Adventitial Stromal Cells Define Group 2 Innate Lymphoid Cell Tissue Niches.

Type 2 lymphocytes promote both physiologic tissue remodeling and allergic pathology, yet their physical tissue niches are poorly described. Here, we used quantitative imaging to define the tissue niches of group 2 innate lymphoid cells (ILC2s), which are critical instigators of type 2 immunity. We identified a dominant adventitial niche around lung bronchi and larger vessels in multiple tissues, where ILC2s localized with subsets of dendritic and regulatory T cells. However, ILC2s were most intimately associated with adventitial stromal cells (ASCs), a mesenchymal fibroblast-like subset that expresses interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP). In vitro, ASCs produced TSLP that supported ILC2 accumulation and activation. ILC2s and IL-13 drove reciprocal ASC expansion and IL-33 expression. During helminth infection, ASC depletion impaired lung ILC2 and Th2 cell accumulation and function, which are in part dependent on ASC-derived IL-33. These data indicate that adventitial niches are conserved sites where ASCs regulate type 2 lymphocyte expansion and function.

Shaker IR T449 mutants separate C- from U-type inactivation.

Previous studies demonstrated that slow inactivation of the Shaker potassium channel can be made ~100-fold faster or slower by point mutations at a site in the outer pore (T449). However, the discovery that two forms of slow inactivation coexist in Shaker raises the question of which inactivation process is affected by mutation. Equivalent mutations in K(V)2.1, a channel exhibiting only U-type inactivation, have minimal effects on inactivation, suggesting that mutation of Shaker T449 acts on C-type inactivation alone, a widely held yet untested hypothesis. This study reexamines mutations at Shaker T449, confirming that T449A speeds inactivation and T449Y/V slow it. T449Y and T449V exhibit U-type inactivation that is enhanced by high extracellular potassium, in contrast to C-type inactivation in T449A which is inhibited by high potassium. Automated parameter estimation for a 12-state Markov model suggests that U-type inactivation occurs mainly from closed states upon weak depolarization, but primarily from the open state at positive voltages. The model also suggests that WT channels, which in this study exhibit mostly C-type inactivation, recover from inactivation through closed-inactivated states, producing voltage-dependent recovery. This suggests that both C-type and U-type inactivation involve both open-inactivated and closed-inactivated states.

Evading immune cell uptake and clearance requires PEG grafting at densities substantially exceeding the minimum for brush conformation.

Coating nanoparticles with polyethylene glycol (PEG), which reduces particle uptake and clearance by immune cells, is routinely used to extend the circulation times of nanoparticle therapeutics. Nevertheless, due to technical hurdles in quantifying the extent of PEG grafting, as well as in generating very dense PEG coatings, few studies have rigorously explored the precise PEG grafting density necessary to achieve desirable "stealth" properties. Here, using polymeric nanoparticles with precisely tunable PEG grafting, we found that, for a wide range of PEG lengths (0.6-20 kDa), PEG coatings at densities substantially exceeding those required for PEG to adopt a "brush" conformation are exceptionally resistant to uptake by cultured human macrophages, as well as primary peripheral blood leukocytes. Less than 20% of these nanoparticles were cleared from the blood after 2 h (t1/2 ∼ 14 h) in BALB/c mice, whereas slightly less densely PEGylated and uncoated control particles were both virtually eliminated within 2 h. Our results suggest that the stealth properties of PEG-coated nanoparticles are critically dependent on achieving PEG grafting at densities exceeding those required for brush conformation.

Using mechanobiological mimicry of red blood cells to extend circulation times of hydrogel microparticles.

It has long been hypothesized that elastic modulus governs the biodistribution and circulation times of particles and cells in blood; however, this notion has never been rigorously tested. We synthesized hydrogel microparticles with tunable elasticity in the physiological range, which resemble red blood cells in size and shape, and tested their behavior in vivo. Decreasing the modulus of these particles altered their biodistribution properties, allowing them to bypass several organs, such as the lung, that entrapped their more rigid counterparts, resulting in increasingly longer circulation times well past those of conventional microparticles. An 8-fold decrease in hydrogel modulus correlated to a greater than 30-fold increase in the elimination phase half-life for these particles. These results demonstrate a critical design parameter for hydrogel microparticles.

