Influence of Surfactant on the Skin Permeation of Methylisothiazolinone and Methylchloroisothiazolinone.

Patch tests are often used in safety evaluations to identify the substance causing skin irritation, but the same substance can sometimes give positive or negative results depending on the test conditions. Here, we investigated differences in the skin penetration of two test compounds under different application conditions. We studied the effects of the anionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant polysorbate 80 (PS) on skin penetration of the preservatives methylisothiazolinone (MT) and methylchloroisothiazolinone (MCT), which are used in cosmetics such as shampoos. The skin permeation of MT was enhanced by SDS but was unchanged by PS. Skin impedance decreased in the presence of SDS whereas PS had the same effect as the control aqueous solution, suggesting that SDS reduction of the barrier function of skin affects the permeation of MT, a hydrophilic drug. Application of a mixture of MCT and MT in the presence of SDS did not affect the skin permeation of MCT whereas the permeation of MT was enhanced by SDS, indicating that the skin permeation of MCT is less affected by SDS than is MT. Thus, attention should be paid to the possible effect of co-solutes, especially hydrophilic drugs.

Rheological Properties and Composition Affecting the Skin Permeation of a Model of a Hydrophilic Drug in Lecithin Reverse Wormlike Micelles.

We investigated the effect of the rheological properties and composition of lecithin reverse wormlike micelles (LRWs) on the skin permeation of a model of a hydrophilic drug to determine whether LRWs support uniform hydrophilic drug/oil-based formulations and good drug penetrate into skin. Here, we prepared LRWs with D (-)-ribose (RI) or glycerol (GL) as polar compounds, liquid paraffin (LP) or isopropyl myristate (IPM) as oils, and 6-carboxyfluorescein (CF) as a model for a hydrophilic drug, and evaluated the rheological properties and skin penetration characteristics of the preparations. The LRWs showed moderate viscosity at 25 °C, a typical storage temperature, but decreasing viscosity at 32 °C, the surface temperature of human skin, suggesting that the LRWs would penetrate the microstructure of skin (e.g., wrinkles and hair follicles). The highest skin permeability of CF was observed when IPM was used as the oil, suggesting that both the stratum corneum and hair follicle routes are involved in drug permeation. The penetration of CF into hair follicles is influenced not only by the rheology of the formulation but also by the interaction between IPM and sebum in the hair follicles.

Preparation and Application of Decanoic Acid/Arginine Hydrogels to a Transdermal Formulation.

We prepared a supramolecular hydrogel composed of decanoic acid and arginine (C10/Arg gel) and evaluated its application to a transdermal formulation. C10/Arg gel adjusted to pH 7 with 1 M NaOH aq or 1 M HCl aq provided a translucent hydrogel with a lamellar liquid crystal structure in the concentration region of decanoic acid ≥12% and arginine ≤9%. Rheological measurements showed that C10/Arg gel is a viscoelastic material with both solid and liquid properties, with elasticity being dominant over viscosity in the low shear stress region. The skin permeability of hydrocortisone (HC) and indomethacin (IM) from C10/Arg gels was investigated in vitro using hairless mouse skin and compared to control formulation drug suspensions (IM or HC) in water. The cumulative permeation amount of HC and IM from the C10/Arg gel at 10 h after application was approximately 16 and 11 times higher than that of the control, respectively. On the other hand, the flux of IM decreased with increasing arginine concentration, likely due to the acid-base interaction between Arg and IM in C10/Arg gel. Adequate drug skin permeation enhancement by C10/Arg gel requires optimizing the gel composition for each specific drug.

Validation study for in vitro skin irritation test using reconstructed human skin equivalents constructed by layer-by-layer cell coating technology.

