1 Department of Epidemiology, University of North Carolina, Chapel Hill, NC.
2 Department of Pediatrics, University of North Carolina, Chapel Hill, NC.
3 Centers for Disease Control and Prevention, Atlanta, GA.
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ABSTRACT |
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DDE; DDT; DNA; immunity; micronuclei; pesticides
Abbreviations: BHC, hexachlorocyclohexane; con A, concanavalin A; DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene; DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; IgA, immunoglobulin A; IgE, immunoglobulin E; IgG, immunoglobulin G; IgM, immunoglobulin M; PHA, phytohemagglutinin; PWM, pokeweed mitogen
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INTRODUCTION |
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Phase II was undertaken to determine 1) whether or not there was any evidence of human exposure as the result of living near the dumpsites, as indicated by higher levels of organochlorine pesticides in blood, and 2) whether or not living near the sites was associated with immune suppression (as indicated by various markers of immune competence) and DNA damage (as indicated by the presence of lymphocyte micronculei). Results reported elsewhere showed that Aberdeen residents overall and those who lived within a mile of the dumpsites had higher plasma DDE (a metabolite of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT)) levels as well as lower mitogen-induced lymphoproliferative activity controlling for age, race, and smoking than residents of nearby communities (1, 3
). The current study was conducted as part of Phase II.
In a previous pilot study of pet dogs in the area, Backer (4) found some evidence of immune suppression and peripheral lymphocyte chromosome damage in dogs that lived in Aberdeen compared with dogs in nearby communities with no known dumpsites. Specifically, 20 pet dogs from Aberdeen had nonstatistically significant lower CD4/CD8 ratios (an indicator of immune suppression) than did the 21 control dogs in nearby communities (CD4/CD8: 1.54 ± 1.14 vs. 2.05 ± 0.87). In addition, the dogs from Aberdeen had statistically significant higher frequencies of lymphocyte micronuclei (number of micronuclei/1,000 bi-nucleate cells: 24.2 vs. 11.0, p < 0.0001).
Animal studies have shown that organochlorine pesticide exposure (including DDT) can have detrimental effects on humoral and cell-mediated immune responses as well as host resistance to challenge with infectious agents (5, 6
). Much less is known about the immunotoxic effects of organochlorine pesticides in humans. Of particular concern for most people are the effects of chronic low-dose exposures. However, the few studies that have been conducted have been among workers with high-dose exposures. For example, workers exposed to DDT have experienced depressed neutrophil function and increased frequency of upper respiratory infections compared with controls (7
). In in vitro studies, DDT has been found to inhibit human erythrocyte rosette formation as well as phytohemagglutinin (PHA) mitogenesis (8
). The clinical consequences of such effects are not clear. There is some evidence to suggest that people occupationally exposed to pesticides, and organo-chlorine pesticides in particular, have increased risks of cancers of the lymphopoietic and hematopoietic systems, including leukemia and non-Hogkins lymphoma (9
). However, not all studies of workers exposed to organochlorine have found elevated risks of cancer (10
).
One of the steps in the initiation of some cancers is chromosomal damage (e.g., mutations, chromosome breaks, aneuploidy) and one indication that chromosome damage has occurred is the presence of micronuclei in cell cytoplasm. Micronuclei are chromosomes or chromosome fragments that have not been incorporated into daughter nuclei during cell division and can be induced by agents that cause chromosome breaks (clastogens) or agents that interfere with the spindle apparatus during nuclear division (11). Because micronuclei are produced during nuclear division, they are typically indicative of more recent rather than historic exposure to chromosome-damaging agents (12
).
The organochlorines, DDT, heptachlor, and chlordane are considered animal carcinogens by the International Agency for Research on Cancer (13). Most carcinogens have been found to be mutagens (14
). Hagmar et al. (15
) have shown that workers with elevated levels of chromosome aberrations are more likely to develop cancer. Although few studies of the mutagenic potential of pesticides in humans have been conducted, those studies of workers exposed occupationally to complexes of pesticides (including organochlorines) have shown that pesticide exposure is associated with damage to DNA (16
24
). In one of the rare studies with individual exposure information concerning organochlorine levels, a dose-response relation was noted between plasma DDT levels and an increase in chromosome aberrations (25
).
