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 Adsorption Apheresis
Characterization of Newly Identified Viral Genomes and Their Clinical Correlations
A Prospective Study of Anti-Hepatitis C Virus-positive Blood Donors
Dissecting the Molecular Immunology of T-cell-aimed Vaccines
Leukocyte, Platelet, and Red Cell Serology in Autoimmune Lymphoproliferative Syndrome
Evaluation of Nucleic Acid Vaccine as a Preventive and Therapeutic Modality
Viral and Immune Factors that Influence Recovery or Progression of Hepatitis C
Hepatitis C Virus Infection in Infants and Children
The Natural History of Hepatitis C 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
Therapeutic Efficacy of Granulocyte Colony-stimulating Factor-mobilized Granulocyte Concentrates
Facilitation of Peripheral Blood Stem Cell Transplants by the National Marrow Donor Program
Effects of Granulocyte Colony-stimulating Factor on Granulocyte Donors
Structure and Function of Granulocyte Antigens
Ex Vivo Culture and Characterization of Dendritic Cells for Clinical Immunotherapy Trials
Storage of Granulocyte Concentrates
Kinetics of Granulocyte Colony-stimulating Factor-induced Granulocyte Mobilization
Citrate Effects and Calcium Replacement Therapy During Large-volume Leukapheresis
Therapy of Von Willebrand's Disease with Single-donor Cryoprecipitate Collected by Apheresis
Malarial Anemia
Adoptive Immune Therapy for Cytomegalovirus Infection
Molecular Testing Standards and New Amplification Approaches
Characterization of Human Pathogenic Mycoplasma from HIV-infected Patients
Large-volume Versus Standard-volume Leukapheresis Peripheral Blood Stem Cells Collection
Prospective Studies of Phlebotomy Therapy in Hereditary Hemochromatosis
Massive Immune Hemolysis in Peripheral Blood Stem Cell Transplants with Minor ABO Incompatibility
Studies of Prophylactic Calcium Administration in Plateletpheresis Donors
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02005-31 DTM
October 1, 1999 to September 30, 2000
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:
C. Schechterly, B.S., M.T., IDS, DTM
P. Hernandez, R.N., IDS, DTM
B. Williams, R.N., IDS, DTM
M.H. Boone, M.T., A.S.C.P., IDS, DTM
J.W. Shih, Ph.D., IDS, DTM
Collaborating Units:
Children's National Medical Center (N. Luban, M.D.)
NHLBI Reds Allogeneic Donor and Recipient (RADAR) Study
Staff-Years:
3.5
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
x (a1) Minors (a2) Interviews
Summary of Work: This project culminates a series of 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 1960's, 10 to 20 percent in the 1970's, 8 to 12 percent in the early 80's, and 4 to 5 percent in the late 80's. 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.
A new prospective study is being initiated that will follow both adult and pediatric patients for a large variety of potential transfusion-transmitted infections. We will continue to look for HBV, HCV, HIV, and HTLV infections to monitor the continued efficacy of existing donor screening measures. However, the main thrust of the study will be to determine the incidence and clinical outcome of infections for which donor screening is not routinely performed. This would include newly proposed hepatitis viruses (TTV and SEN-V), cytomegalovirus (CMV), parvovirus B-19, and the human herpes virus, type 8 (HHV-8). Other infections such as borrelia (lyme disease), babesia (babesiosis), trypanosoma cruzi (Chagas) and HHV-6 may also be monitored. Although, we will continue to measure serologic responses, the study will focus on molecular assays. A primary goal of the study will be to establish a linked donor and recipient repository that can be used to rapidly assess if a newly emergent agent represents a threat to the blood supply. This repository will be linked to the RADAR repository.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02040-16 DTM
October 1, 1999 to September 30, 2000
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 Personnel:
H.G. Klein, M.D., DTM
S.F. Leitman, M.D., BSS, DTM
J.W. Shih, Ph.D., IDS, DTM
C. Cantilena, M.D., Med. Officer, DTM
C. Schechterly, B.S., M.T., DTM
Collaborating Unit:
Washington Area Red Cross (Chesapeake Region; P. Ness)
Staff-Years:
3.0
Human Subjects:
x (a) Human subjects (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 followup since 1985 (N Engl J Med 321:917, 1989). The cohort is now in its 16th year of followup. At enrollment, 182 subjects were Western blot (WB) positive, including 158 asymptomatic donors, 15 blood recipients, and 9 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-positive subjects, 87 percent were donors, 5 percent sexual partners, and 8 percent recipients. Of the 182 WB positives, 49 are alive and in active followup; 58 (32 percent) are dead, of whom 54 (93 percent) died of AIDS; 75 (41 percent) are lost to followup (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 51 in active followup, 40 (75 percent) are males and 49 (92 percent) were detected at blood donation. Nineteen of the 51 (37 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 10 years of followup and have CD4 counts persistently more 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 non-progression. AIDS or HIV-related phenomena have not developed in any of the 21 WB-indeterminate or 70 WB-negative subjects.
Treatment with HAART therapy is being conducted by personal physicians or through other NIH protocols.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02045-14 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Etiology of Allergic Reactions in Platelet- and Granulocytapheresis Donors
Principal Investigator:
S.F. Leitman, M.D. (Chief)
BSS, CC, DTM, NIH
Bethesda, MD 20892
Collaborating Units:
W.J. Lee, Research Associate
Fenwal Laboratories, Division of Baxter Healthcare, Inc. (Round Lake, IL)
Staff-Years:
0.1
Human Subjects:
(a) Human subjects x (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 ten reactions occurred during granulocytapheresis procedures. Using a combination of skin tests, radioallergosorbent tests (RAST), 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 ten of 16 plateletpheresis donors and eight of ten 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. 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 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 Energy Information Administration test for IgE anti-EO will be performed. A cohort of serum samples stored at the Centers for Disease Control and Prevention 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 National Health and Nutritional Examination Survey study.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02055-12 DTM
October 1, 1999 to September 30, 2000
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:
D. Stroncek, DTM
Collaborating Unit:
NMD, CC (J. Carasquillo, M.D.; C. Chen, M.D.)
Staff-Years:
0.2
Human Subjects:
(a) Human subjects x (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-three studies for the purpose of localizing abscess were performed in Clinical Center patients enrolled in a variety of protocols in the past 8 years. Of these 23, three were positive and received appropriate therapy for salmonella osteomyelitis in a hemophiliac patient with HIV 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 23 indium-labeled allogeneic leukocyte studies were performed in patients with chronic granlomatous 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, in specific, 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. In contrast, studies of indium-labeled autologous, cloned T-cells with anti-melanoma activity did not show any trafficking to tumor sites in 14 patients. The results of the trafficking studies were highly predictive of clinical response, in that none of the 14 patients had regression of disease in response to the cloned anti-melanoma T-cells. On the basis of these studies, the protocol was terminated, and a modified approach, using highly immunosuppressive conditioning prior to infusion of autologous cloned T-cells, will be tested. Trafficking of indium-labeled autologous cells will be used as one of the benchmarks with which to evaluate the response to therapy.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02062-10 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Treatment of Familial Hypercholesterolemia by Dextran Sulfate Adsorption Apheresis
Principal Investigator:
S.F. Leitman, M.D. (Chief)
BSS, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
R. Shamburek, M.D., Molecular Disease Branch, NHLBI
Collaborating Unit:
Kaneka America Corporation (New York, NY; D. Zachary,
Vice President)
Staff-Years:
0.2
Human Subjects:
(a) Human subjects x (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 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 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 percent, respectively, in homozygotes and 61, 76, and 65 percent respectively, in heterozygotes. 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 advantage, compared to simple plasma exchange, of eliminating the need for colloid replacement solutions.
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 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. Two patients are continuing to receive regular biweekly LDL apheresis treatments at the Clinical Center, while a third patient, a child, receives biweekly plasma exchange until his blood volume becomes large enough to tolerate the extracorporeal volume of the LDL apheresis device.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02064-09 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Characterization of Newly Identified Viral Genomes and Their Clinical Correlations
Principal Investigator:
J.W.-K. Shih, Ph.D.
IDS, DTM, NIH
Bethesda, MD 20892
Other Personnel:
R.Y.H. Wang, Ph.D., DTM
A. Matsumoto, M.D., Ph.D., DTM (1997-1999)
T. Umemura, M.D., DTM
Y. Tanaka, M.D., DTM
H.J. Alter, M.D., DTM
Collaborating Unit:
None
Staff-Years:
1.5 (NIH only)
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: There are two components in this project and both are extended from our continuous commitment to the clinical investigation of viral hepatitis. One of these is an effort to respond to the increasing demand for a more precise measurement of relevant genomic information in any viral infection. The knowledge of the presence of 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 disease progression, a more precise and quantitative analysis of the specific gene would be required. This previous research-oriented question can now begin to be answered in 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 for HBV, HCV, and HIV infection. Whenever possible, we would improve the basic PCR technique to become a semi-quantitative procedure. During the last 2 years, we were able to apply the same principles of using PCR as primary study tool for viral infection to several newly identified human hepatitis viruses or suspected hepatitis viruses such as HGV, TTV, and SENV. We found that these viruses were indeed transmissible by blood transfusion but have little or no impact on post-transfusion hepatitis. Although specific HGV RNA was identified in both recipients and paired donors sera, it could also be found in non-transfused controls. It could be found in patients with chronic infection with mild or no observed liver function abnormality but their causative relationship could not be determined. The prevalence of these viruses in blood donors, in general, was higher than that of HCV.