The aim of this study is to validate an in vitro skin irritation test (SIT) using three-dimensional reconstructed human epidermal (RhE) skin equivalents prepared by layer-by-layer (LbL) method (LbL-3D Skin) in a series of interlaboratory studies. The goal of these validation studies is to evaluate the ability of this in vitro test to reliably discriminate skin irritant from nonirritant chemicals, as defined by OECD and UN GHS. This me-too validation study is to assess the within- and between-laboratory reproducibility, as well as the predictive capacity, of the LbL-3D Skin SIT in accordance with performance standards for OECD TG 439. The developed skin model, LbL-3D Skin had a highly differentiated epidermis and dermis, similar to the validated reference methods (VRM) and native human skin. The quality parameters (cell survival in controls, tissue integrity, and barrier function) were similar to VRM and in accordance with OECD TG 439. The LbL-3D Skin SIT validation study was performed by three participating laboratories and consisted of three independent tests using 20 reference chemicals. The results obtained with the LbL-3D Skin demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from nonirritants. The predictive potency of LbL-3D Skin SIT using total 54 test chemicals were comparable to those in other RhE models in OECD TG 439. The validation study demonstrated that LbL-3D Skin has proven to be a robust and reliable method for predicting skin irritation.

Giant Cell Arteritis after COVID-19 Vaccination with Long-Term Follow-Up: A Case Report and Review of the Literature.

Giant cell arteritis (GCA) is a chronic vasculitis that primarily affects the elderly, and can cause visual impairment, requiring prompt diagnosis and treatment. The global impact of the coronavirus disease 2019 (COVID-19) pandemic has been substantial. Although vaccination programs have been a key defense strategy, concerns have arisen regarding post-vaccination immune-mediated disorders and related risks. We present a case of GCA after COVID-19 vaccination with 2 years of follow-up. A 69-year-old woman experienced fever, headaches, and local muscle pain two days after receiving the COVID-19 vaccine. Elevated inflammatory markers were observed, and positron emission tomography (PET) revealed abnormal uptake in the major arteries, including the aorta and subclavian and iliac arteries. Temporal artery biopsy confirmed the diagnosis of GCA. Treatment consisted of pulse therapy with methylprednisolone, followed by prednisolone (PSL) and tocilizumab. Immediately after the initiation of treatment, the fever and headaches disappeared, and the inflammation markers normalized. The PSL dosage was gradually reduced, and one year later, a PET scan showed that the inflammation had resolved. After two years, the PSL dosage was reduced to 3 mg. Fourteen reported cases of GCA after COVID-19 vaccination was reviewed to reveal a diverse clinical picture and treatment response. The time from onset of symptoms to GCA diagnosis varied from two weeks to four months, highlighting the challenge of early detection. The effectiveness of treatment varied, but was generally effective similarly to that of conventional GCA. This report emphasizes the need for clinical vigilance and encourages further data collection in post-vaccination GCA cases.

Effect of the Physicochemical Properties of Liquid Paraffin on the Phase State and Rheological Properties of Lecithin Reverse Wormlike Micelles.

Lecithin reverse wormlike micelles (LRWs) have been studied recently for dermal application dosage use but the effects of the physicochemical properties of oils on the formation and rheological properties of LRWs have not been investigated. We studied the effect of oil on the formation of LRWs using 5 types of liquid paraffin (LP) with kinematic viscosities ranging from 4.00 to 88.0 mm2/s. Partial phase diagrams of lecithin/water/LP systems indicated that LPs with low molecular weights could form LRWs with only a small amount of water, but LPs with high molecular weights could not form LRWs, regardless of the water concentration. The solubility of lecithin in LPs was higher for low molecular weight LPs, thus possibly affecting the formation of LRWs. The zero-shear viscosity and relaxation time of LRWs initially increased with increasing water concentration, and then decreased. The water concentration providing the maximum value was dependent on the molecular weight of the LP, whereas the maximum amount and length of the LRWs were independent of the water concentration. Our results indicate that the molecular weight of LP affects the ease of formation and the viscoelasticity of LRWs.

Incorporation and influence of Leishmania histone H3 in chromatin.

Immunopathologies caused by Leishmania cause severe human morbidity and mortality. This protozoan parasite invades and persists inside host cells, resulting in disease development. Leishmania modifies the epigenomic status of the host cells, thus probably averting the host cell defense mechanism. To accomplish this, Leishmania may change the host cell chromatin structure. However, the mechanism by which the parasite changes the host cell chromatin has not been characterized. In the present study, we found that ectopically produced Leishmania histone H3, LmaH3, which mimics the secreted LmaH3 in infected cells, is incorporated into chromatin in human cells. A crystallographic analysis revealed that LmaH3 forms nucleosomes with human histones H2A, H2B and H4. We found that LmaH3 was less stably incorporated into the nucleosome, as compared to human H3.1. Consistently, we observed that LmaH3-H4 association was remarkably weakened. Mutational analyses revealed that the specific LmaH3 Trp35, Gln57 and Met98 residues, which correspond to the H3.1 Tyr41, Arg63 and Phe104 residues, might be responsible for the instability of the LmaH3 nucleosome. Nucleosomes containing LmaH3 resisted the Mg2+-mediated compaction of the chromatin fiber. These distinct physical characteristics of LmaH3 support the possibility that histones secreted by parasites during infection may modulate the host chromatin structure.

A Method for Measuring the Surface Free Energy of Topical Semi-solid Dosage Forms.

Our aim was to determine the surface free energy (SFE) of semi-solid dosage forms (SSDFs) by establishing a reproducible method for measuring the contact angle of liquids to SSDFs. Four SSDFs were used: petrolatum, an oil/water (O/W) and a water/oil (W/O) cream, and an alcohol-based gel. The SSDFs were evenly spread on a glass slide, and the change in contact angle over time was measured by dropping water, glycerol, diiodomethane and n-hexadecane as the test liquids. Depending on the combination of test liquid and SSDF, the contact angle was either constant or decreased in an exponential manner. Contact angles may have decreased in an exponential manner because the reaction between the test liquid and the SSDF altered the interfacial tension between the two phases and changed the surface tension of the test liquid and the SFE of the SSDF. The contact angle of the test liquid to the SSDF could be determined reproducibly using the initial contact angle immediately after dropping the liquid on the SSDF as the contact angle before reaction. Using the obtained contact angles and the Owens-Wendt-Rabel-Kaelble equation, we calculated the SFE and its component for the SSDFs tested and found that the results reflect the physicochemical properties of SSDFs. Furthermore, the work of adhesion (WA) of the SSDF to Yucatan micropig skin was calculated using the SFE for the SSDFs. Interestingly, the WA values for all SSDFs tested were comparable.

Characteristics of an Emulsion Obtained Using Hydrophobic Hydroxypropyl Methylcellulose as an Emulsifier and a High-Pressure Homogenizer.

Hydrophobically modified hydroxypropyl methylcellulose (HM-HPMC), a polymer in which a small amount of HPMC is stearoxyl substituted, was used as an emulsifier of emulsion-type lotion. A high-pressure homogenizer (microfluidizer) was used. The viscosity of the 1% HM-HPMC aqueous gel decreased after passing through the microfluidizer from 5.5 to 2.7 Pa·s. When liquid paraffin (LP) was used as the oil phase, a stable emulsion was obtained with an LP ratio of 1-40%. The apparent viscosity decreased with LP ratios up to 20%, and then increased with increasing LP concentration. The emulsions with an LP ratio <20% presented a pseudo-viscous flow, similar to that of the diluted polymer solution. HM-HPMC likely adsorbed onto the oil with a stearoxyl group; thus, the interaction between the stearoxyl group, which explained the high viscosity of HM-HPMC, decreased, reducing the viscosity of the emulsion. The LP ratio was 40%, and the emulsion presented a plastic flow, which is typical of concentrated emulsions. The size of the droplet in the emulsion was approximately 1 µm regardless of the LP ratio. When low-viscosity LPs or monoester-type oils such as isopropyl myristate were used, some of the emulsions presented creaming. An emulsion using HM-HPMC as an emulsifier and an appropriate oil homogenized with a microfluidizer is stable, has low viscosity, and can be easily spread on skin.

Application of Pickering Emulsion with Cyclodextrin as an Emulsifier to a Transdermal Drug Delivery Vehicle.

The emulsion prepared with ß-cyclodextrin as an emulsifier (ßCDE) is considered to be a Pickering emulsion. We examined the characteristics of ßCDEs using captopril (CP) as a model drug, and studied the in vitro skin permeation of CP from ßCDEs through hairless mouse skin. The stability of ßCDE was increased with increasing ßCD concentration and conversely decreased with increasing CP concentration. The yield stress value from the rheological measurement results was suggested to be one of the factors determining the stability of the ßCDE, and ßCDEs with higher yield stress values were more stable. We found that the skin permeability of CP could be improved by using ßCDE with isopropyl myristate as the oil phase and that the flux of CP depended on the free CP concentration in the water phase of ßCDE.

The Comparison of Surface Free Energy of Human, Yucatan Micropig, and Hairless Mouse Skins and Influence of Surfactant on Surface Free Energy of the Skin.

Surface free energy (SFE) is an important factor for evaluation of wettability or adhesion. Thus, the SFE of a Yucatan micropig (YMP) skin and a hairless mouse (HM) skin, which are commonly used in skin permeation studies instead of human skin, were compared with the human skin. Contact angles of water and 1-bromo naphthalene to skin were measured and the SFE was calculated using the Owens-Wendt equation. The SFE of the human abdominal skin was 40 mN/m and its polar component σsp was as low as 2 mN/m, which was similar to that of the low sebum skin reported previously. In the case of the YMP skin, σsp was high on the surface but similar to that obtained after the skin was tape-stripped twice. The HM skin showed similar SFE as that of the human skin. When the surfactant was applied on the skin, wiped, and dried, the remaining surfactant increased the SFE in σsp; however, the original SFE was obtained after rinsing with water. The YMP skin and HM skin is similar to the human abdominal skin with a low sebum level. Thus, they are also good skin models for studying wettability or adhesion of a substance.

Crystal Structure and Characterization of Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8.

Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.

Skin Permeation of Testosterone from Viscoelastic Lecithin Reverse Wormlike Micellar Solution.

We evaluated testosterone-containing lecithin reverse wormlike micelles (reverse worms) composed of a polar substance/lecithin/isopropyl myristate for transdermal application. Water, D-ribose, or tetraglycerol were used as the polar substance and were key ingredients for forming the reverse worms. Using the reverse worms, 1 wt% of testosterone could be stably solubilized. When using D-ribose as polar substance, the maximum zero-shear viscosity of the reverse worms solution was higher than that of systems using water or tetraglycerol as the polar substance. The mechanism of skin permeation of testosterone from reverse worms solution was elucidated using skin permeation experiments with hairless mouse skin. When the structure of the reverse worms transitioned to lamellar liquid crystals at the skin/formulation interface, testosterone became supersaturated in the formulations. The structural transition occurred in systems using water or D-ribose as the polar substance, increasing the flux of testosterone. The flux of testosterone from reverse worms solution thus depends on the type of polar substance used.

A method for evaluating nucleosome stability with a protein-binding fluorescent dye.

Nucleosomes are extremely stable histone-DNA complexes that form the building blocks of chromatin, which accommodates genomic DNA within the nucleus. The dynamic properties of chromatin play essential roles in regulating genomic DNA functions, such as DNA replication, recombination, repair, and transcription. Histones are the protein components of nucleosomes, and their diverse modifications and variants increase the versatility of nucleosome structures and their dynamics in chromatin. Therefore, a technique to evaluate the physical properties of nucleosomes would facilitate functional studies of the various nucleosomes. In this report, we describe a convenient assay for evaluating the thermal stability of nucleosomes in vitro.

Observation and density estimation of a large number of skin capillaries using wide-field portable video capillaroscopy and semantic segmentation.

Significance: Skin capillaries are non-invasively observable; their structure and blood flow can reflect tissue and systemic conditions. Quantitative analysis of video-capillaroscopy images yields novel diagnostic methods. Because the capillary structure is heterogeneous, analyzing more capillaries can increase the evaluation reliability. Aim: We developed a system that can observe and quantify numerous capillaries and verified the performance on human skin. Approach: We developed a portable video-capillaroscope with a spatial resolution higher than 3.5 µm and a wide field of view (7.4 mm×5.5 mm) and a method to evaluate capillary numbers and areas using U-Net. The model was trained and tested with 22 and 11 cropped images (2.4 mm×1.9 mm) obtained from 11 participants, respectively. They were then applied to the 7.2 mm×5.3 mm images from four participants. Segmentation results were compared to ground-truth at the pixel level and capillary-region level. Results: Over 1000 capillaries were simultaneously observed using the proposed system. Although pixel-level segmentation performance was low [intersection over union (IoU) = 24.5%], the number and area could be estimated. These values differed among four participants and seven sites, and they changed after skin barrier destruction. Conclusions: The proposed system allows for observing and quantifying numerous skin capillaries simultaneously, suggesting its potential for evaluating tissue and systemic conditions.

Novel decomposition of polycarbonate and effect for marine ecosystem.

Analysis of pollution of the ocean plastics is presently being extensively carried out but special attention should be direct to matters. It is widely believed that plastic dose not decompose in the ocean. Certain contaminants, bisphenol-A (BPA) that serves the material for polycarbonate (PC) and epoxy resin (EPX) both of which may possibly be elute or degrade from commercial products, have often been detected in rivers, lakes and oceans. To clarify in detail the extend of this impact of this situation, purified PC (BPA free) was decomposed at temperatures range 50-230 °C. PC was seen to start decomposing at 50 °C over a 3 day period to generated 11 µg kg-1 BPA. Based on the rate constants of BPA, the activation energy was calculated 42.0 kJ mol-1. Since this value is almost same as the EPX and polystyrene (PS) of each decomposition. Based on the PC decomposition rate and the actual BPA value in the deep sea, the 280 million metric tons (MT) BPA in the world ocean was estimated. Unlike plastics, BPA shows endocrine disrupting in fish. It should thus be considered that degraded PC and EPX pose a serious threat to the marine ecosystem, directly.

Stratum Corneum Ceramide Profiles Provide Reliable Indicators of Remission and Potential Flares in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Prevention of exacerbation of AD is a crucial issue for all physicians. However, exacerbation of AD often is seen during reduction of AD treatment, even with appropriate follow-up by tapered topical corticosteroids and daily topical moisturizers, indicating the need for good indicators of AD remission. We hypothesized that the presence of mutations in FLG or the stratum corneum ceramide profile on AD remission phase may predict the ease of AD exacerbation. This study examined the differences in the frequency of FLG mutations or stratum corneum ceramide profiles (stratum corneum levels and carbon chain length for 11 ceramide classes [ceramides containing nonhydroxy fatty acids and dihydrosphingosines; nonhydroxy fatty acids and sphingosines; nonhydroxy fatty acids and 6-hydroxysphingosines; nonhydroxy fatty acids and phytosphingosines; a-hydroxy fatty acids and dihydrosphingosines; a-hydroxy fatty acids and sphingosines; a-hydroxy fatty acids and 6-hydroxysphingosines; a-hydroxy fatty acids and phytosphingosines; ester-linked fatty acids, o-hydroxy fatty acids, and sphingosines; ester-linked fatty acids, o-hydroxy fatty acids, and 6-hydroxysphingosines; and ester-linked fatty acids, o-hydroxy fatty acids, and phytosphingosines]) at AD remission phase between the two AD study groups: subsequent exacerbation (‒) and (+) of AD. The frequency of FLG mutations did not differ between the study groups. On the other hand, the carbon chain lengths of ceramides containing nonhydroxy fatty acids and dihydrosphingosines, nonhydroxy fatty acids and sphingosines, and nonhydroxy fatty acids and 6-hydroxysphingosines were shorter in the exacerbated AD group than in the maintained-AD group. Thus, the stratum corneum ceramide profile at the remission phase of AD is a potential biomarker, predicting the likelihood of substantial AD remission or subsequent AD exacerbation.

