Volatile organic compounds and ionic substances contamination in cell processing facilities during rest period; a preliminary assessment of exposure to cell processing operators.

Introduction: Cell processing operators (CPOs) use a variety of disinfectants that vaporize in the workspace environment. These disinfectants can induce allergic reactions in CPOs, due to their long working hours at cell processing facilities (CPFs). Ionic substances such as CH3COO- generated from peracetic acid, nitrogen oxides (NOx) and sulfur oxides (SOx) from outdoor environment are also known to pollute air. Therefore, our objective was to assess the air quality in CPFs and detect volatile organic compounds (VOCs) from disinfectants and building materials, and airborne ionic substances from outdoor air. Methods: Sampling was conducted at three CPFs: two located in medical institutions and one located at a different institution. Air samples were collected using a flow pump. Ion chromatographic analysis of the anionic and cationic compounds was performed. For VOC analysis, a thermal desorption analyzer coupled with capillary gas chromatograph and flame ionization detector was used. Results: Analysis of the ionic substances showed that Cl-, NOx, and SOx, which were detected in large amounts in the outdoor air, were relatively less in the CPFs. Ethanol was detected as the main component in the VOC analysis. Toluene was detected at all sampling points. As compared to the other environments, air in the incubator contained larger amounts of VOCs, that included siloxane, tetradecane, and aromatics. Conclusions: No VOCs or ionic substances of immediate concern to the health of the CPOs were detected during the non-operating period. However, new clinical trials of cell products are currently underway in Japan, and a variety of new cell products are expected to be approved. With an increase in cell processing, health risks to CPOs that have not been considered previously, may become apparent. We should continue to prepare for the future expansion of the industry using a scientific approach to collect various pieces of information and make it publicly available to build a database.

A Connecting Link between Hyaluronan Synthase 3-Mediated Hyaluronan Production and Epidermal Function.

Hyaluronan (HA), an essential component of the extracellular matrix of the skin, is synthesized by HA synthases (HAS1-3). To date, epidermal HA has been considered a major player in regulating cell proliferation and differentiation. However, a previous study reported that depletion of epidermal HA by Streptomyces hyaluronidase (St-HAase) has no influence on epidermal structure and function. In the present study, to further explore roles of epidermal HA, we examined effects of siRNA-mediated knockdown of HAS3, as well as conventional HA-depletion methods using St-HAase and 4-methylumbelliferone (4MU), on epidermal turnover and architecture in reconstructed skin or epidermal equivalents. Consistent with previous findings, HA depletion by St-HAase did not have a substantial influence on the epidermal architecture and turnover in skin equivalents. 4MU treatment resulted in reduced keratinocyte proliferation and epidermal thinning but did not seem to substantially decrease the abundance of extracellular HA. In contrast, siRNA-mediated knockdown of HAS3 in epidermal equivalents resulted in a significant reduction in epidermal HA content and thickness, accompanied by decreased keratinocyte proliferation and differentiation. These results suggest that HAS3-mediated HA production, rather than extracellularly deposited HA, may play a role in keratinocyte proliferation and differentiation, at least in the developing epidermis in reconstructed epidermal equivalents.

Antiwrinkle efficacy of 1-ethyl-ß-N-acetylglucosaminide, an inducer of epidermal hyaluronan production.

BACKGROUND: Hyaluronan (HA) has a unique hydration capacity that contributes to firmness and bounciness of the skin. Epidermal HA declines with skin aging, which may lead to clinical signs of aging including skin wrinkles and loss of hydration and elasticity. Recently, we developed a new cosmetic agent 1-ethyl-ß-N-acetylglucosaminide (ß-NAG2), which enhances HA production in cultured human keratinocytes. The aim of this study was to explore antiaging potential of ß-NAG2 in reconstructed human epidermal models and human clinical trial. MATERIALS AND METHODS: The amount of HA in ß-NAG2-treated epidermal models by topical application was analyzed by enzyme-linked immunosorbent assay (ELISA)-like assay. A randomized, double-blind and placebo-controlled study was conducted in Japanese females (n = 33) by topically treating each side of the face with a lotion formulated with ß-NAG2 or placebo for 8 weeks. RESULTS: Topically applied ß-NAG2 dose dependently increased HA production in epidermal models. Treatment with ß-NAG2-formulated lotion significantly improved skin hydration and elasticity and reduced skin wrinkling in crow's foot areas when compared to the placebo formulation. CONCLUSION: Topically applied ß-NAG2 promoted epidermal HA production in vitro and showed antiwrinkle activity in vivo accompanying the improvement in skin hydration and elasticity. Our study provides a novel strategy for antiwrinkle care through ß-NAG2-induced epidermal HA production.

Accelerated human epidermal turnover driven by increased hyaluronan production.