The other part of this project is related to viral discovery. We have always maintained an effort to find other causative viral agents that may be responsible for hepatitis cases with unidentifiable cause. Due to its great resource requirement, we tried to conduct this project with industry partners under CRADA. We divided responsibilities by concentrating our group in confirming initial discovery and clinical characterization. In the past few years, we also engaged in developing cloning techniques for rare event genes that might identify low copy infectious agents from patient sera or tissues. The techniques developed were unique and with the potential to be applied to a large number of specimens at the same time. An invention report has been filed with the NIH technology transfer office and is being considered for possible patent application.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02068-09 DTM
October 1, 1999 to September 30, 2000
Title of Project:
A Prospective Study of Anti-Hepatitis C Virus-positive Blood Donors
Principal Investigator:
H.J. Alter, M.D. (Chief)
IDS, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Cantilena, M.D., Med. Officer, DTM
C. Schechterly, B.S., M.T., DTM
J. Shih, Ph.D., DTM
Collaborating Unit:
Liver Service, NIDDK (J. Hoofnagle, M.D.; J. Liang, M.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, 697 subjects have been enrolled, including 403 recombinant immunoblot assay (RIBA) positives, 182 RIBA indeterminates, and 112 RIBA-negative controls. The early data have been published (New England Journal of Medicine 334:1691,1996) and the trends have remained the same over time. Unexpected findings were the high proportion (41 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, 87 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 19 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 of 116 HCV-infected individuals.
The study continues to follow the natural history 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.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02071-05 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Dissecting the Molecular Immunology of T-cell-aimed Vaccines
Principal Investigator:
F. Marincola, M.D.
SB, NCI, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Unit:
HLA Laboratory, DTM, CC
Staff-Years:
5.0
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Human leukocyte antigen (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 few years 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 more recently robotic sequencing. With these high-resolution methods it has been possible to achieve several categories of results:
With these techniques we are actively investigating variables involved in the algorithm modulating tumor/host interactions in the context of active vaccination protocols.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02074-05 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Leukocyte, Platelet, and Red Cell Serology in Autoimmune Lymphoproliferative
Syndrome
Principal Investigator:
D.F. Stroncek, M.D.
DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Cantilena, M.D., DTM
J. Procter, DTM
Collaborating Unit:
NIAID (S. Straus, M.D.)
Staff-Years:
0.2
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
x (a1) Minors (a2) Interviews
Summary of Work: Patients with autoimmune lymphoproliferative syndrome (ALPS) have an autosomal dominant genetic defect that affects lymphocyte apoptosis associated with chronic non-malignant lymphadenopathy, splenomegaly and autoimmunity particularly affecting blood cells. Direct antiglobulin tests (DAT) were performed on 34 consecutive patients with ALPS and 37 of their clinically unaffected relatives. The effects of age, gender, race, and immunoglobulin levels on the incidence of autoantibodies and clinical hemolysis were assessed. The DAT was positive in 21 (62 percent) of ALPS patients but only in one (3 percent) of their relatives (p=0.001). The DAT was reactive due to IgG alone in 43 percent, complement alone in 5 percent, IgG plus complement in 19 percent, and 33 percent of the patients' cells had a positive reaction with polyspecific reagent only. All ten ALPS patients with a history of hemolytic anemia had a positive DAT. Sixty-percent of patients had only IgG on their cells, 30 percent had IgG and complement, and 10 percent of patients with a history of hemolytic anemia reacted only with polyspecific reagent. Of the 11 patients with a positive DAT and no history of hemolytic anemia, IgG alone was present in 27 percent, complement alone in 9 percent, IgG plus complement accounted for 9 percent, and 55 percent had positive DATs only with polyspecific reagent. Among ALPS patients, those with a positive DAT had greater quantities of TCR a/b+ CD4-CD8- cells and higher IgG levels. The DAT results in ALPS patients are most similar to those found in warm autoimmune hemolytic anemia. The DAT is useful to distinguish affected and unaffected individuals within an ALPS family. Preliminary studies have found that the incidence of ALPS patients with neutrophil and platelet specific antibodies is much lower than the incidence of RBC antibodies.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02076-05 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Evaluation of Nucleic Acid Vaccine as a 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
G.J. Hu, Ph.D., DTM (1996-2000)
X. Jiao, Ph.D., DTM
Z. Feng, Ph.D., DTM
H.J. Alter, M.D., DTM
Collaborating Unit:
None
Staff-Years:
2.8 NIH only
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: This program is extended from our continuing efforts to investigate the immune response to the hepatitis C virus (HCV) in both humans and experimental animals. In extensive earlier studies, we identified immunodominant and neutralizing epitopes on the hepatitis C virus that will now be further investigated to examine the relationship between immune response and persistent infection. In FY 95 and FY 96, we initiated a new project to examine the potential of nucleic acid vaccination for the prevention and/or treatment of HCV infection. The long-term goal of both the basic immunology studies and the DNA vaccine studies is to develop models for immune therapy of chronic viral infections of the liver. 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 major concern is the genetic instability of the infectious agent. There are two hypervariable regions in the putative HCV envelope proteins. Immune escape mutants have been attributed to mutations in these regions. Experimentally infected chimpanzees and HCV-infected patients have been found to repeat bouts of infection with either homologous or new strains of HCV. This failure to develop protective immunity links to the high chronicity rate in HCV infection. Directly inducing strong cell-mediated immunity, especially protective cytotoxic T lymphocyte responses, may not only help in preventing initial HCV infection, but may serve as a mechanism for immune modulation to overcome existing infection. Using the mouse model, we were able to evaluate the induction of antibodies to several different plasmid constructs containing both HCV structural and non-structural genes. We were also able to develop assays to measure both humoral and cell-mediated immune responses, including CTL activities, in the mouse model. In the past year, we have tested the genetic sequences of many HCV-related immunogens to establish the best candidate DNA vaccine. We have also studied methods of vaccine delivery and immunity augmentation procedures; accumulated extensive experience in measuring humoral and cell-mediated immunity; and developed effective immunization strategies in small experimental animals. We believe we are now ready to test our findings in the only animal model susceptible to HCV infection, the chimpanzee. Protocols are being written for DNA vaccination in the chimpanzee using constructs containing genes for HCV core and envelope proteins.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02078-05 DTM
October 1, 1999 to September 2000
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 viral burden (initially and over time), HCV genotype, the number of viral quasi-species (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 since the population is fairly homogeneous for HCV genotype 1. However, there does appear to be a correlation between viral quasi-species and disease outcome. Using rare specimens obtained during the first 16 weeks of HCV infection, we have measured the mean Hamming distance that reflects the extent of viral diversity (the degree of sequence divergence within the viral quasi-species). We have found that the mean Hamming distance 12 to 16 weeks after the onset of acute infection predicts whether the patient will recover from HCV infection or develop persistent infection and chronic liver disease. Patients who recover have a declining Hamming distance as antibody to HCV develops, signifying immunologic containment and then clearance of the virus. In contrast, the majority of patients demonstrate an increased mean hamming distance as antibody appears. This suggests that if the immune response is not sufficient to clear the virus, it paradoxically exerts immune pressure that results in mutations (escape variants) that lead to persistent infection. Interestingly, patients with fulminant hepatitis have a very low degree of viral diversity because they succumb to the infection before the immune system can clear the virus or exert immune pressure. This study has been published (Science 288:339-344.2000).
In the next phase of this study, we are going to measure the quasi-species throughout the long-term course of patients for whom we have stored sera from prior prospective studies. In addition we will identify patients with newly acquired acute hepatitis C so that we can serially measure viral load, viral quasi-species, neutralizing antibody responses and particularly, cell-mediated immune responses. Since such infections are no longer occurring in the transfusion setting, we will follow healthcare workers who sustain needle-stick injuries and commercial plasma donors.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02079-05 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Hepatitis C Virus Infection in Infants and Children
Principal Investigator:
H.J. Alter, M.D. (Chief)
IDS, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
N. Luban, M.D. (Chief)
Blood Transfusion Medicine
Children's National Medical Center
Washington, DC
Collaborating Unit:
None
Staff-Years:
0.2 (NIH only)
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
x (a1) Minors (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 followup 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) has identified infants and children who were transfused at CNMC from 1983 to 1992, the decade just prior to second generation anti-HCV testing. 5546 children, who met eligibility criteria, were transfused at CNMC during this interval. The mean age at transfusion was 1 year (range, birth to 10.7 years). Thus far, 2668 children (49 percent) have been recalled and provided consent/assent. The mean age at testing was 11 years (range 4 to 17 years). Of the 1753 children fully tested for antibodies to HCV and hepatitis G virus (HGV), 36 (2.0 percent) are anti-HCV positive and 100 (5.7 percent) HGV RNA positive. The HCV and HGV prevalence in age-matched non-transfused controls are 0.3 and 6.3 percent, respectively. There is a significant association between HCV infection and transfusion, but the overall prevalence is lower than expected given that these children were transfused prior to HCV donor screening. The 36 HCV infected children have been followed a mean of 24 months. All are asymptomatic. The range of ALT is 29 to 140 IU/ml; 80 percent have at least one ALT value that exceeds 1.5 times the upper limit of normal. In an adjunctive study, liver biopsies have been performed on 25 children, 16 of whom are included in this transfusion look-back study. The average interval from transfusion to biopsy was 10.7 years. The histologic lesions were generally mild, but four (16 percent) had bridging fibrosis. None had cirrhosis. Duration of infection and age at infection did not appear to influence the extent of fibrosis.
