Surface plasmon microscopy by spatial light switching for label-free imaging with enhanced resolution.

In this Letter, we describe spatially switched surface plasmon microscopy (ssSPM) based on two-channel momentum sampling. The performance evaluated with periodic nanowires in comparison with conventional SPM and bright-field microscopy shows that the resolution of ssSPM is enhanced by almost 15 times over conventional SPM. ssSPM provides an extremely simple way to attain diffraction limit in SPM and to go beyond for super-resolution in label-free microscopy techniques.

5 nm Silver Nanoparticles Amplify Clinical Features of Atopic Dermatitis in Mice by Activating Mast Cells.

The triggering effect of silver nanoparticles (NPs) on the induction of allergic reactions is evaluated, by studying the activation of mast cells and the clinical features of atopic dermatitis in a mouse model. Granule release is induced in RBL-2H3 mast cells by 5 nm, but not 100 nm silver NPs. Increases in the levels of reactive oxygen species (hydrogen peroxide and mitochondrial superoxide) and intracellular Ca++ in mast cells are induced by 5 nm silver NPs. In a mouse model of atopic dermatitis induced by a mite allergen, the skin lesions are more severe and appear earlier in mice treated simultaneously with 5 nm silver NPs and allergen compared with mice treated with allergen alone or 100 nm silver NPs and allergen. The histological findings reveal that number of tryptase-positive mast cells and total IgE levels in the serum increase in mice treated with 5 nm silver NPs and allergen. The results in this study indicate that cotreatment with 5 nm silver NPs stimulates mast cell degranulation and induces earlier and more severe clinical alterations in allergy-prone individuals.

Macrophage Differentiation from Monocytes Is Influenced by the Lipid Oxidation Degree of Low Density Lipoprotein.

LDL plays an important role in atherosclerotic plaque formation and macrophage differentiation. However, there is no report regarding the oxidation degree of LDL and macrophage differentiation. Our study has shown that the differentiation into M1 or M2 macrophages is related to the lipid oxidation level of LDL. Based on the level of lipid peroxidation, LDL is classified into high-oxidized LDL (hi-oxLDL) and low-oxidized LDL (low-oxLDL). The differentiation profiles of macrophages were determined by surface receptor expression and cytokine secretion profiles. Low-oxLDL induced CD86 expression and production of TNF-α and IL-12p40 in THP-1 cells, indicating an M1 macrophage phenotype. Hi-oxLDL induced mannose receptor expression and production of IL-6 and monocyte chemoattractant protein-1, which mostly match the phenotype of M2 macrophages. Further supporting evidence for an M2 polarization by hi-oxLDL was the induction of LOX-1 in THP-1 cells treated with hi-oxLDL but not with low-oxLDL. Similar results were obtained in primary human monocytes. Therefore, our results strongly suggest that the oxidation degree of LDL influences the differentiation of monocytes into M1 or M2 macrophages and determines the inflammatory fate in early stages of atherosclerosis.

A Case of Recurrent Renal Infarction Following Transient Resolution: Evidence From Serial Computed Tomography.

Although renal infarction (RI) is not a rare disease, its outcomes have not been well-documented. Furthermore, transient resolution and recurrence of RI have not been captured through imaging. We report a case of idiopathic RI that recurred within a short period following transient resolution, as demonstrated by serial computed tomography (CT). A 53-year-old man diagnosed with RI was transferred to the emergency room. An abdominal CT scan at the local hospital revealed a segmental wedge-shaped perfusion defect in the left kidney and a focal thrombotic filling defect in the anterior segmental branch of the left renal artery. Since his left flank pain improved, another CT scan was performed again 6 hours after the initial CT scan. A repeat CT scan showed that the thrombus in the renal artery remained, but the perfusion defect had spontaneously resolved. We initiated anticoagulant therapy using unfractionated heparin. On the sixth day of hospitalization, the left flank pain recurred, prompting another CT scan. The follow-up CT scan confirmed that RI had recurred in the same area as before. We continued anticoagulant therapy and switched to warfarin. After treatment, his symptoms improved, and he was discharged. RI can recur at any time, even after it has spontaneously resolved, as evidenced by our case. Therefore, it is crucial to closely monitor patients who experience resolution of RI for any recurrence of symptoms, and repeat radiological evaluation should be performed even within a short period.

