Human CD99L2 Regulates a Unique Step in Leukocyte Transmigration.

CD99-like 2 (CD99L2 [L2]) is a highly glycosylated 52-kDa type 1 membrane protein that is important for leukocyte transendothelial migration (TEM) in mice. Inhibiting L2 using function-blocking Ab significantly reduces the recruitment of leukocytes to sites of inflammation in vivo. Similarly, L2 knockout mice have an inherent defect in leukocyte transmigration into sites of inflammation. However, the role of L2 in inflammation has only been studied in mice. Furthermore, the mechanism by which it regulates TEM is not known. To study the relevance to human inflammation, we studied the role of L2 on primary human cells in vitro. Our data show that like PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes. Inhibiting L2 using Ab blockade or genetic knockdown significantly reduces transmigration of human neutrophils and monocytes across endothelial cells. Furthermore, our data also show that L2 regulates a specific, sequential step of TEM between PECAM and CD99, rather than operating in parallel or redundantly with these molecules. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to sites of TEM, specifically downstream of PECAM initiation. Collectively, our data identify a novel functional role for human L2 in TEM and elucidate a mechanism that is distinct from PECAM and CD99.

High Incidence of Barotrauma in Patients With Severe Coronavirus Disease 2019.

OBJECTIVE.: To report the high incidence of barotrauma in critically ill patients admitted to the intensive care unit (ICU) with coronavirus disease 2019 (COVID-19) and to discuss its implications. DESIGN.: Retrospective cohort study. SETTING.: ICU of an academic county hospital in Los Angeles, CA admitted from March 15-June 20, 2020. PATIENTS.: 77 patients with COVID-19 pneumonia. 75 patients met inclusion criteria. RESULTS.: 21% of patients with severe COVID-19 sustained barotrauma (33% of patients receiving IMV, 8% of patients receiving (NIV). There were no differences between the barotrauma and non-barotrauma groups regarding demographics, illness severity, or medications received, nor tidal volume or average/peak airway pressures in those receiving IMV. In the barotrauma group there was a greater proportion of patients receiving therapeutic anticoagulation (81% vs. 47%, p = 0.023) and ventilated using airway pressure release ventilation mode (13% vs. 0%, p = 0.043). Barotrauma was associated with increased likelihood of receiving a tracheostomy (OR 2.58 [0.23-4.9], p = 0.018]), longer median ICU length of stay (17 days vs. 7 days, p = 0.03), and longer median length of hospitalization (26 days vs. 14 days, p < 0.001). There was also a trend toward prolonged median duration of IMV (12.5 days vs 7 days, p = 0.13) and higher average mortality (56% vs 37%, p = 0.25). CONCLUSIONS.: Barotrauma is seen in 5-12% of patients with ARDS receiving IMV and is exceedingly rare in patients receiving NIV. We report a high incidence of barotrauma observed in critically ill patients with COVID-19 requiring either NIV or IMV. While there was a trend toward increased mortality in patients with barotrauma, this did not reach statistical significance. The increased incidence of barotrauma with COVID-19 may be a product of the pathophysiology of this disease state and a heightened inflammatory response causing rampant acute lung injury. Evidence-based medicine and lung-protective ventilation should remain the mainstay of treatment.

Holey-structured tungsten metamaterials for broadband ultrasonic sub-wavelength imaging in water.

Metamaterials exhibiting Fabry-Pérot resonances are shown to achieve ultrasonic imaging of a sub-wavelength aperture in water immersion across a broad bandwidth. Holey-structured metamaterials of different thickness were additively manufactured using a tungsten substrate and selective laser melting, tungsten being chosen so as to create a significant acoustic impedance mismatch with water. Both broadband metamaterial behavior and sub-wavelength imaging in water are demonstrated experimentally and validated with finite element simulations over the 200-300 kHz range.

Ultrasonic Propagation in Highly Attenuating Insulation Materials.

Experiments have been performed to demonstrate that ultrasound in the 100-400 kHz frequency range can be used to propagate signals through various types of industrial insulation. This is despite the fact that they are highly attenuating to ultrasonic signals due to scattering and viscoelastic effects. The experiments used a combination of piezocomposite transducers and pulse compression processing. This combination allowed signal-to-noise levels to be enhanced so that signals reflected from the surface of an insulated and cladded steel pipe could be obtained.

Correction to: Nebulization of RNS60, a Physically-Modified Saline, Attenuates the Adoptive Transfer of Experimental Allergic Encephalomyelitis in Mice: Implications for Multiple Sclerosis Therapy.

The original version of this article unfortunately contained a mistake. The Figure 3, 4, 5 legends have been misplaced. The updated legends along with the figures are corrected with this erratum.

Cutting Edge: CD99 Is a Novel Therapeutic Target for Control of T Cell-Mediated Central Nervous System Autoimmune Disease.

