[Inter-laboratory comparison analysis of noise measurement in 91 occupational hygiene technical service organizations].

Objective: To understand the comparability of noise measurement results of various occupational hygiene technical service organizations in Guangdong Province by conducting inter-laboratory comparison of measuring instruments and personnel operation. Methods: In October 2020, the instrument comparison and personnel comparison among 91 occupational hygiene technical service organizations engaged in noise measurement in Guangdong Province were carried out in the form of fixed-point measurement and simulated workplace measurement, and the results were analyzed and evaluated by using the robust z-ratio score. Results: In the instrument comparison, 6 organizations had 1 or 2 outliers in their z-ratio scores, 2 organizations had 2 problematic values in their z-ratio scores, and a total of 8 organizations (accounting for 8.8%) were judged as unqualified; A total of 83 organizations (accounting for 91.2%) with satisfactory z-ratio scores or only one problematic value were judged as qualified. In the personnel comparison, there were 11 organizations with 1 or 2 outliers in the z-ratio score, and 1 organization with 2 problematic values in the z-ratio score. A total of 12 organizations (13.2%) were judged as unqualified and 79 organizations (accounting for 86.8%) with satisfactory z-ratio scores or only one problematic value were judged as qualified. Through comprehensive judgment, 20 organizations (22.0%) were judged as unqualified, and 71 organizations (78.0%) were judged as qualified. There was no statistically significant difference in the qualified rates of instrument comparison results, personnel comparison results and comprehensive evaluation results of non-private organizations and private organizations (P>0.05). There was no significant difference in the qualified rates of instrument comparison results and comprehensive evaluation results of qualified organizations and unqualified organizations (P>0.05), there was significant difference in the qualified rate of personnel comparison results (P<0.05) . Conclusion: The noise measurement results of some occupational health technical service organizations in Guangdong Province are generally comparable. To carry out inter-laboratory comparison of noise instrument performance and personnel operation ability of occupational hygiene technical service organizations, can comprehensively evaluate the testing process of each organization and find out the problems existing in each organization.

Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation.

Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1ß production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP(L) is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1ß. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1ß production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1ß expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-α was not affected by downregulation in c-FLIP. c-FLIP(L) interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly and NLRP3 mitochondrial localization, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1ß generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIP(L) in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes.

Ezrin is a negative regulator of death receptor-induced apoptosis.

Ezrin links cortical actin filaments with the cell membrane, and has a critical role in many membrane-initiated events. Fas is directly associated with ezrin, but conflicting results have been reported for the involvement of ezrin in Fas-induced cell death. In this study we show that ezrin was associated with Fas in T cells before stimulation and was released shortly after Fas ligand (FasL) engagement. The knockdown of ezrin moderately increased Fas-triggered or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-triggered cell death in normal T lymphocytes and in H9 cells, but had no effect on death receptor-induced apoptosis in type II cells, such as Jurkat and CEM. Expression of a dominant-negative form of ezrin also led to an increased Fas-induced apoptosis in H9 cells. Ezrin deficiency did not affect the internalization of Fas after Fas ligation. Instead, an enhanced formation of death-inducing signaling complex (DISC) was observed in H9 cells with ezrin knockdown, leading to accelerated caspase-8 activation. Together, our results suggest that ezrin has a negative role in the recruitment of Fas into signaling complexes in type I T cells. Loss of ezrin likely removes the constraint imposed by ezrin and facilitates the assembly of death receptor complex in T cells.

Impaired TNFalpha-induced A20 expression in E1A/Ras-transformed cells.

