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Transfusion Medicine Department
Project numbers
A Controlled Prospective Study of Transfusion- associated Hepatitis
Significance of Anti-HIV Antibody in Asymptomatic Donors
Etiology of Allergic Reactions in Platelet- and Granulocytapheresis Donors
Kinetic Studies of Indium-labeled Leukocytes
Treatment of Familial Hypercholesterolemia by Dextran Sulfate Apheresis
Quantitative Analysis of Viral Genomes and Their Clinical Correlations
A Prospective Study of Anti-HCV Positive Blood Donors
Functional Relevance of HLA Polymorphism in Vaccination Protocols
Leukocyte, Platelet, and Red Cell Serology in Autoimmune Lymphoproliferative Syndrome (ALPS)
Evaluation of Nucleic Acid Vaccine as Preventive and Therapeutic Modality
Gene-therapy for Human Hepatitis C Infection: A Chronic Infectious Disease Model
Viral and Immune Factors That Influence Recovery or Progression of Hepatitis C
HCV Infection in Infants and Children
The Natural History of Hepatitis C Virus Infection
Clinical Aspects of Hepatitis G Virus Infection
Studies of Viral Hepatitis and AIDS in the Chimpanzee Model
Quantitation and Characterization of Lymphohematopoietic Cells
Development of Methods for Ex Vivo Cultured and Immunologically and/or Genetically Modified Cells
Methods for Positive and Negative Selection of Hematopoietic Progenitor Cells
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
Acquisition of Hematopoietic Stem Cells for Second Transplants by Apheresis of Filgrastim-stimulated Donors Participating in the National Marrow Donor Program (NMDP)
Clinical Efficacy of Daily G-CSF-recruited Granulocyte Transfusions in Patients with Severe Neutropenia and Life-threatening Infections
Effects of G-CSF on Granulocyte Donors
Drug-dependent Immune-mediated Cytopenias
Structure and Function of Granulocyte Antigens
Storage of Granulocyte Concentrates
INTRAMURAL RESEARCH PROJECT Z01 CL-02005-29 DTM
October 1, 1997 to September 30, 1998
Title of Project: A Controlled Prospective Study of Transfusion-associated Hepatitis
Principal Investigator: H.J. Alter, M.D. (Chief)
IDS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: J. Melpolder, IDS, DTM, CC
J.W. Shih, Ph.D., Microbiologist, DTM, CC
Collaborating Units: NIAID (R. Purcell, M.D.)
Div. of Hematology, Georgetown Univ. Hospital
(Washington, DC; R. Sacher, M.D.)
Fairfax Hospital (Fairfax, VA; V. Rustgi)
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 series studies of transfusion-associated hepatitis (TAH) in prospectively followed transfusion recipients undergoing open-heart surgery. The studies have sequentially shown the efficacy of adopting an all volunteer donor system; testing for the hepatitis B surface antigen; utilizing the surrogate assays, alanine aminotransferase (ALT) and anti-hepatitis B core antibody (anti-HBc); and testing for antibodies to the hepatitis C virus (HCV).
Overall, the studies have shown a decline in TAH incidence from near 30 percent in the 1960s, to 10 to 20 percent in the 1970s, to 8 to 12 percent in the early 80s, and to 4 to 5 percent in the late 80s. Since 1990, the study has focused on the impact of the introduction of donor screening assays to detect carriers of HCV. Following first generation anti-HCV testing, introduced in 1990, the rate of TAH fell from 3 to 4 percent to 1.5 percent. Since the introduction of more sensitive second generation assays in 1992, we have followed over 650 recipients and the overall TAH rate has fallen to 0.2 percent while the rate of transfusion-associated hepatitis C has fallen to zero. The overall rate reflects only a single mild case whose etiology is undetermined. Hence, in this approximate 30-year span, we have documented the virtual disappearance of TAH. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02040-14 DTM
October 1, 1997 to September 30, 1998
Title of Project: Significance of Anti-HIV Antibody in Asymptomatic Donors
Principal Investigator: H.J. Alter, M.D. (Chief)
IDS, DTM, CC, NIH, Bethesda, MD 20892
Other Investigators: H.G. Klein, M.D., DTM, CC
S.F. Leitman, M.D., BSS, DTM, CC
J.W. Shih, Ph.D., IDS, DTM, CC
C. Cantilena, M.D., DTM, CC
J. Melpolder, IDS, DTM, CC
D. Tan, M.D., IDS, DTM, CC
Collaborating Unit: Washington Area Red Cross (Chesapeake Region; P. Ness)
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-human immunodeficiency virus (HIV) positive donors and controls has been under prospective follow-up since 1985 (N Engl J Med 1989; 321:917). The cohort is now in its 13th year of follow-up. At enrollment, 182 subjects were Western blot (WB) positive, including 158 asymptomatic donors, 15 blood recipients, and nine sexual partners. A control population included 70 anti-HIV reactive donors who were WB negative and 21 who were WB indeterminate. Of the 182 WB+ subjects, 87 percent were donors, 5 percent sexual partners, and 8 percent recipients. Of the 182 WB positives, 53 (29 percent) are alive and in active follow-up; 58 (32 percent) are dead, of whom 54 (93 percent) died of AIDS; 73 (40 percent) are lost to follow-up (LTFU). We suspect most LTFU have succumbed to AIDS, but need access to the National Death Index to establish this: 13 of the 73 LTFU were known to have AIDS at the time they left the study.
