You have accessJournal of UrologyCME1 Apr 2023MP14-07 DEVELOPMENT OF A LIQUID BIOPSY USING EXTRACELLULAR VESICLES TO ASSESS THE SYSTEMIC T CELL IMMUNE LANDSCAPE IN BLADDER CANCER Karen Doersch, Samuel Walker, Thomas Osinski, Jonathan Flax, and James McGrath Karen DoerschKaren Doersch More articles by this author , Samuel WalkerSamuel Walker More articles by this author , Thomas OsinskiThomas Osinski More articles by this author , Jonathan FlaxJonathan Flax More articles by this author , and James McGrathJames McGrath More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urothelial carcinoma (UC) is commonly treated with therapeutics that target the immune system, such as checkpoint immunotherapy or Bacillus Calmette-Guerin (BCG). While assessment of T cell function is of utility in predicting response to these therapies, current methods for evaluating systemic T cell immunity or intratumoral immune behavior are limited. As antigenically activated T cells produce extracellular vesicles (EVs) carrying markers of cell subtype and activation state and these are found in the blood, we hypothesized that serum T cell derived EVs could be used to interrogate the systemic antitumor T cell response. The purpose of this study was to evaluate a novel microfluidic strategy ‘catch and display for liquid biopsy’ [CAD-LB] for the rapid assessment of T cell biomarkers on individual EVs from T cell conditioned medium or blood. METHODS: T cell derived EVs from cultured cells. We hypothesized that T cell derived EVs have markers of T cell function that mirror their cell of origin. To test this, we assessed markers of activation and exhaustion on EVs secreted by T cells. Activation and exhaustion were induced in vitro by stimulation with anti-CD3/CD28 beads, mimicking the in vivo stimulation of T cells in an antigen-rich tumor environment. T cell subtype and activation state were assessed by CAD-LB and flow cytometry. T cell markers on serum EVs. We developed an affinity purification method for CD3+ EVs and subsequently evaluated the level of T cell biomarkers on these using CAD-LB in UC specimens from patients. RESULTS: 1. T cell marker expression on EVs from conditioned medium from resting, activated and exhausted cells followed the pattern present on the EV producing T cells. 2. T cell derived EVs from UC patient serum identified markers of activated and functionally exhausted cells, indicating that we can capture the full range of T cell functionality with CAD-LB. 3. Detected T cell EVs with markers of non-circulating tissue-resident cells (present in tumor, secondary lymphoid and peripheral tissue) demonstrate that EVs capture the systemic immune responses. CONCLUSIONS: T cell derived EVs capture the phenotype and identity of their cell of origin. CAD-LB detects a range of T cell populations in serum. Future work will test if T cell EVs capture tumor and systemic antitumor immunity, which may help identify UC patients more likely to respond to immunotherapy. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e184 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karen Doersch More articles by this author Samuel Walker More articles by this author Thomas Osinski More articles by this author Jonathan Flax More articles by this author James McGrath More articles by this author Expand All Advertisement PDF downloadLoading ...

Humans spend 70-90% of their time indoors, however, there is a significant lack of knowledge regarding human exposure to microplastic particles and fibers (MPs) within the indoor environment. Fibers make up more than 90% of household settled dust worldwide and have been found in indoor air. Studies have identified MPs larger than 50 micrometers in indoor dust, but these are unlikely to be respirable and little information is available regarding smaller airborne particles. It is evident that humans are inhaling these particles as they have been found in excised diseased and healthy human lung tissues. We have developed methods to identify plastic particles in settled household dust larger than one micrometer in diameter by distinguishing plastic from cellulosic, proteinaceous, and inorganic materials using two different stains, Nile Red for plastics, and Trypan Blue for cellulosic materials. Proteinaceous and inorganic materials remain unstained. Household settled dust is collected onto silicon nitride nanomembranes where in situ analysis of particles can be characterized. The innovative use of nanomembranes allow for particle capture and multiple analyses to take place on the same substrate. Particle analysis via colorimetric staining and imaging is followed by polymer identification via Raman spectroscopy and subsequently characterized via scanning electron microscopy for size and surface morphology. Using this innovative approach, microplastic particles larger than one micrometer in diameter have been identified in all settled dust samples.

Plastics and their breakdown products, specifically microplastics, are ubiquitously present in the environment from a variety of sources. Analysis of air samples has also revealed the presence of fragmented, spherical, and fibrous plastic particles in a wide range of sizes from the submicrometer scale up to tens of micrometers in length and diameter. However, few studies have addressed the health consequences associated with exposures to airborne microplastics, specifically whether they can enter the respiratory tract, where they will deposit, and how polymer chemistry and morphology are linked to adverse health outcomes. Whether microplastics have unique toxicological properties in comparison to other particle types is also unknown. Our work has sought to address critical questions about the inhalability and respirability of microplastics in indoor and outdoor air via particle size-restricted sampling coupled with morphological assessment and estimation of plastic burdens via Nile red staining. We found Nile red-positive (putative plastic) particles in air samples from indoor environments (office, campus/household laundry rooms, 3-D printing/engineering laboratory), most of which appeared to have a fragment-like morphology. These particles represented ~1% of the total collected sample in terms of number. Importantly, these particles were also found when sampling was restricted to the human-respirable fraction (<4 micrometers in aerodynamic diameter), suggesting that they can reach the gas exchange region. Ongoing work is focused on identifying the polymeric species in these air samples, their distribution across particle size classes, and whether indoor and outdoor microplastic particles are similar in terms of morphology, size, and chemistry.

Abstract Introduction The humanized anti-α4 integrin blocking antibody natalizumab (NTZ) is an effective treatment for relapsing–remitting multiple sclerosis (RRMS) that is associated with the risk of progressive multifocal leukoencephalopathy (PML). While extended interval dosing (EID) of NTZ reduces the risk for PML, the minimal dose of NTZ required to maintain its therapeutic efficacy remains unknown. Objective Here we aimed to identify the minimal NTZ concentration required to inhibit the arrest of human effector/memory CD4+ T cell subsets or of PBMCs to the blood–brain barrier (BBB) under physiological flow in vitro. Results Making use of three different human in vitro BBB models and in vitro live-cell imaging we observed that NTZ mediated inhibition of α4-integrins failed to abrogate T cell arrest to the inflamed BBB under physiological flow. Complete inhibition of shear resistant T cell arrest required additional inhibition of β2-integrins, which correlated with a strong upregulation of endothelial intercellular adhesion molecule (ICAM)-1 on the respective BBB models investigated. Indeed, NTZ mediated inhibition of shear resistant T cell arrest to combinations of immobilized recombinant vascular cell adhesion molecule (VCAM)-1 and ICAM-1 was abrogated in the presence of tenfold higher molar concentrations of ICAM-1 over VCAM-1. Also, monovalent NTZ was less potent than bivalent NTZ in inhibiting T cell arrest to VCAM-1 under physiological flow. In accordance with our previous observations ICAM-1 but not VCAM-1 mediated T cell crawling against the direction of flow. Conclusion Taken together, our in vitro observations show that high levels of endothelial ICAM-1 abrogate NTZ mediated inhibition of T cell interaction with the BBB. EID of NTZ in MS patients may thus require consideration of the inflammatory status of the BBB as high levels of ICAM-1 may provide an alternative molecular cue allowing for pathogenic T cell entry into the CNS in the presence of NTZ.

The COVID-19 pandemic demonstrated the public health benefits of reliable and accessible point-of-care (POC) diagnostic tests for viral infections. Despite the rapid development of gold-standard reverse transcription polymerase chain reaction (RT-PCR) assays for SARS-CoV-2 only weeks into the pandemic, global demand created logistical challenges that delayed access to testing for months and helped fuel the spread of COVID-19. Additionally, the extreme sensitivity of RT-PCR had a costly downside as the tests could not differentiate between patients with active infection and those who were no longer infectious but still shedding viral genomes. To address these issues for the future, we propose a novel membrane-based sensor that only detects intact virions. The sensor combines affinity and size based detection on a membrane-based sensor and does not require external power to operate or read. Specifically, the presence of intact virions, but not viral debris, fouls the membrane and triggers a macroscopically visible hydraulic switch after injection of a 40 μL sample with a pipette. The device, which we call the μSiM-DX (microfluidic device featuring a silicon membrane for diagnostics), features a biotin-coated microslit membrane with pores ∼2-3× larger than the intact virus. Streptavidin-conjugated antibody recognizing viral surface proteins are incubated with the sample for ∼1 hour prior to injection into the device, and positive/negative results are obtained within ten seconds of sample injection. Proof-of-principle tests have been performed using preparations of vaccinia virus. After optimizing slit pore sizes and porous membrane area, the fouling-based sensor exhibits 100% specificity and 97% sensitivity for vaccinia virus (n = 62). Moreover, the dynamic range of the sensor extends at least from 105.9 virions per mL to 1010.4 virions per mL covering the range of mean viral loads in symptomatic COVID-19 patients (105.6-107 RNA copies per mL). Forthcoming work will test the ability of our sensor to perform similarly in biological fluids and with SARS-CoV-2, to fully test the potential of a membrane fouling-based sensor to serve as a PCR-free alternative for POC containment efforts in the spread of infectious disease.