There are many potential markers of immune suppression. In this study, we selected four major classes of markers as a screen: 1) the absolute number and percentages of white blood cells, lymphocytes, and their subsets, 2) the levels of different classes of immunoglobulins, 3) the delayed-type hypersensitivity reaction to six antigens and a control as determined using the Multitest CMI skin test (Pasteur Merieux Serums & Vaccines S.A., Lyon, France, distributed by Connaught Laboratories, Inc., Swiftwater, PA), and 4) mitogen stimulation assays. These markers are broad indicators of both cell-mediated and humoral immune function that are important in the protection from and reaction to infection as well as nonspecific resistance to infection. Classes 13 above include immune markers that are recommended by the National Research Council (26) as first-level tests for individuals suspected of immune deficiency. The Multitest skin test has been advocated as an alternative to intradermal skin testing, since one can simultaneously apply several antigens in a standardized manner. Delayed-type hypersensitivity skin testing is an essential component of the evaluation of immune function reflecting cell-mediated immunity. Because it involves multiple steps including antigen recognition and processing, T-lymphocyte activation and response, cytokine production, and cell migration, it is thought to best represent an individual's response to exogenous antigens. The mitogen stimulation assay is an alternative method to delayed-type hypersensitivity skin testing to evaluate lymphocyte function. This in vitro assay compares the ability of a person's lymphocytes to undergo blastogenesis in response to chemical stimulants (mitogens). The response is compared with a person's own unstimulated response and with that of known controls. Mitogens used in this study include PHA, concanavalin A (con A), and pokeweed mitogen (PWM). These mitogens represent two T-lymphocyte mitogens and a T-cell-dependent B cell mitogen. A positive mitogen test panel requires the functioning of both T- and B-cells as well as macrophages. Suppression of lymphocyte function may result in increased susceptibility to both infection and cancer. Other, more specific immune assays were considered for inclusion in the study (e.g., assays for cytokines), but were not performed because of cost considerations.
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MATERIALS AND METHODS |
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Participants were enrolled in the Phase II study between September 1994 and March 1996. Potentially eligible residents were sent a letter explaining the study. The letter was followed by a telephone call to assess eligibility. If the participant was eligible, two appointments were scheduled: one to answer a 30- minute telephone questionnaire and one to provide 45 ml of blood and to undergo a skin test at a local health clinic from 79:30 a.m. on a Tuesday morning. The skin test was read 48 hours later. A consent form was administered before any laboratory procedures were performed. Study participants were paid $20 for out-of-pocket expenses. This research was approved by the Institutional Review Boards at the University of North Carolina Schools of Public Health and Medicine.
Excluded from participation were those individuals who had a bleeding disorder, those who reported testing positive for the human immunodeficiency virus, those who reported having eczema on the forearms, those who had had an asthma attack in the previous year (which might make them more susceptible to an allergic reaction to the skin test), those who had ever worked in a pesticide-manufacturing company, and those residents from the comparison communities who had worked in Aberdeen for at least 20 hours a week during the previous year. Potential participants were temporarily excluded, i.e., enrollment was deferred, if they reported having had an acute infection within the previous month, or surgery, chemotherapy, radiation therapy, immunizations, or a blood transfusion or having taken immunosuppressive drugs within the previous 2 months. Women who were pregnant or lactating were also temporarily excluded. Eligibility criteria were reassessed during a reminder call the night before the clinic visit. Appointments were rescheduled when necessary. Of the residents eligible for the Phase I Study, 38 percent of those contacted in Aberdeen and 37 percent in the comparison areas participated in the Phase II study. The most common reason for refusal to participate was being too busy to commit to a 30-minute telephone interview and two clinic visits. A more detailed description of participant selection is found in the reports by Vine et al. (1, 3
).
Blood drawing
Forty-five ml of blood were drawn from each study participant into vacutainer tubes by a trained nurse. The blood specimens were analyzed for plasma organochlorine pesticide levels (including DDE) and indicators of immune competence including numbers and percentages of various types of white blood cells, immunoglobulin levels (immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin E (IgE), and immunoglobulin M (IgM)) and degree of mitogen-induced lymphoproliferative activity with three different mitogens. The blood specimens were also analyzed for the presence of DNA damage, as indicated by the presence of lymphocyte micronuclei.