Of the 53 in active follow-up, 40 (75 percent) are males and 49 (92 percent) were detected at blood donation. 17 of 53 (32 percent) have had an AIDS defining event. Others have CD4 counts under 300, but have had a stable course even before treatment. A subset of 13 patients have exceeded 9 years of follow-up and have CD4 counts persistently greater than 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 nonprogression. AIDS or HIV-related phenomena have not developed in any of the 21 WB indeterminate or 70 WB negative subjects. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02045-12 DTM
October 1, 1997 to September 30, 1998
Title of Project: Etiology of Allergic Reactions in Platelet- and Granulocytapheresis Donors
Principal Investigator: S.F. Leitman, M.D. (Chief)
BSS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Unit: Fenwal Laboratories
Division of Baxter Healthcare, Inc.
Box 490, Round Lake, IL 60073
(W.J. Lee, Research Associate)
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. 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. Using a combination of skin tests, radioallergosorbent tests (RAST), and basophil histamine release assays, we found specific IgE-mediated sensitization to ethylene oxide (EO), a gas used to sterilize the plastic disposable apheresis kits, in 10 of 16 plateletpheresis donors and 8 of 10 granulocytapheresis donors experiencing reactions, but in none of 140 nonreacting controls. Donors with documented EO sensitization were permanently deferred from subsequent apheresis donations.
The results of these studies were reported to the manufacturer of the CS-3000 apheresis device and to FDA. Similar reactions were reported by another group in plasmapheresis donors. As a result of these reports, the manufacturer of the CS-3000 disposable apheresis kits changed their sterilization techniques from predominantly EO exposure to predominantly gamma irradiation. Since this change, there have been only two documented cases of EO hypersensitivity reactions in DTM donors, in August 1995 and March 1998. However, in August 1997, a donor in another blood center had an acute fatal anaphylactic reaction during plateletpheresis, and was found on postmortem testing to have high titer IgE anti-EO. The donor also had a strong history of asthma. This was the first report of a lethal allergic reaction to EO in an apheresis donor, and has reopened the question of prospective screening of all apheresis donors for EO sensitization. We have estimated that as many as 1.0 percent of all repeat apheresis donors may become sensitized to EO, although only a fraction of those who are sensitized will have clinically evident allergic reactions. To document the current EO sensitization rate among donors and to compare this rate with individuals who have occupational exposure to EO, we have established a collaborative effort with CBER/FDA. Screening of approximately 500 healthy repeat apheresis donors using both an established RAST test and an experimental EIA test for IgE anti-EO will be performed. A cohort of serum samples stored at the CDC and derived from individuals with allergic reactions presumed to be due to EO will also be tested, as will a large sample of samples derived from the NHANES study. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02055-10 DTM
October 1, 1997 to September 30, 1998
Title of Project: Kinetic Studies of Indium-labeled Leukocytes
Principal Investigator: S.F. Leitman, M.D.
BSS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: D. Stroncek, M.D., NMD, CC
J. Carasquillo, M.D., NMD, CC
C. Chen, M.D., NMD, CC
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 non-invasive studies. In these cases autologous or allogeneic granulocytes, collected by simple phlebotomy or by apheresis, are labeled with 50 uCi of 111-Indium per kg of patient weight, not to exceed 500 uCi total. Twenty-two studies for the purpose of abscess localization were performed in Clinical Center patients enrolled in a variety of protocols in the past 7 years. Three of these 22 were positive, and led to appropriate therapy for a case of salmonella osteomyelitis in a hemophiliac patient with human immunodeficiency virus infection, staphylococcal pneumonia in a patient with aplastic anemia, and diverticular abscess in a patient with Zollinger-Ellison syndrome. In many of the negative studies, unnecessary surgical exploration was avoided. Three of the 22 indium-labeled allogeneic leukocyte studies were performed in patients with chronic granulomatous disease, in one case to document clearance of pulmonary infection, in the second to confirm lack of trafficking in an alloimmunized recipient, and in the third to localize a cause for fever. In all three cases, gamma imaging results were used to guide therapy, specifically, to terminate a course of granulocyte transfusions when they were either no longer necessary or were ineffective, or to prevent an exploratory laparotomy.
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 following TIL infusion. Six studies of indium-labeled autologous cloned T cells with antimelanoma activity revealed a trafficking pattern different from that of TIL cells and further studies of these cloned cells, particularly in patients with disease in imageable sites, are in progress. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02062-08 DTM
October 1, 1997 to September 30, 1998
Title of Project: Treatment of Familial Hypercholesterolemia by Dextran Sulfate Apheresis
Principal Investigator: S.F. Leitman, M.D. (Chief) BBB, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: J.M. Hoeg, M.D., Molecular Disease Branch, NHLBI
R. Shamburek, Molecular Disease Branch, NHLBI
Collaborating Unit: Kaneka Pharma 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 Lp(a) cholesterol levels. Diet and drug therapy can reduce cholesterol concentrations in most patients with heterozygous FH, but a small proportion of heterozygotes and nearly all homozygotes 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 high density lipoprotein (HDL) or albumin levels, thus avoiding the need for colloid replacement solutions. Six FH patients were enrolled at the Clinical Center; the total cohort enrolled nation-wide 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 percent, 81 percent, and 68 percent, respectively, in homozygotes and 61 percent, 76 percent, and 65 percent respectively, in heterozygotes. Time-averaged levels of LDL cholesterol were reduced 53 percent (447 to 310 mg/dl) in homozygous FH patients and 41 percent (243 to 143 mg/dL) in heterozygous ones.