Fatal Hypermagnesemia in Patients Taking Magnesium Hydroxide.

Hypermagnesemia is a rare but potentially fatal electrolyte disorder often overlooked because of its unfamiliarity. Magnesium is regulated through a balance of bone, intestinal absorption, and renal excretion. Hypermagnesemia typically arises from excessive magnesium intake or reduced renal excretion; however, it also occurs in patients with normal kidney function. Herein, we report two cases of hypermagnesemia in patients taking magnesium hydroxide for constipation. The first case involved an 82-year-old woman with end-stage renal disease who developed metabolic encephalopathy due to hypermagnesemia, after taking 3,000 mg of magnesium hydroxide daily for constipation. Her magnesium level was 9.9 mg/dL. Her treatment involved discontinuing magnesium hydroxide and continuing hemodialysis, which led to her recovery. In the second case, a 50-year-old woman with a history of cerebral hemorrhage and mental retardation developed hypermagnesemia despite having normal renal function. She was also taking magnesium hydroxide for constipation, and her magnesium level was 11.0 mg/dL. She experienced cardiac arrest while preparing for continuous renal replacement therapy (CRRT). After achieving return of spontaneous circulation, CRRT was initiated, and her magnesium level showed a decreasing trend. However, vital signs and lactate levels did not recover, leading to death. These cases highlight the importance of prompt diagnosis and intervention for hypermagnesemia and the need to regularly monitor magnesium levels in individuals receiving magnesium-containing preparations, especially those with impaired kidney function.

Increased Interleukin-11 and Stress-Related Gene Expression in Human Endothelial and Bronchial Epithelial Cells Exposed to Silver Nanoparticles.

This article aimed to identify and distinguish the various responses to silver nanoparticles (NPs) of endothelial and epithelial cells. We also assessed the significantly increased gene expression levels, as shown by microarray analysis. We evaluated the median lethal dose of NPs in each cell line and found that each value was different. We also confirmed the toxicity of 5 nm silver NPs. Meanwhile, cell death was not observed in cells exposed to 100 nm silver NPs at a high concentration. We verified that 5 nm silver NPs affected the variation in gene expression in cells through microarray analysis and observed a noticeable increase in interleukin (IL)-8 and IL-11 gene expression in early stages. This study showed noticeable variation in the expression of oxidative stress-related genes in early stages. Microarray results showed considerable variation in cell death-, apoptosis-, and cell survival-related gene expression. Of note, IL-11 gene expression was particularly increased following the exposure of endothelial and epithelial cells to 5 nm silver NPs. In conclusion, this study demonstrated that intracellular genes specifically responded to silver NPs in respiratory epithelial cells and endothelial cells. Among cytokine genes, IL-11 expression was noticeably increased. Additionally, we confirmed that NP toxicity was affected by NP size and dose.

Silver Nanoparticles Induce Neutrophil Extracellular Traps Via Activation of PAD and Neutrophil Elastase.

Silver nanoparticles (AgNPs) are widely used in various fields because of their antimicrobial properties. However, many studies have reported that AgNPs can be harmful to both microorganisms and humans. Reactive oxygen species (ROS) are a key factor of cytotoxicity of AgNPs in mammalian cells and an important factor in the immune reaction of neutrophils. The immune reactions of neutrophils include the expulsion of webs of DNA surrounded by histones and granular proteins. These webs of DNA are termed neutrophil extracellular traps (NETs). NETs allow neutrophils to catch and destroy pathogens in extracellular spaces. In this study, we investigated how AgNPs stimulate neutrophils, specifically focusing on NETs. Freshly isolated human neutrophils were treated with 5 or 100 nm AgNPs. The 5 nm AgNPs induced NET formation, but the 100 nm AgNPs did not. Subsequently, we investigated the mechanism of AgNP-induced NETs using known inhibitors related to NET formation. AgNP-induced NETs were dependent on ROS, peptidyl arginine deiminase, and neutrophil elastase. The result in this study indicates that treatment of 5 nm AgNPs induce NET formation through histone citrullination by peptidyl arginine deiminase and histone cleavage by neutrophil elastase.

