Analysis of single nucleotide polymorphisms in chronic beryllium disease.

Sarcoidosis and chronic beryllium disease (CBD) are phenocopies, however the latter one has a clear trigger factor that is beryllium exposure. This study analyses single nucleotide polymorphisms (SNPs) in a large cohort for beryllium-exposed persons. SNPs were chosen for their relevance in sarcoidosis. Even though one of largest cohorts of beryllium-exposed persons was analysed, no statistically relevant association between any SNP and CBD could be verified. Notably, some SNPs exhibit inverse OR for beryllium sensitization and CBD with nominally statistical significance, which allows hypothesizing about pathophysiological role of genes for the disease triggering and development.

5-Aminosalicylic Acid Modulates the Immune Response in Chronic Beryllium Disease Subjects.

INTRODUCTION: Chronic beryllium disease (CBD) is characterized by accumulation of macrophages and beryllium-specific CD4+ T cells that proliferate and produce Th1 cytokines. 5-Amino salicylic acid (5-ASA) is currently used to treat inflammatory bowel disease and has both antioxidant and anti-inflammatory actions. We hypothesized that 5-ASA may be a beneficial therapeutic in CBD. METHODS: Seventeen CBD patients were randomized 3:1 to receive 5-ASA 500-mg capsules or placebo four times daily for 6 weeks orally. Primary study endpoints included changes in beryllium lymphocyte proliferation (BeLPT). Secondary endpoints included changes in bronchoalveolar lavage (BAL) fluid, cells, serum, and blood cell glutathione (GSH) levels, BAL cell TNF-α levels, lung function, and quality of life measures. RESULTS: 5-ASA decreased BAL cell BeLPT by 20% within the 5-ASA treatment group. No significant changes were observed in serum, PBMCs, BALF, or BAL cell GSH levels in either the 5-ASA or placebo treatment group. 5-ASA treatment decreased ex vivo Be-stimulated BAL cell TNF-α levels within the 5-ASA group and when compared to placebo. Significant improvements were noted in quality of life measurements with 5-ASA treatment. CONCLUSIONS: 5-ASA's ability to decrease BAL cell BeLPT and Be-stimulated BAL cell TNF-α levels suggests that 5-ASA may impact the beryllium-specific immune response in CBD. 5-ASA use in other non-infectious granulomatous lung diseases, such as sarcoidosis, may prove to be a useful alternative treatment to corticosteroids for those with mild to moderate disease.

Identification of multiple public TCR repertoires in chronic beryllium disease.

Chronic beryllium disease (CBD) is a granulomatous lung disease characterized by the accumulation of beryllium (Be)-specific CD4(+) T cells in bronchoalveolar lavage. These expanded CD4(+) T cells are composed of oligoclonal T cell subsets, suggesting their recruitment to the lung in response to conventional Ag. In the current study, we noted that all bronchoalveolar lavage-derived T cell lines from HLA-DP2-expressing CBD patients contained an expansion of Be-responsive Vß5.1(+) CD4(+) T cells. Using Be-loaded HLA-DP2-peptide tetramers, the majority of tetramer-binding T cells also expressed Vß5.1 with a highly conserved CDR3ß motif. Interestingly, Be-specific, Vß5.1-expressing CD4(+) T cells displayed differential HLA-DP2-peptide tetramer staining intensity, and sequence analysis of the distinct tetramer-binding subsets showed that the two populations differed by a single conserved amino acid in the CDR3ß motif. TCR Vα-chain analysis of purified Vß5.1(+) CD4(+) T cells based on differential tetramer-binding intensity showed differing TCR Vα-chain pairing requirements, with the high-affinity population having promiscuous Vα-chain pairing and the low-affinity subset requiring restricted Vα-chain usage. Importantly, disease severity, as measured by loss of lung function, was inversely correlated with the frequency of tetramer-binding CD4(+) T cells in the lung. Our findings suggest the presence of a dominant Be-specific, Vß5.1-expressing public T cell repertoire in the lungs of HLA-DP2-expressing CBD patients using promiscuous Vα-chain pairing to recognize an identical HLA-DP2-peptide/Be complex. Importantly, the inverse relationship between expansion of CD4(+) T cells expressing these public TCRs and disease severity suggests a pathogenic role for these T cells in CBD.

