Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

Interlayer expansion of octacalcium phosphate via forced oxidation of the intercalated molecules within its interlayers.

Octacalcium phosphate (OCP), a precursor to apatite, has a layered structure that allows various molecules to be intercalated within its interlayers. Previous research on the phase conversion process of OCP to apatite indicated that the layered structures typically collapse due to the shrinking of the OCP layers. In contrast, this study presents a novel phenomenon involving OCP layer expansion during phase conversion. This expansion is based on a forced oxidation process of the intercalated molecules within the hydrous layers of OCP. By introducing NaClO to an OCP interlayer containing dithiodiglycolic acid (DSG), the OCP layers are expanded. This process involves DSG decomposition through its reaction with NaClO. Specifically, the process occurs when a DSG-substituted OCP (containing disulfide bonds (-S-S-)) is immersed in a NaClO solution. This is the first study to report the expansion phenomenon during the phase conversion process from OCP to apatite, providing a new perspective to the conventional understanding that these layers only shrink.

Evaluation of the cellular effects of silica particles used for dermal application.

The cellular effects of 5 types of spherical amorphous silica particles whose particle size were 4.2-12.8 µm for cosmetic use and two types of crystalline silica whose particle size were 2.4 and 7.1 µm particles for industrial use were examined. These silica particles were applied to HaCaT human keratinocytes for 24 hr. Crystalline silica enhanced IL-8 and IL-6 expression and caused cell membrane damage. Crystalline silica also enhanced HO-1 gene expression; however, the level of intracellular ROS did not change. Compared with crystalline silica, the cellular effects of the spherical silica employed in this study were minor. Cellular uptake of particles was observed for all of silica particle types. Cellular uptake of crystalline silica was observed 1 hr after exposure, and internalized silica particles were present in the cytoplasm. When HaCaT cells were exposed to crystalline silica for 1 hr and incubated for 23 hr in culture medium without silica particles, IL-8 expression was still detected. In addition, silica particles exerted negligible effects using a 3D skin tissue model. Thus, the following conclusions may be drawn. (1) cellular effects exerted by spherical silica are less compared to crystalline silica. (2) phagocytosis of particles is an important first step in the cellular effects of silica particles. (3) spherical silica particles might exert little, if any, effect on healthy skin attributed to no apparent cellular uptake.

Genome Sequencing Unveils Nomadic Traits of Lactiplantibacillus plantarum in Japanese Post-Fermented Tea.

Post-fermented tea production involving microbial fermentation is limited to a few regions, such as Southeast Asia and Japan, with Japan's Shikoku island being particularly prominent. Lactiplantibacillus plantarum was the dominant species found in tea leaves after anaerobic fermentation of Awa-bancha in Miyoshi City, Tokushima, and Ishizuchi-kurocha in Ehime. Although the draft genome of L. plantarum from Japanese post-fermented tea has been previously reported, its genetic diversity requires further exploration. In this study, whole-genome sequencing was conducted on four L. plantarum strains isolated from Japanese post-fermented tea using nanopore sequencing. These isolates were then compared with other sources to examine their genetic diversity revealing that L. plantarum isolated from Japanese post-fermented tea contained several highly variable gene regions associated with sugar metabolism and transportation. However, no source-specific genes or clusters were identified within accessory or core gene regions. This study indicates that L. plantarum possesses high genetic diversity and that the unique environment of Japanese post-fermented tea does not appear to exert selective pressure on L. plantarum growth.

Contaminant microorganisms in the in vitro evaluation of cellular responses of cellulose nanofibers and their microbial inactivation using gamma irradiation.

Cellulose nanofibers (CNFs) are fibrous nanomaterials produced from plants. Since some nanomaterials are toxic, toxicity evaluation, including in vitro examinations using cultured cells, is essential for the effective use of CNFs. On the other hand, microorganisms in the environment can contaminate CNF suspensions. The contamination of CNF samples and the effects of contaminating microorganisms on in vitro examinations were investigated in this study. Microorganism contamination in CNF samples was examined, and microbial inactivation of CNF suspensions using gamma irradiation was evaluated. After gamma-ray irradiation at absorbed doses of 0.5, 1, 5, and 10 kGy, the cellular effects of CNF suspensions were examined using 6 types of cultured cell, HaCaT, A549, Caco-2, MeT-5A, THP-1, and NR8383 cells. CNF samples were contaminated with bacteria and CNF suspensions exhibited endotoxin activity. Gamma irradiation effectively inactivated the microorganisms contained in the CNF suspensions. When the absorbed dose was 10 kGy, the fiber length of CNF was shortened, but the effect on CNF was small at 1.0 kGy or less. CNF suspensions showed lipopolysaccharides (LPS)-like cellular responses and strongly induced interleukin-8, especially in macrophages. Absorbed doses of at least 10 kGy did not affect the LPS-like activity. In this study, it was shown that the CNF suspension may be contaminated with microorganisms. Gamma irradiation was effective for microbial inactivation of suspension for invitor toxicity evaluation of CNF. In vitro evaluation of CNFs requires attention to the effects of contaminants such as LPS.

Fabrication of interconnected porous Ag substituted octacalcium phosphate blocks based on a dissolution-precipitation reaction.

Here, we introduce Ag substituted octacalcium phosphate (OCP-Ag) blocks with interconnected porous structure and sufficient mechanical strength as bone substitute (i.e., foam). We employed a two-step process for fabrication, which includes a setting reaction for acidic calcium phosphate granules using an acidic phosphate solution and a phase conversion process via dissolution-precipitation method in cocktail ((NH4)2HPO4-NH4NO3-NaNO3-AgNO3) solutions. The Ag contents in the fabricated OCP-Ag foams were 0.08-0.15 at%, which were sufficient in exhibiting contact antibacterial ability. The mechanical strength and porosity of the OCP-Ag foams were about 0.5 MPa and 70%, respectively. These values were sufficient for the application of the OCP-Ag foams as bone substitute. Graphical abstract.

Controlling the microbial composition during the fermentation of Ishizuchi-kurocha.

Ishizuchi-kurocha is a popular postfermented tea in Japan. It is performed by domestic and natural fermentation relied on microorganisms derived from tea leaves or the environment of the manufacturing. Ishizuchi-kurocha undergoes aerobic fermentation of fungi first, then second fermented by anaerobic fermentation of lactic acid bacteria during natural fermentation processing. Aspergillus niger that produces mycotoxin is included in natural fermentation. This research aimed to build a novel fermentation method of Ishizuchi-kurocha by adding industrial koji fungi products and laboratory-cultivated Lactobacillus plantarum (Lactiplantibacillus plantarum) artificially. Thus, safety and quality of tea products could be controlled simply. We found artificial fermentation of Ishizuchi-kurocha could get high lactic acid production within 8 days. Final products only consisted of genus Aspergillus and genus Lactobacillus, while harmful Aspergillus niger was not found. However, artificial fermentation methods also decreased the content of polyphenols when compared with commercial tea.

Proinflammatory response caused by lead nanoparticles triggered by engulfed nanoparticles.

In this study, the cellular effects of lead (Pb) nanoparticles with a primary particle size of 80 nm were evaluated in two types of cell lines: human lung carcinoma A549 and macrophage-differentiated THP-1 cells (dTHP-1). The cellular responses induced by the Pb nanoparticles varied among the cell types. Exposure to Pb nanoparticles for 24 h at a concentration of 100 µg/ml induced interleukin-8 (IL-8) expression in dTHP-1 cells. Induction of IL-8 expression in A549 was lower than dTHP-1 cells. Pb nanoparticles also induced the gene expression of heme oxygenase-1 in dTHP-1 cells but not in A549 cells. Though cellular uptake of Pb nanoparticles was observed in both the cell types, the amount of internalized Pb particles was lower in A549 cells than that in dTHP-1 cells. Gene expression of metallothionein 2A was remarkably enhanced by Pb nanoparticle exposure in dTHP-1 cells. Compared with Pb nanoparticles, induction of cytokines caused by lead nitrate (Pb[NO3 ]2 ), a water-soluble Pb compound, was smaller. In conclusion, the present study revealed that Pb nanoparticles induced a stronger cellular response than Pb(NO3 )2 , primarily by eliciting cytokine production, in a cell type-dependent manner.

Development of fibrin hydrogel-based in vitro bioassay system for assessment of skin permeability to and pro-inflammatory activity mediated by zinc ion released from nanoparticles.

