Size Enlargement Enabled Functional Profiling of Extracellular Vesicle at Single-Particle Level.

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Renal cell carcinoma (RCC) is a fairly common and lethal cancer. The wide variety of RCC histological subtypes constitutes a challenge in treatment decision-making. Exosomes are extracellular membrane vesicles that are produced by all cell types in physiological conditions. Extracellular vesicles (EVs) are now accepted as a mode of intercellular communication and transport proteins, RNAs, DNA, and lipids to surrounding and distant cells. The lipid bilayer membrane of the EVs helps to protect these cargos. EVs are involved in many pathological processes, such as cancer, and can be easily obtained through liquid biopsy. Currently, EVs are rarely considered as candidate biomarkers for kidney cancer. However, improvements in the characterization of tumor-derived EVs could lead to the implementation of blood- and urine-derived EVs as biomarkers in the management of oncologic patients. Since all organs, not just the tumor, contribute to EV population, the thoughtful discrimination of tumor-derived EVs remains an unmet need for the clinical application of this kind of liquid biopsy technology. To determine the contribution of the tumor to blood- and urine-derived EVs, we utilize a novel approach to isolate tissue-derived EVs in parallel with liquid biopsy-derived EVs. To our knowledge, only a handful of studies (only one in RCC) have studied EVs directly derived from tissue. We propose the use tissue-derived EVs to screen for candidate EV biomarkers in plasma and/or urine. We hypothesize that using tissue-derived EVs would increase the tumor specificity for the characterization of EVs as liquid biopsy biomarkers. We optimized a protocol in which we used tissue of RCC patients (normal kidney or tumor) to condition media and isolate tumor-derived EVs alone by ultracentrifugation. Additionally, we isolated plasma- and urine EVs by ultracentrifugation using standard protocols. Nanoparticle Tracking Analysis (NTA) showed normalized concentrations of >2 x 109 particles/mL with a size distribution in the small EV-range. Transmission Electron Microscopy (TEM) images showed typical exosome morphology, with the characteristic cup-shaped membrane vesicles. Western Blot (WB) confirmed the presence of exosome markers. We successfully isolated EVs from human RCC and healthy kidney tissue. We will proceed with screening the EV-samples for candidate miRNA-biomarkers by multiplexed gene expression analysis, followed by confirmation of the candidate markers by RT-qPCR. Citation Format: Richard C. Zieren, Liang Dong, Sarah R. Amend, Philip M. Pierorazio, Theo M. de Reijke, Kenneth J. Pienta. Tumor-derived extracellular vesicles as kidney cancer biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1358.

A novel immunoassay for ApoB-100, the main protein component of lipoproteins, enables the development of methods to enrich extracellular vesicles from human plasma while depleting both lipoproteins and free proteins.

The Population of Circulating Extracellular Vesicles Dramatically Alters after Very Premature Delivery- a Previously Unrecognised Postnatal Adaptation Process?

Clinical Implications of Plasma-Derived Extracellular Vesicles in Myeloproliferative Neoplasms: Unveiling Inflammatory Potential and Biomarkers for Cancer Treatment

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. We used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. We show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level.

Increased expression of CD11b, the beta-integrin marker of microglia, represents microglial activation during neurodegenerative inflammation. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) induced the production of NO and increased the expression of CD11b in mouse BV-2 microglial cells and primary microglia. Either a scavenger of NO (PTIO) or an inhibitor of inducible nitric-oxide synthase (L-NIL) blocked this increase in microglial CD11b expression. Furthermore, co-microinjection of PTIO with LPS was also able to suppress LPS-mediated expression of CD11b and loss of dopaminergic neuronal fibers and neurotransmitters in striatum in vivo. Similarly, other inducers of NO production such as interferon-gamma, interleukin-1beta, human immunodeficiency virus type-1 gp120, and double-stranded RNA (poly(IC)) also increased the expression of CD11b in microglia through NO. The role of NO in the expression of CD11b was corroborated further by the expression of microglial CD11b by GSNO, an NO donor. Because NO transduces many intracellular signals via guanylate cyclase (GC), we investigated the role of GC, cyclic GMP (cGMP), and cGMP-activated protein kinase (PKG) in microglial expression of CD11b. Inhibition of LPS- and GSNO-mediated up-regulation of CD11b either by NS2028 (a specific inhibitor of GC) or by KT5823 and Rp-8-bromo-cGMP (specific inhibitors of PKG), and increase in CD11b expression either by 8-bromo-cGMP or by MY-5445 (a specific inhibitor of cGMP phosphodiesterase) alone suggest that NO increases microglial expression of CD11b via GC-cGMP-PKG. In addition, GSNO induced the activation of cAMP response element-binding protein (CREB) via PKG that was involved in the up-regulation of CD11b. This study illustrates a novel biological role of NO in regulating the expression of CD11b in microglia through GC-cGMP-PKG-CREB pathway that may participate in the pathogenesis of devastating neurodegenerative disorders.

