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1.  Impact of negatively charged patches on the surface of MHC class II antigen-presenting proteins on risk of chronic beryllium disease 
Chronic beryllium disease (CBD) is a granulomatous lung disease that occurs primarily in workers who are exposed to beryllium dust or fumes. Although exposure to beryllium is a necessary factor in the pathobiology of CBD, alleles that code for a glutamic acid residue at the 69th position of the HLA-DPβ1 gene have previously been found to be associated with CBD. To date, 43 HLA-DPβ1 alleles that code for glutamic acid 69 (E69) have been described. Whether all of these E69 coding alleles convey equal risk of CBD is unknown. The present study demonstrates that, on the one hand, E69 alleloforms of major histocompatibility complex class II antigen-presenting proteins with the greatest negative surface charge convey the highest risk of CBD, and on the other hand, irrespective of allele, they convey equal risk of beryllium sensitization (BeS). In addition, the data suggest that the same alleles that cause the greatest risk of CBD are also important for the progression from BeS to CBD. Alleles convey the highest risk code for E26 in a constant region and for E69, aspartic acid 55 (D55), E56, D84 and E85 in hypervariable regions of the HLA-DPβ1 chain. Together with the calculated high binding affinities for beryllium, these results suggest that an adverse immune response, leading to CBD, is triggered by chemically specific metal–protein interactions.
PMCID: PMC2607452  PMID: 17956852
HLA-DP; genetic epidemiology; beryllium sensitization; granulomas; free energy perturbation; molecular dynamics
2.  A Reconsideration of Acute Beryllium Disease 
Environmental Health Perspectives  2009;117(8):1250-1256.
Although chronic beryllium disease (CBD) is clearly an immune-mediated granulomatous reaction to beryllium, acute beryllium disease (ABD) is commonly considered an irritative chemical phenomenon related to high exposures. Given reported new cases of ABD and projected increased demand for beryllium, we aimed to reevaluate the patho physiologic associations between ABD and CBD using two cases identified from a survey of beryllium production facility workers.
Case Presentation
Within weeks after exposure to beryllium fluoride began, two workers had systemic illness characterized by dermal and respiratory symptoms and precipitous declines in pulmonary function. Symptoms and pulmonary function abnormalities improved with cessation of exposure and, in one worker, recurred with repeat exposure. Bronchoalveolar lavage fluid analyses and blood beryllium lymphocyte proliferation tests revealed lymphocytic alveolitis and cellular immune recognition of beryllium. None of the measured air samples exceeded 100 μg/m3, and most were < 10 μg/m3, lower than usually described. In both cases, lung biopsy about 18 months after acute illness revealed noncaseating granulomas. Years after first exposure, the workers left employment because of CBD.
Contrary to common understanding, these cases suggest that ABD and CBD represent a continuum of disease, and both involve hypersensitivity reactions to beryllium. Differences in disease presentation and progression are likely influenced by the solubility of the beryllium compound involved.
Relevance to Practice
ABD may occur after exposures lower than the high concentrations commonly described. Prudence dictates limitation of further beryllium exposure in both ABD and CBD.
PMCID: PMC2721869  PMID: 19672405
acute; beryllium; beryllium disease; granuloma; hypersensitivity; immune sensitization; pneumonitis
3.  Electrostatic potential on human leukocyte antigen: implications for putative mechanism of chronic beryllium disease. 
Environmental Health Perspectives  2003;111(15):1827-1834.
The pathobiology of chronic beryllium disease (CBD) involves the major histocompatibility complex class II human leukocyte antigen (HLA). Although occupational exposure to beryllium is the cause of CBD, molecular epidemiologic studies suggest that specific (Italic)HLA-DPB1(/Italic) alleles may be genetic susceptibility factors. We have studied three-dimensional structural models of HLA-DP proteins encoded by these genes. The extracellular domains of HLA-DPA1*0103/B1*1701, *1901, *0201, and *0401, and HLA-DPA1*0201/B1*1701, *1901, *0201, and *0401 were modeled from the X-ray coordinates of an HLA-DR template. Using these models, the electrostatic potential at the molecular surface of each HLA-DP was calculated and compared. These comparisons identify specific characteristics in the vicinity of the antigen-binding pocket that distinguish the different HLA-DP allotypes. Differences in electrostatics originate from the shape, specific disposition, and variation in the negatively charged groups around the pocket. The more negative the pocket potential, the greater the odds of developing CBD estimated from reported epidemiologic studies. Adverse impact is caused by charged substitutions in positions 55, 56, 69, 84, and 85, namely, the exact same loci identified as genetic markers of CBD susceptibility as well as cobalt-lung hard metal disease. These findings suggest that certain substitutions may promote an involuntary cation-binding site within a putatively metal-free peptide-binding pocket and therefore change the innate specificity of antigen recognition.
PMCID: PMC1241746  PMID: 14630515
4.  Study on the effects of nitrilotriproprionic acid and 4,5-dihydroxy-1,3-benzene disulphonate on the fractionation of beryllium in human serum using graphite furnace atomic absorption spectrometry 
Occupational exposure to beryllium may cause Chronic Beryllium Disease (CBD), a lung disorder initiated by an electrostatic interaction with the MHC class II human leukocyte antigen (HLA). Molecular studies have found a significant correlation between the electrostatic potential at the HLA-DP surface and disease susceptibility. CBD can therefore be treated by chelation therapy. In this work, we studied the effect of two complexing agents, nitrilotriproprionic acid (NTP) and 4,5-dihydroxy-1,3-benzene disulphonate (Tiron), on the fractionation of beryllium in human serum analysed by graphite furnace atomic absorption spectrometry (GFAAS).
We found the average serum beryllium concentration of fourteen non-exposed individuals to be 0.53 (± 0.14) μg l-1, with 21 (± 3)% of the beryllium mass bound to the low molecular weight fraction (LMW), and 79 (± 3)% bound to the high molecular weight fraction (HMW). The addition of Tiron increased the beryllium mass in the HMW fraction, while NTP was not seen to have any influence on the fractionation of beryllium between the two fractions. NTP was, however, shown to complex 94.5% of the Be mass in the LMW fraction. The beryllium GFAAS detection limit, calculated as three times the standard deviation of 10 replicates of the lowest standard (0.05 μg L-1), was 6.0 (± 0.2) ng L-1.
The concentration of beryllium or its fractionation in human serum was not affected by sex or smoking habit. On average, three quarters of the beryllium in serum were found in the HMW fraction. Of the two ligands tested, only Tiron was effective in mobilising beryllium under physiological conditions, thus increasing the Be content in the HMW fraction.
PMCID: PMC2396160  PMID: 18479524
5.  CC chemokine receptor 5 gene polymorphisms in beryllium disease 
CC chemokine receptor 5 (CCR5) is expressed on type-1 T-helper cells, which are involved in the pathogenesis of the granulomatous lung disease chronic beryllium disease (CBD). CCR5 gene (CCR5) polymorphisms are associated with sarcoidosis severity. The present study explores associations between CCR5 polymorphisms and CBD and its disease progression.
Eight CCR5 polymorphisms were genotyped in CBD (n = 88), beryllium sensitisation (BeS; n = 86) and beryllium-exposed nondiseased controls (n = 173) using PCR with sequence-specific primers. Pulmonary function and bronchoalveolar lavage data were examined for associations with genotypes.
There were no significant differences in genotype and allele frequency between CBD, BeS individuals and controls. In CBD, associations were found with decline in forced expiratory volume in 1 s and forced vital capacity and the CCR5 -3458 thymidine (T)T genotype (p<0.0001), and an increase in alveolar–arterial oxygen tension difference at rest (p = 0.003) and at maximum exercise (p = 0.01) and the -5663 adenine allele. Increased bronchoalveolar lavage lymphocyte numbers were associated with CCR5 -2459 guanine/-2135T (p = 0.01) only in the combined CBD and BeS group.
This is the first study showing that CCR5 polymorphisms are associated with worsening pulmonary function over time in CBD, suggesting that CCR5 is important in the progression of pulmonary function in CBD. Further studies would be useful to clarify the mechanism whereby CCR5 polymorphisms affect progression of CBD.
PMCID: PMC3061572  PMID: 20075058
Broncholaveolar lavage; CC chemokine receptor 5; chronic beryllium disease; lung function tests; polymorphism
6.  Chronic Beryllium Disease and Sensitization at a Beryllium Processing Facility 
Environmental Health Perspectives  2005;113(10):1366-1372.
We conducted a medical screening for beryllium disease of 577 former workers from a beryllium processing facility. The screening included a medical and work history questionnaire, a chest radiograph, and blood lymphocyte proliferation testing for beryllium. A task exposure and a job exposure matrix were constructed to examine the association between exposure to beryllium and the development of beryllium disease. More than 90% of the cohort completed the questionnaire, and 74% completed the blood and radiograph component of the screening. Forty-four (7.6%) individuals had definite or probable chronic beryllium disease (CBD), and another 40 (7.0%) were sensitized to beryllium. The prevalence of CBD and sensitization in our cohort was greater than the prevalence reported in studies of other beryllium-exposed cohorts. Various exposure measures evaluated included duration; first decade worked; last decade worked; cumulative, mean, and highest job; and highest task exposure to beryllium (to both soluble and nonsoluble forms). Soluble cumulative and mean exposure levels were lower in individuals with CBD. Sensitized individuals had shorter duration of exposure, began work later, last worked longer ago, and had lower cumulative and peak exposures and lower nonsoluble cumulative and mean exposures. A possible explanation for the exposure–response findings of our study may be an interaction between genetic predisposition and a decreased permanence of soluble beryllium in the body. Both CBD and sensitization occurred in former workers whose mean daily working lifetime average exposures were lower than the current allowable Occupational Safety and Health Administration workplace air level of 2 μg/m3 and the Department of Energy guideline of 0.2 μg/m3.
