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Analytical Methodology: Development and Interpretive Application
Magnesium Metabolism in Humans and Biological Systems
Histidine-rich Glycoprotein: Characterization and Clinical Significance Studies
Assessment of Hydrogen Peroxide Generation in Neutrophils
Clinical Utility of Intravenous Catheter Tip Cultures for Short-term Lines
Detection and Identification of Microsporidia Organisms in Stool by Polymerase Chain Reaction
Identification of Molecular Defects in Patients with Von Willebrand's Disease
Survival of Microsporidia after Exposure to Disinfectants and Environmental Conditions
Use of Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Analysis for Identification of Mycobacteria and Nocardia Species
Development of Polymerase Chain Reaction for Cytomegalovirus, Herpes Simplex Virus, and Varicella-Zoster Virus from Spinal Fluid
Does Removal of Catheters Help Clear Catheter-related Gram-negative Bacteremias?
Development of a Polymerase Chain Reaction Procedure for Quantitative Measurement of Cytomegalovirus in Blood
Markers of Disease Activity in Idiopathic Inflammatory Myopathy
Quantitative Detection of Cytomegalovirus Using a Commercially Developed Research Assay
Polymerase Chain ReactionÐSingle-strand Confirmation Polymorphism for the Detection and Speciation of Microsporidia in Clinical Specimens
Comparison of Microbiologic and Cytologic Results for Bronchoalveolar Lavages
Detection and Identification of Mycobacteria in Clinical Specimens
Platelet-associated Antibodies in Patients with Autoimmune Thrombocytopenic Purpura
Assessment of Peripheral Blood Monocytes in Patients with Recurrent Mycobacterial Infection
Assessment of Lymphocytes in Patients with Autoimmune Lymphoproliferative Syndrome
Genetic Defect in Bernard-Soulier Syndrome
Genetic Abnormalities in Patients with Thrombophilia
Evaluation of an Alternative Pathway for Class I MHC Product-dependent Presentation of Oligopeptide Antigens to CD8 T Cells
The Experimental Treatment of Transfusion Dependent 5q Minus Syndrome with Leucovorin
Heparin Cofactor II Levels in Patients with Paroxysmal Nocturnal Hemoglobinuria
Sepsis in Immunocompromised Patients Caused by Helicobacter-like Organisms
Carbon Source Utilization for Identification of Rapidly Growing Mycobacteria
Biochemical Parameters of Coagulation and Fibrinolysis in Postmenopausal Women with Factor V Leiden or Hyperhomocysteinemia and in Control Women
Resistance to Multiple Fluoroquinolones in a Strain of Streptococcus Pyogenes: Detection of Point Mutations in the parC Subunit of DNA Topoisomerase IV and Gyrase A
Identification of Proteolytic Activity for von Willebrand's Factor
Identification of BCR-ABL in Patients with Chronic Myelogenous Leukemia
Development and Diagnostic Use of Rapid Immunoassays
Development of New Assays for Lipoprotein Testing
Mutation Analysis of Selected Lymphoid-immune Disorders
Analytical Performance and Clinical Utility of Thyroid Function Tests
Analytical Performance and Clinical Utility of Laboratory Tests for the Study of Atherothrombosis
Development and Clinical Application of Molecular Diagnostic Tests
Assessment of Memory B Cells in Immune Disorders
Assessment of B220 Expression on Human Lymphocytes
In Vitro Activities of Fluoroquinolone Antibiotics Against Blood Culture Isolates of Viridans Group of Streptococci from Bone Marrow Transplantation Patients at the NIH Clinical Center
Evaluation of a New Culture Medium for Borrelia Organisms
Antibiotic Susceptibility Profiles of Group B Streptococci Isolated from Neonates (1995-1998)
Viridans Streptococci Bacteremia in Allogeneic Bone Marrow Transplant Patients
Immunoassay of Tumor Markers from Tissue Obtained by Laser-capture Microdissection
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10001-25 CP
October 1, 1999 to September 30, 2000
Title of Project:
Analytical Methodology: Development and Interpretive Application
Principal Investigator:
N.N. Rehak, Ph.D. (Senior Staff)
CCS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
G. Csako, M.D., Senior Staff Physician, CCS, CP
S.A. Cecco, M.T., CCS, CP
Collaborating Unit:
None
Staff-Years:
0.1
Human Subjects:
(a) Human subjects (b) Human Tissue x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Two different mechanisms have been proposed by which dopamine and dobutamine cause negative interference for tests that use peroxidase-catalyzed oxidation of hydrogen peroxide for the generation of chromophores. We further investigated the mechanism of inhibition with dopamine, dobutamine, and structurally related compounds (L-dopa, L-methyldopa, and tyramine) on peroxidase-catalyzed reaction(s) for uric acid, creatinine, total cholesterol, and glycerol-blanked triglycerides. Pretreatment of samples with benzene-boronic acid (in up to 6 times excess of drug concentration) had no effect on the inhibition of the reactions. Spectrophotometric studies indicated that increasing peroxide concentration in the absence of drug or in the presence of a constant drug concentration initially increases then decreases absorbances. At a constant peroxide concentration, increasing drug concentration increased the absorbance. The products of the peroxidase-catalyzed reaction were stable. The complexity of interference with these drugs requires additional studies.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10010-24 CP
October 1, 1999 to September 30, 2000
Title of Project:
Magnesium Metabolism in Humans and Biological Systems
Principal Investigator:
N.N. Rehak, Ph.D. (Senior Staff)
CCS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S.A. Cecco, M.T., CCS, CP
C. Bolan, M.D., DTM S. Leitman, M.D., DTM
Collaborating Unit:
None
Staff-Years:
1.0
Human Subjects:
x (a) Human subjects (b) Human Tissue (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The secretion of parathyroid hormone (PTH) by the parathyroid gland is directly regulated by extracellular calcium ions (Ca2+) via the Ca2+-sensing receptor. However, there are reports of magnesium (Mg2+) effect on PTH secretion: acute hypermagnesemia can suppress PTH and it is suggested that the mechanism of suppression is similar to that of hypercalcemia. During platelet apheresis, the infused citrate forms complexes with both Ca2+ and Mg2+ ions and, therefore, decreases the concentration of the bioactive forms ("ionized" Ca and Mg) of both cations in the blood returned to the donor. The resulting hypocalcemia and concomitant hypomagnesemia could influence the PTH response and could be responsible for the citrate toxicity symptoms observed during apheresis.
We investigated the time course of changes in the concentrations of ionized Mg (iMg) and ionized Ca (iCa), and other electrolytes, which occur during large-volume leukapheresis (LVL) procedures in healthy donors, and their relation to the citrate toxicity symptoms. During the LVL procedure (duration from 2.5 to 6 hours, with and without Ca supplementation [IV Ca chloride, Ca gluconate]), the concentration of citrate increased and iCa, iMg, K, and Pi concentrations decreased. The maximum extent to which these analytes changed was as follows (mean; range): citrate (3.96 mmol/L; 1.14 to 8.64 mmol/L), iCa (-0.29 mmol/L; -0.06 to -0.47 mmol/L), iMg (-0.19 mmol/L; -0.07 to -0.28 mmol/L), potassium (-0.6 mmol/L; -1.2 to 0.1 mmol/L), inorganic phosphate (-0.27 mmol/L; -0.77 to 0.03 mmol/L). The time at which the maximum changes occurred was different for each LVL procedure. Highly significant correlations (p less than 0.0001) occurred between citrate and total and ionized Ca and Mg, inorganic phosphorus, and potassium. The concentration of intact PTH increased within the first 30 minutes of citrate infusion and then gradually declined during the LVL procedure. The maximum extent of the increase was mean, 105 ng/L; range, 9Ð258 ng/L. This study shows that, in addition to the physiologically important changes in serum iCa, the concentrations of iMg and potassium are also significantly affected by the citrate. We are investigating the effect of oral calcium supplementation on acute electrolyte changes during LVL procedures.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10220-08 CP
October 1, 1999 to September 30, 2000
Title of Project:
Histidine-rich Glycoprotein: Characterization and Clinical Significance Studies
Principal Investigator:
M.K. Horne, M.D. (Senior Investigator)
HS, CP, CC, NIH
Bethesda, MD 20892
Other Personnel:
C.-L. Wu, M.D.
Collaborating Unit:
None
Staff-Years:
2.5
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Histidine-rich glycoprotein (HRGP) is a multifunctional plasma protein of unclear physiologic significance. It binds not only proteins (plasminogen, fibrinogen, thrombospondin, vitronectin, immunoglobulin G, complement components) but also heparin, transition metals, and heme, and it binds to several types of cells (T-lymphocytes, macrophages, platelets). Therefore, it may be a modulator of fibrinolysis, an immunoregulator, and a carrier of trace metals. The interaction of HRGP with platelets and the ultimate effect of this interaction on platelet-dependent processes in fibrinolysis are areas of interest.
A method for purifying HRGP from fresh plasma was previously developed, and more recently have been using the protein in a variety of studies. So far, several types of interaction between HRGP and platelets have been discovered: (1) HRGP binds saturably to platelets when it is liganded to a transition metal (e.g., zinc); (2) plasminogen also promotes HRGP binding to platelets, although HRGP reduces the affinity of plasminogen binding to platelets; (3) zinc-liganded HRGP augments plasminogen binding to platelets; and (4) in the presence of HRPG (with or without zinc), platelet-dependent activation of plasminogen by tissue plasminogen activator is increased. These observations suggest a complex role for HRPG in regulating fibrinolysis in a platelet-rich fibrin clot.
