Eunjoo Song

Caspase-8 controls the secretion of inflammatory lysyl-tRNA synthetase in exosomes from cancer cells

Aminoacyl-tRNA synthetases (ARSs), enzymes that normally control protein synthesis, can be secreted and have different activities in the extracellular space, but the mechanism of their secretion is not understood. This study describes the secretion route of the ARS lysyl-tRNA synthetase (KRS) and how this process is regulated by caspase activity, which has been implicated in the unconventional secretion of other proteins. We show that KRS is secreted from colorectal carcinoma cells within the lumen of exosomes that can trigger an inflammatory response. Caspase-8 cleaved the N-terminal of KRS, thus exposing a PDZ-binding motif located in the C terminus of KRS. Syntenin bound to the exposed PDZ-binding motif of KRS and facilitated the exosomic secretion of KRS dissociated from the multi-tRNA synthetase complex. KRS-containing exosomes released by cancer cells induced macrophage migration, and their secretion of TNF-&agr; and cleaved KRS made a significant contribution to these activities, which suggests a novel mechanism by which caspase-8 may promote inflammation.

Optical clearing based cellular-level 3D visualization of intact lymph node cortex.

Lymph node (LN) is an important immune organ that controls adaptive immune responses against foreign pathogens and abnormal cells. To facilitate efficient immune function, LN has highly organized 3D cellular structures, vascular and lymphatic system. Unfortunately, conventional histological analysis relying on thin-sliced tissue has limitations in 3D cellular analysis due to structural disruption and tissue loss in the processes of fixation and tissue slicing. Optical sectioning confocal microscopy has been utilized to analyze 3D structure of intact LN tissue without physical tissue slicing. However, light scattering within biological tissues limits the imaging depth only to superficial portion of LN cortex. Recently, optical clearing techniques have shown enhancement of imaging depth in various biological tissues, but their efficacy for LN are remained to be investigated. In this work, we established optical clearing procedure for LN and achieved 3D volumetric visualization of the whole cortex of LN. More than 4 times improvement in imaging depth was confirmed by using LN obtained from H2B-GFP/actin-DsRed double reporter transgenic mouse. With adoptive transfer of GFP expressing B cells and DsRed expressing T cells and fluorescent vascular labeling by anti-CD31 and anti-LYVE-1 antibody conjugates, we successfully visualized major cellular-level structures such as T-cell zone, B-cell follicle and germinal center. Further, we visualized the GFP expressing metastatic melanoma cell colony, vasculature and lymphatic vessels in the LN cortex.

In vivo longitudinal cellular imaging of small intestine by side-view endomicroscopy

Visualization of cellular dynamics in the gastrointestinal tract of living mouse model to investigate the pathophysiology has been a long-pursuing goal. Especially, for chronic disease such as Crohns disease, a longitudinal observation of the luminal surface of the small intestine in the single mouse is highly desirable to investigate the complex pathogenesis in sequential time points. In this work, by utilizing a micro-GRIN lens based side-view endomicroscope integrated into a video-rate confocal microscopy system, we successfully performed minimally-invasive in vivo cellular-level visualization of various fluorescent cells and microvasculature in the small intestinal villi. Also, with a transgenic mouse universally expressing photoconvertible protein, Kaede, we demonstrated repetitive cellular-level confocal endoscopic visualization of same area in the small intestinal lumen of a single mouse, which revealed the continuous homeostatic renewal of the small intestinal epithelium.

1457: CAPILLARY ENTRAPMENT OF MAC-1+ NEUTROPHIL DISTURBS PULMONARY MICROCIRCULATION IN SEPSIS-INDUCED ARDS

www.ccmjournal.org Critical Care Medicine • Volume 46 • Number 1 (Supplement) Learning Objectives: Ascorbic acid appears to be important for endothelial function and immune function. Ascorbid acid levels fall during critical illness. We hypothesized that considerable disruption of metabolic homeostasis would occur in critical illness relative to plasma ascorbic acid levels. Methods: We performed a metabolomics study on biorepository plasma samples collected from a single academic medical center on 90 adults with systemic inflammatory response syndrome or sepsis. We first generated metabolomic data using gas and liquid chromatography mass spectroscopy. We then utilized multivariable logistic regression to determine the association between plasma ascorbic acid and 28-day mortality. We performed fold change analysis based on false discovery rate adjusted p values to evaluate the distribution of individual metabolite concentrations relative to ascorbic acid levels. We followed this by partial least squaresdiscriminant analysis to identify individual metabolites that discriminated ascorbic acid levels. We then interrogated the entire metabolomics profile using pathway over-representation analysis to identify groups of metabolite pathways that were differential relative to ascorbic acid levels. Results: Ascorbic acid plasma levels were significantly lower in patients who died by 28 days (False Discovery Rate adjusted P = 0.043). Following adjustment for APACHE II score, renal function and sepsis, higher plasma ascorbic acid levels early in the ICU stay were associated with lower 28-day mortality [OR = 0.72 (95%CI 0.52–0.98);P = 0.039]. Metabolomic profiles significantly differed in critically ill patients relative to plasma ascorbic acid levels. In particular, decreased erythrulose (a metabolite of ascorbic acid) was the only strong predictor of low ascorbic acid levels. No metabolite pathways were significantly altered with regard to Ascorbic Acid levels. Conclusions: Low Ascorbic Acid levels early in severe critical illness are a robust predictor of 28day mortality. Differential metabolic profiles during critical illness exist according to Ascorbic Acid levels but limited to direct metabolites of Ascorbic Acid. Metabololite pathyways did not appear to be disrupted relative to differential Ascorbic Acid levels.

Intravital longitudinal wide-area imaging of dynamic bone marrow engraftment and multilineage differentiation through nuclear-cytoplasmic labeling

Bone marrow is a vital tissue that produces the majority of erythrocytes, thrombocytes, and immune cells. Bone marrow transplantation (BMT) has been widely performed in patients with blood disorders and cancers. However, the cellular-level behaviors of the transplanted bone marrow cells over wide-areas of the host bone marrow after the BMT are not fully understood yet. In this work, we performed a longitudinal wide-area cellular-level observation of the calvarial bone marrow after the BMT in vivo. Using a H2B-GFP/β-actin-DsRed double-transgenic mouse model as a donor, a subcellular-level nuclear-cytoplasmic visualization of the transplanted bone marrow cells was achieved, which enabled a direct in vivo dynamic monitoring of the distribution and proliferation of the transplanted bone marrow cells. The same spots in the wide-area of the calvarial bone marrow were repeatedly identified using fluorescently labeled vasculature as a distinct landmark. It revealed various dynamic cellular-level behaviors of the transplanted BM cells in early stage such as cluster formation, migration, and active proliferation in vivo.

In vivo lung imaging in pulmonary disease model

We present an in vivo lung imaging of murine model for pulmonary disease by using custom-design video-rate laser-scanning confocal microscopy integrated with suction chamber for motion stabilization.