In the final analysis, this study will determine the minimal rate of transfusion-transmitted HCV and HGV infection in the decade before anti-HCV testing and will allow for an annualized incidence estimate and a determination of the national burden of transfusion-induced viral hepatitis in children. To date it appears that persistent infection and chronic liver disease are less common in children than adults, but continued long-term followup with serial liver biopsies is necessary before the true disease burden can be ascertained. This study will have major implications for anti-viral therapy programs and might serve to shift emphasis to pediatric populations where response rates may be higher and the long-term benefit greater.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02080-05 DTM
October 1, 1999 to September 30, 2000
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:
C. Schechterly, B.S., MT, DTM
P. Hernandez, R.N., DTM
J. Shih, Ph.D., DTM
Collaborating Units:
Veterans' Administration Hosp. (Washington, DC; L. Seeff, M.D.)
NIDDK (J. Hoofnagle, M.D.)
Staff-Years:
0.5
Human Subjects:
x (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 became chronic carriers and 15 percent resolved 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 followup has been 4 percent.
These NIH patients were incorporated into a multi-center 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 followup 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 followup of 103 HCV-positive 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 longstanding HCV infection.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02082-05 DTM
October 1, 1999 to September 30, 2000
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
Other Personnel:
None
Collaborating Units:
Southwest Foundation for Biomedical Research (K. Murthy)
CDC (K. Krawczynski, M.D.)
Blood Centers of the Pacific (M. Busch)
Staff-Years:
0.1 (NIH only)
Human Subjects:
x (a) Human subjects (b) Human tissues (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:
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02083-05 DTM
October 1, 1999 to September 30, 2000
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:
E. Lazarus, M.D., CPS, DTM
H. Klein, M.D., Chief, DTM
T. Trischmann, Ph.D., CPS, DTM
C. Carter, CPS, DTM
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: Four assay development studies are included in this project:
(1) CD34+ cell quantitation by microvolume fluorimetry: A study comparing the biometric imaging assay, microvolume fluorimetry (MVF), to flow cytometry (FC) for quantitation of 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 decision-making about the timing and duration of stem cell apheresis. Plans to evaluate the final clinical CD34 assay configuration in a clinical trial did not materialize because of device malfunction and unavailability of assay kits (related to patent issues), and later to staffing issues and competing priorities, we plan to reassess these goals and implement this study in the next fiscal year.
(2) Megakaryocyte progenitor cell colony (CFU-Mk) quantitation: A study evaluating the utility of CFU-Mk quantitation by the Megacult system (StemCell Technologies, Inc., Vancouver, BC, Canada) for quality assessment of G-CSF mobilized peripheral blood stem cell (PBSC) products was initiated in December 1998. Preliminary data shows that CFU-Mk content of PBSC products is highly correlated with CD34+ cell content. Ongoing studies will evaluate the relationships between these product assays and platelet engraftment after transplantation, so that the clinical utility of the assay can be assessed. Plans to complete this study were postponed until late FY 2000 because of staffing issues.
(3) Quantitation of dendritic cells by flow cytometry: This study was initiated in FY 2000 in concert with our methods development for generating autologous dendritic cells for use in cancer immunotherapy. Characteristic flow cytometry features of immature dendritic cells were described in our first manuscript (see Z01 CL-02097-02 DTM).
(4) Single platform flow cytometry method for quantitation of CD34+ and CD3+ cells: We plan to begin this evaluation early in FY 2001, after we purchase, install, and have staff trained on a new flow cytometer. This method will need to be compared and validated against our current dual platform method.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02084-05 DTM
October 1, 1999 to September 30, 2000
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. Klein, M.D., Chief, DTM
S. Donnelly, M.D., CPS, DTM
T. Trischmann, Ph.D., CPS, DTM
C. Carter, CPS, DTM
K. Hines, CPS, DTM
V. Fellowes, ETB, NCI
Collaborating Units:
Nexell, Inc. (Irvine, CA)
Aastrom Biosciences, Inc. (Ann Arbor, MI)
Hematology Branch and BMT Program, NHLBI
HIV Clinical Trials Program and Laboratory of Host Defenses, NIAID
Clinical Gene Therapy Branch, NCHGR
Experimental Transplantation Program, NCI
Staff-Years:
1.0
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.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02085-05 DTM
October 1, 1999 to September 30, 2000
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. Klein, M.D., Chief, DTM
T. Trischmann, Ph.D., CPS, DTM
C. Carter, CPS, DTM
R. Butz, CPS, DTM
J. Lee, CPS, DTM
F. Vigue, CPS, DTM
Q. Chau, CPS, DTM
M. Agricola, CPS, DTM
Collaborating Units:
Nexell, Inc. (Irvine, CA)
CellPro, Inc. (Bothell, WA)
Hematology Branch and BMT Unit, NHLBI
Laboratory of Host Defenses, NIAID
Staff-Years:
1.5
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Preclinical and clinical studies of automated closed systems 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:
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02089-04 DTM
October 1, 1998 to September 30, 1999
Title of Project:
Therapeutic Efficacy of Granulocyte Colony-stimulating Factor-mobilized Granulocyte
Concentrates
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
E. Wong, M.D., DTM
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: The efficacy of therapeutic granulocyte transfusions is limited by the relatively small number of cells obtained using standard apheresis techniques. In prior studies, we demonstrated that granulocyte concentrates prepared by granulocyte-colony stimulating factor (G-CSF) or the combination of G-CSF and dexamethasone stimulation of the donor contained 2.3- and 3.5-fold greater numbers of granulocytes than products prepared using dexamethasone alone (product content 2.09 plus or minus 0.68 x 10 to the tenth power cells with dexamethasone alone versus 4.87 plus or minus 1.02 and 7.31 plus or minus 1.56 x 10 to the tenth power cells total with G-CSF and G-CSF plus dexamethasone, respectively) (p less than 0.01 for dexa vs G-CSF alone or D+G). Seventy-two percent of donors getting dexamethasone plus G-CSF had restlessness, insomnia, bone pain, or headache. Ten percent of donors requested discontinuation of participation in the study due to the inconvenience and discomfort of the mobilization regimen.
Thirty-seven Clinical Center patients have received G-CSF mobilized granulocytes. Twenty-four were profoundly neutropenic, including ten patients with severe aplastic anemia (SAA), eight stem cell transplant recipients, five patients with lymphoma, and one with breast cancer. The remaining 13 patients had CGD. In the neutropenic patients, 15 had systemic filamentous fungal infections, one had candidemia, seven had bacterial infections, and one had RSV. The mean increment in granulocyte count 1-hour post-transfusion was 2600/uL, and counts greater than 500/uL above baseline were sustained for 12 to 24 hours. One of the 11 neutropenic, immunosuppressed patients who survived longer than 2 weeks after the initiation of granulocyte transfusions developed HLA allosensitization, as did two of the 13 CGD patients. In the absence of HLA allosensitization, granulocyte transfusions were associated with progressive hypoxia, pulmonary infiltrates, and an ARDS-like event in four of ten SAA patients, versus none of 13 CGD patients. Of the neutropenic patients with tissue molds, eight of 15 stabilized or improved during granulocyte transfusion therapy, but only two of 15 survived hospitalization. In contrast, three of seven with bacterial processes were discharged from hospital. Ten of 13 patients with CGD had complete resolution of their fungal (n=5) or bacterial (n=5) infections. These pilot studies of G-CSF mobilized granulocytes suggest that they may confer survival benefit in carefully selected neutropenic patients with life-threatening infections, but may be associated with significant progressive pulmonary toxicity. A randomized prospective study of the efficacy of G-CSF mobilized granulocyte transfusions in patients severe aplastic anemia hospitalized at the Clinical Center is being designed to further delineate the benefit to risk profile of this therapy.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02091-04 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Facilitation of Peripheral Blood Stem Cell Transplants by 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.S. Stroncek, M.D., Chief, Laboratory Services Section, DTM
R. Ashton, R.N., NIH Marrow Donor Center
Collaborating Unit:
NMDP (Minneapolis, MN; D. Confer, M.D.)