A multiblock copolymer with increased charge density in the hydrophilic phase was synthesized by utilizing a trisulfonated poly(arylene ether sulfone) backbone (SHQS100). To achieve a balance of membrane properties a partially fluorinated poly(arylene ether benzonitrile) hydrophobic phase (6FPAEB) was used in uneven block lengths to form the hydrophilic-hydrophobic multiblock copolymer. Multiblock copolymers were synthesized via nucleophilic aromatic substitution to achieve high molecular weight copolymers and corresponding ductile membranes. Combination of the shorter increased charge density hydrophilic phase and longer partially fluorinated hydrophobic phase resulted in superior proton conductivity and mechanical properties while limiting water uptake and swelling, despite high ion-exchange capacity. The trifunctional SHQSH hydrophilic phase is proposed to produce a more concentrated charge region in the hydrophilic phase, while swelling of the membrane was limited by a longer partially fluorinated hydrophobic phase.

Inflammatory diseases and cancer metastases lack concrete pharmaceuticals for their effective treatment despite great strides in advancing our understanding of disease progression. One feature of these disease pathogeneses that remains to be fully explored, both biologically and pharmaceutically, is the passage of cancer and immune cells from the blood to the underlying tissue in the process of extravasation. Regardless of migratory cell type, all steps in extravasation involve molecular interactions that serve as a rich landscape of targets for pharmaceutical inhibition or promotion. Transendothelial migration (TEM), or the migration of the cell through the vascular endothelium, is a particularly promising area of interest as it constitutes the final and most involved step in the extravasation cascade. While in vivo models of cancer metastasis and inflammatory diseases have contributed to our current understanding of TEM, the knowledge surrounding this phenomenon would be significantly lacking without the use of in vitro platforms. In addition to the ease of use, low cost, and high controllability, in vitro platforms permit the use of human cell lines to represent certain features of disease pathology better, as seen in the clinic. These benefits over traditional pre-clinical models for efficacy and toxicity testing are especially important in the modern pursuit of novel drug candidates. Here, we review the cellular and molecular events involved in leukocyte and cancer cell extravasation, with a keen focus on TEM, as discovered by seminal and progressive in vitro platforms. In vitro studies of TEM, specifically, showcase the great experimental progress at the lab bench and highlight the historical success of in vitro platforms for biological discovery. This success shows the potential for applying these platforms for pharmaceutical compound screening. In addition to immune and cancer cell TEM, we discuss the promise of hepatocyte transplantation, a process in which systemically delivered hepatocytes must transmigrate across the liver sinusoidal endothelium to successfully engraft and restore liver function. Lastly, we concisely summarize the evolving field of porous membranes for the study of TEM.

The influence of UV irradiation on gas permeation properties of random segmented poly (imide-siloxane) (PIS) copolymer films was investigated. Random segmented copolymers were synthesized via ester-acid imidization from 4,4'-(hexafluoroisopropylidene)diphthalic anhydride (6FDA) and 3,3',4,4'-benzophenone tetracarboxylic dianhydride (BTDA) in a 7:3 M ratio with 2,4-diamino mesitylene (DAM) and varying amounts of a diamine terminated polydimethylsiloxane (PDMS) oligomer (M-n = 3000 g/mol). Solvent cast films of these copolymers were UV-irradiated on both sides in air at 365 nm, which induced crosslinking and photooxidation. Pure-gas permeabilities of H-2, O-2, N-2, CH4, and CO2 were measured with upstream pressures ranging from 2 to 18 atm at 35 degrees C. Gas permeability coefficients of a series of PIS copolymers decreased, and selectivity of all gas pairs increased after UV irradiation due to a decrease in free volume by UV crosslinking and photooxidation.

