Age-associated B Cells Appear in Patients with Granulomatous Lung Diseases.

Rationale: A subpopulation of B cells (age-associated B cells [ABCs]) is increased in mice and humans with infections or autoimmune diseases. Because depletion of these cells might be valuable in patients with certain lung diseases, the goal was to find out if ABC-like cells were at elevated levels in such patients.Objectives: To measure ABC-like cell percentages in patients with lung granulomatous diseases.Methods: Peripheral blood and BAL cells from patients with sarcoidosis, beryllium sensitivity, or hypersensitivity pneumonitis and healthy subjects were analyzed for the percentage of B cells that were ABC-like, defined by expression of CD11c, low levels of CD21, FcRL 1-5 (Fc receptor-like protein 1-5) expression, and, in some cases, T-bet.Measurements and Main Results: ABC-like cells in blood were at low percentages in healthy subjects and higher percentages in patients with sarcoidosis as well as at high percentages among BAL cells of patients with sarcoidosis, beryllium disease, and hypersensitivity pneumonitis. Treatment of patients with sarcoidosis led to reduced percentages of ABC-like cells in blood.Conclusions: Increased levels of ABC-like cells in patients with sarcoidosis may be useful in diagnosis. The increase in percentage of ABC-like cells in patients with lung granulomatous diseases and decrease in treated patients suggests that depletion of these cells may be valuable.

Clinical and laboratory factors contributing to uninterpretable beryllium lymphocyte proliferation tests (BeLPT).

BACKGROUND: The beryllium lymphocyte proliferation test (BeLPT), has become the principal clinical test for detecting beryllium sensitization and chronic beryllium disease. Uninterpretable BeLPT results can occur in a small but significant proportion of tests from poor lymphocyte growth (PG) or over proliferation of lymphocytes (OP). The clinical and laboratory causes of uninterpretable results are not known. METHODS: BeLPT data from the US Department of Energy-supported Former Worker Screening Program were analyzed for a 10-year period. Drivers of uninterpretable BeLPTs were investigated using multivariable models and classification techniques. RESULTS: Three participant attributes were significantly associated with PG, while OP showed no significant associations. Serum lot for the lymphocyte growth medium accounted for 21% of the variation in PG and 16% in OP. CONCLUSION: Serum lots influence the likelihood of having uninterpretable BeLPT. To better understand uninterpretable results and possibly reduce their occurrence, additional laboratory-related factors should be addressed.

Impaired function of CTLA-4 in the lungs of patients with chronic beryllium disease contributes to persistent inflammation.

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 Ag-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 with 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 the 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.

Beryllium-specific CD4+ T cells in blood as a biomarker of disease progression.

BACKGROUND: 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, T(H)1-type, cytokine-secreting T cells have been characterized. Compared with those seen in beryllium (Be)-sensitized subjects, increased numbers of Be-responsive T cells are present in the blood of patients with CBD. OBJECTIVE: 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. METHODS: Using IFN-γ ELISpot and proliferation-based assays, we determined the frequency and proliferative capacity of Be-responsive T cells in blood. RESULTS: Compared with the Be lymphocyte proliferation test, which detected an abnormal Be-induced proliferative response in 11 (4.2%) of 260 workers from a Be-machining facility, the IFN-γ ELISpot detected a sensitization rate of 10% (χ(2) = 55.7, P < .0001). A significant positive correlation was also noted between the number of Be-responsive CD4(+) T cells in the blood and lung tissue of patients with CBD. Importantly, the transition from Be sensitization to CBD was associated with an increased number of antigen-specific T cells in blood. CONCLUSION: 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.

Search for chronic beryllium disease among sarcoidosis patients in Ontario, Canada.

Chronic beryllium disease (CBD) is clinically similar to other granulomatous diseases such as sarcoidosis. It is often misdiagnosed if a thorough occupational history is not taken. When appropriate, a beryllium lymphocyte proliferation tests (BeLPT) need to be performed. We aimed to search for CBD among currently diagnosed pulmonary sarcoidosis patients and to identify the occupations and exposures in Ontario leading to CBD. Questionnaire items included work history and details of possible exposure to beryllium. Participants who provided a history of previous work with metals underwent BeLPTs and an ELISPOT on the basis of having a higher pretest probability of CBD. Among 121 sarcoid patients enrolled, 87 (72%) reported no known previous metal dust or fume exposure, while 34 (28%) had metal exposure, including 17 (14%) with beryllium exposure at work or home. However, none of these 34 who underwent testing had positive test results. Self-reported exposure to beryllium or metals was relatively common in these patients with clinical sarcoidosis, but CBD was not confirmed using blood assays in this population.