The treatments were very well tolerated. 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 advantages, compared to 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 Food and Drug Administration for treatment of FH in July 1996. Patients are now continuing long-term follow-up on an "LDL-Apheresis Registry" to gather post-licensure data on the effect of long term treatment on development of primary and secondary atherosclerotic events, and on overall survival. A 5-year interim analysis of 49 of the original 64 patients who received long-term LDL-apheresis was performed. There was a 44 percent reduction in cardiovascular events during the 5 years the patients received LDL-apheresis compared with the 5-year period prior to LDL apheresis (3.5 events per 1,000 patient-months of treatment compared with 6.3 events per 1,000 patient-months before LDL-apheresis therapy). These findings support the long-term safety and clinical efficacy of LDL apheresis in patients with FH who are inadequately controlled with drug therapy. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02064-07 DTM
October 1, 1997 to September 30, 1998
Title of Project: Quantitative Analysis of Viral Genomes and Their Clinical Correlations
Principal Investigator: J.W.-K. Shih, Ph. D.
DS, DTM, CC, NIH, Bethesda, MD 20892
Other 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
Collaborating Unit: Univ. of Maryland, Dept. of Pathology (I.C. Hsu, Ph.D.)
Staff-Years: 1.0
Human Subjects: (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: This is a continuous effort in 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, to determine the value of a predictor in the progression of a disease, and to monitor the patient's progression of an infection, a more precise and quantitative analysis of the specific gene would be required. These previously highly research-oriented questions can now begin to be answered in the routine clinical laboratories with the advanced technology of molecular biology such as polymerase chain reaction (PCR) and sequencing and mapping of the restriction nuclease digested fragments. We initiated developmental research in molecular diagnostic technology to meet our clinical study need. The demand for molecular testing for blood products for human immunodeficiency virus, hepatitis C virus (HCV), and hepatitis B virus has grown. Whenever possible, we would improve the basic PCR technique to become a semi-quantitative procedure. During the last few years, we were able to apply the same principles of using PCR as the primary study tool for viral infection such as hepatitis G virus and TT virus. We found that these viruses are transmissible by blood transfusion but cause little or no hepatitis. 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 repairing enzyme, Mut Y, and its clinical application was also initiated as a collaborative project with the University of Maryland. We will continue to evaluate new testing approaches and specifically concentrate on perfecting quantitation procedures, standardization, and application of molecular testing to blood products. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02068-07 DTM
October 1, 1997 to September 30, 1998
Title of Project: A Prospective Study of Anti-HCV Positive Blood Donors
Principal Investigator: H.J. Alter, M.D. (Chief)
IDS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: C. Cantilena, M.D., Atg. Chief, DTM, CC
J. Melpolder, M.T. (ASCP), DTM, CC
Collaborating Unit: NIAID (M. Van Raden, Ph.D.)
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. Thus far, 683 subjects have been enrolled, including 402 recombinant immunoblot assay (RIBA) positives, 145 RIBA indeterminates, and 112 RIBA negative controls. The early data have been published (New Engl J Med 334:1691,1996) and the trends have remained the same over time. Unexpected findings were the high proportion (42 percent) of RIBA+ donors who admitted to prior (remote) intravenous drug use and the strong independent association between cocaine snorting and HCV positivity. Shared paraphernalia for snorting accompanied by epistaxis, may serve as a covert vehicle for parenteral viral transmission. Among anti-HCV+/RIBA positive donors, 86 percent were persistently viremic, but 13 percent appeared to have recovered from prior HCV infection. In those with persistent infection who had liver biopsy, 86 percent had histologic evidence of mild to moderate chronic hepatitis, but only 6 percent had a severe histologic lesion despite prolonged infection, averaging 18 years from the time of exposure. Overall, HCV infection in this cohort was generally asymptomatic and clinically benign. Despite an association of HCV with sexually promiscuous practices, we found no evidence for sexual transmission to the specific partners (greater than 100) of HCV-infected individuals.
The study continues to follow the natural history of HCV infection and is now focusing on histologic progression as assessed in liver biopsies obtained at 5-year intervals. New emphasis is being placed on studies of cell-mediated immune responses to HCV and of treatment responses. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02071-03 DTM
October 1, 1997 to September 30, 1998
Project Title: Functional Relevance of HLA Polymorphism in Vaccination Protocols
Principal Investigator: F. Marincola
SB, NCI, NIH, Bethesda, MD 20892
Other Personnel: None
Collaborating Unit: HLA Laboratory, DTM, CC
Staff-Years: 2
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 polymerease 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. A combination of these techniques allows more efficient and accurate typing. Multiple new HLA alleles have been discovered in the HLA Laboratory last year using this strategy. This will help support peptide-based vaccination protocols ongoing at the NIH.
The HLA laboratory also has developed a comprehensive method for typing T cell receptors (TCR) based on Vb-specific, PCR-based amplification and directed heteroduplex analysis, for purposes of screening for TCR clonality. Most recently, soluble, epitope restricted HLA tetramer technology and automated sequencing have been perfected. These will allow high-resolution/high efficiency characterization of TCR. These techniques are being utilized for the immunologic monitoring of patients undergoing peptide-based vaccination. In vivo expansion of various relevant T cell populations in the peripheral circulation and in the area of specific pathologic interest are characterized. The ultimate goal is the identification of the multiple steps occurring in response to T cell antigen-specific vaccination and their correlation with clinical response. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02074-03 DTM
October 1, 1997 to September 30, 1998
Title of Project: Leukocyte, Platelet, and Red Cell Serology in Autoimmune Lymphoproliferative Syndrome
Principal Investigator: D.F. Stroncek, M.D. (Senior Staff)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: C. Cantilena, M.D., DTM, CC
Collaborating Unit: NIAID (S.E. Strauss, M.D.)
Staff-Years: 0.2
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 overaccumulation 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 lymphadenopathy, hypergammaglobulinemia, autoimmunity, 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. Studies of 25 patients have found that 16 had positive direct antiglobulin tests (DAT), six had platelet specific antibodies, and five had neutrophil specific antibodies. Family members with the Fas mutation who did not have ALPS did not have positive DATs. Among the patients with ALPS, females and younger patients were more likely to have red cell autoantibodies. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02076-03 DTM
October 1, 1997 to September 30, 1998
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
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: FDA (I. Berkower, M.D., Ph.D.)