Nanoparticles (NPs) are promising products in industry and medicine due to their unique physicochemical properties. In particular, zinc oxide (ZnO) NPs are extensively incorporated into sunscreens to protect the skin from exposure to ultraviolet radiation. However, there are several health concerns about skin penetration and the resultant toxicity. As methodologies for evaluating NP toxicity are under development, it is difficult to fully assess the toxicity of ZnO NPs toward humans. In this study, we developed a platform to simultaneously detect skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs. First, we generated a stable reporter cell line expressing green fluorescent protein (GFP) under the control of interleukin-8 (IL-8) promoter activity. The expression levels of GFP induced by zinc reflected the endogenous IL-8 expression levels and the pro-inflammatory responses. Next, we found that fibrin hydrogel can reproduce permeability to zinc ion of a human skin equivalent model and is therefore a promising material to assess skin permeability to zinc ion. Then, we constructed a fibrin hydrogel-based in vitro bioassay system for the simultaneous detection of skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs by using a stable reporter cell line and a fibrin hydrogel layer. This bioassay system is a promising in vitro permeation test due to its technical simplicity and good predictability. Overall, we believe that our bioassay system can be widely used in the cosmetics and pharmaceutical industries.

Changes in lactic acid bacteria and components of Awa-bancha by anaerobic fermentation.

Awa-bancha is a post-fermented tea produced in Naka and Kamikatsu, Tokushima, Japan. We investigated the lactic acid bacteria in each stage of production of Awa-bancha and evaluated the relationships with the components. Lactic acid bacteria were isolated from tea leaves cultured with de Man, Rogosa, and Sharpe (MRS) agar plates, and the species were identified by homology of the 16 S rRNA gene and multiplex polymerase chain reaction (PCR) of the recA gene to distinguish the Lactobacillus plantarum group. As a result, a variety of species were isolated from the raw tea leaves, and Lactobacillus pentosus was isolated most frequently after anaerobic fermentation. Regarding the tea leaf components, organic acids, such as lactic acid, increased, free amino acids decreased, and catechins changed owing to anaerobic fermentation. Our results suggest that the microbial flora mainly composed of L. pentosus is important in the anaerobic fermentation process for flavor formation of Awa-bancha.

Comparison of proinflammatory potential of needle-shaped materials: aragonite and potassium titanate whisker.

Among the crystal forms of calcium carbonate, aragonite has needle-like shape. Although materials with needle-shaped crystals are associated with pulmonary toxicity, the toxic activity of aragonite is unclear. Therefore, proinflammatory potential of aragonite, neutralized aragonite and potassium titanate whisker was evaluated. The cellular effects of aragonite were weaker than those of potassium titanate whisker. Aragonite treatment induced the expression of chemokines in A549 cells and macrophages. Although aragonite exhibited proinflammatory effects in vitro, pulmonary inflammation was not observed in vivo after intratracheal administration of aragonite in mice. We did not observe the induction of inflammatory cytokine secretion or tissue lesion in the lungs of mice after administration of aragonite. Potassium titanate whisker treatment induced chemokine secretion in vitro. An increase in the number of neutrophils was observed in the mice lung tissue after administration of potassium titanate whisker. Aragonite and neutralized aragonite both induced an increase in the levels of intracellular calcium, but the levels were significantly higher in cells treated with aragonite than in cells treated with neutralized aragonite. These results suggested that intracellular calcium release mediates the cellular effects of aragonite. The toxicity of aragonite based on its needle-like structure was also not observed.

The Truth of Toxicity Caused by Yttrium Oxide Nanoparticles to Yeast Cells.

Yttrium oxide (Y2O3) nanoparticles have widespread applications; however, toxicity due to these nanoparticles has also been reported. In this study, we evaluated the in vitro toxicity of Y2O3 nanoparticles according to the technical specifications published by the International Standard Organization (ISO/TS 19337:2016). We used Saccharomyces cerevisiae as a model microorganism represented the environment. We carried out catch ball analysis of yttrium oxide and yttrium ion toxicities. The result showed that Y2O3 nanoparticles (20 mg/5 ml) and YCl3 (5 mg/5 ml) treatment caused oxidative stress in yeast cells. Based on transcriptome analysis, fluorescent spectroscopy, and solubility analysis of Y2O3 nanoparticles, we conclude that the toxicity is due to yttrium ions derived from the nanoparticles. The ions induce oxidative stress and cause protein denaturation, which in turn induces proteasome formation to eliminate denatured proteins. Yttrium nanoparticles induce oxidative stress, which has associated with heavy metal ions. Thus, the use of yttrium nanoparticles or yttrium ions must be controlled like heavy metals.

Validation of metallothionein, interleukin-8, and heme oxygenase-1 as markers for the evaluation of cytotoxicity caused by metal oxide nanoparticles.

