Ms. Kathy L. Delucas
Los Alamos National Laboratory
Public Affairs Office (PA)
P. O. Box 1663, MS C177
Los Alamos, NM 87544
Phone: 505-667-1455
Fax: 505-665-3910
Email: duke@lanl.gov
Projects are approved by an IRB located at: Los Alamos National Laboratory.
The approving IRB operates under a Multiple Project Assurance (MPA) recognized by DOE or by the Department of Health and Human Services (HHS).
MPA number of the IRB: MPA/DOE-LANL96-2000
Number of Human Subjects Projects reported: 9
Project Identifier: LANL-52-91 LANL 06
Project Title:
Manhattan Project Plutonium Workers Health Study
Principal Investigator:
Dr. George L. Voelz
Project started in: 1952
Project Funding Information:
Project received funding in Fiscal Year 1998.
Project used human subjects in Fiscal Year 1998.
Funding Sources:
Funds are carry over from FY1997
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
IRB Approval Number: 91 LANL 06
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 19
Type of Human Subjects Involvement:
X-rays of chest , pelvis, right upper leg for diagnostic and follow-up studies. All testing done in summer of 1997.
MANHATTAN PROJECT PLUTONIUM WORKERS HEALTH STUDY
This project involves long term medical and dosimetry follow-up of 26 workers exposed to plutonium in 1944-1945. In 1998, the work on this project consisted of analyzing the information gathered from medical examinations, urinalysis for plutonium, and chest counts completed in 1997. Some telephone contact with member of the study group and /or their families continues. Contact with family members occurred because of terminal illness and the deaths of three (3) study members at ages 77, 86, and 90 in 1998. Sixteen of the 26 workers are still living. An update on vital status ascertainment was conducted for a comparison cohort of 800+ former Los Alamos workers who also started work in the 1944-1945 era. These data are needed for comparison of mortality rates of exposed and unexposed workers. The principal 1998 objective is to collect and analyze the data and produce a report of the 1997 work.
In 1998, no work was conducted on the study subjects personally, so no risks were involved except for keeping records confidential.
Project Identifier: LANL-76-90 LANL 07
Project Title:
Acceptance Testing for Air-Line Supplied Air-Suits
Principal Investigator:
Mr. Bruce D. Reinert
Project started in: 1976
Project Funding Information:
Project received funding in Fiscal Year 1998.
Project used human subjects in Fiscal Year 1998.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
IRB Approval Number: 90 LANL 07
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 6
Type of Human Subjects Involvement:
Aerosol in exposure chamber to which subjects might be exposed.
ACCEPTANCE TESTING FOR AIR-LINE SUPPLIED-AIR SUITS
A. OBJECTIVES: This program primarily provides testing of air-supplied suits used to protect workers in environments containing plutonium and tritium. It is conducted at Los Alamos because air-supplied suits are not eligible for National Institute of Occupational Safety and Health (NIOSH) testing and certification. The program has been operational since 1974. Suites will continue to be retested to ensure that the continued quality and performance of these suites is maintained.
B. METHODOLOGY: Air-supplied suit testing is conducted in accordance with "Acceptance Testing Procedures for Air-Line Supplied-Air Suit" Los Alamos report LA-10156-MS, June 1984.
C. EXPOSURE TO CHEMICALS: Subjects may be exposed to diethylhexylsebecate (DEHS) up to but not to exceed 10% of the chamber concentration. Individuals sensitive to DEHS may experience mild skin and eye irritation. Subjects may be exposed to elevated levels of carbon dioxide in the suites: these levels are not allowed to exceed 5%.
D. INVOLVEMENT OF HUMAN TEST SUBJECTS: Testing consists of having the test subjects don a suit and enter a chamber that contains an aerosol environment and measuring the amount of aerosol that penetrates into the suit. The test subjects conduct moderate stress exercises while in the chamber to simulate movements of workers using the suit at DOE sites. Another test involves turning the air supply to the suit off while the test subjects are running in-place and monitoring oxygen and carbon dioxide levels in the breathing zones of the test subjects. The air supply is turned on when the oxygen level in the suit reaches 16%.
All records of testing are kept confidential. Reports on test results do not contain the identity of test subjects. All test subjects are briefed on the testing, their rights as test subjects, the consent form is read and signed, and all questions are answered before testing begins.
