|
Items in cart [0] |
Questions? Call 888-624-8373 |
| Copyright © 2010. National Academy of Sciences. All rights reserved. Terms of Use and Privacy Statement |
Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page R1
Biologic Markers in Immunotoxicology
Biologic Markers in Immunotoxicology
Subcommittee on Immunotoxicology
Committee on Biologic Markers
Board on Environmental Studies and Toxicology
Commission on Life Sciences
National Research Council
NATIONAL ACADEMY PRESS
Washington, D.C.
1992
OCR for page R2
Biologic Markers in Immunotoxicology
NATIONAL ACADEMY PRESS
2101 Constitution Ave., N.W. Washington, D.C. 20418
NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the committee responsible for the report were chosen for their special competencies and with regard for appropriate balance.
This report has been reviewed by a group other than the authors according to procedures approved by a Report Review Committee consisting of members of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine.
The National Academy of Sciences is a private, nonprofit, self-perpetuating society of distinguished scholars engaged in scientific and engineering research, dedicated to the furtherance of science and technology and to their use for the general welfare. Upon the authority of the charter granted to it by the Congress in 1863, the Academy has a mandate that requires it to advise the federal government on scientific and technical matters. Dr. Frank Press is president of the National Academy of Sciences.
The National Academy of Engineering was established in 1964, under the charter of the National Academy of Sciences, as a parallel organization of outstanding engineers. It is autonomous in its administration and in the selection of its members, sharing with the National Academy Sciences the responsibility for advising the federal government. The National Academy of Engineering also sponsors engineering programs aimed at meeting national needs, encourages education and research, and recognizes the superior achievements of engineers. Dr. Robert M. White is president of the National Academy of Engineering.
The Institute of Medicine was established in 1970 by the National Academy of Sciences to secure the services of eminent members of appropriate professions in the examination of policy matters pertaining to the health of the public. The Institute acts under the responsibility given to the National Academy of Sciences by its congressional charter to be an adviser to the federal government and, upon its own initiative, to identify issues of medical care, research, and education. Dr. Kenneth Shine is president of the Institute of Medicine.
The National Research Council was organized by the National Academy of Sciences in 1916 to associate the broad community of science and technology with the Academy’s purposes of furthering knowledge and advising the federal government. Functioning in accordance with general policies determined by the Academy, the Council has become the principal operating agency of both the National Academy of Sciences and the National Academy of Engineering in providing services to the government, the public, and the scientific and engineering communities. The Council is administered jointly by both Academies and the Institute of Medicine. Dr. Frank Press and Dr. Robert M. White are chairman and vice chairman, respectively, of the National Research Council.
The project was supported by the Environmental Protection Agency; the National Institute of Environmental Health Sciences; and the Comprehensive Environmental Response, Compensation, and Liability Act Trust Fund through cooperative agreement with the Agency for Toxic Substances and Disease Registry, U.S. Public Health Service, Department of Health and Human Services.
