Liposomal Dexamethasone Reduces A/H1N1 Influenza-Associated Morbidity in Mice.

Re-emerging viral threats have continued to challenge the medical and public health systems. It has become clear that a significant number of severe viral infection cases are due to an overreaction of the immune system, which leads to hyperinflammation. In this study, we aimed to demonstrate the therapeutic efficacy of the dexamethasone nanomedicine in controlling the symptoms of influenza virus infection. We found that the A/Wisconsin/WSLH34939/2009 (H1N1) infection induced severe pneumonia in mice with a death rate of 80%, accompanied by significant epithelial cell damage, infiltration of immune cells, and accumulation of pro-inflammatory cytokines in the airway space. Moreover, the intranasal delivery of liposomal dexamethasone during disease progression reduced the death rate by 20%. It also significantly reduced the protein level of tumor necrosis factor-alpha (TNFα), interleukin-1ß (IL-1ß), IL-6, and the C-X-C motif chemokine ligand 2 (CXCL2) as well as the number of infiltrated immune cells in the bronchoalveolar lavage fluids as compared to the control and free dexamethasone. The liposomal dexamethasone was mainly distributed into the monocyte/macrophages as a major cell population for inducing the cytokine storm in the lungs. Taken together, the intranasal delivery of liposomal dexamethasone may serve as a novel promising therapeutic strategy for the treatment of influenza A-induced pneumonia.

Toxicity Assessment of Transfluthrin, Benzyl Butyl Phthalate, and 17ß-Estradiol on the Primary Fibroblast of the Striped Field Mouse, Apodemus agrarius.

Environmental pollution (EP) is a well-known threat to wild animals, but its toxicological impact is poorly understood. In vitro toxicity evaluation using cells of lower predators could be a promising way to assess and monitor the effects of EPs on whole wildlife populations that are related in the food web. Here, we describe EPs' toxic effect and mechanism in the primary fibroblast derived from the embryo of the striped field mouse, Apodemus agrarius. Characterization of the primary fibroblast was via morphology, genetics, immunocytochemistry, and stable culture conditions for optimal toxicity screening. Cell viability assays-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH)-were performed to observe cytotoxicity, and quantitative PCR was conducted to confirm gene alteration by EP exposure. MTT and LDH assays confirmed the cytotoxicity of transfluthrin (TF), benzyl butyl phthalate (BBP), and 17ß-estradiol (E2) with IC50 values of 10.56 µM, 10.82 µM, and 24.08 µM, respectively, following 48-h exposures. mRNA expression of androgen-binding protein, growth hormone receptor, cytochrome C oxidase, and cytochrome P450-1A1 was induced after exposure to TF, BBP, and E2. We unveiled new EP mechanisms at the mammalian cellular level and discovered potential biomarker genes for monitoring of EPs. Based on our findings, we propose the primary fibroblast of A. agrarius as a valuable model to assess the toxicological effects of EP on wildlife.

Protein Kinase A Catalytic Subunit Is a Molecular Switch that Promotes the Pro-tumoral Function of Macrophages.

As current therapies benefit only a minority of cancer patients, additional therapeutic targets are needed. Tumor-associated macrophages (TAMs) have attracted attention for improving therapeutic responses, yet regulatory strategies remain elusive. Here, we show that the protein kinase A catalytic subunit (PKA-C) acts as a molecular switch, inducing a pro-tumoral immunosuppressive macrophage phenotype within tumors. In human and murine breast cancer, overactivated PKA in TAMs creates a detrimental microenvironment for cancer progression by inducing vascular endothelial growth factor A (VEGFA), interleukin-10 (IL-10), and macrophage-derived arginase 1 (ARG1) expression. Macrophages with genetic deletion of PKA-C are prone to be pro-inflammatory, suggesting a possible immunotherapeutic target. Delivery of liposomal PKA inhibitor facilitates tumor regression and abrogates pro-tumoral TAM functions in mice. The therapeutic effect of targeting PKA is pronounced when combined with αCTLA-4 antibody, increasing cluster of differentiation 8 (CD8)+GranzymeB+ T cells by about 60-fold. Our findings demonstrate critical roles of TAM PKA-C in tumor progression and suggest that targeting PKA-C efficiently augments cancer treatment responses.

Pyruvate dehydrogenase kinase is a negative regulator of interleukin-10 production in macrophages.

Interleukin-10 (IL-10) is the most potent anti-inflammatory cytokine in the body and plays an essential role in determining outcomes of many inflammatory diseases. Cellular metabolism is a critical determinant of immune cell function; however, it is currently unclear whether metabolic processes are specifically involved in IL-10 production. In this study, we aimed to find the central metabolic molecule regulating IL-10 production of macrophages, which are the main producers of IL-10. Transcriptomic analysis identified that metabolic changes were predominantly enriched in Kupffer cells at the early inflammatory phase of a mouse endotoxemia model. Among them, pyruvate dehydrogenase kinase (PDK)-dependent acute glycolysis was negatively involved in IL-10 production. Inhibition or knockdown of PDK selectively increased macrophage IL-10 expression. Mechanistically, PDK inhibition increased IL-10 production via profound phosphorylation of adenosine monophosphate (AMP)-activated protein kinase alpha 1 (AMPKα1) by restricting glucose uptake in lipopolysaccharide-stimulated macrophages. AMPKα1 consequently activated p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and cyclic AMP-responsive element-binding protein to regulate IL-10 production. Our study uncovers a previously unknown regulatory mechanism of IL-10 in activated macrophages involving an immunometabolic function of PDK.

Lactic Acid Upregulates VEGF Expression in Macrophages and Facilitates Choroidal Neovascularization.

Purpose: Lactic acid, the end product of glycolysis, has emerged as an immune-modulating metabolite in various diseases. In this study, we aimed to examine whether lactic acid contributes to the disease pathogenesis of choroidal neovascularization (CNV) and to investigate the role of macrophages in CNV pathogenesis. Methods: CNV was induced by laser photocoagulation in C57BL/6J mice. Lactic acid concentration was measured in the RPE-choroid region. Macrophage infiltration and VEGF were quantified by flow cytometry. VEGF-positive areas and CNV lesions were measured by flat-mount immunofluorescence staining. To inhibit lactic acid uptake in vivo, alpha-cyano-4-hydroxycinnamic acid (α-CHC), a monocarboxylate transporter (MCT) blocker, was injected intravitreally 1 day after laser. VEGF productions were measured in ARPE-19, THP-1 cells, and human umbilical vein endothelial cells (HUVECs) by quantitative PCR and ELISA. Angiogenic activity of lactic acid-treated macrophages was assessed by HUVEC tube formation assay. Results: Lactic acid was significantly increased in the RPE-choroid region of CNV-induced mice. Lactic acid upregulated VEGFA mRNA and VEGF protein expressions in THP-1 macrophages, but did not in ARPE-19 or HUVECs. THP-1 macrophages treated with lactic acid increased the angiogenesis of endothelial cells independent of MCT activity. Intravitreal injection of α-CHC substantially reduced the VEGF-positive area that colocalized with F4/80-positive macrophages. CNV lesions were also significantly reduced following α-CHC injection compared with vehicle-injected controls. Conclusions: To our knowledge, these results show for the first time the role of lactic acid in facilitating neovascularization through macrophage-induced angiogenesis. We suggest that targeting macrophage metabolism can be a promising strategy for CNV treatment.

Poly(ether imide)-silica hybrid coatings for tunable corrosion behavior and improved biocompatibility of magnesium implants.

Magnesium (Mg) and its alloys have gained considerable attention as a promising biomaterial for bioresorbable orthopedic implants, but the corrosion behavior of Mg-based implants is still the major issue for clinical use. In order to improve the corrosion stability and implant-tissue interfaces of these implants, methods for coating Mg have been actively investigated. In this study, poly(ether imide) (PEI)-silica hybrid material was coated on Mg, for the tunable degradation and enhanced biological behavior. Homogeneous PEI-silica hybrid materials with various silica contents were coated on Mg substrates without any cracks, where silica nanoparticles were well dispersed in the PEI matrix without significant particle agglomeration up the 30 vol% silica. The hybrid coatings maintained good adhesion strength of PEI to Mg. The corrosion rate of hybrid-coated Mg was increased along with the increment of the silica content, due to improved hydrophilicity of the hybrid coating layers. Moreover, the biocompatibility of the hybrid-coated Mg specimens was significantly improved, mainly due to the higher Mg ion concentrations associated with faster corrosion, compared to PEI-coated Mg. Therefore, PEI-silica hybrid systems have significant potential as a coating material of Mg for load-bearing orthopedic applications by providing tunable corrosion behavior and enhanced biological performance.

Trimethyltin chloride inhibits neuronal cell differentiation in zebrafish embryo neurodevelopment.

Trimethyltin chloride (TMT) is a neurotoxicant widely present in the aquatic environment, primarily from effluents of the plastic industry. It is known to cause acute neuronal death in the limbic-cerebellar system, particularly in the hippocampus. However, relatively few studies have estimated the effects of TMT toxicity on neurodevelopment. In this study, we confirmed the dose-dependent effects of TMT on neurodevelopmental stages through analysis of morphological changes and fluorescence assays using HuC-GFP and olig2-dsRed transgenic zebrafish embryos. In addition, we analyzed the expression of genes and proteins related to neurodevelopment. Exposure of embryos to TMT for 4 days post fertilization (dpf) elicited a concentration-related decrease in body length and increase in axial malformation. TMT affected the fluorescent CNS structure by decreasing pattern of HuC-GFP and olig2-dsRed transgenic zebrafish. In addition, it significantly modulated the expression patterns of Sonic hedgehog a (Shha), Neurogenin1 (Ngn1), Embryonic lethal abnormal vision like protein 3 (Elavl3), and Glial fibrillary acidic protein (Gfap). The overexpression of Shha and Ngn1, and downregulation of Elavl3 and Gfap, indicate repression of proneural cell differentiation. Our study demonstrates that TMT inhibits specific neurodevelopmental stages in zebrafish embryos and suggests a possible mechanism for the toxicity of TMT in vertebrate neurodevelopment.

Morphometric structural diversity of a natural armor assembly investigated by 2D continuum strain analysis.

Many armored fish scale assemblies use geometric heterogeneity of subunits as a design parameter to provide tailored biomechanical flexibility while maintaining protection from external penetrative threats. This study analyzes the spatially varying shape of individual ganoid scales as a structural element in a biological system, the exoskeleton of the armored fish Polypterus senegalus (bichir). X-ray microcomputed tomography is used to generate digital 3D reconstructions of the mineralized scales. Landmark-based geometric morphometrics is used to measure the geometric variation among scales and to define a set of geometric parameters to describe shape variation. A formalism using continuum mechanical strain analysis is developed to quantify the spatial geometry change of the scales and illustrate the mechanisms of shape morphing between scales. Five scale geometry variants are defined (average, anterior, tail, ventral, and pectoral fin) and their functional implications are discussed in terms of the interscale mobility mechanisms that enable flexibility within the exoskeleton. The results suggest that shape variation in materials design, inspired by structural biological materials, can allow for tunable behavior in flexible composites made of segmented scale assemblies to achieve enhanced user mobility, custom fit, and flexibility around joints for a variety of protective applications.

The first case of Capillaria hepatica infection in a nutria (Myocastor coypus) in Korea.

This study reports the first case of Capillaria hepatica infection in a nutria in Korea. Ten nutrias, captured near the Nakdong River, were submitted to our laboratory for necropsy. White-yellowish nodules were found in the liver of 1 of the nutrias at necropsy. Histologically, the lesions were granulomatous, and infiltrations of lipid-laden macrophages, eosinophils, and several multinucleated giant cells were observed. The lesions consisted of numerous eggs and necrotic hepatocytes. The eggs were lemon-shaped and had polar plugs at the ends of both long sides. The eggs were morphologically identified as those of C. hepatica. Worldwide, C. hepatica infection in nutrias is very rare. Nutrias are a kind of livestock, as well as wildlife; therefore, an epidemiological study for parasitic infections needs to be conducted.

High animal fat intake enhances prostate cancer progression and reduces glutathione peroxidase 3 expression in early stages of TRAMP mice.

