Threonyl-tRNA Synthetase Promotes T Helper Type 1 Cell Responses by Inducing Dendritic Cell Maturation and IL-12 Production via an NF-κB Pathway.

Threonyl-tRNA synthetase (TRS) is an aminoacyl-tRNA synthetase that catalyzes the aminoacylation of tRNA by transferring threonine. In addition to an essential role in translation, TRS was extracellularly detected in autoimmune diseases and also exhibited pro-angiogenetic activity. TRS is reported to be secreted into the extracellular space when vascular endothelial cells encounter tumor necrosis factor-α. As T helper (Th) type 1 response and IFN-γ levels are associated with autoimmunity and angiogenesis, in this study, we investigated the effects of TRS on dendritic cell (DC) activation and CD4 T cell polarization. TRS-treated DCs exhibited up-regulated expression of activation-related cell-surface molecules, including CD40, CD80, CD86, and MHC class II. Treatment of DCs with TRS resulted in a significant increase of IL-12 production. TRS triggered nuclear translocation of the NF-κB p65 subunit along with the degradation of IκB proteins and the phosphorylation of MAPKs in DCs. Additionally, MAPK inhibitors markedly recovered the degradation of IκB proteins and the increased IL-12 production in TRS-treated DCs, suggesting the involvement of MAPKs as the upstream regulators of NF-κB in TRS-induced DC maturation and activation. Importantly, TRS-stimulated DCs significantly increased the populations of IFN-γ+CD4 T cells, and the levels of IFN-γ when co-cultured with CD4+ T cells. The addition of a neutralizing anti-IL-12 mAb to the cell cultures of TRS-treated DCs and CD4+ T cells resulted in decreased IFN-γ production, indicating that TRS-stimulated DCs may enhance the Th1 response through DC-derived IL-12. Injection of OT-II mice with OVA-pulsed, TRS-treated DCs also enhanced Ag-specific Th1 responses in vivo. Importantly, injection with TRS-treated DC exhibited increased populations of IFN-γ+-CD4+ and -CD8+ T cells as well as secretion level of IFN-γ, resulting in viral clearance and increased survival periods in mice infected with influenza A virus (IAV), as the Th1 response is associated with the enhanced cellular immunity, including anti-viral activity. Taken together, these results indicate that TRS promotes the maturation and activation of DCs, DC-mediated Th1 responses, and anti-viral effect on IAV infection.

Mst1-Deficiency Induces Hyperactivation of Monocyte-Derived Dendritic Cells via Akt1/c-myc Pathway.

Mst1 is a multifunctional serine/threonine kinase that is highly expressed in several immune organs. The role of Mst1 in the activation of dendritic cells (DCs), a key player of adaptive immunity, is poorly understood. In this study, we investigated the role of Mst1 in GM-CSF-induced bone marrow-derived DCs and the underlying mechanisms. Mst1-/- DCs in response to GM-CSF expressed higher levels of activation/maturation-related cell surface molecules, such as B7 and MHC class II than Mst1+/+ DCs. Furthermore, the expression of proinflammatory cytokines, such as IL-23, TNF-α, and IL-12p40, was increased in Mst1-/- DCs, indicating that Mst1-deficiency may induce the hyperactivation of DCs. Additionally, Mst1-/- DCs exhibited a stronger capacity to activate allogeneic T cells than Mst1+/+ DCs. Silencing of Mst1 in DCs promoted their hyperactivation, similar to the phenotypes of Mst1-/- DCs. Mst1-/- DCs exhibited an increase in Akt1 phosphorylation and c-myc protein levels. In addition, treatment with an Akt1 inhibitor downregulated the protein level of c-myc increased in Mst1-deficient DCs, indicating that Akt1 acts as an upstream inducer of the de novo synthesis of c-myc. Finally, Akt1 and c-myc inhibitors downregulated the increased expression of IL-23p19 observed in Mst1-knockdown DCs. Taken together, these data demonstrate that Mst1 negatively regulates the hyperactivation of DCs through downregulation of the Akt1/c-myc axis in response to GM-CSF, and suggest that Mst1 is one of the endogenous factors that determine the activation status of GM-CSF-stimulated inflammatory DCs.

l-Asparaginase-mediated downregulation of c-Myc promotes 1,25(OH)2 D3 -induced myeloid differentiation in acute myeloid leukemia cells.

Treatment of acute myeloid leukemia (AML) largely depends on chemotherapy, but current regimens have been unsatisfactory for long-term remission. Although differentiation induction therapy utilizing 1,25(OH)2 D3 (VD3) has shown great promise for the improvement of AML treatment efficacy, severe side effects caused by its supraphysiological dose limit its clinical application. Here we investigated the combinatorial effect of l-asparaginase (ASNase)-mediated amino acid depletion and the latent alternation of VD3 activity on the induction of myeloid differentiation. ASNase treatment enhanced VD3-driven phenotypic and functional differentiation of three-different AML cell lines into monocyte/macrophages, along with c-Myc downregulation. Using gene silencing with shRNA and a chemical blocker, we found that reduced c-Myc is a critical factor for improving VD3 efficacy. c-Myc-dependent inhibition of mTORC1 signaling and induction of autophagy were involved in the enhanced AML cell differentiation. In addition, in a postculture of AML cells after each treatment, ASNase supports the antileukemic effect of VD3 by inhibiting cell growth and inducing apoptosis. Finally, we confirmed that the administration of ASNase significantly improved VD3 efficacy in the prolongation of survival time in mice bearing tumor xenograft. Our results are the first to demonstrate the extended application of ASNase, which is currently used for acute lymphoid leukemia, in VD3-mediated differentiation induction therapy for AML, and suggest that this drug combination may be a promising novel strategy for curing AML.

5-diphenylacetamido-indirubin-3'-oxime as a novel mitochondria-targeting agent with anti-leukemic activities.

Current treatment for leukemia largely depends on chemotherapy. Despite the progress in treatment efficacy of chemotherapy, a poor outcome consequent upon chemoresistance against conventional anti-cancer drugs still remains to be solved. In this study, we report 5-diphenylacetamido-indirubin-3'-oxime (LDD398) as a novel mitochondria-targeting anti-leukemic agent, which is a derivative of indirubin used in traditional medicine. Treatment with LDD398 resulted in caspase activation, cell death, and growth arrest at G2/M phases in leukemia cells. Interestingly, LDD398 quickly collapsed mitochondrial membrane potential (MMP) within 1 h, accompanied by cytochrome c release into cytosol and severe depletion of cellular ATP. However, the LDD398-induced cellular events was significantly mitigated by blockage of mitochondrial permeability transition pore (MPTP) opening with chemical and genetic modifications, strongly supporting that LDD398 executes its anti-leukemic activity via an inappropriate opening of MPTP and a consequent depletion of ATP. The most meaningful finding was the prominent effectiveness of LDD398 on primary leukemia cells and also on malignant leukemia cells resistant to anticancer drugs. Our results demonstrate that, among a series of indirubin derivatives, LDD398 induces leukemia cell death via a different mode from indirubin or conventional chemotherapeutics, and can be employed as a potent anti-cancer agent in the treatment for newly diagnosed and relapsed leukemia.

Concentrative nucleoside transporter 3 as a prognostic indicator for favorable outcome of t(8;21)-positive acute myeloid leukemia patients after cytarabine-based chemotherapy.

Although acute myeloid leukemia (AML) exhibits diverse responses to chemotherapy, patients harboring the t(8;21) translocation are part of a favorable risk group. However, the reason why this subgroup is more responsive to cytarabine-based therapy has not been elucidated. In the present study, we analyzed expression levels of cytarabine metabolism-related genes in patients diagnosed with AML with or without t(8;21) and investigated their correlation with clinical outcomes after cytarabine-based therapy. Among the 8 genes studied, expression of the concentrative nucleoside transporter 3 (CNT3) gene was significantly higher in t(8;21)-positive patients compared to the others in the test population and the validation cohort (P<0.001 in Mann-Whitney U test; P<0.002 in Pearson's correlation analysis). Additionally, in both multivariate and univariate analyses, t(8;21)-positive patients categorized in a higher CNT3 expression tertile had longer disease-free survival [hazard ratio (HR), 0.117; 95% confidence interval (CI), 0.025-0.557; P=0.008] and overall survival (HR, 0.062; 95% CI, 0.007-0.521; P=0.010) compared to t(8;21)-positive patients in a lower CNT3 expression tertile. Notably, these trends did not occur in t(8;21)-negative patients. Our results demonstrate that CNT3 expression is associated with overall favorable outcomes and is predictive of clinical outcomes in AML patients with t(8;21). This suggests that CNT3 expression can be used to optimize treatment strategies for AML patients.

Analysis of gene profiles involved in the enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by sesquiterpene lactones identifies asparagine synthetase as a novel target for differentiation-inducing therapy.

All-trans retinoic acid (ATRA) is one of the most useful drugs in the treatment for acute promyelocytic leukemia (APL), but its adverse effects, which include drug resistance and hypercalcemia are obstacles to achieving complete remission. Our previous study showed that some sesquiterpene lactones (STLs), i.e., helenalin (HE) and parthenolide (PA) but not sclareolide (SC), enhance ATRA-induced differentiation of HL-60 APL cells with no unexpected effects, but the precise mechanism on underlying this synergism is not yet fully understood. In this study, we investigated the distinctive transcriptional profile of cells treated with effective STL compounds, which were identified by comparing the profile with that of cells treated with SC. Genome-wide approaches using cDNA microarrays showed that co-treatment with the differentiation-enhancing STLs HE and PA maximized the transcriptional variation regulated by the suboptimal concentration of ATRA in HL-60 cells. Of the genes of interest, asparagine synthetase was remarkably downregulated by ATRA co-treated with either HE or PA, but not with SC. In an additional analysis for the role of asparagine synthetase, ATRA-mediated HL-60 cell differentiation was enhanced when asparagine in the culture media was depleted by an addition of L-asparaginase, indicating that downregulation of asparagine synthetase gene expression may be involved in the enhanced cell differentiation by STL compounds. These results provide useful insight into differentiation-inducing therapy in the treatment of leukemia.

Penetration of fiber versus spherical particles through filter media and faceseal leakage of N95 filtering facepiece respirators with cyclic flow.

This study investigated differences in penetration between fibers and spherical particles through faceseal leakage of an N95 filtering facepiece respirator. Three cyclic breathing flows were generated corresponding to mean inspiratory flow rates (MIF) of 15, 30, and 85 L/min. Fibers had a mean diameter of 1 µm and a median length of 4.9 µm (calculated aerodynamic diameter, d(ae) = 1.73 µm). Monodisperse polystyrene spheres with a mean physical diameter of 1.01 µm (PSI) and 1.54 µm (PSII) were used for comparison (calculated d(ae) = 1.05 and 1.58 µm, respectively). Two optical particle counters simultaneously determined concentrations inside and outside the respirator. Geometric means (GMs) for filter penetration of the fibers were 0.06, 0.09, and 0.08% at MIF of 15, 30, and 85 L/min, respectively. Corresponding values for PSI were 0.07, 0.12, and 0.12%. GMs for faceseal penetration of fibers were 0.40, 0.14, and 0.09% at MIF of 15, 30, and 85 L/min, respectively. Corresponding values for PSI were 0.96, 0.41, and 0.17%. Faceseal penetration decreased with increased breathing rate for both types of particles (p ≤ 0.001). GMs of filter and faceseal penetration of PSII at an MIF of 30 L/min were 0.14% and 0.36%, respectively. Filter penetration and faceseal penetration of fibers were significantly lower than those of PSI (p < 0.001) and PSII (p < 0.003). This confirmed that higher penetration of PSI was not due to slightly smaller aerodynamic diameter, indicating that the shape of fibers rather than their calculated mean aerodynamic diameter is a prevailing factor on deposition mechanisms through the tested respirator. In conclusion, faceseal penetration of fibers and spherical particles decreased with increasing breathing rate, which can be explained by increased capture by impaction. Spherical particles had 2.0-2.8 times higher penetration through faceseal leaks and 1.1-1.5 higher penetration through filter media than fibers, which can be attributed to differences in interception losses.