Staff-Years:
1.2
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, collection, and transplant centers. As of April 30, 2000, 3.9 million donors were participating in the registry and 9,600 unrelated marrow transplants had been facilitated. Peripheral blood stem cell (PBSC) components, harvested by apheresis of filgrastim-stimulated donors, provide larger numbers of progenitor cells which engraft more rapidly than marrow-derived cells, and are being increasingly used instead of marrow in the matched sibling transplant setting. Early favorable outcomes in the related-donor setting, particularly in patients with high-risk leukemia, have led to the cautious adoption of PBSC transplants in the unrelated donor setting. The NIH Marrow Donor Center, one of the largest hospital-based donor centers participating in the NMDP network, with 50,062 donors on its registry, is participating in two nationwide NMDP protocols, one for acquisition of filgrastim-stimulated PBSC's for primary unrelated donor transplant, and one for acquisition of PBSC's for second transplants (necessitated by rejection or tumor recurrence after a first transplant). The objectives of these studies are (1) to monitor the 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 GVHD, and disease free and overall survival. As of April 2000, 215 NIH unrelated donors had undergone marrow harvest and 13 had undergone PBSC donation for NMDP recipients. Sufficient cells for transplant were obtained in one 12 to 20 liter apheresis procedure in ten of these 13 cases. Two of 13 donors had poor CD34 mobilization responses to filgrastim, and needed two consecutive apheresis procedures to collect the requested cell dose. None of the 13 required a central line. All donors experienced G-CSF-induced fatigue, insomnia, bone pain, or headache, although in only 8 percent were these effects considered severe. Peak mean leukocyte counts after filgrastim were 45 plus or minus 12 x 10 to the ninth power/L, and postapheresis thrombocytopenia (less than 100 x 10 to the ninth power/L) occurred in two of 13 donors (15 percent), both of whom underwent two procedures. The mean time to complete recovery from PBSC donation was 1 week, compared with 3 weeks for marrow harvest. Eight of 13 donors preferred G-CSF-stimulated apheresis donations to marrow harvest due to the lack of need for anesthesia and hospitalization; discomfort of the two procedures was considered equivalent. Recipient outcomes, including time to engraftment, GVHD incidence and severity, and overall survival have not yet been evaluated. Administrative and statistical support for this study is provided by the NMDP National Office. Filgrastim is provided under an IND agreement with Amgen (BB-IND #6821).
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02093-04 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Effects of Granulocyte Colony-stimulating Factor on Granulocyte Donors
Principal Investigator:
D.F. Stroncek, M.D.
DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
S.F. Leitman, M.D., DTM
E.J. Read, M.D., DTM
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 (GCSF), with or without dexamethasone, is the standard agent for mobilizing cells in granulocyte donors. The purpose of this study was to compare the effects of these agents on donors and to determine the minimum interval between donations. In this prospective, randomized, double-blinded, and placebo controlled investigation 20 donors were studied three times. Once the donors were given dexamethasone (8 mg, PO), once G-CSF (5 ug/kg SQ) and once both agents. Granulocyte concentrates were collected by apheresis 12 to 18 hours after the mobilizing agents were given. The donors were assessed and blood tests were performed before the collection, the day of the collection, and 1, 2, 7, 14, 21, 28, and 35 days after the collection. More granulocytes were collected when G-CSF was given than when was dexa-methasone was given (41.5 plus or minus 20.5 x 10 to the ninth power versus 22.2 plus or minus 11.2 x 10 to the ninth power, p less than 0.001). The most granulocytes were collected when G-CSF plus dexamethasone were given (65.6 plus or minus 24.2 x 10 to the ninth power, p less than 0.002). When dexamethasone was given, 58 percent of the donors experienced at least one symptom compared to 85 percent when G-CSF was given and 75 percent G-CSF plus dexamethasone was given. Similar proportions of donors in each group experienced fatigue (26 to 35 percent), insomnia (25 to 35 percent), and bone pain (21 to 30 percent). When G-CSF alone or with dexamethasone was given the donors experienced more headache, joint ache, nausea, diarrhea, and sore throat than when only dexamethasone was given. Following the collection, platelet counts fell and remained below baseline levels for 7 to 14 days. The hemoglobin levels also fell and remained below baseline levels for 7 to 28 days. For all three mobilization regimens the leukocyte counts returned to baseline levels within 3 to 7 days, but a mild neutropenia recurred 21 days post donation. When donors were given each of the three mobilizing regimens the following blood chemistries fell: albumin, potassium, calcium, magnesium, bilirubin, SGOT, cholesterol, and triglycerides. When donors were given dexamethasone alone or dexamethasone plus G-CSF, sodium, chloride, and glucose levels rose, but bicarbonate and creatinine levels fell. Calcium, uric acid, and triglycerides levels did not return to baseline levels until 21 days following the collection; albumin, bilirubin, and SGOT until 28 days; and cholesterol, until 35 days after the collection. The administration of G-CSF plus dexamethasone results in greater granulocyte concentrate yields, and causes no more symptoms than G-CSF alone. The administration of all three mobilization regimens and the collection of granulocyte concentrates caused mild changes in blood counts and blood chemistries for up to 28 days after the collection. At least 4 weeks should separate donations to allow blood counts and chemistries to return to baseline levels.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02095-04 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Structure and Function of Granulocyte Antigens
Principal Investigator:
D.F. Stroncek, M.D.
DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
K. Matsuo, M.D., DTM
J. Procter, DTM
Collaborating Unit:
None
Staff-Years:
0.5
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The Fc-gamma-receptor IIIb (FcgRIIIB) genes that encode neutrophil-specific antigens NA1 and NA2 differ at five nucleotides, four that result in amino acid differences among the two alleles. The role of each of these amino acid differences in antigen expression is not known. People with FcgRIIIB genes that differ from the NA1-FcgRIIIB and NA2-FcgRIIIB by a single nucleotide have been described. The purpose of this study was to compare the expression of NA1 and NA2 on granulocytes among people with variant FcgRIIIB genes and healthy blood donors. Reactions of NA1- and NA2-specific monoclonal and alloantibodies with granulocytes were assessed using flow cytometry in 74 healthy blood donors and six people with known variant FcgRIIIB genes. The granulocytes were tested with one NA1-specific monoclonal antibody, one NA2-specific monoclonal antibody, 4 NA1-specific alloantibodies and 4 NA2 specific alloantibodies. Analysis of the granulocytes from people with variant NA genotypes revealed that single base substitutions in FcgRIIIB at 141 and in FcgRIIIB at 349 are important in the expression of NA1 and single base substitutions in FcgRIIIB at 227 and 277 are important in the expression of the NA2. Among blood donors, age, gender, or race did not affect the expression of NA1 and NA2. The NA2-specific monoclonal antibody reacted more intensely with granulocytes from NA2-homozygous cells than heterozygous cells but this was not true for NA2-specific alloantibodies. There was no difference in the mean reactions of the NA1-specific monoclonal and alloantibodies among cells from NA1-homozygous and NA-heterozygous donors. The intensity of reactions of both the NA1-specific monoclonal and alloantibodies were strongly correlated on homozygous cells but not heterozygous cells as were the reactions of the NA2-specific monoclonal and alloantibodies. In fact, granulocytes from seven healthy blood donors who phenotyped as NA-heterozygous with the monoclonal antibodies were phenotyped as NA2-homozygous with the alloantibodies. It is known that variations in FcgRIIIB are common in African Americans, but five of these donors were Caucasian. These results suggest that variations in FcgRIIIB may be common in both Caucasians and African Americans. The expression of NA2 is dependent on the polymorphisms in FcgRIIIB 227 and 277 both of which are involved in an FcgRIIIB N-glycosylation site. Polymorphisms in FcgRIIIB at 141 and 349 appear more important to the expression of NA1.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02097-02 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Ex Vivo Culture and Characterization of Dendritic Cells for Clinical
Immunotherapy Trials
Principal Investigator:
E.J. Read, M.D. (Chief)
Cell Processing Section, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
E. Wong, M.D., CPS, DTM
T. Trischmann, Ph.D., CPS, DTM
C. Carter, CPS, DTM
J. Lee, CPS, DTM
K. Hines, CPS, DTM
Collaborating Units:
Metabolism Branch and Pediatric Oncology Branch, NCI
Nexell, Inc.