Plasma DDE levels
Plasma DDE levels for 301 participants were evaluated as part of an organochlorine pesticide panel that included alpha-hexachlorocyclohexane (BHC), beta-BHC. delta-BHC, lindane, hexachlorobenzene, alpha endosulfan, beta endosulfan, dieldrin, endrin, cis-chlordane, trans-chlordane, oxychlordane, heptachlor, and heptachlorepoxide. Assay methods were developed by LabCorp (Burlington, North Carolina) based on methods reported by Lopez-Avila et al. (27) and Saady and Poklis (28
). Briefly, chlorinated pesticides in plasma were extracted from a methanol-deproteinated sample into hexane, and the extract was cleaned by C18 solid phase extraction. The extract was injected into a gas chromatograph with dual capillary columns and dual electron capture detectors (Perkin Elmer gas chromatograph, Perkin Elmer, Norwalk, Connecticut). Both columns had to indicate a positive result in order for a sample to be considered positive for a pesticide. The gas chromatograph and associated data reduction equipment were calibrated on each day of use, with positive and negative controls run after every tenth sample. Positive controls contained known amounts of every analyte included in the analysis. Negative controls consisted of a serum matrix. The average recovery for all analytes in the assay was estimated to be 100.9 percent. The lower limit of quantification for the assay was 1 ppb (Dr. Richard Earley, Director of Biological Monitoring/Toxicology, LabCorp of America, Burlington, North Carolina, personal communication, 2000; and Peter Wentz, Director of Toxicology, LabCorp of America, Burlington, North Carolina, personal communication, 1996).
The same panel of 20 pesticides was analyzed at a different laboratory (Research Triangle Institute, Research Triangle Park, North Carolina) on duplicate specimens for 14 arbitrarily selected study participants over a 3-week period. Agreement between the two laboratories was excellent (correlation coefficient 5 0.96).
Complete blood cell counts
A complete blood cell count was performed on 298 participants. Standardized LabCorp procedures with Westgard rules of quality control were used (Laura Hale, LabCorp of America, personal communication, 1995).
Lymphocyte phenotypes
Percentages of the following lymphocyte phenotypes were determined at the University of North Carolina Hospitals according to the methods described by Tamul et al. (29): CD3 (total T-cells), CD3 positive/CD4 positive (CD4) (T-helper cells), CD3 positive/CD8 positive (CD8) (T-suppressor cells), CD16 (majority of natural killer cells), CD19 (total B cells), and CD56 (natural killer cells subset). Centers for Disease Control (CDC) guidelines for quality control were followed (30
).
Immunoglobulin levels
Immunoglobulin levels of IgG, IgA, IgM, and IgE were analyzed for 299 study participants. IgG, IgA, and IgM were assayed using a quantitative turbidimetric method based on an antibody-antigen reaction adapted for a centrifugal analyzer (Roche Cobas Mira, Indianapolis, Indiana) with reference ranges determined by LabCorp.
IgE levels were analyzed in a one-step solid-phase immunoassay using two highly specific monoclonal mouse antibodies to IgE (Ciba-Corning Total IgE, Ciba Corning, Atlanta, Georgia) with the Roche Cobas Core IgE instrument. The coefficient of variation for low, medium and high mean values was less than 5 percent (Eric Schett, Technical Director, Allergy Department, LabCorp of America, Burlington, North Carolina, personal communication, 1996).
Mitogen-induced lymphoproliferative activity
To assess mitogen-induced lymphoproliferative activity, mitogen stimulation assays were performed as described by Wilson et al. (31) on 260 study participants using three mitogens: PWM, con A, and PHA. (Reasons for unanalyzed specimens are: 19, unsatisfactory specimen (e.g. clotted); eight, technical difficulties; and 16, laboratory closed for vacation.) Blood samples were maintained under conditions established by the Acquired Immunodeficiency Syndrome (AIDS) Clinical Trials Study Group (30
) during transportation and delivered to the laboratory within 6 hours of blood donation. Lymphocyte separation and cultures were begun shortly thereafter. Addition of tritiated thymidine was performed at 72 hours, and cells were harvested 18 hours later. Radioactive counts per minute were quantitated using a Matrix 96 Direct Beta Counter (Packard Biosciences, Downers Grove, Illinois). Lower counts indicate immunosuppressive effects, i.e., reduced capacity of lymphocytes to proliferate in response to an antigen.