The laboratory is also exploring the possibility of exploiting the heparin-binding capacity of HRGP on a clinical heparin antidote. The interactions of HRGP, heparin, and zinc are being studied in vitro using assays based on thrombin inhibition.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10222-08 CP
October 1, 1999 to September 30, 2000
Title of Project:
Assessment of Hydrogen Peroxide Generation in Neutrophils
Principal Investigator:
T.A. Fleisher, M.D. (Chief)
IS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Unit:
LHD, DIR, NIAID (H. Malech, M.D.)
Staff-Years:
0.1
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Previous work established that the flow cytometric dihydrorhodamine (DHR)-based assay for evaluation of oxidative burst is a sensitive method for evaluating phagocytes in patients with possible chronic granulomatous disease (CGD). It has also been useful as an accurate test for screening possible X-linked CGD carriers and as a preliminary screen for the genotype of the disease. The method has been optimized and is sensitive to one normal cell in 10,000 or fewer abnormal cells.
This project previously demonstrated the capacity to follow normal granulocytes after allogenic leukocyte transfusion in patients with CGD, and this assay has proven to be reliable in establishing duration of transfused cell survival and all-sensitization. The current phase of gene therapy of CGD continues to be directed at patients with X-linked CGD. The DHR assay is being applied as a critical method for establishing the presence of gene-corrected granulocytes in the circulation and for monitoring the duration of production of the gene-corrected cells in the treated patients.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10227-07 CP
October 1, 1999 to September 30, 2000
Title of Project:
Clinical Utility of Intravenous Catheter Tip Cultures for Short-term Lines
Principal Investigators:
V.J. Gill, Ph.D. (Chief)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
D. Landucci, M.D., CCM
F. Stock, B.S., MS, CPD
R. Cunnion, M.D., CCM
Staff-Years:
0.05
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Culture of intravenous catheter tips has been a routine practice for the past 10 years. Based on the findings of Maki et al. (1977), a quantitative technique based on rolling the tip on a blood agar plate and counting the resulting number of colonies has been a standard practice in most laboratories. The validity and value of these cultures has come into increasing debate, but few well-documented data have been published to clarify this issue.
As part of a larger line-maintenance protocol in the Critical Care Medicine Department, our study used these patients to look at colony counts and their predictive value in determining line sepsis from short-term central lines. Approximately 200 patients were enrolled in this study. Organisms from patients who have positive blood cultures and concomitantly positive catheter tip cultures were examined by molecular typing methods to determine if the catheter-tip isolates were identical to the blood-culture isolates, thereby supporting the diagnosis of catheter-related bacteremia.
Patient accrual was completed and molecular analysis of the strains of bacteria (strain typing) isolated from these patients was performed. The rate of infection following placement of these lines was low, which resulted in a limited number of episodes available for assessment. Preliminary review of the data suggested that, although patients with positive blood cultures commonly had more than 15 colonies on the rolled catheter culture, there were instances of positive blood cultures when the colony counts were less than 15. In addition, there were cases where the organism cultured from the catheter tip was not the same as that cultured from the blood, and instances where catheter-tip cultures yielded more than 15 colonies that were not associated with positive blood cultures. This project is considered completed.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10235-07 CP
October 1, 1999 to September 30, 2000
Title of Project:
Detection and Identification of Microsporidia Organisms in Stool by Polymerase
Chain Reaction
Principal Investigator:
D.P. Fedorko, Ph.D.
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
N.A. Nelson, MS, CPD
Collaborating Unit:
None
Staff-Years:
0.20
Human Subjects:
(a) Human subjects (b) Human tissues x(c) Neither
(a1) Minors (a2) Interviews
Summary of Work: There is considerable interest in determining the diagnostic value of stool specimens for detecting gastrointestinal disease caused by Microsporidia organisms, and a variety of staining modalities have been evaluated for their ability to visualize microsporidia in stool preparations. Unfortunately, the various species of microsporidia that infect humans can be identified accurately only by visualizing characteristic subcellular morphologic features of organisms in electron micrographs of tissue specimens. Because the efficacy of treatment regimens currently in development may be somewhat species-specific, and because invasive procedures are necessary to obtain appropriate specimens for electron microscopy (EM), a more widely available approach to identification than EM is required.
We have published a report which describes our polymerase chain reaction (PCR) assay for amplification of microsporidial rRNA genes followed by restriction endonuclease digestion of PCR products as a method for identifying microsporidia in fresh stool. We are continuing to modify the assay to allow detection of microsporidia in formalin-fixed stool specimens. A Southern blot format using PCR-amplified DNA and species-specific DNA probes has been developed for use in microsporidial speciation. The final stage of the project has been to determine the sensitivity of our assay and the length of time the specimens can be stored in formalin and still allow detection of the microsporidia. We have demonstrated that prolonged storage of microsporidia in formalin or polyvinyl alcohol reduces the chances of amplification by PCR. We suggest washing stool specimens after fixation in formalin and storage of the washed specimen in phosphate-buffered saline. We have developed a shorter, cheaper, and simpler method for extraction of microsporidial DNA from stool.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10241-07 CP
October 1, 1999 to September 30, 2000
Title of Project:
Identification of Molecular Defects in Patients with von Willebrand's Disease
Principal Investigator:
M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
D. Krizek, R.M.T., CP
Collaborating Unit:
None
Staff-Years:
1.0
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: One family that has an abnormal von Willebrand factor (vWf) with a defective binding site for factor VIII has been studied, and the genetic defect has been identified. The binding defect was initially evaluated by assessing the ability of the patient's vWf to bind purified factor VIII. Subsequently, DNA was purified from peripheral blood leukocytes, specific regions of the vWf gene were amplified by polymerase chain reaction (PCR), and direct sequencing of the DNA was carried out. A transition of nucleotide 2451 (T to A) was found, which results in the substitution of GLN for HIS at amino acid 54 in the mature vWf subunit. We recently used a PCR mutagenesis technique to insert the mutation into cloned DNA and have expressed the abnormal protein. The latter was tested in an assay for binding factor VIII and was shown to manifest decreased binding of factor VIII. A manuscript containing the expression data is in preparation.
Two unrelated patients with von Willebrand's disease and an abnormal distribution of vWf multimers have been studied, and one new mutation in the A1 region of the vWf gene has been identified in one family. The mutation was cloned into an expression vector and the expressed abnormal vWf is being characterized. The second family is being studied and appears, in preliminary studies, to have a previously unidentified mutation also in the A1 domain of vWf.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10244-06 CP
October 1, 1999 to September 30, 2000
Title of Project:
Survival of Microsporidia after Exposure to Disinfectants and Environmental
Conditions
Principal Investigator:
D.P. Fedorko, Ph.D.
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
N.A. Nelson, MS, CPD
Collaborating Unit:
None
Staff-Years:
0.15
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Microsporidia are ubiquitous parasites causing infections in insects, fish, and mammals. Recently microsporidia have been demonstrated to infect humans. These organisms cause ophthalmic and gastrointestinal infections, primarily in patients with AIDS. Several genera of human pathogens have been cultivated in cell culture. Presently there are no data regarding the ability of the human pathogens Encephalitozoon intestinalis and E. hellem to survive under various environmental conditions. Also, there are no reports regarding the effects of disinfectants on spores of these two species.
