Atopic dermatitis (AD) is the most common chronic skin inflammatory disease, with a profound impact on patients’ quality of life. AD varies considerably in clinical course, age of onset and degree to ...which it is accompanied by allergic and non-allergic comorbidities. Skin barrier impairment in both lesional and nonlesional skin is now recognized as a critical and often early feature of AD. This may be explained by a number of abnormalities identified within both the stratum corneum and stratum granulosum layers of the epidermis. The goal of this review is to provide an overview of key barrier defects in AD, starting with a historical perspective. We will also highlight some of the commonly used methods to characterize and quantify skin barrier function. There is ample opportunity for further investigative work which we call out throughout this review. These include: quantifying the relative impact of individual epidermal abnormalities and putting this in a more holistic view with physiological measures of barrier function, as well as determining whether these barrier-specific endotypes predict clinical phenotypes (e.g. age of onset, natural history, comorbidities, response to therapies, etc). Mechanistic studies with new (and in development) AD therapies that specifically target immune pathways, Staphylococcus aureus abundance and/or skin barrier will help us understand the dynamic crosstalk between these compartments and their relative importance in AD.
Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and ...RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca
-dependent, intact EVs can be easily released from Tim4 by adding Ca
chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations' proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.
The benefits of spontaneous breathing over muscle paralysis have been proven mainly in mild lung injury; no one has yet evaluated the effects of spontaneous breathing in severe lung injury. We ...investigated the effects of spontaneous breathing in two different severities of lung injury compared with muscle paralysis.
Prospective, randomized, animal study.
University animal research laboratory.
Twenty-eight New Zealand white rabbits.
Rabbits were randomly divided into the mild lung injury (surfactant depletion) group or severe lung injury (surfactant depletion followed by injurious mechanical ventilation) group and ventilated with 4-hr low tidal volume ventilation with spontaneous breathing or without spontaneous breathing (prevented by a neuromuscular blocking agent). Inspiratory pressure was adjusted to control tidal volume to 5-7 mL/kg, maintaining a plateau pressure less than 30 cm H2O. Dynamic CT was used to evaluate changes in lung aeration and the regional distribution of tidal volume.
In mild lung injury, spontaneous breathing improved oxygenation and lung aeration by redistribution of tidal volume to dependent lung regions. However, in severe lung injury, spontaneous breathing caused a significant increase in atelectasis with cyclic collapse. Because of the severity of lung injury, this group had higher plateau pressure and more excessive spontaneous breathing effort, resulting in the highest transpulmonary pressure and the highest driving pressure. Although no improvements in lung aeration were observed, muscle paralysis with severe lung injury resulted in better oxygenation, more even tidal ventilation, and less histological lung injury.
In animals with mild lung injury, spontaneous breathing was beneficial to lung recruitment; however, in animals with severe lung injury, spontaneous breathing could worsen lung injury, and muscle paralysis might be more protective for injured lungs by preventing injuriously high transpulmonary pressure and high driving pressure.
Esophageal manometry has shown its usefulness to estimate transpulmonary pressure, that is lung stress, and the intensity of spontaneous effort in patients with acute respiratory distress syndrome. ...However, clinical uptake of esophageal manometry in ICU is still low. Thus, the purpose of review is to describe technical tips to adequately measure esophageal pressure at the bedside, and then update the most important clinical applications of esophageal manometry in ICU.
Each esophageal balloon has its own nonstressed volume and it should be calibrated properly to measure pleural pressure accurately: transpulmonary pressure calculated on absolute esophageal pressure reflects values in the lung regions adjacent to the esophageal balloon (i.e. dependent to middle lung). Inspiratory transpulmonary pressure calculated from airway plateau pressure and the chest wall to respiratory system elastance ratio reasonably reflects lung stress in the nondependent 'baby' lung, at highest risk of hyperinflation. Also esophageal pressure can be used to detect and minimize patient self-inflicted lung injury.
Esophageal manometry is not a complicated technique. There is a large potential to improve clinical outcome in patients with acute respiratory distress syndrome, acting as an early detector of risk of lung injury from mechanical ventilation and vigorous spontaneous effort.
The potential risks of spontaneous effort and their prevention during mechanical ventilation is an important concept for clinicians and patients. The effort-dependent lung injury has been termed ...'patient self-inflicted lung injury (P-SILI)' in 2017. As one of the potential strategies to render spontaneous effort less injurious in severe acute respiratory distress syndrome (ARDS), the role of positive end-expiratory pressure (PEEP) is now discussed.
Experimental and clinical data indicate that vigorous spontaneous effort may worsen lung injury, whereas, at the same time, the intensity of spontaneous effort seems difficult to control when lung injury is severe. Experimental studies found that higher PEEP strategy can be effective to reduce lung injury from spontaneous effort while maintaining some muscle activity. The recent clinical trial to reevaluate systemic early neuromuscular blockade in moderate-severe ARDS (i.e., reevaluation of systemic early neuromuscular blockade (ROSE) trial) support that a higher PEEP strategy can facilitate 'safe' spontaneous breathing under the light sedation targets (i.e., no increase in barotrauma nor 90 days mortality versus early muscle paralysis).
To prevent P-SILI in ARDS, it seems feasible to facilitate 'safe' spontaneous breathing in patients using a higher PEEP strategy in severe ARDS.