Project Identifier: LANL-91-03
Project Title:
Ligand-Receptor G Protein Dynamics and Neutrophil Response (formally LANL-91-91-LANL-03 in FY97 database)
Principal Investigator:
Dr. Larry A. Sklar
Project started in: 1991
Project Funding Information:
Project received funding in Fiscal Year 1998.
Project did not use human subjects in Fiscal Year 1998.
Explanation:
Purchased cell lines with needed receptors are currently being used. Humans subject material may be used later.
Funding Sources:
Human subject involvement & other purchased cell line materials cost about 10% of total funding. Frequently we use cell lines with needed receptors. Human subjects may be used later.
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
IRB Approval Number: 91 LANL 03
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 0
Type of Human Subjects Involvement:
OBJECTIVES: 1) to use human neutrophils to study the binding of a fluorescent peptide to a cell surface receptor. 2) To use this binding interaction as a system for improving flow cytometric instrumentation.
METHODOLOGY: Neutrophils are isolated by centrifugation from fresh human blood (50 mls). Investigations are performed on the isolated cells by flow cytometry.
HUMAN SUBJECTS: Healthy volunteers donate blood by routine venipuncture. An informed consent is read and signed by each donor. Risks are limited to minimal discomfort from drawing the blood sample.
PROGRESS: The National Flow Cytometry Resource (NFCR) has been funded through the period July 1, 1997 to June 30, 2002. The human cells will be an important part of the core Research and Development activities of the NFCR. We expect the project to increase its activities with the arrival of new personnel and instrumentation in the winter of 1998.
Project Identifier: LANL-96-05
Project Title:
Development of Improved Methods for the Detection of Individual Sensitivity to Beryllium (formally LANL-96-96-LANL-05 in FY97 database)
Principal Investigator:
Dr. Babetta L. Marrone
Project started in: 1996
Project Funding Information:
Project received funding in Fiscal Year 1998.
Project used human subjects in Fiscal Year 1998.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
IRB Approval Number: 96 LANL 05
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 150
Type of Human Subjects Involvement:
Los Alamos National Laboratory (LANL) will soon be the primary facility, within the DOE weapon complex, involved in beryllium work. A state-of-the art facility is being developed that will integrate into the workplace the most advanced methods for monitoring airborne beryllium and for minimizing personal exposure. Strict exposure controls are necessary because respiration of beryllium particles can lead to a cell-mediated immune response in susceptible individuals. This response has been linked to the occurrence of Chronic Beryllium Disease (CBD), an interstitial lung disease. CBD occurs in about 2-5% of all beryllium-exposed workers. One feature of CBD that is believed to be an early marker is the development of cellular hypersensitivity to beryllium. In a sensitive individual, the lymphocyte cell fraction in a peripheral blood sample will increase within 5-7 days after a challenge with soluble beryllium. This test, termed the Lymphocyte Proliferation Test (LPT), is currently being used throughout the DOE complex and in industry to test for sensitivity in former and current beryllium workers. Although there is an active medical surveillance program for LANL beryllium workers, the LPT was only recently added to the LANL medical surveillance program.
About 20% of individuals testing positive on the LPT progress to have physical symptoms of CBD. In addition, there is often disagreement in the results from the three (3) commercial laboratories that currently perform the LPT. The high "false positive" rate has prompted LANL to explore the development of more accurate measures of beryllium sensitivity. Specifically because CBD is a disease of the immune system, the LANL LPT will measure beryllium hypersensitivity in specific lymphocyte subsets. In addition, research on the genetic mechanisms underlying the individual development of beryllium hypersensitivity will be pursued in conjunction with the LPT sampling.
A. Objectives: The near term objective of our proposed research plan is to develop cellular and molecular biomarker assays for detecting beryllium sensitivity in beryllium-exposed workers. The longer term objective is to develop new tests that predict beryllium hypersensitivity in exposed individuals and then apply these tests so that individuals who are susceptible to the adverse health effects of beryllium inhalation may be identified before exposure.
B. Methodology: In FY99, we propose to continue testing the LANL beryllium worker cohort using an LPT by flow cytometry. We will also add beryllium workers from other sites including Lawrence Livermore National Laboratory (LLNL), Lawrence Berkeley National Laboratory (LBNL), Brush-Wellman Inc., and Starmet, Inc. Former beryllium workers who are part of beryllium medical surveillance programs at National Jewish Center, Oak Ridge National Laboratory (ORNL), or other DOE sites may also be included in the study.