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 interest in studying 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 the 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 have mutation 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 percent of the infection becomes chronic. Directly inducing strong cell-mediated immunity, especially protective cytotoxic T lymphocytes may not only help in preventing initial HCV infection, but may even serve as immune modulation to overcome the infection. During the past year, we were able to evaluate the induction of antibodies to several different plasmid constructs containing both HCV structural and non-structural genes in mice. We were also able to develop the assay and to measure CTL activities in the mouse model. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02077-03 DTM
October 1, 1997 to September 30, 1998
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
Staff-Years: 1.85
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
x (a1) Minors
x (a2) Interviews
Summary of Work: To investigate gene therapy as a therapeutic approach for human hepatitis C virus (HCV) infection and to develop this approach as a model for chronic infectious diseases, we have set up the specific goals for the initial phase of a long-term investigation as follows: 1.) to establish a functionally measurable gene expression system, most likely immunological assays such as antibody or cytotoxic T lymphocyte (CTL) induction; 2.) to identify immune functional relevant groups in clinical settings; and 3.) to employ the functional groups that relate to cell killing as potential therapeutic targets against chronic HCV infection. In the past year, in a mouse model, we have successfully demonstrated gene delivery with a retrovirus containing HCV structural genes and established CTL assays against HCV core and envelope proteins. In the next phase, we will attempt to identify important CTL epitopes that are assumed to be the important functional groups to be employed as treatment targets. This project will have to be continuously modified according the growing knowledge of the pathogenesis of hepatitis C infection. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02078-03 DTM
October 1, 1997 to September 30, 1998
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: None
Collaborating Unit: NIAID, Laboratory of Infectious Diseases
(P. Farci, M.D.; R. Purcell, M.D.)
Staff-Years: 1.3
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Approximately 15 percent of patients recover from hepatitis C virus (HCV) infection while 85 percent become persistently infected with various degrees of associated chronic liver disease. In this study, comparisons will be made between 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 the following: 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 antibody responses; and, if appropriate technology is available, cytotoxic T-cell responses. The goal is to determine if any of these parameters can predict outcome. Studies to date have shown no correlation with genotype because the population is fairly homogeneous for HCV genotype 1. However, there does appear to be a correlation between viral quasispecies and disease outcome. Using rare specimens obtained during the first 16 weeks of HCV infection, it has been shown that the greater the number of viral variants (quasispecies) and the greater the complexity of the sequence divergence, the more severe the clinical outcome. It is believed that the quasispecies is driven by immune pressure. It may be that immune pressure that is intense, and yet insufficient to clear the virus, leads to more severe liver disease either as the result of cell-mediated cytotoxicity or the selection of more pathogenic viral variants. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02079-03 DTM
October 1, 1997 to September 30, 1998
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., IDS, DTM, CC
J. Shih, Ph.D., 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 15 percent of persons during their first 20 years of infection. It is presumed that the proportion with severe outcomes will increase as the duration of follow-up increases and it may be that those infected at a young age will fare worse because they have 3 to 8 decades for HCV infection to evolve into overt liver disease.
This study, conducted in collaboration with Children's National Medical Center (CNMC), will identify infants and children who were transfused at CNMC from 19831992, the decade just prior to second generation anti-HCV testing. Sixty-five hundred 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 hepatitis G virus (HGV). Subjects found antibody positive on initial screen will be enrolled in long-term laboratory and clinical follow-up. 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. If persistent infection and chronic liver disease are as common in children as 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. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02080-03 DTM
October 1, 1997 to September 30, 1998
Title of Project: The Natural History of Hepatitis C Virus Infection
Principal Investigator: H.J. Alter, M.D. (Chief)
IDS, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: J. Melpolder, M.T., DTM, CC
J. Shih, Ph.D., DTM, CC
Collaborating Units: Veterans' Administration Hospital
(Washington, DC; L. Seeff, M.D.)
NIDDK (J. Hoofnagle, M.D.)
Staff-Years: 0.5
Human Subjects: (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Patients enrolled in NIH prospective studies of transfusion-associated hepatitis have been followed long-term to determine the persistence of hepatitis C virus (HCV) infection and the chronic consequences of that infection. Eighty-five percent of patients infected with HCV become chronic carriers and 15 percent resolve their infection usually within 1 year of onset. The vast majority of patients with persistent viremia have some evidence of chronic hepatitis based on serial alanine aminotransferase (ALT) determinations and liver biopsy. Of those biopsied, approximately 20 percent have histologic evidence of cirrhosis, though only half of those patients have had clinical evidence of cirrhosis. Liver-related mortality within the first 2 decades of follow-up has been 4 percent.