Role of outer-pore residue Y380 in U-type inactivation of KV2.1 channels.

The interpretation of slow inactivation in potassium channels has been strongly influenced by work on C-type inactivation in Shaker channels. Slow inactivation in Shaker and some other potassium channels can be dramatically modulated by the state of the pore, including mutations at outer pore residue T449, which altered inactivation kinetics up to 100-fold. KV2.1, another voltage-dependent potassium channel, exhibits a biophysically distinct inactivation mechanism with a U-shaped voltage-dependence and preferential closed-state inactivation, termed U-type inactivation. However, it remains to be demonstrated whether U-type and C-type inactivation have different molecular mechanisms. This study examines mutations at Y380 (homologous to Shaker T449) to investigate whether C-type and U-type inactivation have distinct molecular mechanisms, and whether C-type inactivation can occur at all in KV2.1. Y380 mutants do not introduce C-type inactivation into KV2.1 and have little effect on U-type inactivation of KV2.1. Interestingly, two of the mutants tested exhibit twofold faster recovery from inactivation compared to wild-type channels. The observation that mutations have little effect suggests KV2.1 lacks C-type inactivation as it exists in Shaker and that C-type and U-type inactivation have different molecular mechanisms. Kinetic modeling predicts that all mutants inactivate preferentially, but not exclusively, from partially activated closed states. Therefore, KV2.1 exhibits a single U-type inactivation process including some inactivation from open as well as closed states.

Management of fetal hydrops due to maternal antibodies against a low incidence paternal red cell antigen: a novel insight from clinical practice.

A rare case of a low incidence red cell antigen causing severe fetal allo-immune red cell disease is presented. Discussion of how this can be diagnosed and successfully managed antenatally using middle cerebral artery Doppler ultrasound and maternal antibody titre levels for fetal surveillance and timing of intervention with intrauterine transfusion.

PEGylated PRINT nanoparticles: the impact of PEG density on protein binding, macrophage association, biodistribution, and pharmacokinetics.

In this account, we varied PEGylation density on the surface of hydrogel PRINT nanoparticles and systematically observed the effects on protein adsorption, macrophage uptake, and circulation time. Interestingly, the density of PEGylation necessary to promote a long-circulating particle was dramatically less than what has been previously reported. Overall, our methodology provides a rapid screening technique to predict particle behavior in vivo and our results deliver further insight to what PEG density is necessary to facilitate long-circulation.

Cd²âº block and permeation of CaV3.1 (α1G) T-type calcium channels: candidate mechanism for Cd²âº influx.

Cd²âº is an industrial pollutant that can cause cytotoxicity in multiple organs. We examined the effects of extracellular Cd²âº on permeation and gating of Ca(v)3.1 (α1G) channels stably transfected in HEK293 cells, by using whole-cell recording. With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, Cd²âº rapidly blocked currents with 2 mM Ca²âº in a voltage-dependent manner. The block caused by Cd²âº was relieved at more-hyperpolarized potentials, which suggests that Cd²âº can permeate through the selectivity filter of the channel into the cytosol. In the absence of other permeant ions (Ca²âº and Na⁺ replaced by N-methyl-d-glucamine), Cd²âº carried sizable inward currents through Ca(v)3.1 channels (210 ± 20 pA at -60 mV with 2 mM Cd²âº). Ca(v)3.1 channels have a significant "window current" at that voltage (open probability, ∼1%), which makes them a candidate pathway for Cd²âº entry into cells during Cd²âº exposure. Incubation with radiolabeled ¹°9Cd²âº confirmed uptake of Cd²âº into cells with Ca(v)3.1 channels.

Fe²âº block and permeation of CaV3.1 (α1G) T-type calcium channels: candidate mechanism for non-transferrin-mediated Fe²âº influx.

Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe²âº on permeation and gating of Ca(V)3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe²âº state (e.g., use of extracellular ascorbate). With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, extracellular Fe²âº rapidly blocked currents with 2 mM extracellular Ca²âº in a voltage-dependent manner, as described by a Woodhull model with K(D) = 2.5 mM at 0 mV and apparent electrical distance δ = 0.17. Extracellular Fe²âº also shifted activation to more-depolarized voltages (by ∼10 mV with 1.8 mM extracellular Fe²âº) somewhat more strongly than did extracellular Ca²âº or Mg²âº, which is consistent with a Gouy-Chapman-Stern model with surface charge density σ = 1 e(-)/98 Ų and K(Fe) = 4.5 M⁻¹ for extracellular Fe²âº. In the absence of extracellular Ca²âº (and with extracellular Na⁺ replaced by TEA), Fe²âº carried detectable, whole-cell, inward currents at millimolar concentrations (73 ± 7 pA at -60 mV with 10 mM extracellular Fe²âº). With a two-site/three-barrier Eyring model for permeation of Ca(V)3.1 channels, we estimated a transport rate for Fe²âº of ∼20 ions/s for each open channel at -60 mV and pH 7.2, with 1 µM extracellular Fe²âº (with 2 mM extracellular Ca²âº). Because Ca(V)3.1 channels exhibit a significant "window current" at that voltage (open probability, ∼1%), Ca(V)3.1 channels represent a likely pathway for Fe²âº entry into cells with clinically relevant concentrations of extracellular Fe²âº.

Evaluation of a two-site, three-barrier model for permeation in Ca(V)3.1 (alpha1G) T-type calcium channels: Ca (2+), Ba (2+), Mg (2+), and Na (+).

We explored the ability of a two-site, three-barrier (2S3B) Eyring model to describe recently reported data on current flow through open Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide range (100 nM: -110 mM: ) while recording whole-cell currents over a wide voltage range (-150 mV to +100 mV) from channels stably expressed in HEK 293 cells. Effects on permeation were isolated using instantaneous current-voltage relationships (IIV) after strong, brief depolarizations to activate channels with minimal inactivation. Most experimental results were reproduced by a 2S3B model. The model described the IIV relationships, apparent affinities for permeation and block for Ca(2+) and Ba(2+), and shifts in reversal potential between Ca(2+) and Ba(2+). The fit to block by 1 mM Mg(2+)(i) was reasonable, but block by Mg(2+)(0) was described less well. Surprisingly, fits were comparable with strong ion-ion repulsion, with no repulsion, or with intermediate values. With weak repulsion, there was a single high-affinity site, with a low-affinity site near the cytoplasmic side of the pore. With strong repulsion, the net charge of ions in the pore was near +2 over a relatively wide range of concentration and voltage, suggesting a knockoff mechanism. With strong repulsion, Ba(2+) preferred the inner site, while Ca(2+) preferred the outer site, potentially explaining faster entry of Ni(2+) and other pore blockers when Ba(2+) is the charge carrier.

Inflation experiments and inverse finite element modelling of posterior human sclera.

The complexity of inverse finite element modelling methods used in ocular biomechanics research has significantly increased in recent years in order to produce material parameters that capture microscale tissue behaviour. This study presents a more accessible method for researchers to optimise sclera material parameters for use in finite element studies where macroscale sclera displacements are required. Five human donor sclerae aged between 36 and 72 years were subjected to cycles of internal pressure up to 61 mmHg using a custom-built inflation rig. Displacements were measured using a laser beam and two cameras through a digital image correlation algorithm. Specimen-specific finite element models incorporating regional thickness variation and sclera surface topography were divided into six circumferential regions. An inverse finite element procedure was used to optimise Ogden material parameters for each region. The maximum root mean squared (RMS) error between the numerical and experimental displacements within individual specimens was 17.5 µm. The optimised material parameters indicate a gradual reduction in material stiffness (as measured by the tangent modulus) from the equator to the posterior region at low-stress levels up to 0.005 MPa. The variation in stiffness between adjacent regions became gradually less apparent and statistically insignificant at higher stresses. The study demonstrated how inflation testing combined with inverse modelling could be used to effectively characterise regional material properties capable of reproducing global sclera displacements. The material properties were found to vary between specimens, and it is expected that age could be a contributing factor behind this variation.

Resident neuroelectrochemical interfacing using carbon nanofiber arrays.

