Department of Transfusion Medicine

Project numbers

Z01 CL-02005-28 DTM A Controlled Prospective Study of Transfusion-
Associated Hepatitis (TAH)

Z01 CL-02040-13 DTM Significance of Anti-HIV Antibody in Asymptomatic Donors

Z01 CL-02045-11 DTM Etiology of Allergic Reactions in Platelet- and
Granulocytapheresis Donors

Z01CL-02055-09 DTM Kinetic Studies of Indium-Labeled Leukocytes

Z01 CL-02058-09 DTM Characterization of Human Pathogenic Mycoplasma Isolated
From HIV Infected Patients

Z01 CL-02062-07 DTM Treatment of Familial Hypercholesterolemia by Dextran
Sulfate Apheresis

Z01 CL-02064-06 DTM Quantitative Analysis of Viral Genomes and Their
Clinical Correlations

Z01 CL-02068-06 DTM A Prospective Study of Anti-HCV Positive Blood Donors

Z01 CL-02069-06 DTM Immune Response of HCV Infection

Z01 CL-02070-03 DTM Cloning and Characterization of the Hepatitis G Virus

Z01 CL-02071-02 DTM Functional Relevance of HLA Polymorphism in
Vaccination Protocols

Z01 CL-02074-02 DTM Leukocyte, Platelet, and RBC Serology in Autoimmune
Lymphoproliferative Syndrome

Z01 CL-02076-02 DTM Evaluation of Nucleic Acid Vaccine as Preventive and
Therapeutic Modality

Z01 CL-02077-02 DTM Gene-Therapy for Human Hepatitis C Infection: A Chronic
Infectious Disease Model

Z01 CL-02078-02 DTM Viral and Immune Factors That Influence Recovery or
Progression of Hepatitis C

Z01 CL-02079-02 DTM HCV Infection in Infants and Children

Z01 CL-02081-02 DTM Clinical Aspects of Hepatitis G Virus Infection

Z01 CL-02082-02 DTM Studies of Viral Hepatitis and AIDS in the
Chimpanzee Model

Z01 CL-02083-02 DTM Quantitation and Characterization of
Lymphohematopoietic Cells

Z01 CL-02084-02 DTM Development of Methods for Ex Vivo Cultured and
Immunologically and/or Genetically Modified Cells

Z01 CL-02085-02 DTM Methods for Positive and Negative Selection of
Hematopoietic Progenitor Cells

Z01 CL-02086-01 DTM Effectiveness of Granulocyte Transfusions

Z01 CL-02089-01 DTM Comparative Studies of Granulocyte-Colony Stimulating
Factor (G-CSF) and Dexamethasone, Alone and in
Combination, to Optimize the Yields of Granulocytapheresis
Procedures in Healthy Volunteer Donors

Z01 CL-02090-01 DTM Effect of Storage Parameters on Efficacy of Filtration
of Red Blood Cell Units

Z01 CL-02091-01 DTM Acquisition of Hematopoietic Stem Cells for Second
Transplants by Apheresis of Filgrastim-Stimulated
Donors Participating in the National Marrow
Donor Program (NMDP)

Z01 CL-02092-01 DTM Clinical Efficacy of Daily G-CSF-Recruited Granulocyte
Transfusions in Patients with Severe Neutropenia
and Life-Threatening Infections

Z01 CL-02093-01 DTM Effects of G-CSF on Granulocyte Donors

Z01 CL-02094-01 DTM Drug-Dependent Immune Mediated Cytopenias

Z01 CL-02095-01 DTM Structure and Function of Granlocyte Antigens

INTRAMURAL RESEARCH PROJECT Z01 CL-02005-28 DTM

October 1, 1996 to September 30, 1997

Title of Project: A Controlled Prospective Study of Transfusion-Associated
Hepatitis (TAH)

Principal Investigator: H.J. Alter, M.D. (Chief)
IDS, DTM, CC, NIH, Bethesda, MD 20892

Other Personnel: J. Melpolder, IDS, DTM, CC
J.W.-K. Shih, Ph.D., Microbiologist, DTM, CC
R. Sacher, M.D., Chief, Blood Bank, Div. of Hematology,
Georgetown University Hospital, Washington, D.C.
R. Purcell, M.D., Chief, Hepatitis Section, NIAID
V. Rustgi, Fairfax Hospital, Fairfax, VA

Collaborating Units: NIAID; Georgetown Univ. Hospital; Fairfax Hospital

Staff-Years: 3.5

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: This project represents a series studies of transfusion-associated hepa-
titis (TAH) in prospectively followed transfusion recipients undergoing of open heart surgery. These studies, conducted since the 1960's, have serially traced the incidence and causes of TAH and have related incidence to a variety of interventive measures to screen blood donors. The studies have sequentially shown the efficacy of adopting an all-volunteer donor system; testing for hepatitis B surface antigen; utilizing the surrogate assays alanine aminotransferase and anti-hepatitis B core antibody; and testing for antibodies to the hepatitis C virus. These studies were originally conducted at NIH, but when the cardiac surgery branch was closed, the patient base was shifted to Fairfax and Georgetown University hospitals. Overall, the studies have shown a decline in TAH incidence from near 30% in the 1960, to 10% to 20%
in the 1970's, 8% to 12% in the early 1980's, and near zero in the 1990's.

Since 1990, the study has focused on the impact of screening assays to detect carriers
of the hepatitis C virus (HCV). The first generation tests for anti-HCV were introduced in 1990 and the second generation in 1992. Before 1990, TAH incidence was in the range of
3% to 4%. Following first generation assay testing, the rate fell to 1.5%. In approximately 700 recipients following since the introduction of more sensitive second-generation assays
in 1992, the TAH rate has fallen to 0.2% and the rate of TAH C has fallen to zero. The
overall rate reflects only a single mild case, which might be due to the newly described hepatitis G virus, to nonviral causes, or to a currently unrecognized human hepatitis virus. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02040-13 DTM

October 1, 1996 to September 30, 1997

Title of Project: Significance of Anti-HIV Antibody in Asymptomatic Donors

Principal Investigator: H.J. Alter, M.D. (Chief)
DTM, CC, NIH, Bethesda, MD 20892

Other Personnel: H.G. Klein, M.D., Chief, DTM, CC
S.F. Leitman, M.D., Chief, DTM, CC
J.W. Shih, Ph.D., DTM, CC
C. Cantilena, M.D., Medical Officer, DTM, CC
J. Melpolder, DTM, CC
P. Ness, Chesapeake Region, American Red Cross
D. Tan, M.D., Fogarty Fellow, DTM, CC

Collaborating Unit: Washington Area Red Cross

Staff-Years: 3.0

Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: A cohort of anti-HIV positive donors and controls has been under prospective followup since 1985. The original analysis of the study was published in the
New England Journal of Medicine (321:917, 1989). The cohort is now in its 12th year
of followup. At enrollment, 182 subjects were Western blot (WB) positive, including 158
asymptomatic donors, 9 sexual partners of these donors, and 15 persons infected by blood transfusion. In addition, we enrolled a control population consisting of 70 donors who
were anti-HIV reactive on the screening enzyme immunoassay (EIA), but WB negative
or WB indeterminate (21). Of the 182 participants who were Western blot positive, 87%
were donors, 5% sexual partners, and 8% recipients. Of the 182 WB positives, 56 (31%)
are alive and in active followup; 57 (31%) are dead, of whom 53 (93%) died of AIDS;
and 71 (39%) are lost to followup (LTFU). We suspect that most LTFU have succumbed
to AIDS, but we need access to the National Death Index to establish this: 11 of the 71
LTFU were known to have AIDS at the time they left the study.

