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Assessment of autoimmune responses associated with asbestos exposure in Libby, Montana, USA.



Systemic autoimmune responses are associated with certain environmental exposures, including crystalline particles such as silica. Positive antinudear antibody (ANA) tests have been reported in small cohorts exposed to asbestos, but many questions remain regarding the prevalence, pattern, and significance of autoantibodies associated with asbestos exposures. The population in Libby, Montana, provides a unique opportunity for such a study because of both occupational and environmental exposures that have occurred as a result of the mining of asbestos-contaminated vermiculite ver·mic·u·lite  
n.
Any of a group of micaceous hydrated silicate minerals related to the chlorites and used in heat-expanded form as insulation and as a planting medium.
 near the community. As part of a multifaceted assessment of the impact of asbestos exposures on this population, this study explored the possibility of exacerbated autoimmune responses. Age- and sex-matched sets of 50 serum samples from Libby and Missoula, Montana (unexposed), were tested for ANA on HEp-2 cells using indirect immunofluorescence. Data included frequency of positive tests, ANA titers, staining patterns, and scored fluorescence intensity, all against known controls. Serum immunoglobulin A immunoglobulin A
n. Abbr. IgA
The class of antibodies produced predominantly against ingested antigens, found in body secretions such as saliva, sweat, and tears, and functioning to prevent attachment of viruses and bacteria to epithelial
 (IgA), rheumatoid factor rheumatoid factor
n. Abbr. RF
Any of the immunoglobulins found in the serum of individuals with rheumatoid arthritis that enhance the agglutination of suspended particles that are coated with pooled human gamma globulin and that are used
, and antibodies to extractable nuclear antigen (ENA ENA Ecole Nationale d'Administration (French)
ENA Emergency Nurses Association
ENA Energy Networks Association (Australia)
ENA Ethiopian News Agency
ENA Energetic Neutral Atom
) were also tested. The Libby samples showed significantly higher frequency of positive ANA and ENA tests, increased mean fluorescence intensity and titers of the ANAs, and higher serum IgA, compared with Missoula samples. In the Libby samples, positive correlations were found between ANA titers and both lung disease lung disease Pulmonary disease Pulmonology Any condition causing or indicating impaired lung function Types of LD Obstructive lung disease–↓ in air flow caused by a narrowing or blockage of airways–eg, asthma, emphysema, chronic bronchitis;  severity and extent of exposure. The results support the hypothesis that asbestos exposure is associated with autoimmune responses and suggests that a relationship exists between those responses and asbestos-related disease processes. Key words: asbestos, ANA, environmental autoimmunity, immunotoxicology. doi:10.1289/ehp.7431 available via http://dx.doi.org/[Online 30 September 2004]

**********

Asbestos-related lung disease (ARD Ard (ärd), in the Bible.

1 Son of Benjamin.

2 Benjamite, perhaps the same as (1.) An alternate form is Addar.
), including fibrosis, pleural Pleural
Pleural refers to the pleura or membrane that enfolds the lungs.

Mentioned in: Pneumothorax


pleural

emanating from or pertaining to the pleura.
 plaques, and cancer, continues to be a serious and significant problem despite increasing awareness of the health hazards of asbestos inhalation. Although the exact mechanisms leading to the progression of these conditions have not been fully explained, there is evidence that some of the lung pathologies seen with both asbestos and silica exposures are immunologically mediated (Hamilton et al. 1996; Holian et al. 1997; Perkins et al. 1993). Silica and asbestos exposures also both appear to exacerbate autoimmune responses. Epidemiologic studies have shown strong associations between silica exposure and several autoimmune diseases Autoimmune diseases
A group of diseases, like rheumatoid arthritis and systemic lupus erythematosus, in which immune cells turn on the body, attacking various tissues and organs.

Mentioned in: Complement Deficiencies, Premature Menopause
, including scleroderma scleroderma
 or progressive systemic sclerosis

Chronic disease that hardens the skin and fixes it to underlying structures. Swelling and collagen buildup lead to loss of elasticity. The cause is unknown.
, systemic lupus erythematosus Systemic Lupus Erythematosus Definition

Systemic lupus erythematosus (also called lupus or SLE) is a disease where a person's immune system attacks and injures the body's own organs and tissues. Almost every system of the body can be affected by SLE.
 (SLE SLE systemic lupus erythematosus.

SLE
abbr.
systemic lupus erythematosus


Systemic lupus erythematosus (SLE) 
), and rheumatoid arthritis rheumatoid arthritis

Chronic, progressive autoimmune disease causing connective-tissue inflammation, mostly in synovial joints. It can occur at any age, is more common in women, and has an unpredictable course.
 (RA) (Koeger et al. 1995; Parks et al. 1999; Powell et al. 1999; Steenland and Goldsmith 1995). Increased serum immunoglobulins, positive antinuclear antibody an·ti·nu·cle·ar antibody
n. Abbr. ANA
An antibody that attacks cell nuclei.


antinuclear antibody,
n
 (ANA) tests, and immune complexes Immune complexes
Clusters or aggregates of antigen and antibody bound together.