These NIH patients were incorporated into a multicenter study of 568 persons with transfusion-associated non-A, non-B hepatitis (predominantly hepatitis C) and 984 matched controls who were transfused, but did not develop hepatitis. After an average follow-up of 18 years, all-cause mortality was 51 percent in the hepatitis group and 52 percent in the controls (NS). There was a slight increase in liver-related mortality in the hepatitis group (3.3 percent vs. 1.4 percent, p=.03). Seventy-one percent of the deaths due to liver disease occurred in patients with associated chronic alcoholism. Twenty-year morbidity follow-up of 103 HCV+ individuals shows that 73 percent have persistent infection, 17 percent have recovered, but maintain antibody to HCV, and 10 percent show no serologic or molecular evidence of their prior HCV infection. Less than 15 percent have developed cirrhosis; in the absence of cirrhosis, there is virtually no clinical evidence of this long-standing HCV infection. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02081-03 DTM
October 1, 1997 to September 30, 1998
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: J. Shih, Ph.D., IDS, DTM, CC
J. Melpolder, M.T., IDS, DTM, CC
Collaborating Unit: Genelabs Technology, Inc. (Redwood City, CA; J. Kim)
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 percent homology with hepatitis C virus (HCV). Its clinical significance is unknown. We have studied HGV and its relation to blood transfusion risk. HGV was found in approximately 1.5 percent of the donor population. It was shown to be transfusion-transmitted by demonstrating the acute appearance of HGV RNA following transfusion and by linkage between HGV positive donors and infected recipients. HGV can establish persistent infection, but the majority (perhaps 80 percent) clear the virus over time. Although 3 of 13 patients (23 percent) with non-ABC 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 aminotransferase elevations. Overall, of 35 observed and 84 projected HGV infections, only 4 percent occurred in patients with hepatitis unrelated to HCV, but as indicated above the hepatitis was probably not due to HGV; 89 percent of HGV infections occurred in recipients with no evidence of hepatitis. Among patients coinfected with HGV and HCV, it was shown that HGV did not worsen the course coexistent hepatitis C. Similarly, we have studied HGV in renal dialysis patients, hemophiliacs, intravenous drug users, and patients with acute and chronic non-ABC hepatitis. Although the prevalence of HGV is very high among parenterally exposed individuals (10 to 20 percent), there is no association with liver disease in these HGV-infected populations. Studies of antibody to the HGV envelope (anti-E2) have shown that HGV RNA and anti-E2 are mutually exclusive and that the latter is a marker of recovery. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02082-03 DTM
October 1, 1997 to September 30, 1998
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 (K. Murthy)
CDC (K. Krawczynski, M.D.)
Staff-Years: 0.l (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 human immunodeficiency virus (HIV) to the chimpanzee and hence to establish an animal model for these infections. Current studies in this model include the following:
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. It was shown that no markers of HIV infection were detectable until the fifth week post-exposure when virus was detectable by HIV RNA, HIV DNA, and viral culture. Anti-HIV and p24 antigen, as used in blood donor screening, were not detectable until 8 weeks post-exposure. Subsequently, the 3-, 4- and 5-week samples were sequentially innoculated into a second chimp. The 3- and 4-week samples were shown to be non-infectious, while the 5-week sample was infectious. Hence, infectivity did not precede the detection of HIV RNA and DNA. This suggests that if such assays were introduced into blood screening they might totally abrogate the infectious window and prevent blood transmission of HIV.
Viral Inactivation: In collaboration with Cerus Corp., the chimp model was used to establish the efficacy of psoralen/UV-inactivated platelets This is the first viral inactivation procedure that maintains the integrity of the cellular components of blood. Three chimpanzees have been exposed to infectious doses of hepatitis C virus (HCV) and hepatitis B virus (HBV) that have been psoralen-UV treated. The study is still in progress, but after 9 months of follow-up, no animal has been infected with either HBV or HCV. These animal studies confirm in vitro efficacy data and set the stage for safety and efficacy trials in humans. This method should have broad application for platelet transfusion therapy, and ultimately, for red cell transfusion as well. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02083-03 DTM
October 1, 1997 to September 30, 1998
Title of Project: Quantitation and Characterization of Lymphohematopoietic Cells
Principal Investigator: E.J. Read, M.D. (Chief)
Cell Processing Section, DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: H.G. Klein, M.D., DTM, CC
T. Trischmann, Ph.D., CPS, DTM, CC
C. Carter, 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 hematopoietic cells was published in August 1997 (Read et al., J Hematotherapy 1997; 6:291301). 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 decision-making about the timing and duration of stem cell apheresis. Over the past year, plans were made to evaluate the final clinical CD34 assay configuration in a clinical trial. However, device malfunction led to postponement of this study until FY '99. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02084-03 DTM
October 1, 1997 to September 30, 1998
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, Cell Processing Section)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: H.G. Klein, M.D., 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: Nexell, Inc. (Irvine, CA)
NHLBI, Hematology Branch and BMT Program
NIAID, HIV Clinical Trials Program
NCHGR, Clinical Gene Therapy Branch
Staff-Years: 0.75
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, have been carried out in collaboration with a number of NIH Institute investigators, and with Nexell, Inc.
Preparation of CD8-depleted, culture-expanded lymphocytes: In collaboration with NIAID and NCHGR, the Cell Processing Section has applied this 10-day process to a clinical trial of human immunodeficiency virus (HIV) gene therapy in the syngeneic twin model. This trial was completed in June 1998, but data are being collected and analyzed with regard to the relative efficacy of the therapeutic and nontherapeutic vectors. In one patient not on antiretroviral therapy, the therapeutic vector-transduced cells had an in vivo survival advantage over the marker gene-transduced cells. Manuscripts on the methodology are in preparation or in press. The processing methods developed will be adapted to a new protocol using autologous mononuclear cells from HIV-infected patients in FY '99.
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. Over the past year, we have successfully developed a reliable method for expansion of selected T cells from patients with chronic myelogenous leukemia, resulting in a product that has minimal contamination with leukemia cells and is capable of stimulating a third-party (donor) immune response to recipient-specific antigens. In the next fiscal year, we will be adapting this process to the Aastrom bioreactor system, and using these stimulator T cells in an MLR, followed by selective depletion of the alloreactive donor cells by anti-CD25 Pseudomonas exotoxin. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02085-03 DTM
October 1, 1997 to September 30, 1998
Title of Project: Methods for Positive and Negative Selection of Hematopoietic Progenitor Cells
Principal Investigator: E.J. Read, M.D. (Chief, Cell Processing Section)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: H.G. Klein, MD, Chief, DTM
T. Trischmann, Ph.D., CPS, DTM
C. Carter, CPS, DTM
R. Butz, CPS, DTM
J. Lee-Fischer, CPS, DTM
F. Vigue, CPS, DTM
Collaborating Units: Nexell, Inc. (Irvine, CA)
CellPro, Inc. (Bothell, WA)
NHLBI, Hematology Branch and BMT Unit
NIAID, Laboratory of Host Defenses
Staff-Years: 1.5
Human Subjects: (a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: We have done preclinical and clinical studies of automated closed systems for positive and negative selection of lymphohematopoietic cells in collaboration with biotechnology firms that have developed systems for potential application to clinical cellular therapies.
Nexell, Inc. Isolex: We continued our studies of the automated Isolex 300i for immunomagnetic selection
of hematopoietic progenitor cells. Starting in the previous fiscal year
and going into this year, we completed 28 full-scale runs on version 1.0 of this system. We demonstrated its utility
in clinical protocols for chronic granulomatous disease gene transfer and autologous transplantation of chronic
ITP. We also initiated evaluation of the new version 2.0 system, completed 12 full-scale runs, and have begun development
of the combined positive/negative selection procedure for T cell depletion. Preliminary results show consistent
T depletion of 5 logs using this method.
CellPro T Cell Depletion System: A clinical evaluation of this 2-step positive (CD34) and negative (CD2) selection system, which uses an immunoadsorption approach, was recently completed. This study randomized 24 allogeneic donors to fresh versus pooled processing of stem cell apheresis products. Results, submitted in abstract form to ASH, demonstrated equivalence between the two study arms in processing and clinical outcomes. Therefore, the pooled processing approach will be used for practical and economic reasons (less processing time, lower costs associated with use of one expensive system versus two). (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02089-02 DTM
October 1, 1997 to September 30, 1998
Title of Project: Comparative Studies of Granulocyte Colony Stimulating Factor 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, Blood Services Section)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: J.M. Oblitas, M.T., Plateletpheresis Center, DTM, CC
Collaborating Units: 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 steroid stimulation of the donor. To define an optimal mobilization schedule that maximizes cell yields while minimizing donor discomfort during granulocytapheresis, we have studied three donor mobilization schedules. Forty healthy donors have undergone three leukapheresis procedures each, receiving either dexamethasone (dexa) 8 mg orally 12 hrs prior to donation, granulocyte colony stimulating factor (G-CSF) 5 ug/kg SQ 16 to 24 hrs prior to apheresis, or dexa 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. For C-CSF-mobilized procedures, a comparison was made between two interface offset (IO) detector settings on the CS-3000 device: an IO of 33 and an IO of 45.
Administration of G-CSF alone led to a 3.8-fold increase. Use of dexamethasone plus C-CSF led to a 4.9-fold increase in donor peripheral blood granulocyte counts compared to dexa alone (from 6.3 ± 2.5 with dexa, to 24.1 ± 4.9 and 30.5 ± 6.2 x10 to the 9th power cells/L with G-CSF and D+G, respectively). Similarly, use of G-CSF alone or the combination of dexa and G-CSF resulted in 2.3- and 3.5-fold increases in granulocyte content in the product compared to dexa alone (from 2.09 ± 0.68 with dexa alone to 4.87 ± 1.02 and 7.31 ± 1.56 x10 to the 10th power cells total with G-CSF and D+G, respectively), p is less than .01 for all comparisons between dexa and either G-CSF or D+G. In addition, granulocyte counts in both the donor's blood and the apheresis product were greater when donors took the combination of D+G vs G-CSF alone (27 percent increase in the blood granulocyte count and 49 percent increase in product granulocyte content when dexa was added to G-CSF) (p is less than .05). Increasing the IO of the device from 33 to 45 did not increase the efficiency of the procedures, and we are continuing to make modifications to the run parameters of the CS-3000 machine to optimize the mechanics of granulocyte harvesting. With dexa alone, 44 percent of donors had insomnia or flushing; with G-CSF, 68 percent had bone pain, headache, insomnia, or fatigue; this increased to 72 percent with combination D+G. Ten of 100 donors requested discontinuation of G-CSF-mobilized donations due to discomfort or inconvenience.
Addition of dexa to G-CSF significantly increases granulocyte yields and is accompanied by a modest but well-tolerated increase in donor inconvenience and discomfort. The availability of granulocyte products with markedly increased numbers of cells is resulting in a renewal of interest in this transfusion component. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02091-02 DTM
October 1, 1997 to September 30, 1998
Title of Project: Acquisition of Hematopoietic Stem Cells for Second Transplants by Apheresis of Filgrastim-stimulated Donors Participating in the National Marrow Donor Program
Principal Investigator: S.F. Leitman, M.D. (Chief, Blood Services Section)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: D. Stroncek, M.D., Laboratory Services Section, DTM, CC
Collaborating Units: NMDP, 3433 Broadway Street, Suite 500
Minneapolis, MN 55413 (D. 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 National Marrow Donor Program (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 1998, 3.2 million donors were participating in the registry and 6,700 unrelated marrow transplants had been performed. In transplant recipients with early graft loss, the best option for therapy is often another dose of stem cells from the original marrow donor. Peripheral blood stem cell (PBSC) components, harvested by apheresis of filgrastim-stimulated donors, provide larger numbers of hematopoietic progenitor cells that engraft more rapidly than marrow-derived cells. For these reasons, a protocol involving all participating NMDP Donor Centers was initiated in February 1997, for the acquisition of PBSCs for second transplants. The objectives of the study are: 1.) to monitor the immediate and long-term safety of filgrastim administration in healthy volunteer donors; 2.) to compare the adverse effects of bone marrow versus PBSC donation; and 3.) to monitor the outcome of matched unrelated-donor PBSC transplants, including time to engraftment, incidence of acute and chronic GVHD, and disease-free and overall survival rates. As of August 1998, 33 unrelated NMDP donors received filgrastim 10 ug/kg/day SQ for 5 days, followed by apheresis. Seventeen donors underwent one procedure, 15 underwent two, and none required central line placement. All donors experienced granulocyte colony stimulating factor-induced fatigue, insomnia, bone pain or headache, although in only 6 percent of donors were these effects considered severe. Peak mean leukocyte counts after filgrastim were 46 ± 12 x 10 to the 9th power/L, and postapheresis thrombocytopenia (less than 100 x 10 to the 9th power/L) occurred 5 donors (15 percent), all of whom underwent two procedures. The mean time to complete recovery from PBSC donation was 1 week, compared with 3 weeks for marrow harvest. Product CD34 content, and recipient outcomes, including time to engraftment, acute and chronic GVHD in the recipient, and overall survival have not yet been evaluated.
The NIH Marrow Donor Center, a participant in the NMDP, has 48,000 donors on its registry. One hundred eighty-five NIH unrelated donors have undergone marrow harvest and six have undergone PBSC donation for NMDP recipients. Sufficient cells for transplant were obtained in one 1218 liter apheresis procedure in all six cases. The availability of same-day CD34 flow analysis permitted exact quantitation of product content and avoided the need for additional apheresis procedures. Five of six donors preferred PBSC donation by apheresis to marrow harvest due to the lack of need for anesthesia and hospitalization. The discomfort of the two procedures was considered equivalent.
Two of the principal investigators for this national study are in the DTM/CC (SFL and DS). Administrative and statistical support for the study are provided by the NMDP National Office. Filgrastim is provided under an Investigational New Drug agreement with Amgen (BB-IND # 6821). (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02092-02 DTM
October 1, 1997 to September 30, 1998
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 Units: None
Staff-Years: 0.2
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: We established a pilot study to evaluate the safety and efficacy of granulocyte (WBC) transfusions using products prepared by granulocyte colony-stimulating factor (G-CSF) plus dexamethasone stimulation of the donor. Neutropenic patients with life-threatening infections were eligible to receive daily G-CSF/steroid-mobilized WBC components. A different volunteer allogeneic, non-HLA matched donor was used each day. As of August 1998, 14 neutropenic patients (four with aplastic anemia, four following allogeneic marrow/peripheral blood stem cell transplant, two with lymphoma, two with LGL-leukemia, and one each with breast cancer and CML) received a median of 6 (range 2 to 28) daily WBC transfusions. Nine of 14 patients had fungal infections. Specific microbial processes included systemic aspergillus infection in six patients, disseminated fusarium in two, and one patient each with disseminated candida, bacterial pneumonia, vancomycin-resistant enterococcemia, pseudomonas skin ulcer, diverticulitis, and RSV pneumonia. Eleven of 14 patients, including all nine with fungal infections, experienced initial clinical improvement, consisting of devervescence in fever, improvement in easily visible skin lesions, and/or radiographic improvement in pulmonary lesions. Five of these 11 either died of other causes (n=3) or had progression of the infectious process after initial improvement (n=2). Only six of the 14 patients who received WBC transfusions were eventually discharged from the hospital, including three of the nine with fungal infections, and three of five with bacterial processes.
The granulocytapheresis products contained 7.2 ± 1.4 x 10 to the 10th power granulocytes in a mean volume of 358 ml. Platelet content of the granulocyte products was 4.8 x 10 to the 11th power, equivalent to a 9-unit platelet transfusion. The mean 1-hour post-transfusion increment in absolute neutrophil count (ANC) was 1.87 + 0.75 in 10 patients without splenomegaly, but decreased to 0.38 and 0.24 x 10 to the 9th power/L in patients with splenomegaly (n=3) and human leukocyte antigens (HLA)-alloimmunization (n=1), respectively. ANCs continued to rise for up to 8 hours after each transfusion and did not decline to pretransfusion levels until 24 to 30 hrs after infusion. The granulocyte transfusions were well tolerated, and coadministration (within 4 hours) of amphotericin did not lead to pulmonary compromise. None of the six surviving patients became HLA-alloimmunized. Donors experienced mild bone pain, headache, and insomnia, but were willing and eager to support such donations again. In summary, transfusions of G-CSF-mobilized granulocyte components were well tolerated and resulted in dramatic and sustained increases in circulating granulocytes. The beneficial effects experienced in our patients in this pilot study suggest that G-CSF mobilized granulocytes deserve evaluation in the form of a large, randomized, prospectively controlled, multicenter trial. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02093-02 DTM
October 1, 1997 to September 30, 1998
Title of Project: Effects of G-CSF on Granulocyte Donors
Principal Investigator: D.F. Stroncek, M.D. (Senior Staff)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: S.F. Leitman, M.D., DTM, CC
E.J. Read, M.D., DTM, CC
Collaborating Units: CBER, FDA (L. Harvath, Ph.D.)
NHLBI (D. Follmann, Ph.D.)
Staff-Years: 0.3
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Granulocyte colony-stimulating factor (G-CSF), alone or in combination with dexamethasone, is becoming a standard mobilizing regimen for granulocyte donors. The effect of these agents on donor granulocyte counts is well known, but effects on other blood elements are not well described. In a prospective, blinded study, we collected granulocyte concentrates from six donors on three occasions each. In random order, the donors received either dexamethasone (8 mg PO), G-CSF (5 ug/kg SQ) or both on day 1. On day 2, granulocyte concentrates were collected by processing 7 liters on a CS3000 blood cell separator.
Donor granulocyte counts returned to baseline after 2 days in all three types of collections, and granulocytopenia did not occur. Donor platelet counts were similar using all three mobilization methods and returned to normal after 1 week.
When dexamethasone was given, potassium, phosphorus, and uric acid levels fell, but BUN increased. When G-CSF was given, potassium levels fell and leukocyte dehydrogenase (LDH) increased. When both were given, potassium and phosphorus levels decreased, but BUN and LDH increased. Alkaline phosphatase levels were not affected by any of the three mobilization regimens. All chemistries returned to baseline 1 week following the collection.
In conclusion, mobilization of granulocytes with either G-CSF or dexamethasone is associated with mild changes in blood chemistries and a mild transient fall in platelet counts. Blood chemistries and platelet counts returned to baseline levels promptly. It is unclear if these changes have any clinical consequences, but they suggest that mobilized collections should not be performed at greater than 1 week intervals. A comparison of symptoms in donors given G-CSF or dexamethasone is ongoing. Additional studies will compare different methods to administer G-CSF. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02094-02 DTM
October 1, 1997 to January 1, 1998
Title of Project: Drug-dependent Immune-mediated Cytopenias
Principal Investigator: D.F. Stroncek, M.D. (Senior Staff)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: J.L. Procter, MEd, MT(ASCP), SBB,
Transfusion Services, DTM, CC
K. Matsuo, M.D., DTM, CC
S. Leitman, M.D., Blood Services Section, DTM, CC
Collaborating Units: None
Staff-Years: 0.2
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: This study has been terminated. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02095-02 DTM
October 1, 1997 to September 30, 1998
Title of Project: Structure and Function of Granulocyte Antigens
Principal Investigator: D.F. Stroncek, M.D. (Senior Staff)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: K. Matsuo, M.D., DTM
Collaborating Units: None
Staff-Years: 0.5
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Neutrophil-specific antigens NA1 and NA2 are located on Fc-gamma-receptor IIIb (FcgRIIIb). The NA1 and NA2 forms of FcgRIIIb differ by four amino acids and their FcgRIIIB genes by five base pairs. The FcgRIIIB genotypes of 232 people were analyzed at four NA polymorphic sites using polymerase chain reaction and restriction enzymes. Two primer pairs were used to amplify genomic DNA. One amplicon was digested in NA1-positive people at base 227 by XmnI and in NA2-positive at 141 by Cac8I. The second was digested in NA2-positve people at two sites, base 147 by HindIII and base 277 by AclI or Bsp1406I.
The NA1 gene frequency in 100 Japanese (0.66) was greater than in 53 African Americans (0.40) and 79 Caucasians (0.32). The number of people whose genotype was not clearly NA1 or NA2 was unexpectedly high16 (7 percent). The frequency of these unusual genotypes was greater in African Americans (19 percent) than Japanese (4 percent) or Caucasians (2.5 percent). Analysis of FcgRIIIB at bases 141 and 227 identified four African Americans with one gene that was identical to NA1 at 227 and NA2 at 141. Further analysis of FcgRIIIB identified six African Americans who had a NA2 gene that lacked an NA2 restriction digest site at 277. Two Japanese and one Caucasian had an NA2 gene without an NA2 restriction site at 141. One Japanese had an NA1 gene with an NA2 restriction site at 141. One Japanese and one Caucasian had an NA2 gene without an NA2 restriction site at 147. The FcgRIIIB genes in two of these people have been sequenced and two new NA alleles have been identified. The NA1 gene is more prevalent in Japanese. Variations in the NA1 and NA2 alleles of FcgRIIIB exist and are more common in African Americans. (Return to project list)
INTRAMURAL RESEARCH PROJECT Z01 CL-02096-01 DTM
October 1, 1997 to September 30, 1998
Title of Project: Storage of Granulocyte Concentrates
Principal Investigator: D.F. Stroncek, M.D. (Senior Staff)
DTM, CC, NIH, Bethesda, MD 20892
Other Personnel: S.F. Leitman, M.D., DTM, CC
E.J. Read M.D., DTM, CC
Collaborating Units: CBER, FDA (L. Harvath, Ph.D.)
NHLBI (D. Follmann, Ph.D.)
Staff-Years: 0.3
Human Subjects: x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Current standards limit granulocyte storage to 24 hours. Since granulocyte colony-stimulating factor (G-CSF) inhibits granulocyte apoptosis, it may be possible to store G-CSF-mobilized granulocytes for longer periods while maintaining cell viability and function. However, G-CSF mobilization increases the yield of granulocytes several-fold and the resulting higher cell concentrations may diminish viability during storage.
Granulocyte donors were given dexamethasone (8 mg PO), G-CSF (5 µg/kg SQ) or both and on the next day granulocyte concentrates were collected using a CS3000 blood cell separator. Component cell counts and pH were measured over time. Significantly more granulocytes were collected when donors were given G-CSF or both G-CSF and dexamethasone compared with dexamethasone alone. Storage had little effect on WBC count, but pH dropped significantly with time in all three types of granulocyte concentrates. Granulocytes mobilized with G-CSF plus dexamethasone were acidic immediately after the collection and pH was below 6.0 after 24 hours.
Because acidic conditions are detrimental to cell survival, four granulocyte concentrates (two dexamethasone, one G-CSF and one G-CSF plus dexamethasone) underwent serial 2-fold dilution in autologous plasma prior to storage. pH was not maintained until the concentrates were diluted 8- and 16-fold. To optimize storage pH, mobilized granulocyte concentrates require an 8- to 16-fold dilution, which is operationally impractical. Studies that will evaluate the ability of clinical grade granulocyte preservative solutions to maintain pH during storage will begin soon. Additional measurements of viability and function will be used to evaluate such solutions. (Return to project list)