Ambient fine and coarse particles in Japan affect nasal and bronchial epithelial cells differently and elicit varying immune response

Ambient particulate matter (PM) epidemiologically exacerbates respiratory and immune health, including allergic rhinitis (AR) and bronchial asthma (BA). Although fine and coarse particles can affect respiratory tract, the differences in their effects on the upper and lower respiratory tract and immune system, their underlying mechanism, and the components responsible for the adverse health effects have not been yet completely elucidated. In this study, ambient fine and coarse particles were collected at three different locations in Japan by cyclone technique. Both particles collected at all locations decreased the viability of nasal epithelial cells and antigen presenting cells (APCs), increased the production of IL-6, IL-8, and IL-1β from bronchial epithelial cells and APCs, and induced expression of dendritic and epithelial cell (DEC) 205 on APCs. Differences in inflammatory responses, but not in cytotoxicity, were shown between both particles, and among three locations. Some components such as Ti, Co, Zn, Pb, As, OC (organic carbon) and EC (elemental carbon) showed significant correlations to inflammatory responses or cytotoxicity. These results suggest that ambient fine and coarse particles differently affect nasal and bronchial epithelial cells and immune response, which may depend on particles size diameter, chemical composition and source related particles types

Novel Platform for Regulation of Extracellular Vesicles and Metabolites Secretion from Cells Using a Multi-Linkable Horizontal Co-Culture Plate

Microfluidics is applied in biotechnology research via the creation of microfluidic channels and reaction vessels. Filters are considered to be able to simulate microfluidics. A typical example is the cell culture insert, which comprises two vessels connected by a filter. Cell culture inserts have been used for years to study cell-to-cell communication. These systems generally have a bucket-in-bucket structure and are hereafter referred to as a vertical-type co-culture plate (VTCP). However, VTCPs have several disadvantages, such as the inability to simultaneously observe samples in both containers and the inability of cell-to-cell communication through the filters at high cell densities. In this study, we developed a novel horizontal-type co-culture plate (HTCP) to overcome these disadvantages and confirm its performance. In addition, we clarified the migration characteristics of substances secreted from cells in horizontal co-culture vessels. It is generally assumed that less material is exchanged between the horizontal vessels. However, the extracellular vesicle (EV) transfer was found to be twice as high when using HTCP. Other merits include control of the degree of co-culture via the placement of cells. We believe that this novel HTCP container will facilitate research on cell-to-cell communication in various fields

Combination immunohistochemistry for CK5/6, p63, GATA6, and HNF4a predicts clinical outcome in treatment-naïve pancreatic ductal adenocarcinoma

Abstract Although sequence-based studies show that basal-like features lead to worse prognosis and chemotherapy-resistance compared to the classical subtype in advanced pancreatic ductal adenocarcinoma (PDAC), a surrogate biomarker distinguishing between these subtypes in routine diagnostic practice remains to be identified. We aimed to evaluate the utility of immunohistochemistry (IHC) expression subtypes generated by unsupervised hierarchical clustering based on staining scores of four markers (CK5/6, p63, GATA6, HNF4a) applied to endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNAB) materials. EUS-FNAB materials taken from 190 treatment-naïve advanced PDAC patients were analyzed, and three IHC patterns were established (Classical, Transitional, and Basal-like pattern). Basal-like pattern (high co-expression of CK5/6 and p63 with low expression of GATA6 and HNF4a) was significantly associated with squamous differentiation histology (p < 0.001) and demonstrated the worst overall survival among our cohort (p = 0.004). IHC expression subtype (Transitional, Basal vs Classical) was an independent poor prognosticator in multivariate analysis [HR 1.58 (95% CI 1.01–2.38), p = 0.047]. Furthermore, CK5/6 expression was an independent poor prognostic factor in histological glandular type PDAC [HR 2.82 (95% CI 1.31–6.08), p = 0.008]. Our results suggest that IHC expression patterns successfully predict molecular features indicative of the Basal-like subgroup in advanced PDAC. These results provide the basis for appropriate stratification for therapeutic selection and prognostic estimation of advanced PDAC in a simplified manner

Aberrant Gene Expression and Sexually Incompatible Genomic Imprinting in Oocytes Derived from XY Mouse Embryonic Stem Cells <em>In Vitro</em>

<div><p>Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) <i>in vivo</i> and <i>in vitro</i>. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis <i>in vivo</i>; however, <i>in vitro</i> differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, <i>Dnmt3L</i>, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.</p> </div

<i>In vitro</i> maturation culture of ESC-derived FLSs.

<p>(A) Size variation of FLSs emerged in 3-day differentiation cultures of clone-G ESCs. The FLSs were separated through membrane filters with three different pore sizes (40, 70, 100 μm). Scale bar, 100 μm. (B) Prolonged culture of FLSs. Following the 3-day differentiation culture, floating FLSs were collected and transferred to another dish for further cultivation. Scale bar, 100 μm. (C) Wide-field view of prolonged FLS culture on day 16. Scale bar, 100 μm. (D) FLSs in prolonged culture on day 6. Arrowhead indicates a collapsed FLS, which underwent abnormal development. Scale bar, 100 μm. (E) FLSs developing abnormally in collagen gels emerged after 10 days of culture. Scale bar, 100 μm. (F) Maturation culture of ovarian follicles using the ‘one-drop, one-follicle’ method. Secondary follicles isolated from mouse ovaries were cultivated for 14 days. Arrowhead indicates an antrum. Scale bar, 100 μm.</p

Immunohistochemistry of ESC-derived FLSs.

<p>(A) Immunostaining of Cx43 and GDF9 in neonatal ovary and clone-G ESC-derived FLSs. Nuclei were stained with Hoechst 33342. Scale bar, 100 μm. (B) Immunostaining of E-Cadherin and ZP3 proteins in neonatal ovary and FLSs. Nuclei were stained with Hoechst 33342. Scale bar, 100 μm.</p

Transplantation of ESC-derived FLSs under the kidney capsule of SCID mice.

<p>(A) Transplantation of newborn mouse ovary under the kidney capsule of a SCID mouse. Arrowhead indicates the grafted ovary 2 weeks after transplantation. (B) PAS staining of neonatal ovary sections before transplantation. No antral follicles were observed. Scale bar, 100 μm. (C) PAS staining of the grafted ovary sections. The progression of folliculogenesis was observed in the graft as shown by antral cavity formation (arrowhead). Scale bar, 100 μm. (D) Teratoma formation following transplantation of clone-G ESC-derived FLSs under the kidney capsule of SCID mouse. (E) Sections of the teratoma derived from transplanted FLSs. The representative images show sections with gastrointestinal cells, cardiac muscle cells, muscle cells and cells that resembled pancreatic cells or glandula mammaria. Scale bar, 100 μm.</p