BACKGROUND: Tumour necrosis factor (TNF) is capable of activating the cell death pathway, and has been implicated in killing transformed cells. However, TNF also activates survival signals, including NF-kappaB activation and the subsequent expression of anti-apoptotic genes, leading to protection against TNF toxicity. METHODS: In this study, we show that, although untransformed mouse embryonic fibroblasts (MEFs) were resistant to TNF killing, E1A/Ras-transformed MEFs were susceptible to extensive apoptosis induced by TNF. The key factors for determining TNF sensitivity were explored by comparing wild-type and E1A/Ras-transformed MEFs. RESULTS: TNF signalling to NF-kappaB and to its target genes such as IkappaBalpha seemed to be mostly intact in E1A/Ras-transformed cells. Instead, the induction of A20 was completely abolished in E1A/Ras-transformed MEFs, although A20 is known to be NF-kappaB dependent. Reintroduction of A20 into E1A/Ras-transformed MEFs rescued these cells from TNF-induced death and reduced the formation of the FADD/caspase-8 complex. This impaired A20 induction in E1A/Ras MEFs was not because of the stabilisation of p53 or a defective TNF-induced p38 and Jun N-terminal kinase (JNK) signalling. Consistently, we found a reduced A20 promoter activity but normal NF-kappaB activity in TNF-treated E1A/Ras MEFs. However, Bcl-3 seemed to have a role in the transactivation of the A20 promoter in E1A/Ras cells. CONCLUSIONS: Our results suggest that specific inhibition of certain survival factors, such as A20, may determine the sensitivity to TNF-induced apoptosis in transformed cells such as E1A/Ras MEFs.

Selective activation of NFAT by promyelocytic leukemia protein.

Promyelocytic leukemia (PML) protein is a tumor suppressor with complicated action mechanisms not yet fully understood. In this study, we found that the nuclear factor of activated T cell (NFAT) is an unexpected partner of PML: PML specifically enhanced the transcription activation of NFAT. In PML-null mouse embryonic fibroblasts, no transcription activity of NFAT could be detected. There was a selective requirement of PML isoform in NFAT activation: PML-I and PML-VI, but not PML-IV, increased NFAT transactivation. PML specifically promoted the expression of many, but not all, NFAT-targeted genes. We found a specific binding of PML to NFATc. The interaction of PML with NFATc in vivo was further confirmed by chromatin immunoprecipitation and DNA affinity precipitation assay analysis. The unexpected coupling of PML with NFAT reveals a novel mechanism underlying the diverse physiological functions of PML.

c-FLICE inhibitory protein expression inhibits T-cell activation.

Cellular FLICE-inhibitory protein (c-FLIP) inhibits death receptor-mediated apoptosis by specific interaction with FADD and procaspase-8, and may thus interfere with activation events mediated by FADD and caspase-8. Recent studies, however, suggest that c-FLIP also transmits activation signals. The role of c-FLIP on T-cell activation was examined here using several transgenic mice with variable c-FLIP expression. In all c-FLIP-transgenic mice, Fas-mediated apoptosis and in vitro activation-induced T-cell death were suppressed, and T-cell proliferation and IL-2 production were inhibited. c-FLIP transgene also promoted in vivo thymocyte death. Higher c-FLIP transgene expression was correlated with a more profound suppression of T-cell activation and a prominent disturbance in mature thymocyte development. There was no evidence of increased activation and proliferation in all c-FLIP-transgenic T cells examined. Instead, suppression of T-cell activation in c-FLIP-transgenic T cells could be a combinatory effect of FADD/caspase-8-dependent signals and c-FLIP-specific activities.

CREB is one component of the binding complex of the Ces-2/E2A-HLF binding element and is an integral part of the interleukin-3 survival signal.