Staff-Years:
1.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The goal of this project is to develop and evaluate methods for manufacturing dendritic cells (DCs) for clinical immunotherapy trials. In FY 1999, we developed and optimized a full-scale GMP method for 5-day flask culture of autologous DCs in RPMI, autologous plasma or allogeneic serum, IL4 and GMCSF, starting with peripheral blood monocytes collected by apheresis and purified by elutriation. The immature DCs generated are then available for further manipulations (e.g., peptide pulsing) prior to clinical administration. This manufacturing method has been successfully used in three clinical trials, two for pediatric sarcoma and one for colon cancer. A manuscript describing this method has been accepted by Cytotherapy. Because of our interest in developing closed systems and eliminating reagents that are difficult to standardize, we evaluated a 7-day culture system in a protein-defined, serum-free medium (XVIVO15) starting with monocytes from elutriation vs. negative immunomagnetic selection using the Isolex 300I, in bags vs. flasks. We demonstrated that the 2 different isolation methods for monocytes product equivalent immature DC populations, and that bags were equivalent to flasks. Furthermore, historical comparison showed that serum-free medium was equivalent and perhaps even superior to serum-containing medium for generation of immature DCs. A manuscript describing this work is in preparation. Ongoing development studies are now evaluating culture conditions for generating mature DCs using CD40 ligand after culture in IL4 and GMCSF; this will result in a process that will be put into clinical trial by early FY 2001. This project has required simultaneous development and evaluation of assays for quantitation and characterization of these cell populations. Extensive flow cytometric phenotyping of these cultured cells has led to the development of a flow cytometric panels that will continue to be evaluated in the context of clinical trials.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02098-02 DTM
October 1, 1998 to September 30, 1999
Title of Project:
Storage of Granulocyte Concentrates
Principal Investigator:
D.F. Stroncek, M.D.
DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
S.F. Leitman, M.D., DTM
T. Lightfoot, M.D., DTM
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: High cell counts in granulocyte concentrates especially granulocyte colony-stimulating factor (G-CSF) mobilized granulocytes are detrimental to concentrate storage. An eight-fold dilution with autologous plasma improves storage, but this method is impractical. Granulocytes collected from donors given dexamethasone (8 mg PO) and/or G-CSF (5 mg SQ) were diluted eight-fold in plasma, cell culture media: X-Vivo 10, Dulbecco's Modified Eagle's Medium (DMEM), Iscoves Modified Dulbecco's Medium (IMDM) or infusible solutions: Plasma-Lyte A, Normosol R, and lactated Ringer's, supplemented with 1 percent human serum albumin and 50 mM histidine (LRAH) or Plasma-Lyte A supplemented with 50 mm histidine or 25 mM HEPES buffer plus 1 percent human serum albumin. The granulocytes were stored for 48 hours at room temperature. White blood cell (WBC) counts, WBC viability, and pH were measured after approximately 2 hours, 24 hours, and 48 hours of storage. Cell counts, viability, and pH were maintained after 2 hours, 24 hours, and 48 hours in cells stored in the three cell culture media. The pH and cell counts fell after 24 hours and 48 hours in granulocyte concentrates stored in Plasma-Lyte A and Normosol. The cell counts of concentrates diluted in LRAH were stable for 48 hours. The pH fell in cells diluted in LRAH, but was greater than granulocyte concentrates diluted in Plasma-Lyte A or Normosol. The pH of granulocyte concentrates diluted with Plasma-Lyte A plus histidine or HEPES was better maintained than the pH of granulocytes diluted in Plasma-Lyte A alone. Cell counts were better maintained in granulocyte concentrates diluted in Plasma-Lyte A plus albumin and histidine or HEPES than those in diluted with Plasma-Lyte A alone or in Plasma-Lyte A plus histidine or HEPES. Infusible solutions are not buffered adequately, but infusible solutions such as lactacted Ringer's solution or Plasma-Lyte A supplemented with buffers and albumin hold promise as an effective solution for granulocyte storage.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02099-02 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Kinetics of Granulocyte Colony-stimulating Factor-induced Granulocyte Mobilization
Principal Investigator:
D.F. Stroncek, M.D.
DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
S.F. Leitman, M.D., DTM
T. Lightfoot, M.D., DTM
Collaborating Unit:
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: The administration of granulocyte colony-stimulating factor (G-CSF) to increase the white blood cell count in granulocyte donors prior to donation is becoming an increasingly common practice. G-CSF is given subcutaneously to the donor on the day prior to donation, generally 12 to 24 hours before the start of apheresis. It would be advantageous to be able to give G-CSF and collect granulocytes on the same day. However, the single most important factor in optimizing granulocyte collection is the donor's pre-collection granulocyte count. Therefore, any decrease in count would result in a less cellular component. The purpose of this study is to assess granulocyte counts in healthy subjects during an 8-hour period after a single 5 mg/kg intravenous dose of G-CSF with or without dexamethasone. Sixteen subjects will be studied. Each donor is being studied four separate times. The four mobilization protocols are G-CSF 5 mg/kg given intravenously, G-CSF 5 mg/kg subcutaneously, G-CSF 5 mg/kg intravenously plus dexamethasone 8 mg orally, and G-CSF 5 mg/kg subcutaneously plus dexamethasone 8 mg orally. The order of the route of administration is being assigned randomly. White blood cell counts, neutrophil counts and donor symptoms are being measured before G-CSF administration and at 1/2, 1, 2, 4, 6, 8, and 24 hours after administration. The neutrophil counts measured within the first 8 hours after G-CSF are being compared with counts measured 24 hours after G-CSF. This study has just begun; four subjects have been studied. Preliminary results suggest that the granulocyte counts at 6 and 8 hours after the administration of the agents are similar to the counts after 24 hours, granulocyte mobilization with intravenous and subcutaneous G-CSF is similar, granulocyte mobilization with G-CSF plus dexamethasone is better that mobilization with G-CSF alone.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02100-02 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Citrate Effects and Calcium Replacement Therapy During Large-volume Leukapheresis
Principal Investigator:
S.F. Leitman, M.D. (Chief)
Blood Services Section, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
C.D. Bolan, M.D., LTC MC USA, DTM
Collaborating Units:
Chemistry Lab, CPD, CC (N. Rehak, M.D.; A. Remaley, Ph.D.)
Office of the Director, CC (R. Wesley, M.D.)
Staff-Years:
0.7
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Citrate toxicity limits rate of performance and duration of peripheral blood stem cell (PBSC) collections and other large volume leukapheresis (LVL) procedures. Knowledge of the metabolic response to citrate infusions and intravenous calcium (Ca) administration during LVL could increase both donor safety and product yields. We performed detailed clinical and laboratory monitoring in 29 allogeneic donors donating PBSC products for clinical transplant use. Twelve to 25 liters of blood were processed at standard (group A) or high (group B) citrate infusion rates. Citrate-related symptoms were scored as barely noticeable (1), irritating (2), uncomfortable (3), or unbearable (4). Subjects in Group A underwent LVL at standard citrate infusion rates of 1.0, 1.2, 1.4, and 1.6 mg/kg/min (24 total donors, six at each infusion rate). Calcium infusions were administered in group A only for symptom scores greater than or equal to 2. Group B subjects underwent LVL using high citrate infusion rates of 1.6 to 2.5 mg/kg/min, with prophylactic Ca administered at a dose of 0.45-0.6 mg Ca per ml ACD-A (23 total donors, five to six at each infusion rate). Calcium infusions were randomized between equimolar Ca chloride or Ca gluconate. The incidence of grade 1 and 2 citrate-related symptoms in group A increased with increasing citrate infusion rates, as follows: 1/6 and 0/6 donors at 1.0, 3/6 and 1/6 donors at 1.2, 4/6 and 2/6 donors at 1.4, and 5/6 and 2/6 donors at 1.6 mg/kg/min. In contrast, no donor in Group B, the cohort receiving prophylactic Ca infusions, had level 2 symptoms, and only three Group B donors had level 1 symptoms, despite receiving markedly high citrate infusion rates. The mean nadir decrease in ionized Ca was –30 percent in group A and –11 percent in Group B at comparable citrate infusions rates of 1.6 mg/kg/min. Ionized Ca and Mg concentrations were highly correlated with blood citrate levels, which increased with increasing citrate infusion rates and continued to increase throughout the LVL procedure, despite metabolic clearance and urinary excretion. Marked decreases in ionized Mg (20 to 40 percent) occurred, but were not correlated with symptoms. Post-/pre-apheresis urine Ca and Mg ratios became elevated at citrate infusion rates greater than 1.2 mg/kg/min. Equimolar infusions of Ca chloride and Ca gluconate had similar efficacy in mitigating nadir Ca concentrations and preventing symptoms. These studies demonstrate that citrate levels are the main determinant of ionized Ca and Mg levels during LVL. Citrate not only complexes blood Ca and Mg ions, but significantly increases urinary Ca and Mg excretion. Prophylactic Ca infusions, according to a preset algorithm, prevent citrate toxicity and allow higher blood processing rates. These experiments have allowed larger volumes of blood to be processed in shorter times, thus increasing cell yields for transplantation, and have become the basis for new DTM standard operating policies designed to improve collection efficiency, cell yield, and donor comfort during LVL.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02101-02 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Therapy of Von Willebrand's Disease with Single-donor Cryoprecipitate Collected
by Apheresis
Principal Investigator:
S.F. Leitman, M.D. (Chief)
Blood Services Section, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
G.J. Pomper, M.D., DTM
E.J. Read, M.D., Chief, Cell Processing, DTM
Collaborating Unit:
Hematology Section, CP, CC (M.E. Rick, M.D.)
