Acute Systemic Infection with Dengue Virus Leads to Vascular Leakage and Death through Tumor Necrosis Factor-α and Tie2/Angiopoietin Signaling in Mice Lacking Type I and II Interferon Receptors

Severe dengue is caused by host responses to viral infection, but the pathogenesis remains unknown. This is, in part, due to the lack of suitable animal models. Here, we report a non-mouse-adapted low-passage DENV-3 clinical isolate, DV3P12/08, derived from recently infected patients. DV3P12/08 caused a lethal systemic infection in type I and II IFN receptor KO mice (IFN-α/β/γR KO mice), which have the C57/BL6 background. Infection with DV3P12/08 induced a cytokine storm, resulting in severe vascular leakage (mainly in the liver, kidney and intestine) and organ damage, leading to extensive hemorrhage and rapid death. DV3P12/08 infection triggered the release of large amounts of TNF-α, IL-6, and MCP-1. Treatment with a neutralizing anti-TNF-α antibody (Ab) extended survival and reduced liver damage without affecting virus production. Anti-IL-6 neutralizing Ab partly prolonged mouse survival. The anti-TNF-α Ab suppressed IL-6, MCP-1, and IFN-γ levels, suggesting that the severe response to infection was triggered by TNF-α. High levels of TNF-α mRNA were expressed in the liver and kidneys, but not in the small intestine, of infected mice. Conversely, high levels of IL-6 mRNA were expressed in the intestine. Importantly, treatment with Angiopoietin-1, which is known to stabilize blood vessels, prolonged the survival of DV3P12/08-infected mice. Taken together, the results suggest that an increased level of TNF-α together with concomitant upregulation of Tie2/Angiopoietin signaling have critical roles in severe dengue infection.     

Association between Smoking Status and Obesity in a Nationwide Survey of Japanese Adults

Objective

A positive association between the number of cigarettes smoked per day and obesity has been reported, whereas how other smoking-related indices, such as pack-years and duration of smoking, are related with obesity has been less investigated. We analyzed the age-adjusted cross-sectional association between smoking and obesity in a general Japanese population.

Methods

We used data from a nationwide epidemiological study of Japanese adults (N = 23,106). We compared the prevalence of obesity (defined as body mass index ≥ 25kg/m²) among groups classified by smoking behavior, pack-years, number of cigarettes per day, duration of smoking, and duration and time of smoking cessation.

Results

In men, current smokers had a lower odds ratio (OR) for obesity of 0.80 (95% confidence interval (CI): 0.72–0.88) compared to non-smokers, whereas past smokers had a higher OR of 1.23 (95% CI: 1.09–1.37) compared to current smokers. In women, there were no differences in obesity between the three groups classified by smoking behavior. However, in both sexes, the prevalence of obesity tended to increase with pack-years and the number of cigarettes per day, but not with duration of smoking in current and past smokers. Further, in male smokers, the risks for obesity were markedly higher in short-term heavy smokers compared with long-term light smokers, even with the same number of pack-years. Regarding the impact of smoking cessation, female past smokers who quit smoking at an age > 55-years had an elevated OR of 1.60 (95% CI:1.05–2.38) for obesity.

Conclusions

In a general Japanese population, obesity is progressively associated with pack-years and number of cigarettes per day, but not with the duration of smoking. When investigating the association between obesity and cigarette smoking, the daily smoking burden and the duration of smoking require to be independently considered.     

Reappraisal of VAChT‐Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers

VAChT‐Cre.Fast and VAChT‐Cre.Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT‐Cre lines with a reporter line, CAG‐Syp/tdTomato, in which synaptophysin‐tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co‐localized with osteopontin, a recently discovered motor neuron marker for slow‐twitch fatigue‐resistant (S) and fast‐twitch fatigue‐resistant (FR) types. The fluorescence did not preferentially co‐localize with matrix metalloproteinase‐9, a marker for fast‐twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT‐Cre lines are Cre‐drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT‐Cre.Fast and VAChT‐Cre.Slow to VAChT‐Cre.Early and VAChT‐Cre.Late, respectively. The mouse lines will be useful tools to study slow‐type motor neurons, in relation to physiology and pathology.     

Temperature dependence on the pesticide sampling rate of polar organic chemical integrative samplers (POCIS)

Laboratory experiments were performed to determine the sampling rates of pesticides for the polar organic chemical integrative samplers (POCIS) used in Japan. The concentrations of pesticides in aquatic environments were estimated from the accumulated amounts of pesticide on POCIS, and the effect of water temperature on the pesticide sampling rates was evaluated. The sampling rates of 48 pesticides at 18, 24, and 30 °C were obtained, and this study confirmed that increasing trend of sampling rates was resulted with increasing water temperature for many pesticides.     

Combined brain‐derived neurotrophic factor with extinction training alleviate impaired fear extinction in an animal model of post‐traumatic stress disorder

Impaired fear memory extinction (Ext) is one of the hallmark symptoms of post‐traumatic stress disorder (PTSD). However, since the precise mechanism of impaired Ext remains unknown, effective interventions have not yet been established. Recently, hippocampal‐prefrontal brain‐derived neurotrophic factor (BDNF) activity was shown to be crucial for Ext in naïve rats. We therefore examined whether decreased hippocampal‐prefrontal BDNF activity is also involved in the Ext of rats subjected to a single prolonged stress (SPS) as a model of PTSD. BDNF levels were measured by enzyme‐linked immunosorbent assay (ELISA), and phosphorylation of TrkB was measured by immunohistochemistry in the hippocampus and medial prefrontal cortex (mPFC) of SPS rats. We also examined whether BDNF infusion into the ventral mPFC or hippocampus alleviated the impaired Ext of SPS rats in the contextual fear conditioning paradigm. SPS significantly decreased the levels of BDNF in both the hippocampus and mPFC and TrkB phosphorylation in the ventral mPFC. Infusion of BDNF 24 hours after conditioning in the infralimbic cortex (ILC), but not the prelimbic cortex (PLC) nor hippocampus, alleviated the impairment of Ext. Since amelioration of impaired Ext by BDNF infusion did not occur without extinction training, it seems the two interventions must occur consecutively to alleviate impaired Ext. Additionally, BDNF infusion markedly increased TrkB phosphorylation in the ILC of SPS rats. These findings suggest that decreased BDNF signal transduction might be involved in the impaired Ext of SPS rats, and that activation of the BDNF‐TrkB signal might be a novel therapeutic strategy for the impaired Ext by stress.     

Comparison between humic-like peaks in excitation-emission matrix spectra and resin-fractionated humic substances in aquatic environments

Limnology - The intensity of the 340/430-nm peak in the three-dimensional excitation-emission matrix spectra of water samples has been used as an index of the concentration of aquatic humic...     

Cellular responses to disruption of the permeability barrier in a three-dimensional organotypic epidermal model

Repeated injury to the stratum corneum of mammalian skin (caused by friction, soaps, or organic solvents) elicits hyperkeratosis and epidermal thickening. Functionally, these changes serve to restore the cutaneous barrier and protect the organism. To better understand the molecular and cellular basis of this response, we have engineered an in vitro model of acetone-induced injury using organotypic epidermal cultures. Rat epidermal keratinocytes (REKs), grown on a collagen raft in the absence of any feeder fibroblasts, developed all the hallmarks of a true epidermis including a well-formed cornified layer. To induce barrier injury, REK cultures were treated with intermittent 30-s exposures to acetone then were fixed and paraffin-sectioned. After two exposures, increased proliferation (Ki67 and BrdU staining) was observed in basal and suprabasal layers. After three exposures, proliferation became confined to localized buds in the basal layer and increased terminal differentiation was observed (compact hyperkeratosis of the stratum corneum, elevated levels of K10 and filaggrin, and heightened transglutaminase activity). Thus, barrier disruption causes epidermal hyperplasia and/or enhances differentiation, depending upon the extent and duration of injury. Given that no fibroblasts are present in the model, the ability to mount a hyperplastic response to barrier injury is an inherent property of keratinocytes.     

Assembly of lysine 63-linked ubiquitin conjugates by phosphorylated alpha-synuclein implies Lewy body biogenesis

alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of alpha-syn in LBs is phosphorylated at serine 129 (Ser(129)). However, the role of Ser(129)-phosphorylated alpha-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)alpha-syn and Ub, coexpression of phospho-mimic mutant alpha-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D alpha-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT alpha-syn, S129D alpha-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H(2)O(2) and serum deprivation. These results suggest that the contribution of Ser(129)-phosphorylated alpha-syn to the Lys(63)-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies.     

Limited suppression of the interferon-beta production by hepatitis C virus serine protease in cultured human hepatocytes

Toll-like receptors and RNA helicase family members [retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene-5 (MDA5)] play important roles in the induction of interferon-beta as a major event in innate immune responses after virus infection. TRIF (adaptor protein of Toll-like receptor 3)-mediated and Cardif (adaptor protein of RIG-I or MDA5)-mediated signaling pathways contribute rapid induction of interferon-beta through the activation of interferon regulatory factor-3 (IRF-3). Previously, it has been reported that the hepatitis C virus NS3-4A serine protease blocks virus-induced activation of IRF-3 in the human hepatoma cell line HuH-7, and that NS3-4A cleaves TRIF and Cardif molecules, resulting in the interruption of antiviral signaling pathways. On the other hand, it has recently been reported that non-neoplastic human hepatocyte PH5CH8 cells retain robust TRIF- and Cardif-mediated pathways, unlike HuH-7 cells, which lack a TRIF-mediated pathway. In the present study, we further investigated the effect of NS3-4A on antiviral signaling pathways. Although we confirmed that PH5CH8 cells were much more effective than HuH-7 cells for the induction of interferon-beta, we obtained the unexpected result that NS3-4A could not suppress the interferon-beta production induced by the TRIF-mediated pathway, although it suppressed the Cardif-mediated pathway by cleaving Cardif at the Cys508 residue. Using PH5CH8, HeLa, and HuH-7-derived cells, we further showed that NS3-4A could not cleave TRIF, in disagreement with a previous report describing the cleavage of TRIF by NS3-4A. Taken together, our findings suggest that the blocking of the interferon production by NS3-4A is not sufficient in HCV-infected hepatocyte cells.