SELDI-TOF derived serum biomarkers failed to differentiate between patients with beryllium sensitisation and patients with chronic beryllium disease.

BACKGROUND: People exposed to beryllium may develop beryllium sensitisation (BeS) and, in some cases, progress to chronic beryllium disease (CBD). OBJECTIVES: The objective of this study was to test the ability of proteomic technology to identify patterns of serum protein biomarkers that allow differentiation between BeS and CBD and thus remove the need for invasive bronchoscopic procedures. METHODS: Initially, SELDI-TOF methodology and analysis was performed on serum samples from 30 CBD and 31 BeS patients. RESULTS: This 'starter set' yielded two distinct biomarker pattern sets with eight candidate proteins. The first set differentiated between BeS and CBD with 83.3% sensitivity and 82.3% specificity, with 10-fold cross-validation of 75% and 79%, respectively. The second set of biomarkers yielded higher sensitivity (90.0%) and higher specificity (90.3%), with 10-fold cross-validation of 71.7% and 82.3%, respectively. Due to its greater sensitivity and specificity, the second set of biomarkers was used as the framework for differentiating between CBD and BeS in a second set of serum samples from 450 patients with BeS and CBD. When this larger set of samples was subjected to the biomarker framework in a blinded fashion, it yielded a sensitivity of 43.53% and a specificity of 38.93%. CONCLUSIONS: Due to these low sensitivity and specificity values, we have concluded that, currently, the unique set of SELDI-TOF derived biomarkers does not possess the qualities that would allow it to differentiate between a CBD patient and a BeS patient using serum protein biomarkers. Future refinements in sample collection or proteomic technology may be needed to improve biomarker discovery.

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/m(3) time-weighted average with an arithmetic mean of 0.25 µg/m(3) and geometric mean of 0.06 µg/m(3). 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.

[Genotoxic effects caused in workers by beryllium compounds].

The author represents results of studying genotoxic effects caused in workers by beryllium compounds. Increasing age and length of service apperared to correlate with higher level of chromosomal aberration in peripheral WBC. Findings are that obtaining beryllium and its compounds is associated with changed level of chromosomal damages.

Cumulative sensitization and disease in a beryllium oxide ceramics worker cohort.

OBJECTIVE: We followed a cohort of 136 beryllium oxide ceramics workers from 1992 to 2003, including those who left employment, for beryllium sensitization and chronic beryllium disease (CBD). METHODS: We invited the cohort's participation in current worker surveys in 1992, 1998, 2000, and 2002-2003, and in former worker surveys in 2000-2001 and 2003. We calculated 11-year cumulative incidences (after 1992 initial survey) of sensitization and CBD, both crude and corrected for interval censoring; and period prevalences (including 1992 findings), crude and corrected. RESULTS: In 1992, point prevalences were 6% sensitized and 4% CBD. We obtained follow-up on 83% of 128 not sensitized in 1992. Crude cumulative incidences for sensitization and CBD were 13% and 9%, respectively; corrected were 15% and 11%. Crude period prevalences for sensitization and CBD were 16% and 11%, respectively; corrected were 20% and 14%. Corrected period prevalences for pre-1992 machining work were 30% and 20%. CONCLUSIONS: With repeated testing over 11 years, total sensitization and CBD in this cohort were triple initial 1992 survey results.

Beryllium sensitization in aluminum smelter workers.

OBJECTIVE: To determine whether beryllium-related disease exists among aluminum smelter workers. METHODS: A total of 1278 employees from four aluminum smelters determined to have significant beryllium exposure based on 5 years of sampling were invited to participate in medical surveillance that included a respiratory symptoms questionnaire, spirometry, and blood beryllium lymphocyte proliferation test. RESULTS: Of these, 734 employees participated in the program. Beryllium exposure from 965 personal samples ranged from 0.002 to 13.00 microg/m time-weighted average, with a median of 0.05 microg/m, geometric mean of 0.05 microg/m, and arithmetic mean of 0.22 microg/m. Only two employees had confirmed beryllium sensitization (0.27%). CONCLUSION: There is evidence of beryllium sensitization among aluminum smelter workers. When compared with beryllium-exposed workers in other industries, aluminum smelter workers had lower rates of sensitization. The low beryllium sensitization rate observed may be related to work practices and the properties of the beryllium found in this work environment.

Optimizing BeLPT criteria for beryllium sensitization.