Utilizing solution imidization, molecular weight and end group control techniques, soluble, fully cyclized polyimides with very high glass transition temperatures have been developed to meet high temperature applications. Mechanistic aspects are investigated for solution imidization by both the polyamic acid route and by the ester-acid route. Polyimides based on pyromellitic dianhydride and a 3F diamine exhibit glass transition temperatures of 420°C. These polyimides are soluble in polar aprotic solvents and form tough, transparent films which demonstrate mechanical integrity and thermooxidative stability at 700°F. Various processing routes are explored to demonstrate the viability of these materials in high temperature applications. Details of the synthesis and characterization of these materials will be provided.

Endothelial cells (ECs) are an active component of the immune system and interact directly with inflammatory cytokines. While ECs are known to be polarized cells, the potential role of apicobasal polarity in response to inflammatory mediators has been scarcely studied. Acute inflammation is vital in maintaining healthy tissue in response to infection; however, chronic inflammation can lead to the production of systemic inflammatory cytokines and deregulated leukocyte trafficking, even in the absence of a local infection. Elevated levels of cytokines in circulation underlie the pathogenesis of sepsis, the leading cause of intensive care death. Because ECs constitute a key barrier between circulation (luminal interface) and tissue (abluminal interface), we hypothesize that ECs respond differentially to inflammatory challenge originating in the tissue versus circulation as in local and systemic inflammation, respectively. To begin this investigation, we stimulated ECs abluminally and luminally with the inflammatory cytokine tumor necrosis factor alpha (TNF-α) to mimic a key feature of local and systemic inflammation, respectively, in a microvascular mimetic (μSiM-MVM). Polarized IL-8 secretion and polymorphonuclear neutrophil (PMN) transmigration were quantified to characterize the EC response to luminal versus abluminal TNF-α. We observed that ECs uniformly secrete IL-8 in response to abluminal TNF-α and is followed by PMN transmigration. The response to abluminal treatment was coupled with the formation of ICAM-1-rich membrane ruffles on the apical surface of ECs. In contrast, luminally stimulated ECs secreted five times more IL-8 into the luminal compartment than the abluminal compartment and sequestered PMNs on the apical EC surface. Our results identify clear differences in the response of ECs to TNF-α originating from the abluminal versus luminal side of a monolayer for the first time and may provide novel insight into future inflammatory disease intervention strategies.

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide. Most cases are multifactorial in etiology, but some are associated with variants in the myocilin gene, MYOC. Here, we report the identification of a novel MYOC variant, c.1153G>A, in a 24-yr-old female patient with a personal and family history of juvenile/early-onset POAG. Further genetic testing within her family demonstrated that this variant segregates with the POAG phenotype in an autosomal dominant pattern. Identification of this MYOC variant in multiple affected relatives provides evidence for its pathogenicity, supporting previous findings linking MYOC mutations, in particular in the third exon's olfactomedin domain, to juvenile-onset POAG. This case also emphasizes the potential value of genetic testing in families with histories of eye disorders.

A new take on membranes for vascular modeling. In article 1804111, James L. McGrath, Thomas R. Gaborski, and co-workers lithographically fabricate dual-scale (nano- and micro-porous) membranes from ultrathin (100 nm) silicon nitride nanomembranes to support human endothelial cell culture for vascular modeling at the lab bench. Micropores provide transmigration routes for studying immune, cancer, and stem cell egress through the vascular wall.

Nanoscale preconfinement of DNA has been shown to reduce the variation of passage times through solid-state nanopores. Preconfinement has been previously achieved by forming a femtoliter-sized cavity capped with a highly porous layer of nanoporous silicon nitride (NPN). This cavity was formed by sealing a NPN nanofilter membrane against a substrate chip using water vapor delamination. Ultimately, this method of fabrication cannot keep a consistent spacing between the filter and solid-state nanopore due to thermal fluctuations and wrinkles in the membrane, nor can it be fabricated on thousands of individual devices reliably. To overcome these issues, we present a method to fabricate the femtoliter cavity monolithically, using a selective XeF2 etch to hollow out a polysilicon spacer sandwiched between silicon nitride layers. These monolithically fabricated cavities behave identically to their counterparts formed by vapor delamination, exhibiting similar translocation passage time variation reduction and folding suppression of DNA without requiring extensive manual assembly. The ability to form nanocavity sensors with nanometer-scale precision and to reliably manufacture them at scale using batch wafer processing techniques will find numerous applications, including motion control of polymers for single-molecule detection applications, filtering of dirty samples prior to nanopore detection, and simple fabrication of single-molecule nanobioreactors.

