Outer membrane vesicles (OMVs), nanoparticles released by Shiga toxin-producing Escherichia coli (STEC), have been identified as novel efficient virulence tools of these pathogens. STEC O157 OMVs carry a cocktail of virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, flagellin, and lipopolysaccharide. OMVs are taken up by human intestinal epithelial and microvascular endothelial cells, the major targets during STEC infection, and deliver the virulence factors into host cells. There the toxins separate from OMVs and are trafficked via different pathways to their target compartments, i.e., the cytosol (Stx2a-A subunit), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). This leads to a toxin-specific host cell injury and ultimately apoptotic cell death. Besides their cytotoxic effects, STEC OMVs trigger an inflammatory response via their lipopolysaccharide and flagellin components. In this chapter, we describe methods for the isolation and purification of STEC OMVs, for the detection of OMV-associated virulence factors, and for the analysis of OMV interactions with host cells including OMV cellular uptake and intracellular trafficking of OMVs and OMV-delivered toxins.

• MeSH
• Bacterial Toxins metabolism   MeSH
• Endothelial Cells metabolism   microbiology   pathology   MeSH
• Escherichia coli O157 * metabolism   pathogenicity   MeSH
• Virulence Factors metabolism   MeSH
• Humans MeSH
• Cell-Derived Microparticles metabolism   MeSH
• Shiga Toxin 2 metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Neonatal systemic inflammatory response and multiple organ dysfunction syndrome are the main postnatal insults influencing mortality and morbidity. Critically ill newborns with high predicted mortality are supported by extracorporeal membrane oxygenation (ECMO). Biomarkers of inflammatory response and endothelial injury can be used for early diagnosis and treatment of critical neonatal situations. The aim of our study was to explore plasma proteins and endothelial microvesicles as markers of inflammation and endothelial activation in newborns on ECMO and to compare them with healthy neonates. Thirteen newborns on ECMO and 13 healthy newborns were included in the study. Plasma soluble biomarkers were measured using multiplex immunoassay based on Luminex® xMAP multianalyte profiling platform. The total microvesicle count and plasma level of surface antigen-specific microvesicles were determined by flow cytometry. The plasma concentration of cell-derived microvesicles was measured using annexin-V labeling, and the endothelial origin of microvesicles was determined using lineage-specific antigen labeling of endothelial cell/microvesicle markers (endoglin/CD105, PECAM1/CD31, VEGFR2/CD309, and MadCAM1). Inflammatory markers (procalcitonin, IL-1β, IL-6, and IL-22) were increased in the ECMO group (P < 0.01). The assessment of endothelial markers showed higher concentrations of endocan and angiopoietin-2 (P < 0.01) in the ECMO group while VEGF in the ECMO group was significantly lower (P < 0.01). In the ECMO group, the concentration of annexin-V-positive microvesicles (total microvesicles) and endothelial microvesicles expressing mucosal vascular addressin cell adhesion molecule 1 (MadCAM1) was increased (P = 0.05). In summary, we found increased concentrations of soluble inflammatory and endothelial markers in the plasma of critically ill newborns with multiple organ dysfunction. Increased plasma concentrations of microvesicles may reflect the activation or damage of blood cells and vasculature including endothelial cells. The measurement of cell membrane-derived microvesicles may be added to the panel of established inflammatory markers in order to increase the sensitivity and specificity of the diagnostic process in critically ill newborns.

• MeSH
• Biomarkers blood   MeSH
• Cell Membrane metabolism   MeSH
• Endothelium metabolism   MeSH
• Endothelial Cells MeSH
• Critical Illness MeSH
• Humans MeSH
• Cell-Derived Microparticles metabolism   MeSH
• Extracorporeal Membrane Oxygenation * MeSH
• Infant, Newborn MeSH
• Flow Cytometry MeSH
• Inflammation blood   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Human body reacts to physical, chemical and biological insults with a complex inflammatory reaction. Crucial components and executors of this response are endothelial cells, platelets, white blood cells, plasmatic coagulation system, and complement. Endothelial injury and inflammation are associated with elevated blood levels of cell membrane-derived microvesicles. Increased concentrations of microvesicles were found in several inflammatory reactions and diseases including acute coronary syndromes, stroke, vasculitis, venous thromboembolism, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, anti-phospholipid antibody syndrome, inflammatory bowel disease, thrombotic thrombocytopenic purpura, viral myocarditis, sepsis, disseminated intravascular coagulation, polytrauma, and burns. Microvesicles can modulate a variety of cellular processes, thereby having an impact on pathogenesis of diseases associated with inflammation. Microvesicles are important mediators and potential biomarkers of systemic inflammation. Measurement of inflammatory cell-derived microvesicles may be utilized in diagnostic algorithms and used for detection and determination of severity in diseases associated with inflammatory responses, as well as for prediction of their outcome. This review focuses on the mechanisms of release of microvesicles in diseases associated with systemic inflammation and their potential role in the regulation of cellular and humoral interactions.

