Recombinant HLA-DP2 binds beryllium and tolerizes beryllium-specific pathogenic CD4+ T cells.
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Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the alpha-1 and beta-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4 -pulsed, plate-bound rHLA-DP2 molecules induced IFN-gamma secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag. (+info)
Beryllium-induced TNF-alpha production is transcription-dependent in chronic beryllium disease.
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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-alpha secretion. We tested the hypothesis that Be-induced, CBD bronchoalveolar lavage (BAL) T cell, transcription-dependent, TNF-alpha 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-alpha (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-alpha versus 55 pg/ml (range, 0-454) by unstimulated cells. Using real-time RT-PCR, Be-stimulated TNF-alpha production at 6 h was preceded by a 5-fold increase in TNF-alpha pre-mRNA copy number:beta-actin copy number (Be median ratio 0.21; unstimulated median ratio 0.04). The median ratio of mature TNF-alpha mRNA:beta-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-alpha 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-alpha mRNA levels 50% while 2AP decreased levels > 80%, relative to Be exposure alone. Beryllium exposure specifically upregulated transcription factors AP-1 and NF-kappaB. The data suggest that Be exposure induces transcription-dependent TNF-alpha production, potentially due to upregulation of specific transcription factors. (+info)
Antagonistic effects of cofilin, beryllium fluoride complex, and phalloidin on subdomain 2 and nucleotide-binding cleft in F-actin.
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Cofilin/ADF, beryllium fluoride complex (BeFx), and phalloidin have opposing effects on actin filament structure and dynamics. Cofilin/ADF decreases the stability of F-actin by enhancing disorder in subdomain 2, and by severing and accelerating the depolymerization of the filament. BeFx and phalloidin stabilize the subdomain 2 structure and decrease the critical concentration of actin, slowing the dissociation of monomers. Yeast cofilin, unlike some other members of the cofilin/ADF family, binds to F-actin in the presence of BeFx; however, the rate of its binding is strongly inhibited by BeFx and decreases with increasing pH. The inhibition of the cofilin binding rate increases with the time of BeFx incubation with F-actin, indicating the existence of two BeFx-F-actin complexes. Cofilin dissociates BeFx from the filament, while BeFx does not bind to F-actin saturated with cofilin, presumably because of the cofilin-induced changes in the nucleotide-binding cleft of F-actin. These changes are apparent from the increase in the fluorescence intensity of F-actin bound epsilon-ADP upon cofilin binding and a decrease in its accessibility to collisional quenchers. BeFx also affects the nucleotide-binding cleft of F-actin, as indicated by an increase in the fluorescence intensity of epsilon-ADP-F-actin. Phalloidin and cofilin inhibit, but do not exclude each other binding to their complexes with F-actin. Phalloidin promotes the dissociation of cofilin from F-actin and slowly reverses the cofilin-induced disorder in the DNase I binding loop of subdomain 2. (+info)
Enhanced preventive programme at a beryllium oxide ceramics facility reduces beryllium sensitisation among new workers.
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BACKGROUND: A 1998 survey at a beryllium oxide ceramics manufacturing facility found that 10% of workers hired in the previous 6 years had beryllium sensitisation as determined by the beryllium lymphocyte proliferation test (BeLPT). In response, the facility implemented an enhanced preventive programme to reduce sensitisation, including increased respiratory and dermal protection and particle migration control. AIM: To assess the programme's effectiveness in preventing sensitisation. METHODS: In 2000, the facility began testing newly hired workers for beryllium sensitisation with the BeLPT at time of hire and during employment. The sensitisation rate and prevalence for workers hired from 2000 to 2004 were compared with that for workers hired from 1993 to 1998, who were tested in the 1998 survey. Facility environmental conditions for both time periods were evaluated. RESULTS: Newly hired workers in both cohorts worked for a mean of 16 months. Of the 97 workers hired from 2000 to 2004 with at least one employment BeLPT result, four had abnormal results at time of hire and one became sensitised during employment. Of the 69 workers hired from 1993 to 1998 and tested in 1998, six were found to be sensitised. The sensitisation rate for the 2000-4 workers was 0.7-2.7/1000 person-months of employment, and that for the 1993-8 workers was 5.6/1000 person-months, at least 2.1 (95% confidence interval (CI) 0.6 to 8.4) and up to 8.2 (95% CI 1.2 to 188.8) times higher than that for the 2000-4 workers. The sensitisation prevalence for the 2000-4 workers was 1% and that for the 1993-8 workers was 8.7%, 8.4 (95% CI 1.04 to 68.49) times higher than that for the 2000-4 workers. Airborne beryllium levels for production workers for the two time periods were similar. CONCLUSIONS: A comprehensive preventive programme reduced beryllium sensitisation in new workers during the first years of employment, despite airborne beryllium levels for production workers that were similar to pre-programme levels. (+info)
Comparative analysis between CFSE flow cytometric and tritiated thymidine incorporation tests for beryllium sensitivity.