The Ces-2/E2A-HLF binding element (CBE) is recognized by Caenorhabditis elegans death specification gene product Ces-2 and human acute lymphocytic leukemia oncoprotein E2A-HLF. In an attempt to identify a cellular CBE-binding protein(s) that may be involved in apoptosis regulation in mammals, multiple nuclear binding complexes of CBE were identified in various mammalian cell lines and tissues by electrophoretic mobility shift assay. Cyclic AMP (cAMP)-responsive element (CRE)-binding protein (CREB) was present in one major CBE complex of Ba/F3 and TF-1 cells, and both in vitro-translated and Escherichia coli-synthesized CREB bound to CBE. Activation of CREB by cAMP-elevating chemicals or the catalytic subunit of protein kinase A (PKAc) resulted in induction of the CBE-driven reporter gene. Stimulation of Ba/F3 cells with interleukin-3 (IL-3) promptly induced phosphorylation of CREB at serine(133) partially via a PKA-dependent pathway. Consistently, Ba/F3 cell survival in the absence of IL-3 was prolonged by activation of PKA. Conversely, treatment of cells with a PKA inhibitor or expression of the dominant negative forms of the regulatory subunit type I of PKA and CREB overrode the survival activity of IL-3. Last, the bcl-2 gene was demonstrated to be one candidate cellular target of the CREB-containing CBE complex, as mutations in the CRE and CBE sites significantly reduced the IL-3 inducibility of the bcl-2 promoter. Together, our results suggest that CREB is one cellular counterpart of Ces-2/E2A-HLF and is part of IL-3 dependent apoptosis regulation in hematopoietic cells.

c-Jun NH2-terminal kinase activation leads to a FADD-dependent but Fas ligand-independent cell death in Jurkat T cells.

Persistent c-Jun NH2-terminal kinase (JNK) activation induces cell death. Different mechanisms are ascribed to JNK-induced cell death. Most of the JNK-apoptosis studies employ stress stimuli known to activate kinases other than JNK. Here we used overexpression of mitogen-activated protein kinase kinase 7 (MKK7) to activate selectively JNK in T lymphoma Jurkat cells. Similar to that reported previously, Fas ligand (FasL) expression was up-regulated by JNK activation. Dominant negative-FADD and caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu-Thr-Asp effectively inhibited MKK7-induced cell death, supporting a major involvement of FADD cascade. However, MKK7-induced cell death was not prevented by antagonist antibody ZB4 and Fas-Fc, indicating that Fas-FasL interaction is minimally involved. Confocal microscopy revealed that persistent JNK activation led to clustering of Fas. Our results suggest that, in contrast to that reported previously, JNK alone-induced death in Jurkat cells is FADD-dependent but is not triggered by Fas-FasL interaction.

Multiple signals required for cyclic AMP-responsive element binding protein (CREB) binding protein interaction induced by CD3/CD28 costimulation.

The optimal activation of cAMP-responsive element binding protein (CREB), similar to the full activation of T lymphocytes, requires the stimulation of both CD3 and CD28. Using a reporter system to detect interaction of CREB and CREB-binding protein (CBP), in this study we found that CREB binds to CBP only by engagement of both CD3 and CD28. CD3/CD28-promoted CREB-CBP interaction was dependent on p38 mitogen-activated protein kinase (MAPK) and calcium/calmodulin-dependent protein kinase (CaMK) IV in addition to the previously identified extracellular signal-regulated kinase pathway. Extracellular signal-regulated kinase, CaMKIV, and p38 MAPK were also the kinases involved in CREB Ser(133) phosphorylation induced by CD3/CD28. A reconstitution experiment illustrated that optimum CREB-CBP interaction and CREB trans-activation were attained when these three kinase pathways were simultaneously activated in T cells. Our results demonstrate that coordinated activation of different kinases leads to full activation of CREB. Notably, CD28 ligation activated p38 MAPK and CaMKIV, the kinases stimulated by CD3 engagement, suggesting that CD28 acts by increasing the activation extent of p38 MAPK and CaMKIV. These results support the model of a minimum activation threshold for CREB-CBP interaction that can be reached only when both CD3 and CD28 are stimulated.

NF-kappa B-dependent Fas ligand expression.