Insulator-based dielectrophoresis (iDEP) is a simple, scalable mechanism that can be used for directly manipulating particle trajectories in pore-based filtration and separation processes. However, iDEP manipulation of nanoparticles presents unique challenges as the dielectrophoretic force [Formula: see text] exerted on the nanoparticles can easily be overshadowed by opposing kinetic forces. In this study, a molecularly thin, SiN-based nanoporous membrane (NPN) is explored as a breakthrough technology that enhances [Formula: see text] By numerically assessing the gradient of the electric field square [Formula: see text]-a common measure for [Formula: see text] magnitude-it was found that the unique geometrical features of NPN (pore tapering, sharp pore corner and ultrathin thickness) act in favor of intensifying the overall [Formula: see text] A comparative study indicated that [Formula: see text] generated in NPN are four orders of magnitude larger than track-etched polycarbonate membranes with comparable pore size. The stronger [Formula: see text] suggests that iDEP can be conducted under lower voltage bias with NPN: reducing joule heating concerns and enabling solutions to have higher ionic strength. Enabling higher ionic strength solutions may also extend the opportunities of iDEP applications under physiologically relevant conditions. This study also highlights the effects of [Formula: see text] induced by the ion accumulation along charged surfaces (electric-double layer (EDL)). EDL-based [Formula: see text] exists along the entire charged surface, including locations where geometry-based iDEP is negligible. The high surface-to-volume ratio of NPN offers a unique platform for exploiting such EDL-based DEP systems. The EDL-based [Formula: see text] was also found to offset the geometry-based [Formula: see text] but this effect was easily circumvented by reducing the EDL thickness (e.g. increasing the ionic strength from 0.1 to 100 mM). The results from this study imply the potential application of iDEP as a direct, in-operando antifouling mechanism for ultrafiltration technology, and also as an active tuning mechanism to control the cut-off size limit for continuous selectivity of nanomembrane-based separations.

This paper reports water and salt transport properties of sulfonated polysulfone desalination membranes prepared by solvent-free, melt extrusion. The 20mol% disulfonated poly(arylene ether sulfone) (BPS-20K) membranes were prepared by melt processing, using poly(ethyelene glycol) (PEG) M̅n (200 ~ 400g/mol) as plasticizers at concentrations of 20wt% to 30wt%, and different PEG extraction temperatures. Water and salt transport properties of BPS-20K membranes prepared by different processing routes correlated well with water uptake, as expected, based on free volume theory. The melt-extruded BPS-20K membranes show higher water uptake than those of solution cast membranes. As PEG molecular weight and concentration used during extrusion increases and as PEG extraction temperature increases, water uptake also increases. As water uptake increases, water and salt permeabilities and diffusivities increase, consistent with the findings of Yasuda et al. In general, BPS-20K membranes prepared by different processing routes followed the trade-off relationship between water permeability and water/salt permeability selectivity. These results indicate that differences in membrane processing history have significant effects on the transport properties of small molecules in these polymers, similar to other glassy polymers.

Disulfonated poly(arylene ether) and polyimide copolymers can phase separate to form nanoscale hydrophilic and hydrophobic domain morphology. Water in these domains has been assigned to three different states, tightly bound, loosely bound and free water. The states of water were characterized using DSC, NMR relaxation and TGA. The chemical structures of the proton exchange membranes were varied in chemical composition, microstructure (random and block) and also regarding functional sulfonic acid groups fixed to the polymer backbones. A strong dependence of the types of water on the ion content and morphology was observed. For the random copolymers, the formation of free water in the system occurs after reaching a certain ion content. For the multi-blocks, in addition to the ion content, free water also develops with increasing block lengths. The formation of continuous morphology in the copolymers was correlated to the onset of free water. Influences of the states of water on PEM transport properties will be reported separately.