Large field-of-view nanometer-sectioning microscopy by using metal-induced energy transfer and biexponential lifetime analysis.

Total internal reflection fluorescence (TIRF) microscopy, which has about 100-nm axial excitation depth, is the method of choice for nanometer-sectioning imaging for decades. Lately, several new imaging techniques, such as variable angle TIRF microscopy, supercritical-angle fluorescence microscopy, and metal-induced energy transfer imaging, have been proposed to enhance the axial resolution of TIRF. However, all of these methods use high numerical aperture (NA) objectives, and measured images inevitably have small field-of-views (FOVs). Small-FOV can be a serious limitation when multiple cells need to be observed. We propose large-FOV nanometer-sectioning microscopy, which breaks the complementary relations between the depth of focus and axial sectioning by using MIET. Large-FOV imaging is achieved with a low-magnification objective, while nanometer-sectioning is realized utilizing metal-induced energy transfer and biexponential fluorescence lifetime analysis. The feasibility of our proposed method was demonstrated by imaging nanometer-scale distances between the basal membrane of human aortic endothelial cells and a substrate.

Silver nanoparticles induce reactive oxygen species-mediated cell cycle delay and synergistic cytotoxicity with 3-bromopyruvate in Candida albicans, but not in Saccharomyces cerevisiae.

Background: Silver nanoparticles (AgNPs) inhibit the proliferation of various fungi; however, their mechanisms of action remain poorly understood. To better understand the inhibitory mechanisms, we focused on the early events elicited by 5 nm AgNPs in pathogenic Candida albicans and non-pathogenic Saccharomyces cerevisiae. Methods: The effect of 5 nm and 100 nm AgNPs on fungus cell proliferation was analyzed by growth kinetics monitoring and spot assay. We examined cell cycle progression, reactive oxygen species (ROS) production, and cell death using flow cytometry. Glucose uptake was assessed using tritium-labeled 2-deoxyglucose. Results: The growth of both C. albicans and S. cerevisiae was suppressed by treatment with 5 nm AgNPs but not with 100 nm AgNPs. In addition, 5 nm AgNPs induced cell cycle arrest and a reduction in glucose uptake in both fungi after 30 minutes of culture in a dose-dependent manner (P<0.05). However, in C. albicans only, an increase in ROS production was detected after exposure to 5 nm AgNPs. Concordantly, an ROS scavenger blocked the effect of 5 nm AgNPs on the cell cycle and glucose uptake in C. albicans only. Furthermore, the growth-inhibition effect of 5 nm AgNPs was not greater in S. cerevisiae mutant strains deficient in oxidative stress response genes than it was in wild type. Finally, 5 nm AgNPs together with a glycolysis inhibitor, 3-bromopyruvate, synergistically enhanced cell death in C. albicans (P<0.05) but not in S. cerevisiae. Conclusion: AgNPs exhibit antifungal activity in a manner that may or may not be ROS dependent, according to the fungal species. The combination of AgNPs with 3-bromopyruvate may be more useful against infection with C. albicans.

Double-stranded RNA mediates interferon regulatory factor 3 activation and interleukin-6 production by engaging Toll-like receptor 3 in human brain astrocytes.

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. In this study, human brain astrocytes were found to constitutively express TLR3, and this expression was increased by interferon-gamma (IFN-gamma) or double-stranded RNA (dsRNA). Treatment employing dsRNA in astrocytes induced IFN regulatory factor 3 (IRF3) phosphorylation, dimer formation and nuclear translocation followed by STAT1 activation. This treatment also activated nuclear factor-kappaB, p38 and c-Jun N-terminal kinase significantly, while activating extracellular signal-regulated kinase to a lesser extent. Treatment with anti-TLR3 antibody inhibited dsRNA-mediated interleukin-6 (IL-6) production. In the presence of mitogen-activated protein kinase inhibitors, astrocytes failed to secrete IL-6 in response to dsRNA treatment. Therefore, dsRNA-induced IL-6 production is dependent on mitogen-activated protein kinases and type I IFN production is dependent on IRF3 in brain astrocytes. These results suggest that brain inflammation, which produces inflammatory cytokines and type I IFNs, may enhance TLR3 expression in astrocytes. Additionally, upregulated TLR3 might modulate inflammatory processes by producing proinflammatory cytokines.