Of the 56 patients who are in active followup, 42 (75%) are males. Fifty-one (91%) were detected at the time of blood donation, 1 was a blood recipient, and 4 are sexual partners of index donors. Twenty of 56 (36%) have had an AIDS-defining event. Others have CD4 counts under 300, but have had a stable course even before treatment. A subset of 14 patients have exceeded 10 years of followup and have CD4 counts persistently >400 with no AIDS-defining infections and no physical abnormalities except minor adenopathy. Our goal will
be to focus on this group in terms of predictive factors for long-term non-progression.
AIDS or HIV-related phenomena have not developed in any of the 21 WB indeterminate
or 70 WB negative subjects. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02045-11 DTM

October 1, 1996 to September 30, 1997

Title of Project: Etiology of Allergic Reactions in Platelet- and
Granulocytapheresis Donors

Principal Investigator: S.F. Leitman, M.D. (Chief)
DTM, CC, NIH, Bethesda, MD 20892

Collaborating Unit: W.J. Lee, Research Associate, Fenwal Laboratories
Division of Baxter Healthcare, INC.
Box 490, Round Lake, IL 60073

Staff-Years: 0.1

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: In February 1984, the DTM converted from manual to automated platelet collection techniques, acquiring for this purpose four automated cell separation devices. During the next 10 years, 26 donors undergoing apheresis procedures on the
Fenwal CS-3000 device experienced acute hypersensitivity reactions. Sixteen reactions occurred during plateletpheresis, and 10 reactions occurred during granulocytapheresis procedures. The purpose of this study was to identify the allergen responsible for these reactions. Using a combination of skin tests, radioallergosorbent tests, and basophil histamine release assays, specific IgE-mediated sensitization to ethylene oxide, a gas used to sterilize the plastic disposable apheresis kits, was found in 10 of 16 plateletpheresis donors and 8 of 10 granulocytapheresis donors experiencing reactions, but in none of 140 non-
reacting controls. It is estimated that as many as 1.0% of all repeat apheresis donors may become sensitized to ethylene oxide and experience allergic symptoms during donation.
Two of the 10 individuals experiencing reactions during granulocyte donations had positive skin tests to hydroxyethyl starch and appeared to become sensitized to this red cell sedimenting agent during apheresis. Although none of these reactions was life-threatening, the DTM is continuing to test apheresis donors for sensitization to ethylene oxide and hydro-
xyethyl starch, and to defer sensitized individuals from subsequent CS-3000 donations.

The results of these studies were reported to the manufacturer of the CS-3000 apheresis device and to CBER/FDA. Similar reactions were subsequently reported by another group
in plasmapheresis donors. As a result of these reports, the manufacturer of the CS-3000 disposable apheresis kits changed the process by which the kits are sterilized from an ethylene-oxide based system to one involving predominantly gamma sterilization. The manufacturer states that kits manufactured after 1995 no longer contain significant ethylene oxide residuals. The most recent hypersensitivity reaction to occur in a DTM donor was during a granulocytapheresis procedure in August 1995. No reactions have been noted since then, although about 2000 CS-3000 platelet- and granulocytapheresis donor procedures
continue to be performed per year. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02055-O9 DTM

October 1, 1996 to September 30, 1997

Title of Project: Kinetic Studies of Indium-Labeled Leukocytes

Principal Investigator: S.F. Leitman, M.D. (Chief)
BSS, DTM, CC, NIH, Bethesda, MD 20892

Other Personnel: J. Carasquillo, M.D., NMD, CC
C. Chen, M.D., NMD, CC

Collaborating Unit: None

Staff-Years: 0.2

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: The kinetic patterns of fresh, frozen-thawed, or cultured human leukocytes are studied by tagging the cells ex vivo with 111- Indium, a radioisotopic label, and measuring their distribution throughout the body by means of gamma camera imaging and gamma counting of blood samples. This type of study has widespread applications. The most common use of these radiolabeled leukocyte trafficking studies is for abscess localization in cases of suspected infection not definitively diagnosed by other, noninvasive, studies. In these cases autologous or allogeneic granulocytes, collected by simple phlebotomy or by apheresis, are labeled with 50 µCi of 111-Indium per kg of patient weight. Fourteen studies for the purpose of abscess localization were performed in Clinical Center patients enrolled in
a variety of protocols in the past 6 years. Two of these 14 were positive and led to appropriate therapy for a case of Salmonella osteomyelitis in a hemophiliac patient with HIV infection, and resection of a diverticular abscess in a patient with Zollinger-Ellison syndrome. In
many of the negative studies, unnecessary surgical exploration was avoided.

Indium-labeled leukocyte trafficking studies have also been investigated as diagnostic tools is detecting large vessel inflammation in patients with Takayasu's arteritis. A prospective study performed in collaboration with NIAID and NMD/CC showed that radiolabeled autologous mononuclear cell trafficking could not accurately predict whether symptomatic patients with fever and high sedimentation rates had active arteritis, and thus could not be used to determine whether intervention with anti-inflammatory agents should be initiated.

Collaborative trials with the Surgery Branch, NCI, have investigated the diagnostic utility and prognostic application of radiolabeled autologous tumor-infiltrating lymphocyte (TIL) studies in patients with metastatic melanoma. TIL trafficking studies revealed metastatic deposits that were undetected clinically, and TIL trafficking to sites of tumor was strongly correlated with tumor regression in response to infusions of TIL cells and cyclophosphamide.

Most recently, radiolabeled allogeneic granulocyte trafficking studies have been used to determine whether pulmonary infiltrates in patients with chronic granulomatous disease of childhood (CGD) are due to infection or to lung scarring and granuloma formation. Trafficking studies in CGD patients with HLA-alloimmunization have been used to predict whether allogeneic cells will survive following transfusion and thus whether a therapeutic trial of granulocyte transfusions is warranted. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02058-09 DTM

October 1, 1996 to September 30, 1997

Title of Project: Characterization of Human Pathogenic Mycoplasma Isolated
From HIV Infected Patients

Principal Investigators: S.-C. Lo, Ph.D, M.D., AFIP
J.W.-K. Shih, Ph.D.
IDS, DTM, CC, NIH, Bethesda, MD 20892

Other Personnel: R.Y. Wang, Ph.D., DTM, CC
C. Haley, B.S., AFIP
B-X. Zhang, M.D., Ph.D., DTM, CC
H.J. Alter, M.D., DTM, CC
J.G. Tully, Ph.D., NIAID

Collaborating Units: Armed Forces Institute of Pathology (Washington, DC); NIAID

Staff-Years: 1.0

Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: This project is part of a long-term collaborative effort between this laboratory and Dr. Shyh-Ching Lo's at AFIP to investigate the co-factors contributing to
the pathogenesis of AIDS. It has been a fruitful scientific and intellectual collaboration.
The laboratory continues to support work on diagnosis and characterization of myco-
plasma originated from AIDS patients, sexually transmitted disease (STD) patients, and others. We applied serological tests that were developed in this laboratory to several clinical settings, including patients with HIV infection, nongonococcal urethritis (NGU), STDs, and intravenous drug using. We found a high prevalence of antibodies to M. penetrans in patients with Kaposi's sarcoma and antibodies to M. genitalium in patients with NGU. Using paired donor-recipient specimens, we also found M. fermentens and M. genitalium were transmissible through blood transfusion. Recently, we were able to show that the association of anti-
body to M. genitalium with the sexual transmission of HIV was highly significant while
other STDs were not significantly associated. In FY97, we were asked to provide serological tests on specimens from patients who suffered Gulf War syndrome. Over 6,000 paired specimens, including controls, were examined, and we were not able to find any difference between soldiers who served in the Gulf War and controls in antibodies to M. fermentens.
Of future interest for mycoplasma study is the contribution of mycoplasma to neoplasms after long-term chronic infection. We have shown that some species of mycoplasma were able to transform cells in vitro after long-term cocultivation, and several oncogenes were activated. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02062-O7 DTM

October 1, 1996 to September 30, 1997

Title of Project: Treatment of Familial Hypercholesterolemia by Dextran
Sulfate Apheresis

Principal Investigators: S.F. Leitman, M.D. (Chief)
BSS, DTM, CC, NIH, Bethesda, MD 20892

J.M. Hoeg, M.D. (Molecular Disease Branch)
NHLBI, NIH, Bethesda, MD 20892

Collaborating Unit: Kaneka America Corporation, Suite 3603, 800 Third Avenue,
NY, NY 10022 (D. Zachary, Vice President)

Staff-Years: 1.5

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: Patients with familial hypercholesterolemia (FH) type IIa are at high risk of premature coronary artery disease due to elevated low-density lipoprotein (LDL)
and lipoprotein a [Lp(a)] cholesterol levels. Diet and drug therapy can reduce cholesterol concentrations in most patients with heterozygous FH, but a small proportion of heterozygous and nearly all homozygotus patients do not respond to therapy. Selective removal of LDL by dextran sulfate affinity adsorption was evaluated in these patients in a collaborative multicenter U.S. study. The dextran sulfate apheresis system (Liposorber LA-15, Kaneka, Japan) removed LDL and Lp(a) without lowering albumin levels, thus allowing return of autologous plasma without the need for colloid replacement solutions. Furthermore, high-density lipoprotein (HDL), a lipid moiety with highly protective effects against cardiovas-
cular disease, increased over time. Three homozygous and three heterozygous FH patients were enrolled at the Clinical Center; the total cohort enrolled nationwide included 10 homozygotes and 54 heterozygotes. Treatments were administered at 7- to 14-day intervals. Mean acute reductions in total, LDL, and Lp(a) cholesterol levels were 70%, 81%, and
68%, respectively, in homozygotes and 61%, 76%, and 65%, respectively, in heterozygotes. Time-averaged levels of LDL cholesterol were reduced 53% (from 447 to 310 mg/dl) in homozygous FH patients and 41% (from 243 to 143 mg/dL) in heterozygous patients. The treatments were very well tolerated. An adverse association between using angiotensin con-
verting enzyme (ACE) inhibitors and the development of flushing and hypotension was noted, and ACE inhibitor therapy is now routinely discontinued for at least 24 hours prior to each procedure. The results of the multicenter study suggest that dextran sulfate adsorption is
a safe and effective way to clear plasma of LDL cholesterol, and has the advantages, compared with simple plasma exchange, of eliminating the need for colloid replacement
solutions and increasing HDL levels.

