Evaluation of the immunotoxicity potential of nanomaterials using THP-1 cells.

With the expansion of nanomaterials (NMs) usage, concerns about their toxicity are increasing, and the wide variety of NMs makes it difficult to assess their toxicity. Therefore, the development of a high-throughput, accurate, and certified method to evaluate the immunotoxicity of NMs is required. In this study, we assessed the immunotoxicity potential of various NMs, such as nanoparticles of silver, silica, and titanium dioxide, using the human Cell Line Activation Test (h-CLAT) at the cellular level. After exposure to silver nanoparticle dispersions, the expression levels of CD86 and CD54 increased, suggesting the activation of antigen-presenting cells (APCs) by silver nanoparticles. Quantification of silver ions eluted from silver nanoparticles and the activation of APCs by silver ions suggested that it was due to the release of silver ions. Silica nanoparticles also increased the expression of CD86 and/or CD54, and their activation ability correlated with the synthesis methods and hydrodynamic diameters. The ability of titanium dioxide to activate APCs differed depending on the crystal type and hydrodynamic diameter. These results suggest a potential method to evaluate the immunotoxicity potential of various NMs based on their ability to activate APCs using human monocytic THP-1 cells. This method will be valuable in assessing the immunotoxicity potential and elucidating the immunotoxic mechanisms of NMs.

Cellular Membrane Localization of Innate Immune Checkpoint Molecule CD47 Is Regulated by Radixin in Human Pancreatic Ductal Adenocarcinoma Cells.

In the past decade, immune checkpoint inhibitors have exhibited potent antitumor efficacy against multiple solid malignancies but limited efficacy against pancreatic ductal adenocarcinoma (PDAC). Cluster of differentiation (CD) 47, a member of the immunoglobulin G superfamily, is overexpressed in the surface membrane of PDAC and independently correlates with a worse clinical prognosis. Furthermore, CD47 functions as a dominant macrophage checkpoint, providing a potent "do not eat me" signal to enable cancer cells to evade the innate immune system. Thus, the blockade of CD47 is a promising immunotherapeutic strategy for PDAC. In this study, we determined whether ezrin/radixin/moesin (ERM) family members, which post-translationally modulate the cellular membrane localization of numerous transmembrane proteins by crosslinking with the actin cytoskeleton, contribute to the cellular membrane localization of CD47 in KP-2 cells derived from human PDAC. Immunofluorescence analysis showed that CD47 and ezrin/radixin were highly co-localized in the plasma membrane. Interestingly, gene silencing of radixin but not ezrin dramatically decreased the cell surface expression of CD47 but had little effects on its mRNA level. Furthermore, CD47 and radixin interacted with each other, as determined by a co-immunoprecipitation assay. In conclusion, radixin regulates the cellular membrane localization of CD47 as a scaffold protein in KP-2 cells.

Controlling lamellarity and physicochemical properties of liposomes prepared using a microfluidic device.

The function of liposomal drugs and cosmetics is not only controlled by the lipid composition/formulation, but also by the liposome size and internal structure/properties (uni- and multi-lamellae) and membrane rigid/fluidic properties. Although the preparation of liposomes using microfluidic devices offers precise size control and easy scale-up in a continuous manufacturing system, their lamellarity and physicochemical property differences have not been investigated. We therefore prepared different paclitaxel (PTX)-loaded liposomes by changing two process parameters and investigated their physicochemical properties. The liposome size and drug loading were modified by changing the initial lipid concentration and flow rate ratio (FRR) of the aqueous and ethanol phases introduced into the microfluidic channels. Small-angle X-ray scattering and transmission electron microscopy revealed that the liposomes comprised a uni- or multi-lamellar structure that could be controlled by changing the FRR and initial lipid concentration. We also found that these structural differences affected the drug release profiles. Furthermore, the dissolution kinetics of the latter half of the drug release test could be modulated by the membrane fluidity of the liposomes. These differences in the drug release rates were consistent with the results of the in vitro cell viability assay, confirming that the multilamellar liposomes showed milder activity than the PTX solution by allowing the extended release of PTX. Thus, we concluded that the preparation of liposomes using microfluidic devices allows the liposome size, DL%, and drug release profiles to be adjusted as required.

Eliminating the contribution of lipopolysaccharide to protein allergenicity in the human cell-line activation test (h-CLAT).

We previously developed a test for detecting naturally occurring protein-induced skin sensitization based on the markers and criteria of the human cell-line activation test (h-CLAT) and showed that the h-CLAT was useful for assessing the allergenic potency of proteins. However, test proteins were contaminated with varying amounts of lipopolysaccharide (LPS), which might have contributed to the stimulation of CD86 and CD54 expression. In this study, we developed a method to exclude the effects of LPS in the assessment of skin sensitization by naturally occurring proteins. We tested two inhibitors [the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk; hereafter referred to as YVAD), which can mitigate the LPS-induced increases in CD54 expression, and polymyxin B (PMB), which suppresses the effect of LPS by binding to its lipid moiety (i.e., the toxic component of LPS)]. After a 24 hr exposure, YVAD and PMB reduced LPS-induced CD86 and CD54 expression. In particular, the effect of PMB was dependent upon pre-incubation time and temperature, with the most potent effect observed following pre-incubation at 37°C for 24 hr. Moreover, only pre-incubation with cell-culture medium (CCM) at 37°C for 24 hr showed an inhibitory effect similar to that of PMB, with this result possibly caused by components of CCM binding to LPS. Similar effects were observed in the presence of ovalbumin (with 1070 EU/mg LPS) and ovomucoid, and lysozyme (with 2.82 and 0.234 EU/mg LPS, respectively) in CCM. These results indicated that PMB and CCM effectively eliminated the effects of LPS during assessment of protein allergenicity, thereby allowing a more accurate evaluation of the potential of proteins to induce skin sensitization.

Suppression of the rice heterotrimeric G protein ß-subunit gene, RGB1, causes dwarfism and browning of internodes and lamina joint regions.

In the present study, we investigated the function of the heterotrimeric G protein ß-subunit (Gß) gene (RGB1) in rice. RGB1 knock-down lines were generated in the wild type and d1-5, a mutant deficient for the heterotrimeric G protein α-subunit (Gα) gene (RGA1). Both transgenic lines showed browning of the lamina joint regions and nodes that could be attributed to a reduction of RGB1 function, as the abnormality was not observed in d1-5. The RGB1 knock-down lines generated in d1-5 were shorter, suggesting RGB1 to be a positive regulator of cellular proliferation, in addition to RGA1. The number of sterile seeds also increased in both RGB1 knock-down lines. These results suggest that Gßγ and Gα cooperatively function in cellular proliferation and seed fertility. We discuss the potential predominant role of RGB1 in G protein signaling in rice.

[Progression of gastric cancer observed for three and a half years under S-1 administration].

A 60-year-old male patient noticed a sudden epigastric pain and visited our hospital for treatment. Active gastric ulcer was discovered by a routine gastrointestinal endoscopy. Moderately differenciated tubular adenocarcinoma was confirmed by biopsy and was advised to take surgical treatment. However, he refused to go on with gastrectomy and selected to take S-1 as an oral anti-tumor drug instead. Despite many approaches to convince the patient to have surgery, three and a half years passed. Macroscopic findings have changed from 0-IIc type to infiltrating advanced type cancer. Finally, we persuaded him to take a surgical resection, but peritoneal dissemination was evident. Using S-1 against gastric cancer is common. However, S-1 should not be used before another evidence-based curative treatment is available. Furthermore, drugs should have been changed to others, if not effective. A rare clinical course is discussed.