Comparison of workplace protection factors for different biological contaminants.

This study compared workplace protection factors (WPFs) for five different contaminants (endotoxin, fungal spores, (1→3)-ß-D-glucan, total particle mass, and total particle number) provided by an N95 elastomeric respirator (ER) and an N95 filtering facepiece respirator (FFR). We previously reported size-selective WPFs for total particle numbers for the ER and FFR, whereas the current article is focused on WPFs for bioaerosols and total particle mass. Farm workers (n = 25) wore the ER and FFR while performing activities at eight locations representing horse farms, pig barns, and grain handling facilities. For the determination of WPFs, particles were collected on filters simultaneously inside and outside the respirator during the first and last 15 min of a 60-min experiment. One field blank per subject was collected without actual sampling. A reporting limit (RL) was established for each contaminant based on geometric means (GMs) of the field blanks as the lowest possible measurable values. Depending on the contaminant type, 38-48% of data points were below the RL. Therefore, a censored regression model was used to estimate WPFs (WPF(censored)). The WPF(censored) provided by the two types of respirators were not significantly different. In contrast, significant differences were found in the WPF(censored) for different types of contaminants. GMs WPFs(censored) for the two types of respirators combined were 154, 29, 18, 19, and 176 for endotoxin, fungal spore count, (1→3)-ß-D-glucan, total particle mass, and total particle number, respectively. The WPF(censored) was more strongly associated with concentrations measured outside the respirator for endotoxin, fungal spores, and total particle mass except for total particle number. However, when only data points with outside concentrations higher than 176×RL were included, the WPFs increased, and the association between the outside concentrations and the WPFs became weaker. Results indicate that difference in WPFs observed between different contaminants may be attributed to differences in the sensitivity of analytical methods to detect low inside concentrations, rather than the nature of particles (biological or non-biological).

Airborne microorganisms, endotoxin, and (1→3)-ß-D-glucan exposure in greenhouses and assessment of respiratory symptoms among workers.

OBJECTIVES: Greenhouse operations are an important sector of the horticulture industry, also known as the Green Industry. The objectives of this study were (i) to investigate exposure levels to airborne culturable fungi, bacteria (total culturable bacteria and actinomycetes), endotoxin, and (1→3)-ß-D-glucan in three Midwest greenhouses during summer and winter using multiple exposure assessment methods; (ii) characterize the load of microorganisms on greenhouse floors and determine potential microbial source strengths of the floors for aerosolizing microbial biocontaminants, and (iii) to estimate the prevalence of rhinitis, wheezing, asthma, and other respiratory symptoms/conditions among greenhouse workers. METHODS: Stationary inhalable aerosol samples were collected from each greenhouse using Button Inhalable Aerosol Samplers. Control samples were collected from offices and nearby outdoor locations. A microbial source strength tester was used to examine the aerosolization potential of microbial contaminants from greenhouse floors. Additionally, surface samples were collected by sterile cotton swabs. Temperature, relative humidity, and wind velocity were recorded. Airborne culturable fungi, bacteria, and actinomycetes were analyzed in the extracts from field samples by cultivation in nutrient agar media. Endotoxin and (1→3)-ß-D-glucan in the extracts from field samples were analyzed by specific kinetic chromogenic Limulus amebocyte lysate assays. The prevalence of respiratory symptoms among greenhouse workers (n = 35) and control subjects (office workers; n = 14) was estimated with a standardized questionnaire. RESULTS AND CONCLUSIONS: The collected data indicate that workers employed in Midwest greenhouses may be exposed to elevated levels of inhalable culturable microorganisms (fungi and bacteria collectively on the order of 10(2)-10(5) CFU m(-3)), endotoxin (10(1)-10(3) EU m(-3)), and (1→3)-ß-D-glucan (10(1)-10(2) ng m(-3)). Seasonal variations were observed for some bioaerosol components. The prevalence of self-reported respiratory symptoms was generally higher among greenhouse workers compared to controls; however, the differences were not statistically significant, likely due to the relatively low statistical power of the study.