PMCID: PMC1281281  PMID: 16203248
beryllium; chronic beryllium disease; epidemiology; exposure–response; lymphocyte proliferation testing
7.  Low Prevalence of Chronic Beryllium Disease Among Workers at aNuclearWeaponsResearchandDevelopmentFacility 
To study the prevalence of beryllium sensitization (BeS) and chronic beryllium disease (CBD) in a cohort of workers from a nuclear weapons research and development facility.
We evaluated 50 workers with BeS with medical and occupational histories, physical examination, chest imaging with high-resolution computed tomography (N = 49), and pulmonary function testing. Forty of these workers also underwent bronchoscopy for bronchoalveolar lavage and transbronchial biopsies.
The mean duration of employment at the facility was 18 years and the mean latency (from first possible exposure) to time of evaluation was 32 years. Five of the workers had CBD at the time of evaluation (based on histology or high-resolution computed tomography); three others had evidence of probable CBD.
These workers with BeS, characterized by a long duration of potential Be exposure and a long latency, had a low prevalence of CBD.
PMCID: PMC3313491  PMID: 20523233
8.  Linking Genetic Susceptibility and T Cell Activation in Beryllium-induced Disease 
Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by beryllium (Be) exposure in the workplace. It is characterized by the accumulation of Be-specific CD4+ T cells in the lung as well as persistent lung inflammation, culminating in the development of lung fibrosis. CBD occurs in 2 to 16% of Be-exposed workers depending on the individuals' genetic susceptibility and the characteristics of the exposure. Genetic susceptibility to Be-induced disease has been linked to major histocompatibility complex class II molecules. In particular, HLA-DP alleles possessing a glutamic acid at the 69th position of the β-chain (βGlu69) are most strongly linked to disease susceptibility. The HLA-DP alleles that present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. However, the structural features of βGlu69-containing HLA-DP molecules that explain the disease association remain unknown. We have recently crystallized HLA-DP2, which is the most prevalent of the βGlu69-containing HLA-DP molecules. Its unique structure, which includes surface exposure of βGlu69, provides an explanation of the genetic linkage between βGlu69-containing HLA-DP alleles and Be-induced disease.
PMCID: PMC3266018  PMID: 20427584
beryllium; granuloma; human; lung; T cells
9.  Significance of the blood beryllium lymphocyte proliferation test. 
Environmental Health Perspectives  1996;104(Suppl 5):953-956.
The blood beryllium lymphocyte proliferation test (BeLPT) is an in vitro measure of the beryllium antigen-specific cell-mediated immune response. This response to beryllium is now understood to play a central role in the immunopathogenesis of chronic beryllium disease (CBD). Although there remain some unresolved methodologic issues with testing, the blood BeLPT has already undergone sufficient development and field assessment to lead to a number of important conclusions: a) The BeLPT identifies beryllium sensitization and CBD earlier and better than any other clinical test presently available. b) The CBD cases identified with the blood test are clinically significant. c) A subset of the people identified by the BeLPT who do not yet have clinical disease will progress and require treatment with corticosteroids for impairing illness. d) The BeLPT can be used to improve clinical diagnostic accuracy and to correct mistaken diagnoses. e) The blood test can be used in screening large numbers of exposed workers because it is sensitive and specific and has high positive and negative predictive value for CBD. f) In every workforce studied to date, the BeLPT has identified beryllium sensitization and CBD that had been missed by conventional screening efforts. g) Worker populations that have been characterized using the BeLPT can help to elucidate the role of exposure genetics and dysregulated inflammation in the genesis of occupational lung disease.
PMCID: PMC1469695  PMID: 8933041
10.  Modulation of Lymphocyte Proliferation by Antioxidants in Chronic Beryllium Disease 
Rationale: Occupational exposure to beryllium (Be) can result in chronic granulomatous inflammation characterized by the presence of Be-specific CD4+ T cells. Studies show that oxidative stress plays a role in the pathogenesis of chronic inflammatory disorders.
Objectives: We hypothesized that Be-induced oxidative stress modulates the proliferation of Be-specific CD4+ T cells.
Methods: Thirty-three subjects with chronic beryllium disease (CBD), 15 subjects with beryllium sensitization, and 28 healthy normal control subjects were consecutively enrolled from the Occupational and Environmental Health Clinic of the National Jewish Medical and Research Center.
Measurements and Main Results: All studies were performed with Ficoll-Hypaque–isolated peripheral blood mononuclear cells from subsets of the study subjects. Decreased intracellular levels of the thiol antioxidants, glutathione and cysteine, were observed in peripheral blood mononuclear cells from subjects with beryllium sensitization and CBD, as compared with healthy control subjects. Beryllium stimulation decreased intracellular thiol antioxidants by more than 40%, accompanied by increased reactive oxygen species levels and the proliferation of Be-specific blood CD4+ T cells from subjects with CBD. Be-induced T-cell proliferation was inhibited by treatment with the thiol antioxidant N-acetylcysteine or the catalytic antioxidant manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP). MnTBAP treatment also inhibited T-cell proliferation in response to the unrelated, MHC class II–restricted antigen tetanus toxoid. Treatment of CBD blood lymphocytes, but not antigen-presenting cells, with MnTBAP decreased Be-induced T-cell proliferation by more than 40%.
Conclusions: Beryllium can mediate a thiol imbalance leading to oxidative stress that may modulate the proliferation and clonal expansion of Be-specific blood CD4+ T cells. These data suggest that Be-induced oxidative stress plays a role in the pathogenesis of granulomatous inflammation in CBD.
PMCID: PMC2361422  PMID: 18218990
T cells; reactive oxygen species; glutathione; N-acetylcysteine; oxidative stress
11.  Chronic Beryllium Disease: Revealing the Role of Beryllium Ion and Small Peptides Binding to HLA-DP2 
PLoS ONE  2014;9(11):e111604.
Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed [1], the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work [1]. In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade.
PMCID: PMC4219729  PMID: 25369028
12.  Risks of beryllium disease related to work processes at a metal, alloy, and oxide production plant. 
OBJECTIVES: To describe relative hazards in sectors of the beryllium industry, risk factors of beryllium disease and sensitisation related to work process were sought in a beryllium manufacturing plant producing pure metal, oxide, alloys, and ceramics. METHODS: All 646 active employees were interviewed; beryllium sensitisation was ascertained with the beryllium lymphocyte proliferation blood test on 627 employees; clinical evaluation and bronchoscopy were offered to people with abnormal test results; and industrial hygiene measurements related to work processes taken in 1984-93 were reviewed. RESULTS: 59 employees (9.4%) had abnormal blood tests, 47 of whom underwent bronchoscopy. 24 new cases of beryllium disease were identified, resulting in a beryllium disease prevalence of 4.6%, including five known cases (29/632). Employees who had worked in ceramics had the highest prevalence of beryllium disease (9.0%). Employees in the pebble plant (producing beryllium metal) who had been employed after 1983 also had increased risk, with a prevalence of beryllium disease of 6.4%, compared with 1.3% of other workers hired in the same period, and a prevalence of abnormal blood tests of 19.2%. Logistic regression modelling confirmed these two risk factors for beryllium disease related to work processes and the dependence on time of the risk at the pebble plant. The pebble plant was not associated with the highest gravimetric industrial hygiene measurements available since 1984. CONCLUSION: Further characterisation of exposures in beryllium metal production may be important to understanding how beryllium exposures confer high contemporary risk of beryllium disease.
PMCID: PMC1128986  PMID: 9326165
13.  Beryllium-Induced TNF-α Production Is Transcription-Dependent in Chronic Beryllium Disease 
Beryllium (Be)-antigen presentation to Be-specific CD4+ T cells from the lungs of patients with chronic beryllium disease (CBD) results in T cell proliferation and TNF-α secretion. We tested the hypothesis that Be-induced, CBD bronchoalveolar lavage (BAL) T cell, transcription-dependent, TNF-α secretion was accompanied by specific transcription factor upregulation. After 6 h of Be stimulation, CBD BAL cells produced a median of 883 pg/ml TNF-α (range, 608–1,275 pg/ml) versus 198 pg/ml (range, 116–245 pg/ml) by unstimulated cells. After 12 h CBD BAL cells produced a median of 2,963 pg/ml (range, 99–9,424 pg/ml) TNF-α versus 55 pg/ml (range, 0–454) by unstimulated cells. Using real-time RT-PCR, Be-stimulated TNF-α production at 6 h was preceded by a 5-fold increase in TNF-α pre-mRNA copy number:β-actin copy number (Be median ratio 0.21; unstimulated median ratio 0.04). The median ratio of mature TNF-α mRNA:β-actin mRNA was upregulated 1.4-fold (Be median ratio 0.17; unstimulated median ratio 0.12). Be exposure in the presence of the transcription inhibitor pentoxifylline (PTX) decreased CBD BAL cell TNF-α pre-mRNA levels > 60%, whereas treatment with the mRNA splicing inhibitor 2-aminopurine (2AP) decreased levels 40% relative to Be exposure alone. PTX treatment decreased mature TNF-α mRNA levels 50% while 2AP decreased levels > 80%, relative to Be exposure alone. Beryllium exposure specifically upregulated transcription factors AP-1 and NF-κB. The data suggest that Be exposure induces transcription-dependent TNF-α production, potentially due to upregulation of specific transcription factors.
