Tumor cell derived MVs promote the proliferative impact of anti-CD3/CD28 mAb stimulation on PBMC.

<p>Freshly isolated human PBMC were kept for 3 days in the presence of CD3/CD28, CD3/CD28/MVs and MVs alone. As control untreated cells were used. For the last 14-18 h of cell culture the BrdU compound (Roche) was added. Then the cell proliferation assay was performed according to the manufacturer’s protocol. Experiments were performed in triplicates and results presented are expressed as means of OD 450 nm ± SD. The data show one representative result of four independent repeats of the experiment. The difference between the control group and the CD3/CD28 and CD3/CD28/MVs treated groups as well as the CD3/CD28 and CD3/CD28/MVs treated groups were determined by student t-test (*p≤0.005 and **p≤0.0065, respectively; n=3).</p

Colon tumor epithelial cells release MVs in response to serum starvation.

<p>A) Transmission electron micrographs of HT29 cells grown under normal conditions (a) and serum starved (b) for 48 h. After culture, cells fixed, embedded in mold, cut in thin sections and analyzed by transmission electron microscopy. B) The amount of MVs released by HT29 cells cultured in media with or without serum was counted by flow cytometry (n=3).</p

Introduction of a filtration step into the MVs isolation protocol significantly improves pureness of the isolates.

<p>A) Transmission electron micrographs of MVs isolated from serum starved HT29 supernatants by centrifugation without (a) or with (b) prior 0.8 µm filtration step. Samples were fixed, embedded in mold, cut in thin sections and observed using transmission electron microscopy. B) MVs filtered and isolated from serum starved HT29 cells were analyzed with the nanoparticle tracking analysis, allowing estimating the average size distribution of MVs.</p

Pervanadate and peroxide treatment induce tyrosine phosphorylation in cells but not in corresponding MVs.

<p>Confluent HT29 cells and corresponding MVs were treated with pervanadate or H₂O₂, respectively, or left untreated. Western blot analyzes were performed using mAb 4G10 for detecting tyrosine phosphorylation in A) the CEACAM1 immunoprecipitates, and B) the whole cell and MVs lysates. In A) CEACAM1 detection and in B) beta-actin detection served as loading control. The data show one representative result of three independent repeats of the experiment.</p

MVs released by CEACAM expressing cell types reveal a parental cell like CEACAM expression pattern.

<p>Human epithelial cell (HT29, T102/3), human endothelial cells (AS-M.5), mouse endothelial cells (bEND3), CHO and CHO-CEACAM1 cells were serum starved for 48 h. Subsequently culture supernatants were filtered (0.8 µm) before MVs were isolation. Harvested cells and MVs were analyzed by flow cytometry (A). Cells were stained for CEACAM1 (thick line). Background fluorescence was determined by incubating the cells with control IgG antibody instead of anti-CEACAM1 antibody (thin line). (B) Parts of obtained samples were lysed and then analyzed by Western blot using anti-CEACAM1-, CEACAM5- and CEACAM6- specific mAbs. Beta-actin detection served as control for equally loading. The experiments shown are representative for three independent repeats.</p

CEACAM1-positive MVs significantly increase the anti-CD3 and anti-CD3/CD28 mAb triggered T-cell proliferation.

<p>Freshly isolated human PBMC were labeled with CFSE and cultured for 4 days in the presence of anti CD3 and anti CD3/CD28 with and without CHO- and CHO-CEACAM1 derived MVs (A). B) CFSE labeled PBMC were cultured for 4 days in the presence and absence of antiCD3 and antiCD3/CD28 with and without HT29-derived MVs. Untreated treated cells served as control. In indicated cases samples were co-cultured with antiCEACAM1 mAb18/20 (50 µg/ml) or isotype matched control IgG (50 µg/ml). Then PBMCs were analyzed utilizing the Accuri C6 flow cytometer system. The histograms depict PBMCs that have divided 1-3 times based on CFSE dilution peaks and reflex the cell proliferation rate given in %. The data shown are representative for three independent repeats of the experiment.</p

CHO- and CHO-CEACAM1 derived MVs induce the CEACAM1-L tyrosine phosphorylation in confluent HT29 cells.

<p>CEACAM1 immunoprecipitates of confluent HT29 cells treated for 15 min with CHO- and CHO-CEACAM1 derived MVs were probed for tyrosine phosphorylation (upper panel) and CEACAM1 (lower panel) as control for equal loading. Untreated cells were used as negative control. Pervanadate was used to induce CEACAM1-L tyrosine phosphorylation. The data show one representative result of three independent repeats of the experiment.</p

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<p>Prematurely born infants are highly susceptible to various environmental factors, such as inflammation, drug exposure, and also high environmental oxygen concentrations. Hyperoxia induces perinatal brain injury affecting white and gray matter development. It is well known that mitogen-activated protein kinase signaling is involved in cell survival, proliferation, and differentiation. Therefore, we aim to elucidate cell-specific responses of neuronal overexpression of the small GTPase Ras on hyperoxia-mediated brain injury. Six-day-old (P6) synRas mice (neuronal Ras overexpression under the synapsin promoter) or wild-type littermates were kept under hyperoxia (80% oxygen) or room air (21% oxygen) for 24 h. Apoptosis was analyzed by Western blot of cleaved Caspase-3 and neuronal and oligodendrocyte degeneration via immunohistochemistry. Short-term differentiation capacity of oligodendrocytes was assessed by quantification of myelin basic protein expression at P11. Long-lasting changes of hyperoxia-induced alteration of myelin structures were evaluated via transmission electron microscopy in young adult animals (P42). Western blot analysis of active Caspase-3 demonstrates a significant upregulation in wild-type littermates exposed to hyperoxia whereas synRas mice did not show any marked alteration of cleaved Caspase-3 protein levels. Immunohistochemistry revealed a protective effect of neuronal Ras overexpression on neuron and oligodendrocyte survival. Hyperoxia-induced hypomyelination in wild-type littermates was restored in synRas mice. These short-term protective effects through promotion of neuronal survival translated into long-lasting improvement of ultrastructural alterations of myelin sheaths in mice with neuronal overexpression of Ras compared with hyperoxic wild-type mice. Our data suggest that transgenic increase of neuronal Ras activity in the immature brain results in secondary protection of oligodendrocytes from hyperoxia-induced white matter brain injury.</p

Associations between <i>BRAF</i> mutational status and NI in PTCs.

(A) The diagram depicts a significant association between BRAF mutational status and the number of NI/case with p = 0.042. (B-F) Significant correlation between BRAF mutational status and immunoreactivity for autophagy-associated proteins in NI is shown. (B) p62 positive NI/case vs. BRAFV600E: p = 0.024, (C) LC3B positive NI/case vs. BRAFV600E: p = 0.003, (D) Ubiquitin positive NI/case vs. BRAFV600E: p = 0.032, (E) Cathepsin B positive NI/case vs. BRAFV600E: p = 0.035 and (F) Cathepsin D positive NI/case vs. BRAFV600E: p = 0.013. Results are significant at *p≤0.05, **p≤0.01, and ***p≤0.001.</p

Patient’s characteristics: Number of cases, mean age and sex of each group of patients.

Patient’s characteristics: Number of cases, mean age and sex of each group of patients.</p