Library of Congress Catalog Card Number 91-67588
International Standard Book Number 0-309-04389-1
Additional copies of this report are available from the
National Academy Press,
2101 Constitution Avenue, N.W., Washington, D.C. 20418
S538
Printed in the United States of America
First Printing, February 1992
Second Printing, June 1992
Third Printing, July 1992
Fourth Printing, October 1992
Fifth Printing, January 1996
OCR for page R3
Biologic Markers in Immunotoxicology
Subcommittee on Immunotoxicology
DAVID W. TALMAGE, Chairman,
University of Colorado Health Sciences Center, Denver
DAVID E. BICE,
Lovelace Inhalation Toxicology Research Institute, Albuquerque
JOHN CHARLES BLOOM,
Lilly Research Laboratories, Greenfield, IN
LOREN D. KOLLER,
College of Veterinary Medicine, Oregon State University, Corvallis
MICHAEL E. LAMM,
Institute of Pathology Case Western Reserve University, Cleveland
MICHAEL I. LUSTER,
National Institute of Environmental Health Sciences, Research Triangle Park
WILLIAM J. MEGGS,
East Carolina School of Medicine, Greenville, NC
ALBERT E. MUNSON,
Medical College of Virginia, Richmond
KATHLEEN E. RODGERS,
Livingston Laboratories, Los Angeles
NOEL R. ROSE,
Johns Hopkins University, Baltimore
PAUL A. SCHULTE,
National Institute for Occupational Safety and Health, Cincinnati
CURTIS C. TRAVIS,
Office of Risk Analysis, Oak Ridge National Laboratory, Oak Ridge
ERNEST S. TUCKER,
California Pacific Medical Center, San Francisco
ROBERT F. VOGT, JR.,
Centers for Disease Control, Atlanta
THOMAS A. WALDMANN,
National Cancer Institute, Bethesda, MD
Technical Adviser
GARY R. BURLESON,
U.S. Environmental Protection Agency, Research Triangle Park
Staff
RICHARD D. THOMAS, Program Director
ROBERT P. BELILES, Program Officer
MARVIN A. SCHNEIDERMAN, Senior Staff Scientist
KATE KELLY, Editor
RUTH E. CROSSGROVE, Information Specialist
DANIELLE CORRIVEAU, Project Assistant (until 1/91)
JOYCE WALZ, Project Assistant
Sponsors
National Institute of Allergy and Infectious Disease
National Institute of Environmental Health Sciences
U.S. Environmental Protection Agency
U.S. Public Health Service, Agency for Toxic Substances and Disease Registry
OCR for page R4
Biologic Markers in Immunotoxicology
Committee on Biologic Markers
BERNARD GOLDSTEIN, Chairman,
UMDNJ-Robert Wood Johnson Medical School, Piscataway
JAMES GIBSON,
Dow-Elanco, Indianapolis
ROGENE F. HENDERSON,
Lovelace Biomedical and Environmental Research Institute, Albuquerque
JOHN E. HOBBIE,
Marine Biological Laboratory, Woods Hole, MA
PHILIP J. LANDRIGAN,
Mount Sinai Medical Center, New York
DONALD R. MATTISON,
University of Arkansas for Medical Sciences and National Center for Toxicological Research, Little Rock
FREDERICA PERERA,
Columbia University, New York
EMIL A. PFITZER,
Hoffmann-La Roche, Inc., Nutley, NJ
ELLEN K. SILBERGELD,
Environmental Defense Fund, Washington, DC
Staff
RICHARD D. THOMAS, Program Director
OCR for page R5
Biologic Markers in Immunotoxicology
Board on Environmental Studies and Toxicology
PAUL G. RISSER (Chairman),
University of New Mexico, Albuquerque
GILBERT S. OMENN (Immediate Past Chairman),
University of Washington, Seattle
FREDERICK R. ANDERSON,
Washington School of Law, American University
JOHN C. BAILAR, III,
McGill University School of Medicine, Montreal
LAWRENCE W. BARNTHOUSE,
Oak Ridge National Laboratory, Oak Ridge
GARRY D. BREWER,
Yale University, New Haven
EDWIN H. CLARK,
Department of Natural Resources & Environmental Control, State of Delaware, Dover
YORAM COHEN,
University of California, Los Angeles
JOHN L. EMMERSON,
Lilly Research Laboratories, Greenfield, Indiana
ROBERT L. HARNESS,
Monsanto Agricultural Company, St. Louis
ALFRED G. KNUDSON,
Fox Chase Cancer Center, Philadelphia
GENE E. LIKENS,
The New York Botanical Garden, Millbrook
PAUL J. LIOY,
UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey
JANE LUBCHENCO,
Oregon State University, Corvallis
DONALD MATTISON,
University of Pittsburgh, Pittsburgh
GORDON ORIANS,
University of Washington, Seattle
NATHANIEL REED,
Hobe Sound, Florida
MARGARET M. SEMINARIO,
AFL/CIO, Washington, DC
I. GLENN SIPES,
University of Arizona, Tucson
WALTER J. WEBER, JR.,
University of Michigan, Ann Arbor
Staff
JAMES J. REISA, Director
DAVID J. POLICANSKY, Associate Director and Program Director for Applied Ecology and Natural Resources
RICHARD D. THOMAS, Associate Director and Program Director for Human Toxicology and Risk Assessment
LEE R. PAULSON, Program Director for Information Systems and Statistics
RAYMOND A. WASSEL, Program Director for Environmental Sciences and Engineering
OCR for page R6
Biologic Markers in Immunotoxicology
Commission on Life Sciences