PMCID: PMC2176111  PMID: 16980557
granuloma; T lymphocytes; cytokines; gene regulation; lung
14.  Skin as a route of exposure and sensitization in chronic beryllium disease. 
Environmental Health Perspectives  2003;111(9):1202-1208.
Chronic beryllium disease is an occupational lung disease that begins as a cell-mediated immune response to beryllium. Although respiratory and engineering controls have significantly decreased occupational beryllium exposures over the last decade, the rate of beryllium sensitization has not declined. We hypothesized that skin exposure to beryllium particles would provide an alternative route for sensitization to this metal. We employed optical scanning laser confocal microscopy and size-selected fluorospheres to demonstrate that 0.5- and 1.0- micro m particles, in conjunction with motion, as at the wrist, penetrate the stratum corneum of human skin and reach the epidermis and, occasionally, the dermis. The cutaneous immune response to chemical sensitizers is initiated in the skin, matures in the local lymph node (LN), and releases hapten-specific T cells into the peripheral blood. Topical application of beryllium to C3H mice generated beryllium-specific sensitization that was documented by peripheral blood and LN beryllium lymphocyte proliferation tests (BeLPT) and by changes in LN T-cell activation markers, increased expression of CD44, and decreased CD62L. In a sensitization-challenge treatment paradigm, epicutaneous beryllium increased murine ear thickness following chemical challenge. These data are consistent with development of a hapten-specific, cell-mediated immune response following topical application of beryllium and suggest a mechanistic link between the persistent rate of beryllium worker sensitization and skin exposure to fine and ultrafine beryllium particles.
PMCID: PMC1241575  PMID: 12842774
15.  T Cell Recognition of Beryllium 
Current opinion in immunology  2013;25(6):10.1016/j.coi.2013.07.012.
Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by a hypersensitivity to beryllium and characterized by the accumulation of beryllium-specific CD4+ T cells in the lung. Genetic susceptibility to beryllium-induced disease is strongly associated with HLA-DP alleles possessing a glutamic acid at the 69th position of the β-chain (βGlu69). The structure of HLA-DP2, the most prevalent βGlu69-containing molecule, revealed a unique solvent-exposed acidic pocket that includes βGlu69 and represents the putative beryllium binding site. The delineation of mimotopes and endogenous self-peptides that complete the αβTCR ligand for beryllium-specific CD4+ T cells suggests a unique role of these peptides in metal ion coordination and the generation of altered self-peptides, blurring the distinction between hypersensitivity and autoimmunity.
PMCID: PMC3883670  PMID: 23978481
16.  4-1BB Enhances Proliferation of Beryllium-Specific T Cells in the Lung of Subjects with Chronic Beryllium Disease1 
In contrast to naive T cells, reactivation of memory cells is less dependent on CD28-mediated costimulation. We have shown that circulating beryllium-specific CD4+ T cells from chronic beryllium disease patients remain CD28-dependent, while those present in the lung no longer require CD28 for T cell activation. In the present study, we analyzed whether other costimulatory molecules are essential for beryllium-induced T cell function in the lung. Enhanced proliferation of a beryllium-responsive, HLA-DP2-restricted T cell line was seen after the induction of 4-1BB ligand expression on the surface of HLA-DP2-expressing fibroblasts. Following beryllium exposure, CD4+ T cells from blood and bronchoalveolar lavage of chronic beryllium disease patients up-regulate 4-1BB expression, and the majority of beryllium-responsive, IFN-γ-producing CD4+ T cells in blood coexpress CD28 and 4-1BB. Conversely, a significant fraction of IFN-γ-producing bronchoalveolar lavage (BAL) T cells express 4-1BB in the absence of CD28. In contrast to blood, inhibition of the 4-1BB ligand-4-1BB interaction partially blocked beryllium-induced proliferation of BAL CD4+ T cells, and a lack of 4-1BB expression on BAL T cells was associated with increased beryllium-induced cell death. Taken together, these findings suggest an important role of 4-1BB in the costimulation of beryllium-responsive CD4+ T cells in the target organ.
PMCID: PMC3057106  PMID: 18768897
17.  Beryllium Lymphocyte Proliferation Test Surveillance Identifies Clinically Significant Beryllium Disease 
Workplace surveillance identifies chronic beryllium disease (CBD) but it remains unknown over what time frame mild CBD will progress to a more severe form.
We examined physiology and treatment in 229 beryllium sensitization (BeS) and 171 CBD surveillance-identified cases diagnosed from 1982 to 2002. Never smoking CBD cases (81) were compared to never smoking BeS patients (83) to assess disease progression. We compared CBD machinists to non-machinists to examine effects of exposure.