In this paper we examine a nonstationary discrete time, infinite horizon growth model with uncertainty. Under very general hypotheses on the data of the model, we establish the existence of an optimal program and we show that the values of the finite horizon problems tend to that of the infinite horizon as the end of the planning period approaches infinity.

Extracellular Vesicles: How a Circulating Biomarker Can Double As a Regulator of Blood Pressure.

Background: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. Methods: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. Results: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. Conclusion: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells.

The significance of nitric oxide (NO) in man was first investigated in the late 1980s, and NO has subsequently received great attention from biologists. Initially, this highly reactive gaseous molecule was seen as a mere noxious air pollutant. Closer investigation of its function in physiological processes, however, revealed that it took part in many different biologic processes. This multifunctionality led to its declaration as the molecule of the year in 1992. We now know NO to be a smooth-muscle relaxant in blood vessels, an inhibitor of platelet aggregation, a neurotransmitter, and a mediator in local defense (2, 3). In the airways, NO is an important molecule with different functions such as stimulation of ciliary motility, mediation in inflammation, bacteriostatic and virostatic activity, and regulation of bronchial airway tone and even pulmonary vascular tone (4–7). Further studies on other systems will probably reveal more processes in which NO plays a key role. Studies in healthy adults indicate that NO in nasal air is mainly produced in the epithelial cells of the nasal cavity, particularly in the paranasal sinuses (8). Many factors, such as smoking, drugs, physio-logical factors, and nasal and paranasal disorder, influence the level of NO measured in nasal air (6, 9, 10). The measurement technique is also of great importance (10, 11). NO measurement has begun to be used in experimental clinical settings, in order to clarify the clinical value of NO in diagnostic problems and therapeutic strategies for disorders such as primary ciliary dyskinesia (PCD) and various forms of sinusitis and allergy. The use of NO as a noninvasive diagnostic and therapeutic tool is the ultimate goal. Many cells within the (upper and lower) respiratory tract can produce NO, including endothelial cells, epithelial cells, neutrophils, and (alveolar) macrophages (12). First, l-arginine is taken up by the cells via cationic transporters (CAT) (Fig. 1). CAT1 is constitutively expressed (housekeeping), while CAT2 is induced by cytokines. Second, l-arginine is N-hydroxylated into NG-hydroxy-l-arginine (NOHA). Subsequently, a three-electron oxidation takes place, resulting in NO and l-citrulline. While NO diffuses to the lumen, l-citrulline can be reconverted to l-arginine via arginosuccinate inside the cell (13). NO metabolic pathway (13) (reproduced with permission). This pathway of generation of NO is regulated by a family of enzymes called nitric oxide synthases (NOS). Three isoforms of NOS have now been identified in man and are differentially distributed in organs and tissues (14). Constitutively expressed nitric oxide synthase (cNOS) consists of two isoforms, nNOS (NOS type 1) and eNOS (NOS type 3), respectively expressed in neurons and vascular endothelium. The activity of nNOS and eNOS is regulated by intracellular calcium/calmodulin concentrations. These isoforms have been localized in human alveolar type II cells and in transformed and primary cultures of human bronchial epithelial cells (15). Inducible NOS (iNOS or NOS type 2) is probably present in every (epithelial) cell, and is activated by proinflammatory cytokines and/or bacterial products (2). The inducible form of NOS is calcium independent. LPS alone increases the production of NO in human epithelial cells, but IFN-γ acts synergistically to enhance this response (15). Immunohistochemical and mRNA in situ hybridization show that NO synthase is expressed apically in the paranasal sinus epithelium, in contrast to the epithelium of the nasal cavity, where only weak NO synthase activity was found (16). The NOS of the paranasal sinuses most closely resembles the inducible isoform but has different characteristics from iNOS expressed elsewhere. These isoforms seem to be constantly expressed and active, and to be resistant to steroids. These properties are associated with constitutive, rather than with inducible, isoforms of NOS (16). A new nonenzymatic pathway has been discovered in man that produces NO by reduction of inorganic nitrite under specific conditions (17). These nonenzymatic reactions take place in the stomach, on the surface of the skin, in the ischemic heart, and in infected nitrite-containing urine. NO generated by this mechanism is likely to play a role in similar biologic events, as when produced from l-arginine by NO synthases. The exact origin of NO measured in nasal air and the relative contribution from other sources are not fully known. Not only is there the production within the nasal cavity and the paranasal sinuses, but there is also a contribution from other sources such as the ambient air and, more important, the lower respiratory tract (6–8, 10, 18, 19). Most studies indicate that the main production of nasal NO is in the paranasal sinuses (16, 20, 21). The first indication is the observation that there is a transient decrease in nasal NO measured from one nostril when air is continuously removed from one maxillary sinus, while air injected into the same sinus results in a transient elevation of nasal NO. This suggests a continuous flow of NO from the maxillary sinus to the nasal cavity (20). Another indication is the reduction of NO release from the paranasal sinuses by instillation of NO synthase inhibitor (L-NAME) into the maxillary sinus. Administration of L-NAME in the nasal cavity results in only a slight reduction of nasal NO levels (20). In patients who have impaired ostial patency, significantly lower nasal NO levels are measured. Impairment of ostial patency and thus lower nasal NO levels are seen in disorders such as Kartagener's syndrome and cystic fibrosis. In these cases, there is probably a lower