Direct assessment of individual skin barrier components by electrical impedance spectroscopy.

BACKGROUND: Expression of the tight junction proteins Cldn1 and 4 is altered in skin diseases such as atopic dermatitis, and Cldn1 deficiency affects skin barrier formation. Impedance spectroscopy (IS) has been proven to allow detection of alterations in the skin barrier but is currently unable to separate effects on viable epidermis (VE) and stratum corneum (SC). METHODS: Effects of siRNA-mediated Cldn1 and 4 knockdown in reconstructed human epidermis (RHE) on VE and SC barrier function were investigated with Ussing chamber-based IS. Barrier components were sequentially altered, employing iron oxide nanoparticles and EGTA, to identify their contribution to the impedance spectrum. Resistance changes due to apically applied hyperosmolar electrolyte were used to identify barrier defects non-invasively. RESULTS: IS of RHE yielded two relaxation frequencies, representing the barrier properties of the SC (~1000 Hz) and VE (~100 Hz). As proof of concept, it was shown that the Cldn1 knockdown-induced resistance drop arises from the impairment of both SC and VE, indicated by a shift of both relaxation frequencies. Hyperosmolar electrolyte penetration allowed non-invasive detection of Cldn1 knockdown via time-dependent frequency shifts. The absence of Cldn4 knockdown-induced changes revealed the weaknesses of transepithelial electrical resistance analysis. CONCLUSION: In conclusion, the present technique allows to separately measure the barrier properties of SC and VE and further evaluate the Cldn1 and 4 knockdown impact on the skin barrier. As the measurement with agarose-embedded electrolyte allowed non-invasive identification of the Cldn1 knockdown, this opens the way to detailed in vivo skin barrier assessment.

Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells.

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.

Glutathione-conjugated sulfanylalkanols are substrates for ABCC11 and γ-glutamyl transferase 1: a potential new pathway for the formation of odorant precursors in the apocrine sweat gland.

We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH.

Equivalent Behavioral Facilitation to Tactile Cues in Children with Autism Spectrum Disorder.

The alerting network, a subcomponent of attention, enables humans to respond to novel information. Children with ASD have shown equivalent alerting in response to visual and/or auditory stimuli compared to typically developing (TD) children. However, it is unclear whether children with ASD and TD show equivalent alerting to tactile stimuli. We examined (1) whether tactile cues affect accuracy and reaction times in children with ASD and TD, (2) whether the duration between touch-cues and auditory targets impacts performance, and (3) whether behavioral responses in the tactile cueing task are associated with ASD symptomatology. Six- to 12-year-olds with ASD and TD participated in a tactile-cueing task and were instructed to respond with a button press to a target sound /a/. Tactile cues were presented at 200, 400, and 800 ms (25% each) prior to the auditory target. The remaining trials (25%) were presented without tactile cues. Findings suggested that both groups showed equivalent alerting responses to tactile cues. Additionally, all children were faster to respond to auditory targets at longer cue-target intervals. Finally, there was an association between rate of facilitation and RRB scores in all children, suggesting that patterns of responding to transient phasic cues may be related to ASD symptomatology.

Electrophysiological Measures of Tactile and Auditory Processing in Children With Autism Spectrum Disorder.

Behavioral differences in responding to tactile and auditory stimuli are widely reported in individuals with autism spectrum disorder (ASD). However, the neural mechanisms underlying distinct tactile and auditory reactivity patterns in ASD remain unclear with theories implicating differences in both perceptual and attentional processes. The current study sought to investigate (1) the neural indices of early perceptual and later attentional factors underlying tactile and auditory processing in children with and without ASD, and (2) the relationship between neural indices of tactile and auditory processing and ASD symptomatology. Participants included 14, 6-12-year-olds with ASD and 14 age- and non-verbal IQ matched typically developing (TD) children. Children participated in an event-related potential (ERP) oddball paradigm during which they watched a silent video while being presented with tactile and auditory stimuli (i.e., 80% standard speech sound/a/; 10% oddball speech sound/i/; 10% novel vibrotactile stimuli on the fingertip with standard speech sound/a/). Children's early and later ERP responses to tactile (P1 and N2) and auditory stimuli (P1, P3a, and P3b) were examined. Non-parametric analyses showed that children with ASD displayed differences in early perceptual processing of auditory (i.e., lower amplitudes at central region of interest), but not tactile, stimuli. Analysis of later attentional components did not show differences in response to tactile and auditory stimuli in the ASD and TD groups. Together, these results suggest that differences in auditory responsivity patterns could be related to perceptual factors in children with ASD. However, despite differences in caregiver-reported sensory measures, children with ASD did not differ in their neural reactivity to infrequent touch-speech stimuli compared to TD children. Nevertheless, correlational analyses confirmed that inter-individual differences in neural responsivity to tactile and auditory stimuli were related to social skills in all children. Finally, we discuss how the paradigm and stimulus type used in the current study may have impacted our results. These findings have implications for everyday life, where individual differences in responding to tactile and auditory stimuli may impact social functioning.

Attentional Disengagement and the Locus Coeruleus - Norepinephrine System in Children With Autism Spectrum Disorder.

