Novel protein pathways in development and progression of pulmonary sarcoidosis.

Pulmonary involvement occurs in up to 95% of sarcoidosis cases. In this pilot study, we examine lung compartment-specific protein expression to identify pathways linked to development and progression of pulmonary sarcoidosis. We characterized bronchoalveolar lavage (BAL) cells and fluid (BALF) proteins in recently diagnosed sarcoidosis cases. We identified 4,306 proteins in BAL cells, of which 272 proteins were differentially expressed in sarcoidosis compared to controls. These proteins map to novel pathways such as integrin-linked kinase and IL-8 signaling and previously implicated pathways in sarcoidosis, including phagosome maturation, clathrin-mediated endocytic signaling and redox balance. In the BALF, the differentially expressed proteins map to several pathways identified in the BAL cells. The differentially expressed BALF proteins also map to aryl hydrocarbon signaling, communication between innate and adaptive immune response, integrin, PTEN and phospholipase C signaling, serotonin and tryptophan metabolism, autophagy, and B cell receptor signaling. Additional pathways that were different between progressive and non-progressive sarcoidosis in the BALF included CD28 signaling and PFKFB4 signaling. Our studies demonstrate the power of contemporary proteomics to reveal novel mechanisms operational in sarcoidosis. Application of our workflows in well-phenotyped large cohorts maybe beneficial to identify biomarkers for diagnosis and prognosis and therapeutically tenable molecular mechanisms.

p38 Mitogen-Activated Protein Kinase in beryllium-induced dendritic cell activation.

Dendritic cells (DC) play a role in the regulation of immune responses to haptens, which in turn impact DC maturation. Whether beryllium (Be) is able to induce DC maturation and if this occurs via the MAPK pathway is not known. Primary monocyte-derived DCs (moDCs) models were generated from Be non-exposed healthy volunteers as a non-sensitized cell model, while PBMCs from BeS (Be sensitized) and CBD (chronic beryllium disease) were used as disease models. The response of these cells to Be was evaluated. The expression of CD40 was increased significantly (p<0.05) on HLA-DP Glu69+ moDCs after 100 µM BeSO4-stimulation. BeSO4 induced p38MAPK phosphorylation, while IκB-α was degraded in Be-stimulated moDCs. The p38 MAPK inhibitor SB203580 blocked Be-induced NF-κB activation in moDCs, suggesting that p38MAPK and NF-κB are dependently activated by BeSO4. Furthermore, in BeS and CBD subjects, SB203580 downregulated Be-stimulated proliferation in a dose-dependent manner, and decreased Be-stimulated TNF-α and IFNγ cytokine production. Taken together, this study suggests that Be-induces non-sensitized Glu69+ DCs maturation, and that p38MAPK signaling is important in the Be-stimulated DCs activation as well as subsequent T cell proliferation and cytokine production in BeS and CBD. In total, the MAPK pathway may serve as a potential therapeutic target for human granulomatous lung diseases.

Developing effective health and safety training materials for workers in beryllium-using industries.

OBJECTIVE: Despite reduced workplace exposures, beryllium sensitization and chronic beryllium disease still occur. Effective health and safety training is needed. METHODS: Through an Occupational Safety and Health Administration (OSHA) Targeted Topic Training grant and company partners, we developed a training program. Evaluation and validation included knowledge and training reaction assessments and training impact survey. RESULTS: We describe herein the iterative, five-pronged approach: (1) needs assessment; (2) materials development; (3) pilot-testing, evaluation, and material revisions; (4) worker training; and (5) evaluation and validation. Mean posttraining test score increased 14% (82% to 96%; P < 0.005) and were unchanged at 90-day follow-up (94%; P = 0.744). In addition, 49% reported making changes in work practices. CONCLUSIONS: The use of a five-pronged training program was effective and well received and resulted in improved work practices. These materials are available on the OSHA Web site.

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.

Interpreting borderline BeLPT results.