BACKGROUND: Hyaluronan (HA) is an essential component of extracellular matrix in the skin, but its functions in the epidermis remain elusive. OBJECTIVE: We examined the interaction of increased HA production mediated by 1-ethyl-ß-N-acetylglucosaminide (ß-NAG2), a newly developed highly selective inducer of HA production which is intracellularly converted to UDP-N-acetylglucosamine, a substrate of HA, with epidermal proliferation and differentiation. METHODS: The amount, molecular size and epidermal tissue distribution of HA and expression of CD44, a cell surface receptor for HA, were analyzed in ß-NAG2-treated organ cultured human skin, reconstructed human skin equivalents or cultured human skin keratinocytes. The relationship between HA and epidermal proliferation or differentiation was examined. RESULTS: ß-NAG2 significantly increased HA production in the epidermis of skin explants or skin equivalents without affecting molecular size of HA (>2000 kDa) or CD44 mRNA expression. Histochemical experiments revealed that ß-NAG2 enhances HA signals in the basal to granular layers of the epidermis of skin equivalents, accompanying increased epidermal stratification. Immunohistochemical experiments demonstrated that signals of Ki67, transglutaminase 1 and filaggrin are increased in ß-NAG2-treated skin equivalents, and these observations were confirmed by the data showing that mRNA expression of PCNA, transglutaminase 1 (TGM1) and filaggrin (FLG) is significantly up-regulated by ß-NAG2 in skin equivalents. Importantly, blockade of HA production by inhibiting conversion of ß-NAG2 to UDP-NAG abolished ß-NAG2-mediated up-regulation of PCNA, TGM1 and FLG mRNA expression in cultured keratinocytes. CONCLUSION: These results suggest that increased epidermal HA production plays a key role in epidermal morphogenesis and homeostasis by accelerating keratinocyte proliferation and differentiation.

Impaired Tight Junctions in Atopic Dermatitis Skin and in a Skin-Equivalent Model Treated with Interleukin-17.

Tight junction (TJ) dysfunction in the stratum granulosum leads to aberrant barrier function of the stratum corneum (SC) in the epidermis. However, it is unclear whether TJs are perturbed in atopic dermatitis (AD), a representative aberrant SC-related skin disease, and whether some factors related to AD pathogenesis induce TJ dysfunction. To address these issues, we investigated the alterations of TJs in AD skin and the effects of Th2 and Th17 cytokines on TJs in a skin-equivalent model. The levels of TJ proteins were determined in the epidermis of nonlesional and lesional skin sites of AD. Western blot and immunohistochemical analyses revealed that the levels of zonula occludens 1 were decreased in the nonlesional sites of AD, and the levels of zonula occludens 1 and claudin-1 were decreased in the lesional sites relative to the levels in skin from healthy subjects. Next, we examined the effects of interleukin (IL)-4, tumor necrosis factor-α, IL-17, and IL-22 on the TJ barrier in a skin-equivalent model. Only IL-17 impaired the TJ barrier. Furthermore, we observed a defect in filaggrin monomer degradation in the IL-17-treated skin model. Thus, TJs are dysfunctional in AD, at least partly, due to the effect of IL-17, which may result in an aberrant SC barrier.

Origin of inner ear hair cells: morphological and functional differentiation from ciliary cells into hair cells in zebrafish inner ear.

Auditory and vestibular functions in vertebrates depend on the transduction of sound vibration or head acceleration into electrical responses in inner ear hair cells. Mechanoelectrical transduction occurs at the tip of stereocilia, which are polarized to form an orientational arrangement that determines directional sensitivity. It remains to be clarified when and how premature hair cells acquire their specialized structure and function in living animals. The developmental origin of inner ear hair cells has been studied in vivo in zebrafish embryos. Tether cells, a small number of ciliated cells associated with an "ear stone" (or otolith) in the embryonic zebrafish inner ear, are believed to be precocious hair cells. However, whether or not tether cells acquire hair bundles and mechanosensitivity remains unknown. In the present study, we investigated the morphological and functional development of tether cells. Immunohistochemical examination revealed that stereocilia appeared on the tether cell apex in a polarized arrangement at 22 h postfertilization (hpf). Labeling with FM1-43, a marker of functional mechanotransduction channels, and the in vivo electrophysiological recording of mechanotransducer responses in the developing inner ear demonstrated that tether cells acquired direction-selective mechanosensitivity at 23 hpf. These results revealed that tether cells begin to function as hair cells within an hour after the appearance of a polarized array of stereociliary bundles. Thus, the ciliary cells morphologically and functionally differentiate into the first sensory hair cells in the inner ear of the zebrafish.

Auditory input to CNS is acquired coincidentally with development of inner ear after formation of functional afferent pathway in zebrafish.

Auditory perception in vertebrates depends on transduction of sound into neural signals in the inner ear hair cells (HCs) and on transmission of these signals to the brain through auditory (VIIIth) nerve afferents. To investigate the developmental acquisition of auditory inputs by the CNS, we have electrophysiologically and morphologically examined the process of acquisition of auditory responsiveness by zebrafish macular HCs and the Mauthner cells (M-cells) in vivo. The M-cells are a paired large reticulospinal neurons in the hindbrain; they receive direct inputs from the VIIIth nerve afferents and initiate an acoustic startle response. Whole-cell recordings from the M-cells showed that sound-evoked postsynaptic currents were first observed around 40 h postfertilization (hpf); during subsequent development, onset latency decreased and amplitude increased. The appearance and development of microphonic potentials in the inner ear coincided with those of the acoustic responses of the M-cell, whereas the functional auditory circuits from the macular HCs to the M-cell were already formed at 27 hpf. These results suggest that the functional maturation of inner ear after formation of the auditory pathway is a critical process in the acquisition of auditory inputs by CNS neurons.