Small extracellular vesicles (SEVs) secreted from various cells are lipid bilayer vesicles, 30-150 nm in size, that carry proteins, nucleic acids, and lipids as cargos to other cells. They include ...exosomes, which are generated in multivesicular endosomes (MVEs) and secreted upon fusion of MVEs with plasma membranes and a part of microvesicles, which directly bud from plasma membranes. SEVs have attracted attention as diagnostic and drug discovery targets, since it has been demonstrated that SEVs are involved in the intercellular communication in many diseases and physiological phenomena such as cancer, neurodegenerative diseases, and immunity. There are five isolation methods for SEVs, which include ultracentrifugation, density gradient ultracentrifugation, polymer precipitation, affinity isolation, and size-exclusion chromatography. The affinity isolation, which isolates SEVs using magnetic beads conjugated with binding molecules such as antibodies, has the ability to isolate highly pure SEVs in character. However, the population of SEVs is limited by the binding molecules and it is difficult to elute intact SEVs from the antibody beads. In this chapter, we present a TIM4-affinity isolation method that targets phosphatidylserine (PS), a component of the SEV membrane. TIM4 binds to PS in a Ca
-dependent manner, which enables the elution of intact SEVs from TIM4-beads in the presence of the chelating reagent ethylenediaminetetraacetic acid (EDTA). The TIM4-affinity isolation method helps overcome the disadvantages of the affinity isolation method and enables the isolation of heterogeneous SEVs at high purity. This method will facilitate the functional analysis of SEVs, development of diagnostic methods, and drug development of engineered SEVs.
Spontaneous respiratory effort during mechanical ventilation has long been recognized to improve oxygenation, and because oxygenation is a key management target, such effort may seem beneficial. ...Also, disuse and loss of peripheral muscle and diaphragm function is increasingly recognized, and thus spontaneous breathing may confer additional advantage. Reflecting this, epidemiologic data suggest that the use of partial (vs. full) support modes of ventilation is increasing. Notwithstanding the central place of spontaneous breathing in mechanical ventilation, accumulating evidence indicates that it may cause-or worsen-acute lung injury, especially if acute respiratory distress syndrome is severe and spontaneous effort is vigorous. This Perspective reviews the evidence for this phenomenon, explores mechanisms of injury, and provides suggestions for clinical management and future research.
A growing evidence shows that injurious spontaneous breathing, either too weak or too strong, may injure lung and diaphragm. The purpose of review is to understand why we need monitoring for safe ...spontaneous breathing, and to know the target value of each monitoring to preserve safe spontaneous breathing during assisted ventilation.
Lung protection sometimes goes counter to diaphragm protection. For instance, silence of respiratory muscle activity is necessary to minimize lung injury from vigorous spontaneous effort in acute respiratory distress syndrome, but it may also have a risk of diaphragm atrophy. Thus, our current goal is to preserve spontaneous breathing activity at modest level during assisted ventilation. To achieve this goal, several monitoring/techniques are now available at the bedside (e.g., plateau pressure measurement, airway occlusion pressure, end-expiratory airway occlusion, esophageal balloon manometry, electrical impedance tomography). The target value of each monitoring is vigorously being investigated, facilitating 'safe' spontaneous breathing effort from the perspective of lung and diaphragm protection.
We summarize why we need monitoring for safe spontaneous breathing during assisted ventilation and what the target value of each monitoring is to facilitate 'safe' spontaneous breathing during assisted ventilation.
Exosomes have recently gained interest as mediators of cell-to-cell communication and as potential biomarkers for cancer and other diseases. They also have potential as nanocarriers for drug delivery ...systems. Therefore, detailed structural, molecular, and biomechanical characterization of exosomes is of great importance for developing methods to detect and identify the changes associated with the presence of cancer and other diseases. Here, we employed three-dimensional atomic force microscopy (3D-AFM) to reveal the structural and nanomechanical properties of exosomes at high spatial resolution in physiologically relevant conditions. The substructural details of exosomes released from three different cell types were determined based on 3D-AFM force mapping. The resulting analysis revealed the presence of distinct local domains bulging out from the exosome surfaces, which were associated with the exosomal membrane proteins present on the outer surface. The nanomechanical properties of individual exosomes were determined from the 3D-force maps. We found a considerably high elastic modulus, ranging from 50 to 350 MPa, as compared to that obtained for synthetic liposomes. Moreover, malignancy-dependent changes in the exosome mechanical properties were revealed by comparing metastatic and nonmetastatic tumor cell-derived exosomes. We found a clear difference in their Young's modulus values, suggesting differences in their protein profiles and other exosomal contents. Exosomes derived from a highly aggressive and metastatic k-ras-activated human osteosarcoma (OS) cell line (143B) showed a higher Young's modulus than that derived from a nonaggressive and nonmetastatic k-ras-wildtype human OS cell line (HOS). The increased elastic modulus of the 143B cell-derived exosomes was ascribed to the presence of abundant specific proteins responsible for elastic fiber formation as determined by mass spectroscopy and confirmed by western blotting and ELISA. Therefore, we conclude that exosomes derived from metastatic tumor cells carry an exclusive protein content that differs from their nonmetastatic counterparts, and thus they exhibit different mechanical characteristics. Discrimination between metastatic and nonmetastatic malignant cell-derived exosomes would be of great importance for studying exosome biological functions and using them as diagnostic biomarkers for various tumor types. Our findings further suggest that metastatic tumor cells release exosomes that express increased levels of elastic fiber-associated proteins to preserve their softness.
3D-AFM showed the presence of distinct nanodomains bulging out from the membrane surface, which can be attributed to the presence of membrane-associated proteins.
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