BACKGROUND: The beryllium lymphocyte proliferation test (BeLPT) identifies persons sensitized to beryllium (BeS) and thus at risk for chronic beryllium disease (CBD). BeLPT test results are abnormal (AB), borderline (BL), or normal (NL). This manuscript addresses the predictive value and interpretation of BL BeLPT results. METHODS: The various three-result combinations that meet or exceed a nominal referral criteria of 1 AB + 1 BL are assessed with probability modeling and compared. RESULTS: At 2% prevalence, the three-result combinations that meet or exceed this referral criteria and associated probabilities of BeS are: (a) 1 AB + 1 BL + 1 NL (72%); (b) 3 BL (91%); (c) 2 AB + 1 NL (95%); (d) 1 AB + 2 BL (99%); (e) 2 AB + 1 BL (100%); and (f) 3 AB (100%). CONCLUSION: These results suggest that BL results are meaningful and that three BL results predict BeS across a broad range of population prevalences. An analysis of longitudinal BeLPT results and clinical findings from an actual surveillance program is warranted to confirm the model's predictions.

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.

Fas promoter polymorphisms: genetic predisposition to sarcoidosis in African-Americans.

Apoptosis may perpetuate some forms of inflammation. Of the apoptotic pathway proteins, Fas is particularly overexpressed in sarcoidosis. We hypothesized that Fas promoter single nucleotide polymorphisms (SNPs) contribute to the development and severity of sarcoidosis. Associations of known Fas promoter SNPs (-670, -690 and -1377) and deduced haplotypes with sarcoidosis and sarcoidosis severity were evaluated using matched case-control (n = 656 pairs) and case-comparison (n = 656) studies, respectively, using conditional logistic regression. Hardy-Weinberg equilibrium was confirmed for all three polymorphisms in African-Americans (AA), and for the -670 and -1377 in whites. Genotype and allele frequencies were significantly different between whites and AA. Race-stratified analysis revealed that a common haplotype, -1377G/-690T/-670G, was associated with sarcoidosis [odds ratio (OR) = 1.78, P = 0.05] only in AA. The haplotype -1377G/-690C/-670A was negatively associated with sarcoidosis (OR = 0.39, P = 0.03) only in AA. In conclusion, the consistency of these findings suggests that Fas promoter genetic variants may be related to sarcoidosis disease risk in AA.

Tumour necrosis factor gene polymorphisms are associated with COPD.

Tumour necrosis factor (TNF)-alpha has been shown to be an important factor in animal models of chronic obstructive pulmonary disease (COPD). However, human studies of TNF polymorphisms in COPD have been equivocal. Six TNF single nucleotide polymorphisms (-1031C/T, -863C/A, -857C/T, -237G/A, -308G/A and +487G/A) and their haplotypes were investigated in 423 Caucasian smokers (298 patients with spirometric evidence of COPD and 125 without airflow obstruction). The -308 minor allele (A) had a higher odds ratio (OR) of being associated with COPD in multivariate analysis (controlling for age, sex, pack-yrs; OR 1.9, 95% confidence interval (CI) 1.1-3.2) and was also associated with worse forced expiratory volume in one second/forced vital capacity. The -237 minor allele (A) had a lower OR of being associated with COPD (OR 0.40, 95% CI 0.19-0.86). In COPD patients, the -857 minor allele (T) had a lower OR of being associated with severe stages of COPD (Global Initiative for Obstructive Lung Disease stage III and IV versus stage I and II, OR 0.46, 95% CI 0.24-0.88). Other TNF single nucleotide polymorphisms were not associated with COPD but the -1031/-863 haplotype CC/TC had a lower OR in COPD patients versus smoking controls (OR 0.22, 95% CI 0.05-0.97). The present study adds further evidence that tumour necrosis factor genotypes play a role in susceptibility to cigarette smoke.

BTNL2 allele associations with chronic beryllium disease in HLA-DPB1*Glu69-negative individuals.