Unique Chemokine Profiles of Lung Tissues Distinguish Post-chemotherapeutic Persistent and Chronic Tuberculosis in a Mouse Model.

There is a substantial need for biomarkers to distinguish latent stage from active Mycobacterium tuberculosis infections, for predicting disease progression. To induce the reactivation of tuberculosis, we present a new experimental animal model modified based on the previous model established by our group. In the new model, the reactivation of tuberculosis is induced without administration of immunosuppressive agents, which might disturb immune responses. To identify the immunological status of the persistent and chronic stages, we analyzed immunological genes in lung tissues from mice infected with M. tuberculosis. Gene expression was screened using cDNA microarray analysis and confirmed by quantitative RT-PCR. Based on the cDNA microarray results, 11 candidate cytokines genes, which were obviously up-regulated during the chronic stage compared with those during the persistent stage, were selected and clustered into three groups: (1) chemokine genes, except those of monocyte chemoattractant proteins (MCPs; CXCL9, CXCL10, CXCL11, CCL5, CCL19); (2) MCP genes (CCL2, CCL7, CCL8, CCL12); and (3) TNF and IFN-γ genes. Results from the cDNA microarray and quantitative RT-PCR analyses revealed that the mRNA expression of the selected cytokine genes was significantly higher in lung tissues of the chronic stage than of the persistent stage. Three chemokines (CCL5, CCL19, and CXCL9) and three MCPs (CCL7, CCL2, and CCL12) were noticeably increased in the chronic stage compared with the persistent stage by cDNA microarray (p < 0.01, except CCL12) or RT-PCR (p < 0.01). Therefore, these six significantly increased cytokines in lung tissue from the mouse tuberculosis model might be candidates for biomarkers to distinguish the two disease stages. This information can be combined with already reported potential biomarkers to construct a network of more efficient tuberculosis markers.

Dielectric imaging for differentiation between cancer and inflammation in vivo.

In this study, we develop an in vivo dielectric imaging technique that measures capacitance using pin-type electrode arrays. Compared to normal tissues, cancer tissues exhibit higher capacitance values, allowing us to image the cancer region and monitor the chemotherapeutic effects of cancer in real-time. A comparison with the histopathological results shows that the in vivo dielectric imaging technique is able to detect small tumors (<3 mm) and tumor-associated changes. In addition, we demonstrate that cancer and inflammation may be distinguished by measuring the capacitance images at different frequencies. In contrast, the positron emission tomography using 2-[18F]-fluoro-2-deoxy-D-glucose was not capable of discriminating between cancer and inflammation.

Apoptosis of macrophages induced by Trichomonas vaginalis through the phosphorylation of p38 mitogen-activated protein kinase that locates at downstream of mitochondria-dependent caspase activation.

Trichomonas vaginalis, a flagellated protozoan parasite, is the causative organism of trichomoniasis. We have recently demonstrated that T. vaginalis induces apoptotic cell death via a Bcl-x(L)-dependent pathway in RAW264.7 macrophages. In this study, we attempted to characterize in detail the signaling cascades resulting in T. vaginalis-induced macrophage apoptosis, focusing particularly on mitochondrial changes and the role of p38 mitogen-activated protein kinase (p38 MAPK) activation. We found that T. vaginalis induced mitochondrial changes including the release of cytochrome c and the serial activation of caspases, leading to the activation of p38 MAPK in macrophages. These biochemical changes culminated in the apoptosis of the host cells. Caspase inhibitors induced a significant inhibition of T. vaginalis-induced nuclear damage, as well as the activation of p38 MAPK. Treatment with the p38 MAPK inhibitor, SB203580, or the overexpression of kinase-inactive p38 MAPK, induced an attenuation of T. vaginalis-induced apoptosis but not cytochrome c release, the activation of caspase-9 and caspase-3, or PARP cleavage. Furthermore, SB203580 treatment to human macrophages consistently blocked T. vaginalis-induced apoptosis. Collectively, our findings indicate that p38 MAPK signaling cascade is requisite to apoptosis of T. vaginalis-infected macrophage, and this apoptotic process occurs via the phosphorylation of p38 MAPK, which is located downstream of mitochondria-dependent caspase activation, conferring insight into the plausible molecular mechanism of T. vaginalis-immune evasion from macrophage attack.