BACKGROUND: The beryllium lymphocyte proliferation test (BeLPT) is used to identify persons sensitized to beryllium. ATSDR convened an expert panel of physicians and scientists in April 2006 to discuss this test and to consider what BeLPT test results actually establish beryllium sensitization. The three criteria proposed by panel members were (1)one abnormal result, (2)one abnormal and one borderline result, and (3)two abnormal results. METHODS: Complete algorithms were developed for each of the three proposed criteria. Using single-test outcome probabilities developed by Stange et al. [2004. Am J Ind Med 46:453-462], we calculated and compared the sensitivity, specificity, and positive predictive values (PPVs) for each set of criteria. RESULTS: The overall sensitivity and specificity of the three criteria were similar. When the criteria required confirmation of an abnormal result the PPV was higher--whether the requirement was satisfied by a borderline result, or only by another abnormal result. Confirmation also reduced the likelihood of false positives. The differences between the three criteria decreased as the prevalence of sensitization increased. CONCLUSIONS: A single unconfirmed abnormal is usually insufficient to establish sensitization for an apparently healthy person. When the prevalence of beryllium sensitization in a group is high, however, even a single abnormal BeLPT can be a strong predictor.

Use of beryllium lymphocyte proliferation testing for screening of asymptomatic individuals: an evidence-based assessment.

OBJECTIVE: We reviewed published data describing use of beryllium lymphocyte proliferation testing (BeLPT) to determine the appropriateness of BeLPT for screening asymptomatic individuals. METHODS: Published studies were identified by computerized literature searches and hand searches of relevant bibliographies and cited references. Critical assessment of evidence focused on five elements essential to judging effectiveness of preventive services: 1) burden of suffering, 2) accuracy and reliability of screening tests, 3) effectiveness of early detection, 4) harms of screening, and 5) benefits outweighing harms. RESULTS: Important gaps and deficiencies in the evidence were found. The prevalence of beryllium sensitization and chronic beryllium disease in asymptomatic individuals are unknown. The accuracy and reliability of BeLPT are uncertain. Marked intra- and interlaboratory variability has been reported. The clinical benefits of early intervention have not been confirmed or quantified in asymptomatic individuals. CONCLUSIONS: There is currently insufficient scientific evidence to support the use of BeLPT for routine screening of asymptomatic individuals.

Frequency of beryllium-specific, central memory CD4+ T cells in blood determines proliferative response.

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+ T(EM) 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.

Beryllium-ferritin: lymphocyte proliferation and macrophage apoptosis in chronic beryllium disease.

A beryllium (Be)-ferritin adduct containing 270 pm of Be stimulated proliferation of bronchoalveolar lavage (BAL) lymphocytes from subjects with chronic beryllium disease (CBD) at concentrations 5-6 logs lower than the amounts of beryllium sulfate (BeSO4) needed to induce proliferation. We observed increased apoptotic CBD BAL macrophages after exposure to both BeSO4 (50 +/- 6%, mean +/- SEM, P <0.05 versus unstimulated controls) and Be-ferritin (40 +/- 2%), whereas only 2.0 +/- 0.2% of BAL lymphocytes underwent activation-induced cell death. Be-ferritin also induced apoptosis in BAL macrophages from subjects with Be sensitization (25 +/- 3%) and in the H36.12j hybrid macrophage cell line (15 +/- 2%). Be-ferritin induced lung macrophage CD95 (Fas) expression and the activation of intracellular caspase-3, -8 and -9. Thus, lung macrophages take up Be-ferritin, delivering physiologically relevant levels of Be that promote Be antigen presentation and macrophage apoptosis. Be-ferritin thereby serves as a "Trojan Horse," triggering proliferation of Be-ferritin-specific CBD BAL T cells. We hypothesize that Be-ferritin exposure may result in persistent antigen exposure inducing Be-specific T cell clonal expansion and T cell helper type 1-type cytokine production and potentially explains the chronicity of CBD and its development years after environmental Be exposure has ceased.

Chronic beryllium disease: immune-mediated destruction with implications for organ-specific autoimmunity.

Chronic beryllium disease (CBD) is caused by exposure to beryllium in the workplace and is characterized by an accumulation of beryllium-specific CD4+ T cells with granulomatous inflammation in the lung. Owing to its unique physical properties, beryllium is used in a variety of high-technology industries, and CBD continues to be an important public health concern. CBD develops in up to 16% of exposed workers, depending on genetic susceptibility and the nature of the exposure. Increased susceptibility has been associated with particular HLA-DP alleles, especially those possessing negatively charged residues at certain positions of the peptide-binding pocket. The mechanism for this disease association lies in the ability of certain HLA-DP molecules, with associated peptides, to bind and present beryllium to pathogenic CD4+ T cells. In patients with CBD, large numbers of effector memory CD4+ T cells are compartmentalized to the lung, and these cells are poised to release T helper 1-type cytokines upon beryllium recognition. In the same patients, however, beryllium-specific T cells are barely detectable in the circulation. As opposed to those present in blood, beryllium-specific cells in the lung no longer require the engagement of CD28 for optimal T-cell activation and in fact frequently lose the expression of CD28. These findings in CBD have important implications for studies in autoimmune diseases, including those in which the antigen is unknown and the target organ is inaccessible.