Apoptosis of lymphocytes is triggered by different stimuli through the induced expression of Fas and Fas ligand (FasL). Using T cell activation-induced Fas/FasL expression as a model system, we observed a differential regulation of the induction of Fas and FasL. cAMP inhibited activation-induced apoptosis by an effective suppression of TCR-coupled FasL expres sion. In contrast, cAMP weakly interfered with activation-induced Fas expression, and the remaining Fas molecules on cAMP-treated T cells still mediated apoptosis. Among the major transcription elements on the FasL promoter, the activation of NF-kappaB, but not of NF-AT and AP-1, was suppressed by cAMP. The prominent role of NF-kappaB was further demonstrated by a better activation of the FasL promoter and an elevated expression of FasL induced by p65 (RelA) overexpression than those induced by AP-1 or NF-AT. Our results demonstrate the essential role of NF-kappaB for the expression of the death receptor ligand FasL, and suggest a direct link between NF-kappaB activation and the expression of FasL. NF-kappaB may be the common mediator in the induction of FasL through TCR activation and by various stress stimuli.

p38 mitogen-activated protein kinase is involved in Fas ligand expression.

p38 mitogen-activated protein kinase (MAPK) is activated by T cell receptor engagement. Here we showed that T cell receptor activated p38alpha but not p38delta. Inhibition of p38alpha by the specific inhibitor SB 203580 prevented activation-induced cell death in T cells. SB 203580 had no effect on Fas-initiated apoptosis. Instead, SB 203580 preferentially inhibited activation-induced Fas ligand (FasL) expression. The inhibition on FasL expression by SB 203580 was correlated with the suppression on the FasL promoter activation. Overexpression of active MAPK kinase 3b, the activator of p38 MAPK, led to activation of FasL promoter and induction of FasL transcripts in T cells. Stress stimulation of T cells by anisomycin also induced FasL expression in a p38 MAPK-dependent manner. The induction of FasL expression in nonlymphoid cells such as 293T also required activation of p38 MAPK. Our results suggest that p38 MAPK is essential for FasL expression.

Antagonism of p53-dependent apoptosis by mitogen signals.

p53-mediated apoptosis is antagonized by growth factor stimulation. Here, we show that p53-dependent cell death induced by DNA damage was effectively prevented by mitogen activation. The levels of Bcl-2, Bcl-xL, and Bax were not altered by cisplatin treatment and mitogen rescue. Instead, the protection against p53-regulated apoptosis was mediated by at least three distinct signaling pathways. Either phosphatidylinositol (PI) 3-kinase or mitogen-activated protein kinase kinase (MEK) antagonized p53-induced apoptosis, and an additive preventive effect was observed when both kinases were activated. However, the combination of PI 3-kinase and MEK was not sufficient to completely prevent apoptosis induced by DNA damage. Mitogen activation further suppressed cisplatin-induced p53 expression, and the inhibition was mainly dependent on the Ca2+ pathway. Our results demonstrate that effective antagonism of p53-dependent apoptosis by mitogenic activation requires the presence of multiple signal pathways, including PI 3-kinase, MEK, and Ca2+.

Mitogen-activated protein kinase kinase antagonized fas-associated death domain protein-mediated apoptosis by induced FLICE-inhibitory protein expression.

Fas and Fas-associated death domain (FADD) play a critical role in the homeostasis of different cell types. The regulation of Fas and FADD-mediated cell death is pivotal to many physiological functions. The activation of T lymphocytes by concanavalin A (Con A) inhibited Fas-mediated cell death. We identified that among the several activation signals downstream of Con A stimulation, mitogen-activated protein (MAP) kinase kinase (MKK) was the major kinase pathway that antagonized Fas-triggered cell death. MKK1 suppressed FADD- but not caspase-3- induced apoptosis, indicating that antagonism occurred early along the Fas-initiated apoptotic cascade. We further demonstrated that activation of MKK1 led to expression of FLIP, a specific inhibitor of FADD. MKK1 inhibition of FADD-induced cell death was abrogated if induction of FLIP was prevented, indicating that FLIP mediates MKK1 suppression of FADD-mediated apoptosis. Our results illustrate a general mechanism by which activation of MAP kinase attenuates apoptotic signals initiated by death receptors in normal and transformed cells.

Atypical signaling defects prevent IL-2 gene expression in lpr/lpr CD4-CD8- cells.

T cells with CD4-CD8- (double negative, DN) phenotype in MRL-lpr/lpr mouse serve as a model to establish the correlation between the extremely low IL-2 gene expression and the specific signaling inactivation. The extent of nonresponsiveness in lpr DN cells was distinctive in several unusual defects. First, the poor IL-2 production in lpr DN cells could not be restored by supplement of signals known to augment IL-2 response in normal T cells. Second, the activations of both mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK) were attenuated in lpr DN cells upon direct activation by TPA/A23187. Third, IL-2 mRNA was degraded much faster in lpr DN cells than that in normal T cells. Fourth, of the four major transcriptional elements on IL-2 promoter, only AP-1 and nuclear factor of activated T cells (NFAT)-binding activities were suppressed in lpr DN T cells. Altogether, these results suggest that an extremely low level of IL-2 production in lpr DN T cells was due to both the increased instability of mRNA and the reduced activation of IL-2 gene promoter, the latter defect could be attributed to the inactivation of AP-1 and NF-AT as well as the poor activation of the upstream MAP kinase and JNK.

Apoptotic signal of Fas is not mediated by ceramide.

Ceramide has been suggested as the secondary messenger mediating the apoptotic signal for Fas engagement. By using different inhibitors, we demonstrated here that ceramide is unlikely a mediator of Fas-initiated apoptosis. First, cAMP prevented cell death induced by ceramide but not by Fas. Second, ceramide-triggered, but not Fas-triggered, apoptosis was antagonized by the free radical scavenger C60. Third, the metal chelator pyrrolidinedithiocarbamate suppressed ceramide-initiated DNA fragmentation but had no effect on the Fas-induced cell death. Fourth, the SAPK/ERK kinase dominant negative mutant, which attenuated ceramide-induced cell death, did not prevent Fas-induced apoptosis. Finally, activation of NF-kappaB inhibited ceramide-induced but not Fas-initiated apoptosis. The fact that many antagonists of ceramide-induced apoptosis could not suppress Fas-mediated cell death clearly indicates that ceramide is not the mediator for Fas-initiated apoptotic signal.

Nitric oxide increased interleukin-4 expression in T lymphocytes.

Nitric oxide (NO) is a regulator of many biological functions including T helper 1 (Th1)/T helper 2 cells balance. It has been demonstrated that NO inhibits the secretion of interleukin-2 (IL-2) and interferon-gamma on Th1 cells. Here we showed that, in addition to the suppression of IL-2 production, NO-generating agents sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) increased the secretion of IL-4 both in Th2 clones and EL4 T cells. The additive effect was dependent on the dose of SNP and SNAP. Augmentation of IL-4 production was detected with 1 microM SNP, and up to threefold increase in IL-4 secretion could be observed with higher concentrations of SNP/SNAP. NO also weakly increased the activation of IL-4 promoter. In contrast, NO markedly inhibited the induction of IL-2 promoter, which could account for most of the reduction in IL-2 production. Analysis of the transcriptional elements on IL-2 and IL-4 promoters revealed a selective inactivation of NF-kappa B and NF-AT. It is suggested that despite the complex feedback network regulating NO production, the enhanced IL-4 expression would lead to the expansion of Th2 cells once NO is generated.

Overexpression of mitogen-activated protein kinase kinase kinase reversed cAMP inhibition of NF-kappaB in T cells.

cAMP inhibits T cell activation by acting as an antagonist for selective kinases and transcriptional factors. We have recently demonstrated that cAMP inhibited c-Jun N-terminal kinase (JNK) but left the mitogen-activated protein (MAP) kinase cascade almost unaffected in T lymphocytes. In accordance with recent reports, we also observed a selective suppression of nuclear factor NF-kappaB activation by cAMP. The possible link between the JNK cascade and NF-kappaB activation was demonstrated by the fact that the active form of MAP kinase kinase kinase (deltaMEKK), a constitutive activator of JNK, induced NF-kappaB but not AP-1, Oct, and NF-AT in T cells. In contrast, the induction of MAP kinase kinase (MEK)-MAP kinase did not stimulate NF-kappaB activity. The specific activation of NF-kappaB by a single MEKK-JNK cascade was thus unusual, given that the activation of other transcriptional elements in T cells requires at least two signal pathways. This was further confirmed by the fact that cAMP inhibition of NF-kappaB activation was reversed by overexpression of deltaMEKK.