Micronucleus assay with peripheral blood lymphocytes
Of the 302 study participants, results for the micronucleus assay were available for 254 participants. (Reasons for unanalyzed specimens are: 14, unsatisfactory specimen; 11, technical difficulties; and 23, laboratory closed for vacation.) The peripheral blood lymphocytes were cultured as described by Erexson et al. (32, 33
). All assays were cultured by one technician, and slides from the 254 participants were read by three different scorers trained by one person. Scorers noted the total number of micronuclei per 1,000 bi-nucleates as well as the percent of cells that contained micronuclei. Scorers were unaware of the characteristics of the individuals whose slides they were scoring. Despite quality control efforts, one scorer tended to produce lower values than the other two scorers, requiring us to control for scorer in the statistical analyses.
Skin test
Two-hundred-ninety-seven adults underwent a skin test using the Multitest CMI skin test. The Multitest CMI skin test consists of a disposable plastic applicator containing eight sterile test units preloaded with seven delayed hypersensitivity skin test antigens and a glycerine control. Seven antigens were used in this study: tetanus toxoid, diphtheria toxoid, Candida, Trichophyton, Streptococcus, and Proteus. The tuberculin unit was detached from the applicator prior to use because 1) increased rates of mycobacteria exposure in the South (where the study took place) would likely have increased reactions to the tuberculin test, and 2) follow-up of false-positive skin tests with further evaluation and treatment would have been unduly burdensome to the study participants. Five persons who reported having previous widespread or systemic reactions to prior skin tests or prior tetanus or diphtheria vaccination were excluded from the skin test studies.
A trained nurse applied the skin test on the inner forearm of each study participant between 7:00 and 9:30 am on a Tuesday morning and then read the skin test results 48 hours later. For each antigen site, the indurated area was measured to the nearest 0.5 mm across two diameters perpendicular to each other. The overall diameter of the indurated area was calculated as the mean of the two diameters. In normal individuals previously exposed to one of the antigens, an indurated area should occur on the forearm, indicating that the immune system recognized the antigen. A negative test result could mean that the immune system did not recognize the antigen or that the person had never been previously exposed.
Telephone questionnaire
A 30-minute telephone questionnaire was administered by trained interviewers to all participants within the two weeks prior to their appointment at the clinic to provide blood and undergo the skin test. The questionnaire assessed residential, recreational, and occupational exposures to pesticides, other chemicals, and metals. In addition, residents were asked about their residential history; sources of tap water; water consumption; dietary intake of caffeine, alcohol, fruits, vegetables, meat, and dairy products; smoking habits; medical history; reproductive history (women only); allergies; perceived stress; and perceptions concerning health risk due to environmental exposures in their neighborhood.
Statistical analysis methods
In order to evaluate dose-response patterns, plasma DDE levels were grouped by approximate quartiles in the overall study population (1 or undetectable, >12, 24.3, >4.3 ppb) or into five groups where the top quartile was split into two groups at the median 7.6 ppb. Crude associations between the outcome measures and DDE levels were evaluated by comparing mean values by exposure group using t-tests. Adjusted least-squares estimates (34) of mean levels of each of the markers by plasma DDE level were calculated using PROC GLM (SAS, Cary, North Carolina) controlling for potential confounders. The p-values for linear trend were determined for increasing DDE exposure levels based on the statistical significance of DDE as an ordinally coded variable from a separate, but comparable, model.
The number and percent of positive responses to all skin tests combined and to each antigen separately were also calculated by DDE level (high (5 ppb), low (<5 ppb)) for males and females, separately, using chi-square analyses. (Males were more likely than females to respond positively to skin test antigens (35
)). Multivariate logistic regression analyses stratified by sex were performed to assess differences in skin test positivity across plasma DDE categories controlling for potential confounders.