contribution of NO flowing from the paranasal sinuses into the nose, in addition to a possibly decreased production of NO (8, 22). Moreover, nasal NO levels are high in man and other primates with paranasal sinuses, while, in contrast, the baboon, a primate which lacks paranasal sinuses, has very low nasal NO levels (21). The strong constitutive expression of iNOS in the sinus epithelium and the lack of expression in the nasal epithelium are another indication (16). There are indications that nasal NO levels in children rise until the age of 10 years, when they reach the normal value as in adults. This may be a sign of increasing pneumatization of the developing paranasal sinuses in growing children (16, 23). The role of bacteria in the production of nasal NO has also been suggested; however, most studies showed nasal NO release to be independent of the presence of bacteria, since systemic antibiotics had no effect on the nasal NO values of healthy adults, and the sterile nasal cavities of neonates delivered by cesarean section had measurable nasal NO levels (7, 24, 25). As, in recent years, a wide variety of physiological processes in which NO is involved have been thoroughly investigated, it became clear that NO is important within the system where it is produced. Although initially considered a noxious air pollutant, many scientists now agree on the important roles of NO in different organ systems, such as those of a neurotransmitter in the nervous system, a smooth-muscle relaxant, and an inhibitor of platelet aggregation in the cardiovascular system (6, 16, 26). In the airways, NO seems to be of great importance in local host defense and is a major mediator in many physiological and pathophysiological events, although the exact role of this pluripotent gas is far from fully known. It participates in host defense and inflammation, and as an airborne messenger in bronchial tonus and pulmonary vascular resistance. The role of NO in inflammation is contradictory. Some studies indicate a harmful role of NO in inflammation, whereas others indicate a positive influence (18). There is evidence that NO production is enhanced at sites of inflammation, leading to local increased NO levels, as in asthma, cystitis, and inflammatory bowel disease (18, 27). The harmfulness of NO may be due to extensive production of NO by iNOS in some inflammatory circumstances such as pertussis and asthma, leading to autotoxicity in the affected area (18). However, basal NO production in the upper respiratory tract by a continuous expressed iNOS, leading to fairly high NO levels, has no destructive effect on local airway epithelium, and is even physiological (16). On the contrary, NO production in the upper respiratory tract seems to serve as an important protection against local attack, not as a mere inflammatory mediator, but as a regulator of various protective activities in host defense. A remarkable illustration of the positive role of NO in inflammation was given by McCafferty et al., who found worse inflammation in iNOS knockout mice than in wild-type mice in an animal model of colon inflammation (28). The enhanced production of NO during local aggression against the airway epithelium suggests a role of NO in host defense. NO concentration in normal paranasal sinuses and even in the nasal cavity exceeds greatly NO concentrations that are bacteriostatic (i.e., 100 ppb) (6, 16, 29). Children who have low NO production, as in primary ciliary dyskinesia (PCD) and cystic fibrosis, also have recurrent airway infections, a fact which may be an indication of the (host) protective effect of NO. NO may also have virostatic activities, as indicated in a mouse model (30). There are also indications that NO is active against fungi and parasites, and it may also protect against tumor cells (31). NO is also a regulator of ciliary beat frequency in the upper airway epithelium (4, 5, 32). The lack of NO in nasal air in diseases caused by profound ciliary dysfunction, such as PCD, strongly suggests a relation between NO and ciliary motility with clinical implications. For example, in infection, increased NO production can lead to enhanced ciliary activity, resulting in an effective clearance of aggressive organisms and potentially noxious metabolic products. This can have beneficial results in host defense. Other findings suggest that NO enhances blood flow in the human nasal mucosa (33). Although its possible protective effect is not clear yet, further studies on this subject may elucidate the meaning of this finding. NO produced in the upper respiratory tract follows the airstream to the lower airways and lungs with inhalation. This supports the hypothesis that NO derived from the upper airways has physiological effects in the lung and acts as an aerocrine messenger. There is some evidence that inhaled (exogenous) NO, at concentrations as low as 100 ppb, significantly decreases pulmonary vascular resistance and improves arterial oxygenation in subjects with severe pulmonary disease (33). Other studies suggest that NO helps to decrease the bronchial tonus, although this might be a central rather than a peripheral airway effect (7). NO in gas phase at low concentrations, as in the human airways, is fairly stable and therefore can be detected and quantified. The most widely used technique for measurement of NO in exhaled air is the chemiluminescence method. This highly sensitive technique is based on the emission of electromagnetic radiation from excited NO2*. NO reacts with an excess of ozone (O3), resulting in NO2 with an electron in an excited state (NO2*), which returns to its basic energy by emitting a photon. The quantity of light emitted is proportional to the NO concentration and can be displayed online on-screen. The lower limit of measurement is 1 ppb. Nasal NO measurement is based on the same method as exhaled NO, but sampling can be done directly or indirectly from the nose (6, 10). Other methods that have been used to measure NO in human exhaled air are mass spectrometry and gas chromatography–mass spectrometry (6). The measurement technique that is used in a particular experiment is very important for the eventual value of the nasal NO level (10, 34). Even in the same population the NO level is dependent on the measurement technique (11). The most important factors are ambient NO; the method of measuring (i.e., sampling while