Crystal structure of HLA-DP2 and implications for chronic beryllium disease.

Chronic beryllium disease (CBD) is a fibrotic lung disorder caused by beryllium (Be) exposure and is characterized by granulomatous inflammation and the accumulation of Be-responsive CD4(+) T cells in the lung. Genetic susceptibility to CBD has been associated with certain alleles of the MHCII molecule HLA-DP, especially HLA-DPB1*0201 and other alleles that contain a glutamic acid residue at position 69 of the beta-chain (betaGlu69). The HLA-DP alleles that can 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. The structure of HLA-DP2 and its interaction with Be are unknown. Here, we present the HLA-DP2 structure with its antigen-binding groove occupied by a self-peptide derived from the HLA-DR alpha-chain. The most striking feature of the structure is an unusual solvent exposed acidic pocket formed between the peptide backbone and the HLA-DP2 beta-chain alpha-helix and containing three glutamic acids from the beta-chain, including betaGlu69. In the crystal packing, this pocket has been filled with the guanidinium group of an arginine from a neighboring molecule. This positively charged moiety forms an extensive H-bond/salt bridge network with the three glutamic acids, offering a plausible model for how Be-containing complexes might occupy this site. This idea is strengthened by the demonstration that mutation of any of the three glutamic acids in this pocket results in loss of the ability of DP2 to present Be to T cells.

HLA-DP, HLA-DQ, and HLA-DR have different requirements for invariant chain and HLA-DM.

The MHC is central to the adaptive immune response. The human MHC class II is encoded by three different isotypes, HLA-DR, -DQ, and -DP, each being highly polymorphic. In contrast to HLA-DR, the intracellular assembly and trafficking of HLA-DP molecules have not been studied extensively. However, different HLA-DP variants can be either protective or risk factors for infectious diseases (e.g. hepatitis B), immune dysfunction (e.g. berylliosis), and autoimmunity (e.g. myasthenia gravis). Here, we establish a system to analyze the chaperone requirements for HLA-DP and to compare the assembly and trafficking of HLA-DP, -DQ, and -DR directly. Unlike HLA-DR1, HLA-DQ5 and HLA-DP4 can form SDS-stable dimers supported by invariant chain (Ii) in the absence of HLA-DM. Uniquely, HLA-DP also forms dimers in the presence of HLA-DM alone. In model antigen-presenting cells, SDS-stable HLA-DP complexes are resistant to treatments that prevent formation of SDS-stable HLA-DR complexes. The unexpected properties of HLA-DP molecules may help explain why they bind to a more restricted range of peptides than other human MHC class II proteins and frequently present viral peptides.

[Structural and functional-metabolic criteria for studies of bronchial lavage cells in beryllium production workers].

The article presents results of cytomorphologic and cytochemical studies of bronchoalveolar lavage cells in experimental animals and in beryllium production workers having beryllium intoxication signs. Findings are increased counts of degenerated neutrophils and macrophages in bronchial lavage, high degree of microbial (streptococcal and staphylococcal) contamination in workers having atrophic bronchitis, low levels of catecholamines and esterase activity, high content of phospholipids, if compared to apparently healthy individuals. These tests are expedient to use in early diagnosis among beryllium production workers.

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.

[Cytochemical parameters in nasal mucosal and peripheral blood cells in beryllium production workers].

Nasal mucosal and peripheral blood cells from beryllium production workers underwent cytochemical tests for catecholamines and phospholipids and the activity of esterase and lysosomal cationic proteins. The established peripheral blood metabolic changes reflect reduced cell resistance in response to poor occupational factors and may be used in the rapid hygienic assessment of cytotoxicity of toxic substances.