Carbon nanofiber electrode architectures are used to provide for long-term, neuroelectroanalytical measurements of the dynamic processes of intercellular communication between excitable cells. Individually addressed, vertically aligned carbon nanofibers are incorporated into multielement electrode arrays upon which excitable cell matrixes of both neuronal-like derived cell lines (rat pheochromocytoma, PC-12) and primary cells (dissociated cells from embryonic rat hippocampus) are cultured over extended periods (days to weeks). Electrode arrays are characterized with respect to their response to easily oxidized neurotransmitters, including dopamine, norepinephrine, and 5-hydroxytyramide. Electroanalysis at discrete electrodes following long-term cell culture demonstrates that this platform remains responsive for the detection of easily oxidized species generated by the cultured cells. Preliminary data also suggests that quantal release of easily oxidized transmitters can be observed at nanofiber electrodes following direct culture and differentiation on the arrays for periods of at least 16 days.

Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel.

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.

Comment on "Local impermeant anions establish the neuronal chloride concentration".

Glykys et al. (Reports, 7 February 2014, p. 670) conclude that, rather than ion transporters, "local impermeant anions establish the neuronal chloride concentration" and thereby determine "the magnitude and direction of GABAAR currents at individual synapses." If this were possible, perpetual ion-motion machines could be constructed. The authors' conclusions conflict with basic thermodynamic principles.

Correction factors for Goldmann Tonometry.

PURPOSE: The purpose of the study was to evaluate the in vitro accuracy of correction factors in decreasing the error in the intraocular pressure (IOP) measurements obtained using the Goldmann Applanation Tonometer (GAT). METHODS: Nineteen donor corneas, from individuals aged between 57 and 99 years (mean 75.7 years, standard deviation±11.4 years) were subjected to posterior pressure simulating in vivo true IOP (IOPT) using an inflation test rig. Central corneal thickness and corneal curvature were measured. The posterior pressure was set at 25 different pressure levels between 5 and 45 mm Hg and IOP was measured using the GAT. Five different correction equations were applied to the IOP measurements obtained using the GAT to determine corrected IOP. The multiparameter correction equations applied were derived by Elsheikh, Ehlers, Chihara, Shimmyo et al, and Orssengo and Pye. The differences between IOPT and the IOP measured using the GAT were recorded as uncorrected errors, whereas the differences between IOPT and each of the corrected IOP were the tonometry errors after correction. RESULTS: The mean and standard deviation of error in tonometry before correction was +2.25±0.62 mm Hg. The mean errors in tonometry after correction using the Elsheikh and Chihara equations were +0.78±0.62 and +1.08±0.61 mm Hg, respectively. The mean errors in tonometry for the Ehlers, Shimmyo et al, and Orssengo and Pye equations were negative, indicating an overcorrection; the values were -0.75±2.28, -1.27±1.85, and -0.77±1.83 mm Hg, respectively. CONCLUSIONS: The Elsheikh and the Chihara et al's equations considerably decreased error in IOP measurements obtained by the GAT when compared with IOPT and were more consistent than other correction equations. The 2 equations may be of clinical utility in obtaining estimates of IOPT.

Intravital imaging of a spheroid-based orthotopic model of melanoma in the mouse ear skin.

Multiphoton microscopy is a powerful tool that enables the visualization of fluorescently tagged tumor cells and their stromal interactions within tissues in vivo. We have developed an orthotopic model of implanting multicellular melanoma tumor spheroids into the dermis of the mouse ear skin without the requirement for invasive surgery. Here, we demonstrate the utility of this approach to observe the primary tumor, single cell actin dynamics, and tumor-associated vasculature. These methods can be broadly applied to investigate an array of biological questions regarding tumor cell behavior in vivo.

Nanoparticle clearance is governed by Th1/Th2 immunity and strain background.

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol onto particles (PEGylation) extend circulation times; however, these particles are still cleared, and the processes involved in this clearance remain poorly understood. Here, we present an intravital microscopy-based assay for the quantification of nanoparticle clearance, allowing us to determine the effect of mouse strain and immune system function on particle clearance. We demonstrate that mouse strains that are prone to Th1 immune responses clear nanoparticles at a slower rate than Th2-prone mice. Using depletion strategies, we show that both granulocytes and macrophages participate in the enhanced clearance observed in Th2-prone mice. Macrophages isolated from Th1 strains took up fewer particles in vitro than macrophages from Th2 strains. Treating macrophages from Th1 strains with cytokines to differentiate them into M2 macrophages increased the amount of particle uptake. Conversely, treating macrophages from Th2 strains with cytokines to differentiate them into M1 macrophages decreased their particle uptake. Moreover, these results were confirmed in human monocyte-derived macrophages, suggesting that global immune regulation has a significant impact on nanoparticle clearance in humans.

Age-related variations in the biomechanical properties of human sclera.

This study examined age-related changes in biomechanical behaviour in the anterior, equatorial and posterior regions of the human sclera (white of the eye). Circumferential strip specimens were extracted from areas close to the limbus, equator and posterior pole in 45 donor scleras ranging in age between 51 and 84 years. The strips were subjected to cycles of uniaxial tension loading at a strain rate of 8% per minute while monitoring their load-deformation behaviour. All specimens demonstrated nonlinear behaviour with an initially low tangent modulus (a measure of material stiffness) increasing under higher stresses. The average ratios between the tangent modulus at a high stress of 1 MPa and that at a low stress of 0.05 MPa were 11.2±1.7, 12.0±1.7 and 12.4±1.5 for anterior, equatorial and posterior specimens, respectively. Stiffening was observed with age in all regions, but it was statistically significant only in the anterior region (P<0.01). Anterior specimens showed the largest stiffness growth with advancing age in both the initial, matrix regulated phase of behaviour (0.32 MPa/decade), and the final, collagen regulated phase (3.97 MPa/decade), followed by equatorial (0.27 and 2.15 MPa/decade) then posterior specimens (0.14 and 0.26 MPa/decade). The stress-strain behaviour of scleral tissue exhibits increasing stiffness with higher age. In addition to a regional variation of material stiffness, the rate of stiffness growth with age also varies between regions.

An exploratory trial of Cyclamen europaeum extract for acute rhinosinusitis.

OBJECTIVES/HYPOTHESIS: An exploratory US trial in patients with acute rhinosinusitis was conducted to evaluate the efficacy and safety of Cyclamen europeaum extract, a product marketed in Europe that causes reflex nasal discharge and subsequent decongestion. STUDY DESIGN: Prospective, randomized, placebo-controlled, double-blind, and parallel group. METHODS: Outpatients (n = 29) with cardinal symptoms of acute rhinosinusitis and both endoscopic and radiographic (computed tomography [CT] scan) evidence at 25 US centers were randomized to receive intranasal, lyophilized, reconstituted Cyclamen europeaum extract (Cyclamen) or placebo spray for 7 days. Primary outcomes were reduction in percent sinus opacification on CT scans and reduction in PM predose instantaneous total symptom scores measured on a six-point scale. Secondary outcomes included other measures of symptom score change and endoscopic signs of mucopurulence and inflammation. RESULTS: Cyclamen treatment significantly reduced sinus opacification compared with placebo treatment (P < .045). Although Cyclamen treatment reduced total symptom scores from baseline more than placebo treatment (-2.4 vs. -1.4), there were no significant treatment group differences (P = .312). Cyclamen treatment was well tolerated. CONCLUSIONS: Cyclamen treatment significantly reduced sinus opacification in patients with acute rhinosinusitis. Further exploration of Cyclamen treatment in larger patient populations is warranted.

The effect of particle size on the biodistribution of low-modulus hydrogel PRINT particles.

There is a growing recognition that the deformability of particles used for drug delivery plays a significant role on their biodistribution and circulation profile. Understanding these effects would provide a crucial tool for the rational design of drug delivery systems. While particles resembling red blood cells (RBCs) in size, shape and deformability have extended circulation times and altered biodistribution profiles compared to rigid, but otherwise similar particles, the in vivo behavior of such highly deformable particles of varied size has not been explored. We report the fabrication of a series of discoid, monodisperse, low-modulus hydrogel particles with diameters ranging from 0.8 to 8.9 µm, spanning sizes smaller than and larger than RBCs. We injected these particles into healthy mice, and tracked their concentration in the blood and their distribution into major organs. These deformable particles all demonstrated some hold up in filtration tissues like the lungs and spleen, followed by release back into the circulation, characterized by decreases in particles in these tissues with concomitant increases in particle concentration in blood. Particles similar to red blood cells in size demonstrated longer circulation times, suggesting that this size and shape of deformable particle is uniquely suited to avoid clearance.