breathholding or tidal breathing, soft palate closure, etc.); and the characteristics of the chemiluminescence analyzer, the sampling flow, and the intranasal flow (10, 11). For comparison of different values, it is important to have a notion of these factors. In 1997, the European Respiratory Society Task Force tried to determine a standard method in order to obtain more comparable and reliable values (10). However, scientists continue to use different experimental settings, and one should be aware of this in order to interpret and compare NO values from different studies. The values of oral and nasal NO in the exhaled air of controls measured by the chemiluminescence method vary among laboratories: oral NO ranges from 4 to 160 ppb, while nasal NO varies from 200 to 2000 ppb (12, 22, 23, 35–38). Another remarkable feature is that NO levels are always higher in the upper respiratory tract than in the lower airways in normal subjects (6, 8, 10, 12, 22, 24, 36, 38). The variety of NO values in different studies is due to different factors such as measurement techniques, physiological variations, and pathologic changes (9–11, 16, 23, 34, 39–41). A summary of the influences on nasal NO is given in Table 1. Nasal NO levels rise from birth until the age of 10 years, when they reach the normal adult level. This finding supports the paranasal origin of nasal NO, as in children development of paranasal sinuses results in higher nasal NO levels until the age of 10 years, when they reach their final constitution (16, 23, 43). Interestingly, Schedin et al. found nasal NO already present at birth, including those neonates delivered by cesarean section (25). When nasal NO levels were correlated with body surface, the concentration in children around 10 years of age was approximately twice as high as the nasal NO concentration in adults. The following two possible explanations have been proposed: 1)the surface of paranasal sinuses in children develops faster than the body surface 2)children excrete a larger proportion of NO in the nasal mucosa (16). Another study found that nasal NO levels in adults between 20 and 90 years of age were similar (23). Artlich et al. related levels of nasal NO to the body surface in preterm children and found that the NO excretion is similar to that of adults (about 3 nl/kg/min−1). They concluded that the lower NO levels in preterm children are due to the smaller volume of ventilated sinuses and smaller epithelial surface at that age (43). Mammals without sinuses have no age-related increase in nasal NO (44). Recently, Qian et al. contradicted Lundberg et al.'s conclusions. They showed that intranasal flow had a great influence on the result of NO measurement (16, 34). As there are many differences in ventilation and measurement techniques between children and adults, intranasal flow will not always be comparable. More work needs to be done to make measurements in children and adults more comparable, in order to draw conclusions about age-dependent NO differences (34). There is no evidence that nasal NO levels are sex-related (10, 34, 39). Variation in nasal NO levels in relation to the menstrual cycle has not yet been studied. Several studies show that nasal NO decreases during physical exercise (6, 10, 45). Lundberg et al. (6) showed that nasal NO decreased by 47% after 1 min of physical exercise. A maximal reduction of 76% was found at the end of the exercise period; thereafter, NO levels slowly increased. They reached normal basal levels in about 15–20 min. There are several possible reasons for this decrease in nasal NO. Firstly, changes in nasal cavity volume could result in lower NO levels by dilution of nasal air (46). This possibility has been rejected by a recent finding that nasal NO is independent of nasal cavity volume (47). Secondly, NO could be destroyed by reactive agents produced in the nasal mucosa during physical exercise. Thirdly, changes in NO could be caused by a reduction of blood flow in the nasal mucosa with a concomitant decrease in substrate supply to the highly producing NOS type 2 in the paranasal sinuses (6, 46). Smoking control subjects have somewhat lower exhaled NO and nasal NO values than age- and sex-matched nonsmokers. The reason for this could be related to the toxic effect of inhaled smoke on the downregulation in NOS and/or the disruption of NO-producing cells (6, 10, 23). When evaluating the effect of drugs on nasal NO, one should be aware of interactions among drugs, patients, and diseases. It is not always easy to determine whether the changes in nasal NO are caused by the drug or by the disease itself. Topical and systemic glucocorticoids showed no effect on the nasal NO levels in healthy people (6, 8, 48, 49). Antibiotics in healthy persons do not alter nasal NO levels (6, 8). Topical nasal decongestants, such as oxymetazoline, result in a decrease of nasal NO levels (6, 10, 40, 47, 50). The reason for this may be a reduction, caused by vasoconstriction, in substrate supply to the high-output NOS type 2 in the sinuses. Histamine seems to have no influence on nasal NO levels (51). Nasal NO levels in people suffering from an upper respiratory tract infection (URTI) do not differ from nasal NO levels in healthy people. Specifically, Ferguson & Eccles (50) and Lindberg et al. (23) found no significant differences in nasal NO levels during and after an episode of URTI. Lindberg et al. (23) found similar nasal NO levels in patients with URTI and healthy controls (23). Baraldi et al. reached the same conclusion when comparing children with and without URTI (41). The effect of allergic rhinitis on nasal NO is not consistent. Some researchers report higher nasal NO levels in patients with allergic rhinitis (9, 40, 42). This may be due to an upregulation of iNOS by local infection, resulting in higher NO production (9). Kharitonov et al. found that nasal NO levels in patients suffering from allergic rhinitis and treated with topical nasal glucocorticoids are even lower than nasal NO levels in controls (9). This led to the hypothesis that iNOS in nasal epithelial cells gives rise to increased nasal NO levels in allergic rhinitis and contributes to the normal NO production in basal circumstances, since topical nasal glucocorticoids normally do not reach the sinus cavity and decrease nasal NO values in allergic rhinitis to levels lower than nasal