• MeSH
• Humans MeSH
• Cell-Derived Microparticles metabolism   MeSH
• Sepsis pathology   MeSH
• Systemic Inflammatory Response Syndrome pathology   MeSH
• Inflammation blood   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

BACKGROUND Paracrine factors secreted by adipose-derived stem cells can be captured, fractionated, and concentrated to produce therapeutic factor concentrate (TFC). The present study examined whether TFC effects could be enhanced by combining TFC with a biological matrix to provide sustained release of factors in the target region. MATERIAL AND METHODS Unilateral hind limb ischemia was induced in rabbits. Ischemic limbs were injected with either placebo control, TFC, micronized small intestinal submucosa tissue (SIS), or TFC absorbed to SIS. Blood flow in both limbs was assessed with laser Doppler perfusion imaging. Tissues harvested at Day 48 were assessed immunohistochemically for vessel density; in situ hybridization and quantitative real-time PCR were employed to determine miR-126 expression. RESULTS LDP ratios were significantly elevated, compared to placebo control, on day 28 in all treatment groups (p=0.0816, p=0.0543, p=0.0639, for groups 2-4, respectively) and on day 36 in the TFC group (p=0.0866). This effect correlated with capillary density in the SIS and TFC+SIS groups (p=0.0093 and p=0.0054, respectively, compared to placebo). A correlation was observed between miR-126 levels and LDP levels at 48 days in SIS and TFC+SIS groups. CONCLUSIONS A single bolus administration of TFC and SIS had early, transient effects on reperfusion and promotion of ischemia repair. The effects were not additive. We also discovered that TFC modulated miR-126 levels that were expressed in cell types other than endothelial cells. These data suggested that TFC, alone or in combination with SIS, may be a potent therapy for patients with CLI that are at risk of amputation.

• MeSH
• Extracellular Matrix metabolism   MeSH
• Ischemia genetics   pathology   therapy   MeSH
• Stem Cells cytology   MeSH
• Extremities blood supply   pathology   MeSH
• Rabbits MeSH
• Skin pathology   MeSH
• Laser-Doppler Flowmetry MeSH
• Middle Aged MeSH
• Humans MeSH
• MicroRNAs genetics   metabolism   MeSH
• Cell-Derived Microparticles metabolism   MeSH
• Disease Models, Animal MeSH
• Perfusion MeSH
• Gene Expression Regulation MeSH
• Reperfusion Injury pathology   therapy   MeSH
• Intestinal Mucosa physiology   MeSH
• Intestine, Small physiology   MeSH
• Stem Cell Transplantation * MeSH
• Adipose Tissue cytology   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Middle Aged MeSH
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

This review focuses on the formation, composition and function of endothelial microvesicles (MV), often called microparticles (MP). MV release is a controlled event and is considered a hallmark of cellular activation or alteration. MV may affect the function of target cells through surface interaction and receptor activation, cellular fusion and the delivery of intravesicular cargo. Endothelial MV are released as a consequence of endothelial activation during inflammation and have been described to affect hemostasis, various aspects of inflammatory reaction, vessel formation, apoptosis and cell survival, endothelial cell differentiation and function. Recent data suggest the potential use of MV in diagnostics, assessment of severity and prediction of outcomes in inflammatory diseases and their utilization as targets, mediators and vectors in therapy.

• MeSH
• Apoptosis MeSH
• Biomarkers metabolism   MeSH
• Endothelial Cells cytology   metabolism   MeSH
• Exosomes metabolism   MeSH
• Extracellular Vesicles metabolism   MeSH
• Humans MeSH
• Cell-Derived Microparticles metabolism   MeSH
• Neovascularization, Pathologic MeSH
• Thrombosis pathology   MeSH
• Inflammation pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Left ventricular assist devices (LVAD), currently used in treatment of terminal heart failure, are working on principle of rotary pump, which generates continuous blood flow. Non-pulsatile flow is supposed to expose endothelial cells to high stress and potential damage. Therefore, we investigated longitudinal changes in concentration of circulating endothelial microparticles (EMP) as a possible marker of endothelial damage before and after implantation of LVAD. Study population comprised 30 patients with end-stage heart failure indicated for implantation of the Heart Mate II LVAD. Concentrations of microparticles were measured as nanomoles per liter relative to phosphatidylserine before and 3 months after implantation. At 3 months after implantation we observed significant decrease in concentration of EMP [5.89 (95 % CI 4.31-8.03) vs. 3.69 (95 % CI 2.70-5.03), p=0.03] in the whole group; there was no difference observed between patients with ischemic etiology of heart failure (n=18) and with heart failure of non-ischemic etiology (n=12). In addition, heart failure etiology had no effect on the rate of EMP concentration decrease with time. These results indicate possibility that LVAD do not cause vascular damage 3 months after implantation. Whether these results suggest improvement of vascular wall function and of endothelium is to be proved in long-term studies.