Confounder selection for exposure-outcome analyses
Because of the many exposure-outcome associations evaluated, for ease and consistency of reporting, we tried to develop a reasonable set of confounders. Variables evaluated as possible confounders included age; race; sex; ever breastfed; smoking; education; household income; marital status; employment status; number living in household; children less than age 6 years (yes/no); and consumption of alcohol, caffeine, fruits, vegetables, meat, and dairy products. Excluded from consideration as potential confounders were medical history factors that could, theoretically, have been a consequence of immune function. Variables were retained in models as confounders when inclusion changed the adjusted mean value of the outcome by more than 10 percent within categorized levels of DDE. Final models included: age (<40, 4049, 5059, and 60 years), sex (male/female), and smoking expressed as pack-years (non-/ex-smoker, <10, 1038, and
39 pack-years).
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RESULTS |
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Skin test results.
There were no consistent trends in skin test positivity associated with elevated plasma DDE levels. Multivariate logistic regression modeling controlling for household income of more than $20,000, race, and pack-years indicated that men with increased plasma DDE levels were more likely to react positively to diphtheria, Candida, and Proteus, whereas women were more likely to react positively to diphtheria (table 6). There were too few positive skin test results to model Streptococcus, Trichophyton, Proteus, or Candida among women and Trichophyton among either men or women.
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DISCUSSION |
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Lowered mitogen-induced lymphoproliferative activities indicate that both T- and B-cells are less responsive to stimulation by particular mitogens and, therefore, suggest that they are less able to initiate or augment an immune response to foreign agents. In states where there is substantial suppression of T- and B-cell function, there is increased susceptibility to infection (for example, by viral and fungal agents) and to cancer. Modest increases in white blood cells, lymphocytes, and IgA levels in association with increasing plasma DDE levels suggest that DDT exposure elicits an immune response. The consequences, if any, of such a response are not clear.
Decreased mitogen-induced lymphoproliferative activity in association with organochlorine exposure has been noted in other studies. McConnachie and Zahalsky (37) reported an association between organochlorine exposure (chlordane) and decreased response to mitogen stimulation (PHA, con A, and PWM) among people exposed at home or at work 210 years prior to the study. In our study, counts for con A, for example, dropped from 74,218 cpm in the lowest DDE-exposed category to 55,880 cpm in the highest DDE-exposed group. McConnachie and Zahalsky (37
) noted that counts for con A dropped from 99,691 cpm among the controls to 39,057 cpm among the exposed (p = 0.001). DDT has been found to inhibit human lymphocyte PHA mitogenesis in occupationally exposed workers (4
). Blood levels of DDT and DDE were also inversely correlated with PHA and con A mitogenesis in free-living bottle-nosed dolphins (38
).
As in this study, Khan and Ali (39) noted an elevation in white blood cells in association with occupational pesticide exposure. Hermanowicz et al. (7
) reported an increased incidence of tonsillitis among workers exposed to organochlorine pesticides. In this study, individuals with DDE levels above the median (2 ppb) were more likely to have had their tonsils or adenoids removed (>2 ppb: 74 of 145 (51 percent); 2 ppb: 58 of 156 (37 percent); p = 0.01).
Studies assessing the relation between organochlorine exposure and immunoglobulin levels have produced mixed results. Most human studies have involved workers with high dose exposures rather than individuals with residential exposures. In one study of workers exposed to chlorinated pesticides, exposure was associated with decreases in IgM levels and increases in IgG levels (40). In another study, exposure to hexachlorocyclohexane was associated with an elevation in IgM levels, but no change in IgG or IgA levels (41
). Klucinski et al. (42
) noted elevated IgM, IgG, and IgA levels among workers exposed to a variety of pesticides, including chlorinated hydrocarbons. For example, IgA levels were 219 mg/dl among control females and 313 mg/dl among female workers exposed to pesticide (p = 0.001). Among male workers, IgA levels were nonstatistically significantly higher than control levels (294 vs. 263 mg/dl).