The survival of microsporidial spores after exposure to disinfectants, such as chlorine, alcohol, and quaternary ammonium compounds, and environmental conditions, such as elevated temperature and dessication, will be studied. Cultivation of microsporidia in the shell vial system using various cell lines has been investigated and microsporidia appear to replicate in both fibroblast and epithelial cell lines. A monoclonal antibody produced by Dr. T. Nash (NIAID, LPD) has replaced Giemsa staining for detection of infected cells. Although the monoclonal antibody makes detection of infected cells easier, the assay is still cumbersome and tedious. In an effort to replace staining of infected cell cultures, a method for quantitation of microsporidial DNA has been developed. This assay uses polymerase chain reaction (PCR) and a europium-labeled DNA probe and is semiquantitative. A recent report described a new cell line for E. intestinalis in which growth is extremely rapid; however, we found no enhanced growth compared with that of our routine cell line. We will use fluorescent antibody staining and PCR methods to study survival of microsporidial spores.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10247-06 CP
October 1, 1999 to September 30, 2000
Title of Project:
Use of Polymerase Chain Reaction and Restriction Fragment Length Polymorphism
Analysis for Identification of Mycobacteria and Nocardia Species
Principal Investigator:
F.G. Witebsky, M.D. (Senior Investigator)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
P.S. Conville, MS, CPD
S.H. Fischer, M.D., Ph.D., MS, CPD
Collaborating Unit:
None
Staff-Years:
0.80
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Polymerase chain reaction (PCR) amplification of a portion of the genome of both rapidly growing Mycobacteria and Nocardia species, followed by restriction fragment length polymorphism (RFLP) analysis of the amplification products, has proven to be a useful technique in the diagnostic laboratory. Identification of these organisms at the species level can be obtained within a few days of organism isolation, as compared with the month or more required for conventional identification based on biochemical testing. In addition, these molecular procedures allow more accurate discrimination among species and subspecies than is possible with biochemical testing. Our work with two different areas of the Nocardia genome (a portion of the gene for 16S ribosomal RNA and a portion of the gene for the heat-shock protein) has suggested the existence of hitherto unrecognized Nocardia species; work is ongoing to characterize these organisms further. We are just beginning a study of the feasibility of distinguishing among these organisms by high-performance liquid chromatography. A manuscript describing our methodology and summarizing our findings to date has been published.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10260-05 CP
October 1, 1999 to September 30, 2000
Title of Project:
Development of Polymerase Chain Reaction for Cytomegalovirus, Herpes Simplex
Virus, and Varicella-Zoster Virus from Spinal Fluid
Principal Investigator:
S.H. Fischer, M.D., Ph.D. (Senior Staff)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
G. Fahle, M.T., MS
V.J. Gill, Ph.D., MS
Collaborating Unit:
None
Staff-Years:
0.3
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Diagnosis of meningitis in immunocompromised hosts, such as those with AIDS or cancer, is difficult because of the unavailability of sensitive methods for detecting the most likely pathogens. Organisms in these settings, such as cytomegalovirus (CMV), Herpes simplex virus (HSV), and varicella-zoster virus (VZV), are difficult to grow from colony-stimulating factor (CSF), and no effective antigen detection techniques are available. Over the past few years, it has become increasingly clear that detecting these viral agents by the polymerase chain reaction (PCR) method may provide the most sensitive and specific diagnostic tests. Therefore, we have undertaken development of a battery of in-house PCR assays for CMV, HSV, and VZV so that they will be available as routine service tests for CSF from NIH patients.
In 1997, development and clinical validation of the PCR assay for CMV in spinal fluid was completed and the test began to be offered as a routine molecular diagnostic assay. During 1998, final development and validation of the PCR assay for HSV in spinal fluid was completed and the test is now also offered as a routine molecular diagnostic assay. The HSV assay on spinal fluid will specifically determine the presence of HSV1, HSV2, or both viral DNAs. Development of a VZV assay with the same performance characteristics as the existing CMV and HSV assays will continue. All three assays are designed to use internal mimics to evaluate the efficiency of amplification within individual PCR tubes and to detect PCR inhibition to eliminate this source of false-negative results. End-point detection by europium-labeled fluorescence hybridization probes allows a lower limit of detection of three to five viral genome equivalents. Evaluations of the CMV and HSV assays show excellent specificity.
The sensitivity and reliability of molecular diagnostic techniques depend not only on the efficiencies of the amplification and detection methods, but also on the performance of the sample preparation methods. In the past year, a large-scale project was undertaken to investigate the relative efficiencies and reliabilities of six different methods for preparing nucleic acids from human spinal fluid, respiratory samples, or blood. We have identified a sample preparation method that appears to be highly reliable and offers improved efficiency for DNA recovery. This method has now been incorporated into the clinical molecular diagnostic assays offered.
A PCR assay for the detection of VZV in patient CSF samples was developed and validated. This assay is now routinely offered for patient testing along with the previously developed CMV, HSV-1, and HSV-2 assays for spinal fluid.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10264-05 CP
October 1, 1999 to September 30, 2000
Title of Project:
Does Removal of Catheters Help Clear Catheter-related Gram-negative Bacteremias?
Principal Investigator:
V.J. Gill, Ph.D.
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
A. Freifeld, M.D., DCS, NCI
L. Shay, Internal Medicine, CC
F. Gill, M.D., Internal Medicine, CC
Collaborating Unit:
None
Staff-Years:
0.05
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Line-related bacteremias associated with long-term catheters (Hickman/Broviac, Groshong, pulmonary artery, and peripherally inserted central catheters) are being encountered regularly now that these catheters are used so commonly. Earlier studies indicated that these catheter-related episodes did not necessitate removing the offending line if the infecting organism was a coagulase-negative Staphylococcus organisms, most commonly S. epidermidis. Antibiotic treatment with the line left in place generally eradicated the organism. For other organisms, however, particularly gram-negative rods, this issue has not yet been resolved.
We would like to examine whether removal of the catheter results in more reliable initial clearance of the gram-negative rod than nonremoval, and also whether the incidence of recurrent infection with the same organism increases in patients in whom the catheter was left in place. Approximately 70 episodes of catheter-related, gram-negative bacteremia seen over the past 2.5 years are being examined to determine whether there are any significant differences between patients with catheters removed and those with catheters left in place.
The clinical collaborators on this project have had insufficient time to continue with this investigation. This project has been discontinued.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10265-05 CP
October 1, 1999 to September 30, 2000
Title of Project:
Development of a Polymerase Chain Reaction Procedure for Quantitative Measurement
of Cytomegalovirus in Blood
Principal Investigator:
S.H. Fischer, M.D., Ph.D. (Senior Investigator)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
G.A. Fahle, M.T., MS, CPD
V. Thaker, Ph.D., MS, CPD
S. Yan, Ph.D., MS, CPD
J. Canosa, M.T., MS, CPD
Collaborating Units:
CCM (H. Masur, M.D.)
DIR, NIAID (M. Polis, M.D.; J.E. Bennett, M.D.)
Staff-Years:
0.3
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Cytomegalovirus (CMV) disease is a relatively frequent, and often serious, complication in immunocompromised, CMV-infected patients. In the past few years, it has become apparent that to differentiate between subclinical viral shedding and large-scale viral replication, occurring during the prodrome before the onset of active disease, it is necessary to use sequential monitoring with a quantitative assay. Several studies have shown that CMV quantitative polymerase chain reaction (PCR) assays are more sensitive than buffy coat CMV antigen detection assays. This extra sensitivity can, in some cases, give an additional week of warning before the onset of CMV disease. Instituting antiviral therapy at an earlier time point in the prodromal stage may decrease the chance of the patient developing active CMV disease.
We have completed development of a competitive quantitative PCR assay for the detection of CMV in buffy coat cells. The assay can detect as few as three to five viral genome equivalents in an amplification reaction tube. The coefficient of variance of this assay is about 40 percent, in line with other published descriptions of assays of this type.
To have an assay with improved precision and, therefore, better potential predictive value for disease onset or progression, we have developed a real-time CMV PCR assay. This assay utilizes frequency resonance energy transfer fluorescence probes and is designed to run on the Roche LightCycler. Amplification and detection of the assay can be completed within 45 to 50 minutes.
In addition to continuing the developmental work on a real-time PCR assay, we have begun performing an evaluation of the Organon Teknika NASBA pp67 CMV assay. This commercially available assay amplifies CMV RNA in an isothermal reaction using reverse transcriptase and RNA polymerase. By targeting viral mRNA, it should permit detection of replicating virus without the generating positive signal from integrated viral DNA copies that may be present. We have collected sequential blood sample sets from several patients for an evaluation of the NASBA pp67 CMV assay performance against previously obtained pp65 CMV antigenemia results. We also will use the LightCycler real-time PCR to test these sample sets so that we can compare its performance with the other two methods.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10269-05 CP
October 1, 1999 to September 30, 2000
Title of Project:
Markers of Disease Activity in Idiopathic Inflammatory Myopathy
Principal Investigator:
M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
P. Merryman, M.T., CP
A. Cullinane, M.T., CP
Collaborating Units:
IRP, NIAMS (P. Plotz, M.D.)
CEBR/FDA (L. Rider, M.D.)
Staff-Years:
0.15
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors (a2) Interviews
Summary of Work: This study is designed to aid in the evaluation of clinical disease activity in patients with idiopathic inflammatory myopathies, a diverse group of diseases that include inflammation in skeletal muscle. Since the pathology includes primary muscle capillary endothelial cell damage, we have assessed markers of activation and injury to endothelial cells and activation of coagulation factors, including complexes of thrombin-antithrombin, plasmin-antiplasmin, tPa, and thrombomodulin. We have studied 38 patients and are currently analyzing the data to determine clinical correlations with disease activity. A subset of patient shows an increase in thrombin-antithrombin complexes, which is a sensitive assay for activation of coagulation factors.
This project has been awaiting input from investigators in NIAMS. Further testing is planned to resume in late summer 2000.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10277-04 CP
October 1, 1999 to September 30, 2000
Title of Project:
Quantitative Detection of Cytomegalovirus Using a Commercially Developed Research
Assay
Principal Investigator:
D.P. Fedorko, Ph.D. (Senior Investigator)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
L. Calhoun, M.T. (ASCP), MS, CPD
Collaborating Units:
American Medical Laboratories (R. Clark)
Microbiology Reference Laboratories (H.D. Engler)
Staff-Years:
0.2
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Cytomegalovirus (CMV) can be devastating in the patient who has undergone an allogenic bone marrow transplant. Rapid detection of CMV infection in these patients can be lifesaving, allowing the prompt administration of anti-CMV drugs. There is also a clinical need for a quantitative measure of the CMV present in the clinical specimen to allow for monitoring therapy and predicting emergence of drug-resistant variants. A research assay has been developed by a commercial vendor that allows the use of quantitative polymerase chain reaction (PCR) to measure the CMV DNA extracted from whole blood. We are evaluating this PCR assay using peripheral blood leukocytes collected by American Medical Laboratories and comparing the results with those of the CMV antigenemia quantitative assay (performed at American Medical Laboratories) that detects the presence of CMV pp65 early structural protein in polymorphonuclear leukocytes and monocytes. Specimens with discrepant results will be sent to Microbiology Reference Laboratories to be evaluated using a second PCR assay. Results thus far indicate that the commercial PCR is at least as sensitive as the CMV antigenemia assay. More specimens need to be tested for a statistically valid comparison to be made.
