Journal: Nanoscale

Article Title: Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

doi: 10.1039/c5nr01851j

Figure Lengend Snippet: Figure 1: Trigger-dependent Microvesicle Shedding. Scanning electron micrograph (a) and size-distribution assessed by NTA (b) of PMN-derived microvesicles originating from PMNs incubated with plasma-opsonized S. aureus bacteria, E. coli, LPS, heat-inactivated bacteria bioparticles or vehicle (HBSS). PMN-derived CD11β/CD18 and CD11β/CD177-double positive events assessed by flow cytometry as a function of bacterial triggering agent (n = 3) (c). Scanning (d,e) and transmission electron micrographs (f,g) of PMNs showing pronounced membrane budding and shedding of microvesicles following incubation with opsonised S. aureus particles for 30 minutes (arrow indicates S. aureus particle) (e,g) compared to PMNs incubated with HBSS (d,f). 3D-tomographies and outer surface reconstructions of PMN incubated with S. aureus further confirmed the constriction of vesicles from the outer membrane seen in TEM (h). Raman spectroscopy maps of PMN incubated with (top, I) or without (bottom, II, control) bacteria showed lipid droplets and peri-membranous accumulation of glycogen granules in stimulated PMNs (I) compared to control (II) (i). PMNs exposed to S. aureus compared to resting PMNs (Figure 1d,e). Transmission electron micrographs of thin sections of PMNs containing phagocytised S. aureus bacteria confirmed increased membrane budding and formation of microvesicles (Figure 1f,g). Formation of glycogen granule clusters, translocation and peri- membranous massing of glycogen granule aggregates, and shipping of cytoplasmatic microvesicles containing glycogen granules were observed in PMNs exposed to bacteria, while glycogen granules remained well-dispersed in the cytoplasm of unstimulated PMNs (Figure 1f,g). 3D-tomography of PMNs further confirmed

Article Snippet: For flow cytometry, FITC mouse anti-rat CD18 (WT3, IgG1, AbD Serotec) and Alexa-647 anti-rat CD11β (OX-42, IgG2a, κ, BioLegend) were used for double staining at a concentration of 1 μg mL−1.

Techniques: Derivative Assay, Incubation, Clinical Proteomics, Bacteria, Flow Cytometry, Transmission Assay, Membrane, Raman Spectroscopy, Control, Translocation Assay, Tomography

Journal: Nanoscale

Article Title: Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

doi: 10.1039/c5nr01851j

Figure Lengend Snippet: Figure 3. Microvesicles in Plasma Samples from an Experimental Sepsis Model. Caecal ligation and puncture (CLP) procedure in rats (a). Time-dependent concentration of neutrophil- derived CD11β/CD18-double positive microvesicles assessed by flow cytometry (b). Aggregation of S. aureus bacteria standard with microvesicle isolates from animal plasma at the 24 and 48 hour time point (c) and corresponding ROC curves (d). Characterization of Microvesicle-Bacteria Aggregates In order to better understand the nature of the microvesicle-bacteria aggregates, we used an in vitro analysis to further characterize their properties. The CD11β-positivity of the aggregating human PMN- derived vesicles was confirmed by immunostaining (Figure 4a) and transmission electron micrographs of microvesicle-bacteria aggregates were recorded (Figure 4b). The microvesicle- concentration dependence of bacteria aggregation was confirmed by serially diluting microvesicle isolates from PMNs exposed to S.

Article Snippet: For flow cytometry, FITC mouse anti-rat CD18 (WT3, IgG1, AbD Serotec) and Alexa-647 anti-rat CD11β (OX-42, IgG2a, κ, BioLegend) were used for double staining at a concentration of 1 μg mL−1.

Techniques: Clinical Proteomics, Ligation, Concentration Assay, Derivative Assay, Flow Cytometry, Bacteria, In Vitro, Immunostaining, Transmission Assay

Journal: PLoS ONE

Article Title: Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4 + T Cells from Patients with Vogt-Koyanagi-Harada Syndrome

doi: 10.1371/journal.pone.0014616

Figure Lengend Snippet: The differently expressed proteins identified based on single peptides in CD4 + T cells between active VKH patients and normal individuals.

Article Snippet: Membranes were incubated with antibodies at dilutions of 1∶1000 for both anti-human CD18 mAb (R&D systems, Minneapolis, MN) and anti-human AKNA mAb (Genway Biotech, San Diego, CA).

Techniques: Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4 + T Cells from Patients with Vogt-Koyanagi-Harada Syndrome

doi: 10.1371/journal.pone.0014616

Figure Lengend Snippet: Antibodies were used at a dilution of 1∶1000 for the anti-human CD18 monoclonal antibody and anti-human AKNA monoclonal antibody. Proteins were detected using the Phototope-HRP Western blot detection system(A). The immunoreactive band intensities were quantitated and were presented as intensity volumes (vol%). The results showed that both CD18 and AKNA were significantly down-regulated in VKH patients as compared to normal controls (B). VKH: VKH patients, NC: normal controls.

Article Snippet: Membranes were incubated with antibodies at dilutions of 1∶1000 for both anti-human CD18 mAb (R&D systems, Minneapolis, MN) and anti-human AKNA mAb (Genway Biotech, San Diego, CA).

Techniques: Western Blot