Staff-Years:
0.2
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Von Willebrand's Disease (vWD) is the most common inherited bleeding disorder. We have studied the efficacy and feasibility of treating a child with type III severe vWD solely with cryoprecipitate prepared by repeated DDAVP-stimulated plasma exchange donation from a single, dedicated, paternal donor. An infant presented with massive hemorrhage at circumcision. The child was found to have FVIII:C 4 percent, FVIII:Ag 20 percent, vWF:RCo 21 percent, vWF:Ag 3 percent, indicative of severe vWD. His father carried an allele with a defect at the level of vWF mRNA expression but had a negative bleeding history with normal coagulation values. Cryoprecipitate was prepared from serial DDAVP-stimulated plasma exchange donation using peripheral venous access, ACD-A anticoagulant, and autologous cryoprecipitate-depleted plasma as replacement fluid. Exchange volume was 9,620 plus or minus 2,191 (m plus or minus SD, range 4,715-13,500) ml during the first 37 plasma exchange donations that the donor made during the 13 years since the child's birth. Repeated plasma exchange donation was well tolerated, with adverse effects including mild headache and flushing due to the DDAVP, and citrate toxicity. Cryoprecipitate was stored for less than 1 to 102 months at –70 (C. 90 percent of the cryoprecipitate was transfused after 1 year of storage, with a mean collection to transfusion interval of 2.4 years. Cryoprecipitate tested after 13 to 77 months of storage showed 48 to 124 percent of the original FVIII activity; decreased activity was noted with increasing length of storage. Over 13 years, 114,309 units of FVIII were collected in this manner. Most recently, we have standardized plasma exchange donation to involve processing of exactly 4,500 ml of plasma, which yields a mean of 14 bags of cryoprecipitate, each having a FVIII content of 262 plus or minus 62 units). Manufacture of plasma exchange donation-derived FVIII resulted in an estimated 50 percent cost reduction compared with similar doses of commercial factor concentrates. All bleeding episodes that occurred in the patient since birth were successfully managed with cryoprecipitate derived by this method. Remarkably, at age 13, the child has received only one donor exposure throughout his entire life, that of the paternal donor of his cryoprecipitate. Cryoprecipitate prepared by repeated plasma exchange donation of a vWD carrier provided excellent hemostatic function, even after prolonged storage intervals of greater than 1 year. Plasma exchange donation of a committed donor may be the safest option for long-term management of vWD, and provides a cost-effective alternative to commercial factor concentrates.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02102-01 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Malarial Anemia
Principal Investigator:
D.F. Stroncek, M.D.
DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
J. Procter, MeEd, M.T. (A.S.C.P.), SBB
Collaborating Units:
NIAID (A. Magill, M.D.)
Malaria Program, Naval Medical Research Center (S.L. Hoffman, M.D.; T. Jones,
Ph.D.)
Staff-Years:
0.1
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Malaria remains a significant health problem in tropical countries. Malaria is the leading cause of death in African children less than 5 years of age. In hypoendemic areas, cerebral malaria is most problematic and affects children between 36 and 60 months of age. In holoendemic areas, such as sub-Saharan Africa, severe malarial anemia is the leading cause of death in children between 6 and 24 months of age. Anemia in these children does not appear to be related to severe infection, since only a small fraction of all red blood cells (RBCs) are infected. The anemia appears to be due to hemolysis and to be immune-mediated. Severely anemic children with P falciparum infections often have a positive direct antiglobulin test (DAT) due to RBCs coated with IgG and complement. P falciparum infections also change the membranes of RBCs. The expression of complement regulatory proteins CR1 (CD35) and decay accelerating factor (CD55) are decreased on RBCs from children with severe P falciparum anemia, but the expression of the RBC membrane inhibitor of reactive lysis (CD59) is increased. These results suggest that both autoantibodies and RBC membrane changes may contribute to the severe malaria anemia of childhood. The purpose of this study is to investigate the role of RBC autoantibodies and RBC membrane changes in malarial anemia. In collaborative studies with Dr. Stephen Hoffman and Dr. Trevor Jones, malarial anemia is being studied in an aotus monkey model. Monkeys previously immunized by infection or vaccination to P falciparum protein EBA-175 were challenged with low levels of parasites. Animals that experienced low levels or parasitemia experienced a 50 percent fall in hematocrit over 7 to 14 days. The DAT was used to assess the binding of IgG to the RBCs, but none was detected. Further studies are underway which will investigate anemia in infected aotus monkeys using a DAT that will detect C3d and lower levels of IgG. In other studies, in order to assess the effects of P falciparum RBC antigens, RBCs collected from healthy donors will be infected in a laboratory setting with P falciparum. Following infection and culture of the RBCs with P falciparum, RBCs containing parasites will be separated from uninfected RBCs and these RBCs will be tested with a panel of monoclonal and alloantibodies to determine if RBC antigens are changed. The expression of antigens on RBCs that were in culture with P falciparum will be compared with the expression of antigens on RBCs from the same person that were not exposed to P falciparum.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02103-01 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Adoptive Immune Therapy for Cytomegalovirus Infection
Principal Investigator:
D.F. Stroncek, M.D.
DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
M. Provenzano, M.D.
S.-W. Kwon, M.D.
Collaborating Unit:
NCI (F. Marincola, M.D.)
Staff-Years:
1.0
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Cytomegalovirus (CMV) infections remain a serious problem in hematopoietic stem cell transplant patients. Following transplantation CMV infections can cause peumonititis, hepatitis, enteritis, and marrow failure. CMV seropositive transplant recipients can be treated with antiviral agents such as ganciclovir at the onset of infection or at the time of stem cell engraphment, but ganciclovir therapy is associated with renal toxicity and suppression of neutrophil counts. Preliminary studies have found that adoptive immunetherapy using CMV-reactive cytoxic T lymphocytes (CTL) may be an effective and less toxic alternative to prevent CMV infection in seropositive recipients of marrow transplants. The purpose of this study is to develop new treatment strategies for producing CMV-reactive CTLs that can be used for adoptive immunetherapy and to better understand the cellular immune response to CMV. CMV contains over 200 proteins, but several investigators have shown that the CTLs in most HLA-A*0201 CMV seropositive healthy donors reacted most intensely with a single CMV protein, pp65 a lower matrix protein. Furthermore within pp65, HLA-A*0201 CTLs from most people recognize only a single peptide, a nanomer pp65 495-503. Currently, most protocols being developed to treat CMV with donor-derived CTLs are using pp65 or pp65 495-503 to expand CMV-reactive CTLs from HLA-A*0201 donors in vitro. We used peripheral blood mononuclear cells collected by apheresis from HLA-A*0201 CMV seropositive donors and cell culture techniques to confirm that pp65 495-503 can be used to expand CMV reactive CTLs. We also developed a method to detect the activation of CMV-reactive CTL by CMV peptides without using long-term cultures. This technique detects lymphocyte activation by measuring interferon gamma production using quantitative real time PCR. Within 3 to 6 hours of adding CMV pp65 495-503 to lymphocytes from CMV seropositive HLA-A*0201 donors, elevations in interferon-g mRNA levels can be detected using real time quantitative PCR. This technique allows the rapid assessment of other peptides within CMV protein pp65 for their ability to stimulated lymphocytes from seropositive donors with HLA-A*0201 genotypes and other HLA types. This technique has been used to identify a new CMV pp65 epitope recognized by HLA-A*24 CTLs. This technique is also being use to identify epitopes within other CMV proteins recognized by CTLs.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02104-01 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Molecular Testing Standards and New Amplification Approaches
Principal Investigator:
J.W.-K. Shih, Ph.D.
IDS, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
R.Y.H. Wang, Ph.D., DTM
Collaborating Units:
Dept. of Pathology, Univ. of Maryland (C. Hsu, Ph.D.)
Medical Analysis Systems (Camerillo, CA)
Staff-Years:
0.2 (NIH only)
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: There are two components within this program. The first is to produce an ideal internal standard that resembles the target encapsulated viral particles being tested. In order to control all processing steps in molecular detection, this standard should have the same composition of target sequence and use the same primer set for amplification. This internal standard would be most valuable in assuring the validity of negative specimens during large-scale screening assays. The second component is to develop a new molecular amplification platform by combining two independent technologies using Mut-Y mismatch enzyme and TCR target cycling-based amplifications. These two technologies were brought together by a three-way CRADA. The end point for this collaboration is to develop a prototype test and to demonstrate its utility with clinical specimens.
We have constructed a particulate HCV internal standard (IS) based on murine amphotropic retrovirus. To achieve this, we went through a stepwise process including mutating HCV genome by inserting a 36 nucleotide base at nt 272 position in the 5'-UTR and then creating a retroviral vector clone pXT-HCV-NCC-D8 containing 948 bases of the HCV sequence. This vector was used to transfect a retrovirus packaging cell line, PA317.
From the transfected cells, G418 resistant recombinant retrovirus producer clones were established and further characterized. Using sequence specific primers, we were able to show that HCV sequence-containing particles were produced. Both wild type clones with HCV sequence and mutant clones with additional inserts were prepared and isolated. We were able to determine the insertion sequence length as expected by gene analyzer.