Metal oxide nanoparticles have an industrial value, although their harmful effects are also known. Induction of respiratory inflammation through their inhalation is a serious indicator of their toxicity. Although the phenomenon of metal ion release is involved in the induction of inflammation, all metal ions are not necessarily toxic. However, currently, no particular index to evaluate cytotoxicity caused by nanoparticles exists. An index based on biological response is critical. In the present study, we examined the gene expression-based index for nanoparticle-derived cytotoxicity. The cellular effects of six kinds of metal oxide nanoparticles, ZnO, NiO, CuO, MgO, Bi2O3, and MoO3 on A549 cells were examined. It was seen that lactate dehydrogenase (LDH) assay, which is one of the most important assays for assessing cell membrane damage, is inhibited by metal ions released from the metal oxide nanoparticles. In some cases, enzyme activity-based assay was not suitable for the evaluation of cytotoxicity of nanoparticles. ZnO and CuO nanoparticles displayed severe cytotoxicity and enhanced gene expression of heme oxygenase-1 (HO-1) and interleukin-8 (IL-8). The IL-8 gene expression was also increased from Bi2O3 exposure. Additionally, the gene expression of metallothionein 2A (MT2A) was enhanced in the ZnO, CuO, and Bi2O3 exposed cells. These results suggest that these nanoparticles released metal ions in the cells. The enhancement of HO-1, IL-8, and MT2A gene expressions was related to the cytotoxic activity of metal oxide nanoparticles. Thus, the expression level of these genes is a good indicator of nanotoxicology of metal oxide nanoparticles.

Early diagnosis of type 2 diabetes based on multiple biomarkers and non-invasive indices.

We previously reported that type 2 diabetes risk, early impaired glucose tolerance and insulin resistance can be predicted by measuring the fasting levels of certain biomarkers. Here we validated these findings in randomly recruited healthy volunteers (n = 101) based on biomarker expression as well as various non-invasive indices. Weight, body mass index, waist circumference and visceral fat differed between individuals with impaired fasting glucose and/or impaired glucose tolerance, and normal subjects. Fasting plasma levels of glycated hemoglobin, leptin, pro-insulin and retinol binding protein 4 differed between impaired fasting glucose/impaired glucose tolerance and normal subjects group and between newly detected diabetes and normal subjects group. Insulin resistance was correlated with fasting levels of insulin and leptin/adiponectin (r = 0.913); of insulin, retinol binding protein 4 and leptin/adiponectin (r = 0.903); and of insulin, glycated albumin, and leptin/adiponectin (r = 0.913). Type 2 diabetes risk, early impaired glucose tolerance and insulin resistance were predicted with >98% specificity and sensitivity by comparing fasting glucose levels to the estimated Matsuda Index based on fasting levels of insulin, adiponectin and leptin with or without oxidative lineolate metabolites. Non-invasive indices are slightly correlated with glucose tolerance and insulin resistance but do not increase the accuracy of predicting type 2 diabetes risk.

Effect of calcium carbonate particle shape on phagocytosis and pro-inflammatory response in differentiated THP-1 macrophages.

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO3)-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO3 particles.

Ascorbic acid prevents zinc oxide nanoparticle-induced intracellular oxidative stress and inflammatory responses.

Exposure to zinc oxide nanoparticles (ZnO NPs) promotes acute pulmonary toxicity through oxidative stress and inflammation. Furthermore, dissolved zinc from ZnO NPs induces the formation of intracellular reactive oxygen species (ROS). We previously reported that supplemental ascorbic acid (AA) inhibits ZnO NP-induced acute pulmonary toxicity in a rat model; however, the mechanism of this action remains unclear. Therefore, we investigated the effects of AA on ZnO NP-induced cytotoxicity in human lung carcinoma A549 cells. AA was found to suppress intracellular production of ROS, and thus reduce the subsequent inflammation of ZnO NPs. However, intracellular Zn2+ concentrations were higher in AA-treated cells than in AA-untreated cells. AA was found to react with Zn2+ but not with the ZnO NPs themselves. These results suggest the possibility that AA-chelated extracellular Zn2+ and the Zn-AA complex was readily taken up into cell. Even if the intracellular Zn2+ level was high, cytotoxicity might be reduced because the Zn-AA complex was stable. Co-treatment of AA to A549 inhibited ROS production and subsequent intracellular inflammatory responses. These results are consistent with those previously reported from an in vivo model. Thus, two possibilities can be considered about the cytotoxicity-reducing the effect of AA: antioxidant efficacy and chelating effect.

Cytotoxicity of CdSe-based quantum dots incorporated in glass nanoparticles evaluated using human keratinocyte HaCaT cells.

CdSe quantum dots (QDs) are potential fluorescent reagents, but leakage of Cd and Se often induces cytotoxicity. Here we prepared CdSe-based QDs with glass to reduce their leakage and examined their cytotoxicity using keratinocyte cells. The cytotoxicity of the QDs with glass was obviously lower than that of the commercial QDs with polymer, suggesting their safety for biological applications.