Controlled molecular weight poly(arylene ether sulfone) oligomers with aromatic amine end groups and systematically varied degrees of disulfonation were synthesized by direct polymerization of disulfonated and non-sulfonated 4,40-dichlorodiphenylsulfone. The oligomers were crosslinked with a tetrafunctional epoxy curing agent in the membrane casting process. Water uptake and IEC were investigated to understand how the structure and ion content affected the fixed charge concentrations (moles of ions/L of sorbed water). The hydrated mechanical properties of these copolymer networks were also studied in light of their ion contents and water uptake. At similar IECs, membranes with shorter similar to 5000 Da oligomers absorbed less water than those with similar to 10,000 Da blocks. The salt permeabilities correlated with water uptake and fixed charge density. Among the crosslinked membranes, the one with the 10,000 Da oligomer and with 50% disulfonation (mB5-10) had an excellent combination of water uptake, hydrated mechanical properties, fixed charge density, and low salt permeability. (C) 2017 Published by Elsevier Ltd.

This manuscript represents a second part to the investigation of multiblock disulfonated poly(arylene ether sulfone) and poly(arylene ether nitrile) copolymers. In Part 1 it was shown that the multiblock copolymers had high ion exchange capacities (IECs), phase separation, strong membrane integrity and moderate water uptake. In this second part, the electrochemical properties of the membranes are presented and compared against those of the perfluorosulfonated Nafion 212 membrane. The disulfonated poly(arylene ether sulfone) and poly(arylene ether nitrile) multiblock copolymer membranes were fabricated into membrane electrode assemblies (MEAs), and tested in four different humidity environments at the electrodes (95/95%, 100/50%, 50/50%, and 75/25%). The hydrocarbon MEA had similar performance to the Nafion® 212 MEA at low current density, and outperformed the Nafion MEA at high current density for high humidities, and at lower humidities had almost comparable performance to the perfluorosulfonated MEA. The cell testing of the MEAs agreed well with the relative humidity conductivity results obtained for the membranes.

Poly(arylene ether sulfone) and poly(ethylene oxide) (PEO)-based segmented polyurethanes were synthesized as potential gas separation membrane materials. Poly(arylene ether sulfone) oligomers with controlled molecular weights (15,000 and 20,000 g/mol M-n) were prepared with phenol endgroups by nucleophilic aromatic substitution step growth polymerization of 4,4'-dichlorodiphenylsulfone with a calculated excess of 4,4'-(propane-2,2-diyl)diphenol (bisphenol A). The oligomers with phenolic endgroups were subsequently reacted with ethylene carbonate to obtain aliphatic hydroxyethyl terminal functionality. The hydroxyethyl terminated polysulfone oligomers and hydroxy-terminated PEO were reacted with 4,4'-methylene diphenyl diisocyanate (MDI) to obtain segmented polyurethanes. Compositions with high poly(arylene ether sulfone) content relative to the hydrophilic PEO blocks were of particular interest due to their mechanical integrity. Size exclusion chromatograms confirmed that the polyurethanes had high molecular weights with unimodal molecular weight distributions. DMA and DSC thermograms revealed polyurethanes containing 30 wt % PEO have two distinct Tg's. Pure gas permeabilities of membranes were measured using a series of gases having different kinetic diameters including H-2, He, CO2, O-2, N-2, and CH4. Gas transport measurements showed changes in permeabilities as the PEO content in the polyurethane increased. The CO2/N-2 and CO2/CH4 gas selectivity of membranes increased systematically as PEO content increased in the polyurethanes. (C) 2017 Elsevier Ltd. All rights reserved.

Two series of high molecular weight disulfonated poly(arylene ether sulfone) random copolymers were synthesized as proton exchange membranes for high-temperature water electrolyzers. These copolymers differ based on the position of the ether bonds on the aromatic rings. One series is comprised of fully para-substituted hydroquinone comonomer, and the other series incorporated 25 mol % of a meta-substituted comonomer resorcinol and 75 mol % hydroquinone. The influence of the substitution position on water uptake and electrochemical properties of the membranes were investigated and compared to that of the state-of-the-art membrane Nafion. The mechanical properties of the membranes were measured for the first time in fully hydrated conditions at ambient and elevated temperatures. Submerged in water, these hydrocarbon-based copolymers had moduli an order of magnitude higher than Nafion. Selected copolymers of each series showed dramatically increased proton conductivities at elevated temperature in fully hydrated conditions, while their H2 gas permeabilities were well controlled over a wide range of temperatures. These improved properties were attributed to the high glass transition temperatures of the disulfonated poly(arylene ether sulfone)s.