This project has been terminated.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10278-04 CP
October 1, 1999 to September 30, 2000
Title of Project:
Polymerase Chain Reaction—Single-strand Confirmation Polymorphism for the Detection
and Speciation of Microsporidia in Clinical Specimens
Principal Investigator:
D.P. Fedorko, Ph.D.
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
N.A. Nelson, MS, CPD
R. Delgado, CS, CPD
Collaborating Unit:
Tulane Univ. Medical Center (E.S. Didier, Ph.D.)
Staff-Years:
0.20
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: There are many options for the detection and speciation of microsporidia in clinical specimens. Light microscopy allows detection of the parasites, but does not allow speciation. Electron microscopy is the gold standard for speciation, but is not as sensitive a method for the detection of microsporidia. Polymerase chain reaction (PCR) is a sensitive technique with many different methods for confirming a positive result and for determining the genus and species causing an infection. Speciation can be achieved by using species-specific primers in the PCR assay, or by using DNA probes or restriction endonucleases to determine the species after the PCR assay is performed.
Single-strand confirmation polymorphism (SSCP) combined with PCR (PCR-SSCP) has been used to identify bacteria, fungi, and viruses. We will use our PCR assay for the detection of microsporidia in stool specimens and apply SSCP to determine the specific genus and species of the parasite. Organisms from cell culture will also be used to validate the method.
The project has been expanded to apply SSCP to the detection of microsporidial genotypes in epidemiologic studies. Newly published primers allow speciation when the PCR products are digested with restriction enzymes. SSCP analysis will make this task easier and more sensitive than restriction enzyme analysis. We have identified two different genotypes among the Enterocytozoon bieneusi detected in patients with microsporidiosis at the NIH, which will be used to finish this study.
This project has been completed.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10279-04 CP
October 1, 1999 to September 30, 2000
Title of Project:
Comparison of Microbiologic and Cytologic Results for Bronchoalveolar Lavages
Principal Investigator:
F.G. Witebsky, M.D. (Senior Investigator)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
F. Stock, B.S. MS, CPD
V.J. Gill, Ph.D., MS, CPD
Collaborating Unit:
Cytopathology Section, LP, DCS, NCI (A.D. Abati-Scott, M.D.)
Staff-Years:
0.05
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Bronchoalveolar lavage specimens are usually split between cytology and microbiology laboratory analysis. Because different methodologies are used by these two laboratories in the work-up of these specimens, we thought it would be useful to review the results obtained on these specimens by each laboratory. Such a review might help define the relative sensitivities of the different procedures used, suggest areas of redundancy that might be candidates for elimination, and help identify the procedures most likely to produce clinically significant results.
Results from the data analyzed thus far indicate that cytology preparations are more sensitive for the direct detection of significant fungal pathogens than the smears prepared in microbiology, presumably because of the larger volume of material used for preparation of smears in cytology. The data for approximately 7 years have been collected and partially analyzed to assess the relative sensitivities of the procedures performed in the two laboratories not only for fungi but also for the detection of other pathogens such as Mycobacterium tuberculosis and Pneumocystis carinii. Further analysis of the data has been temporarily postponed to deal with more pressing projects; we hope to complete the analysis within the next year, perhaps with the inclusion of recent data.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10281-04 CP
October 1, 1999 to September 30, 2000
Title of Project:
Detection and Identification of Mycobacteria in Clinical Specimens
Principal Investigator:
S.H. Fischer, M.D., Ph.D. (Senior Staff)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
P. Conville, M.S., MS, CPD
F.G. Witebsky, M.D., MS, CPD
G.A. Fahle, M.T., MS, CPD
J. Rampal, Ph.D., Beckman Corp.
Collaborating Unit:
None
Staff-Years:
0.3
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Detection and identification of acid-fast bacilli of Mycobacterium species by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. These methods may take days to 1 or more months. The development of a few highly specific molecular probes for testing cultures growing acid-fast bacilli has greatly reduced the time to identification of some mycobacterial isolates. Recently, the polymerase chain reaction and isothermal nucleic acid amplification techniques have been used in assays that offer a high degree of specificity and reasonable sensitivity for detection of Mycobacterium tuberculosis in clinical samples. At present, no commercially available amplification assay systems are capable of detecting multiple Mycobacterium species while excluding cross-reactive signals from other bacteria commonly present in clinical samples.
We have initiated a new effort to investigate the usefulness of a pressure cycling technique to improve the efficiency of nucleic acid release from mycobacterial cells.
The collaboration with Dr. Jang B. Rampal of Beckman Corporation to develop a better endpoint detection method for the 20-some most commonly encountered Mycobacterium species from human samples has continued. Experiments have been successfully performed with a method that simultaneously detects the presence or absence of nucleic acid amplification products from six common clinically isolated Mycobacterium species.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10283-04 CP
October 1, 1999 to September 30, 2000
Title of Project:
Platelet-associated Antibodies in Patients with Autoimmune Thrombocytopenic
Purpura
Principal Investigator:
M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Rivera, M.D.
M. Riordan, M.T., CP
K. Hansmann, M.T., CP
Collaborating Unit:
NHLBI (C. Dunbar, M.D.; R. Huhn, M.D.; R. Nakamura, M.D.)
Staff-Years:
0.25
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Autoimmune (idiopathic) thrombocytopenic purpura (ITP) is a disease caused by autoantibodies directed against platelets, but the demonstration of specific antibodies has been difficult for a variety of reasons. In general, when the antibodies can be demonstrated, there is an inverse correlation with the platelet count. We have set up two assays, one a screening assay and one an assay for specific platelet glycoproteins, to aid in the diagnosis and treatment monitoring of patients with ITP. We will use the tests particularly for the followup of patients before and after treatment in a pilot study with NHLBI in the treatment setting of T-cell-depleted auto-stem cell transplantation in patients with severe ITP. Sixteen patients have been studied. A verbal presentation was given at the national meeting of the American Society of Hematology in December 1999.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10286-04 CP
October 1, 1999 to September 30, 2000
Title of Project:
Assessment of Peripheral Blood Monocytes in Patients with Recurrent Mycobacterial
Infection
Principal Investigator:
T.A. Fleisher, M.D.
(Chief, Immunology Service) IS, CP, CC, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Unit:
LHD, DIR, NIAID (S. Holland, M.D.)
Staff-Years:
0.2
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Monocytes are being characterized for the expression levels of CD40, CD80, CD86, CD120b, and the gamma interferon receptor alpha chain. These studies have identified two patients with gamma interferon receptor alpha chain deficiency, and the molecular level of this defect is being actively examined. In addition, interferon gamma receptor beta chain deficiency has also been characterized in one patient at the molecular level. As a consequence of these studies, an intracellular flow cytometric method for evaluating STAT protein phosphorylation has been developed and validated. This approach continues to be applied to new patients and is also being used to evaluate patients after bone marrow transplantation.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10287-04 CP
October 1, 1999 to September 30, 2000
Title of Project:
Assessment of Lymphocytes in Patients with Autoimmune Lymphoproliferative Syndrome
Principal Investigator:
T.A. Fleisher, M.D. (Chief)
IS, CP, CC, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Unit:
LCI, DIR, NIAID (S. Straus, M.D.)
Staff-Years:
0.2
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors (a2) Interviews
Summary of Work: An extensive flow cytometric evaluation continues of patients with autoimmune lymphoproliferative syndrome (ALPS) and their extended family members, on the basis of characterization of the expanded double-negative T-cell and B-cell populations. Double-negative T-cells have been demonstrated to be alpha beta TcR, CD57+, HLA-DR+, and CD45RA+. This study has been extended to characterize the double-negative T-cells more completely including gamma-delta TcR T-cells in all ALPS patients. In addition, we have initiated expanded characterization of the B cells, directed at memory B cells, in these patients.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10289-03 CP
October 1, 1999 to September 30, 2000
Title of Project:
Genetic Defect in Bernard-Soulier Syndrome
Principal Investigator:
M. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Rivera, M.D., CP
D. Krizek, R.M.T., CP
Collaborating Unit:
None
Staff-Years:
0.25
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
x (a1) Minors (a2) Interviews
Summary of Work: A patient with thrombocytopenia and large platelets was discovered to have absent platelet membrane glycoprotein Ib-IX complex by flow cytometry. DNA was isolated from his peripheral blood leukocytes and glycoprotein IX gene sequences were amplified and sequenced, revealing a new mutation. His parents' DNA was also isolated and sequenced, and both are heterozygotes for this mutation. More than 100 healthy subjects have been studied for this polymorphism, and a manuscript has been submitted for publication.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10290-03 CP
October 1, 1999 to September 30, 2000
Title of Project:
Genetic Abnormalities in Patients with Thrombophilia
Principal Investigator:
M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
D. Krizek, R.M.T., CP
K. Hansmann, R.M.T., CP
S. Williams, R.M.T., CP
Collaborating Unit:
None
Staff-Years:
0.25
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Patients with recurrent venous thromboembolic disease, thrombotic disease at an early age, and/or a family history of this disease are at higher risk for recurrences of thrombosis; their family members are also potentially at higher risk than healthy subjects. These patients are being studied for genetic abnormalities that may predispose them to thrombosis, including abnormalities in the factor V gene (factor V Leiden), prothrombin gene abnormality 20210, and the mutation leading to labile 5, 10 methylenetetrahydrofolate reductase (which increases plasma levels of homocysteine, leading to thrombosis). Postmenopausal women who take hormonal replacement therapy may also be at risk for thrombotic disease, and selected subjects are being screened. DNA is isolated from peripheral blood leukocytes, and the DNA is analyzed using polymerase chain reaction and restriction enzyme techniques. More than 170 patients have been studied thus far for these abnormalities.
INTRAMURAL
Research Project
Z01 CL-10291-03 CP
October 1, 1999 to
September 30, 2000
Title of Project:
Evaluation of an Alternative Pathway for Class I MHC Product-dependent Presentation
of Oligopeptide Antigens to CD8 T Cells
Principal Investigator:
R.J. Kurlander, M.D. (Investigator)
HS, CP, CC, NIH
Bethesda, MD, 20892
Other Personnel:
E. Chao, Technologist
J. Fields, Technologist
Collaborating Unit:
LI, NIAID (C.A. Piccirillo, Ph.D.)
Staff-Years:
3.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Antigen-specific CD8 cells could have clinical usefulness in a variety of infectious, neoplastic, and transplantation-related diseases. Attempts to induce CD8 responses by vaccination with protein antigens, however, often yield disappointing results. One factor potentially contributing to these poor responses is the limited capacity of antigen presenting cells (APCs) to present exogenous protein antigens to CD8 cells. In seeking a new approach for enhancing exogenous presentation, we have studied the processing of lemA, a hydrophobic 185 amino acid product of the bacteria Listeria monocytogenes. LemA is of interest because its amino terminal hexapeptide can very effectively be processed and presented to CD8 T cells by professional and nonprofessional APCs. In addition, it is also extraordinarily resistant to degradation by extracellular proteases, a potential advantage in increasing antigen survival in vivo.
We hypothesize that these properties are linked to the presence of an extremely hydrophobic membrane-spanning domain immediately adjacent to the immunogenic portion of the molecule. To test this hypothesis, we have expressed and purified a truncated 35 amino acid fragment of lemA lemA1-33 containing only the immunogenic lemA1-6 and the adjacent hydrophobic portion (25 amino acids) of the lemA molecule. In in vitro studies this truncated molecule can be processed by APC and can resist proteolytic degradation like the intact parent molecule. We subsequently prepared chimeric molecules containing an unrelated passenger peptide Ova257-264 inserted immediately in front (s-lemA) or after (lemS) the distinctive lem7-33 element, and demonstrated that this inserted immunogenic element also could be presented to Ova257-264-immune CD8 effectors by a range of APCs. The Ova257-264 insert in these constructs, like the lemA1-6 in lemA, was extremely resistant to degradation by the extremely aggressive protease, proteinase K. Extending our findings, we have examined the impact of these constructs in vivo. To study their ability to elicit CD8 responses, we inoculate animals with 25 µg doses of lemA and lemS, and monitor the number of lemA1-6 or Ova257-264 immune CD8 cells generated using ELISPOT and intracytoplasmic cytokine staining techniques. We find that recombinant truncated lemA is extremely immunogenic. About 5 to 10 percent of total CD8 cells in animals injected subcutaneously in the tail with lemA or lemS are lemA-specific on day 7 after infection. Stimulation is obtained without recourse to an adjuvant. Animals inoculated with lemS also generate an Ova257-264 specific response, albeit less extensive than the lemA1-6-immune response. Ovalbumin or Ova257-264 injected in the same manner do not elicit a Ova257-264 specific response.
To assess quantitatively the magnitude and kinetics of antigen presentation in vivo, we have exploited the availability of OT-1 transgenic mice, which constitutively express a T-cell receptor specific for Ova257-264 on all of their CD8 T-cells. When OT1 cells are labeled with the fluorescent dye CFSE, their subsequent proliferative response to antigen stimulation can be detected as incremental reductions in cell fluorescence. By adoptively transferring CFSE-labeled OT1 cells into na•ve mice, we can measure Ova257-264-specific proliferation in vivo in response to potential antigens. These methods confirm that the Ova257-264 epitope in lemS is processed and presented in vivo. Perhaps because of the extreme protease resistance of this product, interestingly antigen presentation does not begin until 1 to 2 days after inoculation and persists for 2 to 3 days. In parallel experiments, both Ova257-264 and ovalbumin were presented much more rapidly, and at much higher levels than lemS. These results are surprising because (as noted above) both Ova257-264 and ovalbumin fail to stimulate Ova257-264-immune CD8 cells in vivo. These discrepancies suggests either that lemS generates greater co-stimulatory signals than these other antigens, or alternatively that differences in the timing and intensity of antigen stimulation may potently affect the magnitude CD8 response. If the latter interpretation is correct, the unresponsiveness of Ova257-264 and ovalbumin treated animals may paradoxically reflect deletion of overstimulated cells and not a failure in antigen presentation. Further explorations of the relationship between the intensity of antigen presentation and T-cell responsiveness may provide essential insights into why exogenous protein antigens often generate poor CD8 responses in vivo.
During the coming year, we will (1) address further the relationship between lemA and lemS processing and T-cell responses in vivo. These studies will address the impact of variations in antigen dose and co-stimulatory signals on the yield and avidity of specific CD8 effectors. (2) In addition, we will probe the relationship between antigen presentation and CD8 T-cell responsiveness in mice treated with Ova257-264 and ovalbumin. In particular, we will examine the relative importance of alterations in antigen presentation and costimulation in explaining how adjuvants increase CD8 T-cells' responses.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10293-03 CP
October 1, 1999 to September 30, 2000
Title of Project:
The Experimental Treatment of Transfusion Dependent 5q Minus Syndrome with Leucovorin
Principal Investigator:
C.E. Rivera, M.D. (Senior Staff Physician)
CP, CC, NIH
Bethesda, MD 20892
Other Personnel:
D.J. Mayo, R.N., B.S.N., NURS
Collaborating Units:
NCI
NHLBI
Staff-Years:
2.0
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The objective of this protocol is to determine whether leucovorin treatment can normalize hematopoietic cell growth and differentiation in patients with 5q-syndrome which may lack the gene for dihydrofolate reductase (DHFR) enzyme. The patients will be treated with oral leucovorin for 3 months and will be monitored for improvements in their counts. In addition, bone marrow colony assays will be performed in the presence and absence of leucovorin. The DHFR gene will be sequenced in each patient to screen for DHFR mutations. FISH analysis for the DHFR will also be performed on the cytogenetic material to assess the presence and copy number of the DHFR gene.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10294-03 CP
October 1, 1999 to September 30, 2000
Title of Project:
Heparin Cofactor II Levels in Patients with Paroxysmal Nocturnal Hemoglobinuria
Principal Investigator:
C.E. Rivera, M.D. (Senior Staff Physician)
CP, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Williams, R.M.T., CP
K. Hansmann, R.M.T., CP
Collaborating Unit:
NHLBI
Staff-Years:
3.0
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Thromboembolic events are the most common cause of mortality in patients with paroxysmal nocturnal hemoglobinuria (PNH). Low plasma heparin cofactor II (HCII) levels have been shown to occur in a variety of hemolytic conditions including thalassemia intermedia and sickle cell disease. The level of HCII is related to the degree of hemolysis. A correlation between low HCII levels and thrombosis has been demonstrated in some of these patients. Because of the association between PNH and thrombosis, we are exploring the possible association between HCII levels and PNH. We have already demonstrated that PNH patients have low normal baseline levels of HCII. We will now focus our efforts on measuring HCII in patients with a significant history of thrombosis. Other coagulation parameters including ATIII, Prot C, Prot S, APCR, Prothrombin 20210, and Methylenetetrahydrofolate polymorphisms will also be measured.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10296-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Sepsis in Immunocompromised Patients Caused by Helicobacter-like Organisms
Principal Investigator:
V.J. Gill, Ph.D.
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Weir, Ph.D., Visiting Fellow, MS, CPD
S. Fischer, M.D., Ph.D., MS, CPD
Collaborating Units:
NIAID (B. Cuccherini; W. Strober)
NCID, CDC (A. Whitney; J. MacGregor; A. Steigerwalt; M. Daneshvar; R. Weyant)
Georgia Dept. of Human Resources (Atlanta, GA; M. Ray)
Medical College of Georgia (Augusta, GA; B. Wray; J. Steele)
VA Medical Center (Washington, DC; C. Gibert; F. Gordin)
Staff-Years:
0.05
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Three patients with bacteremia caused by unusual gram-negative bacteria came to our attention. Two were patients with Bruton's agammaglobulinemia patients (NIAID); the third was an HIV patient for whom the organism was referred to us for identification (VAMC). The three organisms shared features of Helicobacter-like organisms based on growth characteristics and gram stain morphology but biochemically were not compatible with described species. We initiated multiple studies to fully characterize these organisms, including rRNA gene sequencing. The organisms were extremely fastidious, microaerobic, curved, and helical bacteria that required specialized conditions to obtain satisfactory growth. Extensive biochemical studies and electron microscopy were performed to provide full descriptions of each isolate. Both patients with Bruton's disease had organisms that, by rRNA gene sequencing, aligned most closely with an unusual organism, Flexispira (Helicobacter) rappini. However, DNA hybridization studies (CDC) showed that at least one of the isolates was a new taxon, distinct from F. rappini as well as from other known Helicobacter species. It is likely that the second F. rappini-like organism is similar but not identical to the first, based on a comparison of their rRNA sequences.
The third organism, thought to be a Campylobacter organism by the referring hospital, did not fit known species of Campylobacter or Helicobacter. Sequencing of the rRNA gene of this organism showed it to be most closely related to a recently described Helicobacter species thus far only described from two cases in Australia. These cases help to establish that fastidious species of Helicobacter (non-H. pylori) can be significant pathogens in the immunocompromised host, causing septicemia with notable recurrences if not detected and treated appropriately. The septic-causing species are a closely related group by rRNA sequencing and cannot be accurately identified by sequencing alone. We have shown that organisms with a more than 99 percent base pair identity in the 16 S rRNA gene may show less than 70 percent homology by total genomic DNA hybridization, which is indicative of a species-level difference. The difficulty in detection and identification of these organisms is a problem for diagnostic microbiology laboratories, because special culture methods must be used to grow the organisms, and molecular methods must be used for definitive identification.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10297-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Carbon Source Utilization for Identification of Rapidly Growing Mycobacteria
Principal Investigator:
F.G. Witebsky, M.D. (Senior Investigator)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
P.S. Conville, M.S., MS, CPD
Collaborating Unit:
None
Staff-Years:
0.20
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Some molecular studies we had been doing for the identification of rapidly growing mycobacteria led us to examine more carefully the parameters involved in the carbon source utilization test for the identification of these organisms. This phenotypic test has been considered useful for distinguishing among certain species of clinically significant rapid growers, especially in those institutions that do not have the capability for molecular analysis. However, our data suggest that carbon source utilization does not always allow definitive discrimination among certain species. This study is nearing completion, and a manuscript summarizing our findings will be prepared.
INTRAMURAL
RESEARCH PROJECT
Z01 CC-10298-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Biochemical Parameters of Coagulation and Fibrinolysis in Postmenopausal Women
with Factor V Leiden or Hyperhomocysteinemia and in Control Women
Principal Investigator:
M. Horne, M.D. (Senior Investigator)
HS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
M. Rick, M.D., HS, CPD
C. Rivera, M.D., HS, CPD
D. Mayo, R.N., HS, CPD
P. Merryman, M.T. (ASCP), HS, CPD
A. Cullinane, M.T. (ASCP), HS, CPD
S. Williams, M.T. (ASCP), HS, CPD
G. Hortin, M.D., Ph.D., CCS, CPD
Collaborating Unit:
Cardiology Branch, NHLBI (R. Cannon, M.D.; M. Waclawiw, M.D.)
Staff-Years:
2.0
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Although hormone replacement therapy (HRT) for postmenopausal women is associated with significant health benefits, it also carries a two- to fourfold increased risk of venous thromboembolism (VTE). This protocol is the first step toward determining whether this increased risk is concentrated in the population of women who carry either of two relatively common genetic risk factors for thrombosis, factor V Leiden (FVL) or hyperhomocysteinemia. We are exploring the feasibility of using a battery of laboratory parameters to assess the effect of HRT on coagulation and fibrinolysis in women with these traits. To do this we are first determining the time-dependent variability of these parameters in women with FVL or hyperhomosteinemia as well as in control women to see whether HRT-related effects are likely to be detected using realistically achievable numbers of study subjects. Therefore, we are screening as many as 300 postmenopausal women to identify ten who have FVL and ten who have hyperhomocysteinemia as their only risk factor for thrombosis and 20 control women. These three groups undergo extensive blood testing on four occasions over 3 months. The variability of each test over time will be calculated. On the basis of these results, tests will be selected for a future study of the effects of HRT. So far we have screened 160 women and have identified 23 who have entered the sequential testing phase of the protocol.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10299-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Resistance to Multiple Fluoroquinolones in a Strain of Streptococcus Pyogenes:
Detection of Point Mutations in the parC Subunit of DNA Topoisomerase IV and
Gyrase A
Principal Investigator:
D.P. Fedorko, Ph.D.
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Yan, Ph.D., MS, CPD
V.J. Gill, Ph.D., MS, CPD
C. Fukuda, B.S., MS, CPD
Collaborating Unit:
None
Staff-Years:
0.20
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: A strain of Staphylococcus pyogenes (NIH-R01-GAS) was isolated from the blood of an 18-year-old patient with Job's syndrome who had recently received various antibiotic treatments including levofloxacin. Susceptibility testing revealed the isolate was resistant to the fluoroquinolone antibiotics.
Quinolone resistance-determining regions (QRDRs) of both the parC subunit of topoisomerase and DNA gyrase A are conserved in a number of bacteria belonging to different genera. We designed polymerase chain reaction (PCR) primers flanking the QRDRs of these two genes from S. pyogenes on the basis of the amino acid sequence homologies among S. pneumoniae, E. coli, and S. aureus. The amino acid sequences deduced from the amplified gene products from S. pyogenes demonstrated significantly high homology to the same regions of these two genes in S. pneumoniae. Compared with the quinolone-sensitive ATCC strain, the quinolone-resistant isolate of S. pyogenes presented point mutations in DNA gyrase A and in the parC subunit of topoisomerase IV.
Recently, we acquired another clinical strain of S. pyogenes (NIH-R02-GAS) with increased MICs to some of the fluoroquinolone antibiotics. Amino acid sequences of ParC and GyrA of this isolate demonstrated point mutations only slightly different from those of the first isolate, with serine-79 of the ParC changed to alanine rather than tyrosine, and no mutation of serine-81 of GyrA. Mutation of methionine-99 to leucine was common to GyrA of both isolates. We propose that the minor change in point mutations in the gyrA and uncommon point mutation in the parC gene in strain NIH-R02-GAS may explain the striking differences in fluoroquinolone susceptibilities in these two isolates of S. pyogenes. Sequencing of other regions will be performed.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10300-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Identification of Proteolytic Activity for von Willebrand's Factor
Principal Investigator:
M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
D. Krizek, R.M.T., CP
Dr. D. Aronson, Special Volunteer
Collaborating Unit:
None
Staff-Years:
1.0
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work:
A patient has been identified whose plasma mediates breakdown (presumably proteolysis)
of normal von Willebrand's factor (vWF). Proteolysis of vWF normally occurs
through the action of a plasma enzyme that has recently been characterized;
it accounts for the small quantities of cleavage products normally present in
the circulation. Other enzymes, such as plasmin and leukocyte elastase, also
can cleave vWF. Preliminary studies of this patient's plasma have shown that
there appear to be differences in cation and pH requirements of the patient's
enzyme when compared with the previously characterized enzyme. Further purification
is being performed to better characterize the activity. We have developed a
better and more rapid assay to evaluate the cleavage of vWF multimers and have
characterized the dose response (rough quantitation of the enzyme in the patient's
plasma).
During the past 9 months, we have developed a collagen-binding assay for the "normal" vWF protease, and have established preliminary steps for purification of this enzyme using an affinity chromatography step, as well as more routine plasma fractionation steps. We are also taking steps to set up a collaboration in order to obtain patient plasmas for testing as a special clinical laboratory test.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10301-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Identification of BCR-ABL in Patients with Chronic Myelogenous Leukemia
Principal Investigator:
M.E. Rick, M.D. (Senior Investigator)
HS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
K. Hansmann, R.M.T., CP
Collaborating Unit:
NHLBI (J. Barrett, M.D.)
Staff-Years:
0.5
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: More than 90 percent of patients with chronic myelogenous leukemia (CML) carry a chromosomal abnormality, termed bcr-abl, that is important in the pathogenesis of the disease. Identification of this chromosomal abnormality is significant in the initial diagnosis of these patients and in following their course after treatment. Messenger RNA is isolated from their peripheral blood and reverse transcribed, and identification of bcr-abl is done on the cDNA using a nested polymerase chain reaction strategy and specific primers followed by gel electrophoresis. Approximately 70 studies have been performed on patients with CML.
This project is completed and has been transferred as a "routine" test to the main hematology laboratory, with the need for approval by the senior staff.
INTRAMURAL RESEARCH PROJECT
Z01 CL-10302-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Development and Diagnostic Use of Rapid Immunoassays
Principal Investigator:
A.T. Remaley, M.D., Ph.D.
CCS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
M. Sampson, M.T., CPD
Collaborating Unit:
None
Staff-Years:
0.25
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Several endocrine tumor markers are routinely used in the diagnosis and management of various cancers. The current assays, however, typically take several hours to perform, which precludes their use in the intraoperative management of patients. Most endocrine tumor markers have a half-life in the circulation of less than 5 minutes, thus making it feasible, if a rapid assay were available, for monitoring the concentration of the hormones during surgery or localization procedures. The primary indication for such assays would be to assess the extent of residual tumor after surgery and to localize tumors by selective venous or arterial sampling. In the past year, we have developed rapid assays for ACTH, cortisol, and insulin. In the upcoming year, we plan to evaluate the clinical utility of these rapid tests and to develop a rapid test for gastrin.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10303-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Development of New Assays for Lipoprotein Testing
Principal Investigator:
A.T. Remaley, M.D., Ph.D.
CCS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
M. Sampson, M.T., CPD
Collaborating Unit:
None
Staff-Years:
0.25
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Lipoprotein fraction analysis is a valuable tool in estimating the risk for coronary artery disease. The current procedure, however, requires multiple tests and has several manual steps. In order to reduce the complexity and cost of lipoprotein fraction analysis, we have developed a single-tube homogenous assay for measuring serum high-density lipoprotein (HDL) cholesterol, total cholesterol, and triglyceride. Low-density lipoprotein (LDL) cholesterol can then be calculated from these parameters using the Friedewald equation. The assay uses an anti-apoB antibody to block the reactivity of the reporter enzymes to LDL-cholesterol. The assay is performed in a sequential manner so that after the HDL-cholesterol is determined, a detergent is added to disrupt the antibody complex, which allows the subsequent measurement of total cholesterol. Next, the reporter enzymes for measuring total triglyceride are added.
In the upcoming year, we plan to test the compatibility of the method with alternative approaches for blocking the reactivity of LDL-cholesterol. We also plan to develop a homogenous assay for LDL-cholesterol, followed by sequential assays for total cholesterol and triglyceride.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10304-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Mutation Analysis of Selected Lymphoid-immune Disorders
Principal Investigator:
T.A. Fleisher, M.D. (Chief)
IS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Units:
NIAID (S. Straus, M.D.)
NHGRI (J. Puck, M.D.)
Staff-Years:
0.5
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: This project represents an extension of a long-standing series of collaborative studies performed to better characterize and understand immune deficiency. Mutations involving the genes for the common gamma chain (X-SCID) and fas (ALPS) are being evaluated using dideoxyfinger printing (ddF) and direct gene sequencing. These studies have continued to identify a number of new mutations and these data will be submitted for publication. In addition, this project has provided valuable experience in the critical approaches to molecular diagnosis of genetic disorders. This information together with the procedure manuals, are being used to assist with the NIH CLIA resource program.
INTRAMURAL RESEARCH PROJECT
Z01 CL-10305-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Analytical Performance and Clinical Utility of Thyroid Function Tests
Principal Investigator:
G. Csako, M.D. (Assistant Chief)
CCS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
A. Remaley, M.D., CCS, CPD
R. Costello, M.T., R.T., CCS, CPD
Collaborating Units:
PHARM, CC (F. Pucino, PharmD.)
HEB, CC (W. Robert, Ph.D.)
NIDDK (N. Sarlis, M.D., Ph.D.; M. Skarulis, M.D.)
NICHD (L. Niemann, M.D.)
Staff-Years:
0.5
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: In a multi-institute collaborative study, we assessed the potential clinical utility of thyroid hormone suppression therapy on benign multinodular thyroid disease. The study was performed using our previously developed method of quantitative research synthesis. This approach involved systematic review of the literature for relevant clinical studies and expert opinions, meta-analysis of selected interventional studies, surveys of NIH endocrinology practitioners for therapeutic decisions in hypothetic patients with benign multinodular thyroid disease, practice validation in NIH patients treated with levothyroxine suppression for benign multinodular thyroid disease, and application of Hill's criteria to assess causality between thyroid suppression therapy and regression of benign nodules. Similar to our earlier observation for the effect of thyroid suppression therapy on benign solitary thyroid nodules, we found that a subset of patients with benign multinodular thyroid disease responded to this medical therapy with clinically significant reduction in nodule size. Consequently, an initial trial of this therapy is warranted in patients with benign multinodular thyroid disease in the absence of contraindication(s) to levothyroxine administration. In another study, we evaluated a case in which increased doses of levothyroxine replacement therapy were required to achieve euthyroid status in a patient with nephrotic syndrome. The clinical picture thus mimicked an "inappropriate" thyroid-stimulating hormone (TSH) secretion syndrome.
We analyzed the effect of various blood collection tubes on the measurements of free thyroxine (FT4), free iodothyronine (FT3), and TSH (thyroid-stimulating hormone or thyrotropin) with different nonisotopic methods. We observed clinically significant effects only with one type of collection tube on some assays. Nevertheless, the findings indicate that routine evaluation of these tubes is warranted before their use for thyroid function testing.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10306-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Analytical Performance and Clinical Utility of Laboratory Tests for the Study
of Atherothrombosis
Principal Investigator:
G. Csako, M.D. (Assistant Chief)
CCS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
R. Costello, M.T., R.T., CCS, CPD
Collaborating Units:
CB, NHLBI (R.O. Cannon III, M.D.; A.A. Quyyumi, M.D.)
PD, CC (F. Pucino)
DTM, CC (S. Leitman, M.D.)
HEB, CC (W. Robert, Ph.D.)
Staff-Years:
1.8
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: In collaborative studies, we continued studying atherogenic plasma constituents and markers with respect to their analytic and diagnostic performance and clinical utility. Since analysis of serum lipid and lipoprotein constituents is not always possible in freshly collected specimens, we further investigated the effect of various storage conditions and repeated freezing-thawing on the analytic recoveries of total cholesterol, glycerol-blanked triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, apolipoprotein(a), and lipoprotein(a)-cholesterol. Using a variety of analytic methods, we found that most of these constituents are stable in serum when stored frozen at -70 degrees C and freeze-thawed only once.
We also studied the effect of prolonged oral L-arginine administration on endothelium-dependent vasodilatation and markers of inflammation (including C-reactive protein [CRP]) in healthy postmenopausal women and in patients with coronary artery disease (CAD) on medical management.
In addition, we studied the effect of oral L-arginine supplementation on the plasma amino acid composition in healthy postmenopausal women and in men with established CAD. In collaborative studies, we investigated the hormonal, lipoprotein, and vascular effects of the selective estrogen receptor modulator raloxifene in hypercholesterolemic men and the effects of estrogen and raloxifene on markers of inflammation in postmenopausal women.
In other studies, we continued studying the clinical significance of various viral (e.g., hepatitis A, cytomegalovirus) and bacterial infections (e.g., Helicobacter pylori) with respect to the development and progression of atherosclerotic disease, the effect of various interleukins (IL-6, IL-10) on plasma lipids, lipoproteins, and apolipoproteins, and the possible benefit of frequent blood donations on atherosclerosis.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10307-02 CP
October 1, 1999 to September 30, 2000
Title of Project:
Development and Clinical Application of Molecular Diagnostic Tests
Principal Investigator:
G. Csako, M.D. (Assistant Chief)
CCS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
R. Delgado, M.T., R.T., CCS, CPD
R. Costello, M.T., R.T., CCS, CPD
Collaborating Units:
CMS, CPD, CC (S.H. Fischer, M.D., Ph.D.)
GPB, NIMH (T. Sunderland, M.D.)
LCI, NIAID (A. Marques, M.D.)
Staff-Years:
0.8
Human Subjects:
x (a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: An increasing number of genes have been linked to Alzheimer's disease (AD). Recently, a new polymorphism of the human alpha2-macroglobulin gene (A2M) was proposed to be a risk factor for AD. This polymorphism is due to an A to G transition in exon 24, at position 1000 based on the cDNA sequence or at position 976 based on the mature protein. This point mutation changes Ile (ATC) to Val (GTC) near the thiolester site of alpha2-macroglobulin. The resultant polymorphism is of high frequency with an allele distribution of 60 to 70 percent A vs. 30 to 35 percent G in white populations. We developed a practicable method for detecting this polymorphism. The new method is based on semi-automated polymerase chain reaction (PCR)-single strand sequence polymorphism (SSCP) analysis and is considerably faster and/or simpler than earlier approaches such as PCR-restriction fragment length polymorphism (RFLP) analysis and direct DNA sequencing. Parallel analysis of human peripheral blood specimens with the three methods showed that the new method is as reliable diagnostically as are the earlier methods. The new method is thus particularly advantageous for large-scale studies of the relationship between this novel point mutation and AD risk.
In addition to gene abnormalities, a number of infectious agents such as herpes simplex type 1 virus (HSV-1) have been suggested to participate in the development of AD. We re-investigated the possible role of this virus in the etiology of AD. We found that only a small percentage of coded brain samples from the temporal and frontal cortex of elderly AD patients and controls were positive for HSV-1 DNA. Furthermore, HSV-1 DNA was not more common in the brains of patients with AD and apolipoprotein E4 than in unaffected patients, indicating an unlikely role of HSV-1 in AD.
In other collaborative studies, we continued studying the possible pathogenetic role of apolipoprotein(a) isoforms and alleles in patients with atherosclerotic heart disease and systemic lupus erythematosus.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10308-01 CP
October 1, 1999 to September 30, 2000
Title of Project:
Assessment of Memory B Cells in Immune Disorders
Principal Investigator:
T.A. Fleisher, M.D. (Chief)
IS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Unit:
NIAMS (P. Lipsky, M.D.)