Butyrophilin-like 2 (BTNL2) polymorphisms have been associated with sarcoidosis. We hypothesized that BTNL2 variants might confer a human leukocyte antigen (HLA)-independent risk for chronic beryllium disease (CBD), a granulomatous lung disease with similar clinical, radiological, and pathological features to sarcoidosis. Genomic DNA was obtained from CBD (n= 88), beryllium sensitized (BeS, n= 86), and beryllium exposed nondiseased control subjects (Be-exp, n= 173). Six BTNL2 polymorphisms, HLA-DPB1, DRB1, and DQB1 alleles were determined by sequence-specific primer-PCR. All BTNL2 polymorphisms were in Hardy-Weinberg equilibrium. No significant differences were found between BTNL2 polymorphisms or haplotypes and CBD, BeS, or Be-exp. In HLA-DPB1*Glu69-negative subjects (n= 10 CBD, n= 13 BeS, n= 102 Be-exp), DRB1*13 and BTNL2 rs3117099TT homozygosity were increased in CBD (70% and 40%, respectively) vs Be-exp (16%, P= 0.001 and 2.9%, P= 0.001, respectively). The BTNL2 rs3117099T-HLA-DRB1*13 combination was significantly increased in CBD (50%) compared with Be-exp (6.9%, P= 0.001). In conclusion, both DRB1*13 and rs3117099TT homozygosity are associated with CBD in *Glu69-negative subjects, while DPB1*Glu69 is associated with CBD and BeS compared with Be-exp. As a result of the small sample size and strong linkage disequilibrium between DRB1*13-DQB1*0603/4/9 and the BTNL2 rs3117099T allele, it is difficult to assess the primary association in DPB1*Glu69-negative CBD cases.

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.

High beryllium-stimulated TNF-alpha is associated with the -308 TNF-alpha promoter polymorphism and with clinical severity in chronic beryllium disease.

Beryllium (Be)-antigen stimulates tumor necrosis factor-alpha (TNF-alpha) from bronchoalveolar lavage (BAL) cells in chronic beryllium disease (CBD). This study tested the hypothesis that high concentrations of Be-stimulated TNF-alpha are related to polymorphisms in the TNF-alpha promoter and clinical markers of disease severity in CBD. Demographic and clinical information was obtained from patients with CBD (n = 20). TNF-alpha concentrations were measured in BAL cell culture supernatant by ELISA. A priori, we categorized CBD subjects as either high or low TNF-alpha producers using a cutoff of 1,500 pg/ml. The TNF-alpha promoter sequence, +64 to -1045, was determined by direct sequencing. Human leukocyte-associated antigen (HLA)-DPB1 and -DRB1 genotyping was determined by polymerase chain reaction (PCR). High Be-stimulated TNF-alpha was associated with TNF2 alleles, Hispanic ethnicity, presence of HLA-DPB1 Glu69, and absence of HLA-DR4. Be-stimulated TNF-alpha concentrations correlated with markers of disease severity, including chest radiograph, beryllium lymphocyte proliferation, and spirometry. We found no novel TNF-alpha promoter polymorphisms. These data suggest that the TNF2 A allele at -308 in the TNF-alpha promoter region is a functional polymorphism, associated with a high level of Be-antigen-stimulated TNF-alpha and that these high TNF-alpha levels indicate disease severity in CBD.

Beryllium sensitivity is linked to HLA-DP genotype.

Chronic beryllium disease (CBD) appears to arise from a combination of both exposure and genetic risk factors. A distinguishing feature of CBD is beryllium hypersensitivity, which can be measured in vitro by a lymphocyte proliferation test. The objective of this study was to determine whether certain allelic variations of the HLA-DPB1 gene, which had been observed previously in CBD, could be found in a group of individuals having beryllium hypersensitivity, but no symptoms of CBD. A flow cytometry-based Lymphocyte Proliferation Test combined with immunophenotyping (Immuno-LPT) was used to detect CD4+ and CD8+ T cell proliferation in response to in vitro stimulation with beryllium. The HLA-DPB1 haplotypes of the same individuals were determined by automated DNA sequencing. Twenty-two out of 25 beryllium-sensitive, non-CBD individuals were found to be carriers of the HLA-DPB1 gene having a substitution of a glutamic acid at position 69 in Exon 2 (Glu69), and a significantly high percentage (24%) were Glu69 homozygotes. Most of the CD4+ responders on the Immuno-LPT (10/14) carried rare, non-*0201 Glu69 DPB1 alleles; while most of the non-CD4+ responders (9/11) were common Glu69 carriers (*0201 or *0202) or non-Glu69 individuals (non-Glu69/non-Glu69). This is the first direct evidence that HLA-DP genotype is linked to a phenotypic response that occurs in beryllium sensitization in the absence of clinical CBD.

