MGL2+ Dermal Dendritic Cells Are Sufficient to Initiate Contact Hypersensitivity In Vivo

BACKGROUND:Dendritic cells (DCs) are the most potent antigen-presenting cells in the mammalian immune system. In the skin, epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs) survey for invasive pathogens and present antigens to T cells after migration to the cutaneous lymph nodes (LNs). So far, functional and phenotypic differences between these two DC subsets remain unclear due to lack of markers to identify DDCs. METHODOLOGY/PRINCIPAL FINDINGS:In the present report, we demonstrated that macrophage galactose-type C-type lectin (MGL) 2 was exclusively expressed in the DDC subset in the skin-to-LN immune system. In the skin, MGL2 was expressed on the majority (about 88%) of MHCII(+)CD11c(+) cells in the dermis. In the cutaneous LN, MGL2 expression was restricted to B220(-)CD8alpha(lo)CD11b(+)CD11c(+)MHCII(hi) tissue-derived DC. MGL2(+)DDC migrated from the dermis into the draining LNs within 24 h after skin sensitization with FITC. Distinct from LCs, MGL2(+)DDCs localized near the high endothelial venules in the outer T cell cortex. In FITC-induced contact hypersensitivity (CHS), adoptive transfer of FITC(+)MGL2(+)DDCs, but not FITC(+)MGL2(-)DCs into naive mice resulted in the induction of FITC-specific ear swelling, indicating that DDCs played a key role in initiation of immune responses in the skin. CONCLUSIONS/SIGNIFICANCE:These results demonstrated the availability of MGL2 as a novel marker for DDCs and suggested the contribution of MGL2(+) DDCs for initiating CHS

Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology

細胞膜上の分子がバレエの群舞のように見えてきた： 1蛍光分子の感度で、究極速度で撮像できるカメラを開発. 京都大学プレスリリース. 2023-06-06.The spatial resolution of fluorescence microscopy has recently been greatly enhanced. However, improvements in temporal resolution have been limited, despite their importance for examining living cells. Here, we developed an ultrafast camera system that enables the highest time resolutions in single fluorescent-molecule imaging to date, which were photon-limited by fluorophore photophysics: 33 and 100 µs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore we identified. Using theoretical frameworks developed for the analysis of single-molecule trajectories in the plasma membrane (PM), this camera successfully detected fast hop diffusion of membrane molecules in the PM, previously detectable only in the apical PM using less preferable 40-nm gold probes, thus helping to elucidate the principles governing the PM organization and molecular dynamics. Furthermore, as described in the companion paper, this camera allows simultaneous data acquisitions for PALM/dSTORM at as fast as 1 kHz, with 29/19 nm localization precisions in the 640 × 640 pixel view-field

Mobile monitoring along a street canyon and stationary forest air monitoring of formaldehyde by means of a micro gas analysis system

A micro-gas analysis system (μGAS) was developed for mobile monitoring and continuous measurements of atmospheric HCHO. HCHO gas was trapped into an absorbing/reaction solution continuously using a microchannel scrubber in which the microchannels were patterned in a honeycomb structure to form a wide absorbing area with a thin absorbing solution layer. Fluorescence was monitored after reaction of the collected HCHO with 2,4-pentanedione (PD) in the presence of acetic acid/ammonium acetate. The system was portable, battery-driven, highly sensitive (limit of detection = 0.01 ppbv) and had good time resolution (response time 50 s). The results revealed that the PD chemistry was subject to interference from O3. The mechanism of this interference was investigated and the problem was addressed by incorporating a wet denuder. Mobile monitoring was performed along traffic roads, and elevated HCHO levels in a street canyon were evident upon mapping of the obtained data. The system was also applied to stationary monitoring in a forest in which HCHO formed naturally via reaction of biogenic compounds with oxidants. Concentrations of a few ppbv-HCHO and several-tens of ppbv of O3 were then simultaneously monitored with the μGAS in forest air monitoring campaigns. The obtained 1 h average data were compared with those obtained by 1 h impinger collection and offsite GC-MS analysis after derivatization with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBOA). From the obtained data in the forest, daily variations of chemical HCHO production and loss are discussed

Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics

細胞膜上の分子がバレエの群舞のように見えてきた： 1蛍光分子の感度で、究極速度で撮像できるカメラを開発. 京都大学プレスリリース. 2023-06-06.Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized. They revealed the dynamic nanoorganization of the focal adhesion (FA), leading to the compartmentalized archipelago FA model, consisting of FA-protein islands with broad diversities in size (13–100 nm; mean island diameter ≈30 nm), protein copy numbers, compositions, and stoichiometries, which dot the partitioned fluid membrane (74-nm compartments in the FA vs. 109-nm compartments outside the FA). Integrins are recruited to these islands by hop diffusion. The FA-protein islands form loose ≈320 nm clusters and function as units for recruiting FA proteins