The test uses total lymphocytes isolated from a heparinized blood sample by Ficoll gradient. The isolated lymphocytes are then cultured in complete medium in the presence of serum (either commercial human serum, fetal calf serum or patient's own serum) or plasma. The LPT that we have developed (termed the Immuno-LPT) measures the growing cell fraction of specific lymphocyte subsets in response to a 5 or 7 day challenge with soluble beryllium (beryllium sulfate, beryllium fluoride, etc.). Additional samples may be used to test the growing cell fraction in response to beryllium alloys (beryllium aluminum, beryllium copper, etc.). The growing cell fraction is measured by labeling the cultured cells with either a DNA-binding dye, (to analyze cell cycle stage by total DNA content) or with bromo-deoxyuridine (BrDU) incorporation by cell labeling with antibodies to BrDU(to measure cells in the process of DNA synthesis). In either case, the labeled cells are also labeled with antibodies for lymphocytes subsets: CD4 (T helper), CD8 (T suppresser/cyotoxic); or others in order to interpret the growing cell fraction measure in context of a specific lymphocyte subset. The CD4+ subset is believed to be the beryllium-responsive cell in CBD. The results of the Immuno-LPT are given in terms of the percentage of proliferating cells in a particular lymphocyte subset, usually CD4+.
As part of the Immuno-LPT, a sample of blood is analyzed on the day of collection for baseline immunophenotype. This is done by doing a Complete Blood Count (CBC), followed by immunolabelling for lymphocyte subsets (usually CD3, CD4, CD8, and CD56) and multiparameter flow cytometry. The results are given as percentages and absolute numbers of lymphocytes in each subset. Immunophenotyping results falling outside of the normal ranges are indication of an underlying immune system disorder, which may influence the Immuno-LPT results, and should be taken in consideration for follow-up in the worker's medical surveillance program. The research goals of the immunophenotyping are to better understand the role of lymphocyte subsets in beryllium sensitivity, and to determine whether immunophenotyping information can be used diagnostically in CBD.
In addition, we use peripheral blood samples from the same individuals to develop other methods for detecting beryllium sensitivity even earlier in the course of its development. Specifically, there is evidence that there may be a genetic susceptibility to CBD. One test that we will carry out on the LANL beryllium cohort will be to look for a genetic correlate to beryllium sensitivity as measured by the LPT. This will be done by DNA sequencing in regions believed to be involved in the etiology of CBD. Another test that we will apply occasionally will examine a possible functional endpoint of the genetic susceptibility factor that can be measured by flow cytometry as a cellular metabolic response.
Results on these two measures (LPT and genotype or genetic susceptibility) will be analyzed in relation to beryllium exposure history. If our results support a genetic susceptibility to beryllium sensitivity, then a future goal will be to develop and apply a simple test for screening workers before beryllium exposure.
C. Human subjects will not be exposed to any chemical or radioactive substances as a part of this project.
D. 1. A peripheral blood sample from each subject will be acquired as a part of the project procedure.
Participants will be asked to complete a questionnaire as part of the procedure. Each subject signs an informed consent form before blood withdrawal.
2. The risks are minimal, and will be those normally associated with venipuncture (blood withdrawal).
3. The researchers receive only coded/de-identified blood samples. Only the results of the Immuno-LPT and the Immunophenotyping are provided to Dr. Hugh Smith, LANL Occupational Medicine Group/ESH-2. The samples are de-coded by Dr. Smith and he gives the results to the subjects. The signed consent forms, test results, and codes are kept and protected at ESH-2, and do not become part of the individual's medical records. The results of the genetic susceptibility studies are NOT provided to Dr. Smith or to the subjects.
Project Identifier: LANL-97-02
Project Title:
Prevention of Stress and the Health Consequences of Workplace Downsizing and Reorganization (formally LANL-95-97-LANL-02 in FY97 database)
Principal Investigator:
Dr. Lewis D. Pepper
Principal Investigator's Institution: Boston University School of Public Health
Project started in: 1997
Project Funding Information:
Project received funding in Fiscal Year 1998.