At baseline, CBD and BeS cases did not differ significantly in exposure time or physiology. CBD patients were more likely to have machined beryllium. Of CBD cases, 19.3% went on to require oral immunosuppressive therapy. At 30 years from first exposure, measures of gas exchange were significantly worse and total lung capacity was lower for CBD subjects. Machinists had faster disease progression as measured by pulmonary function testing and gas exchange.
Medical surveillance for CBD identifies individuals at significant risk of disease progression and impairment with sufficient time since first exposure.
PMCID: PMC3063521  PMID: 19681064
beryllium; chronic beryllium disease; medical surveillance
18.  Beryllium-Specific CD4+ T Cells in Blood as a Biomarker of Disease Progression 
CD4+ T cells are responsible for the progressive lung damage seen in patients with chronic beryllium disease (CBD), a granulomatous lung disorder in which antigen-specific, Th1-type cytokine-secreting T cells have been characterized. Compared to beryllium (Be)-sensitized subjects, an increased number of Be-responsive T cells are present in the blood of CBD patients.
The aim of this study was to determine whether the number of Be-specific T cells in blood predicted the development of CBD in a cohort of Be-exposed subjects.
Using IFN-γ ELISPOT and proliferation-based assays, we determined the frequency and proliferative capacity of Be-responsive T cells in blood.
Compared with the Be lymphocyte proliferation test which detected an abnormal Be-induced proliferative response in 11 of 260 (4.2%) workers from a Be-machining facility, IFN-γ ELISPOT detected a sensitization rate of 10% (χ2 = 55.7; P < 0.0001). A significant positive correlation was also noted between the number of Be-responsive CD4+ T cells in blood and lung of CBD patients. Importantly, the transition from Be sensitization to CBD was associated with an increased number of antigen-specific T cells in blood.
These findings have important implications for Be-induced disease and potentially other immune-mediated disorders, suggesting that the frequency of antigen-specific T cells in blood can serve as a noninvasive biomarker to predict disease development and severity of the Be-specific CD4+ T cell alveolitis.
Clinical implications
These findings suggest that the number of Be-responsive T cells in the circulation can serve as a biomarker of disease progression and as an estimate of the severity of Be-induced lung inflammation.
PMCID: PMC3205205  PMID: 21943943
Human; Lung; CD4-Positive T-Lymphocytes; Beryllium; Cytokines; Granuloma; ELISPOT
19.  Effects of Beryllium on Human Serum Immunoglobulin and Lymphocyte Subpopulation 
Toxicological Research  2013;29(2):115-120.
To investigate the effects of short-term exposure of beryllium on the human immune system, the proportion of T-lymphocytes such as CD3+, CD4+, CD8+, CD95, and NK cells, andthe proportion of B cells and TNFα level in peripheral blood and immunoglobulins in the serum of 43 exposed workers and 34 healthy control subjects were studied. External exposure to beryllium was measured by atomic absorption spectrometer as recommended by the NIOSH analytical method 7300. T lymphocyte subpopulation analysis was carried out with flow cytometer. The working duration of exposed workers was less than 3 months and the mean ambient beryllium level was 3.4 μg/m3, 112.3 μg/m3, and 2.3 μg/m3 in molding (furnace), deforming (grinding), and sorting processes, respectively (cited from Kim et al., 2008). However, ambient beryllium level after process change was non-detectable (< 0.1 μg/m3). The number of T lymphocytes and the amount of immunoglobulins in the beryllium-exposed workers and control subjects were not significantly different, except for the total number of lymphocytes and CD95 (APO1/FAS). The total number of lymphocytes was higher in the beryllium-exposed individuals than in the healthy control subjects. Multiple logistic regression analysis showed lymphocytes to be affected by beryllium exposure (odd ratio = 7.293; p < 0.001). These results show that short-term exposure to beryllium does not induce immune dysfunction but is probably associated with lymphocytes proliferation.
PMCID: PMC3834450  PMID: 24278637
Beryllium; Short-term exposure; T lymphocyte subpopulation; Immunoglobulins
20.  Frequency of beryllium-specific, central memory CD4+ T cells in blood determines proliferative response 
Journal of Clinical Investigation  2005;115(10):2886-2893.