Staff-Years:
0.2
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: A project to develop the complete immunophenotype of memory B cell has been initiated. This is being done evaluating normal subjects and patients with a number of immune disorders. In addition, the immunophenotypic data is being compared with Ig gene recombination results generated in Dr. P. Lipsky's laboratory (NIAMS).
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10309-01 CP
October 1, 1999 to September 30, 2000
Title of Project:
Assessment of B220 Expression on Human Lymphocytes
Principal Investigator:
T.A. Fleisher, M.D. (Chief)
IS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Unit:
None
Staff-Years:
0.1
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: A project to evaluate the CD45 alternative isoform, B220, expression on lymphocytes has been initiated. This is being done in healthy subjects and patients with autoimmune lymphoproliferative syndrome (ALPS) as well as other patients with lymphocyte disorders. Preliminary data suggest that this isoform is expressed at different levels in certain lymphocyte disorders and may serve as a characteristic finding in subtypes of ALPS.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10310-01 CP
October 1, 1999 to September 30, 2000
Title of Project:
In Vitro Activities of Fluoroquinolone Antibiotics Against Blood Culture
Isolates of Viridans Group of Streptococci from Bone Marrow Transplantation
Patients at the NIH Clinical Center
Principal Investigator:
D. P. Fedorko, Ph.D.
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
S. Yan, Ph.D., MS, CPD
V.J. Gill, Ph.D., MS, CPD
C. Fukuda, B.S., MS, CPD
Collaborating Unit:
None
Staff-Years:
0.20
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Thirty-seven strains of viridans streptococci isolated from blood cultures on bone marrow transplant (BMT) recipients during the past 4 years and 39 strains of viridans streptococci isolated from non-BMT patients were tested for fluoroquinolone susceptibility. Of the five strains from BMT patients that were resistant to levofloxacin, trovafloxacin, and/or grepafloxacin, one isolate (NIH-(R02) demonstrated high-level resistance to multiple fluoroquinolones tested. DNA gyrase A is one of the major targets for quinolone antibiotics. The amino acid sequence within the quinolone resistance-determining region (QRDR) of the gyrase A of NIH-(R02) was determined. When compared with ten sensitive strains of S. mitis, NIH-(R02) carried a single point mutation in the "GKYHPHGDS" box within the QRDR of the gyrA gene (serine to leucine replacement). The mechanism of fluoroquinolone resistance in this isolate of S. mitis is thus likely similar to the previously reported mechanisms in S. pneumoniae.
We will sequence the QRDR of the parC subunit of topoisomerase IV (parC gene) of NIH-(R02) and the ten sensitive strains to search for additional point mutations that may confer resistance to the quinolone antibiotics in the viridans group of streptococci.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10312-01 CP
October 1, 1999 to September 30, 2000
Title of Project:
Evaluation of a New Culture Medium for Borrelia Organisms
Principal Investigator:
V. J. Gill, Ph.D. (Chief)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
F. Stock, MS, CPD
Collaborating Unit:
NIAID (A. Marques, M.D.)
Staff-Years:
0.05
Human Subjects:
(a) Human subjects x (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Lyme disease (LD) is a complex multisystem infection caused by Borrelia burgdorferi. Chronic Lyme disease (CLD) describes patients who suffer from chronic symptoms after what is thought to be adequate antibiotic therapy. Diagnosis of a persistent infection is difficult because of the poor sensitivity of currently available methods. Although the organism can be cultured in enriched artificial medium (BSK), culture has a very low sensitivity outside of samples for the acute disease, specifically the lesions of erythema migrans in which the reported culture sensitivity ranges from 20 to 90 percent. In 1998, Philips et al. described a new medium (MPM) and methods for culture by which they cultured Borrelia organisms from 43 of 47 patients with CLD, all of whom had relapsed after long-term oral and intravenous antibiotics. This new culture medium and method would be a major advance in the laboratory diagnosis of CLD.
We tested 19 blood samples from 18 patients at the NIH seen in 1999 using the new media and methods of Philips et al. Cases included 11 patients with CLD, five with Lyme arthritis, one patient recovered from LD, and one with early LD. In addition, comparative studies using a control strain of Borrelia species were used to compare the growth characteristics of MPM to those of BSK. We were unable to recover the Borrelia spirochete from any of the blood cultures using either MPM or BSK. In addition, serial dilution studies using a control strain of Borrelia showed that BSK was more sensitive than MPM for the detection of Borrelia organisms.
INTRAMURAL RESEARCH PROJECT
Z01 CL-10313-01 CP
October 1, 1999 to September 30, 2000
Title of Project:
Antibiotic Susceptibility Profiles of Group B Streptococci Isolated from Neonates
(1995-1998)
Principal Investigators:
V.J. Gill, Ph.D. (Chief)
MS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
None
Collaborating Units:
NICHD (F.C. Lin, M.D.)
Children's Hospital Medical Center of Northern California (Oakland, CA)
Baylor College of Medicine (Houston, TX)
Univ. of Alabama at Birmingham (Birmingham, AL)
Columbia Univ. Health Sciences (New York, NY)
Univ. of Florida (Gainesville, FL)
Univ. of Dentistry and Medicine of New Jersey (Piscataway, NJ)
Staff-Years:
0.05
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Group B streptococci (GBS) specimens were collected prospectively from infants with early-onset disease and infants who were colonized but did not develop GBS disease between 1995 and 1998 at six U.S. academic centers. The isolates were verified as GBS at the NIH, and antibiotic susceptibilities of all isolates (119 invasive and 227 colonizing strains) were determined. All strains were sensitive to penicillin, vancomycin, chloramphenicol, and cefotaxime. The resistance rates were 20.2 percent to erythromycin and 7.2 percent to clindamycin. (These figures are the overall resistance rates of the isolates collected from 1995-1998). Resistance to erythromycin increased from 15 percent in 1995 to 26 percent in 1997. Type V strains were more resistant to erythromycin than were types Ia, Ib, and III. Resistance was highest in strains from California (32 percent to erythromycin and 12 percent to clindamycin) as compared with a low of 8.5 percent and 2.7 percent, respectively, in Florida. Penicillin continues to be the drug of choice for GBS infection. For women that are penicillin-intolerant, however, selection of an alternative antibiotic should be guided by contemporary regional resistance patterns.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10314-01 CP
October 1, 1999 to September 30, 2000
Title of Project:
Viridans Streptococci Bacteremia in Allogeneic Bone Marrow Transplant Patients
Principal Investigators:
V.J. Gill, Ph.D. (Chief)
MS, CPD, CC, NIH
Bethesda, MD 20892
J.E. Bennett, M.D.
NIAID, NIH
Bethesda, MD 20892
Other Personnel:
C.D. Fukuda, MS, CPD
Collaborating Units:
LCM, NIAID (C. Graber; K.N.F. Almeida)
NIDCR (J.C. Atkinson; D. Javaheri)
HB, NHLBI (A.J. Barrett)
Staff-Years:
0.05
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: In 61 consecutive myeloablative allogeneic hematopoietic stem cell transplant recipients, viridans streptococci were the most common cause of bacteremia, accounting for 19 of 31 (61 percent) cases of bacteremia occurring during the period of post- transplant neutropenia. Seven patients had more than one species of viridans streptococcus in the blood. The most common species was Streptococcus mitis. Most of the streptococcal isolates were resistant to norfloxacin, an antibiotic used for prophylaxis. A comparison of these 19 patients with a group of 23 transplant recipients with fever and neutropenia, but no identified focus of infection, revealed that patients with viridans streptococcal bacteremia were more likely to have severe intraoral pathology.
Results of this analysis suggest that poor underlying dental health and the use of norfloxacin for prophylaxis predispose this patient population to viridans streptococcal bacteremia.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-10315-01 CP
October 1, 1999 to September 30, 2000
Title of Project:
Immunoassay of Tumor Markers from Tissue Obtained by Laser Capture Microdissection
Principal Investigator:
A.T. Remaley, M.D., Ph.D.
CCS, CPD, CC, NIH
Bethesda, MD 20892
Other Personnel:
T. Fleisher, M.D., Ph.D., CPD
Collaborating Unit:
NCI (L. Liotta, M.D., Ph.D.)
Staff-Years:
0.1
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Immunohistochemistry is a commonly used method for determining the presence of various markers, such as tumor markers, on tissue. One major limitation of immunohistochemistry is that it is a qualitative test and is therefore subject to significant variability in the interpretation of the test result. Quantitative immunoassays, however, can not be directly applied to tissue because most tissues are comprised of various cell types, which may not all express a particular marker. We have developed a procedure for coupling laser capture microdissection (LCM) with quantitative immunoassays. LCM enables the user to collect individual cells of interest from a stained tissue section. After solubilization, cells collected by LCM can then be analyzed by quantitative soluble immunoassays. The feasibility of this approach was shown by collecting epithelial cells from prostate cancer tissue and then measuring the level of PSA, using an automated immunoassay analyzer. The PSA content from as little as ten cells could be accurately measured in this way. In the upcoming year, we plan to extend this type of analysis to other clinically relevant tumor markers, such as Her2/Neu.
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