Hydrogen peroxide upregulates TNF-related apoptosis-inducing ligand (TRAIL) expression in human astroglial cells, and augments apoptosis of T cells.

The brain is particularly vulnerable to oxygen free radicals, and these radicals have been implicated in the pathology of several neurological disorders. In this study, the modulation of TNF-related apoptosis-inducing ligand (TRAIL) expression by oxidative stress was shown in LN215 cells, an astroglioma cell line. Hydrogen peroxide (H2O2) treatment increased TRAIL expression in LN215 cells and H2O2-induced TRAIL augmented apoptosis in Peer cells, a cell line sensitive to TRAIL- mediated cell death. Our findings suggest that the upregulation of TRAIL in astroglial cells may abrogate immune cell effector functions.

Human brain astrocytes mediate TRAIL-mediated apoptosis after treatment with IFN-gamma.

TNF-related apoptosis inducing ligand (TRAIL) expressions were studied in primary human brain astrocytes in response to pro-inflammatory cytokines. When astrocytes were treated with IL-1beta, TNF-alpha or IFN-gamma, TRAIL was induced in cultured fetal astrocytes. In particular, IFN-gamma induced the highest levels of TRAIL in cultured astrocytes. When astrocytes were pre-treated with IFN-gamma, they induced apoptosis in TRAIL-sensitive Peer cells. Our results suggest that IFN-gamma modulates the expression of TRAIL in astrocytes, which may enhance cytotoxic sensitivity of infiltrating immune cells or brain cells other than astrocytes during inflammation of brain.

Do Helper T Cell Subtypes in Lymphocytic Thyroiditis Play a Role in the Antitumor Effect?

BACKGROUND: Papillary thyroid carcinoma (PTC) is frequently accompanied by lymphocytic thyroiditis (LT). Some reports claim that Hashimoto's thyroiditis (the clinical form of LT) enhances the likelihood of PTC; however, others suggest that LT has antitumor activity. This study was aimed to find out the relationship between the patterns of helper T cell (Th) cytokines in thyroid tissue of PTC with or without LT and the clinicopathological manifestation of PTC. METHODS: Fresh surgical samples of PTC with (13 cases) or without (10 cases) LT were used. The prognostic parameters (tumor size, extra-thyroidal extension of PTC, and lymph node metastasis) were analyzed. The mRNA levels of two subtypes of Th cytokines, Th1 (tumor necrosis factor α [TNF-α], interferon γ [IFN-γ ], and interleukin [IL] 2) and Th2 (IL-4 and IL-10), were analyzed. Because most PTC cases were microcarcinomas and recent cases without clinical follow-up, negative or faint p27 immunoreactivity was used as a surrogate marker for lymph node metastasis. RESULTS: PTC with LT cases showed significantly higher expression of TNF-α (p = .043), IFN-γ (p < .010), IL-4 (p = .015) than those without LT cases. Although the data were not statistically significant, all analyzed cytokines (except for IL-4) were highly expressed in the cases with higher expression of p27 surrogate marker. CONCLUSIONS: These results indicate that mixed Th1 (TNF-α, IFN-γ , and IL-2) and Th2 (IL-10) immunity might play a role in the antitumor effect in terms of lymph node metastasis.

Real-time monitoring of 3D cell culture using a 3D capacitance biosensor.