• MeSH
• Endothelium, Vascular metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Longitudinal Studies MeSH
• Cell-Derived Microparticles metabolism   MeSH
• Heart-Assist Devices * trends   MeSH
• Aged MeSH
• Heart Failure blood   diagnosis   surgery   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

OBJECTIVES: Recent technological breakthroughs in the design of reliable systems for long term non-pulsatile mechanical heart support offer the possibility to study the effect of continuous blood flow in the vascular system. Generally, it is assumed that the absence of physiological pulsatile flow leads to prothrombogenic and proatherogenic changes. We investigated the change in the circulating endothelial microparticle concentration as a marker of endothelial damage in patients implanted with a continuous-flow left ventricle assist device (LVAD). METHODS: Endothelial microparticles were measured in 8 males (mean age 54.1±11.5 years) with terminal heart failure before and 3 months after implantation of an LVAD. The group consisted of 3 patients with dilated cardiomyopathy, 3 patients with ischemic cardiomyopathy, 1 patient with both conditions and 1 patient with congenital valvular disease. The concentration of endothelial microparticles was determined by ELISA Zymutest MP activity test. RESULTS: We did not observe a significant change in the concentration of circulating endothelial microparticles measured before and 3 months after implantation (p=0.669). High inter-individual variability in response to implantation was found. However, no association between a change in endothelial microparticle concentration and heart failure aetiology or a significant clinical complication attributed to LVAD implantation was observed. CONCLUSION: Results from this preliminary pilot study do not indicate that LVADs contribute to short-term vascular damage as defined by an increase in circulating endothelial microparticles.

• MeSH
• Biomarkers blood   MeSH
• Endothelium, Vascular metabolism   pathology   MeSH
• Cardiomyopathy, Dilated blood   surgery   MeSH
• Adult MeSH
• Hemodynamics physiology   MeSH
• Myocardial Ischemia blood   surgery   MeSH
• Middle Aged MeSH
• Humans MeSH
• Cell-Derived Microparticles metabolism   pathology   MeSH
• Pilot Projects MeSH
• Heart-Assist Devices adverse effects   MeSH
• Prospective Studies MeSH
• Aged MeSH
• Heart Failure blood   surgery   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Clinical Trial MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Diagnostic Imaging methods   instrumentation   utilization   MeSH
• Diffusion Magnetic Resonance Imaging methods   utilization   MeSH
• Elasticity Imaging Techniques methods   instrumentation   utilization   MeSH
• Extracellular Space physiology   metabolism   MeSH
• Cell Physiological Phenomena MeSH
• Tomography, Emission-Computed, Single-Photon methods   utilization   MeSH
• Medical Oncology methods   instrumentation   trends   MeSH
• Humans MeSH
• Magnetic Resonance Imaging methods   utilization   MeSH
• Metabolism MeSH
• Cell-Derived Microparticles physiology   metabolism   pathology   MeSH
• Neoplasms diagnosis   etiology   metabolism   MeSH
• Perfusion Imaging methods   utilization   MeSH
• Tomography, X-Ray Computed methods   utilization   MeSH
• Positron-Emission Tomography methods   utilization   MeSH
• Body Water metabolism   MeSH
• Check Tag
• Humans MeSH

BACKGROUND AIMS: Microvesicles (MV) shed from the plasma membrane of eukaryotic cells, including human embryonic stem cells (hESC), contain proteins, lipids and RNA and serve as mediators of cell-to-cell communication. However, they may also contain immunogenic membrane domains and infectious particles acquired from xenogenic components of the culture milieu. Therefore, MV represent a potential risk for clinical application of cell therapy. METHODS: We tested the ability of hESC and their most commonly used feeder cells, mouse embryonic fibroblasts (MEF), to produce MV. We found that hESC are potent producers of MV, whereas mitotically inactivated MEF do not produce any detectable MV. We therefore employed a combined proteomic approach to identify the molecules that constitute the major components of MV from hESC maintained in a standard culture setting with xenogenic feeder cells. RESULTS: In purified MV fractions, we identified a total of 22 proteins, including five unique protein species that are known to be highly expressed in invasive cancers and participate in cellular activation, metastasis and inhibition of apoptosis. Moreover, we found that hESC-derived MV contained the immunogenic agents apolipoprotein and transferrin, a source of Neu5Gc, as well as mouse retroviral Gag protein. CONCLUSIONS: These findings indicate that MV represent a mechanism by which hESC communicate; however, they also serve as potential carriers of immunogenic and pathogenic compounds acquired from environment. Our results highlight a potential danger regarding the use of hESC that have previously been exposed to animal proteins and cells.

• MeSH
• Antigens, Heterophile immunology   MeSH
• Antigens, Neoplasm immunology   metabolism   MeSH
• Apolipoproteins immunology   metabolism   MeSH
• Cell Line MeSH
• Microscopy, Electron MeSH
• Embryonic Stem Cells cytology   immunology   metabolism   MeSH
• Fibroblasts cytology   immunology   metabolism   MeSH
• Gene Products, gag immunology   metabolism   MeSH
• Coculture Techniques MeSH
• Humans MeSH
• Cell-Derived Microparticles immunology   metabolism   MeSH
• Mice MeSH
• Apoptosis Regulatory Proteins immunology   metabolism   MeSH
• Proteomics MeSH
• Risk-Taking MeSH
• Cattle MeSH
• Tandem Mass Spectrometry MeSH
• Tissue Therapy, Historical adverse effects   MeSH
• Transferrin immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH