsy. Muscle Nerve 1978; 1, 461-

466. Schmutzhard E, Jitpimolmard S, Boongird P, Vejjajiva A. Peripheral eosinophilia in the course of treatment of

cryptococcal meningitis. Mykosen 1987; 30, 601-604. Schmutzhard E, Boongird P, Vejjajiva A. Eosinophilic meningitis and radiculomyelitis in Thailand, caused by

CNS invasion of Gnathostoma spinigerum and Angiostrongylus cantonensis. J Neurol Neurosurg Psychiatry 1988; 51, 80-87.

Schmutzhard E, Boongird P, Gerstenbrand F, Jitpimolmard S, Ponglikitmongkol S, Vejjajiva A. Is cryptococcal meningoencephalitis in the tropics a distinct entity? A retrospective study from Thailand. Trop Geogr Med 1990; 42, 133-139.

Gandour J, Ponglorpisit S, Khunadorn F, Dechongkit S, Boongird P, Boonklam R. Timing characteristics of speech after brain damage: vowel length in Thai. Brain Lang 1992; 42, 337-345.

Gandour J, Ponglorpisit S, Khunadorn F, Dechongkit S, Boongird P, Boonklam R, Potisuk S. Lexical tones in Thai after unilateral brain damage. Brain Lang 1992; 43, 275-307.

Gandour J, Dechongkit S, Ponglorpisit S, Khunadorn F, Boongird P. Intraword timing relations in Thai after unilateral brain damage. Brain Lang 1993; 45, 160-179.

Gandour J, Ponglorpisit S, Dechongkit S, Khunadorn F, Boongird P, Potisuk S. Anticipatory tonal coarticulation in Thai noun compounds after unilateral brain damage. Brain Lang 1993; 45, 1-20.

Boongird P, Soranastaporn S, Menken M, Vejjajiva A. The practice of neurology in Thailand. A different type of medical specialist. Arch Neurol 1993; 50, 311-312.

Charoenpan P, Muntarbhorn K, Boongird P, Puavilai G, Ratanaprakarn R, Indraprasit S, Tanphaichitr V, Likittanasombat K, Varavithya W, Tatsanavivat P. Nocturnal physiological and biochemical changes in sudden unexplained death syndrome: a preliminary report of a case control study. Southeast Asian J Trop Med Public Health 1994; 25, 335-340.

Gandour J, Potisuk S, Ponglorpisit S, Dechongkit S, Khunadorn F, Boongird P. Tonal coarticulation in Thai after unilateral brain damage. Brain Lang 1996; 52, 505-535.

Gandour J, Ponglorpisit S, Potisuk S, Khunadorn F, Boongird P, Dechongkit S. Interaction between tone and intonation in Thai after unilateral brain damage. Brain Lang 1997; 58, 174-196.

Charoenpan P, Thanakitcharu S, Muntarbhorn K, Kunachak S, Boongird P, Likittanasombat K, Suwansathit W. Sleep apnoea syndrome in Ramathibodi Hospital: clinical and polysomnographic baseline data. Respirology 1999; 4, 371-374.

Boongird, P. Compression & Entrapment Neuropathies: A color atlas for bedside diagnosis and neurolocalization,1996. Swicharn Press Co., LTD, Bangkok, Thailand. A “MAHIDOL AWARD” winning English medical textbook, 1999

Gandour J, Ponglorpisit S, Khunadorn F, Dechongkit S, Boongird P, Satthamnuwong N. Speech timing in Thai left- and right-hemisphere-damaged individuals. Cortex 2000; 36, 281-288.

Kunieda T, Zuscik MJ, Boongird A, Perez DM, Luders HO, Najm IM. Systemic overexpression of the alpha 1B-adrenergic receptor in mice: an animal model of epilepsy. Epilepsia 2002; 43, 1324-1329.

Yun J, Gaivin RJ, McCune DF, Boongird A, Papay RS, Ying Z, Gonzalez-Cabrera PJ, Najm I, Perez DM. Gene expression profile of neurodegeneration induced by alpha1B-adrenergic receptor overactivity: NMDA/GABAA dysregulation and apoptosis. Brain 2003; 126, 2667-2681.

Suksamrarn A, Chotipong A, Suavansri T, Boongird S, Timsuksai P, Vimuttipong S, Chuaynugul A. Antimycobacterial activity and cytotoxicity of flavonoids from the flowers of Chromolaena odorata. Arch Pharm Res 2004; 27, 507-511.


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Follow the sample format for each person. DO NOT EXCEED FOUR PAGES.

NAME TRUMAN R. BROWN

POSITION TITLE

Professor of Radiology and Biomedical Engineering

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE (if applicable)

YEAR(s) FIELD OF STUDY

M.I.T. Cambridge, MA B.S. 1964 Mathematics M.I.T. Cambridge, MA Ph.D. 1970 Physics

A. Positions and Honors. 1970-71 Instructor in Physics, Massachusetts Institute of Technology 1971-83 Member of Technical Staff, Bell Laboratories, Murray Hill, NJ 1983-00 Senior Member, NMR and Medical Spectroscopy, Fox Chase Cancer Center, Philadelphia, PA 1988-Pres. Adjunct Professor, Dept. of Biochemistry & Biophysics, University of Pennsylvania 2000-Pres. Professor, Departments of Radiology and Biomedical Engineering, Director, MR Research, Columbia University B. Selected peer-reviewed publications (in chronological order). Nelson, S.J. And Brown, T.R. The Accuracy Of Quantification From 1D NMR Spectra Using The PIQABLE

Algorithm. J. Magn. Reson. 84:95-109, 1989. Buchthal, S., Thoma, W.T., Taylor, J.S., Murphy-Boesch, J., Nelson, S.J. And Brown, T.R. In Vivo T1 Values Of

Phosphorus Metabolites In Human Liver And Muscle Determined At 1.5 T By Chemical Shift Imaging. NMR In Biomed. 2:298-304, 1989.

Szwergold, B.S., Kappler, F. And Brown, T.R. Identification Of Fructose 3-Phosphate In The Lens Of Diabetic Rats. Science 247:451-454, 1990.

Vigneron, D.B., Nelson, S.J., Murphy-Boesch, J., Kelley, D.A.C., Kessler, H.B., Brown, T.R. And Taylor, J.S. Chemical Shift Imaging Of Human Brain: Axial, Sagittal, And Coronal P-31 Metabolite Images. Radiology 177:643-649, 1990.

Jeneson, J.A.L., Taylor, J.S., Willard, T.S., Vigneron, D., Carvajal, L., Nelson, S.J. And Brown, T.R. 1H MR Imaging Of Anatomical Compartments Within The Finger Flexor Muscles Of The Human Forearm. Magn. Reson. Med. 15:491-496, 1990.

Petersen, A., Szwergold, B.S., Kappler, F., Weingarten, M. And Brown, T.R. Identification Of Sorbitol 3-Phosphate And Fructose 3-Phosphate In Normal And Diabetic Human Erythrocytes. J. Biol. Chem. 265:17424-17427, 1990.

Graham, R.A., Brown, T.R. And Meyer, R.A. An Ex Vivo Model For The Study Of Tumor Metabolism By NMR: The Characterization Of The 31P Spectrum Of The Isolated Perfused Morris Hepatoma 7777. Cancer Research 51:841-849, 1991.

Nelson, S.J., Taylor, J.S., Vigneron, D.B., Murphy-Boesch, J. and Brown, T.R. Metabolite images of the human arm: changes in spatial and temporal distribution of high energy phosphates during exercise. NMR In Biomedicine 4:268-273, 1991.

Nelson, S.J., Vigneron, D.B. and Brown, T.R. Detection of significant features and statistical analysis of 2D and 3D images of 31P metabolites. Magn. Reson. Med. 25: 85-93, 1992.

Petersen, A., Kappler, F., Szwergold, B.S. and Brown, T.R. Fructose metabolism in the human erythrocyte: phosphorylation to fructose 3-phosphate. Biochem. J. 284:363-366, 1992.

Brown, T.R. Practical applications of chemical shift imaging. NMR In Biomed. 5:238-243, 1992.


� PHS 398/2590 (Rev. 05/01) Page __23__ Biographical Sketch Format Page �

Jeneson, J.A., van Dobbenburgh, J.O., van Echteld, C.J., Lekkerkerk, C., Janssen, W.J., Dorland, L., Berger, R. and Brown, T.R. Experimental design of 31P MRS assessment of human forearm muscle function: restrictions imposed by functional anatomy. Magnetic Resonance in Med. 30(5):634-40, 1993.

Murphy-Boesch, J., Stoyanova, R., Srinivasan, R., Willard, T., Vigneron, D., Nelson, S., Taylor, J.S. and Brown, T.R. Proton-decoupled P-31 chemical shift imaging of the human brain in normal volunteers. NMR In Biomedicine 6:173-180, 1993.

Su, B.Y., F. Kappler, B.S. Szwergold and Brown, T.R. Identification of a putative tumor marker in breast and colon cancer. Cancer Res. 53:1751-54, 1993.

Lal, S., Szwergold, B.S., Kappler, F. and Brown, T.R. Detection of fructose-3-phosphokinase activity in intact mammalian lenses by 31P NMR spectroscopy. J. Biol. Chem. 268(11):7763-67, 1993.

Gonen, O., Hu, J., Murphy-Boesch, J., Stoyanova, R. and Brown, T.R. Dual interleaved 1H and proton-decoupled-31P in vivo chemical shift imaging of human brain. Magn. Reson. Med. 32:104-9, 1994.

Lal, S., Szwergold, B.S., Taylor, A.H., Randall, W.C., Kappler, F. and Brown, T.R. Production of fructose and fructose-3-phosphate in maturing rat lenses. Invest Ophthalmol. Visual Sci. 36:969-973, 1995.

Negendank, W.G., Padavic-Shaller, K.A., Li, C.W., Murphy-Boesch, J., Stoyanova, R., Krigel, R.L., Schilder, R.J., Smith, M.R., and Brown, T.R. Metabolic characterization of human non-Hodgkin's lymphomas in vivo using proton-decoupled phosphorus magnetic resonance spectroscopy. Cancer Research 55(15): 3286-3294, 1995.

Stoyanova, R., Kuesel, A.C. and Brown, T.R. Application of PCA for NMR. J. Magn. Reson. 115:265-269, 1995. Jeneson, J.A.L., Westerhoff, H.V., Brown, T.R., Van Echteld, C.J.A. and Berger, R. Quasi-linear relationship

between Gibbs free energy of ATP hydrolysis and power output in human forearm muscle. Am. J. Of Physiology 37 268:C1474-C1484, 1995.

Brown, T.R. and Stoyanova, R. NMR spectral quantitation by principal component analysis II: determination of frequency and phase shifts. J. Magn. Reson. B112:32-43, 1996.

Negendank, W., Li, C.W., Padavic-Shaller, K., Murphy-Boesch, J. and Brown, T.R. Phospholipid metabolites in 1H-decoupled 31P MRS in vivo in human cancer: implications for experimental models and clinical studies. Anticancer Research 16:1539-1544, 1996.

Arias-Mendoza, F., Javaid, T., Stoyanova, R., Brown, T.R. and Gonen, O. Heteronuclear multivoxel spectroscopy of in vivo human brain - two-dimensional proton interleaved with three-dimensional H1-decoupled phosphorus chemical shift imaging. NMR in Biomed. 9(3):105-113, 1996.

Lal, S., Kappler, F., Walker, M., Orchard, T.J., Beisswenger, P.J., Szwergold, B.S. and Brown, T.R. Quantitation of 3-deoxyglucosone levels in human plasma. Arch. of Biochem. Biophy. 2:254-260, 1997.

Murphy-Boesch, J., Jiang, H., Stoyanova, R., Brown, T.R. Quantitation of phosphorus metabolites from chemical shift imaging spectra with corrections for point spread effects and B1 inhomogeneity. MRM 39:429-438, 1998.

Lal, S., Kappler, F., Walker, M., Orchard, T.J., Beisswenger, Paul J., Szwergold, B.S., and Brown, T.R., Production and metabolism of 3-deoxyglucosone in humans. Maillard Reaction in Foods and Medicine. 223:291-298, 1998.

Ochs, M.F., Stoyanova, R.S., Arias-Mendoza, F., and Brown T.R., A New Method for Spectral Decomposition Using a Bilinear Bayesian Approach. J. Magn. Reson. 137:161-176, 1999.

Stoyanova, R., and Brown, T. R. NMR Spectral Quantitation by Principal Component Analysis. NMR Biomed 14:271-277, 2001.

Stoyanova, R., and Brown, T. R. NMR Spectral Quantitation by Principal Component Analysis. III. A Generalized Procedure for Determination of Lineshape Variations. J Magn Resonance 154; 163–175, 2002.

S. Franks, M. Smith, F. Arias-Mendoza, C. Shaller, K. Padavic-Shaller, F. Kappler, Y. Shang, W. Negendank, T. R. Brown, Phosphomonoester concentrations differ between chronic lymphocytic leukemia cells and normal human lymphocytes Leukemia Research 26; 919-926, 2002.

Brown TR, Su B, Brown KA, Schwartz MA, Tobia AM, Kappler F. Modulation of in vivo 3-deoxyglucosone levels. Biochem Soc Trans 2003; 31, 1433-1437.

Arias-Mendoza F, Brown TR. In vivo measurement of phosphorous markers of disease. Dis Markers 2003;19, 49-68. Stoyanova R, Nicholson JK, Lindon JC, Brown TR. Sample classification based on Bayesian spectral decomposition

of metabonomic NMR data sets. Anal Chem 2004; 76, 3666-3674. Stoyanova R, Nicholls AW, Nicholson JK, Lindon JC, Brown TR. Automatic alignment of individual peaks in large

high-resolution spectral data sets. J Magn Reson 2004; 170, 329-335.


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C. Research Support.

Active U01 CA62556 (PI: Brown) 05/18/1995 – 2/28/2002 Predicting Human Tumor Response by 31-P MRS This grant is in a one year extension. Interactive RO1 proposals from eight institutions are cooperating to test the hypothesis that in vivo 31P MRS can predict sensitivity or resistance of human cancers to treatment. UO1 CA62556 (PI: Brown) 03/01/2002 – 2/28/2004 A funded extension of the grant above to complete the ongoing study. P01 CA41078 (PI: Brown) 07/01/1997 – 3/31/2003 NMR Studies of Human Cancer

The major goals of this program are designed to optimize the utility of nuclear magnetic resonance spectroscopy in both basic and clinical research. This Program Project Grant consists of four Projects and four Cores. Project I will develop techniques to 1) Carry out polarization transfer between protons and phosphorous in humans; and 2) Acquire both 31P and 19F spectra from patients undergoing 5 FU therapy. Project II will study patients with head and neck carcinomas who are participating in a trial of correlating p02 levels in their tumors with radio responsiveness. 1H decoupled 31P CSI spectra will be obtained from their tumors and correlated with the hypoxic status of the tumor. Projects III and IV concentrate on studying specific metabolic pathways in well-controlled cell cultures in order to determine the metabolic causes for the elevated phospholipid metabolites observed in virtually all tumors and transformed cell lines. The Chemistry Core provides synthetic organic chemistry support to projects. The Instrument and Computer Core provides both the technical support necessary to develop special purpose NMR coils and probes required in the projects. The Data Analysis Core will provide automatic spectral quantitative software to all the projects. An Administrative Core provides budgetary control and clerical services for the PPG.

R01 DK57209 (PI: Brittenham) 09/30/2003-29/9/2007 Magnetic resonance measurement of heart and liver iron This research project integrates radiological, clinical and laboratory efforts in the development and validation of quantitative magnetic resonance methods for the measurement of heart and liver iron. The overall goal of the research is to acquire a fundamental theoretical and physical understanding of the resonance effects of tissue iron as a guide in the development of novel MR techniques that will provide clinically applicable methods for the quantitative measurement of hepatic and cardiac iron.





NAME Robert L. DeLaPaz

POSITION TITLE Professor of Radiology




University of California, Berkeley CA B.A. 1969 Psychology University of Utah, Salt Lake City UT M.D. 1976 Medicine

A. Positions and Honors 1976-1980 Internship,Residency (Neurology & Radiology) University of Minnesota, Minneapolis MN 1980-1981 Research Fellow, Neuroradiology/CT/PET, SNB, NINCDS, NIH, Bethesda MD 1981-1982 Residency, Radiology, George Washington University, Washington DC 1982-1984 Fellowship, Neuroradiology, Massachusetts General Hospital, Boston MA 1984-1985 Assistant Professor, Radiology, University of California, San Francisco CA 1985-1991 Assistant Professor, Radiology, Stanford University, Stanford CA 1991-1995 Associate Professor, Radiology, Memorial Sloan-Kettering Cancer Center & Cornell University Medical College,

New York NY May 1995- Professor, Radiology, Director of Neuroradiology, Medical Director of High Field Magnetic Resonance Research, and Director of PACS, Columbia University College of Physicians & Surgeons, New York NY

B. Selected peer-reviewed publications 1.DeLaPaz RL, Dickman SR, Grosser BI. Effects of stress on rat brain adenosine 3',5' - monophosphate in vivo, Brain Research

85:171-175 (1975). 2.Di Chiro G, DeLaPaz RL, Brooks RA, Sokoloff L, Kornblith PL, Smith BH, Patronal NJ, Kufta CV, Kessler RM, Johnson GS,

Manning RG, Wolf AP. Glucose utilization of cerebral gliomas measured by (18F)- fluorodeoxyglucose and positron emission tomography, Neurology (NY) 32:1323-1329 (1982).

3.Patronas NJ, Chiro G, Brooks RA, DeLaPaz RL, Kornblith PL, Smith BH, Rizzoli HV, Kessler RM, Manning RG, Channing M, Wolf AP, Conner CM. (18F)- fluorodeoxyglucose and P.E.T in the evaluation of radiation necrosis of the Brain. Radiology 144:885-889 (1982).

4.DeLaPaz RL, Patronas NJ, Brooks RA, Smith BH, Kornblith PL, Milam H, Chiro G. Positron emission tomography study of suppression of gray-matter glucose utilization by brain tumors. Am J Neuroradiology 4:826-829 (1983).

6.DeLaPaz RL, Brady TJ, Buonanno FS, New PFJ, Kistler JP, McGinnis BD, Pykett IL, Taveras JM. Nuclear magnetic resonance (NMR) imaging of Arnold-Chiari type I malformation & hydromyelia. J Comp. Assisted Tomography 7(1):126-129 (1983);

7.DeLaPaz RL, New PFJ, Buonanno, Kistler JP, Oot RF, Rosen BR, Taveras JM, Brady TJ, NMR imaging of intracranial hemorrhage. J Computer Assisted Tomoraphy 8(4):599-607 (1984).

8.Galarraga J, Loreck DJ, Graham N, DeLaPaz RL, Smith BH, Hallgren D. Glucose metabolism in human gliomas: correspondence of in situ and in vitro metabolic rates and altered energy metabolism. Metabolic Brain Disease 1:279-291 (1986).

9.Enzmann DR, Rubin JB, DeLaPaz RL, Wright A. Benefits and pitfalls in MRI scanning from CSF pulsation. Radiology 161:773-778 (1986).

10.Duguid JR, DeLaPaz RL, DeGroot J, MRI of the midbrain in Parkinson's disease. Annals of Neurology 20:744-777 (1986). 11.DeLaPaz RL, Chang PJ, Bernstein R, Dave JV. Approximate fuzzy C-means (AFCM) cluster analysis of medical magnetic

resonance image (MRI) Data, IEEE Cybernetics 2:869-871 (1987). 12.Charness ME, DeLaPaz RL. Mamillary body atrophy in wernicke's encephalopathy: antemortem identification using magnetic

resonance imaging, Annals of Neurology 22:595-600 (1987). 13.Hoehne KH, DeLaPaz RL, Bernstein R, Tayloe RC. Combined surface display and reformatting for the three-dimensional

analysis of tomographic data, Investigative Radiology 22:658-664 (1987). 14.DeLaPaz RL, Bernstein R, Hanson WA, Walker MG. Approximate fuzzy C-means (AFCM) cluster analysis of medical magnetic

resonance image (MRI) data – system for research & education , IEEE Trans. Geoscience and Remote Sensing. Vol. GE-25:815-824 (1987).

15.DeLaPaz RL, Bernstein R. Computerized analysis and information of medical MRI. SPIE Remote Sensing 902:151-4 (1988). 16.Marks MP, DeLaPaz RL, Fabrikant JI, Frankel KA, Phillips MH, Levy RP, Enzmann DR. Imaging of charged-particle

radiosurgery for intracranial vascular malformations part I: Results of therapy. Radiology 168:447-455 (1988). 17.Marks MP, DeLaPaz RL, Fabrikant JI, Frankel KA, Phillips MH, Levy RP, Enzmann DR. Imaging of charged-particle

radiosurgery for intracranial vascular malformations part II: Complications. Radiology 168:457-462 (1988).



18.Steinberg GK, George P, DeLaPaz RL, Shibata DK, gross T. Dextromethorphan protects against cerebral injury following transient focal ischemia. Stroke 19:1112-1118 (1988).

19.Steinberg GK, Saleh J, DeLaPaz RL, Kunis D, Zamegar SR. Pretreatment with the NMDA antagonists dextromethorphan reduces cerebral injury following focal ischemia in rabbits. Brain Research 497:382-386 (1989).

20.DeLaPaz RL, Tetrud J, Langston W. MRI of L-DOPA responsive and nonresponsive Parkinson's disease. Neurology 39:275 (1989).

21. Lo EH, Frankel KA, Poljak A, DeLaPaz RL, Phillips MH, Woodruff KH, Brennan KM, Valk PE, Fabrikant JI. Cerebrovascular and metabolic perturbations in delayed heavy charged particle radiation injury. Brain Research 504:168-172 (1989).

22. DeLaPaz RL, Herskovitz E, DiGesu V, Hanson WA, Bernstein. Cluster analysis of medical magnetic resonance imaging data: diagnostic application and evaluation. SPIE/SPSE Proceedings 1259:176 (1990).

23. DeLaPaz RL, Shibata D, Zamegar SR, Steinberg GK. Cerebral ischemia in Rabbits: correlation of MRI and histopathology. Am J Neuroradiology 12:89-95 (1991).

24. DeLaPaz RL, Steinberg GK, Poljack A, Saleh J, Kunis D. MRI of experimental cerebral ischemia: response to NMDA Antagonist therapy. Neuroradiology 33:60-62 (1991).

25. DeLaPaz RL, Lo E, Frankel KA, Phillips MH, Fabrikant JL. MRI and PET of CNS delayed radiation injury. Neuroradiology 33:146 (1991).