One preparation of virus supernatant from a high virus producer, D8-54 was evaluated extensively. The relative copy number per milliliter was determined by different methods including RT/PCR titer, electron microscope particle counting, infectious colony-forming counts, and end-point infectious titer. We found consistent results with different methods of determination. This demonstrated that this approach could provide ideal particulate IS for HCV. We were also able to apply this IS to a small-scale study with clinical specimens. We were also able to develop a convenient EIA detection system based on insertion specificity. NIH filed a U.S. patent based on this work.
For the second part of this project in finding new amplification approaches, in collaboration with Dr. Hsu of the University of Maryland, we found unique substrate specificity of Mut-Y enzyme that can recognize both DNA and RNA mismatches. The release mechanism for enzyme-substrate complex was determined. The cofactors, which enhance the turnover of substrate-product, were found. Potential target sequences on different strains of HIV were selected and specific probes were designed and synthesized. Ten- to thousand-fold amplification was demonstrated by estimating the probe products. Specificity was shown by a narrow range of strain specific recognition. A U.S. patent was filed and pending jointly by the University of Maryland and NIH based on these observations. To commercialize this patent, an industry partner capable of developing this technology was sought. Medical Analysis System of Camerillo, California presented the target cycle reaction (TCR) technology as an ideal partner. Combination between Mut-Y enzyme and TCR was shown to be compatible. High level of amplification was demonstrated. A U.S. patent based on the conditions set forth by the CRADA is being prepared for this combined technology.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02105-01 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Characterization of Human Pathogenic Mycoplasma from HIV-infected Patients
Principal Investigator:
J.W.-K. Shih, Ph.D.
IDS, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
R.Y. Wang, Ph.D., DTM
S.-C. Lo, Ph.D, M.D., AFIP
C. Haley, B.S., AFIP
Collaborating Unit:
None
Staff-Years:
0.2 (NIH only)
Human Subjects:
(a) Human subjects (b) Human tissues x (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 lab 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 the work on diagnosis and characterization of mycoplasma originating from AIDS patients, sexually transmitted diseases (STDs) patients, and others. We applied serological tests that were developed in this laboratory to patients in several clinical settings, including patients with HIV infection, nongonococcal urethritis (NGU), STDs, and intravenous drug use. 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 the paired donor-recipient specimens, we also found that M. fermentans and M. genitalium were transmissible through blood transfusion. We were able to show that the association of the presence of antibodies to M. genitalium with the sexual transmission of HIV was highly significant, while agents for other STDs were not.
In recent years, we were asked to support Dr. Lo's lab by providing serological tests on specimens from patients who suffered from the Gulf War syndrome or Gulf War infection (GWI). Over 6,000 paired specimens, including controls, were examined. We were not able to find any difference between soldiers who served in the Gulf War and their controls in antibody seroconversion to M. fermentans. To clarify a report that more than 50 percent of veterans with GWI had M. fermentans (strain incognitus) in their blood as measured by a molecular diagnostic technique called nuclear gene tracking, we conducted a large-scale, case-control study to compare the prevalence of antibodies to M. fermentans lipid-associated membrane proteins (LAMPs) between the Gulf War veterans with unexplained illness and a randomly selected, matched group of veterans who did not enroll in the registry for health evaluation. In addition, we analyzed, using banked serum samples obtained on each individual before and after the deployment, the rates of seroconversion for this mycoplasma in these two groups of veterans. Our results showed 4.8 percent of the cases and 5.2 percent of the controls tested positive for M. fermentans-specific antibodies before operation deployment. Most important, there was no difference in rates of seroconversion between cases and controls (1.1 vs. 1.2 percent) to M. fermentans during ODS. Thus, there is no serological evidence that suggests infection by M. fermentans is associated with development of GWI.
We also studied blood, urine, oral swabs, and rectal swabs for evidence of mycoplasmal infection by culture from a group of 149 Gulf War veterans who complained of various illnesses and were enrolled in the second phase of the health evaluation by the Army Comprehensive Clinical Examination Program (CCEP). None of the urine samples, oral swabs, or rectal swabs grew M. fermentans. No mycoplasma organism was isolated from any of the 149 blood samples. PCR study was conducted using RW oligonucleotide primer set (RW004 and RW005), based on the unique sequence of the M. fermentans insertion-sequence-like element. The amplified products were confirmed by southern blot using RW006 as the hybridization probe. Each sample was tested in triplicate at least three times. Three out of 65 (4 percent) blood samples were considered positive. Two of these three patients tested positive for M. fermentans antibodies in the serological study.
In conclusion, our culture study of ODS veterans with GWI revealed isolation of only mycoplasma organisms commonly found in similar samples from healthy individuals. No unusual mycoplasma was identified. Contrary to reported studies from some other laboratories, our PCR and serological studies showed only a low percentage of the veterans having evidence of M. fermentans infection.
One of the future interests for mycoplasma study is its contribution to the development of neoplasms after long-term, low-level chronic infection. We have shown that some species of mycoplasma were able to transform cells in vitro after long-term co-cultivation, and several indicative oncogenes were activated.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02106-01 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Large-volume Versus Standard-volume Leukapheresis for Peripheral Blood Stem
Cell Collection
Principal Investigator:
S.F. Leitman, M.D. (Chief)
Blood Services Section, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
C.D. Bolan, M.D., LTC MC USA, Research Fellow, DTM
E.J. Read, M.D., Chief, Cell Processing, DTM
Collaborating Unit:
Hematology Branch, NHLBI (R. Childs, M.D.)
Staff-Years:
0.3
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: There has been a dramatic increase in allogeneic hematopoietic transplant activity at the Clinical Center during the past 2 years, due to markedly increased numbers of peripheral blood stem cell (PBSC) transplants performed by NHLBI and NCI investigators. Nonmyeloablative transplant protocols piloted by NHLBI have targeted large numbers of CD34-positive stem cells in the donor graft, in order to (1) facilitate rapid and durable engraftment, (2) promote graft-versus-tumor activity, (3) hasten immunologic reconstitution, and (4) permit complex graft engineering. Meeting target PBSC CD34 cell doses requires large volume leukapheresis (LVL) in healthy donors. To optimize use of limited space and staff resources, and minimize donor discomfort and inconvenience, we studied the kinetics of CD34 mobilization, the apheresis product yield, and degree of resource utilization in very large volume vs standard volume leukapheresis. Allogeneic donors were randomly assigned to a single 25-liter PBSC collection or two consecutive daily 15L PBSC procedures. Donors received filgrastim 10 (g/kg/d SC for 5 (25 L x 1) or 6 days (15 L x 2). LVL was performed using a Baxter CS-3000 Plus with blood flow rate of 80-85 ml/min, ACDA ratio of 13:1, and prophylactic intravenous calcium chloride infusion. Blood CD34 counts were measured prior to, after each 5 L processed, and 1 hour after LVL. Seven donors were studied in each group. Donor gender, age, weight, and blood volume were similar in the two groups. However, day 5 preapheresis CD34 counts were higher in the group undergoing 15L LVL x 2 (78 vs 62 cells/(l, p less than 0.05). Despite the higher initial blood CD34 count, the overall CD34 cell yield in the single 25 L procedure that same as that in the two consecutive 15 L procedures (750 vs 820 million CD34 cells, p greater than 0.05). The minimum target dose of 3 x10 to the sixth power CD34 kg recipient weight was met in 7/7 25L x 1 and 6/7 15L x 2 procedures. Circulating CD34 counts decreased by 25 to 30 percent at 15L processed, but increased above pre-apheresis values one hour following LVL in all procedures. Day 5 lymphocyte counts prior to apheresis, and total lymphocyte yield, were the same in both groups. No donor in either group experienced citrate-related symptoms or other adverse effects of apheresis. These studies demonstrate that a single 25-L large volume leukapheresis for allogeneic PBSC collection results in similar CD34 product yields as two consecutive daily 15-liter procedures, but is more convenient for the donor, requires one rather than two hospital visits, less venipunctures, and one less dose of filgrastim. In addition, the single 25-L procedure significantly decreased apheresis staff time and space utilization and halved the costs of apheresis supplies and laboratory testing. These results have had substantial impact in changing DTM policies for the performance of LVL for allogeneic PBSC collection in both related sibling and matched unrelated donor settings.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02107-01 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Prospective Studies of Phlebotomy Therapy in Hereditary Hemochromatosis
Principal Investigator:
S.F. Leitman, M.D. (Chief)
Blood Services Section, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