CD28-costimulation activates cyclic AMP-responsive element-binding protein in T lymphocytes.

Cyclic AMP-responsive element binding protein (CREB) mediates gene expression in response to cAMP stimulation. The transcriptional activity of CREB depends on both the phosphorylation of Ser133 and the recruitment of cofactor for assembly of transcriptional complex. Extensive Ser133 phosphorylation of CREB was induced during T cell activation. This phosphorylation event is essential for IL-2 gene expression. However, phosphorylation of CREB at Ser133 was not sufficient for transcriptional activity by CREB. The presence of a second signal from CD28, a potent costimulatory molecule on T cells, stimulated CREB-mediated gene expression. CD28, an effective costimulator of T cell activation and IL-2 gene expression, is shown to induce CREB activation in the presence of anti-CD3 or O-tetradecanoylphorbol 13-acetate. These two signals together stimulated a CRE-dependent reporter gene, the proliferating cell nuclear Ag promoter, and transactivation by the GAL4-CREB fusion protein. Thus optimal induction of CREB, similar to the full activation of T lymphocytes, may be mediated by two distinct signal transductions. Using the specific kinase inhibitor, one of the two pathways appeared to involve mitogen-activated protein kinase kinase but not protein kinase C, protein kinase A, or p70 S6 kinase.

Different V beta usage in antigen-specific and alloreactive T cells specific for the same MHC elements.

V beta 8 has been shown to be used in the majority of antigen specific T cell hybridomas restricted by I-Ad and I-Ed. The usage of V beta 8 in these T cell responses in vivo was confirmed as V beta 8 depleted BALB/c mice responded weakly to these I-Ad- and I-Ed-restricted antigens. We used this deletion assay to further examine if V beta 8 is similarly dominantly used in alloreactive T cell specific for I-Ad/Ed. The depletion of V beta 8-population in allogenic mice did not affect the alloreactive responses toward I-Ad/Ed. Although specific for the same MHC, there is no apparent overlap on the use of TCR V beta 8 between alloreactive T cells and antigen-specific T cells.

Selective contact during TCR recognition.

Recent structural analysis of the peptide-MHC complex reveals that an antigenic peptide binds to MHC in only one conformation and that side chains anchoring in the binding pocket would not contact TCR. The identification of all the MHC-anchoring residues on an antigenic peptide is a prerequisite to understand how a given peptide interacts with the TCR. In a combination of binding analysis and model simulation, model peptide lambda repressor cl 16-26 was shown to bind to I-Ek through four anchor residues (Leu18, IIe21, Glu23 and Lys26), a pattern found in many I-Ek-binding peptides. TCR reactivity analysis clearly indicates a great variation in the interaction with cl 16-26 by T cells generated from different strains of I-Ek-bearing mice. Most of the T cell generated from A/J mice reacted with the central regions of cl 16-26, while there is a great diversity on the recognition of cl 16-26 by T cells from C3H and B10.BR mice. Despite the diverse interactions with antigenic peptide by these T cells, most TCR-E-k contacts are limited to the central region of the I-Ek beta-chain. T cells recognizing only the N-terminal part of cl 16-26 were found to contact I-Ek at nearly the same residues as T cells interacting with the C-terminal of cl 16-26. TCR-I-Ek recognition was apparently independent of TCR-cl 16-26 contact. The discordant TCR-peptide and TCR-MHC interaction may represent a unique feature of TCR recognition.