26. Steinberg GK, Kunis D, Saleh J, DeLaPaz RL, Protection after Transient Focal Cerebral Ischemia by the N-Methyl-D-Aspartate Antagonist Dextrorphan is Dependent upon Plasma and Brain Levels, J Cerebral Blood Flow & Metab 11:1015-102 (1991)

27. DeLaPaz RL, Echo Planar Imaging, Radiographics 14:1045-1058 (1994). 29. Meyer KL, Kim K, Li T, Tulipano PK, Lee KM, DeLaPaz R, Hirsch J, Ballon D, Sensitivity-enhanced echo-planar MRI at 1.5T

using a 5 x 5 mesh dome resonator, Magn Reson Med 36:606-612, (1996). 30. Chan S, Chin S, Nordli DR, Goodman RR, DeLaPaz RL, Pedley TA, Prospective MR Identification of Non-Balloon and Balloon

Cell Subtypes of Focal Cortical Dysplasia, Ann. Neurology, 44:749-757 (1998). 31. Small SA, Perera GM, DeLaPaz RL, Mayeux R, Stern Y, Differential Regional Dysfunction of the Hippocampus Formation

Among Elderly with Memory Decline and Alzheimer’s Disease, Ann. Neurology, 45:466-472 (1999). 32. Marshall RS, Perera GM, Lazar RM, Krakauer JW, Constantine RC, DeLaPaz RL, The Evolution of Cortical Activation during

Recovery from Corticospinal Tract Infarction, Stroke, 31(3):656-61 (2000). 33. Lazar RM, Marshall RS, Pile-Spellman J, Duong HC, Mohr JP, Young WL, Solomon RL, Perera GM, DeLaPaz RL,

Interhemispheric transfer of language in patients with left frontal cerebral arteriovenous malformation. Neuropsychologia. 38(10):1325-32. (2000).

34. Small SA, Nava AS, Perera GM, Delapaz R, Stern Y, Evaluating the function of hippocampal subregions with high-resolution MRI in Alzheimer's disease and aging. Microsc Res Tech. 51(1):101-8. (2000).

35. Huang J, Mocco J, Choudhri TF, Poisik A, Popilskis SJ, Emerson R, DelaPaz RL, Khandji AG, Pinsky DJ, Connolly ES Jr, A modified transorbital baboon model of reperfused stroke. Stroke. 12:3054-63. (2000).

36. Scott A. Small, Ed X. Wu, Dusan Bartsch, Gerard M. Perera, Clay O. Lacefield, Robert DeLaPaz, Richard Mayeux, Yaakov Stern, and Eric R. Kandel, Imaging Physiologic Dysfunction of Individual Hippocampal Subregions in Humans and Genetically Modified Mice Neuron, 28:653–664 (2000).

37. Small SA, Nava AS, Perera GM, DeLaPaz RL, Mayeux R, Stern Y, Circuit mechanisms underlying memory encoding and retrieval in the long axis of the hippocampal formation, Nature Neuroscience 4: 442-449 (2001).

38. Small SA, Tsai WY, DeLaPaz R, Mayeux R, Stern Y. Imaging hippocampal function across the human life span: is memory decline normal or not. Ann Neurol 51:290-295 (2002).

39 Schumacher HC, Dumoulin CL, Feng L, Mangla S, Meyers PM, Hirsch J, Mohr JP, DeLaPaz RL, Pile-Spellman J. Real-time magnetic resonance imaging for interventional neuroradiological procedures. Surg Technol Int 2003; 11, 183-196.

40. Stern Y, Zarahn E, Hilton HJ, Flynn J, DeLaPaz R, Rakitin B. Exploring the neural basis of cognitive reserve. J Clin Exp Neuropsychol 2003; 25, 691-701.

41. Mack WJ, Komotar RJ, Mocco J, Coon AL, Hoh DJ, King RG, Ducruet AF, Ransom ER, Oppermann M, DeLaPaz R, Connolly ESJ. Serial magnetic resonance imaging in experimental primate stroke: validation of MRI for pre-clinical cerebroprotective trials. Neurol Res 25:846-852 (2003).

42. Bell-McGinty S, Habeck C, Hilton HJ, Rakitin B, Scarmeas N, Zarahn E, Flynn J, DeLaPaz R, Basner R, Stern Y. Identification and differential vulnerability of a neural network in sleep deprivation. Cereb Cortex 14:496-502 (2004).

C. Selected research projects ongoing or completed during the last three years:

ACTIVE R01 AG 07370 (PI: Small) 06/30/99 to 06/30/04 Co-Investigator (5%) NIH $448,354



Title: HIPPOCAMPAL SUBUNIT ACTIVATION WITH NORMAL AND IMPAIRED MEMORY: A FUNCTIONAL ANALYSIS This project will use functional MRI to study normal and impaired memory in elderly and Alzheimer’s subjects. R01 NS 40409-02 (PI: Connolly) 09/30/00 to 06/30/05 Co-Investigator (5%) NIH/NINDS $ 250,000 Title: COMPLEMENT-MEDIATED NEURONAL INJURY IN STROKE This project will investigate primate and murine stroke both in terms of no-reflow and direct neuronal toxicity.

1 R01 CA 89395-01A1 (PI: Bruce) 12/01/01 to 11/30/06 Co-PI (20%) NIH/NCI $ 346,415 Title: TOPOTECAN BY INTRACEREBRAL CLYSIS FOR BRAIN TUMORS This project will investigate the efficacy and safety of topotecan delivered by intracerebral clysis, the application of advanced MR imaging as a noninvasive means of optimizing treatment parameters, and analyze tumor histopathology, topoisomerase I expression, and in vitro drug sensitivity.

P50 AG 08702-13 (PI: Shelanski) 06/01/99 to 05/31/05 Co-Investigator (5%) NIH Alzheimer’s Disease Research Center $113/201/ Project 3: fMRI analysis of hippocampal regions in aging and Alzheimer’s disease. The general goal is to develop activity-evoked fMRI to investigate and diagnose Alzheimer’s Disease.

NIA PO1 AGO7232 (PI: Mayeux) 02/01-89 to 06/30/04 Co-Investigator (5%) $1.514.765 Title: The Epidemiology of Dementia in an Urban Community

A cross-cultural investigation of dementia in a northern Manhattan community. NIH 5R01 NSO 29993-12(PI: Sacco) 01/01/03 to 12/31/07 Co-Investigator (5%) $1,624,926 Title: Stroke incidence and risk factors in a tri-ethnic region The aims are to evaluate the relationship between vascular outcomes to determine if MRI subclinical disease accounts for race-ethnic differences in cognitive impairment. PI: Small 02/01/03 – 01/31/06 Co-Investigator (5%) $90,909





NAME Robert L. DeLaPaz

POSITION TITLE Professor of Radiology




University of California, Berkeley CA B.A. 1969 Psychology University of Utah, Salt Lake City UT M.D. 1976 Medicine

A. Positions and Honors 1976-1980 Internship,Residency (Neurology & Radiology) University of Minnesota, Minneapolis MN 1980-1981 Research Fellow, Neuroradiology/CT/PET, SNB, NINCDS, NIH, Bethesda MD 1981-1982 Residency, Radiology, George Washington University, Washington DC 1982-1984 Fellowship, Neuroradiology, Massachusetts General Hospital, Boston MA 1984-1985 Assistant Professor, Radiology, University of California, San Francisco CA 1985-1991 Assistant Professor, Radiology, Stanford University, Stanford CA 1991-1995 Associate Professor, Radiology, Memorial Sloan-Kettering Cancer Center & Cornell University Medical College,

New York NY May 1995- Professor, Radiology, Director of Neuroradiology, Medical Director of High Field Magnetic Resonance Research, and Director of PACS, Columbia University College of Physicians & Surgeons, New York NY

B. Selected peer-reviewed publications 1.DeLaPaz RL, Dickman SR, Grosser BI. Effects of stress on rat brain adenosine 3',5' - monophosphate in vivo, Brain Research

85:171-175 (1975). 2.Di Chiro G, DeLaPaz RL, Brooks RA, Sokoloff L, Kornblith PL, Smith BH, Patronal NJ, Kufta CV, Kessler RM, Johnson GS,

Manning RG, Wolf AP. Glucose utilization of cerebral gliomas measured by (18F)- fluorodeoxyglucose and positron emission tomography, Neurology (NY) 32:1323-1329 (1982).

3.Patronas NJ, Chiro G, Brooks RA, DeLaPaz RL, Kornblith PL, Smith BH, Rizzoli HV, Kessler RM, Manning RG, Channing M, Wolf AP, Conner CM. (18F)- fluorodeoxyglucose and P.E.T in the evaluation of radiation necrosis of the Brain. Radiology 144:885-889 (1982).

4.DeLaPaz RL, Patronas NJ, Brooks RA, Smith BH, Kornblith PL, Milam H, Chiro G. Positron emission tomography study of suppression of gray-matter glucose utilization by brain tumors. Am J Neuroradiology 4:826-829 (1983).

6.DeLaPaz RL, Brady TJ, Buonanno FS, New PFJ, Kistler JP, McGinnis BD, Pykett IL, Taveras JM. Nuclear magnetic resonance (NMR) imaging of Arnold-Chiari type I malformation & hydromyelia. J Comp. Assisted Tomography 7(1):126-129 (1983);

7.DeLaPaz RL, New PFJ, Buonanno, Kistler JP, Oot RF, Rosen BR, Taveras JM, Brady TJ, NMR imaging of intracranial hemorrhage. J Computer Assisted Tomoraphy 8(4):599-607 (1984).

8.Galarraga J, Loreck DJ, Graham N, DeLaPaz RL, Smith BH, Hallgren D. Glucose metabolism in human gliomas: correspondence of in situ and in vitro metabolic rates and altered energy metabolism. Metabolic Brain Disease 1:279-291 (1986).

9.Enzmann DR, Rubin JB, DeLaPaz RL, Wright A. Benefits and pitfalls in MRI scanning from CSF pulsation. Radiology 161:773-778 (1986).

10.Duguid JR, DeLaPaz RL, DeGroot J, MRI of the midbrain in Parkinson's disease. Annals of Neurology 20:744-777 (1986). 11.DeLaPaz RL, Chang PJ, Bernstein R, Dave JV. Approximate fuzzy C-means (AFCM) cluster analysis of medical magnetic

resonance image (MRI) Data, IEEE Cybernetics 2:869-871 (1987). 12.Charness ME, DeLaPaz RL. Mamillary body atrophy in wernicke's encephalopathy: antemortem identification using magnetic

resonance imaging, Annals of Neurology 22:595-600 (1987). 13.Hoehne KH, DeLaPaz RL, Bernstein R, Tayloe RC. Combined surface display and reformatting for the three-dimensional

analysis of tomographic data, Investigative Radiology 22:658-664 (1987). 14.DeLaPaz RL, Bernstein R, Hanson WA, Walker MG. Approximate fuzzy C-means (AFCM) cluster analysis of medical magnetic

resonance image (MRI) data – system for research & education , IEEE Trans. Geoscience and Remote Sensing. Vol. GE-25:815-824 (1987).

15.DeLaPaz RL, Bernstein R. Computerized analysis and information of medical MRI. SPIE Remote Sensing 902:151-4 (1988). 16.Marks MP, DeLaPaz RL, Fabrikant JI, Frankel KA, Phillips MH, Levy RP, Enzmann DR. Imaging of charged-particle

radiosurgery for intracranial vascular malformations part I: Results of therapy. Radiology 168:447-455 (1988). 17.Marks MP, DeLaPaz RL, Fabrikant JI, Frankel KA, Phillips MH, Levy RP, Enzmann DR. Imaging of charged-particle

radiosurgery for intracranial vascular malformations part II: Complications. Radiology 168:457-462 (1988).



18.Steinberg GK, George P, DeLaPaz RL, Shibata DK, gross T. Dextromethorphan protects against cerebral injury following transient focal ischemia. Stroke 19:1112-1118 (1988).

19.Steinberg GK, Saleh J, DeLaPaz RL, Kunis D, Zamegar SR. Pretreatment with the NMDA antagonists dextromethorphan reduces cerebral injury following focal ischemia in rabbits. Brain Research 497:382-386 (1989).

20.DeLaPaz RL, Tetrud J, Langston W. MRI of L-DOPA responsive and nonresponsive Parkinson's disease. Neurology 39:275 (1989).

21. Lo EH, Frankel KA, Poljak A, DeLaPaz RL, Phillips MH, Woodruff KH, Brennan KM, Valk PE, Fabrikant JI. Cerebrovascular and metabolic perturbations in delayed heavy charged particle radiation injury. Brain Research 504:168-172 (1989).

22. DeLaPaz RL, Herskovitz E, DiGesu V, Hanson WA, Bernstein. Cluster analysis of medical magnetic resonance imaging data: diagnostic application and evaluation. SPIE/SPSE Proceedings 1259:176 (1990).

23. DeLaPaz RL, Shibata D, Zamegar SR, Steinberg GK. Cerebral ischemia in Rabbits: correlation of MRI and histopathology. Am J Neuroradiology 12:89-95 (1991).

24. DeLaPaz RL, Steinberg GK, Poljack A, Saleh J, Kunis D. MRI of experimental cerebral ischemia: response to NMDA Antagonist therapy. Neuroradiology 33:60-62 (1991).

25. DeLaPaz RL, Lo E, Frankel KA, Phillips MH, Fabrikant JL. MRI and PET of CNS delayed radiation injury. Neuroradiology 33:146 (1991).

26. Steinberg GK, Kunis D, Saleh J, DeLaPaz RL, Protection after Transient Focal Cerebral Ischemia by the N-Methyl-D-Aspartate Antagonist Dextrorphan is Dependent upon Plasma and Brain Levels, J Cerebral Blood Flow & Metab 11:1015-102 (1991)

27. DeLaPaz RL, Echo Planar Imaging, Radiographics 14:1045-1058 (1994). 29. Meyer KL, Kim K, Li T, Tulipano PK, Lee KM, DeLaPaz R, Hirsch J, Ballon D, Sensitivity-enhanced echo-planar MRI at 1.5T

using a 5 x 5 mesh dome resonator, Magn Reson Med 36:606-612, (1996). 30. Chan S, Chin S, Nordli DR, Goodman RR, DeLaPaz RL, Pedley TA, Prospective MR Identification of Non-Balloon and Balloon

Cell Subtypes of Focal Cortical Dysplasia, Ann. Neurology, 44:749-757 (1998). 31. Small SA, Perera GM, DeLaPaz RL, Mayeux R, Stern Y, Differential Regional Dysfunction of the Hippocampus Formation

Among Elderly with Memory Decline and Alzheimer’s Disease, Ann. Neurology, 45:466-472 (1999). 32. Marshall RS, Perera GM, Lazar RM, Krakauer JW, Constantine RC, DeLaPaz RL, The Evolution of Cortical Activation during

Recovery from Corticospinal Tract Infarction, Stroke, 31(3):656-61 (2000). 33. Lazar RM, Marshall RS, Pile-Spellman J, Duong HC, Mohr JP, Young WL, Solomon RL, Perera GM, DeLaPaz RL,

Interhemispheric transfer of language in patients with left frontal cerebral arteriovenous malformation. Neuropsychologia. 38(10):1325-32. (2000).

34. Small SA, Nava AS, Perera GM, Delapaz R, Stern Y, Evaluating the function of hippocampal subregions with high-resolution MRI in Alzheimer's disease and aging. Microsc Res Tech. 51(1):101-8. (2000).

35. Huang J, Mocco J, Choudhri TF, Poisik A, Popilskis SJ, Emerson R, DelaPaz RL, Khandji AG, Pinsky DJ, Connolly ES Jr, A modified transorbital baboon model of reperfused stroke. Stroke. 12:3054-63. (2000).

36. Scott A. Small, Ed X. Wu, Dusan Bartsch, Gerard M. Perera, Clay O. Lacefield, Robert DeLaPaz, Richard Mayeux, Yaakov Stern, and Eric R. Kandel, Imaging Physiologic Dysfunction of Individual Hippocampal Subregions in Humans and Genetically Modified Mice Neuron, 28:653–664 (2000).

37. Small SA, Nava AS, Perera GM, DeLaPaz RL, Mayeux R, Stern Y, Circuit mechanisms underlying memory encoding and retrieval in the long axis of the hippocampal formation, Nature Neuroscience 4: 442-449 (2001).

38. Small SA, Tsai WY, DeLaPaz R, Mayeux R, Stern Y. Imaging hippocampal function across the human life span: is memory decline normal or not. Ann Neurol 51:290-295 (2002).

39 Schumacher HC, Dumoulin CL, Feng L, Mangla S, Meyers PM, Hirsch J, Mohr JP, DeLaPaz RL, Pile-Spellman J. Real-time magnetic resonance imaging for interventional neuroradiological procedures. Surg Technol Int 2003; 11, 183-196.

40. Stern Y, Zarahn E, Hilton HJ, Flynn J, DeLaPaz R, Rakitin B. Exploring the neural basis of cognitive reserve. J Clin Exp Neuropsychol 2003; 25, 691-701.

41. Mack WJ, Komotar RJ, Mocco J, Coon AL, Hoh DJ, King RG, Ducruet AF, Ransom ER, Oppermann M, DeLaPaz R, Connolly ESJ. Serial magnetic resonance imaging in experimental primate stroke: validation of MRI for pre-clinical cerebroprotective trials. Neurol Res 25:846-852 (2003).

42. Bell-McGinty S, Habeck C, Hilton HJ, Rakitin B, Scarmeas N, Zarahn E, Flynn J, DeLaPaz R, Basner R, Stern Y. Identification and differential vulnerability of a neural network in sleep deprivation. Cereb Cortex 14:496-502 (2004).

C. Selected research projects ongoing or completed during the last three years:

ACTIVE R01 AG 07370 (PI: Small) 06/30/99 to 06/30/04 Co-Investigator (5%) NIH $448,354



Title: HIPPOCAMPAL SUBUNIT ACTIVATION WITH NORMAL AND IMPAIRED MEMORY: A FUNCTIONAL ANALYSIS This project will use functional MRI to study normal and impaired memory in elderly and Alzheimer’s subjects. R01 NS 40409-02 (PI: Connolly) 09/30/00 to 06/30/05 Co-Investigator (5%) NIH/NINDS $ 250,000 Title: COMPLEMENT-MEDIATED NEURONAL INJURY IN STROKE This project will investigate primate and murine stroke both in terms of no-reflow and direct neuronal toxicity.

1 R01 CA 89395-01A1 (PI: Bruce) 12/01/01 to 11/30/06 Co-PI (20%) NIH/NCI $ 346,415 Title: TOPOTECAN BY INTRACEREBRAL CLYSIS FOR BRAIN TUMORS This project will investigate the efficacy and safety of topotecan delivered by intracerebral clysis, the application of advanced MR imaging as a noninvasive means of optimizing treatment parameters, and analyze tumor histopathology, topoisomerase I expression, and in vitro drug sensitivity.

P50 AG 08702-13 (PI: Shelanski) 06/01/99 to 05/31/05 Co-Investigator (5%) NIH Alzheimer’s Disease Research Center $113/201/ Project 3: fMRI analysis of hippocampal regions in aging and Alzheimer’s disease. The general goal is to develop activity-evoked fMRI to investigate and diagnose Alzheimer’s Disease.

NIA PO1 AGO7232 (PI: Mayeux) 02/01-89 to 06/30/04 Co-Investigator (5%) $1.514.765 Title: The Epidemiology of Dementia in an Urban Community

A cross-cultural investigation of dementia in a northern Manhattan community. NIH 5R01 NSO 29993-12(PI: Sacco) 01/01/03 to 12/31/07 Co-Investigator (5%) $1,624,926 Title: Stroke incidence and risk factors in a tri-ethnic region The aims are to evaluate the relationship between vascular outcomes to determine if MRI subclinical disease accounts for race-ethnic differences in cognitive impairment. PI: Small 02/01/03 – 01/31/06 Co-Investigator (5%) $90,909





NAME POSITION TITLE

Dwip Kitayaporn Professor of Epidemiology EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)


Mahidol University, Bangkok, Thailand B.Sc. 1977 Medical Science Mahidol University, Bangkok, Thailand M.D. 1979 Medicine Mahidol University, Bangkok, Thailand D.T.M.&H. 1983 Tropical Medicine Johns Hopkins University, Baltimore, MD, USA M.Ph.H. 1987 Epidemiology Johns Hopkins University, Baltimore, MD, USA Dr.Ph.H. 1990 Epidemiology

A. Positions and Honors. 1. Professional Appointments and Experience:

1980 General Practitioner, Bang Saphan Community Hospital, Prachuap Khiri Khan, Ministry of Public Health of Thailand.

1981-1985 Acting Director, Bang Saphan Community Hospital,Prachuap Khiri Khan, Ministry of Public Health of Thailand.

1985-1982 Lecturer, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 1991-1999 Medical Epidemilogist, HIV/AIDS Collaboration, joint appointment by the Thai Ministry of

Public Health and the U.S. Centers for Disease Control and Prevention 1992-1996 Assistant Professor, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 1996-2000 Associate Professor of Epidemiology, Department of Social and Environmental Medicine,

Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. 1999- Chief, Data Management Unit, Vaccine Trial Centre, Faculty of Tropical Medicine, Mahidol

University, Bangkok, Thailand. 2000- Professor of Epidemiology, Department of Social and Environmental Medicine, Faculty of

Tropical Medicine, Mahidol University, Bangkok, Thailand. 2000- Head, Department of Social and Environmental Medicine, Faculty of Tropical Medicine,

Mahidol University, Bangkok, Thailand. 2001-2 Director, Thailand Tropical Disease Research Program, T-2, A joint collaboration between

WHO/TDR and Thailand. 2002- Member, Thai AIDS Vaccine Evaluation Group

2. Membership:

1994- Scientific and Ethical Review Group (SERG) for the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization

1998- Bangkok Aids Vaccine Evaluation Group (BVEG).

3. Editorial Board/Editor

Editorial Board, Journal of Tropical Medicine and Parasitology, 2000- , Editorial Board, Southeast Asian Journal of Tropical Medicine and Parasitology, 2001- ,

B. Selected peer-reviewed publications: Kitayaporn D, Nelson KE, Charoenlarp P, Pholpothi T. Haemoglobin-E in the presence of oxidative

substances from fava bean may be protective against Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 1992; 86:240-4.



Kitayaporn D, Uneklabh C, Weniger BG, Lohsomboon P, Kaewkungwal J, Morgan WM, Uneklabh T. HIV-1 incidence determined retrospectively among drug users in Bangkok, Thailand. Aids 1994; 8:1443-50.

Kitayaporn D, Bejrachandra S, Chongkolwatana V, Chandanayingyong D, Weniger BG. Potential deferral criteria predictive of human immunodeficiency virus positivity among blood donors in Thailand. Transfusion 1994; 34:152-7.

Kitayaporn D, Kaewkungwal J, Bejrachandra S, Rungroung E, Chandanayingyong D, Mastro TD. Estimated rate of HIV-1-infectious but seronegative blood donations in Bangkok, Thailand. Aids 1996; 10:1157-62.

Kitayaporn D, Tansuphaswadikul S, Lohsomboon P, Pannachet K, Kaewkungwal J, Limpakarnjanarat K, Mastro TD. Survival of AIDS patients in the emerging epidemic in Bangkok, Thailand. J Acquir Immune Defic Syndr Hum Retrovirol 1996; 11:77-82.

Des Jarlais DC, Vanischseni S, Marmor M, Kitayaporn D. HIV vaccine trials. Science 1998; 279:1433-4. Kitayaporn D, Vanichseni S, Mastro TD, Raktham S, Vaniyapongs T, Des Jarlais DC, Wasi C, Young

NL, Sujarita S, Heyward WL, Esparza J. Infection with HIV-1 subtypes B and E in injecting drug users screened for enrollment into a prospective cohort in Bangkok, Thailand. J Acquir Immune Defic Syndr Hum Retrovirol 1998; 19:289-95.