Preoperative risk stratification of lymph node metastasis for non-functional pancreatic neuroendocrine neoplasm: An international dual-institutional study

BACKGROUND: /Objectives: Although the presence of lymph node metastasis (LNM) defines malignant potential, preoperative prediction of LNM has not been established for non-functional pancreatic neuroendocrine neoplasm (NF-PNEN). We sought to develop a prediction system using only preoperatively available factors that would stratify the risk of LNM for NF-PNEN. METHODS: We retrospectively reviewed patients who underwent R0/1 resection of NF-PNEN at Kyoto University (2007-2019) and the University of California, San Francisco (2010-2019). Risk stratification of LNM was developed using preoperative factors by the logistic regression analysis. Long-term outcomes were compared across the risk groups. RESULTS: A total of 131 patients were included in this study. Lymph nodes were pathologically examined in 116 patients, 23 (20%) of whom had LNM. Radiological tumor size [1.5-3.5 cm (odds ratio: 13.5, 95% confidence interval: 1.77-398) and >3.5 cm (72.4, 9.06-2257) against ≤1.5 cm], <50% cystic component (8.46 × 10^6, 1.68 × 10^106-), and dilatation of main pancreatic duct ≥5 mm (31.2, 3.94-702) were independently associated with LNM. When patients were classified as the low-risk (43 patients), intermediate-risk (44 patients), and high-risk groups (29 patients), proportions of LNM differed significantly across the groups (0%, 14%, and 59%, respectively). Recurrence-free survival (RFS) of the low- and intermediate-risk groups were significantly better than that of the high-risk group (5-year RFS rates of 92.2%, 85.4%, and 47.1%, respectively). CONCLUSIONS: The prediction system using preoperative radiological factors stratifies the risk of LNM for NF-PNEN. This stratification helps to predict malignant potential and determine the surgical procedure and necessity of regional lymphadenectomy

Safety and tolerability of bosutinib in patients with amyotrophic lateral sclerosis (iDReAM study) : A multicentre, open-label, dose-escalation phase 1 trial

Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the loss of motor neurons, and development of effective medicines is urgently required. Induced pluripotent stem cell (iPSC)-based drug repurposing identified the Src/c-Abl inhibitor bosutinib, which is approved for the treatment of chronic myelogenous leukemia (CML), as a candidate for the molecular targeted therapy of ALS. Methods: An open-label, multicentre, dose-escalation phase 1 study using a 3 + 3 design was conducted in 4 hospitals in Japan to evaluate the safety and tolerability of bosutinib in patients with ALS. Furthermore, the exploratory efficacy was evaluated using Revised ALS Functional Rating Scale (ALSFRS-R), predictive biomarkers including plasma neurofilament light chain (NFL) were explored, and single-cell RNA sequencing of iPSC-derived motor neurons was conducted. Patients, whose total ALSFRS-R scores decreased by 1–3 points during the 12 week, received escalating doses starting from 100 mg quaque die (QD) up to 400 mg QD based on dose-limiting toxicity (DLT) occurrence, and all participants who received one dose of the study drug were included in the primary analysis. This trial is registered with ClinicalTrials.gov, NCT04744532, as Induced pluripotent stem cell-based Drug Repurposing for Amyotrophic Lateral Sclerosis Medicine (iDReAM) study. Findings: Between March 29, 2019 and May 7, 2021, 20 patients were enrolled, 13 of whom received bosutinib treatment and 12 were included in the safety and efficacy analyses. No DLTs were observed up to 300 mg QD, but DLTs were observed in 3/3 patients of the 400 mg QD cohort. In all patients receiving 100 mg–400 mg, the prevalent adverse events (AEs) were gastrointestinal AEs in 12 patients (92.3%), liver function related AEs in 7 patients (53.8%), and rash in 3 patients (23.1%). The safety profile was consistent with that known for CML treatment, and ALS-specific AEs were not observed. A subset of patients (5/9 patients) was found to respond well to bosutinib treatment over the 12-week treatment period. It was found that the treatment-responsive patients could be distinguished by their lower levels of plasma NFL. Furthermore, single-cell RNA sequencing of iPSC-derived motor neurons revealed the pathogenesis related molecular signature in patients with ALS showing responsiveness to bosutinib. Interpretation: This is the first trial of a Src/c-Abl inhibitor, bosutinib, for patients with ALS. The safety and tolerability of bosutinib up to 300 mg, not 400 mg, in ALS were described, and responsiveness of patients on motor function was observed. Since this was an open-label trial within a short period with a limited number of patients, further clinical trials will be required