Role of high-affinity HLA-DP specific CLIP-derived peptides in beryllium binding to the HLA-DPGlu69 berylliosis-associated molecules and presentation to beryllium-sensitized T cells.

Berylliosis is driven by the accumulation in the lung of beryllium-specific T helper type 1 (Th1) cells recognizing beryllium as antigen when presented principally by human leucocyte antigen DP molecules carrying a glutamate at position beta69 (HLA-DPGlu69). This study was designed to clarify the precise role of peptides in beryllium binding to the HLA-DP groove's pocket 4 and to identify peptides with higher affinity for pocket 4 that might prevent beryllium presentation and T-cell stimulation. Beryllium/HLA-DP interactions were analysed by the ability of beryllium to compete with CLIP and CLIP-derived peptides to HLA-DPGlu69 soluble molecule. The CLIP-derived low-affinity peptide CLIP-AA, could not outcompete beryllium; while the CLIP-derived high-affinity peptides CLIP-YY, CLIP-QY and CLIP-RF were only marginally influenced by the presence of beryllium in the competition assay. The effect of these CLIP-derived high-affinity peptides on beryllium presentation was determined by measuring interferon-gamma (IFN-gamma) release upon beryllium stimulation of peripheral blood mononuclear cells obtained from beryllium-hypersensitive subjects. CLIP-YY did inhibit beryllium presentation and T-cell activation, while CLIP-QY and CLIP-RF markedly enhanced the IFN-gamma response to beryllium. Anti-HLA-DP monoclonal antibody blocked the beryllium-induced IFN-gamma release in the presence of CLIP-QY (88%) and CLIP-RF (76%). A similar effect was observed for CLIP-YY capability to block IFN-gamma release by beryllium stimulation in the presence of CLIP-QY (79%) and CLIP-RF (76%). Overall, these data support the proposal that HLA-DP high-affinity peptides might be used as a model for specific berylliosis therapy.

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.

Increased levels of nerve growth factor in the airways of patients with sarcoidosis.

OBJECTIVES: Nerve growth factor (NGF) is a potent neuronal growth factor with inflammatory properties that recently has been proposed to be of importance in airway pathology. A role for NGF in the inflammatory granulomatous lung disease sarcoidosis is not well elucidated. The aims of this study were to investigate the secreted levels of NGF in bronchoalveolar lavage fluid (BALF) from sarcoidosis patients compared with patients with resolved disease, patients with another granulomatous disease--chronic beryllium disease (CBD)--and healthy subjects and also to investigate the relationship between NGF levels and markers of inflammation. METHODS AND RESULTS: NGF levels in BALF from 56 patients with active sarcoidosis (22 with Löfgren's syndrome), nine subjects with resolved sarcoidosis, six patients with CBD, and 31 healthy subjects were compared. A 10-fold elevation of NGF levels was found in patients with active sarcoidosis compared with subjects with clinically resolved sarcoidosis, patients with CBD and healthy subjects. In sarcoidosis patients, positive correlations between concentrations of NGF and lymphocytes, eosinophils and interferon-gamma, interleukin (IL)-4, IL-10, IL-12 were found. CONCLUSIONS: We demonstrate that secreted levels of NGF are markedly enhanced in the airways in active pulmonary sarcoidosis. Furthermore, a relationship between NGF and pulmonary inflammation in sarcoidosis is supported.

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-DPbeta1 gene have previously been found to be associated with CBD. To date, 43 HLA-DPbeta1 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-DPbeta1 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.

A quantitative proteomic analysis of soluble bronchoalveolar fluid proteins from patients with sarcoidosis and chronic beryllium disease.