Mentioned in: Wegener's Granulomatosis
 have been reported in small cohorts of individuals exposed to asbestos (Lange 1980; Nigam et al. 1993; Zerva et al. 1989), but to our knowledge no comprehensive study has been undertaken to assess the prevalence, specificity, and significance of autoantibodies associated with asbestos exposures. The population in Libby provides a unique research opportunity because of significant exposures that occurred as a result of the mining of asbestos-contaminated vermiculite near the community. Exposures have been documented not only in the miners but also in their family members, as well as anyone who used the vermiculite or played near the mine tailings Tailings (also known as tailings pile, tails, leach residue, or slickens[1]) are the materials left over[2] after the process of separating the valuable fraction from the worthless fraction of an ore. . Therefore, the Libby asbestos exposures were both occupational and environmental throughout the community (Peipins etal. 2003).

In addition to the ARD in Libby, there have been anecdotal reports of an increased prevalence of systemic autoimmune disease autoimmune disease, any of a number of abnormal conditions caused when the body produces antibodies to its own substances. In rheumatoid arthritis, a group of antibody molecules called collectively RF, or rheumatoid factor, is complexed to the individual's own gamma  (SAID), but verification of these diagnoses is still in progress. When the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry disease registry Public health A surveillance system that collects and maintains structured records on the new cases of a specific disease or condition for a specified time period and population; a DR analyzes, and interprets data those with a common illness or  (ATSDR ATSDR Agency for Toxic Substances & Disease Registry ) performed its screening in Libby during 2000-2001, 494 (6.7%) of 7,307 screening participants indicated that they had been diagnosed with either SLE, scleroderma, or RA (Larson T, personal communication). In contrast, a prevalence of < 1% for these three conditions combined would be expected based on pooled estimates from 43 prevalence studies (Jacobson et al. 1997). These data, along with extensive evidence of silica-associated autoimmunity, provided the impetus to initiate a multifaceted assessment of the impact of asbestos exposures on the population of Libby, Montana, including possible autoimmune responses.

In this article we report on a study designed to assess whether there were humoral hu·mor·al
adj.
1. Relating to body fluids, especially serum.

2. Relating to or arising from any of the bodily humors.


Humoral
Pertaining to or derived from a body fluid.
 alterations in serum samples from an asbestos-exposed population (Libby samples) that might indicate autoimmune responses, providing the rationale for future full-scale studies. Serum samples of subjects from Libby and from a Montana community with no reported asbestos exposure were assayed for a variety of immune parameters, including ANA, immunoglobulin A (IgA), rheumatoid factor (RF), and antibodies to extractable nuclear antigens Extractable Nuclear Antigens are soluble cytoplasmic and nuclear components that are antibody targets with over 100 differerent antigens described. The main 6 used in immunological laboratories for detection are Ro, La, Sm, RNP, Scl-70 and Jo1, which are screened for by Ouchterlony  (ENA).

Materials and Methods

Human samples. All samples were acquired according to approved University of Montana institutional review board protocols, protecting the well-being and confidentiality of all subjects. Appropriate informed consent was obtained from all subjects, and a questionnaire was administered regarding overall health, smoking status, asbestos exposure, age, sex, and recontact information.

Two sample pools were obtained through other studies at the Center for Environmental Health Sciences. Subjects were recruited through flyers and ads in Missoula, Montana, for a study of immune function Immune function
The state in which the body recognizes foreign materials and is able to neutralize them before they can do any harm.

Mentioned in: Herbalism, Traditional Chinese, Stress Reduction
 (E. Putnam), and serum samples were collected at the same time for convenience. Missoula is similar to Libby in that it is located in a mountain valley subject to similar climatic conditions, including winter inversions, dry summers, and exposure to smoke from fall forest fires. Because prevailing winds in Missoula are from the west and Libby is well to the north, there would be no transfer of asbestos from the Libby airshed to the Missoula airshed. Therefore, on a relative basis, although one cannot exclude the possibility that there could have been some minimal asbestos exposure by the Missoula population, it is an acceptable reference population for the Libby subjects who were definitely exposed to asbestos. For this study, we selected 50 samples from subjects with no reported asbestos exposure from the Missoula pool, excluding any who had lived or worked in Libby. Concurrently, subjects were being recruited from Libby, Montana, for a genetic study of ARD susceptibility, (E. Putnam), and the samples were drawn at the Center for Asbestos Related Diseases Clinic in Libby when subjects came for screening or responded to subject recruitment advertisements. From this pool, 50 Libby subjects were selected that were matched to the 50 Missoula subjects to give similar mean age and sex ratios for the two subject sets. Permission was obtained to acquire information about prescription drug prescription drug Prescription medication Pharmacology An FDA-approved drug which must, by federal law or regulation, be dispensed only pursuant to a prescription–eg, finished dose form and active ingredients subject to the provisos of the Federal Food, Drug,  use and ARD status from medical records, and these data were inserted into a coded database.

Both communities from which subjects were drawn are fairly homogeneous in terms of ethnicity; most residents are of northern European descent, with 94.7% white in Lincoln County, (Libby) and 93.6% white in Missoula County (Missoula) according to Montana census data (U.S. Census Bureau 2004). The mean age for both sample sets was 55 years (Missoula, 54.8 [+ or -] 2.5 years; Libby, 55.0 [+ or -] 2.1 years), and the male:female ratio was 25:25 for both sets. An exclusion criterion was the use of medications strongly associated with drug-induced autoimmunity (Fritzler 1994). The presence of diagnosed autoimmune disease did not exclude individuals from either sample set but was noted on intake. None of the Missoula subjects recruited had diagnosed SAID. Of the Libby subjects recruited initially for the genetics study, the percentage with diagnosed SAID was 5.9%, and in the final Libby sample set of 50, there were two with SLE, 1 with multiple sclerosis, ;rod 1 with RA. Because the individual with RA also had SLE, this means that three people in 50 had SAID (6%). These values are consistent with the ATSDR screening data for this community (Larson T, personal communication).

The lung diseases in this community have been previously described (Peipins et al. 2003) and include primarily pleural abnormalities (17.8%) and interstitial abnormalities (< 1%). In our Libby sample set, 12 (24%) had no reported abnormalities, 27 (54%) had pleural abnormalities, 8 (16%) had interstitial abnormalities, and 3 (6%) had a combination of pleural and interstitial abnormalities.

Sample and data collection. The blood samples were collected, and serum samples were obtained and frozen by standardized clinical methods to prevent differences due to handling. The samples were blinded with only sex and age noted, and stored at -80[degrees]C until assayed. After testing, coded information regarding disease status and exposure was obtained from the questionnaire and ATSDR screening data (ATSDR 2002).

ARD and asbestos exposure rankings. ARD status, based on data recorded in the database primarily as a result of the ATSDR screenings, was ranked on a scale of 0-3, as described in Table 1. The rankings were intentionally simplified, based on radiographic radiographic (rā´dēōgraf´ik),
adj relating to the process of radiography, the finished product, or its use.
 evidence of single versus extensive plaques or interstitial abnormalities, as well as spirometry Spirometry

The measurement, by a form of gas meter, of volumes of gas that can be moved in or out of the lungs. The classical spirometer is a hollow cylinder (bell) closed at its top.
 evidence of functional deficits. For example, a subject with a single pleural plaque and no functional deficit would be scored at 1, whereas a subject with bilateral plaques and effects on spirometry was scored at 3. To further break down the sample sets by disease types would have made the subsets too small for statistical analysis. Exposure status was ranked on a scale of 0-4, as described in Table 2. These scores were also simplified in order to focus on duration of exposure and the existence of occupational and/or environmental exposure to asbestos. The rankings of the Libby subjects were performed independently by two of the researchers (J.C.P. and E.A.P.).

Autoantibody autoantibody /au·to·an·ti·body/ (-an´ti-bod?e) an antibody formed in response to, and reacting against, an antigenic constituent of one's own tissues.

au·to·an·ti·bod·y
n.
 testing. A clinical test for nuclear antigens (ANA assay), used to screen for antibodies commonly seen in SAID, was performed at a screening dilution of the sera. All serum samples were diluted 1:40 in phosphate-buffered saline (PBS PBS
 in full Public Broadcasting Service

Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural,
) and tested by indirect immunofluorescence (IIF IIF Institute of International Finance
IIF Irish Insurance Federation
IIF Immediate IF
IIF Innovation Investment Fund (investment supporting R&D new technology/science ventures)
IIF Intuit Interchange Format
) on a single lot of commercially prepared and fixed HEp-2 cells (ImmunoConcepts Inc., Sacramento, CA), according to manufacturer's instructions. The staining pattern and relative fluorescence intensity were compared with known positive and negative controls using a Zeiss fluorescence microscope with 40x objective and recorded as positive (1+ to 4+) or negative (0). The staining pattern was also noted and recorded. The same microscope and settings were used for all samples, and the slides were read by two independent readers. Samples showing homogeneous staining patterns were reevaluated using the Crithidia luciliae substrate (ImmunoConcepts), which specifically detects antibodies to double-stranded DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 (dsDNA), and by enzymelinked immunosorbent immunosorbent /im·mu·no·sor·bent/ (-sor´bent) an insoluble support for antigen or antibody used to absorb homologous antibodies or antigens, respectively, from a mixture; the antibodies or antigens so removed may then be eluted in pure  assay (ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.

ELISA
n.
) to detect antibodies to chromatin chromatin: see chromosome.  (INOVA Diagnostics, San Diego, CA), both according to the manufacturers' instructions. Samples with positive ANAs were also evaluated using a modified ANA test to determine whether any of the anti-ANA antibodies were of the IgA isotype i·so·type
n.
An antigenic marker that occurs in all members of a subclass of an immunoglobulin class.



i
. The samples were tested on HEp-2 slides as above, but instead of the anti-human IgG fluorescein isothiocyanate (FITC FITC

fluorescein isothiocyanate; used as a fluorescent label for proteins, especially antibodies.
) conjugate conjugate /con·ju·gate/ (kon´jdbobr-gat)
1. paired, or equally coupled; working in unison.

2. a conjugate diameter of the pelvic inlet; used alone usually to denote the true conjugate diameter; see
 included with the slides, we used goat anti-human IgA FITC conjugate (Southern Biotech, Birmingham, AL). The slides were read as described above.

IgA ELISA. For detection of serum IgA, the mucosal antibody isotype, 96-wall polysorb plates (Nunc, Rochester, NY) were coated with 1 [micro]g/mL of anti-human kappa light chain (Southern Biotech) in carbonate coating buffer overnight at 4[degrees]C. Wells were then blocked with PBS containing 1% bovine serum albumin serum albumin
n.
See seralbumin.
 (BSA 1. BSA - Business Software Alliance.
2. BSA - Bidouilleurs Sans Argent.
) for 1 hr. Subject samples were diluted 1:4,000, 1:8,000, and 1:16,000 in diluent diluent /dil·u·ent/ (dil´oo-int)
1. causing dilution.

2. an agent that dilutes or renders less potent or irritant.


dil·u·ent
adj.
Serving to dilute.

n.
 buffer (PBS, 1% BSA, 0.1% Tween-20). Samples, standards, and blanks were added to wells to give 100 [micro]L/well. After 1 hr, plates were washed with three changes of PBS containing 0.1% Tween-20. The detection antibody [goat anti-human IgA alpha chain coupled to horseradish peroxidase horse·rad·ish peroxidase
n.
An enzyme used in immunohistochemistry to label the antigen-antibody complex.
 (HRP); Caltag, Burlingame, CA] was added and the plates were incubated for 1 hr at room temperature. The plates were washed again and developed using HRP-tetramethylbenzidine (TMB TMB Tetramethylbenzidine
TMB Technical Management Board
TMB Twisted Metal: Black (video game)
TMB Third Millennium Bible
TMB Touch My Body (song)
TMB Text Me Back
TMB Too Many Birthdays
) substrate (Zymed, San Francisco, CA). The reaction was stopped with 2N [H.sub.2]S[O.sub.4], and the plate was read on a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA). All data were evaluated against a standard curve, using human IgA (Sigma, St. Louis, MO).

RF ELISA. RF in the subjects' serum was measured with an ELISA kit according to the manufacturer's protocol (INOVA Diagnostics). The plates were read on the SpectraMax plate reader. Optical density (OD) values were con> pared with known controls provided with the kit and rated as negative or positive (marginal, moderate, or high).

ENA array. Analyses of antibodies to five extractable nuclear proteins commonly seen in SAID (Sm, RNP RNP
abbr.
ribonucleoprotein



RNP

see ribonucleoprotein.
, SS-A, SS-B, and Scl-70) were performed using an addressable Reachable. When something is addressable, it can be identified and manipulated independently of its surroundings. For example, screen pixels and RAM memory are addressable. Each of the screen's picture elements can be individually turned on and off, and each of the memory's bytes can be  bead array kit (QuantaPlex ENA-5; INOVA Diagnostics) according to the manufacturer's instructions, on a Luminex multiplex system (MiraiBio, Alameda, CA). The values were compared by using MasterPlex software (MiraiBio) to negative and graduated positive control reagents provided with the kit, and determined to be low, moderate, or high positive, or negative.

Statistics. In this study we included analysis of several different types of data, including percentages/frequencies (e.g., ANA frequencies), ordinal (mathematics) ordinal - An isomorphism class of well-ordered sets.  (e.g., disease status assessed on a 4-point scale), and scale (e.g., mg/mL IgA) data. Cousequently, we used the following statistical methods: a) differences in percentages were tested using raw frequencies with Fisher's exact test Fisher's exact test

a statistical test for association in a two-by-two table based on the exact hypergeometric distribution of the frequencies within the table.
; b) contingency tables with 4- and 5-point ordinal level frequency comparisons were made via the chi-square test chi-square test: see statistics. ; and c) independent sample t-tests were used for scale measures. In the Libby samples, comparisons (correlations) between ANA levels and disease and exposure rankings were made using the nonparametric Spearman spear·man  
n.
A man, especially a soldier, armed with a spear.
 rank correlation. In all analyses we used two-tailed, unpaired analyses, and reported 0.05 type I error levels. Data reported in the text are mean [+ or -] SEM.

Results

Frequency and fluorescence intensity of positive ANAs. The 50 serum samples in each set were tested by IIF and were determined to be positive or negative for ANA based on known controls. Figure 1A shows that the relative frequency of positive ANAs was 28.6% higher in the Libby sample set than in the Missoula set (p = 0.006). Because low-titer positive ANAs are fairly common in normal populations, the ANA slides were scored for fluorescence intensity. The scored mean fluorescence intensity of positive ANAs, rated on a scale of 1-4 against known controls, was higher in the Libby sample set (mean = 2.34 [+ or -] 0.153) compared with those from Missoula (1.76 [+ or -] 0.194; p = 0.02), and the distribution of subjects receiving the various scores was shown to be significantly different between the sample sets (p = 0.004; Figure 1B). The scored fluorescence intensity is not a direct quantification of autoantibodies but generally suggests a higher titer titer /ti·ter/ (ti´ter) the quantity of a substance required to react with or to correspond to a given amount of another substance. . Therefore, the positive samples were subsequently titered to 1:1,280 and further analyzed for ANA. In both groups, the ANA scores and titers were highly correlated, suggesting that the scoring provides a close estimation of titer (Missoula: correlation coefficient Correlation Coefficient

A measure that determines the degree to which two variable's movements are associated.

The correlation coefficient is calculated as:
 = 0.502, p < 0.001; Libby: correlation coefficient = 0.828, p < 0.001; Table 3). We found that the distribution of titers for samples in the two sample sets were significantly different (p = 0.036; data not shown). The percentage of subjects having a positive ANA at a titer [greater than or equal to] 320 is shown in Figure 1C, again showing a significant difference between the two sample sets.

[FIGURE 1 OMITTED]

IgA levels and RF. Because other studies of asbestos-exposed populations have shown differences in serum IgA compared with unexposed subjects, serum IgA levels were analyzed by ELISA in our sample sets. The Libby samples showed significantly higher levels of serum IgA than the Missoula samples (Figure 2). Although both sample sets had mean IgA concentrations within normal ranges (~ 0.9-4.5 mg/mL), the Libby mean was at the high end of the range (4.2 mg/mL). The ANA tests were subsequently modified to detect IgA rather than IgG, and all of the ANA tests were negative in both sample sets, indicating that the autoantibodies were most likely primarily IgG and not IgA (data not shown).

[FIGURE 2 OMITTED]

RF consists of IgM or IgG antibodies directed against the constant domain of immunoglobulin. These autoantibodies can lead to immune complex immune complex
n.
Any of various complexes of an antigen and an antibody in the blood, to which complement may also be fixed, and which may form a precipitate.
 deposition in tissues and are associated with a variety of infectious and inflammatory disorders such as RA. The samples were evaluated for IgM RE by ELISA, and there were no differences in either the mean OD calculated for each sample set or the frequency of positive tests for RE (Figure 3).

[FIGURE 3 OMITTED]

Staining patterns on ANAs. To determine the specific targets for the IgG autoantibodies, we analyzed the patterns visible on the ANA tests. In the Missoula samples, a nuclear speckled speck·led  
adj.
1. Dotted or covered with speckles, especially flecked with small spots of contrasting color.

2. Of a mixed character; motley.

Adj. 1.
 staining pattern was most common, as expected in a normal population (speckled, 14%; homogeneous, 12%; nucleolar nucleolar

pertaining to or emanating from nucleolus.
, 10%). Other staining patterns were relatively rare, as expected. However, in the Libby samples, homogeneous (indicative of antibodies to chromatin) and nucleolar staining patterns were more prevalent, although the differences were not statistically significant (speckled, 18%; homogeneous, 22%; nucleolar, 18%). We tested for specific antibodies related to these IIF patterns using an ELISA for chromatin and an IIF assay for anti-dsDNA (Crithidia luciliae test). The results indicated that although 22% of the Libby group had homogeneous staining patterns, less than half of those were positive for either chromatin or dsDNA (Figure 4). This suggests that either individual histones not available for binding in chromatin preparations (i.e., H3, H4) or other components of chromatin may be the targets being recognized by the autoantibodies in these individuals.

[FIGURE 4 OMITTED]

Serum antibodies to ENAs. To further explore possible targets for the autoantibodies, we used a Lumine multiplex analyzer with an addressable laser bead immunoassay Immunoassay

An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus.
 to detect antibodies to five ENAs. Twelve of the Libby samples (24%) had positive ENA tests, with most of the positive samples having more than one of the antibody specificities. The Missoula sample set had only two samples (4%) with positive ENA tests. These differences were statistically significant by Fisher's exact test (p = 0.004; data not shown). Figure 5 shows the distribution of positive tests in each group for all five antigens tested.

[FIGURE 5 OMITTED]

Immune parameter correlations. Table 3 shows statistical correlations among tested immune parameters within the Libby sample set, using Spearman's rho Spearman's rho,
n.pr a statistical test for correlation between two rank-ordered scales. It yields a statement of the degree of interdependence of the scores of the two scales.
 nonparametric test. As might be expected based on the epidemiology of autoimmune diseases, the age of the individual was positively correlated with the ANA titer, ANA score, and RF. RF was also correlated with ANA titer. IgA levels were not correlated with any other parameter, so the physiologic significance of the elevation seen in the Libby samples remains unclear.

Correlation of assay results with extent of exposure and ARD. In addition to the correlations shown above, the immune parameters were analyzed against the scores of exposure levels and ARD status, as described in "Materials and Methods." Table 4 shows that there were significant positive associations between ANA titers and both asbestos exposure and disease status. Because a central hypothesis relating to asbestos toxicology is that asbestos exposure is positively associated with ARD, we tested that correlation using our scoring system. The correlation between disease and exposure was 0.239, consistent with the hypothesis (Table 4).

The analysis for asbestos exposure was performed using the graded system described in Table 2, but in looking at the data, it was apparent that the largest effect on ANA titer was seen in terms of length of exposure rather than the source (occupational or environmental). Figure 6A shows the mean ANA titers for the Libby samples when subgrouped by duration of exposure and demonstrates that the titers were significantly higher for those subjects exposed to asbestos for > 5 years. When samples were separated according to whether the subject was exposed environmentally or occupationally, no significant difference in mean ANA titer was observed in those two groups (Figure 6B). This suggests that the duration of exposure had a greater impact on autoimmune responses than the source of exposure. It should be noted that none of the subjects reported solely occupational exposures; all also had environmental exposures as well.

[FIGURE 6 OMITTED]

Discussion

By demonstrating an association between asbestos exposure and measures of autoimmune responses, this study supports and augments other existing evidence that, like silica, asbestos is an agent of systemic autoimmunity. Asbestos-contaminated vermiculite from Libby has been shipped and processed in many sites in the United States, and this material is still used in many applications. It therefore remains a significant health risk to humans both occupationally and environmentally, and an awareness of an association with autoimmunity could impact necessary monitoring, testing, and treatment regimens for exposed individuals or populations.

In addition, this study establishes the Libby population as an excellent cohort for further study of the immunologic aspects of asbestos toxicology, it is a unique population with both occupational and environmental exposures, excellent ongoing monitoring and demographic data, enthusiasm for participation in these studies, and sufficient numbers of exposed individuals to develop sample sets with adequate power for statistical analyses of many parameters. The objective of this study was to compare the frequency of serum autoantibodies in two matched populations, one of them having significant asbestos exposure. It was designed as an initial study in order to explore the feasibility and justification for a more extensive study of autoimmunity in the Libby population.

Previous studies have measured several immune parameters in populations exposed, primarily occupationally, to asbestos. Nigam et al. (1993) demonstrated increased IgG and IgA and positive ANAs in asbestos-exposed individuals compared with controls, even in the absence of apparent ARD. This finding suggested that immune alterations may precede the onset of ARD. A high frequency of positive ANAs was also found in a Japanese group of asbestos plant workers (Tamura et al. 1993). Interestingly, a 3-year follow-up study of the Japanese group showed significant correlation of positive ANAs with progression of the disease, leading to additional diagnoses of asbestosis asbestosis

Lung disease caused by long-term inhalation of asbestos fibres. A pneumoconiosis found primarily in asbestos workers, asbestosis is also seen in people living near asbestos industries.
 in a previously healthy group (Tamura et al. 1996); this suggests that autoimmunity may play a role in ARD. Because that important observation needs to be confirmed, the present study also forms the basis for a similar progressive study of the Libby population to explore the temporal relationship between autoimmunity and ARD. If autoimmune responses play a role in the progression of ARD, this would become an important target for therapeutic strategies.

A higher frequency of positive ANA was seen in the Libby sample set compared with the samples from Missoula, even though the Missoula ANA positive frequency was fairly high (40%). This high "normal" frequency may be due to the age of the population, the low dilution used (1:40) for the screening, or other unidentified population considerations. Nevertheless, the Libby sample set showed a 28.6% increase above that seen for the Missoula samples. In addition, the mean fluorescence intensity of the ANAs, as well as the titers, were higher in the Libby samples compared with the Missoula samples. Although higher titers of autoantibodies are not necessarily correlated with an autoimmune disease process, in some cases increased titers can indicate an exacerbation, relapse, or stage of an autoimmune process.

Other immune parameters showed differences in the Libby group as well, including increases in serum IgA. Serum IgA is generally found at relatively low levels, but increased levels are associated with some chronic inflammatory disorders, such as occupational lung disease Main Article COPD

Occupational lung diseases are a specific branch of occupational diseases concerned primarily with work related exposures to harmful substances, be they dusts or gases, and the subsequent pulmonary disorders that may occur as a result.
, psoriatic arthritis Psoriatic Arthritis Definition

Psoriatic arthritis is a form of arthritic joint disease associated with the chronic skin scaling and fingernail changes seen in psoriasis.
, Crohn's disease Crohn's disease: see colitis. , and ankylosing spondylitis Ankylosing Spondylitis Definition

Ankylosing spondylitis (AS) refers to inflammation of the joints in the spine. AS is also known as rheumatoid spondylitis or Marie-Strümpell disease (among other names).
 (Hoffman et al. 2003; Lindqvist et al. 2002; Zhestkov 2000). The significance of increased serum IgA levels in the Libby samples is not clear, but it is consistent with other studies of asbestos-exposed subjects (Nigam et al. 1993; Zerva et al. 1989). We demonstrated that the autoantibodies detected by ANA were not of the IgA isotype, so it is possible that the IgA antibodies are simply elicited by nonspecific nonspecific /non·spe·cif·ic/ (non?spi-sif´ik)
1. not due to any single known cause.

2. not directed against a particular agent, but rather having a general effect.


nonspecific

1.
 chronic inflammatory processes in these individuals. This possibility is supported by the lack of correlation between IgA titers and either ANA or asbestos exposure; however, a correlation between IgA titers and disease status was also lacking in our analysis.

We found no difference between the two sample sets in terms of frequency of positive tests for RF, and in general the positive samples in both groups were rated marginal to moderate when compared with known controls (data not shown). There was also no correlation between RF and asbestos exposure. These results suggest that asbestos exposure is not associated with increases in RF, especially because previous studies of asbestos-exposed populations have not consistently reported elevated RF. Interestingly, we found a positive association of RF with lung disease status. This may suggest that the presence of RF is more dependent on the chronic inflammation chronic inflammation
n.
Inflammation that may have a rapid or slow onset but is characterized primarily by its persistence and lack of clear resolution; it occurs when the tissues are unable to overcome the effects of the injuring agent.
 resulting from the ARD than on asbestos exposure itself. Alternatively, because there were positive correlations between RF and both ANA titer and ARD, the threshold of asbestos exposure impacting development of RF may simply be too low to be detected using the simplified exposure scale (Table 2). Therefore, the pathophysiologic significance of these results remains to be determined with further study. Although assaying RF is a good screening tool, it is not specific for RA. An alternative test would be an assay for cyclic citrullinated peptide (CCP (Certified Computer Professional) The award for successful completion of a comprehensive examination on computers offered by the ICCP. See ICCP and certification.
.

1. (language) CCP - Concurrent Constraint Programming.
2.
), a more specific epitope epitope: see immunity.  characteristic of autoantibodies in RA (Bombardieri et al. 2004; Saraux et al. 2003) because CCP can antedate ANTEDATE. To, put a date to an instrument of a time before the time it was written. Vide Date.  clinical RA.

The Libby sample set had a significantly higher number of total positive ENA subjects (24% vs. 4% in the Missoula group). ENAs are defined target antigens in a variety of autoimmune diseases, including systemic lupus lupus (l`pəs), noninfectious chronic disease in which antibodies in an individual's immune system attack the body's own substances. , mixed connective tissue disease mixed connective tissue disease A connective tissue disease with features of SLE, dermatomyositis, rheumatoid arthritis Clinical Pleuritis, Raynaud's phenomenon, sclerodactyly, good response to steroids Lab MCTD has a unique speckled nucleolar pattern due to , systemic sclerosis Systemic sclerosis
A rare disorder that causes thickening and scarring of multiple organ systems.

Mentioned in: Scleroderma

systemic sclerosis 
, and polymyositis Polymyositis Definition

Polymyositis is an inflammatory muscle disease causing weakness and pain. Dermatomyositis is identical to polymyositis with the addition of a characteristic skin rash.
 (Pahor et al. 1998). In HEp-2 cells, most of these antibodies produce a speckled or atypical speckled pattern. Therefore, our combined data suggest that the major responses in asbestos-exposed individuals include antibody development to targets such as chromatin components (i.e., histone histone (hĭs`tōn), any of a class of protein molecules found in the chromosomes of eukaryotic cells. They complex with the DNA (see nucleic acid) and pack the DNA into tight masses of chromatin, which have the structure of coiled coils, much ) or nucleolar components (fibrillarin, DNA topoisomerase DNA topoisomerase /DNA topo·isom·er·ase/ (to?po-i-som´er-as) either of two types of isomerase that catalyze the breakage, passage, and rejoining of one or both DNA strands, type I topoisomerases  I), which give the homogeneous and nucleolar patterns, and several ENAs that give the speckled pattern. There are autoantibodies--other than those detected by the assay used here--that could give the speckled patterns seen in both populations. Analysis of a more comprehensive array of autoantigens may provide more insight into the spectrum of autoantibodies related to asbestos exposure.

The presence of autoantibodies does not necessarily suggest a disease process. However, to begin to explore a possible role of these antibodies in the health of the population in Libby, data regarding the extent of ARD severity were gathered as described in "Materials and Methods." Extent of exposure included both duration and site of exposure (work vs. recreational/home). The data showed that individuals with longer exposures, and especially if exposed both at work and at home, had higher ANA titer than those with shorter exposures (< 5 years). Disease status considered the extent of fibrosis or plaquing, as well as functional deficits. Again, the data showed increasing ANA titer with increasing disease severity. The stronger correlation between ANA titer and disease than between ANA titer and exposure may be due to imprecise scoring systems. Exposure and ARD are assumed to be correlated (Peipins et al. 2003), even though this correlation was only at the 90th percentile using our scoring systems. Therefore, these data do not exclude the possibility that the correlation of ANA titer with exposure may be secondary to the chronic inflammation and tissue damage of the associated ARD. It is interesting to note, however, that of those 494 participants from the Libby Tremolite tremolite: see amphibole.  Asbestos Registry cohort with suspected SAID, 171 (35%) have had pleural and/or interstitial abnormalities indicated on chest radiographs and confirmed by two B-readers (Larson T, personal communication). These cross-sectional findings suggest that the proportion of radiographic abnormalities among participants with suspected SAID (35%) was almost double the proportion of radiographic abnormalities among the entire Libby cohort (~ 18%) (Peipins et al. 2003). Further study is required to determine if, and how, autoimmune responses are related to ARD and whether autoimmunity influences ARE) progression.