Three-dimensional (3D) cell cultures have recently received attention because they represent a more physiologically relevant environment compared to conventional two-dimensional (2D) cell cultures. However, 2D-based imaging techniques or cell sensors are insufficient for real-time monitoring of cellular behavior in 3D cell culture. Here, we report investigations conducted with a 3D capacitance cell sensor consisting of vertically aligned pairs of electrodes. When GFP-expressing human breast cancer cells (GFP-MCF-7) encapsulated in alginate hydrogel were cultured in a 3D cell culture system, cellular activities, such as cell proliferation and apoptosis at different heights, could be monitored non-invasively and in real-time by measuring the change in capacitance with the 3D capacitance sensor. Moreover, we were able to monitor cell migration of human mesenchymal stem cells (hMSCs) with our 3D capacitance sensor.

Silver nanoparticles affect glucose metabolism in hepatoma cells through production of reactive oxygen species.

The silver nanoparticle (AgNP) is a candidate for anticancer therapy because of its effects on cell survival and signaling. Although numerous reports are available regarding their effect on cell death, the effect of AgNPs on metabolism is not well understood. In this study, we investigated the effect of AgNPs on glucose metabolism in hepatoma cell lines. Lactate release from both HepG2 and Huh7 cells was reduced with 5 nm AgNPs as early as 1 hour after treatment, when cell death did not occur. Treatment with 5 nm AgNPs decreased glucose consumption in HepG2 cells but not in Huh7 cells. Treatment with 5 nm AgNPs reduced nuclear factor erythroid 2-like 2 expression in both cell types without affecting its activation at the early time points after AgNPs' treatment. Increased reactive oxygen species (ROS) production was detected 1 hour after 5 nm AgNPs' treatment, and lactate release was restored in the presence of an ROS scavenger. Our results suggest that 5 nm AgNPs affect glucose metabolism by producing ROS.

Two novel mutations of Wiskott-Aldrich syndrome: the molecular prediction of interaction between the mutated WASP L101P with WASP-interacting protein by molecular modeling.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, thrombocytopenia and increased susceptibility of infections, with mutations of the WAS gene being responsible for WAS and X-linked thrombocytopenia. Herein, two novel mutations of WAS at T336C on exon 3, and at 1326-1329, a G deletion on exon 10, resulting in L101P missense mutation and frameshift mutation 444 stop, respectively, are reported. The affected patients with either mutation showed severe suppression of WAS protein (WASP) levels, T cell proliferation, and CFSE-labeled T cells division. Because WASP L101 have not shown direct nuclear Overhauser effect (NOE) contact with the WASP-interacting protein (WIP) in NMR spectroscopy, molecular modeling was performed to evaluate the molecular effect of WASP P101 to WIP peptide. It is presumed that P101 induced a conformational change in the Q99 residue of WASP and made the side chain of Q99 move away from the WIP peptide, resulting in disruption of the hydrogen bond between Q99 WASP and Y475 WIP. A possible model for the molecular pathogenesis of WAS has been proposed by analyzing the interactions of WASP and WIP using a molecular modeling study.

Expression of a novel secreted splice variant of the receptor for advanced glycation end products (RAGE) in human brain astrocytes and peripheral blood mononuclear cells.

The engagement of the receptor for advanced glycation end products (RAGE) on the cell surface induces cellular dysfunction in a number of pathophysiological situations of vascular dysfunction, tumor cell invasion, inflammatory response, and T cell infiltration. The administration of truncated, soluble RAGE can modulate RAGE-mediated perturbations. Here, we report a novel splice variant (delta8-RAGE) of RAGE mRNA, which lacks exon 8 of the genomic RAGE gene and contains an early stop codon in exon 10 due to a frame shift mutation. delta8-RAGE mRNA was found in human primary astrocytes and peripheral blood mononuclear cells (PBMCs). Transient transfection experiments demonstrated that delta8-RAGE mRNA was translated into a secretory protein as deduced. Moreover, two different segments of the spliced variant were identified in PBMCs by RT-PCR. The findings of this study suggest that the diverse splicing variants of RAGE are possible in many tissues and their products may influence the RAGE-mediated pathogenesis and immune modulation.