Project used human subjects in Fiscal Year 1998.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 1,506
Type of Human Subjects Involvement:
PREVENTION OF STRESS AND THE HEALTH CONSEQUENCES OF WORKPLACE DOWNSIZING AND REORGANIZATION
A. The specific aims of this project are to: (a) Identify and measure the effects of downsizing on organizational climate, health and performance using qualitative and quantitative research methods including focus groups, open-ended interviews, structured surveys, and archival data. (b) Identify and measure the characteristics of downsizing which adversely affect organizational climate, health, and performance using similar techniques. (c) Determine and describe the relationship between downsizing and employee health and consider areas for intervention. (d) Develop a health promotion program based on the most significant problems associated with downsizing.
B. Data collection in this phase of the project will include an employee survey and review of summary archival data. The study, which is beginning its fourth year of funding, is scheduled to last five years. Individual participants will be asked to complete the survey and will not be involved in any additional parts of the study. All study participants are non-patients.
The Boston University Workplace Survey has been administered to approximately 10,000 DOE contractor employees at five DOE sites (Idaho National Engineering and Environmental Laboratory, Nevada Test Site, Oak Ridge Y-12, Los Alamos National Laboratory, Pantex) including LANL. Employees were randomly selected based on their work organization, size, and location. The survey, which was developed over a one and one-half year period, is designed to examine organizational and individual consequences of downsizing and restructuring as well as human health. The survey was pilot tested at each of the study sites and modified based on site input.
This survey will take approximately 30 minutes to complete. Surveys have been distributed with a cover letter to explain the purpose of the study and to encourage participation. This letter has been signed by company and employee leaders at LANL and by Boston University principal investigators. An accompanying "Information and Consent" form provides information about the study and informs individuals that by completing the survey they are consenting to participate in the study. This letter, which participants will retain, includes elements found in informed consent forms including information about the risks and benefits of participating, confidentiality of data collected, and that participation is voluntary.
Participants will not be paid nor will they incur any charges because of participation. Participation (in the form of completing the questionnaire) is completely voluntary. Individuals have been informed that there will be no adverse consequences associated with choosing not to participate in the study or withdrawing from the study at any time.
The project's next year will be primarily focused on collecting, storing, and statistically analyzing survey and organizational data. An intervention project will be developed based on the results of this study and potentially implemented at on or more of the study sites. An addendum to this application will be submitted to cover that phase of the research (fall/winter, 1998).
C. Confidentiality: A database system has been developed with the assistance of the Data Coordinating Center (DCC) at Boston University School of Public Health. This database will provide a method of recording each survey. Each survey has a unique study identification number. The participant's name neither appears on the survey nor is entered into the study database.
Organizational outcome data will be stored in either electronic or paper format. No individual identifiers will be included in this database. All information requests to the sites will indicate that individual identifiers not be included with the requested data. Therefore, it will not be possible to link organizational outcome data, such as employee assist program (EAP), with any individual study participant.
The DCC will maintain strict confidentiality of the project database. All employees including research assistants, coders, programmers, and other project staff will be required to sign a confidentiality agreement. Confidentiality will be stressed in staff training as well as in standard procedures for tracing and data collection. Access to the database will be password restricted.
Data will become part of the CDC Privacy Act System (09-20-0147 "Occupational Health Epidemiological Studies") and all records associated with this project will be kept confidential to the extent allowable by law. Project staff reviewing records will not reveal a participant's identity in publication of reports resulting from the study or in any other way.
Project researchers will review and analyze all the responses to the survey and report on the findings. Results will be made available without personal identifiers to all study participants through presentations, written reports and postings in site publications and web sites. In addition, the results of the project will be released in aggregate both in the final project report and in any articles in scientific journals.
Project Identifier: LANL-98-01
Project Title:
Development and Testing of A Fieldable Prototype Noninvasive Intracranial Pressure Measurement System
Principal Investigator:
Dr. William O. Wray
Project started in: 1998
Project Funding Information:
Project received funding in Fiscal Year 1998.
Project did not use human subjects in Fiscal Year 1998.
Explanation:
Technical work began on 7/1/98, but work with human subjects will not begin until the first quarter of FY 99.