BACKGROUND: Sarcoidosis and chronic beryllium disease (CBD) are granulomatous disorders which can lead to development of chronic inflammation and fibrosis. These diseases have several similarities from their clinical aspects. The aim of this study was to compare the protein profile at the site of active inflammation i.e. the lungs of patients with sarcoidosis and CBD. METHODS: Bronchoalveolar lavage (BAL) proteins from patients having sarcoidosis or CBD were studied using two dimensional gel based proteomics. In this study, we used Difference Gel Electrophoresis (DIGE) proteomics approach to analyse the protein expression profiles from sarcoidosis patients (n=4), CBD (n=4) and healthy controls (n = 5). Subsequently, differentially expressed proteins were identified by using mass spectrometry. RESULTS: We found 37 protein-spots with statistically different expression levels, and identified 14 of these proteins. The protein expression levels of peroxiredoxin 5, heat shock protein 70, complement C3, annexin A2 and transthyretin were significantly different in sarcoidosis versus control group. The proteins; hemopexin, beta2-microglobulin, alpha-1 antitrypsin, cystatin B, IgG kappa chain, apolipoprotein A1, and albumin were significantly different between CBD versus control group. When comparing CBD versus sarcoidosis, we found superoxide dismutase and hemoglobin upregulated in the CBD group. CONCLUSION: By using quantitative proteomics, we were able to find proteins with different expression levels in both diseases.

[Toxicologic characteristics of ammonium fluoroberyllate and its distribution in body after various intake methods].

The authors present materials on toxic influence and distribution of ammonium fluoroberyllate after various intake ways in white rats, on decontamination of skin wound surface.

Beryllium in exhaled breath condensate as a biomarker of occupational exposure in a primary aluminum production plant.

OBJECTIVE: Low beryllium exposure can induce pulmonary granulomatosis, so called berylliosis. For occupational health monitoring, it is more relevant to assess the internal dose of Be received by the lungs than urinary or atmospheric Be. Exhaled breath condensate (EBC) is a matrix collected non-invasively that derives from the airway lining fluid. EBC beryllium (Be) levels were evaluated as a marker of occupational exposure in a primary aluminum production plant. METHODS: We collected urine and EBC from controls and workers recently exposed to beryllium in the pot room and the anode repair sectors, and calculated a cumulative beryllium exposure index (CBEI) summing the number of years of employment in each task and multiplying by the estimated average beryllium exposure for the task. Concentrations of beryllium and aluminum were measured in EBC (Be-EBC and Al-EBC) and in urine (Be-U and Al-U) by ICP-MS. RESULTS AND CONCLUSION: We have shown that it was possible to measure Be and Al in workers' EBC. Compared with controls and after adjustment for smoking status, levels of Be-EBC and Al-EBC were higher in pot room workers and exposed subjects, respectively. Due to its relationship with CBEI, but not with Be-U, it appears that Be-EBC could be a promising marker of occupational exposure and provide additional toxicokinetic information in occupational health studies.

Beryllium increases the CD14(dim)CD16+ subset in the lung of chronic beryllium disease.

CD14dimCD16+ and CD14brightCD16+ cells, which compose a minor population of monocytes in human peripheral blood mononuclear cells (PBMC), have been implicated in several inflammatory diseases. The aim of this study was to investigate whether this phenotype was present as a subset of lung infiltrative alveolar macrophages (AMs) in the granulomatous lung disease, chronic beryllium disease (CBD). The monocytes subsets was determined from PBMC cells and bronchoalveolar lavage (BAL) cells from CBD, beryllium sensitized Non-smoker (BeS-NS) and healthy subjects (HS) using flow cytometry. The impact of smoking on the AMs cell phenotype was determined by using BAL cells from BeS smokers (BeS-S). In comparison with the other monocyte subpopulations, CD14dimCD16+ cells were at decreased frequency in PBMCs of both BeS-NS and CBD and showed higher HLA-DR expression, compared to HS. The AMs from CBD and BeS-NS demonstrated a CD14dimCD16+phenotype, while CD14brightCD16+ cells were found at increased frequency in AMs of BeS, compared to HS. Fresh AMs from BeS-NS and CBD demonstrated significantly greater CD16, CD40, CD86 and HLA-DR than HS and BeS-S. The expression of CD16 on AMs from both CBD and BeS-NS was downregulated significantly after 10µM BeSO4 stimulation. The phagocytic activity of AMs decreased after 10µM BeSO4 treatment in both BeS-NS and CBD, although was altered or reduced in HS and BeS-S. These results suggest that Be increases the CD14dimCD16+ subsets in the lung of CBD subjects. We speculate that Be-stimulates the compartmentalization of a more mature CD16+ macrophage phenotype and that in turn these macrophages are a source of Th1 cytokines and chemokines that perpetuate the Be immune response in CBD. The protective effect of cigarette smoking in BeS-S may be due to the low expression of co-stimulatory markers on AMs from smokers as well as the decreased phagocytic function.