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BACKGROUND: In this study, we evaluated alternative possibility for CFSE beryllium flow cytometric test against beryllium blood lymphocyte proliferation test (BeLPT) as a standard radioactive clinical screening method to identify sensitization to beryllium. METHODS: Delta PD (the ratio of divided cell population to the total number of cells with subtracted counts of unstimulated cells) of specific beryllium-induced pathogenic CD3+ CD4+ T-lymphocytes and stimulation index (SI) in CFSE proliferation test was compared with delta counts per minute (mean test CPM minus mean control CPM) and SI in radioactive blood BeLPT. RESULTS: Comparison analysis of CFSE and BeLPT demonstrated excellent agreement between delta PD and delta CPM (kappa = 0.845, P << 0.0001). We determined 6.8% positive subjects in the beryllium-exposed, Be-LPT-negative group. The decreased mean difference of these indexes to percentage of average and the long tail in the plot reflects increased sensitivity. CFSE/CD4+ T-cell proliferation assay has 100% specificity, significantly higher sensitivity and efficiency than BeLPT. CONCLUSIONS: Both delta PD, measured by the precursor frequencies method in CFSE assay and delta CPM, defined by tritiated thymidine in BeLPT, can be used for the enumeration of beryllium specific CD4+ T-cell proliferation and may substantially improve the quality of the early diagnosis of beryllium hypersensitivity. (+info)
Beryllium induces premature senescence in human fibroblasts.
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After cells have completed a sufficient number of cell divisions, they exit the cell cycle and enter replicative senescence. Here, we report that beryllium causes proliferation arrest with premature expression of the principal markers of senescence. After young presenescent human fibroblasts were treated with 3 microM BeSO(4) for 24 h, p21 cyclin-dependent kinase inhibitor mRNA increased by >200%. Longer periods of exposure caused mRNA and protein levels to increase for both p21 and p16(Ink4a), a senescence regulator that prevents pRb-mediated cell cycle progression. BeSO(4) also caused dose-dependent induction of senescence-associated beta-galactosidase activity (SA-beta-gal). Untreated cells had 48 relative fluorescence units (RFU)/microg/h of SA-beta-gal, whereas 3 microM BeSO(4) caused activity to increase to 84 RFU/microg/h. In chromatin immunoprecipitation experiments, BeSO(4) caused p53 protein to associate with its DNA binding site in the promoter region of the p21 gene, indicating that p53 transcriptional activity is responsible for the large increase in p21 mRNA elicited by beryllium. Forced expression of human telomerase reverse transcriptase (hTERT) rendered HFL-1 cells incapable of normal replicative senescence. However, there was no difference in the responsiveness of normal HFL-1 fibroblasts (IC(50) = 1.9 microM) and hTERT-immortalized cells (IC(50) = 1.7 microM) to BeSO(4) in a 9-day proliferation assay. The effects of beryllium resemble those of histone deacetylase-inhibiting drugs, which also cause large increases in p21. However, beryllium produced no changes in histone acetylation, suggesting that Be(2+) acts as a novel and potent pharmacological inducer of premature senescence. (+info)
Crystal structures of the receiver domain of the response regulator PhoP from Escherichia coli in the absence and presence of the phosphoryl analog beryllofluoride.
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The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg(2+). Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation. (+info)
Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization.
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Diguanylate cyclases (DGCs) are key enzymes of second messenger signaling in bacteria. Their activity is responsible for the condensation of two GTP molecules into the signaling compound cyclic di-GMP. Despite their importance and abundance in bacteria, catalytic and regulatory mechanisms of this class of enzymes are poorly understood. In particular, it is not clear if oligomerization is required for catalysis and if it represents a level for activity control. To address this question we perform in vitro and in vivo analysis of the Caulobacter crescentus diguanylate cyclase PleD. PleD is a member of the response regulator family with two N-terminal receiver domains and a C-terminal diguanylate cyclase output domain. PleD is activated by phosphorylation but the structural changes inflicted upon activation of PleD are unknown. We show that PleD can be specifically activated by beryllium fluoride in vitro, resulting in dimerization and c-di-GMP synthesis. Cross-linking and fractionation experiments demonstrated that the DGC activity of PleD is contained entirely within the dimer fraction, confirming that the dimer represents the enzymatically active state of PleD. In contrast to the catalytic activity, allosteric feedback regulation of PleD is not affected by the activation status of the protein, indicating that activation by dimerization and product inhibition represent independent layers of DGC control. Finally, we present evidence that dimerization also serves to sequester activated PleD to the differentiating Caulobacter cell pole, implicating protein oligomerization in spatial control and providing a molecular explanation for the coupling of PleD activation and subcellular localization. (+info)