Funding Sources:
According to Statement of Work, work with human subjects will not begin until 12/1/98. Total amount of funding for this project for FY98 is $150K
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
IRB Approval Number: 98 LANL 01
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 0
Type of Human Subjects Involvement:
We propose to develop a non-invasive Intracranial Pressure (ICP) measurement system that could be used by paramedics to monitor the intracranial pressure of combat casualties while they are being transported to an appropriate medical facility. Medication or other treatment required to regulate the patient's ICP could be administered during transport, thereby improving the survival rate of head trauma cases. The ICP sensor could also be designed to work within the framework of a more general patient status system that would provide vital signs and other data to medical facility personnel via telemetry.
During the first year of the proposed study, we will use a combination of ultrasonic tests, to be conducted on both phantom heads and human subjects' heads, and finite element analysis to determine the most viable frequency range for application of the Swept Frequency Acoustic Interferometry (SFAI) technique to noninvasive ICP measurement. We will primarily be examining low ultrasonic frequencies between 100 kHz and 1MHz. The goal is to identify the specific frequency range that produces response spectra that can be most readily associated with intracranial pressure variations. The LANL head model will be used to simulate the ultrasonic tests and provide an understanding of the mechanisms associated with the various response modes. The maximum spatial resolution of the LANL head model is 2mm, indicating that the model could resolve wavelengths about 10mm. Given a sound speed of 2920m/s in the human skull (F. A. Duck, 1990), a 10mm wavelength implies a frequency of about 250 kHz. Thus, the upper limit of frequency that could be directly simulated by the LANL head model is about 250 kHz. This limit will tend to focus our interest on the lower frequency portion of the ultrasonic range to be investigated.
The number of human subjects to be tested has been set at 10 to 20 in order to provide a good measure of human variability without duly burdening the project with massive data collection and processing requirements.
Initially, each participant will be scheduled for two test sessions. These sessions will take place on two separate days. Dr. Sinha will conduct the testing in his laboratory at TA-3, Bldg. 40, and RM. N161. Each test session will take approximately 20 minutes. Two small piezoelectric transducers, about the size of a nickel, will be placed on the participant's head, and held in place by an elastic band or a small amount of ultrasound gel. The participant will be asked to lie down on a small rollaway bed and relax for 5 minutes. After a five-minute rest period, Dr. Sinha will take a reading with the test device. The time required for each reading is approximately 20 seconds. It may be necessary to repeat a reading two or three times to obtain a satisfactory result. In any case, the total ultrasound exposure time in the supine position will not exceed two minutes. After completion of testing in the supine position, the whole procedure will then be repeated while the participant is in a sitting position and then in a standing position. The total ultrasound exposure will not exceed six minutes total per test session.
After completion of the two scheduled test sessions, the participants may be invited back for up to a maximum of three additional sessions. These additional test sessions will be requested only if there is a real need for additional data.
All test data and other information concerning human subjects will be kept in a locked file drawer in Dr. Sinha's laboratory (TA-3, Bldg. 40, Rm. N161). No publication or any other report of test results, whether written or verbal, will disclose the identity of any subject who participates in the study.
Project Identifier: LANL-98-02
Project Title:
Neuromagnetic Mapping of Functional Centers in the Human Brain
Principal Investigator:
Dr. Robert H. Kraus, Jr.
Project started in: 1998
Project Funding Information:
Project did not receive funding in Fiscal Year 1998.
Project did not use human subjects in Fiscal Year 1998.
Explanation:
Concerning the funding at the time the IRB was submitted, we did not have the NIH grant in hand, and the IRB approval request was for this new proposal. The NIH grant has been approved as of 7/98. First year funding is $299K direct.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
IRB Approval Number: 98 LANL 02
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 0
Type of Human Subjects Involvement:
We propose to complete construction of a novel whole-head superconducting image sensor system for Magnetoencephalography (MEG) of the human brain, to experimentally calibrate and validate the system using physical phantoms, and to demonstrate system efficacy by direct comparison with a commercial whole-head MEG array. A cost-effective whole-head system will provide important capabilities for noninvasive functional human brain measurements for both clinical applications and basic research. MEG directly measures a physical effect of neuronal currents with temporal resolution not limited by the sluggish vascular response; unlike PET and MRI that measure hemotological changes associated with neuronal activity. High temporal resolution is particularly important for studying neurological disorders such as epilepsy where temporal information is a major diagnostic, and for fundamental studies of synchronization and oscillatory brain activity.
