A functional human skin barrier is critical in limiting harmful exposure to environmental agents and regulating the absorption of intentionally applied topical drug and cosmetic products. Inherent ...differences in the skin barrier between consumers due to extrinsic and intrinsic factors are an important consideration in the safety assessment of dermatological products. Race is a concept often used to describe a group of people who share distinct physical characteristics. The observed predisposition of specific racial groups to certain skin pathologies highlights the potential differences in skin physiology between these groups. In the context of the human skin barrier, however, the current data correlating function to race often conflict, likely as a consequence of the range of experimental approaches and controls used in the existing works. To date, a variety of methods have been developed for evaluating compound permeation through the human skin, both
in vivo
and
in vitro
. Additionally, great strides have been made in the development of reconstructed human pigmented skin models, with the flexibility to incorporate melanocytes from donors of different race and pigmentation levels. Together, the advances in the production of reconstructed human skin models and the increased adoption of
in vitro
methodologies show potential to aid in the standardization of dermal absorption studies for discerning racial- and skin pigmentation-dependent differences in the human skin barrier. This review analyzes the existing data on skin permeation, focusing on its interaction with race and skin pigmentation, and highlights the tools and research opportunities to better represent the diversity of the human populations in dermal absorption assessments.
Sphingosine-1-phosphate (S1P) signals to enhance or destabilize the vascular endothelial barrier depending on the receptor engaged. Here, we investigated the differential barrier effects of S1P on ...two influential primary endothelial cell (EC) types, human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). S1PR1 (barrier protective) and S1PR3 (barrier disruptive) surface and gene expression were quantified by flow cytometry and immunofluorescence, and RT-qPCR, respectively. Functional evaluation of EC monolayer permeability in response to S1P was quantified with transendothelial electrical resistance (TEER) and small molecule permeability. S1P significantly enhanced HUVEC barrier function, while promoting HPMEC barrier breakdown. Immunofluorescence and flow cytometry analysis showed select, S1PR3-high HPMECs, suggesting susceptibility to barrier destabilization following S1P exposure. Reevaluation of HPMEC barrier following S1P exposure under inflamed conditions demonstrated synergistic barrier disruptive effects of pro-inflammatory cytokine and S1P. The role of the Rho-ROCK signaling pathway under these conditions was confirmed through ROCK1/2 inhibition (Y-27632). Thus, the heterogeneous responses of ECs to S1P signaling are mediated through Rho-ROCK signaling, and potentially driven by differences in the surface expression of S1PR3.
•S1P has heterogeneous barrier effects on native HUVECs and HPMECs.•Minor population of HPMECs contains high level of S1PR3 expression.•Inflammation exacerbates negative barrier effects of S1P on HPMECs.•Y-27632 inhibition of Rho-ROCK negates S1P-Induced HPMEC barrier disruption.
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 (
= 62). Moreover, the dynamic range of the sensor extends at least from 10
virions per mL to 10
virions per mL covering the range of mean viral loads in symptomatic COVID-19 patients (10
-10
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.
Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network ...(OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 μm non-motile cocci, S. aureus deforms smaller than 0.5 μm in the sub-micron channels of the OLCN. Here we present the μSiM-CA (Microfluidic – Silicon Membrane – Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The μSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the μSiM-CA. However the μSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.