Beryllium exposure can lead to the development of beryllium-specific CD4+ T cells and chronic beryllium disease (CBD), which is characterized by the presence of lung granulomas and a CD4+ T cell alveolitis. Studies have documented the presence of proliferating and cytokine-secreting CD4+ T cells in blood of CBD patients after beryllium stimulation. However, some patients were noted to have cytokine-secreting CD4+ T cells in blood in the absence of beryllium-induced proliferation, and overall, the correlation between the 2 types of responses was poor. We hypothesized that the relative proportion of memory T cell subsets determined antigen-specific proliferation. In most CBD patients, the majority of beryllium-specific CD4+ T cells in blood expressed an effector memory T cell maturation phenotype. However, the ability of blood cells to proliferate in the presence of beryllium strongly correlated with the fraction expressing a central memory T cell phenotype. In addition, we found a direct correlation between the percentage of beryllium-specific CD4+ TEM cells in blood and T cell lymphocytosis in the lung. Together, these findings indicate that the functional capability of antigen-specific CD4+ T cells is determined by the relative proportion of memory T cell subsets, which may reflect internal organ involvement.
PMCID: PMC1199530  PMID: 16151531
21.  Lung function, biological monitoring, and biological effect monitoring of gemstone cutters exposed to beryls 
OBJECTIVES—Gemstone cutters are potentially exposed to various carcinogenic and fibrogenic metals such as chromium, nickel, aluminium, and beryllium, as well as to lead. Increased beryllium concentrations had been reported in the air of workplaces of beryl cutters in Idar-Oberstein, Germany. The aim of the survey was to study the excretion of beryllium in cutters and grinders with occupational exposure to beryls—for example, aquamarines and emeralds—to examine the prevalence of beryllium sensitisation with the beryllium lymphocyte transformation test (BeLT), to examine the prevalence of lung disease induced by beryllium, to describe the internal load of the respective metals relative to work process, and to screen for genotoxic effects in this particular profession.
METHODS—In a cross sectional investigation, 57 out of 100 gemstone cutters working in 12 factories in Idar-Oberstein with occupational exposure to beryls underwent medical examinations, a chest radiograph, lung function testing (spirometry, airway resistance with the interrupter technique), and biological monitoring, including measurements of aluminium, chromium, and nickel in urine as well as lead in blood. Beryllium in urine was measured with a newly developed direct electrothermal atomic absorption spectroscopy technique with a measurement limit of 0.06 µg/l. Also, cytogenetic tests (rates of micronuclei and sister chromatid exchange), and a BeLT were performed. Airborne concentrations of beryllium were measured in three factories. As no adequate local control group was available, the cutters were categorised into those with an exposure to beryls of >4 hours/week (group A) and ⩽4 hours/week (group B).
RESULTS—Clinical, radiological, or spirometric abnormalities indicating pneumoconiosis were detected in none of the gemstone cutters. Metal concentrations in biological material were far below the respective biological limit values, and beryllium in urine was only measurable in subjects of group A. Cytogenetic investigations showed normal values which were independent of the duration of beryllium exposure. In one subject, the BeLT was positive. Beryllium stimulation indices were significantly higher in subjects with detectable beryllium in the urine than in those with beryllium concentrations below the detection limit (p<0.05). In one factory, two out of four measurements of airborne beryllium concentrations were well above the German threshold limit value of 2 µg/m3 (twofold and 10-fold), and all gemstone cutters working in this factory had measurable beryllium concentrations in urine.
CONCLUSION—No adverse clinical health effects were found in this cross sectional investigation of gemstone cutters working with beryls. However, an improvement in workplace hygiene is recommended, accompanied by biological monitoring of beryllium in urine.

Keywords: gemstone cutter; beryllium in urine; lymphocyte transformation test
PMCID: PMC1739907  PMID: 10711282
22.  Target organ localization of memory CD4+ T cells in patients with chronic beryllium disease 
The Journal of Clinical Investigation  2002;110(10):1473-1482.
Chronic beryllium disease (CBD) is caused by exposure to beryllium in the workplace, and it remains an important public health concern. Evidence suggests that CD4+ T cells play a critical role in the development of this disease. Using intracellular cytokine staining, we found that the frequency of beryllium-specific CD4+ T cells in the lungs (bronchoalveolar lavage) of 12 CBD patients ranged from 1.4% to 29% (mean 17.8%), and these T cells expressed a Th1-type phenotype in response to beryllium sulfate (BeSO4). Few, if any, beryllium-specific CD8+ T cells were identified. In contrast, the frequency of beryllium-responsive CD4+ T cells in the blood of these subjects ranged from undetectable to 1 in 500. No correlation was observed between the frequency of beryllium-responsive bronchoalveolar lavage (BAL) CD4+ T cells as detected by intracellular staining and lymphocyte proliferation in culture after BeSO4 exposure. Staining for surface marker expression showed that nearly all BAL T cells exhibit an effector memory cell phenotype. These results demonstrate a dramatically high frequency and compartmentalization of antigen-specific effector memory CD4+ cells in the lungs of CBD patients. These studies provide insight into the phenotypic and functional characteristics of antigen-specific T cells invading other inaccessible target organs in human disease.