BRUCE M. ALBERTS (Chairman),
University of California, San Francisco
BRUCE N. AMES,
University of California, Berkeley
J. MICHAEL BISHOP,
Hooper Research Foundation, University of California Medical Center, San Francisco
MICHAEL T. CLEGG,
University of California, Riverside
GLENN A. CROSBY,
Washington State University, Pullman
LEROY E. HOOD,
California Institute of Technology, Pasadena
DONALD F. HORNIG,
Harvard School of Public Health, Boston
MARIAN E. KOSHLAND,
University of California, Berkeley
RICHARD E. LENSKI,
University of California, Irvine
STEVEN P. PAKES,
Southwestern Medical School, University of Texas, Dallas
EMIL A. PFITZER,
Hoffman-LaRoche, Inc., Nutley, New Jersey
THOMAS D. POLLARD,
Johns Hopkins Medical School, Baltimore
JOSEPH E. RALL,
National Institutes of Health, Bethesda, Maryland
RICHARD D. REMINGTON,
University of Iowa, Iowa City
PAUL G. RISSER,
University of New Mexico, Albuquerque
HAROLD M. SCHMECK, JR.,
Armonk, New York
RICHARD B. SETLOW,
Brookhaven National Laboratory, Upton, New York
CARLA J. SHATZ,
Stanford University School of Medicine, Stanford
TORSTEN N. WIESEL,
Rockefeller University, New York, NY
JOHN E. BURRIS, Executive Director
OCR for page R7
Biologic Markers in Immunotoxicology
Preface
The American people have become increasingly aware of the potential for exposure to toxic material in our environment and of a need for accurate, objective information on the health effects of pollutants. In keeping with that need, the Agency for Toxic Substances and Disease Registry of the U.S. Public Health Service, the Office of Health Research of the U.S. Environmental Protection Agency, the National Institute of Environmental Health Sciences, and the National Institute of Allergy and Infectious Disease requested the Board on Environmental Studies and Toxicology in the National Research Council's Commission on Life Sciences to examine the potential for use of biologic markers in environmental health research. The term "biologic markers" has been used by the National Research Council's Committee on Biologic Markers to refer to indicators of events in biologic systems or samples. It is useful to classify biologic markers into three types—markers of exposure, of effect, and of susceptibility—and to describe the events peculiar to each type.
The Committee on Biologic Markers was organized to consider the areas of environmental research in which the use of biologic markers offered the greatest potential for major contributions. Four biologic systems were chosen: the reproductive system, the respiratory system, the immune system, and the urinary system. A companion report, Environmental Neurotoxicology, emphasizes biologic markers for the nervous system. This report is the product of the Subcommittee on Immunotoxicology, which included clinicians, epidemiologists, toxicologists, pathologists, and biochemists. Our intent was to consider various kinds of basic research that might reveal markers of environmental exposure and disease, even if the original goal of the research had nothing to do with such markers. Eventually, the subcommittee decided to place major emphasis on biologic markers of three types: markers originating from the immune system, markers related to immunosuppressive toxicants of exposure, and markers of effects of environmental pollutants. Markers of susceptibility to environmental materials also were considered important and were included especially if they were of a genetic nature and could serve to identify individuals who are susceptible to autoimmune diseases.
The subcommittee decided to organize this report according to types of action on the
OCR for page R8
Biologic Markers in Immunotoxicology
immune system (hypersensitivity or suppression), rather than according to specific pollutants, on the grounds that it is more important to discuss general approaches than to attempt to compile a list of pollutant-specific markers.
In the course of the subcommittee's deliberations, several additional scientists were called on to provide information. The subcommittee especially wishes to recognize the contributions of Gary Burleson of the U.S. Environmental Protection Agency.
This report could not have been produced without the untiring efforts of the National Research Council staff, especially Robert P. Beliles, the program officer; Joyce Walz, the project assistant; Danielle Corriveau, the administrative secretary; Tania Williams, who prepared the camera copy; Kate Kelly and Norman Grossblatt, the editors of the report; Devra Davis, resident scholar; and Richard D. Thomas, associate director, and James J. Reisa, director of the Board on Environmental Studies and Toxicology.