NIH has supported the whole-head MEG system proposed here during the initial project period of this grant. The system is based on the Los Alamos patented principle of superconducting image surface gradiometry where magnetic sources are imaged on the surface and magnetometers near this surface sense the combined fields as if the sensors were gradiometers. Our whole-head system promises both superior performance and lower cost than conventional whole-head MEG systems. The whole-head MEG system design is complete; fabrication and assembly are about 90% complete. Additional key accomplishments include (A) the superconducting imaging principle was experimentally verified; (B) comparison of niobium and lead imaging surface performance demonstrated superior performance in the lead; (C) a new cryogenic sensor support material was patented; and (D) development of novel software and analog background rejection techniques. Support is now being requested to (1) complete assembly of the whole-head MEG system with all SQUID magnetometers; (2) complete flux-locked loop design implementing our new control and background cancellation techniques; (3) implement a powerful real-time data acquisition system with data management, display, and analysis; (4) use phantom measurements to test and calibrate sensitivity, imaging and noise performance, and shielding characteristics of the sensor array; (5) experimentally verify system efficacy using a sophisticated phantom; and (6) acquire experimental data for human subjects under the same conditions as NIH-funded experimental studies of the visual and somatosensory systems.
The work proposed here will result in a fully functional whole-head MEG system based on new physics application, sensor design, and fabrication techniques that promise to dramatically reduce system cost and complexity while improving system performance. Such a system will be of great value to both basic neuroscience and clinical applications.
Project Identifier: LANL-98-03
Project Title:
Use of Human Peripheral Blood Samples in Structure Function Based Poisoning of Anthrax Toxins
Principal Investigator:
Dr. Bruce E. Lehnert
Project started in: 1998
Project Funding Information:
Project received funding in Fiscal Year 1998.
Project used human subjects in Fiscal Year 1998.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
IRB Approval Number: 98 LANL 03
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 4
Type of Human Subjects Involvement:
USE OF HUMAN PERIPHERAL BLOOD SAMPLES IN STRUCTURE FUNCTION BASED POISONING OF ANTHRAX TOXINS
Bacillus anthracis continues to represent a biological threat to both civilians and military personnel. Although some advances have been made in the area of developing vaccines to protect against the pathogenicity of this organism, the vaccines alone offer limited efficacy and use. Hence, it is evident that new approaches are required to defend against the toxicity of Bacillus anthracis in vivo. Virulence of Bacillus anthracis the production of three proteins, protective antigen, edema factor, and lethal factor, which form the Anthrax toxin complexes. This project focuses on a strategy by which essential steps in the formation and/or functional activation of the toxin complexes may be inhibited for prophylactic/therapeutic advantages. The sequence of events that underlie the pathogenic effects of the Anthrax toxins have become increasingly well understood. It is now recognized that the first step in the process is the binding of Protective Antigen (PA) with a surface receptor on susceptible cells. This first step represents a structurally and correspondingly, functionally based opportunity to disrupt the successful expression of anthrax toxicity.
OBJECTIVE: As a new form of anti-anthrax therapy, this project focuses on protection that may be afforded by competitively inhibiting such protein interactions via relatively low molecular weight antibodies that have high affinities for the relevant binding domains responsible for bringing about the interactions. We are specifically focusing on PA as our target protein in that it is clear that the generation of Anthrax toxins requires the binding of PA to its receptor as the initiating step.
METHODOLOGY: In order to isolate high affinity antibodies, whole blood (~50 ml) from PA-immunized volunteers (LANL employees that received recent immunizations against PA because of the nature of their work on other projects) is isolated by venipuncture. This procedure is no different than what is routinely used for conventional plebotomy. Peripheral blood mononuclear cells (PBMC), which contain the entire antibody repertoire against PA, and RNA from the cells, are isolated. The purified RNA is converted to cDNA using random hexamer primers and commercially available reverse transcriptase. This cDNA library is then used as a polymerase chain reaction (PCR) template for the variable light (VL) and heavy (VH) chains (collectively termed Fv) of gamma globulin-G (IgG). These IgG, VH, and VL domains are inserted into specific cloning vectors which allow expression of single chain Fv as either soluble proteins or as a fusion protein with M13 phage gene 3. Expressed single-chain Fv antibodies are being assessed for their abilities to bind with PA and for their abilities to prevent PA binding to cell receptors.