The data gathered in this study were used as the basis for licensure of the LA-15 system, which was approved by the FDA for treatment of FH in July 1996. Patients are now continuing long-term followup on an "LDL-Apheresis Registry" to gather post-licensure data on the effect of long-term treatment on the development of primary and secondary atherosclerotic events, and on overall survival. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02064-06 DTM

October 1, 1996 to September 30, 1997

Title of Project: Quantitative Analysis of Viral Genomes and Their
Clinical Correlations

Principal Investigator: J.W.-K. Shih, Ph.D.
IDS, DTM, CC, NIH, Bethesda, MD 20892

Others Personnel: R.Y.H. Wang, Ph.D., DTM, CC
A. Matsumoto, M.D., Ph.D., DTM, CC
Y. Nakatsuji, M.D., DTM, CC
H.J. Alter, M.D., DTM, CC
I.C. Hsu, Ph.D., Department of Pathology,
University of Maryland

Collaborating Unit: None

Staff-Years: 1.0

Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: This continuing effort is responding to the increasing demand for a more precise measurement of relevant genomic information in any viral infection. The knowledge of the presence of a specific viral gene will help in identifying the infectious agent. However, to assess the stage of a disease, to evaluate the efficacy of a treatment, or to determine the value of a predictor of the progression of a disease, a more precise and quantitative analysis of the specific gene would be required. These previously research-oriented questions can now begin to be answered in routine clinical laboratories with the advanced technology of molecular biology, such as polymerase chain reaction (PCR) and the sequencing and mapping of restriction nuclease-digested fragments. We initiated developmental research in molecular diagnostic technology to meet our clinical study needs in hepatitis B virus, hepatitis C virus (HCV), and HIV infection. Whenever possible, we would improve the basic PCR technique to become a semiquantitative procedure. During the last 2 years, we were able to use PCR as primary study tool for viral infection by a newly identified human hepatitis virus, HGV. We found that this virus was transmitted by blood transfusion: specific HGV RNA was identified in both recipients and paired donor sera. HGV can cause chronic infection with mild or no observed liver function abnormality. In the preliminary studies, the prevalence of HGV in blood donors was higher than that of HCV. We also initiated a project to construct an internal standard
to be used in RT/PCR for determining HCV RNA. A project to study the unique specificity
of the repairing enzyme Mut Y and its clinical application was also initiated during last year. Our future effort in this program will be to continue to evaluate new testing approaches, specifically, perfecting the quantitation procedures, and standardizing and applying molecular testing to blood products. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02068-06 DTM

October 1, 1996 to September 30, 1997

Title of Project: A Prospective Study of Anti-HCV Positive Blood Donors

Principal Investigator: H.J. Alter, M.D. (Chief)
DTM, CC, NIH, Bethesda, MD 20892

Other Personnel: C. Cantilena, M.D., Atg. Chief, DTM, CC
J. Melpolder, M.T. (ASCP), DTM, CC
M. Van Raden, Ph.D., NIAID

Collaborating Unit: NIAID

Staff-Years: 2.0

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: This protocol is designed to study the natural history and epidemiology of hepatitis C virus (HCV) infection in an asymptomatic blood donor population found to be anti-HCV positive at the time of blood donation. Thus far, 560 subjects have been enrolled, including 305 RIBA positives, 143 RIBA indeterminates, and 112 RIBA negative controls. The accrued data have been published recently (N Engl J Med 334;1691, 1996) and can be summarized as follows: 1) characteristics of HCV-infected donors as compared with controls were a younger age, African-American race, and a lower education level; 2) independently associated risk factors for HCV were transfusion, intravenous drug use (IVDU), cocaine snorting, sexual promiscuity, and ear piercing among males; 3) although it was anticipated that IVDU would be a risk factor, it was a major surprise to find that 42% of RIBA-positive volunteer blood donors admitted to having used intravenous drugs at some point in their lives, generally 10 or more years before the donation; 4) another major surprise was the strong independent association between cocaine snorting and HCV positivity; we postulate that shared paraphernalia for snorting accompanied by frequent cocaine related epistaxis
may serve as a covert vehicle for parenteral viral transmission; 5) 86% of RIBA positive donors were viremic, confirming the persistent nature of this agent; 6) in contrast, 13%
of subjects appeared to have recovered from HCV infection as evidenced by repeatedly negative polymerase chain reaction for HCV RNA and by persistently normal alanine
transaminase; 7) 66% had biochemical evidence of liver disease and 86% of those biopsied
had an abnormal liver histology; however, only 6% had a severe histologic lesion despite prolonged infection (mean 18 years); and 8) despite an association with promiscuous sex practices, when we tested the direct partners of HCV-infected individuals, we could find
no evidence for sexual transmission. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02069-06 DTM

October 1, 1996 to September 30, 1997

Title of Project: Immune Response of HCV Infection

Principal Investigator: J.W.-K. Shih, Ph.D. (Microbiologist)
IDS, DTM, CC, NIH, Bethesda, Maryland 20892

Other Personnel: Z. Chen, M.D., Ph.D., Visiting Fellow, DTM, CC
D. Tan, M.D., Visiting Fellow, DTM, CC
R.Y.-H. Wang, Ph.D., Molecular Biologist, DTM, CC
H.J. Alter, M.D., Chief, Infectious Dis. Sec., DTM, CC
W.M. Ching, Ph.D., NMRI
I. Berkower, M.D., Ph.D., FDA

Collaborating Units: Naval Medical Research Institute; FDA

Staff-Years: 0.5

Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: This is a continuous effort of a collaborative multi-phase project to investigate the immune response in both humans and experimental animals after their infection with human hepatitis C virus. In the initial phase, the commercial testing kits were used to detect antibodies to HCV proteins in the known non-A, non-B hepatitis patient sera. This provided information on the serological data of hepatitis C virus infection and its relationship to other clinical determinations. The second phase of the study will concentrate on the identification of immune dominant epitopes and neutralizing epitopes. The scope of this phase of study will be extensive and time-consuming. The third phase of the project will encompass using the knowledge of the immune response to develop preventive strategy and understanding of the pathogenicity of this viral infection. Special attention will be paid in identifying
the possible relationship between the immune response and the high chronicity in HCV infected patients. One part of this study is to transfer techniques and procedures developed
in the laboratory studies to our clinical studies. We have developed a proliferation assay to measure the immune response in mice to recombinant HCV core proteins. We then adopted the experience learned in working with mice to develop procedures to differentiate chronic infected patients from the recovered ones. During FY '95 and FY '96, we initiated a new project to examine the potential for a nucleic acid vaccine for HCV. The knowledge, the techniques and the material developed in this nucleic acid vaccine study will also complement our continued effort in understanding the immune response in HCV infection. The
long-term goal of both projects will lead to developing models of immune therapy of
chronic viral infection of the liver. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02070-03 DTM

October 1, 1996 to September 30, 1997

Title of Project: Cloning and Characterization of the Hepatitis G Virus

Principal Investigator: H.J. Alter, M.D. (Chief)
DTM, CC, NIH, Bethesda, MD 20892

Other Personnel: J.W.-K. Shih, Ph.D., Microbiologist, DTM, CC
Y. Nakatsuji, Visiting Fellow, DTM, CC
J. Kim, Genelabs, Inc., Emeryville, CA
H. Margolis, CDC, Atlanta, GA

Collaborating Units: CDC; Genelabs

Staff-Years: 4.0

Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: Studies of transfusion-associated hepatitis at NIH and of community-acquired hepatitis at CDC indicated that 10% to 20% of observed hepatitis cases could not be accounted for by the known hepatitis viruses: HAV, HBV, HCV, HDV, and HEV. To investigate the existence of an additional agent or agents of human hepatitis, a CRADA
was established between the Department of Transfusion Medicine of NIH, Genelabs Technologies, Inc. of Emeryville, CA, and the CDC. Suspect clinical materials were sent to Genelabs, where they were amplified following reverse transcription by Sequence Independent Single Primer Amplification and then cloned into E. coli using a gt11 expression vector. A unique clone was identified that was novel to the gene bank and exogenous by hybridization assays. The agent could be recovered from the cloning source and was shown to be a flavivirus distinct from other members of that family. It had approximately 25% homology with HCV. A polymerase chain reaction assay was developed and clinical materials screened. The agent, tentatively designated HGV, is associated with approximately 20% of transfusion and community-associated hepatitis that is unrelated to previously recognized agents, but
its causal role in the development of hepatitis has not been established. HGV is found in
1% to 2% of blood donors and has been proved to be transfusion-transmitted by linked donor-recipient samples.