BACKGROUND: Differences in non-social attentional functions have been identified as among the earliest features that distinguish infants later diagnosed with autism spectrum disorder (ASD), and may contribute to the emergence of core ASD symptoms. Specifically, slowed attentional disengagement and difficulty reorienting attention have been found across the lifespan in those at risk for, or diagnosed with, ASD. Additionally, the locus coeruleus-norepinephrine (LC-NE) system, which plays a critical role in arousal regulation and selective attention, has been shown to function atypically in ASD. While activity of the LC-NE system is associated with attentional disengagement and reorienting in typically developing (TD) individuals, it has not been determined whether atypical LC-NE activity relates to attentional disengagement impairments observed in ASD. OBJECTIVE: To examine the relationship between resting pupil diameter (an indirect measure of tonic LC-NE activation) and attentional disengagement in children with ASD. METHODS: Participants were 21 school-aged children with ASD and 20 age- and IQ-matched TD children. The study consisted of three separate experiments: a resting eye-tracking task and visual and auditory gap-overlap paradigms. For the resting eye-tracking task, pupil diameter was monitored while participants fixated a central crosshair. In the gap-overlap paradigms, participants were instructed to fixate on a central stimulus and then move their eyes to peripherally presented visual or auditory targets. Saccadic reaction times (SRT), percentage of no-shift trials, and disengagement efficiency were measured. RESULTS: Children with ASD had significantly larger resting pupil size compared to their TD peers. The groups did not differ for overall SRT, nor were there differences in SRT for overlap and gap conditions between groups. However, the ASD group did evidence impairments in disengagement (larger step/gap effects, higher percentage of no-shift trials, and reduced disengagement efficiency) compared to their TD peers. Correlational analyses showed that slower, less efficient disengagement was associated with increased pupil diameter. CONCLUSION: Consistent with prior reports, children with ASD show significantly larger resting pupil diameter, indicative of atypically elevated tonic LC-NE activity. Associations between pupil size and measures of attentional disengagement suggest that atypically increased tonic activation of the LC-NE system may be associated with poorer attentional disengagement in children with ASD.

Claudin-1 decrease impacts epidermal barrier function in atopic dermatitis lesions dose-dependently.

The transmembrane protein claudin-1 is a major component of epidermal tight junctions (TJs), which create a dynamic paracellular barrier in the epidermis. Claudin-1 downregulation has been linked to atopic dermatitis (AD) pathogenesis but variable levels of claudin-1 have also been observed in healthy skin. To elucidate the impact of different levels of claudin-1 in healthy and diseased skin we determined claudin-1 levels in AD patients and controls and correlated them to TJ and skin barrier function. We observed a strikingly broad range of claudin-1 levels with stable TJ and overall skin barrier function in healthy and non-lesional skin. However, a significant decrease in TJ barrier function was detected in lesional AD skin where claudin-1 levels were further reduced. Investigations on reconstructed human epidermis expressing different levels of claudin-1 revealed that claudin-1 levels correlated with inside-out and outside-in barrier function, with a higher coherence for smaller molecular tracers. Claudin-1 decrease induced keratinocyte-autonomous IL-1ß expression and fostered inflammatory epidermal responses to non-pathogenic Staphylococci. In conclusion, claudin-1 decrease beyond a threshold level results in TJ and epidermal barrier function impairment and induces inflammation in human epidermis. Increasing claudin-1 levels might improve barrier function and decrease inflammation and therefore be a target for AD treatment.

Biphasic influence of Staphylococcus aureus on human epidermal tight junctions.

Bacterial infections (e.g., with Staphylococcus aureus) are serious problems in skin with a compromised barrier, such as in patients with atopic dermatitis. Previously, it was shown that tight junction (TJ) proteins are influenced by staphylococcal infection, and TJ function is impaired after infection of the keratinocyte cell line HaCaT. However, functional studies in cells or models more similar to human skin are missing. Therefore, we investigated bacterial colonialization and infection with live S. aureus in primary human keratinocytes and reconstructed human epidermis (RHE). We show that short-term inoculation results in increased TJ barrier function-which could not be seen in HaCaT cells-hinting at an early protective effect. This is accompanied by occludin phosphorylation and sustained localization of occludin and claudin-4 at cell membranes. Long-term incubation resulted in decreased presence of claudin-1 and claudin-4 at cell membranes and decreased TJ barrier function. The agr regulon of S. aureus plays a role in the increasing but not in the decreasing effect. Proinflammatory cytokines, which are produced as a result of S. aureus inoculation, influence both phases. In summary, we show here that S. aureus can have short-term promoting effects on the TJ barrier, while in the long term it results in disturbance of TJs.

The role of tight junctions in skin barrier function and dermal absorption.

The skin protects our body from external assaults like pathogens, xenobiotics or UV irradiation. In addition, it prevents the loss of water and solutes. To fulfill these important tasks, a complex barrier system has developed which comprises the stratum corneum, tight junctions, the microbiome, the chemical barrier and the immunological barrier. These barriers do not act separately, but influence each other e.g. after external manipulation or in skin diseases. Especially the two mechanical barriers, i.e. stratum corneum and tight junctions, are of great interest for drug delivery, because they are the first interaction partners of drug delivery systems and play the major role in skin absorption. Tight junctions are of special interest, as they are centrally localized in this complex barrier system in the outermost viable layer - the stratum granulosum of the interfollicular epidermis and the companion cell layer of the hair follicle - and because they can react very quickly to stimuli. We summarize here our current knowledge about tight junction barrier function in mammalian interfollicular epidermis and hair follicles, and the interaction of tight junctions with other skin barrier components in health and disease. Furthermore, we discuss their relevance for drug delivery and provide examples for tight junction modulators.