Beryllium induces apoptosis in human lung macrophages.

The link between metal-induced apoptosis and granulomatous inflammation in human disease pathogenesis is not established. The presence of TUNEL positive nuclei in chronic beryllium disease (CBD) pulmonary granulomas suggested the possibility that beryllium (Be) could induce apoptosis in adherent macrophages from CBD bronchoalveolar (BAL) cells. Apoptosis was measured in un-stimulated and Be-stimulated BAL adherent macrophages from CBD (n = 21) and Be-sensitized (BeS, n = 16) subjects. Be-stimulated CBD and BeS macrophages underwent caspase-dependent nuclear fragmentation, cytoplasmic membrane blebbing and CD14 loss. Be-stimulated adherent macrophage apoptosis was not due to TNF-alpha production. Apoptosis, CD14 loss and TNF-alpha production were not observed in unstimulated BAL macrophages. Thus, Be-stimulated BAL adherent macrophage apoptosis occurred whether cells were derived from patients with granulomatous inflammation or not, was caspase-mediated and occurred independent of TNF-alpha levels. We conclude that Be-induced human lung adherent macrophage apoptosis could contribute to the host's continued re-exposure to Be resulting in chronic granulomatous inflammation.

Role of the HLA-DP Glu 69 and the TNF-alpha TNF-alpha 2 gene markers in susceptibility to beryllium hypersensitivity.

Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of beryllium dusts, characterized by the accumulation of CD4+ T cells and macrophages in the lower respiratory tract. Beryllium presentation to CD4+ T cells from patients with berylliosis results in T cell activation and these Be-specific CD4+ T cells undergo clonal proliferation and Th1-type cytokine production such as interleukin-2, interferon-gamma and tumor necrosis factor-alpha. In exposed workers, genetic susceptibility to this granulomatous disorder is associated with major histocompatibility gene and the TNF-alpha gene. The HLA-DP glutamic 69 residue was shown to be the MHC genetic marker associated with disease susceptibility; furthermore the TNF-alpha TNFA-308*2 allele was found to be independently associated with HLA-DP Glu69 in the determination of berylliosis risk.

Role of Liv-52 in protection against beryllium intoxication.

An ayurvedic medicine, Liv-52, was studied as a prophylactic agent against beryllium-induced toxicity in rats. Administration of beryllium per se caused severe degenerative and necrotic changes in kidneys, liver, and uterus. Beryllium exposure also reduced glycogen content, activities of alkaline phosphatase, succinate-dehydrogenase, and adenosine-triphosphatase in these organs. On the contrary, activities of acid phosphatase and glucose-6-phosphatase showed marginal increase. Liv-52-primed rats exhibited comparatively less marked toxic effects.

[Chronic berylliosis of the lungs]. / Khronicheskii berillioz legkikh.

An autopsy case of the chronic lung berylliosis in a patient having an occupational contact with a beryllium bronze is described. Clinically the disease had no specific features and was diagnosed as chronic obstructive bronchitis and pneumosclerosis. Sclerotic granulomas with giant cells, conchoidal bodies, iron deposition in the pulmonary stroma--all these lung alterations allowed one to establish a diagnosis of lung berylliosis.