Animal studies of organochlorine exposure and immunoglobulin levels have also produced conflicting results. For example, mice (43) and rats (44
) fed high doses of DDT experienced reductions in both IgG and IgM levels. Mice fed B-hexachlorocyclohexane (technical grade lindane) at a dose of 300 mg/kg for 30 days experienced a decrease in T-lymphocyte-mediated cytolysis of tumor targets and a reduction in natural killer cell activity (45
). However, there was no significant reduction in IgM or IgG plaque-forming cells. Chickens fed DDT (100 ppm ad libitum from hatching to 40 days) showed a decrease in IgG but an increase in IgM (46
). It has been suggested that the amount of protein in the diet may affect the immunosuppressive activity of DDT, with low-protein diets being associated with immune suppression in rats (47
). The mechanisms by which DDE might affect the immune system are still speculative.
No consistent differences in skin test positivity were found in association with plasma DDE levels evaluated separately by gender, controlling for age, race, income, and pack-years. There are few studies of this type with which to compare results. One study of Brazilian children exposed to DDT found no association between exposure and diphtheria immunization response (48). Street and Sharma (49
) noted a decreased delayed-type hypersensitivity skin test response to tuberculin in rabbits exposed to DDT. Bernstein and Storms (50
) suggest that the Multitest device may not be as sensitive a measure of delayed-type hypersensitivity testing as intradermal methods during the same time period. For example, in some studies, it took 7296 hours for responses to occur using the Multitest CMI skin test that took only 48 hours to appear using intradermal testing methods.
As per the results of previous studies, factors associated with immune markers in this study included age (51), sex (52
), and smoking (53
55
), although not every immune marker was associated with all three factors. Each factor was included as a potential confounder in analyses for consistency of reporting.
Low overall levels of plasma DDE (median = 2 ppb) limited our ability to detect dose-response relations between current plasma DDE levels and current immune marker measures. It is likely that organochlorine levels were higher in the past, when pesticide manufacturing plants in Aberdeen were operational. Residents who lived in Aberdeen prior to 1985 had higher DDE levels than did those who lived there only after that time (1, 3
). Data from the Second National Health and Nutrition Survey (NHANES II) (56
) conducted between 1976 and 1980 (shortly after DDT was banned for use in the United States in 1972) indicate that national DDE levels were also higher at that time (median = 12.0 ppb for those aged 2544 years and 18.3 ppb for those aged 4574 years). A recent study of organochlorine levels among women in North Carolina noted overall plasma DDE levels that were similar to those found in this study (57
).
Because the immune system has the capacity to recover from injury, past exposures could have resulted in immune changes that were not detectable in this study. For example, immune changes returned to normal within 3 months among a group of occupationally exposed pesticide workers who left employment (39). Furthermore, residents who were most affected by the exposures may not have been available for the study due to illness, death, or having moved away prior to the start of the study, limiting our ability to detect effects. Multiple comparisons and uncontrolled confounding may have accounted for some positive findings. While organochlorines were measured in the blood of study participants, other potential exposures to the dumpsites, such as solvents and metals, were not assessed directly. Distance from the dumpsites was used as a proxy measure of exposure in some analyses (1
, 3
) and showed that those residents who lived within a mile of the sites also had lowered mitogen-induced lymphoproliferative activity. However, dumpsite exposures would have affected only half of the people in this studythose who lived in Aberdeen. Yet, the association between higher plasma DDE levels and decreased mitogen-induced lymphoproliferative activity was also evident for PHA and con A among residents of the comparison community alone (data not shown). Small numbers could have affected results with PKW mitogen. Other environmental immunotoxins, such as polychlorinated biphenyls (which are sometimes correlated with DDE exposure), were also not measured in blood as they were not considered contaminants of the Aberdeen Pesticides Dumps Site.
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Conclusions |
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ACKNOWLEDGMENTS |
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The authors thank Dorothy Sims for performing the mitogen stimulation assay and Carol Morton and Elizabeth Powell for helping to prepare the final manuscript. Special thanks go to Minna Wiley for her tireless data collection efforts and to Wanda Garner Steele for drawing blood and administering the skin tests.
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NOTES |
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REFERENCES |
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