PMCID: PMC151812  PMID: 12438445
23.  Impaired Function of CTLA-4 in the Lungs of Patients with Chronic Beryllium Disease Contributes to Persistent Inflammation1 
Chronic beryllium disease (CBD) is an occupational lung disorder characterized by granulomatous inflammation and the accumulation of beryllium-responsive CD4+ T cells in the lung. These differentiated effector memory T cells secrete IL-2, IFN-γ, and TNF-α upon in vitro activation. Beryllium-responsive CD4+ T cells in the lung are CD28 independent and have increased expression of the coinhibitory receptor, programmed death 1, resulting in antigen-specific T cells that proliferate poorly yet retain the ability to express Th1-type cytokines. To further investigate the role of coinhibitory receptors in the beryllium-induced immune response, we examined the expression of CTLA-4 in blood and bronchoalveolar lavage cells from subjects with CBD. CTLA-4 expression was elevated on CD4+ T cells from the lungs of study subjects compared to blood. Furthermore, CTLA-4 expression was greatest in the beryllium-responsive subset of CD4+ T cells that retained the ability to proliferate and express IL-2. Functional assays show that the induction of CTLA-4 signaling in blood cells inhibited beryllium-induced T cell proliferation while having no effect on the proliferative capacity of beryllium-responsive CD4+ T cells in lung. Collectively, our findings suggest a dysfunctional CTLA-4 pathway in the lung and its potential contribution to the persistent inflammatory response that characterizes CBD.
PMCID: PMC3750981  PMID: 23851684
Human; T Cells; Cell Surface Molecules; Cytokines; Lung
24.  Identification of beryllium-dependent peptides recognized by CD4+ T cells in chronic beryllium disease 
The Journal of Experimental Medicine  2013;210(7):1403-1418.
Identification of peptides that form complexes with beryllium and class II HLA molecules and are recognized by CD4+ T cells from patients with chronic beryllium disease.
Chronic beryllium disease (CBD) is a granulomatous disorder characterized by an influx of beryllium (Be)-specific CD4+ T cells into the lung. The vast majority of these T cells recognize Be in an HLA-DP–restricted manner, and peptide is required for T cell recognition. However, the peptides that stimulate Be-specific T cells are unknown. Using positional scanning libraries and fibroblasts expressing HLA-DP2, the most prevalent HLA-DP molecule linked to disease, we identified mimotopes and endogenous self-peptides that bind to MHCII and Be, forming a complex recognized by pathogenic CD4+ T cells in CBD. These peptides possess aspartic and glutamic acid residues at p4 and p7, respectively, that surround the putative Be-binding site and cooperate with HLA-DP2 in Be coordination. Endogenous plexin A peptides and proteins, which share the core motif and are expressed in lung, also stimulate these TCRs. Be-loaded HLA-DP2–mimotope and HLA-DP2–plexin A4 tetramers detected high frequencies of CD4+ T cells specific for these ligands in all HLA-DP2+ CBD patients tested. Thus, our findings identify the first ligand for a CD4+ T cell involved in metal-induced hypersensitivity and suggest a unique role of these peptides in metal ion coordination and the generation of a common antigen specificity in CBD.
PMCID: PMC3698527  PMID: 23797096
25.  Prevalence of beryllium sensitization among aluminium smelter workers 
Background Beryllium exposure occurs in aluminium smelters from natural contamination of bauxite, the principal source of aluminium.
Aims To characterize beryllium exposure in aluminium smelters and determine the prevalence rate of beryllium sensitization (BeS) among aluminium smelter workers.
Methods A population of 3185 workers from nine aluminium smelters owned by four different aluminium-producing companies were determined to have significant beryllium exposure. Of these, 1932 workers participated in medical surveillance programmes that included the serum beryllium lymphocyte proliferation test (BeLPT), confirmation of sensitization by at least two abnormal BeLPT test results and further evaluation for chronic beryllium disease in workers with BeS.
Results Personal beryllium samples obtained from the nine aluminium smelters showed a range of <0.01–13.00 μg/m3 time-weighted average with an arithmetic mean of 0.25 μg/m3 and geometric mean of 0.06 μg/m3. Nine workers were diagnosed with BeS (prevalence rate of 0.47%, 95% confidence interval = 0.21–0.88%).
Conclusions BeS can occur in aluminium smelter workers through natural beryllium contamination of the bauxite and further concentration during the refining and smelting processes. Exposure levels to beryllium observed in aluminium smelters are similar to those seen in other industries that utilize beryllium. However, compared with beryllium-exposed workers in other industries, the rate of BeS among aluminium smelter workers appears lower. This lower observed rate may be related to a more soluble form of beryllium found in the aluminium smelting work environment as well as the consistent use of respiratory protection.
PMCID: PMC3716328  PMID: 20610489
Aluminium; beryllium; sensitization; smelter; surveillance

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