The induction of lipid peroxidation during the acute oxidative stress response induced by intratracheal instillation of fine crystalline silica particles in rats.

Crystalline silica (SiO2) is an important material for industry but is considered potentially carcinogenic. Inhalation of a crystalline SiO2 aerosol may contribute to serious lung diseases. Crystalline SiO2 particles are commonly used as a positive control in toxicity assays of particulate materials (e.g. nanoparticles). Crystalline SiO2 induces oxidative stress resulting in lipid peroxidation, but the acute oxidative stress response in the lung is not well understood. Lipid peroxidation during the acute stage of oxidative stress after instillation of crystalline SiO2 into rats was examined by bronchoalveolar lavage fluid (BALF) analysis. The levels of 8-iso-prostaglandin F2α and hydroxyoctadecadienoic acid (HODE) in the BALF were measured using liquid chromatography coupled to quadrupole mass spectrometry. The concentration of the antioxidant protein heme oxygenase-1 (HO-1) in the BALF was determined using enzyme-linked immunosorbent assay. Intratracheal instillation of crystalline SiO2 increased the level of HODE and HO-1 in BALF at 24 h after administration. The levels of HODE and HO-1 returned to baseline at 72 h after instillation. Lactate dehydrogenase leakage was observed only after 1 h instillation. These results suggest that the contribution of oxidative stress to the pulmonary toxicity of crystalline SiO2 is minimal in the early acute stage after exposure.

Antioxidative and Antidiabetic Effects of Natural Polyphenols and Isoflavones.

Many polyphenols that contain more than two phenolic hydroxyl groups are natural antioxidants and can provide health benefits to humans. These polyphenols include, for example, oleuropein, hydroxytyrosol, catechin, chlorogenic acids, hesperidin, nobiletin, and isoflavones. These have been studied widely because of their strong radical-scavenging and antioxidative effects. These effects may contribute to the prevention of diseases, such as diabetes. Insulin secretion, insulin resistance, and homeostasis are important factors in the onset of diabetes, a disease that is associated with dysfunction of pancreatic ß-cells. Oxidative stress is thought to contribute to this dysfunction and the effects of antioxidants on the pathogenesis of diabetes have, therefore, been investigated. Here, we summarize the antioxidative effects of polyphenols from the perspective of their radical-scavenging activities as well as their effects on signal transduction pathways. We also describe the preventative effects of polyphenols on diabetes by referring to recent studies including those reported by us. Appropriate analytical approaches for evaluating antioxidants in studies on the prevention of diabetes are comprehensively reviewed.

Does photocatalytic activity of TiO2 nanoparticles correspond to photo-cytotoxicity? Cellular uptake of TiO2 nanoparticles is important in their photo-cytotoxicity.

Titanium dioxide (TiO2) nanoparticles are important industrial nano-objects with wide applications, including as photocatalysts and sunscreen components. Recently, the phototoxicity of TiO2 nanoparticles has been a concern. However, phototoxicity caused by photocatalytic activity may differ between anatase and rutile nanoparticles. In the present study, we compared the phototoxicity of anatase and rutile nanoparticles. Human keratinocyte HaCaT cells were treated with stable TiO2 nanoparticle suspensions. Without UVA irradiation, TiO2 nanoparticles did not affect mitochondrial activity or cell membranes. However, exposure to rutile nanoparticle suspensions inhibited cell growth and induced HO-1 gene expression without UVA irradiation. These effects may be explained by the hydrophobic surface of rutile nanoparticles. Next, TiO2-exposed cells were irradiated with UVA for 4 h and effects of TiO2 nanoparticles on cells were examined. The rutile nanoparticles did not show any cellular effects after UVA irradiation. However, the anatase nanoparticles caused strong phototoxicity. Decreased mitochondrial activity, cell membrane damage and the induction of oxidative stress were observed in the cells exposed to anatase nanoparticles with UVA irradiation. Cellular uptake of the nanoparticles was observed in both anatase- and rutile-exposed cells. These results suggest that internalized anatase nanoparticles are important for phototoxicity. Additionally, the exposure of a 3D skin model to TiO2 nanoparticles did not result in significant toxicity. In conclusion, rutile nanoparticles used in sunscreen did not exhibit phototoxic activity. Despite the strong phototoxic activity of anatase nanoparticles in cell cultures, they demonstrated no phototoxicity using a 3D skin model.