C.D. Bolan, M.D., LTC MC USA, Research Fellow, DTM
C. Conry-Cantilena, M.D., Senior Staff Physician, DTM
Collaborating Unit:
None
Staff-Years:
0.4
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: The recent availability of a genetic test (homozygosity for the C282Y mutation in the HFE gene) for the diagnosis of hereditary hemochromatosis (HH) has focused renewed attention on this disorder and has resulted in the diagnosis being made in younger subjects with smaller iron burdens. However, phlebotomy therapy in HH remains hampered by lack of simple, physiologic laboratory monitoring guides. In addition, phlebotomy therapy has been perceived as wasteful because the blood obtained has not been not used for allogeneic transfusion although many HH subjects meet standards for allogeneic blood donation. Recent regulatory changes now allow increased flexibility in establishing policies for transfusion of blood obtained from HH subjects. In studies performed over the last 12 years, we have developed a simple, physiologically based phlebotomy guide as the basis for a protocol to utilize blood from appropriate hemochromatosis patients for allogeneic transfusion. Prospective studies in the DTM have shown that the red cell mean corpuscular volume (MCV), a physiologic, precisely measured indicator of erythopoietic iron availability, when measured in conjunction with the hemoglobin, provides all the information necessary to manage patients during induction as well as maintenance phlebotomy. Phlebotomy was started weekly and transitioned to less frequent intervals when the MCV decreased to 5 to 10 percent below baseline and the hemoglobin and MCV were decreasing n concert. During maintenance phlebotomy, the hemoglobin was targeted at greater than 13 g/dL and the MCV was kept at 5 to 10 percent below baseline (generally 85 to 90 (3). A stable, asymptomatic, iron-depleted state was obtained in all patients after a median of 38 phlebotomies and removal of 9.0 grams of iron. The MCV was stable for a prolonged period during induction therapy, and then decreased in a smooth, consistent manner, indicating the onset of iron-limited erythropoiesis. Transferrin saturation varied considerably during phlebotomy, but remained below 40 percent during stable MCV-targeted maintenance. Ferritin values were not useful to assess the pace of iron depletion or maintenance phlebotomy. The median stable maintenance interval was 7.5 (range 6 to 16) weeks, corresponding to an average daily iron removal of 35 to 67(g/kg. The data in this study indicate that the red cell MCV is a physiologic, inexpensive guide to phlebotomy therapy in patients with HH. In contrast to standard, more expensive measurements such as ferritin levels, the MCV accurately predicts iron depletion, prevents excessive rebound transferrin saturation and guides maintenance according to each patient's individual iron absorption. The MCV-guided phlebotomy guideline, along with extended rheumatologic and hepatic evaluations, forms the core of a large, omnibus protocol being developed to optimally manage phlebotomy in hemochromatosis patients while utilizing blood from appropriate patients for allogeneic transfusion.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02108-01 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Massive Immune Hemolysis in Peripheral Blood Stem Cell Transplants with Minor
ABO Incompatibility
Principal Investigator:
S.F. Leitman, M.D. (Chief)
Blood Services Section, DTM, CC, NIH
Other Personnel:
C.D. Bolan, M.D., LTC MC USA, Research Fellow, DTM
Collaborating Units:
Hematology Branch, NHLBI (R. Childs, M.D.)
BMT Unit, Hematology Branch, NHLBI (J. Barrett, M.D., Chief)
Staff-Years:
0.2
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors
(a2) Interviews
Summary of Work: Massive immune hemolysis due to minor ABO incompatibility is an underappreciated, potentially fatal complication of allogeneic hematopoietic transplantation. The increased lymphoid content and rapid engraftment seen with peripheral blood stem cell (PBSC) transplants may increase the frequency and severity of this event. In addition, newly developed nonmyeloablative conditioning regimens favor rapid and vigorous donor-type immune reconstitution, relying on donor lymphocytes to mediate both an anti-tumor effect and durable myeloid engraftment. To further the graft versus tumor effect, antiproliferative agents such as methotrexate are frequently omitted from post-transplant anti-GVHD regimens. We observed abrupt, catastrophic hemolysis in the first NIH patient to receive a nonmyeloablative PBSC transplant involving minor ABO incompatibility. We established a protocol for close clinical and laboratory monitoring of the next nine consecutive minor ABO-incompatible, nonmyeloablative PBSC transplants performed on NHLBI and NCI services. Cyclosporine alone was employed to prevent GVHD in all nine cases. Two additional cases of massive immune hemolysis were detected during daily serologic and laboratory monitoring. Hemolysis began 7 to 11 days following stem cell infusion. Both cases responded rapidly to vigorous hydration and prompt donor-compatible red cell transfusions, without adverse clinical consequences. All patients with hemolysis demonstrated a positive direct antiglobulin test (DAT), with eluate reactivity against the relevant recipient blood group (anti-A in two cases, anti-B in one). However, neither the intensity of the DAT nor the donor isohemagglutinin titer distinguished cases with from those without hemolysis. All three cases with hemolysis involved female donors, compared with only two of seven cases without hemolysis. These results demonstrate that isohemagglutinins produced by donor passenger B lymphocytes in minor ABO incompatible, PBSC transplants utilizing cyclosporine alone for GVHD prophylaxis can mediate massive immune hemolysis in a considerable proportion of subjects at risk. Immune hemolysis can adversely impact the otherwise improved safety profile of nonmyeloablative conditioning regimens. Serologic monitoring alone was not sufficient to determine cases with from those without hemolysis. Twice daily blood counts during the period at risk (day 6 to 11 post-transplant), careful monitoring of red cell serologic studies (DAT, IAT), prompt donor-compatible red cell transfusions, and improved awareness can avert serious complications due to minor ABO incompatibility and should be practiced in all such cases.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02109-01 DTM
October 1, 1999 to September 30, 2000
Title of Project:
Studies of Prophylactic Calcium Administration in Plateletpheresis Donors
Principal Investigator:
S.F. Leitman, M.D. (Chief)
Blood Services Section, DTM, CC, NIH
Bethesda, MD 20892
Other Personnel:
C.D. Bolan, M.D., LTC MC USA, DTM
J. Oblitas, MT, Manager, Platelet Center, DTM
Collaborating Units:
CPD, CC (N. Rehak, M.D.; A. Remaley, Ph.D.)
OD, CC (R. Wesley, M.D.)
Staff-Years:
0.4
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Citrate-related symptoms, due to decreased ionized calcium (iCa) levels, are common during plateletpheresis. The role of prophylactic oral calcium administration in preventing such symptoms has not been critically evaluated. We performed a randomized, placebo-controlled, double-blind study of the efficacy of prophylactic oral calcium administration in mitigating symptoms of citrate toxicity during plateletpheresis. Twenty-three donors underwent four 90-minute platelet-pheresis procedures each; performed every 4 to 8 weeks at constant citrate infusion rates of 1.5 mg/kg/min, using a Baxter CS-3000 device. Donors randomly received either calcium carbonate 1 or 2 gm (1C or 2C, supplied as TUMS 500 mg/tablet) or an identical-appearing placebo (1P or 2P) 30 minutes before donation. Symptoms were scored as 1 (barely noticeable), 2 (irritating), 3 (uncomfortable), or 4 (unbearable). Procedure flow rates were decreased by 16 percent for symptoms greater than or equal to grade 2 and stopped for grade 4 symptoms. Serum samples were analyzed before and after tablet ingestion, every 30 minutes during donation, and the day after donation. Urinary solutes were measured in both spot samples and 24-hour collections pre- and post-donation. Investigators were blinded until completion of all four procedures. Concentrations are in mmol/L, statistics are by 2-tailed McNemar and paired t-tests. At 90 minutes, serum total calcium (tCa) was higher after either oral calcium dose compared to placebo, 2.24/2.13 (2C/2P, p less than 0.000003) and 2.20/2.13 (1C/1P, p less than 0.0004), while the absolute and percent decrease in iCa was lower after 2 gm (2C/2P: 0.90/0.85 and 29/33 percent, p less than 0.0006) but not after 1 gm (1C/1P: 0.86/0.85 and 31/33 percent, p greater than 0.27) of oral calcium. Circumoral paresthesias improved significantly with the 2 gm oral calcium dose compared to placebo (unchanged in nine, better in 13, worse in two donors, p less than 0.008), with a trend toward decreased overall symptoms (6/8 grade 2, 4/6 grade 3, and 0/2 grade 4) after either dose of calcium compared to placebo (C/P). Serum citrate levels were equivalent in all groups. Post- versus pre-donation urinary spot calcium excretion increased markedly (greater than seven-fold) in all groups. Laboratory data revealed a trend toward a sustained calcium-avid state in donations without oral calcium: day-after parathyroid hormone (PTH) increased (17 to 34 percent) and urinary calcium excretion decreased (4 to 7 percent) after placebo (1P to 2P), while day-after PTH decreased (5 to 13 percent) and calcium excretion increased (13 to 23 percent) after oral calcium (1C-2C). These data show that ingestion of a commercially available preparation of oral calcium carbonate 30 minutes prior to plateletpheresis reduces donor symptoms and increases serum Ca levels. The more pronounced effect of the 2 gm versus 1 gm dose suggests that higher doses might show even greater efficacy. The changes in PTH response and calcium excretion over 24 hours suggest that future studies of bone metabolism and calcium balance in frequent platelet donors should be initiated.
Index:
Annual Report of Clinical Research Activities FY 2000
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