Zhang K, Fujioka H, Lobo CA, Kitayaporn D, Aikawa M, Kumar N. Cloning and characterization of a new asparagine-rich protein in Plasmodium falciparum. Parasitol Res 1999; 85:956-63.

Subbarao S, Vanichseni S, Hu DJ, Kitayaporn D, Choopanya K, Raktham S, Young NL, Wasi C, Sutthent R, Luo CC, Ramos A, Mastro TD. Genetic characterization of incident HIV type 1 subtype E and B strains from a prospective cohort of injecting drug users in Bangkok, Thailand. AIDS Res Hum Retroviruses 2000; 16:699-707.

Kilmarx PH, Ramjee G, Kitayaporn D, Kunasol P. Protection of human subjects' rights in HIV-preventive clinical trials in Africa and Asia: experiences and recommendations. Aids 2001; 15 Suppl 5:S73-9.

Parekh BS, Hu DJ, Vanichseni S, Satten GA, Candal D, Young NL, Kitayaporn D, Srisuwanvilai LO, Rakhtam S, Janssen R, Choopanya K, Mastro TD. Evaluation of a sensitive/less-sensitive testing algorithm using the 3A11-LS assay for detecting recent HIV seroconversion among individuals with HIV-1 subtype B or E infection in Thailand. AIDS Res Hum Retroviruses 2001; 17:453-8.

Vanichseni S, Kitayaporn D, Mastro TD, Mock PA, Raktham S, Des Jarlais DC, Sujarita S, Srisuwanvilai LO, Young NL, Wasi C, Subbarao S, Heyward WL, Esparza L, Choopanya K. Continued high HIV-1 incidence in a vaccine trial preparatory cohort of injection drug users in Bangkok, Thailand. Aids 2001; 15:397-405.

Hu DJ, Subbarao S, Vanichseni S, Mock PA, van Griensven F, Nelson R, Nguyen L, Kitayaporn D, Young NL, Des Jarlais D, Byers R, Jr., Choopanya K, Mastro TD. Higher viral loads and other risk factors associated with HIV-1 seroconversion during a period of high incidence among injection drug users in Bangkok. J Acquir Immune Defic Syndr 2002; 30:240-7.

Hudgens MG, Longini IM, Jr., Vanichseni S, Hu DJ, Kitayaporn D, Mock PA, Halloran ME, Satten GA, Choopanya K, Mastro TD. Subtype-specific transmission probabilities for human immunodeficiency virus type 1 among injecting drug users in Bangkok, Thailand. Am J Epidemiol 2002; 155:159-68.

Nguyen L, Hu DJ, Choopanya K, Vanichseni S, Kitayaporn D, van Griensven F, Mock PA, Kittikraisak W, Young NL, Mastro TD, Subbarao S. Genetic analysis of incident HIV-1 strains among injection drug users in Bangkok: evidence for multiple transmission clusters during a period of high incidence. J Acquir Immune Defic Syndr 2002; 30:248-56.

Anh PK, Khanh NT, Ha DT, Chien do T, Thuc PT, Luong PH, Kilmarx PH, Wongchotigul V, Kitayaporn D, Rowe PJ. Prevalence of lower genital tract infection among women attending maternal and child health and family planning clinics in Hanoi, Vietnam. Southeast Asian J Trop Med Public Health 2003; 34:367-73.

Choopanya K, Des Jarlais DC, Vanichseni S, Mock PA, Kitayaporn D, Sangkhum U, Prasithiphol B, Hiranrus K, van Griensven F, Tappero JW, Mastro TD. HIV risk reduction in a cohort of injecting drug users in Bangkok, Thailand. J Acquir Immune Defic Syndr 2003; 33:88-95.

Hu DJ, Vanichseni S, Mock PA, Young NL, Dobbs T, Byers RH, Jr., Choopanya K, van Griensven F, Kitayaporn D, McDougal JS, Tappero JW, Mastro TD, Parekh BS. HIV type 1 incidence estimates by detection of recent infection from a cross-sectional sampling of injection drug users in Bangkok: use of the IgG capture BED enzyme immunoassay. AIDS Res Hum Retroviruses 2003; 19:727-30.



1. Ongoing research support: Project: "Phase III Trial, VAC-HIV (vCP1521) Priming With VaxGen gp120 B/E"

Principal Investigator: Dwip Kitayaporn

Agency: Thai Ministry of Public Health

Type: Research Grant

Period: 1 Jul 2003 to 30 Jun 2006

This is the second efficacy trial of HIV-1 candidate vaccines in Thailand in HIV-uninfected Thai adults, using the ALVAC/gp120 prime-boost HIV vaccine. Role: Principal Investigator. Data Management Unit Project: "Research, Development and Research Training in Human Reproduction"

Principal Investigator: Dwip Kitayaporn

Agency: UNDP/UNFPA/WHO World Bank

Type: Research Grant

Period: 1 Jul 1999 to 30 Jun 2004

This project is the Thai component of the WHO Special Programme in Human Reproduction. Role: Principal Investigator





NAME POSITION TITLE

Jiraporn Laothamatas, M.D. Neuroradiologist and Director, AIMC EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)


Chulalongkorn University B.Sc. (Honors) 1976 Medical Science Chulalongkorn Universitye M.D. (Honors) 1981 Medicine

A. Positions and Professional Experience Internship Chulalongkorn Rotating Internship 982 Diploma Hospital, Bangkok

American Board in Columbia University Diagnostic 1985-1989 Diagnostic Radiology College P&S, NYC, NY Radiology

Diploma US Armed Force Radiologic 1988 Institute of Pathology Pathology

Certificate Hahnemann Univ. Fellowship in 1990 Hospital, Philadelphia, Neuroradiology Pennsylvania, USA

Diagnostic radiologist Ireland Army Hospital 1990 at Fort Knox Louisville, Kentucky, USA

Diagnotic radiologist Chulalongkorn Hospital 1991 - 1992 Bangkok, Thailand

Neuroradiologist Ramathibodi Hospital, 1992 - present Mahidol University

Managing Director, Sirikit Medical Building, 1997 - present Advanced Diagnostic Ramathibodi Hospital Imaging and Image-guided Minimal Invasive Therapy Center (AIMC)

B. Professional Memberships

American Society of Neuroradiology Radiological Society of North America Asian and Oceanian Society of Neuroradiology and Head and Neck Radiology (AOSNHNR) The Radiological Society of Thailand Royal College of Radiologists of Thailand Neurological Society of Thailand (Central committee) Epilepsy Society of Thailand (Founding member) Neurosurgical Society of Thailand



C. Selected peer-reviewed publications:

Pulkes T, Sura T, Laothamatas J, Boongird P, G.C. Cowan D.Phill. Trinucleotide repeats in Huntington’s disease : a case report. Rama Med J 1995;18:266-70. Wongsrisoontorn S, Laothamatas J. Re-designing a needle used in pre-biopsy localization of a nonpalpable breast lesion. Rama Med J 1996 ; 19 :146-52. Jongjirasiri S, Chuapetcharasopon C, Wibulpolprasert B, Tapaneeyakorn J, Nitjapanich S, Laothamatas J, Wacharasin R. Clinical value of electron beam CT angiogram in evaluating thoracic and abdominal aneurysm and dissecting aneurysm. Bullentin of Vascular Surgery. 1997 ; 1 : 101-110. Charuvanij A, Laothamatas J, Torcharus K, Sirivimonmas S. Moyamoya disease and protein S deficiency: a case report. Pediatr Neurol. 1997;17:171-173. Thitithanyanont A, Mendoza L, Chuansumrit A, et al. Use of an immunotherapeutic vaccine to treat a life-threatening human arteritic infection caused by Pythium insidiosum. Clin Infect Dis. 1998;27:1394-1400. Visudhiphan P, Bunyaratavej S, Visudtibhan A, et al. Temporal lobectomy for intractable complex partial seizures in pediatric patients. J Med Assoc Thai. 1999;82:778-783. Dhanachai M, Theerapancharoen V, Laothamatas J, et al. Early neurological complications after stereotactic radiosurgery/radiotherapy. J Med Assoc Thai. 2001;84:1729-1737. Kunnatiranont R, Laothamatas J, Asavaphatiboon S, Worapruckjaru L, Kumkrua C, Kampangtip A. Localization of sensorimotor cortex by using functional magnetic resonance imaging: comparison between finger tapping and palm scratching in normal volunteer. J Med Assoc Thai. 2002;85:1264-1272. Ratanakorn D, Laothamatas J, Pongpech S, Tirapanich W, Yamwong S. Pitfall of electron beam computed tomography angiography in diagnosis of subclavian steal syndrome. J Neuroimaging. 2002;12:80-83. Hemachudha T, Laothamatas J, Rupprecht CE. Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges. Lancet Neurol. 2002;1:101-109. Dujneungkunakorn T, Sungkanuparph S, Vibhagool A, et al. Detection of JC virus infection in progressive multifocal leukoencephalopathy: the first documented case in Thailand. J Med Assoc Thai. 2002;85:1139-1144. Warrasak S, Suvaranamani C, Euswas A, Sumetpimolchai V, Laothamatas J. Choroidal osteoma in Oriental patients. J Med Assoc Thai. 2003;86:562-572. Laothamatas J, Hemachudha T, Mitrabhakdi E, Wannakrairot P, Tulayadaechanont S. MR imaging in human rabies. Am J Neuroradiol. 2003;24:1102-1109. Wattanasirichaigoon D, Visudtibhan A, Jaovisidha S, Laothamatas J, Chunharas A. Expanding the phenotypic spectrum of Lenz-Majewski syndrome: facial palsy, cleft palate and hydrocephalus. Clin Dysmorphol. 2004;13:137-142. Phoncharoensri D, Witoonpanich R, Tunlayadechanont S, Laothamatas J. Confusion and paraparesis. Lancet.

2004;363:1954.



BIOGRAPHICAL SKETCH

NAME

SRIRAMA SWAMINATHAN, PH.D. POSITION TITLE

Clinical Scientist, Philips Medical Systems

INSTITUTION AND LOCATION DEGREE

(if applicable)

YEAR(s)

FIELD OF STUDY

University of Madras, Chennai, TN, India Central University of Hyderabad, Hyderabad, AP, India Michigan Technological Institute, Houghton, MI

B.Sc. M.Sc.

Ph.D.

1990 1992

1998

Physics Physics Physics

Professional Experience: 1991-1992 Research Assistant, Central University of Hyderabad, Hyderabad, India 1992-1993 Junior Research Fellow, IUC- Central University of Hyderabad, Hyderabad, India 1993-1994 Graduate Teaching Assistant, MTU, Houghton, MI 1994-1997 Graduate Research Fellow, MTU, Houghton, MI 1996 Summer Intern, New Mexico Institute of Neuroimaging, Albuquerque, NM 1997-1998 Graduate Research Assistant, MTU, Houghton, MI 1998-1999 Postdoctoral Fellow, Fox Chase Cancer Center, Philadelphia, PA 1999-2001 Research Fellow, Mayo Clinic, Rochester, MN 2001-Present Clinical Scientist, Philips Medical Systems, Cleveland, OH Publications: S. Swaminathan and C.S.Sunandana, Rapid Synthesis and characterization of NH4Ag4I5, Solid State Ionics, 70/71, 163, (1994)

S. V. Swaminathan and Bryan Suits, NMR Imaging of solids with very broad lines, Proceedings of the 37th ENC, 1996

V. S. Swaminathan and B. H. Suits, NMR Imaging Using Second Order Quadrupole Broadened Resonances, J. Magn. Reson. 132, 274 (1998).

J. Sepa, R.J. Gorte, D.White, B.H. Suits and V.S. Swaminathan, A 13C NMR and FTIR investigation of Acetonitrile Adsorption in H-MFI between 190 and 523K, Proceedings of the 12th International Zeolite Conference at Baltimore, July 5, 1998. S.V. Swaminathan, O.Gonen and G.Goelman, Non-Echo 3D multivoxel 1H MRS of the human brain using 2D 8th order Hadamard/1D x 16 CSI, Proceedings of the 7th ISMRM, May 1999 O.Gonen, I. Catalaa, L.J.Mannon, S.V. Swaminathan and R.Grossman, Total Brain N-Acetylaspartate concentration and its decline with age in relapsing-remitting multiple sclerosis, Proceedings of 85th RSNA: Supplement to Radiology, 213(P), 1999

O.Gonen, I.Catalaa, S.V. Swaminathan, L.J.Mannon, D.Kolson, and R.Grossman, Quantitating Total Brain NAA and its decline with age in multiple sclerosis using Non-echo 1H MRS, Proceedings of the 7th ISMRM, May 1999 S. V. Swaminathan and B. H. Suits, Reconstructing Powder NQR Images with Real Gradient Coils, Journal of Magnetic Resonance 138,123 (1999).



S.V. Swaminathan, S.B.Fain, R.F.Busse and S.J. Riderer, Multiple time phase CE-3D MR Angiography, Proceedings of 86th RSNA: Supplement to Radiology, 2000 S.V. Swaminathan, A.R. Fuisz, U.Iqbal, and J.R.Osborne, SENSE PVM-A comparitive study with reqular Q-Flow, J. Card. Magn. Reson. 5 (1), pp. 167-183, 2003 U. Iqbal, A.R. Fuisz, L.Satler, A.Pichard, and S.Swaminathan, Qp/Qs In Atrial Septal Defects By Cardiac Magnetic Resonance Imaging Is Comparable To Qp/Qs By Cardiac Catheterization, Proccedings of the 52nd American College of Cardiology, March, 2003. R. Wyche, S. McGlynn, U.Iqbal, S.V. Swaminathan, A.R. Fuisz, G.A.Hirsch, W. P. Ingkanisorn,

A.H. Aletras, and P.Kellman, Safety of MRI Perfusion Imaging Using Adenosine, J. Card. Magn. Reson. 5 (1), pp. 68–118, 2003

S.R. McGlynn, A.R. Fuisz, and S.V. Swaminathan, Preliminary Experience with Combined MR Perfusion and MR Coronary Angiography in the Detection of Coronary Artery Stenoses, J. Card. Magn. Reson. 5 (1), pp. 68–118, 2003 M.Shinnar, and S.V. Swaminathan, Evaluation of time efficiency of real time Navigator against breath hold in respiratory compensated cardiac exams, J. Card. Magn. Reson. 5 (1), pp. 119-143, 2003 J. F. London, G.Tilak, S.V. Swaminathan, and J.Whang, Exercise Cardiac Stress Testing Using Real-Time MRI: A Comparison with Stress Echo in Patient, J. Card. Magn. Reson. 5 (1), pp. 1-67, 2003 J. F. London, G.Tilak, S.V. Swaminathan, A comparison of Dobutamine Stress Function and Stres Perfusion MRI in 260 patients, J. Card. Magn. Reson. 5 (1), pp 1-67, 2003 S. V. Swaminathan and A.R. Fuisz, Non-Breathhold Cardiac functional imaging with Radial k-Space sampling. Proceedings of the 11th ISMRM, July, 2003 S. V.Swaminathan, K. Demarco, L. Boccassini, and R. Hoogeveen, Is Balanced Turbo Field Echo a clinically viable alternative for MOTSA?, Proceedings of the 11th ISMRM, July, 2003 M. Tebeica, S.V.Swaminathan, N.J. Weissman, P. Rai, A.R. Fuisz, Images in cardiovascular medicine. Visualization of left main coronary dissection by magnetic resonance coronary angiography. Circulation. 2003 Aug 26; 108(8): 1034-5. S. V. Swaminathan, D. Owen, and A. R. Fuisz, Rapid Phase Velocity Mapping of the Aortic flow with and without SENSE –Volume and velocity measurements compared to standard techniques, J. Card. Magn. Reson. 6 (1), pp. 235-236, 2004 S.V. Swaminathan, D.I. Paterson, D. Owen, and A. R. Fuisz, Optimization of parameters, SENSE and coil comparison for vulnerable plaque imaging in patients with ACS in a routine clinical practice, J. Card. Magn. Reson. 6 (1), pp. 270-271, 2004 S. V. Swaminathan, C. M. Rattin, D. I. Paterson, M. Breeuwer, R. Wyche, D. Owen, A. R. Fuisz, An assement of free breathing on image quality for visual and semi quantitative cardiac perfusion, Proceedings of the 12th ISMRM, May, 2004 P.F.Friedman, S.V.Swaminathan and R.I. Smith, SENSE Imaging of the Breast, AJR (In Press)





NAME POSITION TITLE

Polrat Wilairatana, M.D., Ph.D. Professor of Tropical Medicine EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)


Thai Medical Council, Thailand Diplomate 1990 Internal Medicine Chulalongkorn University, Bangkok, Thailand M.Sc. 1990 Gastroenterology Mahidol University, Bangkok, Thailand M.D. 1984 Medicine

A. Positions and Honors. 1. Professional Appointments:

1991-1993 Lecturer, Dept. of Clinical Tropical Medicine and Hospital for Tropical Diseases, Faculty of Medicine, Mahidol University, Bangkok, Thailand

1993-1996 Assistant Professor, Dept. of Clinical Tropical Medicine and Hospital for Tropical Disease, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

1993-present Head, Division of Gastroenterology and Hepatology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

1996-1998 Associate Professor, Department of Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

1996-present Director, Hospital for Tropical Diseases, Deputy Dean, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

1997-present Director, International Society of Nephrology (ISN) Renal Sister Center. 1998-present Professor, Department of Clinical Tropical Medicine, Faculty of Tropical Medicine,

Mahidol University, Thailand. 1999-present Head, Clinical Pharmacology Unit, Faculty of Tropical Medicine, Mahidol University,

Bangkok, Thailand.

2. Awards:

1990 First Prize for the Distinguished Research from the Royal College of Physicians, Thailand. 1995 The Mahidol University Braun Prize 1995 for Medicine and Public Health of Thailand from

Mahidol University, Thailand 1996 The Mahidol University Prize 1996 for the Best Research from Mahidol University,

Thailand 1999 The Best Research Award, National Research Council, Thailand.

3. Membership

1984-present Thai Medical Council . 1984-present Gastroenterological Association of Thailand. 1993-present Parasitology and Tropical Medicine Association of Thailand, 1993 - 1994-present New York Academy of Science, USA. 1994-present American College of Gastroenterology, USA; Fellow, 1998. 1998-present Thai Academy of Science and Technology

B. Selected peer-reviewed publications:

Looareesuwan S, Wilairatana P, Vanijanonta S, Kyle D, Webster K. Efficacy of quinine-tetracycline for acute uncomplicated falciparum malaria in Thailand. Lancet 1992;339:369.


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Wilairatana P, Looareesuwan S, Kaocharern S, Vanijanonta S, Charoenlarp P. Gastric emptying in acute uncomplicated falciparum malaria. J Trop Med Hygiene 1995;98:22-24.

Wilairatana P, Looareesuwan S. APACHE II scoring for predicting outcome in cerebral malaria. J Trop Med Hyg 1995;98:256-260.

Looareesuwan S, Wilairatana P, Vanaphan S, Gordeuk VR, Taylor TE, Meshnick SR, Brittenham, GM. Co-administration of desferrioxamine with artesunate in malaria: an assessment of safety and tolerance. Annals Trop Med Parasitol 1996;90:551-554.

Wilairatana P, Viriyavejakul P, Looareesuwan S, Chongsuphajaisiddhi T. Artesunate suppositories: an effective treatment for severe falciparum malaria in rural areas. Ann Trop Med Parasitol 1997; 91:891-6.

Wilairatana P, Looareesuwan S, Riganti M, Teja-Isavadharm P, Keeratithakul D, Eickmeyer S, Walsh DS. Pustular eruption in a malaria patient treated with chloroquine. Int J Dermatol 1997; 36:634-5.

Wilairatana P, Kyle DE, Looareesuwan S, Chinwongprom K, Amradee S, White NJ, Watkins WM. Poor efficacy of antimalarial biguanide-dapsone combinations in the treatment of acute, uncomplicated, falciparum malaria in Thailand. Ann Trop Med Parasitol 1997; 91:125-32.

Wilairatana P, Meddings JB, Ho M, Vannaphan S, Looareesuwan S. Increased gastrointestinal permeability in patients with Plasmodium falciparum malaria. Clin Infect Dis 1997; 24:430-5.

Camacho LH, Gordeuk VR, Wilairatana P, Pootrakul P, Brittenham GM, Looareesuwan S. The course of anaemia after the treatment of acute, falciparum malaria. Ann Trop Med Parasitol 1998; 92:525-37.

Looareesuwan S, Wilairatana P, Vannaphan S, Wanaratana V, Wenisch C, Aikawa M, Brittenham G, Graninger W, Wernsdorfer WH. Pentoxifylline as an ancillary treatment for severe falciparum malaria in Thailand. Am J Trop Med Hyg 1998; 58:348-53.

Wilairatana P, Chanthavanich P, Singhasivanon P, Treeprasertsuk S, Krudsood S, Chalermrut K, Phisalaphong C, Kraisintu K, Looareesuwan S. A comparison of three different dihydroartemisinin formulations for the treatment of acute uncomplicated falciparum malaria in Thailand. Int J Parasitol 1998; 28:1213-8.

Camacho LH, Wilairatana P, Weiss G, Mercader MA, Brittenham GM, Looareesuwan S, Gordeuk VR. The eosinophilic response and haematological recovery after treatment for Plasmodium falciparum malaria. Trop Med Int Health 1999; 4:471-5.

Hutagalung R, Wilairatana P, Looareesuwan S, Brittenham GM, Aikawa M, Gordeuk VR. Influence of hemoglobin E trait on the severity of Falciparum malaria. J Infect Dis 1999; 179:283-6.

Wilairatana P, Silachamroon U, Krudsood S, Singhasivanon P, Treeprasertsuk S, Bussaratid V, Phumratanaprapin W, Srivilirit S, Looareesuwan S. Efficacy of primaquine regimens for primaquine-resistant Plasmodium vivax malaria in Thailand. Am J Trop Med Hyg 1999; 61:973-7.

Wilairatana P, Westerlund EK, Aursudkij B, Vannaphan S, Krudsood S, Viriyavejakul P, Chokejindachai W, Treeprasertsuk S, Srisuriya P, Gordeuk VR, Brittenham GM, Neild G, Looareesuwan S. Treatment of malarial acute renal failure by hemodialysis. Am J Trop Med Hyg 1999; 60:233-7.

Hutagalung R, Wilairatana P, Looareesuwan S, Brittenham GM, Gordeuk VR. Influence of hemoglobin E trait on the antimalarial effect of artemisinin derivatives. J Infect Dis 2000; 181:1513-6.

Wilairatana P, Krudsood S, Treeprasertsuk S, Chalermrut K, Looareesuwan S. The future outlook of antimalarial drugs and recent work on the treatment of malaria. Arch Med Res 2002; 33:416-21.

Ittarat W, Pickard AL, Rattanasinganchan P, Wilairatana P, Looareesuwan S, Emery K, Low J, Udomsangpetch R, Meshnick SR. Recrudescence in artesunate-treated patients with falciparum malaria is dependent on parasite burden not on parasite factors. Am J Trop Med Hyg 2003; 68:147-52.

Krudsood S, Wilairatana P, Vannaphan S, Treeprasertsuk S, Silachamroon U, Phomrattanaprapin W, Gourdeuk VR, Brittenham GM, Looareesuwan S. Clinical experience with intravenous quinine, intramuscular artemether and intravenous artesunate for the treatment of severe malaria in Thailand. Southeast Asian J Trop Med Public Health 2003; 34:54-61.