David Talmage, Chairman
Subcommittee on Immunotoxicology
OCR for page R9
Biologic Markers in Immunotoxicology
Contents
LIST OF TABLES AND FIGURES
xiii
LIST OF ABBREVIATIONS
xv
SUMMARY
1
Hypersensitivity
2
Autoimmunity
2
Immune Suppression
3
Clinical Application of Existing Immunotoxicologic Biomarkers
4
Role of Biologic Markers of Immunotoxicity in Epidemiology
4
Indoor Air Pollution and Multiple Chemical Sensitivity
5
1
INTRODUCTION
9
Biologic Markers
11
Validity of Biologic Markers
19
Uncertainty and Risk
21
Ethical and Practical Issues
21
Structure of the Report
22
2
THE STRUCTURE AND FUNCTION OF THE IMMUNE SYSTEM AND MECHANISMS OF IMMUNOTOXICITY
23
Development and Function of the Immune System
24
Mechanisms of Chemically Induced Immune Disease
26
Effects of Xenobiotics on the Immune System
30
3
BIOLOGIC MARKERS FOR IMMUNE-MEDIATED DISEASE
33
Definition of the Problem
33
Exposure Through Inhalation (Pulmonary Hypersensitivity)
34
Exposure Through Ingestion
37
Dermal Exposure
38
Nonspecific Immune Enhancement
40
Biologic Markers of Hypersensitivity
41
Animal Models for Detecting Chemically Mediated Hypersensitivity
43
Summary
49
OCR for page R10
Biologic Markers in Immunotoxicology
Recommendations for Future Research
50
IgE and Cellular Immunity
51
4
AUTOIMMUNE DISEASES
53
Definition of the Problem
53
Incidence of Autoimmune Diseases
53
Susceptibility Versus Exposure
55
Xenobiotic-Induced Autoimmunity
56
Mechanisms
57
Animal Models
58
Biologic Markers
59
Major Histocompatibility Complex
59
Immunoglobulin Allotypes
59
Other Genetic Markers
59
Rate of Acetylation
60
Summary and Conclusions
61
5
THE CAPACITY OF TOXIC AGENTS TO COMPROMISE THE IMMUNE SYSTEM (BIOLOGIC MARKERS OF IMMUNOSUPPRESSION)
63
Consequences of Immunosuppression
64
Environmental Contaminants
68
Inhalation and Immunosuppression
74
Skin and Immunosuppression
77
Myelotoxicity and Immunosuppression
77
Difficulties in Establishing Human Risk
78
Factors That Affect Susceptibility
78
Importance of Mechanistic Studies
80
Summary
80
Recommendations
81
6
ANIMAL MODELS FOR USE IN DETECTING IMMUNOTOXIC POTENTIAL AND DETERMINING MECHANISMS OF ACTION
83
Animal Immunotoxicity Bioassays
83
Assays of Pulmonary Immunocompetence
90
Assays Requiring Additional Development
91
Use of Immunotoxicity Bioassays
92
Summary
97
Recommendations
97
7
HUMAN IMMUNE-SYSTEM BIOLOGIC MARKERS OF IMMUNOTOXICITY
99
Tests for Assessing Immunity
99
Tests of the Humoral Immune System
100
Cellular Immune System
102
Other Tests
104
Opportunities for Development of Biologic Markers That Assess the Effect of Immunotoxicants
105
Proposed Testing Regimen
108
OCR for page R11
Biologic Markers in Immunotoxicology
Summary
110
Recommendations
111
8
APPLICATION OF BIOLOGIC MARKERS OF IMMUNOTOXICITY IN EPIDEMIOLOGY
113
Epidemiology
113
Contribution of Biologic Markers to Epidemiology
114
Variability in Reference Populations
115
Sensitivity, Specificity, and Predictive Value
115
Authentication of the Event Status
117
Study Design
119
Reference Populations
120
Case Studies
121
Recommendations
125
9
USE OF BIOLOGIC MARKERS IN CONTROVERSIAL AREAS OF ENVIRONMENTAL HEALTH
127
Evidence of Exposure to Organic Chemicals
128
Health Effects of Indoor Air Contaminants
128
Case Definitions of Multiple Chemical Sensitivity Syndrome
132
Immune-System Dysfunction in MCS Patients
134
Biologic Markers of Sensitivity to Chemicals
136
Antibodies to Formaldehyde-Human Serum Albumin Adducts
137
Conclusions
137
Recommendations
138
10
SUMMARY AND RECOMMENDATIONS
141
Chemical-Induced Immunosuppression in Humans
143
Role of Environmental Chemical Exposure in Hypersensitivity and Autoimmune Diseases
145
Animal and In Vitro Models
145
Markers of Skin and Mucosal Responses
147
Education and Training
147
Environmental Exposures and Sensitivity Syndromes
148
REFERENCES
149
GLOSSARY
183
BIOGRAPHIES
193
INDEX
199
ADDENDUM: MULTIPLE CHEMICAL SENSITIVITY SEPARATE VOLUME
OCR for page R12
Biologic Markers in Immunotoxicology
This page in the original is blank.