NO levels in controls. According to this hypothesis, iNOS in the nasal cavity, as its activity is altered by glucocorticoids, must be different from iNOS found in the paranasal sinuses, which is not influenced by glucocorticoids (9, 52). Lundberg et al. (36) and Henriksen et al. (53) found no alterations in nasal NO levels in patients with allergic rhinitis. The cause of these discrepancies is not very clear. One could speculate that the upregulation of iNOS in the nose leads to higher nasal NO levels in rhinitis, as is the case in local infections in the lower airways, such as asthma (9, 52, 54). In contrast, swelling of the nasal mucosa in rhinitis can lead to occluded sinus ostia, which results in a reduced passage of NO from the paranasal sinuses to the nasal cavity, where it is measured (40). An interesting finding supporting this view was made by Arnal et al. (40), who found increased nasal NO levels in patients with allergic rhinitis. But patients without symptoms at the moment of the measurement had even higher nasal NO levels than patients with symptoms. One could postulate that nasal NO levels in patients with symptoms are lower because of a reduced contribution of the NO produced in the paranasal sinuses, as a result of obstructed sinus ostia. In patients without symptoms, ostial patency is mostly better leading to a higher of NO from the paranasal sinuses into the nasal cavity (40). in the nasal NO level measure may be the result of in the may even This must be taken into when a given nasal NO value is Nasal NO levels seem not to be influenced by asthma (18, 22, 36, One can that asthma the upper respiratory tract to a than the lower airways, where increased NO levels are of glucocorticoids can NO levels by reduction of iNOS NO levels are considered to be a of airway measurement of NO levels in the lower airways could indicate the of (6, 22). Nasal NO levels seem to be decreased in patients suffering from but not studies are consistent. Lindberg et al. patients with sinusitis and found that nasal NO production was reduced by more than in comparison with healthy subjects (23). In contrast, Arnal et al. found no significant differences in their study of patients with sinusitis Lindberg et al. found similar nasal NO levels in patients after sinusitis and healthy subjects (23). nasal NO levels were measured by Baraldi et al. in children with These decreased nasal NO levels increased after with systemic nasal NO levels were to the levels of healthy children (41). It has not yet been whether low nasal NO levels in sinusitis result from reduced passage of NO via the sinus ostia, or whether the NO production is reduced in those patients (41). A low production of NO as a cause of low nasal NO levels in sinusitis is by the study of Lindberg et al., who found nasal NO levels to be low sinus or by sinus as by (23). In contrast to Lindberg et al.'s Baraldi et al. found only a reduced NO level in children with a of the sinus in the air derived from the nostril (41). The effect of nasal has not been The of nasal NO and nasal has been in only one Arnal et al. increased nasal NO levels in patients with nasal and to whereas patients with nasal without had significantly lower nasal NO The nasal NO concentration in patients with allergic was significantly higher than in patients with For a similar of sinus nasal NO was higher in allergic than in This that is an important in relation to the level of nasal NO in In the nasal NO concentration was correlated with the of alterations of the paranasal sinuses. This that the of the paranasal sinuses by the decreases the nasal NO with a similar of of the nasal NO levels was that sites of production other than the sinuses also to the nasal NO. It has been that also may to the NO production, as they also iNOS in their epithelial cells (2). can that the of paranasal sinus and the allergic strongly influence the nasal NO level in nasal studies report very low nasal NO levels in patients suffering from cystic (12, 22, This may be the result of reduced NO production by destroyed epithelial cells or reduced NOS An increased NO into the sinus and a reduced NO passage from the sinuses to the nasal cavity may be another possible (12, 22, Kartagener's syndrome is a and They are part of In patients with PCD, nasal NO levels are (8, 12, 52). explanations are reduced NO production by a reduced from the nasal and paranasal and reduced passage of NO via the sinus (8, 12, 22, 52). In studies on PCD, significantly lower nasal NO levels in than in disease controls. nasal NO values, however, do not We found that the in have no significant influence on the nasal NO level et al., of NO can an interesting and diagnostic and therapeutic However, to be done in order to make it a in This noninvasive measurement can be even in It could be used as an easy for the of In the therapeutic may It is to that drugs will be used to or decrease NO production in such a that it can have a positive influence on However, there is to be in the various physiological and pathologic factors, such as that nasal NO, particularly the should on and on measurements more reliable and comparable. NO is a gaseous the significance of which in man to be investigated in the late the it has the attention of many who have revealed its significance in various physiological and pathologic processes. It has functions in the cardiovascular system, the nervous system, and the upper and lower In the airways, NO levels in the upper respiratory tract are higher ppb) than those in the lower respiratory tract The chemiluminescence which is based on a of NO with resulting in the emission of is the most widely used measurement technique for NO. NO has a major influence on airway by mediation in ciliary activity, inflammation, host bronchial and pulmonary vascular resistance. It is also considered to be an aerocrine messenger between the upper and lower such as physical smoking, and some drugs influence physiological nasal NO concentrations. conditions such as allergic rhinitis, nasal cystic fibrosis, and lead to altered nasal NO concentrations. of nasal NO can be at and can be used to for disease or to the effects of However, the clinical of the measurement of nasal NO in different physiological and pathologic conditions to be it can be used as a diagnostic on the function of NO in and is its in diagnostic and therapeutic of some

Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins.

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.

Introduction: Myocardial infarction (MI) is a leading cause of mortality worldwide. The potency of cell-based therapies for MI is increasingly attributed to the release of extracellular vesicles (EVs) which consist of a lipid/protein membrane and encapsulate RNA cargo. Specifically, EVs from ckit+ progenitor cells (CPCs) and mesenchymal stromal cells (MSCs) are shown to be pro-reparative, with clinical trials ongoing. Despite copious research into EV cargo, the role of donor cell type on EV membrane composition and its effects on EV uptake mechanism by recipient cells remain unclear. This is crucial for designing EV-based therapeutics as uptake mechanism dictates the functionality of the cargo. Thus, we hypothesized that (1) EV membrane composition varies by donor cell type and (2) this variation covaries with the mechanism of uptake. Methods: EVs were isolated using differential ultracentrifugation from four cardiac cell types: CPCs, MSCs, cardiac endothelial cells (CECs) and rat cardiac fibroblasts (RCFs) grown in normoxia (18% O 2 ) or hypoxia (1% O 2 ) to mimic ischemic conditions. EVs were characterized for size and concentration. EV lipid membrane profile was assessed through LC/MS/MS. Donor cell’s role on EV uptake mechanism was determined by inhibiting known uptake pathways (clathrin, dynamin, macropinocytosis and caveolae/lipid raft) with small molecules and quantifying CEC/RCF endocytosis of EVs with flow cytometry. Finally, partial least squares regression was used to determine the most important lipids involved in EV uptake mechanism. Results: EVs were successfully isolated and characterized. The EV membrane lipid profiles clustered by donor cell type. Uptake mechanism of EVs varied based on both donor and recipient cell type with dynamin mediated endocytosis being the most common. Further, the uptake mechanism was independent of normoxic/hypoxic conditioning. Finally, supervised learning methods revealed specific lipid classes (sphingolipids and glycerophospholipids) covaried with EV uptake mechanism. Conclusion: This work highlights the importance of the understudied EV membrane and its role in delivering therapeutic cargo. Active donor cell selection for efficient EV uptake will allow for more potent EV-based MI therapies.