BACKGROUND: Prostate cancer is the most frequently diagnosed cancer in Western men, and more men have been diagnosed at younger ages in recent years. A high-fat Western-style diet is a known risk factor for prostate cancer and increases oxidative stress. METHODS: We evaluated the association between dietary animal fat and expression of antioxidant enzymes, particularly glutathione peroxidase 3 (GPx3), in the early stages of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Six-week-old male nontransgenic and TRAMP mice were placed on high animal fat (45% Kcal fat) or control (10% Kcal fat) diets and sacrificed after 5 or 10 weeks. RESULTS: The histopathological score increased with age and high-fat diet consumption. The histopathological scores in dorsal and lateral lobes increased in the 10-week high-fat diet group (6.2±0.2 and 6.2±0.4, respectively) versus the 10-week control diet group (5.3±0.3 and 5.2±0.2, respectively). GPx3 decreased both at the mRNA and protein levels in mouse prostate. GPx3 mRNA expression decreased (∼36.27% and ∼23.91%, respectively) in the anterior and dorsolateral prostate of TRAMP mice fed a high-fat diet compared to TRAMP mice fed a control diet. Cholesterol treatment increased PC-3 human prostate cancer cell proliferation, decreased GPx3 mRNA and protein levels, and increased H2 O2 levels in culture medium. Moreover, increasing GPx3 mRNA expression by troglitazone in PC-3 cells decreased cell proliferation and lowered H2 O2 levels. CONCLUSIONS: Dietary fat enhances prostate cancer progression, possibly by suppressing GPx3 expression and increasing proliferation of prostate intraepithelial neoplasia (PIN) epithelial cells.

Direct quantification of the mechanical anisotropy and fracture of an individual exoskeleton layer via uniaxial compression of micropillars.

A common feature of the outer layer of protective biological exoskeletons is structural anisotropy. Here, we directly quantify the mechanical anisotropy and fracture of an individual material layer of a hydroxyapatite-based nanocomposite exoskeleton, the outmost ganoine of Polypterus senegalus scale. Uniaxial compression was conducted on cylindrical micropillars of ganoine fabricated via focused ion beam at different orientations relative to the hydroxyapatite rod long axis (θ = 0°, 45°, 90°). Engineering stress versus strain curves revealed significant elastic and plastic anisotropy, off-axial strain hardening, and noncatastrophic crack propagation within ganoine. Off-axial compression (θ = 45°) showed the lowest elastic modulus, E (36.2 ± 1.6 GPa, n ≥ 10, mean ± SEM), and yield stress, σ(Y) (0.81 ± 0.02 GPa), while compression at θ = 0° showed the highest E (51.8 ± 1.7 GPa) and σ(Y) (1.08 ± 0.05 GPa). A 3D elastic-plastic composite nanostructural finite element model revealed this anisotropy was correlated to the alignment of the HAP rods and could facilitate energy dissipation and damage localization, thus preventing catastrophic failure upon penetration attacks.

Threat-protection mechanics of an armored fish.

It has been hypothesized that predatory threats are a critical factor in the protective functional design of biological exoskeletons or "natural armor", having arisen through evolutionary processes. Here, the mechanical interaction between the ganoid armor of the predatory fish Polypterus senegalus and one of its current most aggressive threats, a toothed biting attack by a member of its own species (conspecific), is simulated and studied. Finite element analysis models of the quad-layered mineralized scale and representative teeth are constructed and virtual penetrating biting events simulated. Parametric studies reveal the effects of tooth geometry, microstructure and mechanical properties on its ability to effectively penetrate into the scale or to be defeated by the scale, in particular the deformation of the tooth versus that of the scale during a biting attack. Simultaneously, the role of the microstructure of the scale in defeating threats as well as providing avenues of energy dissipation to withstand biting attacks is identified. Microstructural length scale and material property length scale matching between the threat and armor is observed. Based on these results, a summary of advantageous and disadvantageous design strategies for the offensive threat and defensive protection is formulated. Studies of predator-prey threat-protection interactions may lead to insights into adaptive phenotypic plasticity of the tooth and scale microstructure and geometry, "adaptive stalemates" and the so-called evolutionary "arms race".

Materials design principles of ancient fish armour.

Knowledge of the structure-property-function relationships of dermal scales of armoured fish could enable pathways to improved bioinspired human body armour, and may provide clues to the evolutionary origins of mineralized tissues. Here, we present a multiscale experimental and computational approach that reveals the materials design principles present within individual ganoid scales from the 'living fossil' Polypterus senegalus. This fish belongs to the ancient family Polypteridae, which first appeared 96 million years ago during the Cretaceous period and still retains many of their characteristics. The mechanistic origins of penetration resistance (approximating a biting attack) were investigated and found to include the juxtaposition of multiple distinct reinforcing composite layers that each undergo their own unique deformation mechanisms, a unique spatial functional form of mechanical properties with regions of differing levels of gradation within and between material layers, and layers with an undetectable gradation, load-dependent effective material properties, circumferential surface cracking, orthogonal microcracking in laminated sublayers and geometrically corrugated junctions between layers.