Interstitial fibrosis following hydrogen sulfide exposure.

This case report describes a patient with interstitial fibrosis 4 years after poisoning by hydrogen sulfide. Hydrogen sulfide causes pulmonary edema and is also toxic to the nervous system. Long-term pulmonary sequelae of hydrogen sulfide poisoning has not been reported frequently in the literature.

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.

Beryllium health effects in the era of the beryllium lymphocyte proliferation test.

The beryllium lymphocyte proliferation test (BeLPT) has revolutionized our approach to the diagnosis, screening, and surveillance of beryllium health effects. Based on the development of a beryllium-specific cell-mediated immune response, the BeLPT has allowed us to define early health effects of beryllium, including beryllium sensitization (BeS), and chronic beryllium disease (CBD) at a subclinical stage. The use of this test as a screening tool has improved our understanding of these health effects. From a number of studies it is apparent that BeS precedes CBD and develops after as little as 9 weeks of beryllium exposure. CBD occurs within 3 months and up to 30 years after initial beryllium exposure. Exposure-response variables have been associated with BeS/CBD, including work as a machinist, chemical or metallurgical operator, laboratory technician, work in ceramics or beryllium metal production, and years of beryllium exposure. Recent studies have found BeS and CBD in workplaces in which the majority of exposures were below the 2 microg/m3 OSHA time-weighted average (TWA). Ideally, the BeLPT would be used in surveillance aimed at defining other risk-related processes, determining exposure variables which predict BeS and CBD, and defining the exposure level below which beryllium health effects do not occur. Unfortunately, the BeLPT can result in false negative tests and still requires an invasive procedure, a bronchoscopy, for the definitive diagnosis of CBD. Thus, research is needed to establish new tests to be used alone or in conjunction with the BeLPT to improve our ability to detect early beryllium health effects.

Efficacy of serial medical surveillance for chronic beryllium disease in a beryllium machining plant.

There is limited information on the use of the blood beryllium lymphocyte proliferation test (BeLPT) at regular intervals in medical surveillance. Employees of a beryllium machining plant were screened with the BeLPT biennially, and new employees were screened within 3 months of hire. Of 235 employees screened from 1995 to 1997, a total of 15 (6.4%) had confirmed abnormal BeLPT results indicating beryllium sensitization; nine of these employees were diagnosed with chronic beryllium disease. Four of the 15 cases were diagnosed within 3 months of first exposure. When 187 of the 235 employees participated in biennial screening in 1997 to 1999, seven more had developed beryllium sensitization or chronic beryllium disease, increasing the overall rate to 9.4% (22 of 235). The blood BeLPT should be used serially in beryllium disease surveillance to capture new or missed cases of sensitization and disease. Beryllium sensitization and chronic beryllium disease can occur within 50 days of first exposure in modern industry.

Beryllium particulate exposure and disease relations in a beryllium machining plant.

We examined the relationship between exposure to beryllium and the presence of beryllium sensitization (BeS) and chronic beryllium disease (CBD) in a cohort of workers in a beryllium precision machining facility. Twenty workers with BeS or CBD (cases) were compared with 206 worker-controls in a case-control study. Exposure for each job title was measured using cascade impactors placed in the workers' breathing zone to measure total beryllium exposure and exposure to particles < 6 microns and < 1 micron in aerodynamic diameter. Cumulative exposure was calculated as sigma (job title exposure estimate x years in job title). Individual lifetime-weighted (LTW) exposure was calculated as sigma [(job title exposure x years in job title) divided by total years employment)]. Workers in the case group were more likely to have worked as machinists (odds ratio, 4.4; 95% confidence interval, 1.1 to 17.5) than those in the control group. The median cumulative exposure was consistently greater in the cases compared with the controls for all exposure estimates and particle size fractions, although this was not statistically significant. The median cumulative exposure was 2.9 micrograms/m3-years in the cases versus 1.2 micrograms/m3-years in the controls for total exposure, and 1.7 micrograms/m3-years in the cases versus 0.5 microgram/m3-years in the controls for exposure to particles < 6 microns in diameter. With cumulative exposure categorized into low-, intermediate-, and high-exposure groups, the odds ratios were 2.4 (95% confidence interval, 0.7 to 8.2) for the intermediate-exposure group and 1.2 (95% confidence interval, 0.4 to 4.2) for the high-exposure group compared with the low-exposure group. The median LTW exposure was 0.25 microgram/m3 in both groups. The median LTW exposure to particles < 6 microns was 0.20 microgram/m3 in the cases compared with 0.14 microgram/m3 in the controls. The differences in cumulative and LTW exposure were not statistically significant. None of the 22 workers with LTW exposure < 0.02 microgram/m3 had BeS or CBD. Twelve workers (60%) in the case group had LTW exposures > 0.20. In conclusion, increased cumulative and LTW exposure to total and respirable beryllium was observed in workers with CBD or BeS compared with the controls. These results support efforts to control beryllium exposure in the workplace.

Beryllium-stimulated apoptosis in macrophage cell lines.

In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

Aerosols generated during beryllium machining.

Some beryllium processes, especially machining, are associated with an increased risk of beryllium sensitization and disease. Little is known about exposure characteristics contributing to risk, such as particle size. This study examined the characteristics of beryllium machining exposures under actual working conditions. Stationary samples, using eight-stage Lovelace Multijet Cascade Impactors, were taken at the process point of operation and at the closest point that the worker would routinely approach. Paired samples were collected at the operator's breathing zone by using a Marple Personal Cascade Impactor and a 35-mm closed-faced cassette. More than 50% of the beryllium machining particles in the breathing zone were less than 10 microns in aerodynamic diameter. This small particle size may result in beryllium deposition into the deepest portion of the lung and may explain elevated rates of sensitization among beryllium machinists.

Differential susceptibilities to chronic beryllium disease contributed by different Glu69 HLA-DPB1 and -DPA1 alleles.

Chronic beryllium disease (CBD) is associated with the allelic substitution of a Glu69 in the HLA-DPB1 gene. Although up to 97% of CBD patients may have the Glu69 marker, about 30-45% of beryllium-exposed, unaffected individuals carry the same marker. Because CBD occurs in only 1-6% of exposed workers, the presence of Glu69 does not appear to be the sole genetic factor underlying the disease development. Using two rounds of direct automated DNA sequencing to precisely assign HLA-DPB1 haplotypes, we have discovered highly significant Glu69-containing allele frequency differences between the CBD patients and a beryllium-exposed, nondiseased control group. Individuals with DPB1 Glu69 in both alleles were almost exclusively found in the CBD group (6/20) vs the control group (1/75). Whereas most Glu69 carriers from the control group had a DPB1 allele *0201 (68%), most Glu69 carriers from the CBD group had a non-*0201 DPB1 Glu69-carrying allele (84%). The DPB1 allele *0201 was almost exclusively (29/30) associated with DPA1 *01 alleles, while the non-*0201 Glu69-containing DPB1 alleles were closely associated with DPA1 *02 alleles (26/29). Relatively rare Glu69-containing alleles *1701, *0901, and *1001 had extremely high frequencies in the CBD group (50%), as compared with the control group (6.7%). Therefore, the most common Glu69-containing DPB1 allele, *0201, does not seem to be a major disease allele. The results suggest that it is not the mere presence of Glu69, per se, but specific Glu69-containing alleles and their copy number (homozygous or heterozygous) that confer the greatest susceptibility to CBD in exposed individuals.