In summary, HGV is a new member of the Flaviviridae family that is prevalent in the general population and transfusion transmissible. Further studies are required to establish
its disease burden and particularly, to define whether it causes hepatitis or is merely associated with hepatitis because of shared transmission patterns with another agent(s) that is currently undetectable. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02071-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Functional Relevance of HLA Polymorphism in
Vaccination Protocols

Principal Investigator: F.M. Marincola, M.D. (Associate Director for Science)
HLA Laboratory, DTM, CC, NIH, Bethesda, MD 20892

Collaborating Units: Surgery Branch, NCI; HLA Laboratory, DTM

Staff-Years: 2.0

Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: HLA genes are the most polymorphic genes in the human genome. Knowledge about HLA polymorphism in relation to possible peptide-based, T-cell-restricted vaccination protocols is important for understanding the physiology of T-cell recognition and to improve strategies of T-cell antigen-specific vaccination. During the last year the HLA laboratory has developed and perfected techniques for high-resolution typing of HLA class I and class II molecules using polymerase chain reaction (PCR) techniques and comparing the yield and accuracy of information to other techniques, which include directed heteroduplex analysis and automated sequencing of genomic DNA. Preliminary results suggest that a combination of these techniques will allow more efficient and more accurate typing in support
of peptide-based vaccination protocols presently being developed at different Institutes at
NIH. Furthermore, the HLA laboratory has been involved in the development of a comprehensive method for typing T-cell receptors (TCR) based on Vb-specific, PCR-based amplification in association with directed heteroduplex analysis, for purposes of screening for TCR clonality as well as direct sequencing using automated techniques for further characterization of TCR of specific functional or clinical interest.

In the future, these techinques will be utilized for the immunologic monitoring of patients undergoing peptide-based vaccination with the purpose of analyzing the in vivo expansion
of various relevant T-cell populations in the peripheral circulation as well as in the area
of specific pathologic interest. The ultimate goal will be to identify the steps occurring in response to T-cell antigen-specific vaccination and their correlation with clinical response. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02074-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Leukocyte, Platelet, and RBC Serology in Autoimmune

Lymphoproliferative Syndrome
Principal Investigator: C. Conry-Cantilena, M.D. (Medical Officer)

DTM, CC, NIH, Bethesda, Maryland 20892
Other Personnel: D. Stroncek, M.D., Chief, Laboratory Services Section, DTM, CC

J.L. Procter, MEd, MT (ASCP), SBB, Technical Specialist,
Transfusion Services, DTM, CC
S.E. Straus, M.D., NIAID
L. Barreda, Medical Technologist (ASCP), DTM, CC
Collaborating Unit: None

Staff-Years: 0.1

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: The process of apoptosis or programmed cell death of activated lymphocytes is critical for immune homeostasis. The cell surface expression of the protein Fas and its ligand are pivotal in regulating lymphocyte apoptosis. In mice defective Fas expression results in a severe over accumulation of mature lymphocytes and autoimmune disease. Mutations of the Fas gene have been described in humans. Clinically, this defect in Fas expression results in autoimmune lymphoproliferative syndrome (ALPS). Several kindreds with ALPS have been identified and followed for several years. Clinically, massive splenomegaly and lymphadenoopathy, hypergammaglobulinemia, autoimmuntiy, B cell lymphocytosis, and expansion of an unusual population of CD4 through CD8 T cells characterize ALPS. Commonly, the autoimmune phenomena include warm autoimmune hemolytic anemia, neutropenia, and thrombocytopenia. Characterization of features of autoimmunity in these patients and their family pedigrees is under way. They are being tested for antibodies
to red cells, platelets, neutrophils and HLA antigens. They are also being typed for HLA
class I and II antigens. The results of the antibody tests and HLA typings are being corre-
lated with clinical features. Studies of 11 patients have found that 9 had positive direct antiglobulin tests (DAT), 7 had platelet specific antibodies and 3 had neutrophil specific antibodies. Newly diagnosed patients will be tested to determine if the presence of anti-
bodies correlates with anemia, thrombocytopenia or neutropenia. Attempts will be made
to identify the antigenic targets of the autoantibodies. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02076-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Evaluation of Nucleic Acid Vaccine as Preventive and

Therapeutic Modality
Principal Investigator: J.W.-K. Shih, Ph.D.

IDS, DTM, CC, NIH, Bethesda, MD 20892
Others Personnel: R.Y. Wang, Ph.D., DTM, CC

D. Han, Ph.D., DTM, CC
G.J. Hu, Ph.D., DTM, CC
H.J. Alter, M.D., DTM, CC
I. Berkower, M.D., Ph.D., FDA
Collaborating Unit: FDA

Staff-Years: 1.2

Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: This program was initiated to elucidate a newly recognized modality of vaccination and to extend our long-term study of the immune response and clinical sequelae of hepatitis C virus (HCV) infection. One of the advantages of genetic immunization is that the endogenously expressed proteins can be recognized by class I MHC molecules and expressed on the cell surface. The MHC-antigen complex on the cell surface can be recognized by cytotoxic T lymphocytes (CTL), which in turn are activated and attack infected cells. The possibility of inducing an immune response to HCV core protein using DNA immunization provides an attractive alternative to classic vaccination. There are many problematic issues related to vaccine development for hepatitis C. One of the major concerns is the genetic stability of the infectious agent, HCV. There are two hypervariable regions in the putative HCV envelope proteins. Immune escape mutants observed were attributed to mutations in these regions. Experimentally infected chimpanzees and HCV patients were found to have repeated bouts of infection with either homologous or new strains of HCV. This could also be one of the reasons that more than 80% of the infections become chronic. Directly inducing strong cell-mediated immunity, especially protective cytotoxic T lymphocytes, may not only help in preventing initial HCV infection, but may also produce immune
modulation to overcome existing infection. During the past year, we have constructed
several different plasmids containing HCV genes and were able to evaluate the induction
of antibodies to HCV core proteins in mice. We are in the process of developing assays to measure CTL activity in the mouse model. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02077-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Gene-Therapy for Human Hepatitis C Infection: A Chronic

Infectious Disease Model
Principal Investigator: J.W.-K. Shih, Ph.D.