Indirect and direct gas exchange at maximum exercise in beryllium sensitization and disease.

STUDY OBJECTIVES: To determine whether pulse oximetry accurately estimates arterial blood gas measurements during exercise in the assessment of chronic beryllium disease (CBD) and beryllium sensitization (BeS). DESIGN: Participants underwent maximal exercise physiology testing in a clinical-practice setting. Oxygen saturation in the blood was measured through an indwelling arterial line and by pulse oximetry. SETTING: All exercise physiology tests were performed in the pulmonary physiology unit of the National Jewish Medical and Research Center (NJMRC) between December 1985 and November 1998. PATIENTS: We analyzed the exercise physiology data for 168 individuals who were referred to NJMRC for evaluation of possible CBD and underwent exercise testing. On evaluation, they subsequently received diagnoses of either CBD or BeS. RESULTS: In BeS subjects, the percentage of oxygen saturation as measured by pulse oximetry (SpO(2)) often underestimated the percentage of arterial oxygen saturation (SaO(2)) (mean [+/- SD] underestimation, 0.88 +/- 4.6%) at maximum exercise and showed no significant correlation (r = -0.13; p = 0.3). The use of SpO(2) misclassified 14.9% of BeS subjects as having abnormal gas exchange levels (< 90%) that were normal by arterial blood gas measurement. In contrast, SpO(2) and SaO(2) values correlated at maximum exercise in CBD subjects (r = 0.55 [corrected]; p = 0.0001) without exhibiting SpO(2) underestimation of SaO(2), and misclassification occurred in only 5.9%. CONCLUSIONS: These data suggest that pulse oximetry cannot be used reliably to distinguish between CBD and BeS and, thus, is not an adequate substitute for arterial blood gas analysis with exercise.

IL-4 fails to regulate in vitro beryllium-induced cytokines in berylliosis.

Bronchoalveolar lavage (BAL) cells from patients with chronic beryllium disease (CBD) have been used to evaluate the beryllium-specific immune response and potential immunotherapeutics. Beryllium induces interferon-gamma (IFN-gamma), interleukin-2 (IL-2), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-10 (IL-10) from BAL cells. An antibody to IL-2 and recombinant human (rHu) IL-10 is able to partially suppress the beryllium-stimulated immune response. To obtain BAL cells, bronchoscopy is required, providing risk to the patient and a limited number of cells to study the immune response. As a result, the objectives of the study were to determine 1) whether CBD peripheral blood mononuclear cells (PBMNs) stimulated with beryllium would produce a similar cytokine pattern as BAL cells, and 2) whether this response could be modulated by interleukin-4 (IL-4), an immunomodulatory cytokine. CBD and normal individuals' PBMN and BAL cells were stimulated with and without beryllium sulfate. To modulate this antigen-stimulated response, we added rHu IL-4 to the unstimulated and beryllium-stimulated cells. IFN-gamma, IL-2, TNF-alpha, IL-6 and IL-10 cytokine concentrations were determined from cell supernatants by enzyme-linked immunosorbent assays (ELISA), while IL-4 messenger ribonucleic acid (mRNA) was assessed using polymerase chain reaction (PCR). Beryllium did not stimulate any of these cytokines from normal PBMNs. Increasing levels of IL-6 and TNF-alpha were produced constituitively by CBD PBMNs over time. Compared to the unstimulated CBD PBMNs, beryllium stimulated significant IFN-gamma, TNF-alpha, IL-2, IL-6 and IL-10 production. This response was similar to that stimulated from CBD BAL cells, although of a much lower magnitude. Low levels of IL-4 mRNA were found in CBD and control PBMNs, which were not increased with beryllium stimulation. The beryllium-stimulated cytokine levels were not decreased by the addition of IL-4. IL-4 was unable to downregulate any of these beryllium-stimulated cytokines from CBD BAL cells or increase IL-4 mRNA from either CBD PBMN or BAL cells, and thus is an unlikely immunomodulatory agent in CBD. From the data, it was concluded that chronic beryllium disease peripheral blood mononuclear cells provide a model to study the beryllium-stimulated immune response. Interleukin-4's inability to downregulate any of the beryllium-stimulated cytokines makes it an unlikely therapeutic candidate in chronic beryllium disease.