Nagamine Y, Hayano M, Kashiwamura S, Okamura H, Nakanishi K, Krudsod S, Wilairatana P, Looareesuwan S, Kojima S. Involvement of interleukin-18 in severe Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 2003; 97:236-41.

Nagao Y, Chavalitshewinkoon-Petmitr P, Noedl H, Thongrungkiat S, Krudsood S, Sukthana Y, Nacher M, Wilairatana P, Looareesuwan S. Paroxysm serum from a case of Plasmodium vivax malaria inhibits the maturation of P. falciparum schizonts in vitro. Ann Trop Med Parasitol 2003; 97:587-92.

Looareesuwan S, Imwong M, Wilairatana P. Chlorproguanil-dapsone for malaria in Africa. Lancet. 2004;363:1838-1839.

Nacher M, Silachamroon U, Singhasivanon P, et al. Comparison of artesunate and chloroquine activities against Plasmodium vivax gametocytes. Antimicrob Agents Chemother. 2004;48:2751-2752.


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C. Research Support 1. Ongoing research support: Project: "Pathogenesis of severe malarial anemia in Thailand"


Agency: NIAID

Type: R01 (AI51310, Years 1-4)


The proposed research project is designed to examine the pathogenesis of severe malarial anemia in Thailand to define those elements attributable to infection with Plasmodium falciparum. Role: Co-Investigator, Mahidol University subcontract Project: " Malaria control in northwestern Thailand"


Agency: Gates Foundation

Type: Joint Grant

Period: 1 Dec 2000 to 30 Nov 2005

The Global Health Program of the Bill & Melinda Gates Foundation has provided a joint grant for the Mahidol University Faculty of Tropical Medicine, the Wellcome Mahidol Oxford Unit and the Communicable Disease Control Department (CDC) of the Thai Ministry of Public Health to support a trial of combined antimalarial therapy for control of falciparum malaria in northwestern Thailand. Role: Co-Investigator Project: “Effective antimalarial combination therapy”


Agency: Thai Ministry of University Affairs

Type: Grant


This project provides for research and development ofeffective antimalarial drugs in combination for widespead use in the treatment of malaria. Role: Co-Investigator

2. Completed research support: Project: “Cerebral malaria: role of iron in pathogenesis and therapy.”


Agency: NIAID

Type: U01 (AI35827, Years 1-6)

Period: 1 July 1994 to 30 Sept 2000

The overall goals of this project, based in Bangkok, Thailand, were to define the role of iron in the pathogenesis of cerebral malaria and to determine if the iron chelator deferoxamine could reduce morbidity and mortality in patients with this disorder. The underlying hypothesis was that the pathophysiology of cerebral malaria includes the release of free hemoglobin, hemozoin and iron, leading to the generation of oxygen-derived free radicals which cause lipid peroxidant damage to cellular and subcellular membranes of the central nervous system. Role: Co-Investigator


PHS 398 (Rev. 05/01) Page __41__ Resources Format Page

RESOURCES FACILITIES: Specify the facilities to be used for the conduct of the proposed research. Indicate the performance sites and describe capacities, pertinent capabilities, relative proximity, and extent of availability to the project. Under “Other,” identify support services such as machine shop, electronics shop, and specify the extent to which they will be available to the project. Use continuation pages if necessary.

Laboratory:

Facilities include a (1) Central Research Laboratory, available for use by expert staff from any department; Clinical laboratories of the Hospital for Tropical Diseases (2 stories), two fully equipped immunology laboratories; two laboratories for the culture in vitro of malarial parasites. Clinical: The Bangkok Hospital for Tropical Diseases is a specialized hospital for tropical diseases which was founded in 1961. The Hospital is now a 300 bed facility admitting both adult and pediatric patients. Of the hospital beds, 250 are allocated for adult patients, 50 for children. The hospital has a well equipped intensive care unit with four beds which has been operated for more than ten years. The equipment for this facility includes cardiac monitors, volume respirators and hemodialysis units. Animal: Two floors of accredited research animal housing.

Computer: A total of 10 networked PC’s are available with up-to-date word processing and statistical software, with notebook computers for data entry and 5 network printers. Office: Ample office facilities include a suite of clinical offices, two data storage room and two meeting rooms.

Other: A Data Management Unit is available for research use. The Faculty maintains three field research stations, in Ratchaburi, Kanchanaburi and Tak Provinces. A Phillips 3.0T Intera Master Magnetic Resonance scanner is available to the Faculty of Tropical Medicine for research use at the nearby Ramathibodi Hospital.

MAJOR EQUIPMENT: List the most important equipment items already available for this project, noting the location and pertinent capabilities of each.

Analytical equipment and facilities available in the Central Research Laboratory include: Automated DNA sequencer, Pharmacia Model ALFexpress., Electrophoresis system and accessories, Bio-Rad , Gel Dryer, Bio-Rad 583, Thermal cycler, Perkin Elmer, UV-Vis Spectrophotometer, Automatic high speed refrigerated centrifuge, Hitachi CR20B2, Ultracentrifuge, Hitachi HiMaG, Vacuum centrifuge, Labconco, Table-top refrigerated centrifuge, Hitachi, Densitometer, Bio-Rad GS-670, High Performance Liquid Chromatograph ( HPLC), Water Inc., Gas Chromatograph – Mass Spectrometer (GC-MS), Fisons, Atomic Absorption Spectrometer ( AAS), Hitachi, Auto-sampler, Pharmacia, ELISA reader, Deep freezers ( 2 units ), - 80 oC , Freezer –20oC, Refrigerators ( 2 units ) , Compound microscope, Shaking water bath, Precision Scientific 360, Thermal bath, Simadzu TB85, Cooling bath, Pharmacia LKB, Laboratory water bath, Sonicator, Analytical balance, Oerting NA164, Electronic balance, Rotator platform, Thomas, Cold room facility, CO2 Incubator , Incubator ( 2 units ), pH meter, ORION, Automatic autoclaves ( 2 units ), Liquid nitrogen tank, Hot plate/stirrers ( 2 units ).Thermotek, Hot air ovens ( 2 units ), Memmert, Mettler Electronic Inc., Fraction collector, Fume hoods ( 2 units ), Laminar flow cabinets ( 2 units ). An Advia 120 Hematology Analyzer has recently been installed in the Central Hematology Laboratory.


PHS 398 (Rev. 5/01) Page __42__ Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.

Research Plan: RBC sequestration in cerebral malaria: MRI measurement

a. Specific Aims

The proposed research will use quantitative magnetic resonance methods to determine the role of red blood cell sequestration in the pathogenesis of cerebral malaria. Cerebral malaria is a major life-threatening complication of infection with Plasmodium falciparum, diagnosed clinically as unrousable coma in the absence of other attributable causes. Without treatment, cerebral malaria is almost inevitably fatal. Even with parenteral antimalarials and intensive management of complications, mortality is 20% overall and, with associated organ dysfunction, rises to 50% or more. Remarkably, most survivors recover rapidly and are left without apparent long-term neurological damage. The pathological basis for cerebral malaria is complex, multifaceted and poorly understood. At autopsy, the hallmark of cerebral malaria is congestion of cerebral capillaries and postcapillary venules with parasitized and non-parasitized red blood cells. This sequestration of infected red blood cells in the relatively hypoxic venous beds provides P. falciparum with conditions that both promote optimal growth and prevent destruction of the parasites by the spleen. For the past decade, investigators have held deeply divergent views of the precise role of microvascular sequestration of parasitized red blood cells in the pathogenesis of cerebral malaria. The sequestration theory holds that the presence of infected red blood cells in the microvasculature of the brain is the indispensable initial event that is the prerequisite for the development of cerebral malaria. Conversely, the cytokine theory considers that cerebral malaria is the consequence of the excess production of pro-inflammatory cytokines in response to P. falciparum infection but that sequestration of parasitized erythrocytes is neither necessary nor sufficient for the production of coma. Obtaining evidence to differentiate between the sequestration and cytokine theories has been near impossible, in part because determination of the extent of microvascular sequestration has been restricted to pathological studies at autopsy. An improved understanding of the pathogenetic role of red cell sequestration is urgently needed as a basis for rational design of effective treatments for patients with cerebral malaria.

We propose non-invasive high-field (3.0 Tesla) magnetic resonance (MR) studies to determine the extent of microvascular red blood cell sequestration in conjunction with measurements of plasma cytokines in patients with cerebral malaria, in patients with other forms of severe and uncomplicated malaria, and in uninfected controls in Bangkok, Thailand. This research program will take advantage of a unique convergence of resources and expertise at Mahidol University in Bangkok: (i) the Bangkok Hospital for Tropical Disease, Faculty of Tropical Medicine, a world-renowned malarial research facility, (ii) the Ramathibodi Hospital, Faculty of Medicine, Department of Radiology, fully equipped with a Phillips Intera 3.0 Tesla MR system, and (iii) an established, decade-long collaborative relationship with investigators at Columbia University, now enhanced by a scientific linkage with the Hatch Magnetic Resonance Research Center. Our prospective, case-control study of adults admitted to the Bangkok Hospital for Tropical Disease will compare each patient with cerebral malaria to controls with severe (but non-cerebral) malaria, to controls with uncomplicated falciparum malaria, and to healthy volunteers. On admission, after patients are clinically stable following initial antimalarial and supportive treatment, magnetic resonance studies, detailed below, and determinations of plasma cytokines will be carried out. Magnetic resonance and cytokine measurements will then be repeated at the time of recovery from coma and at the end of the 28 day follow-up that is standard at the Bangkok Hospital for Tropical Disease. The proposed research has three specific aims:

(1) to test the hypothesis that the extent of red blood cell sequestration in the cerebral vasculature is greater in cerebral malaria than in other forms of severe malaria, as determined by time of flight MR angiography and perfusion studies using arterial spin labeling techniques;

(2) to test the hypothesis that cerebral metabolic dysfunction is greater in cerebral malaria than in other forms of severe malaria, as determined by proton (1H) MR spectroscopic measurements of ventricular lactate and of an indicator of axonal injury, N-acetylaspartate, in brain tissue; and


By determining definitively the role of cerebral sequestration of P. falciparum-infected red blood cells, the proposed studies would advance the understanding of the pathogenesis of cerebral malaria and help guide development of effective therapeutic interventions.



b. Background and Significance

ICIDR Overview: Our application requests support for the Mahidol-Columbia International Collaboration in Infectious Disease Research (ICIDR) as a single project cooperative agreement (U01) for multidisciplinary collaborative research that would continue the decade-long linkage between investigators in the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, and in the Department of Pediatrics, Columbia University College of Physicians and Surgeons, New York, N.Y., U.S.A. Our application is focused on the pathogenesis of cerebral malaria, a major life-threatening complication of infection with Plasmodium falciparum. The proposed research in the Mahidol-Columbia ICIDR will be conducted in its entirety in Bangkok, Thailand, using the facilities of the Hospital for Tropical Diseases of the Faculty of Tropical Medicine, and of the Ramathibodi Hospital of the Faculty of Medicine. Sornchai Looareesuwan, M.D., Professor of Clinical Tropical Medicine, Dean of the Faculty of Tropical Medicine, and Secretary General/Coordinator of the Southeast Asian Ministers of Education Organization, Regional Tropical Medicine and Public Health (SEAMEO TROPMED) Network, will serve as our Major Foreign Collaborator. Dr. Looareesuwan is a leading, internationally renowned investigator in malaria and tropical disease who has authored more than 400 peer-reviewed publications in these areas, including many of the seminal observations on the pathophysiology of cerebral malaria. During his tenure as Dean, he has lead the Mahidol Faculty of Tropical Medicine to its acknowledged rank as “Asia’s leader in Tropical Medicine.” In accordance with the policy of Mahidol University, after completing his second term of service in the fall of 2004, Dr. Looareesuwan will step down as Dean of the Faculty of Tropical Medicine and he will then return to a full-time commitment to research. As Major Foreign Collaborator, 40% of the professional effort of Dr. Looareesuwan would be devoted to the Mahidol-Columbia ICIDR. We emphasize that the proposed ICIDR would provide a natural extension of our current NIH research project, R01 AI51310 "Pathogenesis of severe malarial anemia in Thailand" (9/1/2002 to 6/30/2006) and of our current NIH research training program, D43 TW006240 "International Malarial Anemia Research Training Program" (3/1/2003 to 2/28/2007) and continue collaborative efforts that began more than a decade ago. b1. Overall ICIDR Research Goals and Objectives: The overall research goal of our project is to contribute to the development of improved diagnostic and therapeutic approaches to cerebral malaria, a major life-threatening complication of infection with Plasmodium falciparum. Our specific research objective is to take advantage of an unprecedented research opportunity to develop and apply to cerebral malaria a leading edge technology, high field (3.0 Tesla) MRI to clarify the pathogenesis of cerebral malaria, to guide the development of new therapeutic initiatives and to develop a new diagnostic tool that will provide novel means for the rapid evaluation of candidate therapies for cerebral malaria. b1.1 Potential importance of the proposed research to the health and well being of the Thai population: In Thailand, malaria is endemic and the P. falciparum is the most drug-resistant in the world. As in most of southeast Asia, the frequency of malarial transmission is low, with the consequences that acquired immunity provides little protection against symptomatic and devastating disease, asymptomatic parasitemia is unusual, and acute and cerebral malaria may occur at any age. The proposed research could lead to a major advance in understanding the pathogenesis of cerebral malaria, improve methods of diagnosis and hasten evaluation of candidate therapeutic interventions. b1.2 Organization of the Mahidol-Columbia ICIDR: The Mahidol-Columbia ICIDR will be constituted by an interinstitutional agreement between Mahidol and Columbia Universities developed in accordance with NIH guidelines for consortium arrangements (see Letter of Intent in Appendix). These research work will be carried out in Bangkok, Thailand, using the facilities of the Hospital for Tropical Diseases of the Faculty of Tropical Medicine, and of the Ramathibodi Hospital of the Faculty of Medicine. A detailed plan for the organizational structure is set forth below, in section b1.8.



b1.3 Participating institutions: The institutions participating in the Mahidol-Columbia ICIDR are: (i) Mahidol University, with the involvement of the Faculty of Tropical Medicine and the Hospital for Tropical Diseases, and of the Faculty of Medicine and the Ramathibodi Hospital, (ii) Columbia University, with the involvement of the Departments of Radiology and Pediatrics, and (iii) New York University (through a subcontract to Columbia University), with the involvement of the Department of Radiology. b1.4 Roles of consortium members: The Office of Grants and Contracts, Columbia University College of Physicians and Surgeons, will have administrative authority and be responsible for the fiscal management of the project. The Financial and Procurement Unit in the Office of the Dean Support Services, Faculty of Medicine, Mahidol University will exercise overall fiscal and grants administrative oversight in Thailand. As a sub-grantee, the Office of Sponsored Programs Administration, New York University School of Medicine, will be responsible for programmatic and administrative oversight and management. b1.5 Strategic Plan of the Mahidol-Columbia ICIDR: b1.5.1 Strengthening research capacity in the Mahidol Faculty of Tropical Medicine: Our ICIDR efforts to strengthen research capacity in the Mahidol Faculty of Tropical Medicine have as their rationale the specific need to develop new, flexible multidisciplinary approaches to equip young Thai investigators with the diverse array of skills, knowledge and expertise required for sustainable independent research in malaria. Scientific breakthroughs are most likely to come from individuals able to cross disciplinary boundaries to solve investigative problems. Our ICIDR program takes advantage of the breadth and depth of opportunities for participation and training in malaria research offered by the Mahidol Faculty of Tropical Medicine. To the greatest extent possible, we plan mentored research training efforts in our program in the context of longstanding collaborative research between the applicants. A primary goal of the ICIDR is the development of innovative means for the participation of young Thai investigators in collaborative malaria research projects and intervention trials within Thailand. If needed, the ICIDR Program will also be able to draw upon the expertise in all areas of basic science at the Columbia University College of Physicians and Surgeons (including genetics, biophysics, cellular and molecular biology, physiology, immunology, pharmacology, and development) and at the nearby Colleges of Letters, Science and Engineering on the Morningside Campus (including chemistry, bioengineering, computer science, mathematics and statistics). Both Mahidol and Columbia Universities are institutions encouraging strong interactions between basic and clinical scientists which bridge or transcend traditional departmental boundaries. Their alliance will help foster a training environment ideally suited to the development of investigators able to apply interdisciplinary approaches to fundamental problems in the pathogenesis of malaria infection. The ultimate aims of our ICIDR effort to strengthen research capacity are to provide clinical and basic research training in the fundamental aspects of modern scientific disciplines and to equip young Thai scientists with the knowledge, skills and aptitudes needed for sustained, effective and independent investigation of malaria in Thailand. b1.5.2 Disseminating research findings to host country health leaders: Throughout the course of the project, the major results of the research effort will be verified, documented and filed in an easily retrievable manner. At the end of the research project, a cleaned, edited and documented data tape, copies of all study forms, and a copy of the final research project protocol will be prepared and archived. Manuscripts describing the results and recommendations of the research project will be prepared and submitted for publication to peer-reviewed medical journals of high quality. A report of the principal results of the study will be prepared in lay language, translated into Thai and sent by letter to study participants. A conference or series of conferences presenting the results of the study will be held for all members of the Research Staffs at the Hospital for Tropical Diseases and at the Ramathibodi Hospital. b1.5.3 Outcome evaluation plan for capacity building and research and training objectives: As described below in section b1.8, the ICIDR Steering Committee with the assistance of an External Advisory Committee will evaluate annually our initiatives for capacity building and our research training accomplishments.



b1.6 Strengths: Faculty of Tropical Medicine, Mahidol University: The Faculty of Tropical Medicine, Mahidol University is a principal strength of the ICIDR application. Under the leadership of Dr. Looareesuwan, our Major Foreign Collaborator, the Faculty of Tropical Medicine has progressed to its generally acknowledged rank as “Asia’s leader in Tropical Medicine.” The Faculty of Tropical Medicine was established by the Thai Government on April 5, 1960 with three main functions: teaching and training, research, and medical services. Today, the Faculty has approximately 700 staff members working in 11 departments and other supporting units, namely, the Departments of Clinical Tropical Medicine, Helminthology, Medical Entomology, Microbiology and Immunology, Pathology, Protozoology, Social and Environmental Medicine, Tropical Hygiene, Tropical Nutrition and Food Science, Tropical Pediatrics, Tropical Radioisotopes, the Bangkok School of Tropical Medicine, the Bangkok Hospital for Tropical Diseases, Vaccine Trial Center, Central Equipment and Laboratory Unit, Research and Academic Affairs Unit, Library, Information and Computer Unit and Museum and Reference Center. The Faculty joined the Southeast Asian Ministers of Education Organization, Regional Tropical Medicine and Public Health Network (SEAMEO TROPMED) Project in 1967, and was designated by the Thai Government as the TROPMED National Center of Thailand. In 1994, the national status of the program was elevated to the regional level, thus becoming the SEAMEO TROPMED Regional Center of Tropical Medicine. In 1999, the Faculty was designated as the Training Center for Parasite Control for Asia, in collaboration with other agencies, including SEAMEO-TROPMED, under the title “The Asian Center of International Parasite Control” (ACIPAC). Research activities of the Faculty of Tropical Medicine are directed towards the recognition and solution of health problems commonly encountered in Thailand and other tropical countries including: malaria, amebiasis, liver fluke infections, paragonimiasis, soil transmitted helminthiasis, schistosomiasis, filariasis, dengue infection, Japanese encephalitis, rabies, HIV/AIDS, food borne mutagens and carcinogens, nutritional problems, diarrhea and enteric fever, melioidosis, leprosy, diagnosis of tropical diseases, vaccine development and vector control. Many research projects involve productive collaborations with scientists and researchers both nationally and internationally. The variety of collaborations and scientific linkages of the Faculty of Tropical Medicine provide remarkable opportunities to establish high quality research, and highlight the capacity of the Mahidol research team to function independently through the ICIDR collaboration. b1.7 Research Focus: b1.7.1 Translation into improved diagnosis and treatment of cerebral malaria: If successful, our research project could lay the foundation for the development of new means of avoiding the misdiagnosis of other forms of coma for cerebral malaria and could provide a more rapid and efficient means of evaluating new candidate treatments. b1.7.2 Potential impact on future public health policy: The Faculty of Tropical Medicine has a pivotal role in the development of public health policy in Thailand because of the excellence of its research and faculty and the productive partnerships and linkages with other programs with valuable research resources, including (i) the Southeast Asian Ministers of Education Organization, Regional Tropical Medicine and Public Health Network (SEAMEO TROPMED), as a Regional Center for Tropical Medicine, (ii) the World Health Organization (WHO), as a Collaborating Center for Environmental Management for Vector Control, (iii) the Wellcome Mahidol Oxford Tropical Medicine Program, (iv) the Mahidol Vaccine Trial Center and Data Management Unit, (v) the Asian Center of International Parasite Control (ACIPAC), and, most recently, (vi) the Bill & Melinda Gates Foundation. This array of collaborative relationships provides evidence of the authoritative involvement of the Faculty of Tropical Medicine at Mahidol University in formulating clinical treatment and prevention policies with respect to malaria locally, nationally and internationally. b1.8 Structure of the Mahidol-Columbia ICIDR: Columbia University will serve as the grantee institution with overall administrative authority and responsibility for the fiscal management of the project. The participation of both Mahidol and New York Universities will be through consortium agreements to be established with each institution. With respect to the scientific direction of the ICIDR, Sornchai Looareesuwan,



M.D., the Major Foreign Collaborator, and Gary M. Brittenham, M.D., Principal Investigator, will serve as ICIDR co-directors, with the advice and assistance of a Steering Committee. The ICIDR Steering Committee will be chaired by Dr. Looareesuwan and composed of Dr. Brittenham, Prasert Boongird, M.D., Professor of Neurology, Faculty of Medicine, Rachanee Udomsangpetch, Ph.D., Professor of Pathobiology, and Dwip Kitayaporn, M.D., D.T.M.&H., M.P.H., Dr.P.H., Professor of Epidemiology and Chief of the Data Management Unit of the Mahidol Vaccine Trial Center. The ICIDR Steering Committee will have the responsibility of advising the ICIDR co-directors and of evaluating the performance of ICIDR faculty and staff. Twice yearly, the Committee will also formally monitor the progress and accomplishments of the research program. The overall performance of the ICIDR Program will have a two-tiered system of review, with both an Internal and an External Advisory Committee to provide regular examination and evaluation. The Internal Advisory Committee is made up of Professor and Dean Emeritus Tan Chongsuphajaisiddhi (Chair), Polrat Wilairatana, M.D., Ph.D., Director of the Hospital for Tropical Diseases, and Francois Nosten, M.D., of the Wellcome Unit. The Internal Advisory Committee will meet every 6 months to assess overall ICIDR performance, review research progress and provide a written evaluation to the Steering Committee. An External Advisory Committee is composed of Christine E. McLaren, Ph.D., Professor of Epidemiology and Biostatistics, University of California, Irvine (Chair), Victor R. Gordeuk, M.D., Professor of Medicine, Howard University, and Mario Cazzola, Professor of Hematology, University of Pavia, Italy. The External Advisory Committee meets once each year to review written summaries of progress prepared by the ICIDR investigators and any publications that have resulted. To strengthen the research effort, the External Advisory Committee will evaluate the overall performance and progress of the research project and make explicit recommendations to the Steering Committee with respect to any suggested changes. A Publications Committee, to be named, will have responsibility for developing ICIDR policies with respect to data sharing, publication agreements and publication goals. An organizational diagram for the proposed Mahidol-Columbia ICIDR has been prepared in tabular form, incorporating a narrative description of the roles and responsibilities of all scientific and administrative staff and their level of effort, and is presented in the Appendix. b2. Collaborative Arrangements and Capacity Building: A letter of intent to establish written inter-institutional agreements in accordance with NIH Guidelines for Establishing and Operating Consortium Grants for the Mahidol-Columbia ICIDR is included in the Appendix, signed by the appropriate institutional officials, and documenting the willingness of Sornchai Looareesuwan, M.D., to serve as the Major Foreign Collaborator for the project and as the Principal Investigator of the Mahidol University subcontract. A similar letter of intent is included for the subcontract between Columbia and New York University to provide for the participation of Jens H. Jensen, Ph.D. with the goal of developing novel MRI techniques for the detection in vivo of malarial pigment (hemozoin). b3. Administrative requirements

b3.1 Professional effort of ICIDR personnel: Gary M. Brittenham, M.D., the Principal Investigator at Columbia University, will devote 25% of his professional effort to the ICIDR program and will spend at least 2 months annually at Mahidol University. He will coordinate regular visits by Truman R. Brown, Ph.D., and Robert L. DeLaPaz, M.D., the Columbia ICIDR scientists, and for Jens H. Jensen, Ph.D., the New York University ICIDR scientist, for long-term collaborative efforts with investigators at Mahidol University. Sornchai Looareesuwan, M.D., will serve as the Major Foreign Collaborator for the project and the Principal Investigator of the Mahidol University subcontract, devoting 40% of his professional effort to this role. Dwip Kitayaporn, M.D., Ph.D., Professor of Epidemiology, will serve as the designated biostatistician for the project, devoting 10% of his professional effort to these duties. Jaranit Kaewkungwal, Ph.D., Assistant Professor of Epidemiology, will serve as the designated full time data manager for the project, devoting 100% of his professional effort to these duties.



b3.2 Administrative plan: The administration of the ICIDR program will be the responsibility of the administrative staff of Dr. Looareesuwan, which is experienced and proficient in the management of research collaborations. In lieu of a description in this section, the Annual Review 2004 of the Faculty of Tropical Medicine is included in the Appendix, with a full organizational chart (page 6) and identification of the administrative components and personnel (pages 93-98). b3.3 Data management system: As detailed below in the Research Plan (section d-3), a full service Data Management Unit was established at the Faculty of Tropical Medicine in 1998 to provide data management and data analysis services to research projects. The Data Management Unit fully meets the ICIDR requirements for security features for controlled access to project data, for a tracking system for data forms and activities, for double data entry of data forms, for date and time of stamping of all data records with electronic signatures, and for audit trails to track all changes made to data records. The Faculty of Tropical Medicine Data Management Unit is compliant with FDA requirements for electronic records and signatures. b3.4 ICIDR Executive Committee Meetings: Time and budgetary allocations have been made to provide for attendance at ICIDR Executive Committee meetings, by Dr. Brittenham, the Principal Investigator, by Dr. Looareesuwan, the Major Foreign Collaborator, and by additional ICIDR staff as required. b3.5 ICIDR Opportunity Pool: In accordance with RFA instructions, no budgetary allowances have been made for the ICIDR Opportunity Pool but the applicants look forward to participation in the Pool to respond to unanticipated scientific needs and opportunities in tropical medicine. b3.6 Pathogen Study Groups: Within the Mahidol University administrative budget (under “Other Expenses”), $15,000 has been allocated annually for Pathogen Study Group activities such as conference calls, internet connectivity, attending special training courses or workshops, and organizing interest group meetings. b3. Scientific Background and Significance Our ICIDR application is focused on the pathogenesis of cerebral malaria, a major life-threatening complication of infection with Plasmodium falciparum. In this section, we will (i) briefly consider the present epidemiology of infection with P. falciparum and the consequences for cerebral malaria, (ii) summarize the clinical and pathological manifestations of cerebral malaria, with an emphasis on southeast Asia, and (iii) briefly compare and contrast the sequestration and cytokine theories of the pathogenesis of cerebral malaria and their importance as guides for the development of new and effective therapeutic interventions for patients with cerebral malaria. Finally, we will (iv) describe the potential for detection of paramagnetic hemozoin (malarial pigment) by magnetic resonance (MR) studies, providing a possible new, specific means of following the course of sequestration in patients with cerebral malaria and thereby provide vital new information about the cause and course of coma.

Present epidemiology of infection with P. falciparum: Over the past 4 decades, the incidence of malaria has increased 2- to 3-fold1. Nearly half the world’s population now lives in regions endemic for malaria in Asia, Africa and South America. Each year, as many as 500 million people are now infected and almost 3 million die1. Of the four protozoan species of the genus Plasmodium that cause infection in humans, P. falciparum is responsible for almost all severe illness and death. In many parts of the world, cerebral malaria is the most common clinical presentation and cause of death in patients with falciparum malaria2. Clinical manifestations of falciparum malaria depend upon the intensity of malaria transmission3. Where transmission of P. falciparum is both stable and intense, as in much of sub-Saharan Africa, symptomatic malaria is primarily a disease of young, non-immune children with chronic malarial infection. Repeated episodes of malarial infection in childhood eventually result in resistance to the clinical effects of malaria. Older children, adolescents and adults are asymptomatic by virtue of acquired immunity but most or all of the population has persistent parasitemia. In regions with lower frequencies of malarial transmission, immunity declines and older individuals may develop symptomatic and severe disease. In areas of low malaria transmission, as in Thailand



and most of southeast Asia, acquired immunity is still less protective, asymptomatic parasitemia is unusual, and acute malaria may occur at any age. Thailand offers a unique setting for the study of cerebral malaria. In Thailand, malaria is endemic and the P. falciparum is the most drug-resistant in the world. Thai populations have some of the highest frequencies known of some of the genes that affect host susceptibility to malaria, especially those for the thalassemias and hemoglobinopathies. The site for the proposed studies, the Hospital for Tropical Diseases, Mahidol University, Bangkok, Thailand, has a long and distinguished history of fundamental contributions to our understanding of all aspects of malaria. The applicants have collaborated for more than a decade in a series of studies of the pathophysiology of malarial infection, the role of iron in malaria, and the influence of the thalassemias and hemoglobinopathies.

Figure b-1: Life Cycle of Malaria Parasites. Malaria is transmitted by the bite of an infected female mosquito of the genus Anopheles. The sporozoites enter the bloodstream, travel to the liver and multiply. In P. vivax and P. ovale, some sporozoites remain dormant as hypnozoites for months or years before proliferation. Infected hepatocytes rupture releasing merozoites that enter the bloodstream and invade red blood cells where the intraerythrocytic asexual cycle of reproduction yields more merozoites which then invade new red cells. A small proportion of asexual parasites converts to gametocytes that transmit the infection onward through female anopheline mosquitoes. (Modified from Daily and Waldron, New Engl J Med 2004;349:287-295).

Clinical and pathological manifestations of cerebral malaria: Malarial disease is the consequence of the asexual cycle of parasite reproduction within red blood cells4. As shown in Figure b-1, malaria is transmitted by the bite of an infected female mosquito of the genus Anopheles. The parasites, called sporozoites at this stage of development, are injected into subcutaneous tissue or directly into the bloodstream, travel to the liver and eventually enter hepatocytes. Within the hepatocyte, each sporozoite then develops into tens of thousands of merozoites. After rupture of the hepatocyte and release from the liver, each merozoite can then enter an erythrocyte. As shown in Figure b-2, P. falciparum then develops over 48 hours within the red blood cell, passing successively through intraerythrocytic stages identified as ring forms, trophozoites and schizonts. The red blood cell is then ruptured, releasing about 20 merozoites, each able to invade another red blood cell and reinitiate the cycle. A small proportion of asexual parasites converts to gametocytes that are critical for the transmission of the infection through female anopheline mosquitoes. Neither the gametocytes nor the hepatic phase of the infection cause disease.



P. falciparum alters the red blood cell surface so that infected erythrocytes are sequestered by binding to the walls of blood vessels4. During the first half of the 48 hour reproductive cycle, infected erythrocytes circulate freely. During the second 24 hours, the parasitized red blood cells adhere to endothelial cells lining the microvasculature of vital organs throughout the body. Erythrocytes containing gametocytes also adhere to the endothelium. Consequently, only ring forms of P. falciparum are found in erythrocytes circulating in the peripheral blood; these red blood cells have a smooth surface. By contrast, as shown in Figure b-3, the surface of erythrocytes containing trophozoites and schizonts is covered with small cup-shaped, electron-dense protrusions called knobs that are about 30 to 40 nm high and 100 nm in diameter 5. These knobs are principal mediators of adhesion. Sequestration protects the parasite from destruction in the spleen; non-adherent erythrocytes containing trophozoites or schizonts are rapidly cleared. Other studies have suggested that sequestration also may be a means used by P. falciparum to suppress host immune responses. Infected erythrocytes adhere to dendritic cells and inhibit their maturation, induce a defect in dendritic cell antigen processing and reduce the capacity of dendritic cells to stimulate T cells 6.

Figure b-2: Representative parasites (magnification, 31000) from a highly synchronized culture in vitro of Plasmodium falciparum. The stage of development is assessed from the overall size of the parasite, the ratios of the area and diameter of the nucleus to that of the cytoplasm, the amount of visible hemozoin (malarial pigment), and, in mature parasites, the number of nuclei. Hemozoin (called pigment in the figure legend above) is first apparent in early trophozoites and the amount steadily increases until schizont rupture. (Reproduced from Dondorp et al., Acta Tropica 2004; 89:309–317)

The molecular basis of adhesion is a receptor-mediated interaction of ligands in the red blood cell membrane with host receptors on the surface of vascular endothelial cells4. Parasitized erythrocytes are sequestered in vital organs including the brain, heart, lung, liver, kidney, subcutaneous tissues, and placenta. The types and amounts of host receptors expressed by the vascular endothelial cells in these organs and by syncytiotrophoblasts in placenta influence the binding of infected red blood cells. Conversely, infected erythrocytes can bind to a considerable number of host receptors. A single parasite protein, the P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed at the infected erythrocyte surface, is the chief mediator of binding to the various receptors. Different parasites have a variety of affinities to diverse combinations of host receptors. Accordingly, the pattern of the tissue distribution of sequestration in a falciparum malarial infection is the product of the interplay of these host and parasite factors.



A

B

Figure b-3: A. Scanning electron micrograph showing knobs (arrow) over the surface of a P. falciparum infected erythrocyte. Bar = 1 µm. (Modified from Aikawa et al.. J Parasitol 1983;69:435–7) B. Transmission electron micrograph showing adherence (arrows) between a P. falciparum (P) infected erythrocyte via knobs and an endothelial cell (EC) of a cerebral microvessel. Bar = 1 µm. (Modified from Fujioka and Aikawa, Microbial Pathogenesis 1996;20:63–72)

The consequences of the sequestration of parasitized erythrocytes for microcirculatory blood flow are illustrated in Figure b-4, showing small vessels obstructed by parasitized red blood cells adherent to the endothelium. The effects of microcirculatory obstruction are amplified by a number of other features of malarial infection 7. Parasitized red cells become rigid, impeding their flow through capillaries whose lumen has already been reduced by sequestered erythrocytes. Recent studies further indicate that rigidity is increased even in uninfected erythrocytes. In addition, adhesive interactions between infected red cells (auto-agglutination), between infected and uninfected red cells (rosetting) and between uninfected erythrocytes (aggregation) may also impair microcirculatory flow 8.

A

B

Figure b-4: A. Microphotograph of cross section of a retinal vessel with a central core of well-hemoglobinized red blood cells surrounded by parasitized red blood cells containing schizonts and little remaining hemoglobin (hematoxylin-eosin, original magnification x200; modified from Lewallen et al., Arch Ophthalmol 2000;118:924-928). B. Microphotograph of a longitudinal section of brain vessels from a case of fatal falciparum malaria showing accumulations of schizonts with abundant pigment. (Modified from Silamut et al., Am J Pathol 1999;155:395–410)

Sequestered parasitized red blood cells, by causing microcirculatory obstruction, are believed to be a critical factor in the pathogenesis of all forms of severe and fatal malaria. In general, the severity of disease in P. falciparum malaria seems to be more closely related to the number of sequestered parasites than to the number



circulating in the peripheral blood7. Sequestration of parasitized erythrocytes in the microcirculation of vital organs may cause great divergence between the peripheral blood parasite count and the total body parasite burden9. Consequently, the parasite count in the peripheral blood is a poor and unreliable guide to sequestered parasite mass. Sequestration theory of the pathogenesis of cerebral malaria: Evidence for the essential role of sequestration in the pathogenesis of severe malaria is strongest for cerebral malaria, the most lethal complication of falciparum malaria. Cerebral malaria is a rapidly progressive encephalopathy with a mortality of up to 50% with standard therapy; without treatment the condition is almost inevitably fatal. A recent quantitative electron microscopic study of patients who died of severe malaria in Thailand and Vietnam has interpreted the findings as evidence that sequestration is the single most important factor in the pathogenesis of cerebral malaria9. To some extent, sequestration in the brain probably develops in all cases of falciparum malaria9. Nonetheless, in the brains of patients with cerebral malaria, sequestration of parasitized red blood cells in the microvasculature was significantly greater than that in the brains of patients with non-cerebral malaria. When cerebral sequestration was compared with peripheral parasitemia pre mortem, there were 26 times more parasitized red blood cells in the brain microvasculature than in the peripheral blood. No differences were found in parasite clearance from cerebral vessels or in parasite load between patient groups treated with artemether or quinine9. This finding contrasts sharply with the results of parasite counts in the peripheral blood, where clearance is much more rapid after treatment with artemesinin derivatives. The pathological findings also indicated that parasitized red blood cells, once bound to an endothelial cell, did not appear to recirculate and die in-situ. Finally, the ghosts of parasitized red cells membranes that were left after schizogony and erythrocyte rupture and their associated hemozoin remained cytoadherent to the endothelium 9. Cytokine theory of the pathogenesis of cerebral malaria: For more than a decade, other investigators, while acknowledging that cerebral sequestration is “often present and sometimes blocks vessels”10, have developed a sustained argument that sequestration is not essential for the onset of the cerebral manifestations of malaria11. The cytokine theorists maintain that “cerebral malaria can occur without significant sequestration10, 12. Instead, cerebral malaria is regarded as a type of septic encephalopathy13 whose pathogenesis is the consequence of an imbalance in cytokines leading to excessive pro-inflammatory production11, 14, 15. In brief, cytokines originating from T-helper cells, natural killer cells and macrophages are major mediators of the body’s response to parasitic infection 16. Two CD 4+ T-helper cell subsets exist in mice17 and probably in man, each of which produces a typical set of cytokines that regulate different immune effector functions and cross-react with each other. T-helper type 1 (Th-1) cells produce IFN-γ, IL-2 and TNF-β. These cytokines activate macrophages, thus contributing to the formation of pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6, and the induction of cytotoxic immune effector mechanisms of macrophages. By contrast, Th-2 cells produce IL-4, IL-5, IL-10, and IL-13, cytokines which induce a strong antibody response but also inhibit various macrophage functions. The balance between Th-1 and Th-2 cell-mediated immune effector mechanisms is of central importance for the host response to parasitic infections in mice and probably in humans16. While Th-1 derived cytokines such as IFN-γ and IL-2 are crucial for effective host defense in the acute phase of certain parasitic infections, increased activity of Th-2 derived cytokines such as IL-4, IL-10 and IL-13 heightens susceptibility to these infections and causes exacerbations. The latter effects may be due to an inhibitory function of IL-4, IL-10 or IL-13 on the production of Th-1 cytokines and on macrophage activation. Such Th-2-mediated inhibition of Th-1 pathways would reduce cell-mediated antimicrobial cytotoxicity, e.g. by suppressing the induction of nitric oxide synthase II12. Evidence for the detrimental role to the host of Th-2 mediated immune function during acute parasitic infections has been shown in several models17. (i) IL-4 deficient mice become more susceptible to acute parasitic infections after re-introducing the IL-4 gene into their genome. (ii) The inhibitory effects of IL-4 and IL-10 on cell-mediated immunity are due in part to depression of IFN-γ production. (iii) In parasitic infections Th-1 cells default to the Th-2 pathway in the absence of endogenous IFN-γ. (iv) Various infections are less



severe and have reduced mortality in IL-4 deficient-mice. (v) Plasma levels of IL-10 increase with disease severity in human malaria. Based on these and several other studies, Th-1 mediated immune effector function appears to be beneficial during early stages of plasmodial infections. In contrast, Th-2 derived cytokines appear to exert a protective role later in chronic infection with plasmodia or in the recovery period 16. Magnetic resonance (MR) studies and severe malaria: Obtaining evidence to differentiate between the sequestration and cytokine theories has been near impossible, in part because determination of the extent of microvascular sequestration has been restricted to pathological studies at autopsy. Despite both the clinical and pathophysiological importance of the adherence of parasitzed red blood cells to the endothelium of the microvasculature, no direct method is available to detect or measure the extent of sequestration in living patients. Clinically, counts of the number of circulating parasitzed red blood cells in peripheral blood may be very misleading9. Reliable means of measuring the sequestered parasite population are urgently needed to improve the clinical care of patients with severe falciparum malaria and to advance our understanding of the pathophysiology of malarial infection.

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Figure b-5: A. Scanning electron micrograph of hemozoin, isolated from mature trophozoites of P. falciparum. The isolated malaria pigment crystals appear as long needles (dimensions approximately: 1.0 µm x 0.6 µm x 0.2 µm) and the predominant angle between the long and short faces of the crystals is 135°. (Modified from Hempelmann et al., Trends Parasitol 2003;19:23-26). B. Transmission electron microscopy and electron spectroscopic imaging of P. falciparum showing the distribution of elemental iron within a schizont. Left panel: Transmission electron microscopy of a schizont infected erythrocyte and distribution of elemental iron. Right panel: Electron spectroscopic imaging; the iron distribution coincides exactly with haemozoin crystals. (Modified from Egan et al., Biochem J 2002;365:343-347).

Our project will take advantage of the unprecedented opportunity at the Bangkok Hospital for Tropical Diseases and the Ramathibodi Hospital to use established clinical MRI methods to seek evidence of microvascular obstruction (perfusion studies using non-invasive arterial spin labeling studies), to examine patients with cerebral malaria for any obstruction of larger vessels (using MR angiography), and to assess the degree of metabolic dysfunction (chemical shift imaging studies) during and after recovery from coma. We also hope to develop a novel MR method, magnetic field correlation imaging, that has the potential to detect the cerebrovascular deposition of paramagnetic hemozoin. In the remainder of this section, we will examine the detailed information already accumulated about the biology and biophysics of hemozoin. In the following section describing preliminary studies, we will summarize the theoretical basis for this approach to developing a means of monitoring a specific marker of falciparum infection in living patients.



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Figure b-6: A: Two views of the structure of β-hematin determined by solution of the X-ray powder diffraction pattern. Both views show the iron-oxygen and propionic acid linkages between dimeric pairs of hemes. The five coordinate high spin iron, S = 5/2, is located 0.47 Å out of the plane of the porphyrin and forms a relatively short bond, 1.889 Å , with one of the oxygens of the paired protoporphyin-IX propionic acid substituents. (Reproduced from Bohle et al., Acta Cryst 2002; D58:1752-6). B: Packing diagram of the crystal structure of β-hematin. The [131] face, which is roughly parallel to the porphyrin rings, is in the plane of the paper. Lighter shaded atoms (C, grey; O, red; N, green; Fe, blue; H, not shown) are nearer the viewer. The chains of hydrogen-bonded dimers extend from left to right. (Reproduced from Pagola et al., Nature 2000;404:307-310)

Inside a sequestered erythrocyte, the malarial parasite ingests as much as 80% of the hemoglobin into an acidic food vacuole where the globin protein is digested and the heme released18. Almost all the reactive free heme is detoxified by aggregation into an insoluble, chemically inert crystalline hemozoin. Hemozoin crystals isolated from mature trophozoites of P. falciparum, are shown in Figure b-5A; viewed in polarized light, the crystals are birefringent. As illustrated in Figure b-5B, careful studies using chemical analysis, 57Fe-Mössbauer spectroscopy and electron spectroscopic imaging have established that more than 95% of the heme iron released from host hemoglobin by P. falciparum is incorporated into hemozoin18. Hemozoin is chemically, spectroscopically and crystallographically identical to synthetic β-hematin (FeIII-protoporphyrin-IX) whose crystal structure has been determined by analysis of powder diffraction data obtained with synchrotron radiation18. As shown in Figure b-6, the molecules are linked into dimers through reciprocal iron–carboxylate bonds to one of the propionic side chains of each porphyrin, and the dimers form chains linked by hydrogen bonds in the crystal19. Determination of the structure of hemozoin, together with studies using variable temperature EPR and Mössbauer spectroscopies, have definitively established that hemozoin has a single high-spin S = 5/2 iron environment. The paramagnetic moment of hemozoin is of sufficient magnitude to allow the isolation of parasitzed erythrocytes using an external magnetic field. The first use of the magnetic properties of parasitized erythrocytes as a method to separate the infected red blood cells from whole blood was reported in 194620 and a series of investigations using higher fields has followed21. A “magnet test” for malaria was described in 1995 to separate and concentrate infected red blood cells22.

MRI may be able to detect and measure the amount of paramagnetic high-spin Fe (III) in the hemozoin within sequestered parasitized red blood cells. MRI examinations of patients with malaria have been reported 23 but



the conditions used were not intended or optimized for detection of paramagnetic hemozoin. At the Hospital for Tropical Diseases, we have previously carried out a prospective study of MRI of the brain in twenty-four adults with cerebral malaria 24. Twenty-two of the twenty-four patients had no evidence of cerebral edema but MRI revealed that brain volume during acute cerebral malaria was slightly greater than that during the convalescent phase of the disease. This difference was attributed to an increase in the volume of intracerebral blood. The cerebral volume was lower during early convalescence than several months later. These results suggested that the volume of the brain in patients with cerebral malaria is increased but that the increased volume is probably the result of sequestration of parasitized erythrocytes and compensatory vasodilatation rather than from edema.

The overall purpose of this study is to determine the feasibility of using MRI to detect sequestered parasitized red blood cells within the microvasculature of patients with cerebral, severe or uncomplicated falciparum malaria. The development of means to monitor non-invasively the sequestration of parasitized red blood cells in patients with falciparum malaria would enhance our ability to diagnose malarial complications and provide a new investigational tool for studies of the pathogenesis of severe and fatal malaria.

c. Preliminary Studies The argument could be made that, over the past quarter century, investigators at the Bangkok Hospital for Tropical Diseases have contributed more to the understanding of the clinical manifestations, pathology and pathogenesis of cerebral malaria than any other research group in the world. In this section, we will first acknowledge the distinguished past history of investigations of cerebral malaria, many of the most significant lead by our Major Foreign Collaborator, Sornchai Looareesuwan, M.D., with brief summaries of some of the most important work pertinent to our research plan. We will then highlight the extraordinary research opportunity provided by the availability of a state-of-the-art high-field (3.0 Tesla) magnetic resonance instrument immediately adjacent a world-class institution dedicated to the study of malaria. After briefly illustrating the wealth of information about cerebral anatomy, function and metabolism that can be obtained with the new generation of MR instrumentation, we will show a single clinical example of the application of MRI in a patient with hyperparasitemia. Finally, we will conclude by summarizing the theoretical basis for a new approach to monitoring malarial infection, the use of magnetic field correlation imaging to detect paramagnetic hemozoin. Brief history of pivotal studies of cerebral malaria at the Hospital for Tropical Diseases, Faculty of Medicine, Mahidol University, Bangkok, Thailand. The results of the first study of cerebral malaria to be considered in this section changed the approach to the treatment of malaria worldwide25. At the time, high-dose dexamethasone was routinely administered to patients with cerebral malaria. In a placebo-controlled, double-blind trial involving 100 comatose patients with strictly defined cerebral malaria, dexamethasone had no effect on the risk of death but significantly prolonged the duration of coma among survivors and more than doubled the rate of complications. These results demonstrated that, contrary to the widely held belief at the time, dexamethasone is deleterious in cerebral malaria and should no longer be used therapeuticallly25. Dr. Looareesuwan subsequently led a study that used the then advanced technique of computed tomography of the brain to directly determine the contribution of cerebral edema to coma in cerebral malaria26. A total of 10 patients were studied; 5 died. Cerebral edema was found in only 2 of the fatal cases. These results showed that while cerebral edema may sometimes develop in severe cerebral malaria that swelling of the brain is not consistently found in living patients and consequently cannot always be the cause of their coma26. As discussed above, the first (and still only) MRI study of a series of malaria patients, carried out in 24 patients with cerebral malaria with a 0.2 Tesla scanner in 1995, has since confirmed these findings24. As will be illustrated below, substantially more information about cerebral anatomy, function and metabolism is readily available with MRI at 3.0 Tesla.



Studies at the Hospital for Tropical Diseases helped first establish the prognostic significance of cerebrospinal-fluid (CSF) lactate concentrations in cerebral malaria27. CSF lactate concentrations was elevated in all but 1 of 45 patients with cerebral malaria and were significantly higher in patients who died than in those who survived. All patients with high concentrations of lactate (> 6 mmol/L) died. These results demonstrated the prognostic value of measurements of CSF lactate27. These older findings suggest the potential value of serial determinations of ventricular lactate in patients with cerebral malaria by MR chemical shift imaging (CSI), especially in combination with measurements of brain lactate and of a brain tissue indicator of axonal injury, N-acetylaspartate (NAA).

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Figure c-1: MR images of a normal subject illustrating anatomic detail available at 3.0 Tesla. A. Image obtained with a short T1 inversion recovery (STIR) sequence. B. Image obtained with a T1-weighted fluid-attenuated inversion recovery (T1WFLAIR) sequence.

The first reported measurements of cerebral blood flow in cerebral malaria were conducted at the Hospital for Tropical Diseases28. Using a tracer technique, cerebral blood flow in 12 comatose patients with cerebral malaria was found to be within the range found in healthy controls, despite an increase in cerebral vascular resistance. Importantly, cerebral oxygen consumption was decreased while cerebral venous pO2 was increased, observations consistent with the microvascular shunting considered in our research plan. Notably, both arterial lactate and cerebral lactate production declined with recovery from coma. Autopsy studies of patients who died of cerebral malaria at the Hospital for Tropical Diseases have made fundamental contributions to understanding the pathogenesis of coma. Importantly, a study of 39 autopsies in patients who had died of cerebral malaria found that the degree of parasitized red blood cell sequestration correlated closely with the severity of coma judged clinically29. In a later study, histopathologic and immunohistologic assays at autopsy have suggested that fatal cerebral malaria may be associated with focal accumulation of cytokines30. Most recently, in the largest published series of patients who died of severe



malaria, ultrastructural studies in 65 cases have found a quantitative association between the degree of sequestration of P. falciparum-infected erythrocytes in the cerebral microvasculature and pre-mortem coma9. The results suggest the potential investigative rewards that can be expected by using noninvasive MR methods to serially measure the extent of sequestration in living patients. Anatomic, functional and metabolic information available with high field (3.0T) MR studies: Our project seeks to use established clinical MRI methods to seek evidence of microvascular obstruction (perfusion studies using non-invasive arterial spin labeling studies), to examine patients with cerebral malaria for any obstruction of larger vessels (using MR angiography), and to assess the degree of metabolic dysfunction (chemical shift imaging studies) during and after recovery from coma. As illustration of the level of anatomic detail available, Figure c-1 shows images from a normal subject acquired at 3.0 T using two of the sequences to be used in our proposed studies (see below): c-1A, obtained with a short T1 inversion recovery (STIR) sequence, and c-1B, obtained with a T1-weighted fluid-attenuated inversion recovery (T1WFLAIR) sequence. Figure c-2 shows chemical shift imaging (CSI) spectra, also obtained from a normal subject.

Figure c-2: Chemical shift image (CSI) spectra obtained from a normal subject, acquired from a 3xs grid, shown in the small box in the overlay on the right. The spectra are from 15x15x15 mm3 volumes with an echo time (TE) of 143 ms and a repetition time (TR) of 1000 ms. The spectra were acquired in about 3 minutes using a circular k-space sampling scheme. The spectra show indicate that choline, creatine and N-acetylaspartate (NAA) concentrations can be measured with an accuracy of a few percent. In this normal volunteer, no lactate is visible but precise and reproducible measurements of elevated lactate concentrations are readily available31-33.

Magnetic Resonance (MR) angiography of a patient after admission with hyperparasitemia. As described in Section b, Background and Significance, in falciparum malaria only ring-forms are seen in circulating red blood cells, indicating that the trophozoite and schizont stages are sequestered. Although detailed examination of tissue sequestration has been possible only at autopsy and almost always only in patients who have been treated with antimalarials, virtually all pathologic descriptions of cerebral malaria describe the sequestration of large numbers of infected red blood cells in the microvasculature and to a lesser extent the margination of infected erythrocytes in the medium and large vessels 9. Little is known with certainty about the pattern of sequestration in patients with acute malaria who are successfully treated and survive but sequestration in the brain seems



likely to occur in all cases of falciparum malaria9. Evidence for margination of infected blood cells in the cerebral arteries is shown in Figure c-3 on a 3D time of flight MR angiogram obtained for clinical indications using a 1.5 Tesla Siemens scanner at the Bangkok Neurological Institute. The patient was a 16 year old male admitted with a 6 day history of high fever and drowsiness who was found to have hyperparasitemia. The MR angiogram with gadolinium contrast obtained shortly after admission showed moderately severe focal narrowing at the proximal A1 segment of the right anterior cerebral artery, mild narrowing and decreased flow-related enhancement of the distal left posterior cerebral artery and mild irregular narrowing of the M2 segment of the right middle cerebral. No localizing neurological findings were found on physical examination and the patient recovered uneventfully with antimalarial therapy. Four weeks later, follow up MR angiography found complete resolution of the narrowings in the cerebral arteries. Please note than in our proposed studies, 3D time of flight MR angiograms will be obtained without use of contrast agents.

Figure c-3: A 3D time of flight MR angiogram using gadolinium contrast in a 16 year old male with hyperparasitemia. This study was done in the Neurological Institute, Bangkok, using a 1.5 Tesla Siemens Magnetom Vision plus.

Magnetic field correlation (MFC) imaging for detection of malarial hemozoin. This new magnetic resonance imaging (MRI) contrast technique, devised by our co-investigator , Jens H. Jensen, Ph.D., is based on a quantity termed the magnetic field correlation (MFC). As detailed below, the MFC is strongly dependent on the spatial distribution of microscopic magnetic field inhomogeneities, providing a sensitive probe of tissue microstructure that gives information beyond what is contained in the standard nuclear magnetic resonance relaxation times such as T2 and T2*. The MFC was originally developed for the detection of microscopic magnetic field inhomogeneities, such as those associated with ferritin iron localized in glial cells or with a paramagnetic contrast agent confined to an extracellular compartment. The MFC may have application in evaluation of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases, that have associated iron



abnormalities, and in the diagnosis and grading of tumors, using paramagnetic contrast agents. In this proposal, we describe the use of MFC imaging for the detection of hemozoin within the cerebral microvasculature of patients with falciparum malaria. The available preliminary information indicates that MFC imaging will permit detection and measurement of paramagnetic hemozoin (with its five coordinate high spin iron, S = 5/2, as described in Section b above) within sequestered parasitized red blood cells. Here, we summarize the work already accomplished in the development of MRC imaging methods and present MFC images from a normal subject obtained with a 3.0 Tesla MR instrument (see Figure c-5).

Figure c-4. Asymmetric CPMG sequence with N = 3. The 180º refocusing pulses are shifted by a time

st from

their conventional positions, while the signal acquisition times are unchanged. The MFC is defined as the product of the magnetic field shifts experienced by a water molecule at two different times, averaged over all the water molecules within a specified region of interest. The field shifts are the difference between the magnitude of the field and the magnitude of the uniform background field (which is often nearly the same as the external field magnitude). If the biological tissue has linear magnetic characteristics, as is usually the case, then the MFC varies as the square of the external field. By contrast, the MFC dependence on the difference in the two sampling times is purely an intrinsic tissue property. In practice, the MFC is most sensitive to the spatial dependence of the field shifts over length scales of about 10 µm, which is comparable to the size of many cell types. In brief, the MFC is defined with respect to a tissue is exposed to a strong, uniform external magnetic field, as is the case in a typical MRI experiment. Because of spatial variations in its magnetic susceptibility, the tissue will generate an inhomogeneous secondary field. The susceptibility variations could be due to structures such as hemozoin or vessels with deoxygenated blood. Now consider a water molecule diffusing through the tissue. Because of the field inhomogeneities, the field experienced by the water molecule will vary with time. Let δB(t) be the difference at a time t between the magnitude of the field at the water molecule and the uniform background field. The MFC is then defined by

( ) ( ) ( )tBtBttK !""=!# , [1]

where the angle brackets indicate an average over all the water molecules in a specified region of interest (in practice this would be an MRI voxel). The temporal behavior of the MFC, which we will indicate with the symbol K in mathematical expressions, involves only the time difference tt !" , as follows from an assumption that the state of the tissue is time invariant. Note that, by definition, the MFC depends quadratically on the magnitude of the field inhomogeneities. The pulse sequence used for MFC imaging is an asymmetric dual spin echo (ADSE; see Figure c-4); both standard (spin warp) and echo planar imaging (EPI) versions have been developed. The physical significance of the MFC is essentially twofold. First, the magnitude of the MFC provides a measure of the amplitude of the magnetic field inhomogeneities within a tissue. In particular, as follows from Eq. [1], K(0) is the variance of the field. Second, the decay of the MFC with time contains information about



the spatial variation of the field inhomogeneities. More precisely, the temporal rate of change of the MFC at a time t (i.e., )(tK ! ) is primarily sensitive to field inhomogeneities with a length scale of about Dt6 , where D is the water diffusion constant and Dt6 is the average distance a water molecule diffuses over a time t. Measuring the MFC with MRI is an ideal method for probing inhomogeneities on a length scale of a few tens of micrometers, because MRI echo times are typically in the range of 10 to 100 ms and the water diffusion constant in biological tissues is about /msm0.1

2ì . Consequently, the temporal behavior of the measured MFC

is sensitive to spatial variations in the range of 8 to 25 µm. Biologically, these dimensions encompass the size of capillaries and erythroctyes, making the MFC well suited for the detection of the paramagnetic effects of hemozoin within sequestered parasitized red blood cells.

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Figure c-5: MR images from a normal subject at 3 Tesla. A. Spin echo image. B. Magnetic field correlation (MFC) image; note the enhancement of the iron-rich substantia nigra and red nuclei (arrows, zoomed insert); modified from Helpern JA, Jensen J, Lee SP, Falangola MF. Quantitative MRI assessment of Alzheimer's disease. J Mol Neurosci 2004; 24, 45-48.



d Research Design and Methods Rationale: The proposed research project is designed to determine the role of sequestration of red blood cells in the pathogenesis of cerebral malaria by using quantitative magnetic resonance (MR) methods coordinated with measurements of inflammatory cytokines in serum. Among human malarias, P. falciparum is responsible for almost all severe disease and death. Sequestration of P. falciparum-infected erythrocytes occurs in all falciparum malaria, uncomplicated or severe, as evidenced by the lack of circulating trophozoite and schizont forms on peripheral blood smears. The characteristic pathological feature of falciparum malaria is the adherence of erythrocytes containing mature forms of the parasite to venous and capillary endothelium in the brain, heart, kidney and other organs. At merogony (schizogony), the adherent parasitized erythrocyte bursts, releasing daughter merozoites into the blood stream and leaving behind a red cell ghost. As emphasized above, despite intensive investigation the precise role of microvascular sequestration of parasitized red blood cells in the pathogenesis of cerebral malaria remains uncertain. The overall goal of the investigations proposed here is to provide definitive evidence with respect to the relative roles of red blood cell sequestration and of falciparum-induced excess production of pro-inflammatory cytokines in the pathogenesis of cerebral malaria by using high-field (3.0 Tesla) magnetic resonance studies to determine the extent of microvascular red blood cell sequestration in conjunction with assays of plasma cytokines. d1 Research Plan: d1.1 Disease entity proposed for study: Cerebral malaria. d1.2 Study hypotheses: This prospective, case-control study of adults admitted to the Bangkok Hospital for Tropical Diseases will compare each patient with cerebral malaria to matched controls with severe (but non-cerebral) malaria, to controls with uncomplicated falciparum malaria, and to healthy volunteers. The specific aims of the research are to test three hypotheses:

(1) to test the hypothesis that the extent of red blood cell sequestration in the cerebral vasculature is greater in cerebral malaria than in other forms of severe malaria, as determined by time of flight MR angiography and perfusion studies using arterial spin labeling techniques;

(2) to test the hypothesis that cerebral metabolic dysfunction is greater in cerebral malaria than in other forms of severe malaria, as determined by proton (1H) MR spectroscopic measurements of ventricular lactate; and


d1.3 Proposed methodology: Cytokine studies: To provide a systematic and comprehensive assessment of patterns of changes in circulating inflammatory cytokines, we will use a new cytometric bead array method (BD Biosciences, San Diego, Calif.) fluorescence detection by flow cytometry to examine the patterns of the inflammatory cytokines IL-1 , IL-6, IL-8, IL-10, IL-12(p70), and TNF-α in serum. The use of this measurement technique requires small sample volumes (1 mL whole blood) but provides accurate and reproducible results. Another advantage is that the same techniques has been used in a recent case-control study of 248 cases of severe Plasmodium falciparum malaria among children aged 3 months to 14 years in Mali, matched to cases of uncomplicated malaria and healthy controls16. These data will greatly facilitate comparison of our results, to be obtained in adults in Thailand, with those from African children. In brief, patient whole blood (1 mL) will be collected into sterile Eppendorf tubes on admission and prior to institution of antimalarial therapy and subsequently at the time of each MR examination. Blood will be refrigerated at 4°C and then allowed to coagulate for 4 to 6 h prior to processing via centrifugation. Sera will preserved at 70° C until analysis in the Immunology Laboratory of the



Hospital for Tropical Diseases. Levels of IL-1 , IL-6, IL-8, IL-10, IL-12(p70), and TNF-α in serum were determinedaccording to the manufacturer’s recommendations. For analysis, 40 µl of bead populations with discrete fluorescent intensities of Peridinin chlorophyll protein (PerCP)-Cy5.5 and coated with cytokine-specific capture antibodies will be added to 40 µl of patient sera and 40 µl of phycoerythrin-conjugated anti-human inflammatory cytokine antibodies. Simultaneously, standards for each cytokine (0 to 5,000 pg/ml) will be similarly mixed with cytokine capture beads and phycoerythrin-conjugated reagent. The vortexed mixtures will then be allowed to incubate for 3 days, to enhance the lower limit of detection. Flow cytometric analysis will be performed and analyzed by a single operator and standard curves will be derived from the cytokine standards. For results above the upper limit of detection, 1:4, 1:8, and 1:16 dilutional experiments will be performed to accurately determine cytokine levels. MRI Examinations: MRI studies of the brain will be acquired using a Philips 3.0T Intera Master in the Sirikit Medical Building, Department of Radiology, Ramathibodi Hospital, Faculty of Medicine, Mahidol University in Bangkok, Thailand, under the supervision of Jiraporn Laothamatas, M.D. Neuroradiologist and Managing Director of the Advanced Diagnostic Imaging and Image-guided Minimal Invasive Therapy Center (AIMC). The Philips Intera 3.0T has all neurological sequences, a complement of phased array coil detectors and a wide range of single and multiple coils, along with research keys to permit new sequence development; this instrument will be upgraded to a Philips 3.0T Achieva system in January 2005. A Philips workstation, which runs the Philips sequence development software, is located near the MR console. The workstation is connected to the Mahidol University network by a 100MHz link, simplifying data transfer for offline analysis. After patient identifiers have been replaced by study identifiers, encrypted images and data will be securely transmitted electronically to the Hatch Magnetic Resonance Research Center at Columbia University using Digital Imaging and Communications in Medicine (DICOM) protocols. The Hatch MR Research Center is fully equipped with Philips MR instrumentation, including a 1.5T Intera and a 3.0T Achieva system, now being installed. Srirama Swaminathatn, Ph.D., a co-investigator and Clinical Scientist, Philips Medical Systems, will help insure data transfers and software transparency between the Mahidol and Columbia sites. The MR examination will consist of one series of scans designed to obtain clinical and anatomic data and another series designed to acquire functional and metabolic data regarding local perfusion, the presence of hemozoin, and metabolite levels, particularly N-acetylaspartate (NAA) and lactate. The series are distinguished solely for descriptive purposes; all will be completed in a single continuous imaging session. The clinical and anatomic series of scans will be acquired in the axial plane along the anterior commissure-posterior commissure (AC-PC) line seen on sagittal T1 weighted images. The anterior-posterior center of the field of view (FOV) will be centered at the midpoint of the AC-PC line to ease comparisons among the different examinations in a single subject over time and among study groups. Specifically, in the clinical and anatomic series of scans, we will acquire: (1) a T1-weighted fluid-attenuated inversion recovery (T1WFLAIR) sequence (2) a short T1 inversion recovery (STIR) sequence, (3) a T2*-weighted (T2*W) sequence, (4) a T2-weighted fluid-attenuated inversion recovery (T2WFLAIR) sequence, (5) a double-echo T2-weighted (DET2W) sequence, (6) a diffusion-weighted imaging echo-planar imaging (DWI-EPI) sequence, and (7) a time of flight MR angiogram (TOF-MRA). The functional and metabolic series of scans will consist of: (8) an Arterial Spin Labeled (ASL) perfusion study to measure brain perfusion,



(9) a Chemical Shift Image (CSI) study through the ventricles to measure the lactate concentration in the CSF and the levels of choline, creatine, NAA and lactate in brain tissue, and

(10) a magnetic field correlation (MFC) study to detect hemozoin in infected red blood cells and red blood cell ghosts adhering to the cerebral microvasculature.

Table d-1 lists the specific acquisition parameters and times for these studies. The entire series of clinical, anatomic, functional and metabolic studies will be completed in a single imaging session lasting approximately 55 minutes. The full MR examination will be repeated on three occasions for each study patient:

(i) on admission, after patients are clinically stable following initial antimalarial and supportive treatment,

(ii) shortly after recovery from coma for patients with cerebral malaria or at a similar time for patients with severe but not cerebral malaria or with uncomplicated malaria, and

(iii) at the end of the four-week hospitalization that is standard for patients in the Bangkok Hospital for Tropical Diseases.

Uninfected healthy control subjects will have the MR examination only once. All MR scans will be promptly reviewed by Dr. Laothamatas and her colleagues and any clinical finding or abnormality reported directly to the responsible physician at the Bangkok Hospital for Tropical Diseases. Specific attention will be paid to the diffusion-weighted (DWI) images with their associated calculated Apparent Diffusion Coefficient (ADC) maps to identify acute ischemia. The T2-weighted fluid-attenuated inversion recovery (T2WFLAIR) studies and the double-echo T2-weighted (DET2W) images will be used to assess structural lesions and vasogenic edema. The T2*-weighted (T2*W) images will be specifically examined for signs of low-signal acute hemorrhage or venous thrombosis and the T1-weighted fluid-attenuated inversion recovery (T1WFLAIR) images reviewed for signs of subacute hemorrhage. For research purposes, the images from the first series of studies will be randomly ordered and scored independently by Dr. Laothamatas at Mahidol University and Dr. DeLaPaz at Columbia University. The T1-weighted fluid-attenuated inversion recovery (T1WFLAIR) images will be acquired to maintain high grey matter-white matter (GM-WM) contrast because at 3.0T the GM-WM contrast is reduced in the Spin Echo T1-weighted images (SET1W) used for this purpose at 1.5T. The time of flight MR angiogram (TOF-MRA) will be reconstructed using a maximum intensity projection and will be correlated with the diffusion-weighted (DWI) images and Arterial Spin Labeled (ASL) images to identify any macroscopic vascular stenosis and effects on distal cerebral perfusion (e.g., diffusion-perfusion mismatch). Our experience with 3T scans has indicated that the short T1 inversion recovery (STIR) images better separate the pixel intensities of gray and white matter so that we can segment the brain as discussed below. Arterial spin labeling (ASL) estimates local cerebral perfusion by subtracting an arterial labeled image from a control image to determine the amount of labeled blood which is transported to the tissue. The results will be expressed in units of mL/min/100g. The short T1 inversion recovery (STIR) images will be used to segment the brain into gray and white matter and cerebrospincal fluid (CSF), based on the high signal contrast between these tissues (low white matter, intermediate gray matter, high signal CSF). The distributions of perfusion values from gray and white matter will be examined for changes over time. As detailed below, because of the spatially heterogeneous, temporally shifting pattern of the vascular blockage anticipated with cerebral malaria, we expect that the distribution of voxel by voxel cerebral perfusion found while the patient is comatose will show considerably more variation (higher CV) than in the scans after recovery from coma and at the end of the four week hospitalization. We will both examine the distributions for visible changes (e.g. extensive wings, etc.) and calculate the voxel by voxel coefficient of variation of the different distributions and compare these over time and across patient groups.



Table d-1: Cerebral Magnetic Resonance Imaging Protocol Summary

MR Sequence

Name

Acquisition Parameters Flip TE/TR/TI Angle

Resolution

Field of View

Acquisition time

(with setup)

Slice thickness/gap

(mm)

Number of

slices

T1-weighted fluid-

attenuated inversion recovery

(T1FLAIR)

10/3000/1200 90 288x192 220x170

2 min 4/0 30

Short T1 inversion recovery (STIR)

26/3000/400 90 288x224 220x170

4 min

4/0 30

T2*-weighted (T2*W)

40/750 15 228x192 220x170

4 min 4/0 30

T2-weighted fluid-

attenuated inversion recovery

(T2WFLAIR)

100/8000/1200 90 228 x 192 220 x 170

2 min 5/0 23

Double-echo T2-weighted

(DET2W)

7/87/4800 90 288x224 220x170

2 min 5/0 23

Diffusion-weighted

echo-planar imaging

(DWI-EPI)

30/700 (b = 1000)

90 128 x 128 220x170

1 min 5/0 23

Time of flight MR

angiogram (TOF-MRA)

[SENSE]

3.5/31 20 832 x 572 250 x 250

10242 (recon)

5 min [SENSE

factor 2.5]

1/0 0.5 over

contiguous

150 (3 stacks)

32

Chemical shift image (CSI)

143/1000 90 32x32 (circular k space)

15 min

10/3

4

Arterial spin labeled

perfusion (ASL)

20/5000 90 96x64

10 min 5/2 15-18

Asymmetric spin echo

echo-planar imaging

(ASE-EPI)

32/64/2000 90 128x128

10 min 1.8 20



The major metabolites in the Chemical Shift Image (CSI) datasets are expected to be choline, creatine, N-acetylaspartate (NAA) and lactate. Spectral arrays will be overlaid on the structural images and voxel shifted to obtain at least one spectrum from entirely with the ventricles using a program developed at Columbia University to allow careful registration of spectral and anatomic information to validate the location of the spectra to the ventricles for accurate estimation of the lactate levels. If more than one spectrum is available, these will be averaged to provide a single representative spectrum of the CSF. Normal brain regions will be similarly averaged to produce a representative spectrum of brain tissue. Peak areas of the changes in metabolites of interest will be estimated in these spectra and quantitated by comparison to a fixed sized point-resolved spectroscopy (PRESS) box of unsuppressed water34. In patients with cerebral malaria, we expect that the lactate levels will be reduced as brain metabolism is returned to normal. Lactate levels in brain tissue will also be measured and compared across the three MR measurements. These proposed measurements would be the first MR metabolic studies in patients with malaria. Magnetic field correlation (MFC) images will be obtained using a segmented echo planar imaging asymmetric spin echo (ASE-EPI) sequence. As described in the section on Preliminary Studies, we have recently demonstrated that the magnetic field correlation (MFC) can be measured using asymmetric spin echoes35-37. The MFC is strongly affected by microscopic magnetic field inhomogeneities, such as those expected from accumulation of paramagnetic species of iron in the cerebral microvasculature in the form of hemozoin or excess deoxyhemoglobin. We will explore this technique as a tool for evaluating cerebral malaria, as the MFC is potentially a more specific indicator of iron abnormalities than either R2 or R2*. We will use an echo-planar imaging (EPI) sequence to acquire the images to minimize both the acquisition time and any motion artifacts. The EPI acquisition will be segmented into 3 shots (EPI factor = 43) to allow for shorter echo times (TE’s) and to reduce image distortion. Two TE’s (32 ms and 64 ms) will be used to permit the MFC to be calculated at two separate times. The time dependence of the MFC will yield information on the spatial scale of the field inhomogeneities and help to establish their origin. The MFC will be calculated on a voxel by voxel basis by performing a least squares fit to formula:

( )2,0 *MFC*2exp

isitSS != , i=0,1,…,4, [?]

where ts,i is the value of the ith pulse shift and Si is the corresponding signal intensity. Choice of MR measure of perfusion: Because no MR studies at 3.0T have been reported in patients with cerebral malaria, we have no direct data to assess the potential sensitivity MR methods to detect changes in the mean CV of cerebral perfusion of at least the 33.4% magnitude used in our estimate of sample size. The most commonly used MR approach to measure local changes in blood flow is dynamic susceptibility contrast (DSC) MR perfusion imaging. With this method, a bolus of susceptibility agent (typically a gadolinium or dysprosium chelate) is injected and the response in the form of a transient alteration in MR signal is monitored38, 39. Classical tracer kinetic methods can then be used to measure cerebral blood volume, mean transit time and cerebral blood flow. Contrast-based methods have superior signal-to-noise ratios and provide the best estimates of transit delay but require rapid injection of a bolus of contrast agent39. Although gadolinium and other MR contrast agents have an extraordinary record of safety in studies of patients with cerebral vascular disease, we were concerned about the unknown risks of injections of material into comatose patients with cerebral malaria. Consequently, we have chosen not to use contrast-based MR approaches in the proposed studies.

We will use arterial spin labeling (ASL) studies for the detection of changes in the coefficient of variation (CV)



of cerebral perfusion, determined voxel by voxel. Perfusion imaging with ASL contrast uses magnetically labeled arterial blood water as an endogenous tracer to provide quantitative cerebral blood flow (CBF) measurements40. MR perfusion studies using ASL are completely noninvasive and can be repeated without risk39. Results of direct comparisons of ASL with contrast-based methods have demonstrated highly comparable perfusion results in normal brain tissue41 and in patients with cerebrovascular disease39. Studies of patients with brain tumors have found that ASL provides a reliable assessment of microvascular perfusion that is closely correlated with results of susceptibility contrast studies after administration of gadolinium with a power injector. In particular, ASL was able to demonstrate heterogeneous blood flow distribution within individual tumors38. ASL perfusion measurements are typically derived from pairwise subtractions of temporally adjacent images acquired with and without spin labeling. This pairwise subtraction and the subsequent calibration process provides an absolute measure of cerebral blood flow40.

Figure d-1: Voxels with significant change in perfusion in response to visual stimulation (projected image of a flashing checkerboard) as determined in an arterial spin labeling (ASL) study. From Aguirre et al. Experimental design and the relative sensitivity of BOLD and perfusion fMRI. Neuroimage 2002; 15, 488-500. In the absence of data from patients with malaria, applications of ASL to functional magnetic resonance imaging (fMRI) studies provide an indication of the potential usefulness of this method for detecting changes in cerebral perfusion in patients in cerebral malaria. In brief, fMRI experiments use ASL perfusion studies to detect changes in cerebral blood flow as a measure of the spatial distribution of neural activity42. For example, Figure d-1 shows the distribution of voxels with significant changes in perfusion in response to visual stimulation. Changes in cerebral blood flow may be determined with considerable reproducibility. For example, with a bilateral finger-tapping task, cerebral blood flow values in a defined region of interest (motor hand area in the precentral gyrus, a region approximately 0.8 x 1.7 x1.9 cm43) were (mean ± SD) 52.8 ± 1.2 mL/100g/min [CV = 2.2%] at rest and 66.4 ± 1.1 mL/100g/min [CV = 1.7%]40. The coefficient of variation reliably detected with ASL perfusion studies (~ 2% in this study) suggests that this technique will readily be able to detect a minimum difference in the mean CV of cerebral perfusion of 33.4% between patients with cerebral malaria and control subjects with severe, but not cerebral, malaria. d1.4 Study site and patient population: This hospital-based study will be carried out at the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. The Bangkok Hospital for Tropical Diseases is a specialized hospital for tropical diseases which was founded in 1961. The Hospital is now a 300 bed facility admitting both adult and pediatric patients. Of the hospital beds, 250 are allocated for adult patients and 50 for children. The hospital has a well equipped intensive care unit



with four beds which has been operated for more than ten years. The equipment for this facility includes cardiac monitors, volume respirators and hemodialysis units. Each year, more than 3500 patients are admitted to the Bangkok Hospital for Tropical Disease, and about 60% of them (2000 cases) are malaria cases. Half of the malaria cases, or about 1000 patients per year, have falciparum malaria and, of these, 35% or 350 patients per year, have severe falciparum malaria as defined by the World Health Organization, including severe anemia, cerebral malaria, pulmonary edema, acute renal failure, hyperparasitemia, jaundice and aesthenia. Of the 350 patients with severe falciparum malaria, approximately 10 percent, or about 35 to more than 50 patients each year have cerebral malaria on admission. Accordingly, adequate numbers of patients for the proposed studies will be available at the Hospital for Tropical Diseases. d2 Concept proposal for clinical study d2.71 Study title: RBC sequestration in cerebral malaria: MRI measurement d2.72 Hypothesis to be tested: Our primary study hypothesis is that the extent of red blood cell sequestration in the cerebral vasculature is greater in patients with cerebral malaria than in patients with other forms of severe malaria, as determined by cerebral perfusion studies using arterial spin labeling techniques, d2.73 Study objectives: This prospective, case-control study of adults admitted to the Bangkok Hospital for Tropical Diseases will compare each patient with cerebral malaria to matched controls with severe (but non-cerebral) malaria, to controls with uncomplicated falciparum malaria, and to healthy volunteers. In addition to our primary study hypothesis, a number of subsidiary hypotheses will be tested, including (i) the hypothesis that cerebral metabolic dysfunction is greater in cerebral malaria than in other forms of severe malaria, as determined by proton (1H) MR spectroscopic measurements of ventricular lactate and (ii) that cerebral hemozoin deposition is greater in cerebral malaria than in other forms of severe malaria, as determined by a novel MR method, magnetic field correlation imaging. d2.74 Study site and patient population: This study population will be recruited from patients admitted to the Bangkok Hospital for Tropical Diseases with cerebral malaria, with severe but not cerebral malaria, with uncomplicated falciparum malaria, and healthy volunteers. The Hospital is a specialized facility for tropical diseases which was founded in 1961. The Hospital is now a 300 bed facility admitting both adult and pediatric patients. Of the hospital beds, 250 are allocated for adult patients and 50 for children. The hospital has a well equipped intensive care unit with four beds which has been operated for more than ten years. The equipment for this facility includes cardiac monitors, volume respirators and hemodialysis units. Each year, more than 3500 patients are admitted to the Bangkok Hospital for Tropical Disease, and about 60% of them (2000 cases) are malaria cases. Half of the malaria cases, or about 1000 patients per year, have falciparum malaria and, of these, 35% or 350 patients per year, have severe falciparum malaria as defined by the World Health Organization, including severe anemia, cerebral malaria, pulmonary edema, acute renal failure, hyperparasitemia, jaundice and aesthenia. Of the 350 patients with severe falciparum malaria, approximately 10 percent, or about 35 to more than 50 patients each year have cerebral malaria on admission. Accordingly, adequate numbers of patients for the proposed studies will be available at the Hospital for Tropical Diseases. d2.75 Clinical sites: The Hospital for Tropical Diseases will be the sole study site. d2.76 Comparators: Our primary outcome variable, the coefficient of variation (CV) of cerebral perfusion, determined voxel by voxel within a strictly defined region of white matter using arterial spin lableing (ASL), has been chosen to provide the most sensitive non-invasive measure of microvascular blood flow. While we will measure perfusion within both white and grey matter, we have chosen perfusion within white matter as our primary outcome variable because we will also be assessing evidence of axonal damage in these patients by measurements of N-acetylaspartate44.



d2.77 Regimen: All patients will be treated with the standard antimalarial regimens used at the Hospital for Tropical Diseases and will receive intensive management of complications as needed. d2.78 Study design: This will be a prospective case-control study that will compare each patient with cerebral malaria admitted to the Hospital for Tropical Diseases to controls with severe but not cerebral malaria, with ,uncomplicated falciparum malaria, and to healthy volunteers with respect to each of the study endpoints. d2.781 Eligibility/exclusion criteria: Study inclusion and exclusion criteria will be based on WHO clinical definitions45. Patients must be at least 18 years of age and willing to participate in the study. For all patients, exclusion criteria will include: medical condition too unstable for MRI examination. Children are excluded to focus on a homogeneous population with a common pathogenetic basis for cerebral malaria. In addition, the number of children admitted to the Hospital for Tropical Diseases each year with cerebral malaria is not sufficient for study.Specific criteria for inclusion and exclusion for each of the three study groups will be detailed. --Patients with cerebral malaria: inclusion criteria include: age ≥ 18 years, peripheral blood positive for asexual forms of P. falciparum and Glasgow coma score <10 in the absence of other causes of coma. Exclusion criteria include an abnormal cerebrospinal fluid examination and other concomitant major infections such as pneumonia or meningitis. -- Patients with severe malaria: inclusion criteria include: age ≥ 18 years, peripheral blood positive for asexual forms of P. falciparum, and severe malaria strictly defined as the presence of one or more of the following WHO major criteria at admission: (i) anemia with hemoglobin of less than 5 g/L; (ii) renal failure with serum creatinine of more than 265 µmol/L; (iii) pulmonary edema with the presence of criteria for acute respiratory distress syndrome or acute lung injury; (iv) hypoglycemia with blood glucose of less than 2.2 mM; (v) circulatory collapse with systolic blood pressure of less than 80 mm Hg despite adequate volume repletion; (vi) spontaneous bleeding and/or disseminated intravascular coagulation; (vii) repeated generalized seizures; (viii) acidemia (pH of less than 7.25) or acidosis (serum bicarbonate of less than 15 mM); and (ix) macroscopic hemoglobinuria if definitely related to acute malaria. Exclusion criteria include Glasgow coma score <10 and other concomitant major infections such as pneumonia or meningitis. --Patients with uncomplicated malaria: inclusion criteria include age ≥ 18 years, peripheral blood positive for asexual forms of P. falciparum and Glasgow coma score of 15. Exclusion criteria include coma, and other concomitant major infections such as pneumonia or meningitis. Restriction of participation by gender or ethnicity: Participation in the study will be unrestricted with respect to gender and ethnicity. d2.782 Randomization/stratification plan: Not applicable. d2.783 Number of subjects: 200; 50 patients in each of the four study groups, or a total of 200 subjects. Please see sample size calculation below. d2.784 Study duration: Four years. Training in study procedures will be carried out in the first six months of the five year project and final data analysis will be completed in the last six month. d2.785 Interim study progress and safety monitoring plan: Please see section b1.8 above for details of the composition and schedule of monitoring by the ICIDR Internal and External Advisoray Committees.



d2.786 Study outcomes d2.7861 Primary study outcomes: Our primary study outcome will be a comparison will be between patients with cerebral malaria and patients with other forms of severe malaria, with respect to the coefficient of variation (CV) of cerebral perfusion as determined voxel by voxel within a strictly defined region of white matter by an arterial spin labeling perfusion study. d2.7862 Secondary study outcomes: Subsidiary analyses will be made with respect to patients with other forms of severe and of uncomplicated malaria and with normal control subjects with respect to cerebral metabolic dysfunction, cerebral hemozoin deposition, and a number of other variables. d2.788 Sample size justification and data analyses planned Our primary study hypothesis is that the extent of red blood cell sequestration in the cerebral vasculature is greater in patients with cerebral malaria than in patients with other forms of severe malaria. Our primary outcome variable, the coefficient of variation (CV) of cerebral perfusion, determined voxel by voxel within a strictly defined region of white matter using arterial spin lableing (ASL), has been chosen to provide the most sensitive non-invasive measure of microvascular blood flow. While we will measure perfusion within both white and grey matter, we have chosen perfusion within white matter as our primary outcome variable because we will also be assessing evidence of axonal damage in these patients by measurements of N-acetylaspartate44. As described earlier, red blood cell sequestration seen at autopsy in patients who have died of cerebral malaria is spatially extremely heterogeneous46. One capillary or venule may be tightly packed with infected parasitized red blood cells while the adjacent vessel has none9. Because of local increases in lactic acid and pCO2, combined with acidosis, hypoglycemia and release of vasoactive mediators, nearby arterioles and venules may be maximally vasodialated46. As a consequence, while flow through one capillary or group of capillaries may be partially occluded, increased flow through immediately neighboring vessels may more than compensate, resulting in no change or even an increase in regional blood flow. Moreover, temporally the pattern of microvascular obstruction is constantly changing. Schizpont rupture of adherent parasitzed red blood cells relieves obstruction in one set of vessels with the release of merozoites to infect another crop of erythrocytes. Almost concurrently, previously infected red blood cells newly adhere to a different subset of capillaries and venules7. Recent studies suggest that even after administration of quinine, two distinct parasite broods continue to develop in antiphase, producing peaks of infection at 24 hour intervals, at least during the first three days of treatment47. In patients with cerebral malaria, sequestration almost surely occurs to some extent at all levels of the vascular tree, as evidenced by the transient narrowing of the right middle cerebral artery in the patient with hyperparasitiemtia shown in Figure 8 above and by the minority of survivors who are left with permanent neurological damage48. Nonetheless, obstruction within the microvasculature is identified by sequestration theory as the prerequisite for the development of cerebral malaria. While the voxel volume of our MRI studies is much greater than the volume occupied by a single segment of the microcirculation, the shifting, heterogeneous pattern of microvascular obstruction should be detectable as increased variability in perfusion as measured voxel by voxel within a strictly defined region of white matter (see below). Because the proposed perfusion studies using arterial spin labeling would be the first such investigations in patients with malaria, no magnetic resonance data are available to provide a basis for calculation of sample size. Nonetheless, because the results of measurements of cerebral blood flow by arterial spin labeling are comparable to those obtained with dynamic susceptibility-weighted contrast enhanced MRI41 which are, in turn, correlated with those by transcranial Doppler studies49, earlier studies of cerebral blood flow in patients with cerebral malaria can be used to derive an estimate. As emphasized above, the first studies of cerebral blood flow in patients with cerebral malaria were conducted at the Bangkok Hospital for Tropical Diseases by Dr.



Looareesuwan almost two decades ago, using measurements of arterio-jugular venous differences of tracers. In 12 patients, the cerebral blood flow was within the range for healthy controls 28. The largest published series of measurements of cerebral blood flow in cerebral malaria is that using transcranial Doppler sonography in examinations of 50 Kenyan children with cerebral malaria and of 115 conscious controls of similar age50. As in the earlier study in Bangkok, no significant differences in mean cerebral blood flow velocity were found between the patients with cerebral malaria and the control subjects. Nonetheless, substantial differences were seen in the variability of cerebral blood flow, which we have calculated as the coefficient of variation of cerebral blood flow velocity as measured in the basilar artery. We have used the measurements in the basilar artery for our estimates because considerable differences were found in the cerebral blood flow velocity measured in the right and left middle cerebral arteries in more than 50% of children on admission50. Basilar artery flow measurements were available in 34 children (20 who survived without sequelae, 9 who survived with sequelae, and 5 who died); the mean coefficient of variation of cerebral blood flow velocity (CV) was 51.3%, with a standard deviation (SD) of 30.5%. Among 38 control children, the mean CV of cerebral blood flow velocity was 17.9%. We recognize that these measurements are of macro- rather than microvascular blood view and will use these data for the estimation of sample size with caution. With this background, as indicated above, our primary study hypothesis is that the extent of red blood cell sequestration in the cerebral vasculature is greater in patients with cerebral malaria than in patients with other forms of severe malaria. Our primary study comparison will be between patients with cerebral malaria and patients with other forms of severe malaria, with respect to the coefficient of variation (CV) of cerebral perfusion as determined voxel by voxel within a strictly defined region of white matter. Accordingly, the sample size has been estimated only with respect to this primary comparison. Subsidiary analyses will be made with respect to patients with other forms of severe and of uncomplicated malaria and with normal control subjects with respect to cerebral metabolic dysfunction, cerebral hemozoin deposition, and a number of other variables, but these comparisons have not entered into the sample size calculation. As just described, the data of Newton and colleagues from children with cerebral malaria50 yield an estimate of the coefficient of variation of cerebral perfusion of a mean CV = 51.3 ± 30.5% (SD). For purposes of sample size estimation, we assume that patients with severe, but not cerebral, malaria will have a mean CV that is not different from control subjects, or 17.9%. In the absence of comparable measurements in patients with severe, non-cerebral malaria, this assumption seems reasonable, especially because some patients who survive after treatment of cerebral malaria have a CV of cerebral blood flow velocity that is within the normal range on admission50. Accordingly, the minimum difference in the mean CV of cerebral perfusion that we would like to detect is estimated as the difference between the CV in patients with cerebral malaria and that in controls, or (51.3% - 17.9%) = 33.4%. For our estimate of the standard deviation of the CV, we use the estimated value from the same study50, or 30.5%. Given these assumptions, to detect a difference in the mean CV of cerebral perfusion of at least 33.4% between patients with cerebral malaria and those with severe but non-cerebral malaria, using an unpaired two-sample t-test with a two-sided significance level of 0.01 and a β error specification of 0.90, about 25 subjects will be needed in each group:

n = 2 (zα - zβ)2σ2

δ2 = 2

!

(2.576 +1.282)2(33.4)

(30.5)2

2

= 24.8

where n indicates the sample size in each study group, zα and zβ respectively denote the upper α and lower β percent points of the normal distribution, σ is the standard deviation expected for the study groups, and δ denotes the difference in the mean CV of cerebral perfusion between the groups. Because of the imperfections in our estimate of σ, a sensitivity analysis was carried out to evaluate the influence of errors in this estimate on the sample size calculation. If the value of σ = 30.5 were an overestimate by as much as 20% (i.e. actual value = 24.4), then the sample size needed would be 16. If σ were underestimated by as much as 20% (i.e. actual value = 36.6), than the sample size would need to be 36. Again, aware of the limitations in the



data used to estimate σ and to be cautious in our estimate of the sample size needed for the study, we have chosen the upper estimate of 36. In addition, allowing for as much as a 25% loss of participants during the 4 weeks of study (due to patients not being clinically stable for the MRI examination, initial misdiagnosis, development of complications such as bacterial infection or withdrawal for any reason), then our sample size estimate is 36/0.75 = 47.6, rounded for administrative convenience to about 50 patients in each of the four study groups, or a total of 200 subjects over the four years of study enrollment. This sample size estimate is almost certainly very conservative. Although the subsidiary analyses planned have not entered into the calculation of the sample size required for the proposed study, both the conservative estimate and the rigorous values used for the significance level and the β-error specification should help assure adequate power for the subsidiary comparisons planned. In addition, the three controls (one each with severe but not cerebral malaria, one with uncomplicated falciparum malaria, and a healthy control) included for each patient with cerebral malaria should also help provide adequate power for the subsidiary comparisons. d3 Data Management and Biostatistical support requirements The Data Management Unit (DMU) was established in late 1998 and is located on the 9th floor of the Anek Prasong Building, Faculty of Tropical Medicine, to fulfill national requirements under the Thailand National AIDS Committee. Its establishment and the early-year operation have been sponsored by VaxGen Inc., Brisbane, CA, U.S.A. The primary objective of the Unit is to provide data management and data analysis services to research projects, particularly clinical trials; meanwhile, its current commitment is put mainly into the Phase III Trial to determine the efficacy of AIDSVAXTM B/E vaccine in injecting drug users in Bangkok, Thailand. In September 2002, its Chief and Deputy Chief received technological transfer from VaxGen on preparation of data for the Data and Safety Monitoring Board (DSMB) meeting to consider interim efficacy analysis of this trial. In the meantime, five additional staff were recruited in preparation for the second-generation HIV vaccine efficacy trial. This will be the largest HIV vaccine efficacy trial in the world to test the prime-boost concept of HIV candidate vaccines. The trial, A Phase III Trial of Aventis Pasteur Live Recombinant ALVAC-HIV (vCP1521) Priming With VaxGen gp120 B/E (AIDSVAXTM B/E) Boosting in HIV-uninfected Thai Adults, is scheduled for launch in early 2003. The Data Management Unit also renders service on data management for biomedical research through Mahidol University Applied and Technological Service Center.

CONCLUSION

We believe that the research plan in this application provides a logical approach to definitively determining the the role of cerebral sequestration of P. falciparum-infected red blood cells in the pathogeneis of cerebral malaria. For the past decade, investigators have held deeply divergent views of the precise role of microvascular sequestration of parasitized red blood cells in the pathogenesis of cerebral malaria. The sequestration theory holds that the presence of infected red blood cells in the microvasculature of the brain is the indispensable initial event that is the prerequisite for the development of cerebral malaria. Conversely, the cytokine theory considers that cerebral malaria is the consequence of the excess production of pro-inflammatory cytokines in response to P. falciparum infection but that sequestration of parasitized erythrocytes is neither necessary nor sufficient for the production of coma. Obtaining evidence to differentiate between the sequestration and cytokine theories has been near impossible, in part because determination of the extent of microvascular sequestration has been restricted to pathological studies at autopsy. An improved understanding of the pathogenetic role of red cell sequestration is urgently needed as a basis for rational design of effective treatments for patients with cerebral malaria. Clinical research in developing countries can be challenging and fraught with unforeseen obstacles and problems. We have planned to maximize the possibilities of success of this project by working in an institution that has a long history of excellence in malaria research from the clinic to the laboratory.



e. Human Subjects Research 1. Risks to the subjects:

Human Subjects Involvement and Characteristics: Patients admitted for treatment of cerebral, severe or uncomplicated falciparum malaria to the Bangkok Hospital for Tropical Diseases, Mahidol University, Bangkok, Thailand, who meet the criteria for inclusion and exclusion for the study will be identified as potential study subjects. Over the five years of the study, about 200 subjects will be recruited for the study with appoximately equal numbers of patients with cerebral, severe and uncomplicated malaria and of uninfected healy controls. Patients must be at least 18 years of age and willing to participate in the study. For all patients, exclusion criteria will include: medical condition too unstable for MRI examination, any antimalarial treatment in the 10 days preceding admission and concomitant P. vivax infection. Specific criteria for inclusion and exclusion for each of the three study groups will be detailed. (1) Patients with cerebral malaria: inclusion criteria include: age ≥ 18 years, peripheral blood positive for asexual forms of P. falciparum and Glasgow coma score <10 in the absence of other causes of coma. Exclusion criteria include an abnormal cerebrospinal fluid examination and other concomitant major infections such as pneumonia or meningitis. (2) Patients with severe malaria: inclusion criteria include: age ≥ 18 years, peripheral blood positive for asexual forms of P. falciparum, and severe malaria strictly defined as the presence of one or more of the following WHO major criteria at admission: (i) anemia with hemoglobin of less than 5 g/L; (ii) renal failure with serum creatinine of more than 265 µmol/L; (iii) pulmonary edema with the presence of criteria for acute respiratory distress syndrome or acute lung injury; (iv) hypoglycemia with blood glucose of less than 2.2 mM; (v) circulatory collapse with systolic blood pressure of less than 80 mm Hg despite adequate volume repletion; (vi) spontaneous bleeding and/or disseminated intravascular coagulation; (vii) repeated generalized seizures; (viii) acidemia (pH of less than 7.25) or acidosis (serum bicarbonate of less than 15 mM); and (ix) macroscopic hemoglobinuria if definitely related to acute malaria. Exclusion criteria include Glasgow coma score <10 and other concomitant major infections such as pneumonia or meningitis. (3) Patients with uncomplicated malaria: inclusion criteria include age ≥ 18 years, peripheral blood positive for asexual forms of P. falciparum and Glasgow coma score of 15. Exclusion criteria include coma, and other concomitant major infections such as pneumonia or meningitis. Participation of “vulnerable” populations: Pregnant women, mental patients, prisoners, elderly persons and persons who are institutionalized will be excluded from the study. Children are excluded both to focus on a homogeneous population and because the number of children admitted to the Hospital for Tropical Diseases each year with malarial infection is not sufficient for study as a separate group. Restriction of participation by gender or ethnicity: Participation in the study will be unrestricted with respect to gender and ethnicity.

Sources of research materials Demographic data and data from the medical history and physical

examination will be recorded on standardized research forms; these data will be needed for routine patient care but they will also be made use of by the research project. The blood sample drawn on Day 0 will be needed for patient care, in terms of determining the parasite count, complete blood count, serum chemistries, and type and cross match. Sera, plasma, red blood cells, and white blood cells that remain after completing the blood work necessary for patient care will be saved and used in this research project. On Days 3, 7, 21 and 28, in addition to the blood samples needed for routine medical care, a total of about 20 ml of peripheral blood will be drawn



by venipuncture for research. Stool and urine samples will be collected on Days 0 and 28 for routine medical care and the results will also be recorded for research purposes.

Potential Risks: With appropriate precautions to exclude individuals with magnetic materials in their bodies, magnetic resonance studies have no known risks. Detailed questioning, medical histories and screening procedures will be used to exclude individuals with magnetic materials in their bodies from participation in the magnetic resonance examinations. The U.S. Food and Drug Administration (FDA) has classified the MR procedure as having a “non-significant risk” for the subject of study. When blood is drawn from a vein in the arm. there may be some temporary discomfort and the minimal risk of local bruising, infection or blockage of the vein. Suitable precautions will be taken to avoid these risks.

2. Adequacy of protection against risks:

Subject recruitment and informed consent procedures: Patients admitted for treatment of cerebral, severe or uncomplicated falciparum malaria to the Bangkok Hospital for Tropical Diseases, Mahidol University, Bangkok, Thailand, who meet the criteria for inclusion and exclusion for the study will be identified as potential study subjects.

Protection against risk: The research project will center around the enrollment of adults with cerebral, severe or uncomplicated malaria. Written informed consent will be obtained from study subjects, or, for patients who present for treatment of cerebral malaria, from a family member who will be asked to provide permission for their relative with cerebral malaria to enter this study. The family member must be legally able to provide consent for participation in the study of their relative with cerebral malaria. Detailed questioning, medical histories and screening procedures will be used to exclude individuals with magnetic materials in their bodies from participation in the magnetic resonance examinations. All data will be kept confidential at all times; digital data will be kept in password protected systems and paper files will be kept in locked storage cabinets. If a published paper presents results from these studies, volunteer names will not be used. Appropriate venipuncture precautions will be taken.

3. Potential benefits of the proposed research to the subjects and others: There may be no direct benefit to volunteers who participate in this study. Study subjects may not derive any direct benefits from their participation in this research. Subjects potentially may benefit from identification of previously unrecognized cerebral abnormalities on brain MRI examinations but such benefits cannot be predicted in advance or assured. 4. Importance of the knowledge to be gained: An improved understanding of the pathogenetic role of red cell sequestration is urgently needed as a basis for rational design of effective treatments for patients with cerebral malaria. Given the minimal risks associated with all of the study procedures, these risks seem reasonable in view of the medical and scientific importance of the knowledge that reasonably may be expected to result. Inclusion of women in the proposed clinical research: Female participants of all ages will be included in the proposed studies with the sole exception of children under 18 years of age as detailed below in the section “Inclusion of children”. Both healthy women and women with malaria will be recruited. Inclusion of minority populations in the proposed clinical research: Members of minority groups and their subpopulations of all ages will be included in the proposed studies with the sole exception of children under 18 years of age as detailed below in the section “Inclusion of children”. Both normal controls and subjects with malaria will be recruited. No exclusions of any minority group or subpopulation are planned.



Inclusion of children in the proposed clinical research: This study does not include children under 18 years of age Composition of the proposed study population: Subject selection criteria: Enrollment of equal numbers of male and female subjects is anticipated and active efforts will be undertaken to assure the participation of minority populations. Exclusion of any sex/gender or racial/ethnic group: Not applicable; no potential participant will be excluded by reason of sex/gender or of being a member of any racial /ethnic group. Proposed dates of enrollment: Enrollment of patients will begin in Year 1, month 1, of the project and continue through Year 4, month 9, of the project, leaving the final three months for completion of data collection, analysis and manuscript preparation. Proposed outreach programs for recruiting women and members of minority populations: Not applicable. Proposed study sample composition: Please see the “Targeted /Planned Enrollment Format Page” on page 52 below. f. Vertebrate Animals: Not applicable. g. Literature Cited 1. Hartl DL. The origin of malaria: mixed messages from genetic diversity. Nat Rev Microbiol 2004; 2, 15-22. 2. Organization WH. Severe and complicated malaria. Trans R Soc Trop Med Hyg 1990; 84 Suppl 2, 1-65. 3. Luxemburger C, Thwai KL, White NJ, Webster HK, Kyle DE, Maelankirri L, Chongsuphajaisiddhi T,

Nosten F. The epidemiology of malaria in a Karen population on the western border of Thailand. Transact Royal Soc Trop Med Hygiene 1996; 90, 105-111.

4. Miller LH, Baruch DI, Marsh K, Doumbo OK. The pathogenic basis of malaria. Nature 2002; 415, 673-679. 5. Fujioka H, Aikawa M. The molecular basis of pathogenesis of cerebral malaria. Microb Pathog 1996; 20, 63-

72. 6. Urban BC, Ferguson DJ, Pain A, Willcox N, Plebanski M, Austyn JM, Roberts DJ. Plasmodium falciparum-

infected erythrocytes modulate the maturation of dendritic cells. Nature 1999; 400, 73-77. 7. Dondorp AM, Pongponratn E, White NJ. Reduced microcirculatory flow in severe falciparum malaria:

pathophysiology and electron-microscopic pathology. Acta Trop 2004; 89, 309-317. 8. Dondorp AM, Omodeo-Sale F, Chotivanich K, Taramelli D, White NJ. Oxidative stress and rheology in

severe malaria. Redox Rep 2003; 8, 292-294. 9. Pongponratn E, Turner GD, Day NP, Phu NH, Simpson JA, Stepniewska K, Mai NT, Viriyavejakul P,

Looareesuwan S, Hien TT, Ferguson DJ, White NJ. An ultrastructural study of the brain in fatal Plasmodium falciparum malaria. Am J Trop Med Hyg 2003; 69, 345-359.

10. Clark IA, Rockett KA. The cytokine theory of human cerebral malaria. Parasitol Today 1994; 10, 410-412. 11. Clark IA, Alleva LM, Mills AC, Cowden WB. Pathogenesis of malaria and clinically similar conditions.

Clin Microbiol Rev 2004; 17, 509-539.



12. Clark IA, Awburn MM, Harper CG, Liomba NG, Molyneux ME. Induction of HO-1 in tissue macrophages and monocytes in fatal falciparum malaria and sepsis. Malar J 2003; 2, 41.

13. Papadopoulos MC, Davies DC, Moss RF, Tighe D, Bennett ED. Pathophysiology of septic encephalopathy: a review. Crit Care Med 2000; 28, 3019-3024.

14. Clark IA, Cowden WB. The pathophysiology of falciparum malaria. Pharmacol Ther 2003; 99, 221-260. 15. Brown H, Turner G, Rogerson S, Tembo M, Mwenechanya J, Molyneux M, Taylor T. Cytokine expression

in the brain in human cerebral malaria. J Infect Dis 1999; 180, 1742-1746. 16. Lyke KE, Burges R, Cissoko Y, Sangare L, Dao M, Diarra I, Kone A, Harley R, Plowe CV, Doumbo OK,

Sztein MB. Serum Levels of the Proinflammatory Cytokines Interleukin-1 Beta (IL-1{beta}), IL-6, IL-8, IL-10, Tumor Necrosis Factor Alpha, and IL-12(p70) in Malian Children with Severe Plasmodium falciparum Malaria and Matched Uncomplicated Malaria or Healthy Controls. Infect Immun 2004; 72, 5630-5637.

17. de Souza JB, Riley EM. Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis. Microbes Infect 2002; 4, 291-300.

18. Bohle DS, Kosar AD, Stephens PW. Phase homogeneity and crystal morphology of the malaria pigment beta-hematin. Acta Crystallogr D Biol Crystallogr 2002; 58, 1752-1756.

19. Pagola S, Stephens PW, Bohle DS, Kosar AD, Madsen SK. The structure of malaria pigment beta-haematin. Nature 2000; 404, 307-310.

20. Heidelberger M, Mayer MM, Demarest CR. Studies in human malaria. J Immunol 1946; 52, 325-330. 21. Paul F, Roath S, Melville D, Warhurst DC, Osisanya JO. Separation of malaria-infected erythrocytes from

whole blood: use of a selective high-gradient magnetic separation technique. Lancet 1981; 2, 70-71. 22. Nalbandian RM, Sammons DW, Manley M, L. X, Sterling CR, Egen NB, Gingras BA. A molecular-based

magnet test for malaria. Am J Clin Pathol 1995; 103, 57-64. 23. Cordoliani YS, Sarrazin JL, Felten D, Caumes E, Leveque C, Fisch A. MR of cerebral malaria. AJNR Am J

Neuroradiol 1998; 19, 871-874. 24. Looareesuwan S, Wilairatana P, Krishna S, Kendall B, Vannaphan S, Viravan C, White NJ. Magnetic

resonance imaging of the brain in patients with cerebral malaria. Clin Infect Dis 1995; 21, 300-39. 25. Warrell DA, Looareesuwan S, Warrell MJ, Kasemsarn P, Intaraprasert R, Bunnag D, Harinasuta T.

Dexamethasone proves deleterious in cerebral malaria. A double-blind trial in 100 comatose patients. N Engl J Med 1982; 306, 313-319.

26. Looareesuwan S, Warrell DA, White NJ, Sutharasamai P, Chanthavanich P, Sundaravej K, Juel-Jensen BE, Bunnag D, Harinasuta T. Do patients with cerebral malaria have cerebral oedema? A computed tomography study. Lancet 1983; 1, 434-437.

27. White NJ, Warrell DA, Looareesuwan S, Chanthavanich P, Phillips RE, Pongpaew P. Pathophysiological and prognostic significance of cerebrospinal-fluid lactate in cerebral malaria. Lancet 1985; 1, 776-778.

28. Warrell DA, White NJ, Veall N, Looareesuwan S, Chanthavanich P, Phillips RE, Karbwang J, Pongpaew P, Krishna S. Cerebral anaerobic glycolysis and reduced cerebral oxygen transport in human cerebral malaria. Lancet 1988; 2, 534-538.

29. Riganti M, Pongponratn E, Tegoshi T, Looareesuwan S, Punpoowong B, Aikawa M. Human cerebral malaria in Thailand: a clinico-pathological correlation. Immunol Lett 1990; 25, 199-205.



30. Udomsangpetch R, Chivapat S, Viriyavejakul P, Riganti M, Wilairatana P, Pongponratin E, Looareesuwan S. Involvement of cytokines in the histopathology of cerebral malaria. Am J Trop Med Hyg 1997; 57, 501-506.

31. Pavlakis SG, Kingsley PB, Kaplan GP, Stacpoole PW, O'Shea M, Lustbader D. Magnetic resonance spectroscopy: use in monitoring MELAS treatment. Arch Neurol 1998; 55, 849-852.

32. Shungu DC, Mao X, Kaufmann P. Comparison of in vitro and in vivo CSF lactate in A3243G MELAS patients: a viable method for absolute quantitation of CSF lactate by 1H MRSI. Proc Int Soc Magn Reson Med 2002; 10, 695.

33. Lin DD, Crawford TO, Barker PB. Proton MR spectroscopy in the diagnostic evaluation of suspected mitochondrial disease. AJNR Am J Neuroradiol 2003; 24, 33-41.

34. Knight-Scott J, Haley AP, Rossmiller SR, Farace E, Mai VM, Christopher JM, Manning CA, Simnad VI, Siragy HM. Molality as a unit of measure for expressing 1H MRS brain metabolite concentrations in vivo. Magn Reson Imaging 2003; 21, 787-797.

35. Jensen JH, Chandra R. Method for measuring the magnetic field correlation function for water protons in biological tissues. Proc Intl Soc Magn Reson Med 2002; 10, 2297.

36. Jensen JH, Johnson G, Chandra R, Helpern JA. MRI measurement of magnetic field correlation in a cell suspension. Proc Intl Soc Magn Reson Med 2003; 11, 1120.

37. Helpern JA, Jensen J, Lee SP, Falangola MF. Quantitative MRI assessment of Alzheimer's disease. J Mol Neurosci 2004; 24, 45-48.

38. Warmuth C, Gunther M, Zimmer C. Quantification of blood flow in brain tumors: comparison of arterial spin labeling and dynamic susceptibility-weighted contrast-enhanced MR imaging. Radiology 2003; 228, 523-532.

39. Wolf RL, Alsop DC, McGarvey ML, Maldjian JA, Wang J, Detre JA. Susceptibility contrast and arterial spin labeled perfusion MRI in cerebrovascular disease. J Neuroimaging 2003; 13, 17-27.

40. Wang J, Aguirre GK, Kimberg DY, Roc AC, Li L, Detre JA. Arterial spin labeling perfusion fMRI with very low task frequency. Magn Reson Med 2003; 49, 796-802.

41. Weber MA, Gunther M, Lichy MP, Delorme S, Bongers A, Thilmann C, Essig M, Zuna I, Schad LR, Debus J, Schlemmer HP. Comparison of arterial spin-labeling techniques and dynamic susceptibility-weighted contrast-enhanced MRI in perfusion imaging of normal brain tissue. Invest Radiol 2003; 38, 712-718.

42. Aguirre GK, Detre JA, Zarahn E, Alsop DC. Experimental design and the relative sensitivity of BOLD and perfusion fMRI. Neuroimage 2002; 15, 488-500.

43. Yousry TA, Schmid UD, Alkadhi H, Schmidt D, Peraud A, Buettner A, Winkler P. Localization of the motor hand area to a knob on the precentral gyrus. A new landmark. Brain 1997; 120, 141-157.

44. Medana IM, Day NP, Hien TT, Mai NT, Bethell D, Phu NH, Farrar J, Esiri MM, White NJ, Turner GD. Axonal injury in cerebral malaria. Am J Pathol 2002; 160, 655-666.

45. Organization WH. Severe falciparum malaria. Trans R Soc Trop Med Hyg 2000; 94, 1-90. 46. White NJ. Cerebral perfusion in cerebral malaria. Crit Care Med 1999; 27, 478-479. 47. Davis TM, Supanaranond W, Pukrittayakamee S, Silamut K, White NJ. Evidence for continued two-brood

replication of Plasmodium falciparum in vivo during quinine treatment. Acta Trop 2003; 89, 41-45. 48. Newton CR, Warrell DA. Neurological manifestations of falciparum malaria. Ann Neurol 1998; 43, 695-

702.



49. Apruzzese A, Silvestrini M, Floris R, Vernieri F, Bozzao A, Hagberg G, Caltagirone C, Masala S, Simonetti G. Cerebral hemodynamics in asymptomatic patients with internal carotid artery occlusion: a dynamic susceptibility contrast MR and transcranial Doppler study. AJNR Am J Neuroradiol 2001; 22, 1062-1067.

50. Newton CR, Marsh K, Peshu N, Kirkham FJ. Perturbations of cerebral hemodynamics in Kenyans with cerebral malaria. Pediatr Neurol 1996; 15, 41-49.

h. Consortium/Contractual Arrangements: The Columbia University College of Physicians and Surgeons, New York, New York, USA, the applicant organization, and the collaborating institutions, Mahidol University, Bangkok, Thailand, and New York University, New York, N.Y., are prepared to establish in writing consortium agreement in compliance with the NIH publication "Guidelines for Establishing and Operating Consortium Grants", NIH Guide for Grants and Contracts.

Programmatic considerations: Gary M. Brittenham, M.D., as the Principal Investigator and representative of the Columbia University College of Physicians and Surgeons, will retain the responsibility for overall supervision of the project. Truman R. Brown, Ph.D., will have the responsibility for the design and interpretation of the magnetic resonance studies and is designated as one of the Key Personnel. Sornchai Looareesuwan, M.D., will be the Major Foreign Collaborator and have the responsibility for the supervision of the research work at Mahidol University and is designated as one of the Key Personnel. Jens Jensen, Ph.D., will have the responsibility for the supervision of the research work at New York University and is designated as one of the Key Personnel.

Fiscal and administrative considerations: Monthly invoices shall be submitted in duplicate to Columbia University College of Physicians and Surgeons and reference the FS Grant number. Invoices shall reflect summary detail, by budget category, of the costs incurred. Invoices will be reviewed by the Principal Investigator and the Department Administrator prior to payment. The total cost, both direct and indirect, of performing the research work shall not exceed the amounts shown in the proposed budget. The agreements made shall be subject to applicable Public Law and DHHS regulations, policies and required assurances. i. Letter of support from consultant: Not applicable.


PHS 398 (Rev. 05/01) Page _______ Checklist Form Page

CHECKLIST TYPE OF APPLICATION (Check all that apply.)

NEW application. (This application is being submitted to the PHS for the first time.)

SBIR Phase I SBIR Phase II: SBIR Phase I Grant No. _ ______________________ SBIR Fast Track

STTR Phase I STTR Phase II: STTR Phase I Grant No. _ ______________________ STTR Fast Track

REVISION of application number:

(This application replaces a prior unfunded version of a new, competing continuation, or supplemental application.)

COMPETING CONTINUATION of grant number: INVENTIONS AND PATENTS (Competing continuation appl. and Phase II only)

(This application is to extend a funded grant beyond its current project period.) No Previously reported

SUPPLEMENT to grant number: Yes. If “Yes,” Not previously reported (This application is for additional funds to supplement a currently funded grant.)

CHANGE of principal investigator/program director.

Name of former principal investigator/program director:

FOREIGN application or significant foreign component.

1. PROGRAM INCOME (See instructions.) All applications must indicate whether program income is anticipated during the period(s) for which grant support is request. If program income is anticipated, use the format below to reflect the amount and source(s).

Budget Period Anticipated Amount Source(s)

2. ASSURANCES/CERTIFICATIONS (See instructions.) The following assurances/certifications are made and verified by the signature of the Official Signing for Applicant Organization on the Face Page of the application. Descriptions of individual assurances/ certifications are provided in Section III. If unable to certify compliance, where applicable, provide an explanation and place it after this page.

•Human Subjects; •Research Using Human Embryonic Stem Cells• •Research on Transplantation of Human Fetal Tissue •Women and Minority Inclusion Policy •Inclusion of Children Policy• Vertebrate Animals•

•Debarment and Suspension; •Drug- Free Workplace (applicable to new [Type 1] or revised [Type 1] applications only); •Lobbying; •Non-Delinquency on Federal Debt; •Research Misconduct; •Civil Rights (Form HHS 441 or HHS 690); •Handicapped Individuals (Form HHS 641 or HHS 690); •Sex Discrimination (Form HHS 639-A or HHS 690); •Age Discrimination (Form HHS 680 or HHS 690); •Recombinant DNA and Human Gene Transfer Research; •Financial Conflict of Interest (except Phase I SBIR/STTR) •STTR ONLY: Certification of Research Institution Participation.

3. FACILITIES AND ADMINSTRATIVE COSTS (F&A)/ INDIRECT COSTS. See specific instructions.

DHHS Agreement dated: No Facilities And Administrative Costs Requested.

DHHS Agreement being negotiated with Regional Office.

No DHHS Agreement, but rate established with Date CALCULATION* (The entire grant application, including the Checklist, will be reproduced and provided to peer reviewers as confidential information.)

a. Initial budget period: Amount of base $ x Rate applied % = F&A costs $ b. 02 year Amount of base $ x Rate applied % = F&A costs $ c. 03 year Amount of base $ x Rate applied % = F&A costs $ d. 04 year Amount of base $ x Rate applied % = F&A costs $ e. 05 year Amount of base $ x Rate applied % = F&A costs $

TOTAL F&A Costs $ *Check appropriate box(es):

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4. SMOKE-FREE WORKPLACE Yes No (The response to this question has no impact on the review or funding of this application.)

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