OCR for page R13
Biologic Markers in Immunotoxicology
Tables and Figures
TABLES
1-1
Health Effects Associated with Immune Dysfunction,
14
1-2
Factors Influencing the Immune System and Associated Markers,
17
2-1
Immunologic Reactions,
28
3-1
Methods of Detecting Chemicals That Produce Contact Hypersensitivity,
45
4-1
Xenobiotics Incriminated in Human Autoimmunity,
54
4-2
Autoimmune Diseases Related to Specific Xenobiotic Exposure,
55
5-1
Consequences of Immunosuppression,
65
5-2
Species Comparison of Immune Responses Suppressed by Cyclosporin A,
67
5-3
Classes and Examples of Chemicals Causing Immunological Changes,
70
5-4
EPA Survey Concentrations of Groundwater Contaminants and Composition of a Complex Chemical Mixture Representing a Contaminated Groundwater Sample,
75
6-1
Approaches to Animal Immunotoxicity Testing,
85
6-2
Validated Rodent Immunoassays,
86
7-1
Tier 1 (All Persons Exposed to Immunotoxicants),
108
7-2
Tier 2 (All Persons with Abnormal Tier 1 Results and a Fraction of the Total Exposed Population to be Determined by Statistician),
109
7-3
Tier 3 (To be Considered for Those with Abnormalities in Tier 2 Tests or for a Random Fraction of the Entire Population in Tier 2),
110
8-1
Three examples of the Relationship between Exposed Subjects and the Presence (+) or Absence (-) of Markers Illustrating the Interaction of Prevalence, Sensitivity, Specificity, and Predictive Value,
116
8-2
Interrelationship Among Prevalence, Sensitivity, and Specificity,
118
9-1
Randolph's Characterization of MCS,
129
9-2
Agents Reported to Cause Symptoms in Chemically Sensitive Individuals,
133
9-3
Cullen's MCS Case Definition,
134
OCR for page R14
Biologic Markers in Immunotoxicology
FIGURES
1-1
Simplified Flow Chart of Classes of Biologic Markers,
12
2-1
Cellular Interactions Involved in the Generation of an Immune Response,
25
2-2
Model of the Competent Immune System, Depicting Normal Interrelation of the Major Components,
26
2-3
Model of the Competent Immune System, Depicting Sites of Potential Effects on the Major Components by Toxins
3-1
Sensitization and Elicitation,
47
3-2
Selected Methods Over Time for Detecting Chemicals That Produce Contact,
48
OCR for page R15
Biologic Markers in Immunotoxicology
List of Abbreviations
ADCC
Antibody-dependent cell-mediated cytotoxicity
ACGIH
American Conference of Governmental Industrial Hygienists
ACTH
Adrenal cortical trophic hormone
AIDS
Acquired immune deficiency syndrome
ATSDR
Agency for Toxic Substances and Disease Registry
BALF
Bronchoalveolar lavage fluid
B-cell system
Humoral immune system
B16F10
Mouse tumor cell model (melanoma)
C1-C9
Complement system components
cAMP
Cyclic adenosine monophosphate
CD
Cluster of differentiation, e.g. CD3
CD3
T-cell surface marker associated with the T-cell receptor for antigen
CD4
T-cell surface marker identifying the helper (or inducer) subset of T cells
CD8
T-cell surface marker identifying the suppressor (or cytotoxic) subset of T cells
CD4:CD8
Helper/suppressor cell (ratio)
CD19