Conclusion

We have established that there are significant differences in frequency and titer of positive ANA tests, frequency of positive ENA tests, and higher levels of serum IgA when an asbestos-exposed Libby cohort was compared with one from Missoula with no reported asbestos exposures. We have also shown significant associations between asbestos exposure and ANA titer. These data support the hypothesis that asbestos exposure is associated with autoimmune responses. The correlations of ARD with ANA titer, ANA score, and RF suggest the possibility that autoimmunity could play a role in the progression of ARD. This study provides the foundation and justification for a larger and more extensive study, planned to explore these associations much more rigorously. These studies will increase our understanding of the immune components of ARD and could lead to improved interventions as an ultimate goal of the discovery of interrelated in·ter·re·late  
tr. & intr.v. in·ter·re·lat·ed, in·ter·re·lat·ing, in·ter·re·lates
To place in or come into mutual relationship.



in
 pathologies.

Address correspondence to J.C. Pfau, Center for Environmental Health Sciences, Department of Biomedical bi·o·med·i·cal
adj.
1. Of or relating to biomedicine.

2. Of, relating to, or involving biological, medical, and physical sciences.
 and Pharmaceutical Sciences, Skaggs 154, University of Montana, Missoula, MT 59812 USA. Telephone: (406) 243-4529. Fax: (406) 243-2807. E-mail: jean.pfau@umontana.edu

We thank the subjects, families, and the Center for Asbestos Related Diseases Clinic in Libby, MT; R. Hamilton [Center for Environmental Health Sciences (CEHS CEHS College of Education and Human Services
CEHS Center for Environmental Health Sciences (University of California, Davis)
CEHS Canadian Economic History Society
CEHS Civilian Employee's Health Service
) biostatistics], C. Noonan (CEHS epidemiology), and M. Fritzler, University of Calgary (rheumatology rheumatology /rheu·ma·tol·o·gy/ (-tol´ah-je) the branch of medicine dealing with rheumatic disorders, their causes, pathology, diagnosis, treatment, etc.

rheu·ma·tol·o·gy
n.
) for their expertise; and T. Larson, Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry, for Tremolite Asbestos Registry data.

This work was supported by National Institutes of Health grants ES-04804 (J.C.P.) and ES-11676 (E.A.P.) and National Research Service Award ES-11249 (J.C.P.).

The authors declare they have no competing financial interests.

Received 16 July 2004; accepted 30 September 2004.

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Jean C. Pfau, Jami J. Sentissi, Greg Weller, and Elizabeth A. Putnam

Center for Environmental Health Sciences, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana, USA
Table 1. Simple classification of ARD severity.

Disease                                                      Ordinal
severity   Criteria used                                      value

None       No reported lung pathology                           0
Limited    Unilateral radiograph abnormality                    1
Moderate   Bilateral abnormality, minimal functional deficit    2
Severe     Bilateral abnormality, severe or progressive
             functional deficit                                 3

Table 2. Asbestos exposure scores determined from screening data.

Asbestos                                                     Ordinal
exposure   Criteria used                                      value

None       No reported occupational or environmental            0
             asbestos exposure
Minimal    < 5 years, only occupational or environmental        1
Low        < 5 years, both occupational and environmental       2
Moderate   > 5 years, only occupational or environmental        3
High       > 5 years, both occupational and environmental       4

Table 3. Correlation coefficients (CC) with immune
parameters in the Libby cohort.

Parameter analyzed               Age      ANA titer

ANA score
  CC                           0.399 **    0.828 **
  Significance (two-tailed)    0.004       0.00
  No.                            51          50
ANA titer
  CC                           0.381 **    1.00
  Significance (two-tailed)    0.006
  No.                            50
IgA (mg/mL)
  CC                          -0.009      -0.278
  Significance (two-tailed)    0.953       0.075
  No.                            43          42
RF (OD)
  CC                           0.331 *     0.351 *
  Significance (two-tailed)    0.030       0.023
  No.                            43          42

* Correlation is significant at the 0.05 level (two-tailed
Spearman's rho test). ** Correlation is significant at the
0.01 level (two-tailed Spearman's rho test).

Table 4. Correlation coefficients (CC) between
immune parameters and scores both of asbestos
exposure and of asbestos-related disease.

Parameter analyzed            Exposure      ARD

Age
  CC                          -0.015       0.304 *
  Significance (two-tailed)    0.920       0.034
  No.                            51          49
ANA score
  CC                           0.252       0.295 *
  Significance (two-tailed)    0.074       0.040
  No.                            51          49
ANA titer
  CC                           0.366 **    0.392 **
  Significance (two-tailed)    0.009       0.006
  No.                            50          48
RF (OD)
  CC                           0.129       0.388 *
  Significance (two-tailed)    0.410       0.010
  No.                            43          43
Exposure
  CC                           1.00        0.239
  Significance (two-tailed)                0.098
  No.                                        49
IgA
  CC                          -0.167      -0.090
  Significance (two-tailed)    0.283       0.567
  No.                            43          43

* Correlation is significant at 0.05 (two-tailed Spearman's rho
test). ** Correlation is significant at 0.01 (two-tailed
Spearman's rho test).
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Title Annotation:Research
Author:Putnam, Elizabeth A.
Publication:Environmental Health Perspectives
Date:Jan 1, 2005
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