HUMAN EXPOSURE: NONE
INVOLVEMENT OF HUMAN SUBJECTS: Human subjects are only used as a source of peripheral blood mononuclear cells. The only risks are those that may occur as a result of conventional phlebotomy. All participants are required to sign an informed consent form. The identities of those participants are not relevant to the project's objectives. Peripheral blood samples are processed immediately after collection, i.e., samples are not stored or maintained in a repository for further use or dissemination. The project is not a study of individual human responses to bacterial toxins per se, but instead, white blood cells from volunteer donors are used to generate cDNA libraries with the ultimate goal being to develop antibodies that can inhibit the binding of PA to its cell surface receptor in a manner that can preclude the downstream events that underlie the cytotoxicity of the anthrax toxins. We have selected human cells for this purpose versus developing antibodies from immunized laboratory animals because ligands that inhibit the actions of the anthrax toxins ultimately may be used as prophylactic/therapeutic agents in humans. Using human antibodies expectedly will minimize immune responses that otherwise can occur when humans are administered foreign proteins.
Project Identifier: LANL-98-04
Project Title:
Use of Human Peripheral Blood Samples in Structural Biology of Bacterial Toxins
Principal Investigator:
Dr. Bruce E. Lehnert
Project started in: 1998
Project Funding Information:
Project received funding in Fiscal Year 1998.
Project used human subjects in Fiscal Year 1998.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: January 22, 1998
IRB Approval Number: 98 LANL 04
Number of Human Subjects who participated in this project/protocol during
FY 1998 (10/1/97 - 9/30/98): 4
Type of Human Subjects Involvement:
USE OF HUMAN PERIPHERAL BLOOD SAMPLES IN STRUCTURAL BIOLOGY OF BACTERIAL TOXINS
Bacterial toxin superantigens and bipartite toxins continue to pose a major health problem. Superantigens such as the Staphylococcal enterotoxins (SEA, SEB, SEC1-3, SED, and SEE), which are single unit proteins, can simultaneously interact with class II MHC receptors present on antigen presenting cells (APC) and T cell receptors present on the surfaces of T lymphocytes. Direct outcomes of such binding include the excessive release of the cytokine interleukin-2 (IL-2) and the proliferation of T cells. Subsequent cytokine effects and downstream cytokine networks then medicate a variety of clinical symptoms, e.g., fever, shock. Bipartite AB5 toxins like those produced by E. coli and Vibrio Colerae mediate their effects upon binding to specific receptors (ganglioside receptors, GM1) on target intestinal epithelial cells, resulting in an excessive stimulation of adenyl cyclase and a pathologic production of cyclic adenosine monophosphate (cAMP). With both types of toxins, the initial step in the mediation of their disease-causing effects requires binding to specific receptors.
OBJECTIVE: In a project funded by DARPA (entitled "Structural Biology of Bacterial Toxins"), we are developing non-pathogenic mimics of superantigens and AB5 toxins that should serve as competing, but pathogenically inactive ligands for receptor structures that are recognized as binding sites by the toxins. The goal here is to develop therapeutic strategies that will inhibit the binding of superantigens to their cell receptors and thereby prevent their toxic effects.
METHODOLOGY: To date, three rationally designed superantigens mimics have been synthesized. Using human lymphocytes, we have found that mimics can inhibit both the superantigen (SEB)-stimulated production of Interleukin-2 and the T cell proliferative response. These mimics are being further optimized structurally, and we are generating new mimics to inhibit the toxic actions of other superantigens. As indicated above, we are assessing the ability of the mimics to interfere with superantigen-induced T cell proliferation and the production of Interleukin-2.
HUMAN TREATMENTS: None. Human subjects are only used as a source of peripheral blood to obtain mononuclear leukocytes for in vitro testing.
HUMAN SUBJECTS: Human peripheral blood is sampled by conventional phlebotomy. Mononucleated white blood cells are obtained using density gradient centrifugation. Cells are cultured in the presence of superantigens and mimics. Cell counts are performed. Cell proliferation is also determined by flow cytometry to assess the percentages of cells in S and G2M phases of the cell cycle. Interleukin-2 concentrations in cell culture supernatants are determined by immunoassay.
RISKS: The project poses no risks other than those that may be encountered by conventional, routine phlebotomy.
CONSENT: Participants are volunteers. Before phlebotomy, volunteers are advised about the project's objectives and how their blood will be used. The volunteers must sign an informed consent form before participating in the project as blood donors. Individual identities of the volunteers are irrelevant to the project's objectives.