B-cell surface marker
CD20
B-cell surface marker
CD22
B-cell marker present on the membrane of mature B cells and in the cytoplasm of immature B cells
CD25
T-cell surface marker identifying marker for IL-2 receptor
CFU
Colony-forming unit
CFU-B
Colony-forming unit, basophils
CFU-G
Colony-forming unit, granulocytes
CFU-GM
Colony-forming unit, granulocytes and macrophages
CH50
Hemolytic complement
CMI
Cell-mediated immunity
C1q
Subunit of first component of complement
Con A
Concanavalin A
CsA
Cyclosporin A
OCR for page R16
Biologic Markers in Immunotoxicology
CSF
Colony-stimulating factor
CTL
Cytotoxic T lymphocyte
DT
Diphtheria-tetanus (vaccine)
DPT
Diphtheria-pertussis-tetanus (vaccine)
DTH
Delayed-type hypersensitivity
EAE
Experimental allergic encephalomyelitis
EBV
Epstein-Barr virus
EI
Environmental illness
ELISA
Enzyme-linked immunosorbent assay
Fc
Fragment cystalline- The fragment of an antibody that is responsible for binding to antibody receptors on cells and the C1q component of complement
FEV-1
Forced expiratory volume in one second
Gm
Gammaglobulin
GM-CSF
Granulocyte-macrophage colony-stimulating factor
Gmfb
Gammaglobulin allotype
GVH
Graft versus host
HAH
Halogenated aromatic hydrocarbon
HIV
Human immunodeficiency virus
HLA
Human leukocyte antigen
HLA-B27
HLA associated with ankylosing spondylitis
HLA-Rw4
HLA associated with Pemphigus vulgaris
HSA
Human serum albumin
IFN
Interferon
Ia
Murine class II major histocompatibility complex antigen
Ig
Immunoglobulin class; A, D, E, G, M
IL-1 -IL-8
Interleukin, one through eight
IU
International unit
K562
Sensitive cell target for NK cell assay (leukemic cell line)
kg
Kilogram
KLH
Keyhole limpet hemocyanin
LAK
Lymphokine-activated killer (cells)
LALN
Lung-associated lymph nodes
LPS
Lipopolysaccharide, a B-cell-specific mitogen
MCMV
Murine cytomegalovirus
MCS
Multiple chemical sensitivity
MDI
Methylene diphenyl diisocyanate
mg
Milligram
MHC
Major histocompatibility complex
MLC
Mixed-lymphocyte culture
MLR
Mixed-leukocyte response
mm3
Cubic millimeter
NK
Natural killer cell
PAF
Platelet activating factor
PAH
Polycylic aromatic hydrocarbon
PBB
Polybrominated biphenyl
PCB
Polychlorinated biphenyl
PFC
Plaque-forming cell
OCR for page R17
Biologic Markers in Immunotoxicology
PHA
Phytohemagglutinin, T-cell mitogen
PHSC
Pluripotent hematopoietic stem cell
PPD
Purified protein derivative
ppm
Parts per million
PRP
Polyribose phosphate
PWM
Pokeweed mitogen, a T-and B-cell mitogen
PYB6
Mouse tumor cell model (fibrosarcoma)
RBC
Red blood cells
SAC
Staphylococcus aureus Cowen strain activator
SBS
Sick building syndrome
SCID
Severe combined immunodeficiency
SLE
Systemic lupus erythematosus
SRBC
Sheep red blood cell
T-cell system
Cellular immune system
TEAM
Total Exposure Assessment Methodology study
Thy-1
T-cell marker related to thymic maturation
VOC
Volatile organic compound
YAC-1
Mouse tumor cell model (lymphoma) used to test NK activity
OCR for page R18
Biologic Markers in Immunotoxicology
This page in the original is blank.