Effect of particle size on respiratory protection provided by two types of N95 respirators used in agricultural settings.

This study compared size-selective workplace protection factors (WPFs) of an N95 elastomeric respirator (ER) and an N95 filtering facepiece respirator (FFR) in agricultural environments. Twenty-five healthy farm workers ranging in age from 20 to 30 years voluntarily participated in this study. Altogether, eight farms were included representing three different types: two horse farms, three pig barns, and three grain handling sites. Subjects wore the ER and FFR while performing their daily activities, such as spreading hay, feeding livestock, and shoveling. Aerosol concentrations in an optical particle size range of 0.7-10 µm were determined simultaneously inside and outside the respirator during the first and last 15 min of a 60-min experiment. For every subject, size-selective WPFs were calculated in 1-min intervals and averaged over 30 min. For the ER, geometric mean WPFs were 172, 321, 1013, 2097, and 2784 for particle diameters of 0.7-1.0, 1.0-2.0, 2.0-3.0, 3.0-5.0, and 5.0-10.0 µm, respectively. Corresponding values for the FFR were 67, 124, 312, 909, and 2089. The 5th percentiles for the ER and FFR were higher than the assigned protection factor of 10 and varied from 28 to 250 and from 16 to 223, respectively. Results show that the N95 ER and FFR tested in the study provided an expected level of protection for workers on agricultural farms against particles ranging from 0.7 to 10 µm. WPFs for the ER were higher than the FFR for all particle size ranges. WPFs for both respirator types increased with increasing particle size.

Large particle penetration through N95 respirator filters and facepiece leaks with cyclic flow.

The aim of this study was to investigate respirator filter and faceseal penetration of particles representing bacterial and fungal spore size ranges (0.7-4 mum). First, field experiments were conducted to determine workplace protection factors (WPFs) for a typical N95 filtering facepiece respirator (FFR). These data (average WPF = 515) were then used to position the FFR on a manikin to simulate realistic donning conditions for laboratory experiments. Filter penetration was also measured after the FFR was fully sealed on the manikin face. This value was deducted from the total penetration (obtained from tests with the partially sealed FFR) to determine the faceseal penetration. All manikin experiments were repeated using three sinusoidal breathing flow patterns corresponding to mean inspiratory flow rates of 15, 30, and 85 l min(-1). The faceseal penetration varied from 0.1 to 1.1% and decreased with increasing particle size (P < 0.001) and breathing rate (P < 0.001). The fractions of aerosols penetrating through the faceseal leakage varied from 0.66 to 0.94. In conclusion, even for a well-fitting FFR respirator, most particle penetration occurs through faceseal leakage, which varies with breathing flow rate and particle size.

Investigation of a mercury speciation technique for flue gas desulfurization materials.

Most of the synthetic gypsum generated from wet flue gas desulfurization (FGD) scrubbers is currently being used for wallboard production. Because oxidized mercury is readily captured by the wet FGD scrubber, and coal-fired power plants equipped with wet scrubbers desire to benefit from the partial mercury control that these systems provide, some mercury is likely to be bound in with the FGD gypsum and wallboard. In this study, the feasibility of identifying mercury species in the FGD gypsum and wallboard samples was investigated using a large sample size thermal desorption method. Potential candidates of pure mercury standards including mercuric chloride (HgCl2), mercurous chloride (Hg2Cl2), mercury oxide (HgO), mercury sulfide (HgS), and mercuric sulfate (HgSO4) were analyzed to compare their results with those obtained from FGD gypsum and dry wallboard samples. Although any of the thermal evolutionary curves obtained from these pure mercury standards did not exactly match with those of the FGD gypsum and wallboard samples, it was identified that Hg2Cl2 and HgCl2 could be candidates. An additional chlorine analysis from the gypsum and wallboard samples indicated that the chlorine concentrations were approximately 2 orders of magnitude higher than the mercury concentrations, suggesting possible chlorine association with mercury.