Coinhibitory receptors, such as CTLA-4 and PD-1, play a critical role in maintaining immune homeostasis by dampening T cell responses. Recently, they have gained attention as therapeutic targets in ...chronic disease settings where their dysregulated expression contributes to suppressed immune responses. The novel coinhibitory receptor TIGIT (T cell Ig and ITIM domain) has been shown to play an important role in modulating immune responses in the context of autoimmunity and cancer. However, the molecular mechanisms by which TIGIT modulates immune responses are still insufficiently understood. We have generated a panel of monoclonal anti-mouse TIGIT Abs that show functional properties in mice in vivo and can serve as important tools to study the underlying mechanisms of TIGIT function. We have identified agonistic as well as blocking anti-TIGIT Ab clones that are capable of modulating T cell responses in vivo. Administration of either agonist or blocking anti-TIGIT Abs modulated autoimmune disease severity whereas administration of blocking anti-TIGIT Abs synergized with anti-PD-1 Abs to affect partial or even complete tumor regression. The Abs presented in this study can thus serve as important tools for detailed analysis of TIGIT function in different disease settings and the knowledge gained will provide valuable insight for the development of novel therapeutic approaches targeting TIGIT.
Over the last two decades, usage of neonicotinoid (NEO) insecticides has increased due to their high selectivity for insects versus mammals and their effectiveness for extermination of insects ...resistant to conventional pesticides such as pyrethroids and organophosphates (OPs). However, historical change of the NEO exposure level in humans is poorly understood. The aim of this study is to reveal changes in the levels of NEO and OP exposure in the human body over the last two decades using biomonitoring technique. We quantified urinary concentrations of 7 NEOs (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) and 4 metabolites of OPs (dimethylphosphate, dimethylthiophosphate, diethylphosphate, and diethylthiophosphate) in 95 adult females aged 45–75 in 1994, 2000, 2003, 2009, and 2011 (n = 17–20 different individuals in each year). The results show that the detection rates of urinary NEOs in Japanese women increased significantly between 1994 and 2011, suggesting that intakes of NEOs into the human body rose during that period. In contrast, exposure to OPs having O,O-dimethyl moieties decreased steadily according to a finding that geometric means of urinary dimethylphosphate concentrations kept diminishing considerably. These changes may reflect the amounts of NEOs and OPs used as insecticides in Japan.
Abstract: Biological Monitoring Method for Urinary Neonicotinoid Insecticides Using LC-MS/MS and Its Application to Japanese Adults: Jun UEYAMA, et al. Department of Pathophysiological Laboratory ...Sciences, Field of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine - Objectives: Agricultural use of neonicotinoid (NEO) insecticides has been increasing in recent years, but their biological monitoring methods have been scarcely reported. In this study, we developed and validated a rapid and sensitive method for quantifying urinary NEO concentrations using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods: After phosphate-induced acidification of a urine sample, urinary NEOs were trapped by a solid-phase extraction column and eluted with methanol for acetamiprid, imidacloprid, thiacloprid, thiamethoxam, clothianidin and dinotefuran and with an acetonitrile and methanol solution (1:1, v/v) containing 5% NH3 for nitenpyram. A separation analysis was performed by LC-MS/MS within 10 minutes for the sample. This method was applied to first morning urine obtained from 52 Japanese (40.9 +- 10.5 years old, mean +- standard deviation) without occupational NEO exposure. Results: The linear dynamic ranges and their limit of quantification (LOQ, signal to noise ratio=10) levels were 0.3-20 or 50 μg// (r=0.998-0.999) and 0.05-0.36 μg//, respectively. The absolute recovery was 64-95%, and the intra- and inter-day precisions were less than 16.4% (relative standard deviation, %RSD). This method was successfully applied for analysis of NEOs in human urine samples obtained from 52 adults. The frequencies of individuals who showed more than LOD levels was above 90% for imidacloprid, thiamethoxam, clothianidin and dinotefuran, more than 50% for acetamiprid and thiacloprid and 29% for nitenpyram. Conclusions: These results indicated that our new method could be applied to biological monitoring of NEO exposure even at environmental exposure levels in Japanese adults without occupational spraying histories.
The use of neonicotinoid (NEO) insecticides has increased over the past decade not only in Japan but also worldwide, while organophosphate (OP) and pyrethroid (PYR) insecticides are still ...conventionally used in agriculture and domestic pest control. However, limited data are currently available on the NEO exposure levels, especially in children, who are particularly vulnerable to environmental toxicants. Thus, the purpose of this study was to characterize the exposure to NEOs, as well as OPs and PYRs, in three-year-old Japanese children by assessing the range, distribution, and seasonal differences of the urinary concentrations of seven NEOs (acetamiprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, imidacloprid, and nitenpyram); four OP metabolites (dialkylphosphates DAPs), including dimethylphosphate, dimethylthiophosphate, diethylphosphate, and diethylthiophosphate; and three PYR metabolites (3-phenoxybenzoic acid, trans-chrysanthemumdicarboxylic acid, and 3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropane carboxylic acid). Urine samples were collected from 223 children (108 males and 115 females) in the summer and winter months. The detection rates of NEOs were 58% for dinotefuran, 25% for thiamethoxam, 21% for nitenpyram, and <16% for all other NEOs. The median and maximum concentrations of the sum of the seven NEOs (ΣNEO) were 4.7 and 370.2nmol/g creatinine, respectively. Urinary ΣNEO, dimethylphosphate, and all PYR metabolite concentrations were significantly higher in the summer than in the winter (p<0.05). The creatinine-adjusted concentration of ΣNEO significantly correlated with those of all DAPs (p<0.05) but not with those of the PYR metabolites. Moreover, the NEO-detected group showed higher urinary ΣDAP (sum of four OP metabolites) concentrations than the group without NEO detection. These findings suggest that children in Japan are environmentally exposed to the three major insecticide lines, and that the daily exposure sources of NEOs are common to those of OPs.
•Urine insecticide metabolites were monitored in 3-year-old children.•The detection rate of dinotefuran ranked first among the seven neonicotinoids (NEO).•Organophosphate (OP) and pyrethroid metabolites were detected in almost all children.•For most biomarkers, the concentrations were higher in summer than winter.•Urinary concentrations of NEOs and OP metabolites were positively correlated with each other.
Objectives: Currently available questionnaires have limited ability to measure physical activity (PA) using accelerometers as a gold standard. This study aimed to develop a PA questionnaire for ...middle-aged Japanese workers and propose a PA scoring system for predicting low moderate-to-vigorous PA (MVPA).Methods: A total of 428 participants (median age 49 years; 75.8% men) participated in a 7-day PA measurement using an accelerometer and a questionnaire. The association between questionnaire responses and low MVPA (<150 min/wk) was assessed by logistic regression analysis. A score was assigned to each response based on the correlation coefficients of the multivariate model. The ability of the sum score to predict low MVPA was assessed using the area under the receiver operating characteristic curve (AUC).Results: Five questionnaire items were used for measuring PA scores (range: 0-50; higher scores indicated a higher probability of low MVPA). The AUC was 0.741 (95% CI, 0.689-0.792), and the sensitivity and specificity at the optimal cut-off value were 66.7% and 68.2%, respectively. This predictive ability was slightly increased by body mass index (AUC 0.745 95% CI, 0.693-0.796; sensitivity 69.9%; specificity 66.9%). These predictive values were greater than those of conventional questionnaires used in health checkups in Japan (P < .05).Conclusions: This questionnaire-based PA scoring system showed moderate accuracy in predicting low MVPA. It is useful for screening physically inactive workers and promoting PA.
Background
In patients with heart failure (HF), physical frailty should be assessed to enable risk stratification. No conventional frailty criteria have so far been developed considering HF‐specific ...outcomes. This study aimed to propose a frailty‐based prognostic score using a nationwide cohort study of Japanese patients with HF.
Methods
We analysed 2721 patients hospitalized for HF and capable of walking at discharge (median age: 76 years interquartile range 67–83, men: 60.5%). Physical frailty was evaluated at discharge using four quantitative measures: usual walking speed, grip strength, Performance Measure for Activities of Daily Living‐8 (PMADL‐8), and Self‐Efficacy for Walking‐7 (SEW‐7). The primary outcome was a composite of HF rehospitalization and all‐cause mortality within 2 years. A cut‐off point was identified for each measure using receiver operating characteristic analysis in a derivation cohort (n = 1778). Cox proportional hazards model was used to assign a score to each frailty domain according to the correlation with the endpoint. Patients were divided into four categories according to the sum score, and survival was compared by analysing the Kaplan–Meier curve and Cox proportional hazards model. Cumulative incidences of the events according to frailty categories were compared between the derivation cohort and a validation cohort (n = 943).
Results
The cut‐off value and assigned score of each indicator was determined as follows: usual walking speed < 0.98 m/s = 4 points; grip strength < 30.0 kg (men) or 17.5 kg (women) = 5 points; PMADL‐8 ≥ 21 points = 2 points; SEW‐7 ≤ 20 points = 3 points. We stratified patients into four categories according to the sum score: Category I, ≤3 points; Categories II, 4–8 points; Category III, 9–13 points; and Category IV, 14 points. The prevalence and cumulative incidence of the composite outcome for Categories I to IV in the derivation cohort were 27.4%, 25.2%, 26.4%, and 21.0%, and 9.5, 16.3, 26.3, and 36.8/100 person‐years, respectively. Similar results were confirmed in the validation cohort. In Cox proportional hazards model, frailty categories were associated with the composite outcome independent of potential confounders (hazard ratio 95% confidence interval in reference to Category I: Categories II, 1.51 0.84–2.72, P = 0.169; Category III, 2.37 1.32–4.23, P = 0.004; Category IV, 2.66 1.45–4.89, P = 0.002).
Conclusions
The frailty‐based prognostic score proposed in this study was well associated with prognosis and will serve for risk stratification in patients with HF.
CD4+ T cells have Th cell function and include two major functional subsets, Th1 and Th2. However, there are a restricted number of studies concerning phenotypic classification of human CD4+ T cells. ...Here by using seven- and eight-color flow cytometric analysis, we investigated the function of the subsets classified by four markers, CD27, CD28, CD45RA and CCR7. Five major subsets were identified by using these markers. These subsets showed different patterns of cytokine production after they were stimulated with phorbol myristate acetate and ionomycin. The analyses of cytokine production suggested that CCR7+CD45RA+CD27+CD28+, CCR7+CD45RA−CD27+CD28+ and CCR7−CD45RA−CD27+CD28+ subsets were naive, central memory and effector memory T cells, respectively, whereas CCR7−CD45RA−CD27−CD28+ and CCR7−CD45RA−CD27−CD28− subsets included Th1 and Th2 cells. The analysis of cytokine production by these subsets stimulated with anti-CD3 and anti-CD28 mAbs or with human cytomegalovirus antigens showed that IFN-γ production was significantly higher in the CCR7−CD45RA−CD27−CD28− subset than in other subsets and that both CCR7−CD45RA−CD27−CD28+ and CCR7−CD45RA−CD27−CD28− subsets produced a higher level of IL-4 than did other subsets. Our analyses demonstrated that the CCR7−CD45RA−CD27−CD28− subset predominantly included Th1 effector cells and that CCR7−CD45RA−CD27−CD28+ subsets included Th1 and Th2 effector memory/effector cells as well as unclassified cells. The analysis of classification by using these four markers also suggested the differentiation pathway of human CD4+ T cells.
Th17 cells are key players in defense against pathogens and maintaining tissue homeostasis, but also act as critical drivers of autoimmune diseases. Based on single-cell RNA-seq profiling of ...pathogenic versus nonpathogenic Th17 cells, we identified protein C receptor (PROCR) as a cell surface molecule expressed in covariance with the regulatory module of Th17 cells. Although PROCR expression in T cells was controlled by the cooperative action of the Th17 lineage-specific transcription factors RORγt, IRF4, and STAT3, PROCR negatively regulated Th17 differentiation. CD4
T cells from PROCR low expressor mutant mice readily differentiated into Th17 cells, whereas addition of the PROCR ligand, activated protein C, inhibited Th17 differentiation in vitro. In addition, PROCR acted as a negative regulator of Th17 pathogenicity in that it down-regulated expression of several pathogenic signature genes, including IL-1 and IL-23 receptors. Furthermore, T cell-specific deficiency of PROCR resulted in the exacerbation of experimental autoimmune encephalomyelitis (EAE) and higher frequencies of Th17 cell in vivo, indicating that PROCR also inhibits pathogenicity of Th17 cells in vivo. PROCR thus does not globally inhibit Th17 responses but could be targeted to selectively inhibit proinflammatory Th17 cells.
Four pyrethroids (PYRs), metofluthrin, profluthrin, tefluthrin, and transfluthrin, which were newly developed and have relatively high vapor activity at ambient temperature, are now playing a key ...role in safely controlling insects in our daily lives. We developed a sensitive and high-throughput determination method for urinary metabolites derived from the newly developed PYR, e.g., 2,3,5,6-tetrafluoro-1,4-benzenedimethanol (HOCH
2
-FB-Al), 2,3,5,6-tetrafluorobenzyl alcohol (FB-Al), and other PYR metabolites such as
trans
-chrysanthemumdicarboxylic acid (
trans
-CDCA) and 3-phenoxybenzoic acid (3PBA). After high temperature acid hydrolysis of 2 mL urine sample in 24-deep well plate, the PYR metabolites were extracted by semi-automated liquid-liquid extraction with
tert
-butyl methyl ether.
N
,
O
-Bis (trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilane or 1,1,1,3,3,3-hexafluoroisopropanol were used for the derivatization of PYR metabolites, and the derivatized metabolites were analyzed separately by GC-MS/MS equipped with dual injector system (DB-5MS and mid- to high-polarity phase Rtx-65 columns). The derivatization and evaporation conditions were mainly optimized for improving sensitivity and reproducibility. The mean within-run day precisions were less than 18.4% (relative standard deviation, %RSD) with low detection limits ranging from 0.01 μg/L for HOCH
2
-FB-Al to 0.06 μg/L for
trans
-CDCA. This method was successfully applied to urine samples obtained from 50 3-year-old children with high detection frequencies (e.g., 82% for HOCH
2
-FB-Al and 84% for FB-Al). This method may be a pivotal tool for developing risk assessment from PYR exposure in the general population.
Age-related macular degeneration (AMD) is a major cause of blindness in developed countries and is closely related to oxidative stress, which leads to lipid peroxidation. Malondialdehyde (MDA) is a ...major byproduct of polyunsaturated fatty acid (PUFA) peroxidation. Increased levels of MDA have been reported in eyes of AMD patients. However, little is known about the direct relationship between MDA and AMD. Here we show the biological importance of MDA in AMD pathogenesis. We first confirmed that MDA levels were significantly increased in eyes of AMD patients. In ARPE-19 cells, a human retinal pigment epithelial cell line, MDA treatment induced vascular endothelial growth factor (VEGF) expression alternation, cell junction disruption, and autophagy dysfunction that was also observed in eyes of AMD patients. The MDA-induced VEGF increase was inhibited by autophagy–lysosomal inhibitors. Intravitreal MDA injection in mice increased laser-induced choroidal neovascularization (laser-CNV) volumes. In a mouse model fed a high-linoleic acid diet for 3 months, we found a significant increase in MDA levels, autophagic activity, and laser-CNV volumes. Our study revealed an important role of MDA, which acts not only as a marker but also as a causative factor of AMD pathogenesis-related autophagy dysfunction. Furthermore, higher dietary intake of linoleic acid promoted CNV progression in mice with increased MDA levels.
Display omitted
•Malondialdehyde accumulated in the sera and eyes of patients with AMD.•VEGF secretion in RPE cells was dysregulated by malondialdehyde.•Malondialdehyde induced RPE cell junction disruption and autophagy dysfunction.•Administration of malondialdehyde increased CNV volumes in mice.•Higher dietary intake of linoleic acid promoted CNV progression in mice.
PDF