We successfully developed the μSiM-CA (Microfluidic – Silicon Membrane – Canalicular Array) platform to distinguish the phenotype of mutant S. aureus strains based on their propagation through nanopores that mimic canaliculi invaded during chronic osteomyelitis. In doing so, we demonstrated the application of the μSiM-CA as a predictive tool for canalicular invasion by studying a putative virulence regulator of S. aureus, the accessory gene regulator, in the μSiM-CA and in an in vivo model of implant-associated osteomyelitis. Display omitted
Skin permeation is a primary consideration in the safety assessment of cosmetic ingredients, topical drugs, and human users handling veterinary medicinal products. While excised human skin (EHS) ...remains the ‘gold standard’ for in vitro permeation testing (IVPT) studies, unreliable supply and high cost motivate the search for alternative skin barrier models. In this study, a standardized dermal absorption testing protocol was developed to evaluate the suitability of alternative skin barrier models to predict skin absorption in humans. Under this protocol, side-by-side assessments of a commercially available reconstructed human epidermis (RhE) model (EpiDerm-200-X, MatTek), a synthetic barrier membrane (Strat-M, Sigma-Aldrich), and EHS were performed. The skin barrier models were mounted on Franz diffusion cells and the permeation of caffeine, salicylic acid, and testosterone was quantified. Transepidermal water loss (TEWL) and histology of the biological models were also compared. EpiDerm-200-X exhibited native human epidermis-like morphology, including a characteristic stratum corneum, but had an elevated TEWL as compared to EHS. The mean 6 h cumulative permeation of a finite dose (6 nmol/cm2) of caffeine and testosterone was highest in EpiDerm-200-X, followed by EHS and Strat-M. Salicylic acid permeated most in EHS, followed by EpiDerm-200-X and Strat-M. Overall, evaluating novel alternative skin barrier models in the manner outlined herein has the potential to reduce the time from basic science discovery to regulatory impact.
Introduction
The pathophysiological increase in microvascular permeability plays a well-known role in the onset and progression of diseases like sepsis and atherosclerosis. However, how interactions ...between neutrophils and the endothelium alter vessel permeability is often debated.
Methods
In this study, we introduce a microfluidic, silicon-membrane enabled vascular mimetic (μSiM-MVM) for investigating the role of neutrophils in inflammation-associated microvascular permeability. In utilizing optically transparent silicon nanomembrane technology, we build on previous microvascular models by enabling
in situ
observations of neutrophil-endothelium interactions. To evaluate the effects of neutrophil transmigration on microvascular model permeability, we established and validated electrical (transendothelial electrical resistance and impedance) and small molecule permeability assays that allow for the
in situ
quantification of temporal changes in endothelium junctional integrity.
Results
Analysis of neutrophil-expressed β
1
integrins revealed a prominent role of neutrophil transmigration and basement membrane interactions in increased microvascular permeability. By utilizing blocking antibodies specific to the β
1
subunit, we found that the observed increase in microvascular permeability due to neutrophil transmigration is constrained when neutrophil-basement membrane interactions are blocked. Having demonstrated the value of
in situ
measurements of small molecule permeability, we then developed and validated a quantitative framework that can be used to interpret barrier permeability for comparisons to conventional Transwell™ values.
Conclusions
Overall, our results demonstrate the potential of the μSiM-MVM in elucidating mechanisms involved in the pathogenesis of inflammatory disease, and provide evidence for a role for neutrophils in inflammation-associated endothelial barrier disruption.
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.
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
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
platforms. In addition to the ease of use, low cost, and high controllability,
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
platforms.
studies of TEM, specifically, showcase the great experimental progress at the lab bench and highlight the historical success of
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.
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.
Selective cellular transmigration across the microvascular endothelium regulates the innate and adaptive immune responses, stem cell localization, and cancer cell metastasis. Integration of ...traditional microporous membranes into microfluidic vascular models permits the rapid assay of transmigration events but suffers from poor reproduction of the cell permeable basement membrane. Current microporous membranes in these systems have large non-porous regions between micropores that inhibit cell communication and nutrient exchange on the basolateral surface reducing their physiological relevance. Here we present the use of 100 nm thick continuously nanoporous silicon nitride membranes as a base substrate for lithographic fabrication of 3 μm pores, resulting in a highly porous (~30%), dual-scale nano- and micro-porous membrane for use in an improved vascular transmigration model. Ultrathin membranes were patterned using a precision laser writer for cost-effective, rapid micropore design iterations. The optically transparent dual-scale membranes enabled complete observation of leukocyte egress across a variety of pore densities. We also discovered a maximal density of ~ 14 micropores per cell beyond which cell-substrate interactions are compromised giving rise to endothelial cell losses under flow. Addition of a subluminal extracellular matrix rescued cell adhesion, allowing for creation of shear-primed endothelial barrier models on nearly 30% continuously porous substrates.
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