Immune checkpoint inhibitors (ICIs) have provided new hope for melanoma patients, however, not all patients benefit. Furthermore, ICI-related therapies cause significant immune-related adverse events that adversely affect patient outcomes. Therefore, there is a pressing need for reliable biomarkers to identify patients most likely to benefit from these treatments. In this study, we employed an extracellular vesicles (EVs) protein expression array to explore the longitudinal membrane protein profiles of plasma-derived EVs from 32 melanoma patients receiving anti-PD-1 and anti-angiogenesis therapy at baseline and early treatment. We found that the dynamic changes in PD-L2 on the EV membrane were associated with treatment response and patient survival. The dynamic change of EV PD-L2 as an indication of treatment efficacy was validated in an independent cohort of melanoma patients treated with anti-PD-1 monotherapy. Plasma-derived PD-L2+ EVs from patients with mucosal melanoma significantly reduced the frequency of granzyme B+ CD8 T cells within the peripheral blood mononuclear cells (PBMCs) of healthy individuals. The inhibitory effect of PD-L2+ EVs on CD8 T cells was further validated using human melanoma cell lines and the B16-F10 mouse model. Although intratumoural injection of PD-L2+ EVs could promote melanoma growth in vivo, tumours with PD-L2+ EVs showed a higher response to anti-PD-1 than those without PD-L2+ EVs. Collectively, our study demonstrates that PD-L2+ EVs inhibit CD8 T cell activation and promote melanoma growth, and changes in PD-L2 on circulating EVs during early treatment could serve as a biomarker for ICI-based therapy.