Leukocyte trafficking into the CNS is a prominent feature driving the immunopathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Blocking the recruitment of inflammatory leukocytes into the CNS represents an exploitable therapeutic target; however, the adhesion molecules that specifically regulate the step of leukocyte diapedesis into the CNS remain poorly understood. We report that CD99 is critical for lymphocyte transmigration without affecting adhesion in a human blood-brain barrier model. CD99 blockade in vivo ameliorated experimental autoimmune encephalomyelitis and decreased the accumulation of CNS inflammatory infiltrates, including dendritic cells, B cells, and CD4(+) and CD8(+) T cells. Anti-CD99 therapy was effective when administered after the onset of disease symptoms and blocked relapse when administered therapeutically after disease symptoms had recurred. These findings underscore an important role for CD99 in the pathogenesis of CNS autoimmunity and suggest that it may serve as a novel therapeutic target for controlling neuroinflammation.

Nebulization of RNS60, a Physically-Modified Saline, Attenuates the Adoptive Transfer of Experimental Allergic Encephalomyelitis in Mice: Implications for Multiple Sclerosis Therapy.

Developing a new and effective therapeutic approach against multiple sclerosis (MS) is always an important area of research. RNS60 is a bioactive aqueous solution generated by subjecting normal saline to Taylor-Couette-Poiseuille flow under elevated oxygen pressure. Recently we have demonstrated that RNS60, administered through intraperitoneal injection, ameliorated clinical symptoms and disease progression of experimental allergic encephalomyelitis (EAE), an animal model of MS. Since the intravenous route is not preferred for treating a chronic condition, we tested if nebulization of RNS60 could attenuate the disease process of adoptively-transferred EAE in mice. Although we could not directly image RNS60 after nebulization, nebulized Alexa680 reached spleen, spinal cord and different parts of the brain. Nebulization of RNS60 starting from the acute phase attenuated clinical symptoms of relapsing-remitting EAE in female SJL/J mice. RNS60 nebulization also inhibited perivascular cuffing, maintained the integrity of blood-brain and blood-spinal cord barriers, suppressed inflammation, normalized the expression of myelin genes, and blocked demyelination in the CNS of EAE mice. On the immunomodulatory front, nebulization of RNS60 to EAE mice led to the enrichment of anti-autoimmune regulatory T cells (Tregs) and suppression of autoimmune Th17 cells. Together, these results suggest that nebulization of RNS60 may be used to control aberrant immune responses in MS and other autoimmune disorders.

4D intravital microscopy uncovers critical strain differences for the roles of PECAM and CD99 in leukocyte diapedesis.

Leukocyte transendothelial migration (TEM) is an essential component of the inflammatory response. In vitro studies with human cells have demonstrated that platelet/endothelial cell adhesion molecule (PECAM) functions upstream of CD99 during TEM; however, results in vivo with mice have been apparently contradictory. In this study we use four-dimensional (4D) intravital microscopy to demonstrate that the site and order of function of PECAM and CD99 in vivo are dependent on the strain of mice. In FVB/n mice, PECAM functions upstream of CD99, as in human cells in vitro, and blocking antibodies against either molecule arrest neutrophils before they traverse the endothelium. However, in C57BL/6 mice, PECAM and CD99 appear to function at a different step, as the same antibodies arrest leukocyte migration through the endothelial basement membrane. These results are the first direct comparison of PECAM and CD99 function in different murine strains as well as the first demonstration of the sequential function of PECAM and CD99 in vivo.

Phospholipids at the interface: current trends and challenges.

Phospholipids are one of the major structural elements of biological membranes. Due to their amphiphilic character, they can adopt various molecular assemblies when dispersed in water, such as bilayer vesicles or micelles, which give them unique interfacial properties and render them very attractive in terms of foam or emulsion stabilization. This article aims at reviewing the properties of phospholipids at the air/water and oil/water interfaces, as well as the recent advances in using these natural components as stabilizers, alone or in combination with other compounds such as proteins. A discussion regarding the challenges and opportunities offered by phospholipids-stabilized structure concludes the review.

Optimised polymer trapped-air lenses for ultrasound focusing in water exploiting Fabry-Pérot resonance.

The concept of employing air volumes trapped inside polymer shells to make a lens for ultrasound focusing in water is investigated. The proposed lenses use evenly-spaced concentric rings, each having an air-filled polymer shell construction, defining concentric water-filled channels. Numerical simulations and experiments have shown that a plane wave can be focused, and that the amplification can be boosted by Fabry-Pérot resonances within the water channels with an appropriate choice of the lens thickness. The effect of the polymer shell thickness and the depth of the channels is discussed, as these factors can affect the geometry and hence the frequency of operation. The result was a lens with a Full Width at Half Maximum value of 0.65 of a wavelength at the focus. Results obtained on a metal-based counterpart are also shown for comparison. An advantage of this polymeric design is that it is easily constructed via additive manufacturing. This study shows that trapped-air lenses made of polymer are suitable for ultrasound focusing in water near 500 kHz.

Trapped air metamaterial concept for ultrasonic sub-wavelength imaging in water.

Acoustic metamaterials constructed from conventional base materials can exhibit exotic phenomena not commonly found in nature, achieved by combining geometrical and resonance effects. However, the use of polymer-based metamaterials that could operate in water is difficult, due to the low acoustic impedance mismatch between water and polymers. Here we introduce the concept of "trapped air" metamaterial, fabricated via vat photopolymerization, which makes ultrasonic sub-wavelength imaging in water using polymeric metamaterials highly effective. This concept is demonstrated for a holey-structured acoustic metamaterial in water at 200-300 kHz, via both finite element modelling and experimental measurements, but it can be extended to other types of metamaterials. The new approach, which outperforms the usual designs of these structures, indicates a way forward for exploiting additive-manufacturing for realising polymer-based acoustic metamaterials in water at ultrasonic frequencies.

Lack of improvement in antimicrobial prescribing after a diagnosis of Clostridium difficile and impact on recurrence.

BACKGROUND: Antimicrobial use is one of the largest modifiable risk factors for development of Clostridium difficile infection (CDI). We sought to determine if a recent diagnosis of CDI affected the appropriateness of subsequent antimicrobial prescribing. METHODS: This study is a retrospective electronic chart review of the Greater Los Angeles Veterans Administration. Medication administration records were reviewed for all patients with new CDI from 2015-2016 to determine the appropriateness (drug choice, duration, and dosage) of all non-CDI antimicrobials prescribed within 90 days pre- and post-initial CDI (iCDI) positive testing. RESULTS: Of the 210 patients diagnosed with new-onset iCDI, 140 met inclusion criteria. Of antimicrobial courses prescribed, 40.6% of pre-iCDI were inappropriate compared with 43.1% of post-iCDI, demonstrating no difference in prescribing habits (P = .717). Thirty-three patients developed recurrent CDI (rCDI). After adjustment for other known risk factors, inappropriate antimicrobial use was associated with a significant increased risk of recurrence compared with appropriate use alone (odds ratio [OR], 6.19; 95% confidence interval [CI], 1.45-26.42). Antimicrobial use in general was associated with increased recurrence compared with no antimicrobial use post-iCDI (OR, 2.6; 95% CI, 1.16-5.84); however, after adjustment, it was no longer significant (OR, 2.13; 95% CI, 0.90-5.04). CONCLUSIONS: The appropriateness of antimicrobial prescribing was not affected by the diagnosis of recent CDI. Inappropriate antimicrobial use after iCDI was associated with higher risk of rCDI.

Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM.

GSK3beta and beta-catenin modulate radiation cytotoxicity in pancreatic cancer.

BACKGROUND: Knowledge of factors and mechanisms contributing to the inherent radioresistance of pancreatic cancer may improve cancer treatment. Irradiation inhibits glycogen synthase kinase 3beta (GSK3beta) by phosphorylation at serine 9. In turn, release of cytosolic membrane beta-catenin with subsequent nuclear translocation promotes survival. Both GSK3beta and beta-catenin have been implicated in cancer cell proliferation and resistance to death. METHODS: We investigated pancreatic cancer cell survival after radiation in vitro and in vivo, with a particular focus on the role of the function of the GSK3beta/beta-catenin axis. RESULTS: Lithium chloride, RNAi-medicated silencing of GSK3beta, or the expression of a kinase dead mutant GSK3beta resulted in radioresistance of Panc1 and BxPC3 pancreatic cancer cells. Conversely, ectopic expression of a constitutively active form of GSK3beta resulted in radiosensitization of Panc1 cells. GSK3beta silencing increased radiation-induced beta-catenin target gene expression as measured by studies of AXIN2 and LEF1 transcript levels. Western blot analysis of total and phosphorylated levels of GSK3beta and beta-catenin showed that GSK3beta inhibition resulted in stabilization of beta-catenin. Xenografts of both BxPC3 and Panc1 with targeted silencing of GSK3beta exhibited radioresistance in vivo. Silencing of beta-catenin resulted in radiosensitization, whereas a nondegradable beta-catenin construct induced radioresistance. CONCLUSIONS: These data support the hypothesis that GSK3beta modulates the cellular response to radiation in a beta-catenin-dependent mechanism. Further understanding of this pathway may enhance the development of clinical trials combining drugs inhibiting beta-catenin activation with radiation and chemotherapy in locally advanced pancreatic cancer.

Rheofluorescence technique for the study of dilute MEH-PPV solutions in Couette flow.

A novel rheofluorescence technique has been developed that permits the study of fluorescent polymers in a near-uniform shear field. The system has been used to examine the effects of shear flow on dilute solutions of two commercially available samples of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) in toluene and xylene. A detailed description of the instrument is provided, along with data that confirm a small probe molecule, Rhodamine 6G, is not affected by simple shear flow. MEH-PPV solutions were examined over two decades of concentration for rheochromism indicative of changes in segment length, and shear-induced orientation revealed by measurements of the steady state emission anisotropy. It is demonstrated that these dilute samples were not influenced by shear rates in the range 100-1000 s(-1). In contrast, MEH-PPV dispersed in a concentrated polystyrene solution showed evidence of shear-induced orientation and rheochromism. This new technique shows promise for investigating the impact of shear flow on the conformation of conjugated polymers employed in organic optoelectronic devices.