IDS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: R.Y. Wang, Ph.D., DTM, CC

D. Han, Ph.D., DTM, CC
G.J. Hu, Ph.D., DTM, CC
H.J. Alter, M.D., DTM, CC
Collaborating Unit: None

Staff-Years: 1.85

Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: About a year ago a new initiative started to develop a comprehensive research and development program to meet the challenging demand of future transfusion services. As part of our service responsibility, we will be required to provide state-of-the-art quality supports and consultation to the other sectors within the institute. To complement our expertise and experience in viral hepatitis research, and to be selective and focused, we chose to build this new R&D program on an infectious disease model. To investigate gene therapy as a therapeutic approach for human hepatitis C infection and to develop this approach as a model for chronic infectious diseases, we have set up the following goals: (a) to establish
a functionally measurable gene expression model in hematopoietic cells using a virally-mediated gene delivery system for the hepatitis C virus (HCV) gene; (b) to develop lineage-specific gene expression in hematopoietic cells for HCV genes; (c) to demonstrate the
efficacy of lineage-specific gene expression by functional assessment with CTL assays
and tumor-killing assays directed to HCV-specific epitopes; and, (d) to establish lineage-specific gene expression as a therapeutic modality against chronic HCV infection. In the
past year, in a mouse model, we have evaluated a viral gene delivery system and established CTL assays against HCV core and envelope proteins. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02078-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Viral and Immune Factors That Influence Recovery or

Progression of Hepatitis C
Principal Investigator: H.J. Alter, M.D. (Chief)

IDS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: P. Farci, M.D., Laboratory of Infectious Diseases, NIAID

R. Purcell, M.D., Chief, Laboratory of Infectious Diseases, NIAID
Collaborating Unit: NIAID

Staff-Years: 1.3

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: Approximately 15% of patients recover from hepatitis C virus (HCV) infection while 85% become persistently infected, resulting in various degrees of associated chronic liver disease. In this study comparisons will be made among patients who rapidly recover, those who have delayed recovery, those with persistent infection and stable chronic disease, and those with rapidly progressive, fatal infection. The parameters measured will

be viral burden (initially and over time), HCV genotype, the number of viral quasispecies (extent of viral heterogeneity) at the time of infection and subsequently, neutralizing anti-
body responses and, if appropriate technology is available, cytotoxic T cell responses.
The goal is to determine if any of these parameters can predict outcome and hence serve
as adjuncts to therapeutic decisions, particularly decisions regarding early intervention. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02079-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: HCV Infection in Infants and Children

Principal Investigator: N. Luban, M.D. (Chief)

Pediatrics, Children's Hospital National Medical Center
Washington, DC
Other Personnel: H.J. Alter, M.D., Chief, IDS, DTM, CC

J. Shih, Ph.D., Microbiologist, DTM, CC
Collaborating Unit: None

Staff-Years: 0.2 (NIH only)

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

x (a1) Minors

x (a2) Interviews

Summary of Work: It has become apparent from multiple studies that hepatitis C virus (HCV) infection is very indolent and that serious sequelae (cirrhosis, carcinoma) occur in less than 10% of persons during their first 20 years of infection. It is presumed that the proportion with severe outcomes will increase as the duration of followup increases. A corollary to these findings is that most persons who acquire this infection late in life will not be seriously affected by their HCV infection, whereas those who acquire the infection
in childhood and young adulthood may be at increased risk because they will have 3 to 8 decades for HCV infection to produce liver damage. This study is thus geared to shift attention to HCV-infected children. It is principally conducted by the Children's Hospital National Medical Center (CNMC) in Washington, DC. The DTM, NIH is a collaborating unit.
The study will identify infants and children who were transfused at CNMC from 1983 to 1992, the decade just prior to second-generation anti-HCV testing. This period was selected because rates of transfusion-associated hepatitis were still high at that time and because identified subjects would be younger than 15 at the time of enrollment and thus less likely

to have had sexual contact or IV drug use as confounding sources of infection. A total of 6,500 children who meet eligibility criteria have been transfused at CNMC. The entire cohort will be contacted and asked to provide a blood sample that will be tested for antibodies to HCV and hepatits G virus (HGV). Subjects found antibody-positive on initial screen will be enrolled in long-term laboratory and clinical followup. In those with biochemical evidence
of chronic hepatitis, a liver biopsy may be performed. The study will determine the minimal rate of transfusion-transmitted HCV and HGV infection and will allow for an annualized incidence estimate and a determination of the national burden of transfusion-induced viral hepatitis in children. Using archival samples, the study will also determine the duration of infection and the rate of viral persistence. Liver biopsy will establish the extent of disease
in those chronically infected. If persistent infection and chronic liver disease are as common in children as in adults, this study will have major implications for antiviral therapy programs and might serve to shift emphasis to pediatric populations where response rates may be higher and long-term benefit would be greater. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02081-02 DTM

October 1, 1995 to September 30, 1996

Title of Project: Clinical Aspects of Hepatitis G Virus Infection

Principal Investigator: H.J. Alter, M.D. (Chief)

IDS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: Y. Nakatsuji, M.D., Visiting Fellow, DTM, CC

J. Shih, Ph.D., Microbiologist, IDS, DTM, CC
J. Melpolder, M.T., IDS, DTM, CC
Collaborating Unit: Jungsuh Kim, Genelabs Technology, Inc., Redwood City, CA

Staff-Years: 1.5

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: The hepatitis G virus (HGV) is a newly discovered member of the Flaviviridae family that has approximately 25% homology with hepatitis C virus.
Its clinical significance is unknown. We have studied HGV and its relation to blood transfusion risk. HGV was found in approximately 1.5% of the donor population and was shown to be transfusion-transmitted by demonstrating the acute appearance of HGV RNA following transfusion, by demonstrating linkage between HGV-positive donors and infected recipients, and by showing a higher incidence in transfused subjects than in nontransfused controls. HGV was shown to persist in the majority of infected subjects, but near 85% appear to clear the virus over time. Although 3 of 13 patients (23%) with non-A, non-B, non-C transfusion-associated hepatitis had acute HGV infection, the causal role of HGV in these cases is not clear because there was no definite temporal relationship between HGV RNA level and alanine transaminase (ALT) elevations, and because one case had alternative explanations
for the ALT abnormalities. Overall, of 35 observed and 84 projected HGV infections, only 4% occurred in patients with hepatitis (the 3 cases described above), 7% occurred in those with coexisting hepatitis C where the ALT elevations were shown to be HCV related, 16% were in recipients who had such minor ALT elevations that they were not classified as having hepatitis, and 73% occurred in recipients with no biochemical or clinical evidence of hepatitis. Hence, the vast majority of HCV-infected patients have no evidence of liver disease,
and in the few that do, causality cannot be proved.
Similarly, we have studied HGV in renal dialysis patients, hemophiliacs, intravenous drug users, and patients with acute and chronic non-A, B, C, D, E hepatitis. Although the prevalence of HGV is very high among parenterally exposed individuals (10% to 20%),
there is no association with liver disease in these HGV-infected populations. Among
HGV and HCV coinfected patients, it was shown that HGV did not worsen the course
of coexistent hepatitis C. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02082-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Studies of Viral Hepatitis and AIDS in the Chimpanzee Model

Principal Investigator: H.J. Alter, M.D. (Chief)

IDS, DTM, CC, NIH, Bethesda, MD 20892
Collaborating Units: Southwest Foundation for Biomedical Research (Kristina Murthy);

CDC (Kristof Krawczynski, M.D.)
Staff-Years: 0.1 (NIH only)

Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: This laboratory was the first to transmit non-A, non-B hepatitis (subsequently proved to be hepatitis C), and HIV to the chimpanzee and hence to establish
an animal model for these infections. Current studies in this model include the following:
(1) The Early Events of HIV Infection: In this study a chimpanzee was infected with HIV
and serial apheresis units were obtained during the window period between exposure and
the first detection of anti-HIV antibody. No markers of HIV infection were detectable until the fifth week post-exposure, when virus was detectable by measurement of HIV RNA, HIV DNA, and viral culture. In contrast, anti-HIV and p24 antigen, used in blood donor screening, were not detectable until 8 weeks post-exposure. In a second phase of the experiment, the 3, 4, and 5 week samples were sequentially inoculated into a second chimp. The 3- and 4-week samples were shown to be noninfectious, while the 5-week sample was infectious. Hence, infectivity did not precede the detection of HIV RNA and DNA, suggesting that if such assays were introduced into blood screening they might totally abrogate the infectious
window and prevent blood transmission of HIV. (2) Hepatitis G Virus (HGV)-Hepatitis C Virus (HCV) Interactions: In collaboration with CDC, this study will infect HCV carrier chimps with HGV and simultaneously infect two chimps with HCV and HGV to determine if HGV facilitates the clearance of HCV as has been suggested in human studies. (3) Fulminant Hepatitis: Using samples from rare cases of fulminant hepatitis C, this study will seek to identify a virulent strain of HCV by chimp inoculation and serial passage. (4) Viral Inactivation: This study will use the chimp model to establish the safety of psoralen/UV light-inactivated platelets. This is the first viral inactivation procedure that maintains the integrity of the cellular components of blood. In vitro studies indicate that the method inactivates a broad array of viruses including HIV, hepatoviruses, and flaviviruses, the latter serving as models for HBV and HCV. If inactivation is confirmed in vivo, this method should have broad application for platelet transfusions and possibly for red cells. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02083-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Quantitation and Characterization of Lymphohematopoietic Cells

Principal Investigator: E.J. Read, M.D. (Chief)

CPS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: H.G. Klein, M.D., Chief, DTM

T. Trischmann, Ph.D., CPS, DTM, CC
C. Carter, CPS, DTM, CC
J. Lee-Fischer, CPS, DTM, CC
Q. Chau, CPS, DTM, CC
Collaborating Unit: Biometric Imaging, Inc., Mountain View, CA

Staff-Years: 0.5

Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: A study comparing microvolume fluorimetry (MVF) to flow cytometry (FC) for quantitation of CD34+ hematopoietic cells was published in August 1997 (Read et al., J Hematotherapy 1997;6:291-301). This study demonstrated the potential utility of the MVF method as a simpler, more rapid alternative to standard FC methods for product quality control and for clinical decisionmaking about the timing or duration of stem cell apheresis. An extension of this study was carried out this year to validate the use of a new capillary configuration (plastic, with larger volume) and modified analytic software for the MVF CD34+ cell assay on samples of peripheral blood, leukapheresis collections, and bone marrow. Beginning in October 1997, we will evaluate a new MVF assay for rapid quantitation of CD3+ lymphocytes in hematopoietic progenitor cell collections. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02084-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Development of Methods for Ex Vivo Cultured and

Immunologically and/or Genetically Modified Cells
Principal Investigator: E.J. Read, M.D. (Chief)

CPS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: H.G. Klein, M.D., Chief, DTM, CC

S. Donnelly, M.D., CPS, DTM, CC
T. Trischmann, Ph.D., CPS, DTM, CC
C. Carter, CPS, DTM, CC
V. Fellowes, CPS, DTM, CC
K. Hines, CPS, DTM, CC
Collaborating Units: Baxter Healthcare, Inc., Irvine, CA; Hematology Branch

and BMT Program, NHLBI; HIV Clinical Trials Program,
NIAID; Clinical Gene Therapy Branch, NCHGR
Staff-Years: 1.5

Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: Preclinical development of complex processing systems for ex vivo culture-expanded lymphohematopoietic cells, with subsequent immunologic and/or genetic manipulation, has been carried out in collaboration with a number of NIH investigators,

and with Baxter Healthcare, Inc.
A. Preparation of CD8-depleted, culture-expanded lymphocytes: In collaboration with Drs. Robert Walker, Michael Blaese, and Richard Morgan, this process involves CD8 cell depletion of peripheral blood lymphocytes using a Baxter antibody and Max Sep, an immuno-magnetic system for large-scale negative selection, prior to ex vivo culture-expansion and gene transduction. To date, there have been 19 large-scale runs (5 preclinical and 14 clinical) for this 10-day process, which is currently being applied to normal syngeneic donor lymphocytes in an HIV clinical gene therapy trial. This trial is ongoing and beginning to yield important data on the relative efficacy of a therapeutic gene vs a marker gene. Process changes this year were based on studies of paramagnetic bead concentrations for the CD8-negative selection.

B. Preparation of allogeneic donor lymphocytes selectively depleted for alloreactive T cells: This process is being developed, in collaboration with Dr. John Barrett and colleagues, for application to clinical allogeneic hematopoietic transplantation, especially in the HLA-mismatched setting. It involves (1) preparation of recipient (stimulator) lymphocytes, (2) preparation of donor (responder) lymphocytes, and (3) coculture and preparation of stimulator and responder lymphocytes, followed by selective depletion of alloreactive responder T lymphocytes with an anti-CD25-Pseudomonas exotoxin construct. This year we have concentrated on developing culture-expansion methods that will eliminate leukemia cells from the normal recipient (stimulator) cells, including methods for culture-expansion of selected lymphocyte populations. Further studies will continue into next year, as we prepare for a clinical trial. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02085-02 DTM

October 1, 1996 to September 30, 1997

Title of Project: Methods for Positive and Negative Selection of

Hematopoietic Progenitor Cells
Principal Investigator: E.J. Read, M.D. (Chief)

CPS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: H.G. Klein, M.D., Chief, DTM, CC

T. Trischmann, Ph.D., CPS, DTM, CC
C. Carter, CPS, DTM, CC
R. Butz, CPS, DTM, CC
F. Vigue, CPS, DTM, CC
Collaborating Units: Baxter Healthcare, Inc., Irvine, CA; CellPro, Inc., Bothell, WA;

Hematology Branch, BMT Unit, NHLBI; Laboratory of Host
Defenses, NIAID
Staff-Years: 1.2

Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: Preclinical development of automated closed system methods for positive and negative selection of lymphohematopoietic cells have been done in collaboration with biotechnology firms which have developed systems for potential application to clinical cellular therapies.

A. Baxter Isolex: Over 50 preclinical evaluations were carried out with the Isolex 300SA system for immunomagnetic positive selection of CD34+ hematopoietic progenitor cells. These evaluations accomplished the requisite development and validation of this methodology prior to incorporation into a clinical trial of gene therapy for chronic granulomatous disease (PI, Harry Malech). Current studies are focusing on development and validation of the new Isolexi, the improved, more automated version of this system. In ongoing studies of this device, we are processing granulocyte colony-stimulating factor (G-CSF) mobilized normal donor leuka pheresis collections and evaluating the yield of CD34+ cells, the depletion of CD3+ T cells, and the overall utility of the system. Results of this evaluation, and comparison with evaluations of other systems (see below), will determine its applicability for specific clinical trials.

B. CellPro T Cell Depletion system: A preclinical evaluation of this 2-step positive (CD34) and negative (CD2) selection system, which uses an immunoadsorption approach, was recently completed. For eight GCSF-mobilized leukapheresis products from normal donors, mean depletion of CD3+ T cells was 3.75 logs, and mean CD34+ cell yield was 44%. This system is ready for incorporation into clinical trials of allogeneic T-depleted hematopoietic transplantation after required regulatory applications to FDA. These
results will also be evaluated in relationship to those from the ongoing Baxter Isolex
system evaluation (see above). (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02086-01 DTM

October 1, 1996 to September 30, 1997

Title of Project: Effectiveness of Granulocyte Transfusions

Principal Investigator: S.F. Leitman, M.D. (Chief, Blood Services Section)

DTM, CC, NIH, Bethesda, Maryland 20892
Other Personnel: E. Kicklighter, M.D., Transfusion Medicine Senior Fellow, DTM

J. Obitas, DTM, CC
M.A. Proscham, Ph.D., EC, NHLBI
D.F. Stroncek, M.D., Chief, Laboratory Services Section, DTM, CC
N.S. Young, M.D., IR, NHLBI
T.J. Walsh, M.D., DCT, NCI
Collaborating Unit: None

Staff-Years: 0.2

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: The results of clinical trials to treat neutropenic adults with bacterial or fungal infections are controversial. Some studies have found that granulocyte transfusions are effective in treating bacterial infections but several other have not. While granulocyte transfusions have been effective in treating fungal infections in neutropenic animal models, they have not been effective in treating humans. The ineffectiveness of granulocyte transfusions is likely due in part to the limited quantity of granulocytes that can be collected. Recently, it has been shown that the number of granulocytes collected can be increased two to five-fold by giving donors granulocyte colon-stimulating factor (G-CSF). G-CSF mobilized granulocyte transfusions may be effective in treating bacterial and fungal infections in neutropenic adults. The purpose of this study is to determine if severely neutropenic aplastic anemia patients with bacterial or fungal infections benefit from granulocyte transfusions. Eligible patients will be randomly assigned to either receive conventional antimicrobial therapy or G-CSF mobilized granulocyte transfusions plus conventional antimicrobial therapy. The response of their infection to the treatments as well as their survival will be monitored. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02089-01 DTM

October 1, 1996 to September 30, 1997

Title of Project: Comparative Studies of Granulocyte-Colony Stimulating Factor

(G-CSF) and Dexamethasone, Alone and in Combination, to
Optimize the Yields of Granulocytapheresis Procedures in
Healthy Volunteer Donors
Principal Investigator: S.F. Leitman, M.D. (Chief)

BSS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: J.M. Oblitas, M.T., Plateletpheresis Center, DTM, CC

Collaborating Unit: None

Staff-Years: 0.2

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: The efficacy of therapeutic granulocyte transfusions is limited bythe relatively small number of cells obtained using standard starch and steroid stimulation
of the donor. To determine an optimal mobilization schedule that would maximize cell yields and minimize donor discomfort during granulocytapheresis, we designed a pair-controlled study of three mobilization schedules. Twenty healthy donors underwent three leukapheresis procedures each, receiving either dexamethasone (8 mg orally 12 hrs prior to donation), G-CSF (5 µg/kg SQ 16-24 hrs prior to apheresis), or dexamethasone plus G-CSF (D+G in the same doses). Seven liters of whole blood were processed on the CS-3000 Plus device using hetastarch as the sedimenting agent. All products were transfused to patients with qualitative or quantitative granulocyte deficiencies and life-threatening infections.

Administration of G-CSF alone led to a 3.5-fold increase and the combination of dexamethasone plus C-CSF led to a 4.8-fold increase in donor peripheral blood granulocyte counts compared with dexamethasone alone (from 6.0 + 2.3 with dexamethasone, to 21.3 + 5.2 and 28.9 + 6.8 x109 cells/L with G-CSF and D+G, respectively). Similarly, use of G-CSF alone or the combination of dexamethasone and G-CSF resulted in 2.4- and 3.4-fold increases in granulocyte content in the product compared with dexamethasone alone (from 2.16 + 0.71 with dexamethasone alone to 5.08 + 1.10 and 7.25 + 1.54 x1010 cells total with G-CSF
and D+G, respectively), p < 0.01 for all comparisons between dexamethasone and either
G-CSF or D+G. In addition, both peripheral blood and product granulocyte counts were greater (p < 0.05) when donors took combination D+G vs G-CSF alone (36% increase in
the blood granulocyte count and 43% increase in product granulocyte content when dexa
was added to G-CSF). With dexa alone, 25% of donors had insomnia or flushing; with
G-CSF, 65% had bone pain, headache, insomnia, fatigue, or diaphoresis; this increased to 85% with combination D+G. Two donors requested discontinuation of G-CSF-mobilized donations due to the discomfort and inconvenience of the mobilization regimen.

Addition of dexamethasone to G-CSF significantly increases granulocyte yields at the expense of a moderate increase in donor inconvenience and discomfort. The availability of granulocytapheresis products with markedly increased numbers of highly functional cells is likely to result in a resurgence of interest in this transfusion component. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02090-01 DTM

October 1, 1996 to September 30, 1997

Title of Project: Effect of Storage Parameters on Efficacy of Filtration

of Red Blood Cell Units
Principal Investigator: S.F. Leitman, M.D. (Chief, Blood Services Section)

DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: J.D. Smith, M.T. (SBB) ASCP, DTM, CC

Collaborating Unit: None

Staff-Years: 0.2

Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: Several studies have suggested that the efficacy of leukocyte (white blood cell, WBC) removal by filtration is affected by storage time, temperature, and rate of filtration. However, these studies did not clearly differentiate among storage, interdonor, and interfilter variables. This study was designed to determine the influence of time, tem-
perature, and expression of leukocyte adhesion molecules on the efficacy of leukoreduction
of red cell units in a controlled format.

Ten donors underwent whole blood phlebotomy on each of three occasions. Units underwent filtration using a standard, commercially available RCXL-1filter (Pall Corp.)
as follows: (1) prestorage filtration at 22°C using laboratory conditions and gravity flow rates; (2) filtration after 14 days of storage at 4°C, using laboratory conditions and gravity flow rates; and (3) filtration after 14 days of storage at 4°C, using mock bedside conditions. Mock bedside conditions consisted of flow through an IMED electromechanical pump at 22°C at a rate that would infuse the product over 2 hours. Pre- and postfiltration red and white cell counts and leukocyte CD11a expression were assessed on Days 0 and 14. Post filtration white cell counts were determined by propidium iodide staining and Neubauer chamber counting.
WBC content pre- and postfiltration was lower in the two poststorage groups (residual WBCs 1.02 x 104 for in-laboratory vs. 2.31 x 104 for bedside filtration) than in the prestorage group (52.8 x 104 residual WBCs), and log10 reduction in WBCs was significantly greater in the two poststorage vs. prestorage groups (4.59 vs. 3.83 log10 reduction). Mean postfiltration RBC recovery was greater than 80% with all three filtration techniques. Leukocyte expression of LFA-1 as measured by CD11a was similar in all three groups.

Our study demonstrates that the efficacy of leukoreduction is significantly greater in red cell units undergoing post-storage rather than prestorage RCXL-1 filtration. In poststorage filtered units, neither the temperature nor rate at which filtration was performed affected the degree of leukodepletion, although a greater range of residual WBCs was seen with bedside filtration. These findings suggest that optimal leukodepletion of red cell units is accomplished in the laboratory rather than at the bedside, and that leukodepletion is least effective if filtration is performed immediately after the units are collected. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02091-01 DTM

October 1, 1996 to September 30, 1997

Title of Project: Acquisition of Hematopoietic Stem Cells for Second Transplants

by Apheresis of Filgrastim-Stimulated Donors Participating in
the National Marrow Donor Program (NMDP)
Principal Investigator: S.F. Leitman, M.D. (Chief, Blood Services Section)

DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: D. Stroncek, M.D., Chief, Laboratory Services Section, DTM, CC

Collaborating Unit: NMDP, 3433 Broadway Street, Suite 500, Minneapolis, MN 55413

(Dennis Confer, M.D., Medical Director)
Staff-Years: 0.2

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: The NMDP was established in 1987 to (1) create a registry of volunteer, tissue-typed, unrelated bone marrow donors and (2) facilitate matched unrelated donor marrow transplants through a coordinated circuit of Donor Centers, Collection Centers, and Transplant Centers. As of July 1997, 2.7 million donors were participating in the registry and 5,700 unrelated marrow transplants had been performed. The rate of nonengraftment and graft rejection in unrelated-donor transplants varies from 6% to 40%, depending on the degree of HLA matching between the donor and recipient and on whether graft engineering to remove T cells was performed. In recipients with nonengraftment or early graft loss, the best option for therapy is often another dose of stem cells from the original marrow donor. For the first 9 years of the program, consent was sought from the original donor for a second marrow harvest in this setting. More recently, it has been appreciated that peripheral blood-derived stem cell (PBSC) transplants, harvested by apheresis of filgrastim (granulocyte colony-stimulating factor) stimulated donors, can provide larger numbers of hematopoietic progenitor cells that engraft more rapidly than marrow-derived cells. This is particularly true in NMDP donors undergoing a second donation: their marrow is still somewhat depleted of progenitor cells, the sites of marrow harvest are still quite painful, and the recipient is often critically ill and needs hematopoietic reconstitution as rapidly as possible. For these reasons, a protocol involving all participating NMDP Donor Centers was initiated in February 1997 for the acquisition of PBSC's for second transplants. The objectives of the study are (1) to monitor the immediate and long-term safety/sequelae of filgrastim administration in healthy volunteer donors; (2) to compare donor tolerance of, psychosocial response to, and adverse effects of first donations of marrow versus second donations of PBSCs; (3) to determine the efficacy and stem cell content of filgrastim-mobilized PBSC collections; and (4) to monitor the outcome of matched unrelated-donor PBSC transplants, in terms of time to engraftment, incidence of acute and chronic GVHD, and disease-free and overall survival. Donors will be given filgrastim 10 ucg/kg/day SQ for 5 days, with apheresis performed on days 5 and 6. Followup clinical and laboratory analysis of the donor will continue annually for a total of 5 years. Thus far, only 4 NMDP donors have donated PBSC's on this protocol, with insufficient data for analysis at this time.

The NIH Marrow Donor Center, a participant in the NMDP, has 44,000 donors on its registry.168 NIH unrelated donors to date have undergone marrow harvest for an NMDP recipient. It is estimated that 2 to 3 NIH donors per year will be enrolled in this second donation study. Two of the principal investigators for this study are in the DTM, CC (S.F.L. and D.S.). Administrative and statistical support for the study is provided by the NMDP National Office. Filgrastim is provided under an IND agreement with Amgen (BB-IND # 6821). The IND is held by the NMDP National Office. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02092-01 DTM

October 1, 1996 to September 30, 1997

Title of Project: Clinical Efficacy of Daily G-CSF-Recruited Granulocyte

Transfusions in Patients With Severe Neutropenia and
Life-Threatening Infections
Principal Investigator: S.F. Leitman, M.D. (Chief, Blood Services Section)

DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: J.M. Oblitas, M.T., Plateletpheresis Center, DTM, CC

Collaborating Unit: None

Staff-Years: 0.2

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: The efficacy of therapeutic granulocyte transfusions is limited by the relatively small number of cells obtained using standard starch and steroid stimulation
of the donor. We evaluated the safety and efficacy of daily granulocyte colony stimulating
factor (G-CSF)-stimulated granulocyte transfusions in two patients, a 55 kg female with
T cell large granular lymphocytic leukemia and a sigmoid phlegmon, and a 131 kg male
with systemic aspergillus flavus infection on day 10 following T cell depleted marrow allo-
grafting for myeloma. Both patients had ANCs < 0.1x109/L, and were receiving G-CSF
10 µg/kg/d and systemic antibiotics and antifungal agents. Healthy volunteer allogeneic
apheresis donors were given a single dose of G-CSF 5 µg/kg SQ 12 to 24 hours prior to
apheresis as well as a single dose of dexamethasone 8 mg orally 12 hours prior to donation. Seven liters of whole blood were processed on the Fenwal CS-3000 device using Hetastarch
as the sedimenting agent. A different non-HLA matched donor was used each day. Four irradiated granulocyte transfusions were given to the first patient and 12 to the second patient prior to recovery of the patients' own neutrophils. Mean donor white blood cell count prior to apheresis (m + sd) was 29.9 + 3.8 x109/L (94% granulocytes), range 25.1-37.6 x109/L. The granulocytapheresis products contained 7.4 + 1.3 x 1010 granulocytes in a mean volume of
379 mL. Platelet content of the granulocyte products was 5.0 x1011, equivalent to a 9-unit platelet transfusion. The mean number of granulocytes transfused per kg was 1.2 x109/kg in
the first and 0.6 x109 /kg in the second patient. Dramatic one-hour posttransfusion increments
in circulating neutrophil counts were routinely seen in both patients (immediate ANC increments
of 1.99 and 1.87 x109/L, respectively). ANCs continued to rise for up to 8 hours after each transfusion and did not decline to pretransfusion levels until 24-30 hours after infusion. Both patients became afebrile and exhibited stabilization and/or improvement in clinical, hemodynamic, and radiographic indicators of infection. The granulocyte transfusions were well tolerated, and coadministration (within 12 hours) of amphotericin did not lead to pulmonary compromise. Neither patient became HLA-alloimmunized. Donors experienced mild bone pain, headache, and insomnia but were willing and eager to support such donations again. In summary, G-CSF-recruited granulocyte components contained four-fold greater numbers of cells than historical non-G-CSF mobilized products, were well tolerated, and appeared to have a markedly prolonged circulatory half-life, perhaps due to G-CSF-related inhibition of apoptosis or recruitment of younger granulocytes. The beneficial effects experienced in our two patients suggest that this new transfusion component deserves broader study. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02093-01 DTM

October 1, 1996 to September 30, 1997

Title of Project: Effects of G-CSF on Granulocyte Donors

Principal Investigator: D. Stroncek, M.D. (Chief, Laboratory Services Section)

DTM, CC, NIH, Bethesda, Maryland 20892
Other Personnel: S. Leitman, M.D., Chief, Blood Services Section, DTM, CC

E. Read, M.D., Chief, Cell Processing Center, DTM, CC
L. Harvath, Ph.D., DH, CBER
D. Follmann, Ph.D., EC, NHLBI
Collaborating Unit: CEBR, FDA

Staff-Years: 0.2

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: For several years people that donate granulocytes for transfusion have been given dexamethasone to increase the number of cells that can be collected. Recently, it has been recognized that treating granulocyte donors with single dose of granulocyte-colony-stimulating factor (C-CSF) alone or in combination with dexamethasone increases the number of cells collected two to five-fold. While G-CSF is becoming the standard agent for mobilizing granulocytes, its effects on donors are not clear. It is known that 4 to 5 daily doses of G-CSF induces several symptoms including bone pain, headache and fatigue; changes in blood chemistries including LDH, alkaline phosphatase, and potassium; and changes in blood counts including thrombocytopenia. In order to provide HLA compatible granulocytes for some patients, donors are often asked to donate granulocytes repeatedly. The purpose of this study is to determine how a single dose of G-CSF, dexamethasone or G-CSF plus dexamethasone and the collection of one granulocyte concentrate effects how donors feel, their blood chemistries and blood counts. In addition, the data obtained will be used to determine the minimum interval between each donation and the cells collected will be used to investigate the storage of G-CSF mobilized granulocytes concentrates. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02094-01 DTM

October 1, 1996 to September 30, 1997

Title of Project: Drug-Dependent Immune Mediated Cytopenias

Principal Investigator: D. Stroncek, M.D. (Chief, Laboratory Services Section)

DTM, CC, NIH, Bethesda, Maryland 20892
Other Personnel: J.L. Procter MEd, MT(ASCP), SBB, Technical Specialist,

Transfusion Services, DTM, CC
K. Matsuo, M.D., Fogarty Fellow, DTM, CC
S. Leitman, M.D., Chief, Blood Services Section, DTM, CC
Collaborating Unit: None

Staff-Years: 0.2

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: Drug-dependent antibodies are a rare by serious cause of hemolytic anemia, but little is know of the nature of the antigens recognized by the drug-dependent antibodies. Precise identification of the antigens recognized by drug-dependent antibodies will lead to a better understanding to mechanisms of antibody formation, better methods to detect drug-dependent antibodies and improved treatment of drug-dependent hemolysis. The purpose of these studies is to use serologic and biochemical techniques to determine the nature of the antigens recognized by drug-dependent antibodies. The antibodies will be tested against red cells treated by proteolytic or glycolytic enzymes to determine if the antigens are located on carbohydrates or proteins. Immunoprecipitation or monoclonal antibody capture assays as be used to more precisely define the molecules identified by the drug-dependent antibodies. To confirm the identity of the epitope, the antibody will be tested against red cells that lack the suspected target molecule. Tolmetin-, cefotetan- and quinine-dependent antibodies will be tested. Initial studies show that reactions of the quinine-dependent antibodies are inhibited by proteolytic enzymes, but those of tolmetin- and cefotetan-dependent antibodies are enhanced. (Back to the project list.)

INTRAMURAL RESEARCH PROJECT Z01 CL-02095-01 DTM

October 1, 1996 to September 30, 1997

Title of Project: Structure and Function of Granulocyte Antigens

Principal Investigator: D. Stroncek, M.D. (Chief, Laboratory Services Section)

DTM, CC, NIH, Bethesda, Maryland 20892
Other Personnel: K. Matsuo, M.D., Fogarty Fellow, DTM, CC

J.L. Procter, MEd, MT(ASCP), SBB, Technical Specialist,
Transfusion Services, DTM, CC
Collaborating Unit: Neutrophil Serology Reference Laboratory, American Red Cross,

North Central Blood Services, St. Paul, Minnesota
Staff-Years: 0.8

Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither

(a1) Minors

(a2) Interviews

Summary of Work: Two very similar genes encode for the Fc-gð-receptor III (FcgðRIII, CD16). FcgðRIIIA is expressed on NK cells and macrophages and FcgðRIIIB on neutrophils. FcgðRIIIB has two clinically important polymorphisms, NA1 and NA2. In autoimmune neutropenia of childhood neutrophil antibodies are most commonly directed to NA1 or NA2. Neutrophils from people that are NA1 homozygous are more effective at the phagocytosis of opsonized red cells than neutrophils from NA2 homozygous people. Assays using sequence specific primers and the polymerase chain reaction (PCR) have been used to differentiate

the two alleles. We developed a new method to genotype NA1 and NA2 with PCR and restriction enzymes. Portions of both FcgðRIIIA and FcgðRIIIB were amplified by PCR,
but only the FcgðRIIIB NA1 PCR product was digested by restriction enzyme XmnI and
the FcgðRIIIB NA2 PCR product by Cac8I. To validate the assay the NA phenotypes of neutrophils from 45 white people were determined by granulocyte agglutination and were compared with their NA genotype determined by PCR and restriction enzymes. The NA phenotypes and genotypes were the same in all 45 people. The method will used to compare the NA1 and NA2 genotypes of Japanese, African American and white people. If unusual genotypes are identified, than donor's neutrophils will be phenotyped and the molecular
basis of the polymorphism will be determined. Initial studies show that the NA1 and NA2 gene frequencies in Japanese are 0.70 and 0.30 compared to 0.30 and 0.70 in African
Americans and whites. (Back to the project list.)

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Questions about the Clinical Center? OCCC@nih.gov Or call: (301) 496-2563 National Institutes of Health, Warren Grant Magnuson Clinical Center, Bethesda, Maryland 20892. Last Modified 3/98