Stroke volume variation obtained with Vigileo/FloTrac™ system during bleeding and fluid overload in dogs.

PURPOSE: Stroke volume variation (SVV) is a parameter for estimating fluid responsiveness. Recently, the Vigileo™ and the Flo-Trac™ sensor (Edwards Lifesciences, Irvine, CA, USA) were made available for clinical use to estimate SVV. The aim of this study was to investigate the relationship between the circulating blood volume and SVV, measured by the Vigileo-FloTrac™ system (SVV-FloTrac) or by central venous pressure (CVP), during a dynamic change in circulating blood transfusion volume, using a continuous constant bleeding and fluid-overload model in dogs. METHODS: Ten anesthetized and mechanically ventilated beagles were used. SVV-FloTrac and CVP were measured during a bleeding period (2 ml/kg/min, 15 min), a stabilization period (15 min), a blood transfusion period (2 ml/kg/min, 15 min), and a 6% hydroxyethyl starch solution overload period (2 ml/kg/min, 15 min). RESULTS: SVV-FloTrac changed significantly when more than 8 ml/kg blood was withdrawn or when more than 8 ml/kg blood was transfused. The change in SVV-FloTrac directly reflected the circulating blood volume change during continuous bleeding and blood transfusion. CVP decreased significantly when more than 4 ml/kg blood was withdrawn or when more than 10 ml/kg was infused, and this indicated that the CVP change did not directly reflect the level of the circulating blood volume change. During the stable circulating blood volume period after blood withdrawal, SVV-FloTrac changed significantly but CVP remained constant. During the fluid overload period, CVP, but not SVV-FloTrac, changed significantly. CONCLUSION: SVV-FloTrac is a sensitive indicator of the dynamic circulating blood volume change during both bleeding and transfusion, but not during either the stable circulating blood volume period after blood withdrawal or the fluid-overload period, in mechanically ventilated dogs.

Case Report: A Rare Case of Esophagogastric Junctional Squamous Cell Carcinoma After the Successful Treatment of Neuroendocrine Carcinoma: Clonal Tumor Evolution Revealed by Genetic Analysis.

Neuroendocrine carcinoma (NEC) of the esophagogastric junction (EGJ) is a rare disease with no established treatments. Herein, we describe a case of recurrent squamous cell carcinoma (SCC) after achieving complete response to chemotherapy against NEC of the EGJ. A 67-year-old man was referred to our hospital because of epigastric discomfort. Computed tomography imaging and esophagogastroduodenoscopy revealed ulcerated tumors at the EGJ. Endoscopic biopsy revealed small tumor cells with a high nuclear/cytoplasmic ratio, suggesting small-cell NEC. Immunohistochemistry (IHC) analysis showed tumor cells with an MIB-1 index of 80%. The patient achieved complete response after 10 cycles of chemotherapy. Follow-up endoscopic examination revealed small red-colored mucosal lesions in the center of the cicatrized primary lesion. Re-biopsy detected cancer cells harboring large eosinophilic cytoplasm with keratinization and no evidence of NEC components. IHC of the cells were cytokeratin 5/6-positive and p53-negative. The tumor persisted without evidence of metastases after chemoradiotherapy, and total gastrectomy with lymph node dissection was performed. Pathological assessment of the resected specimens revealed SCC, without evidence of NEC. The patient survived without a recurrence for >3 years after the initial presentation. Somatic mutation profiles of the primary NEC and recurrent SCC were analyzed by targeted amplicon sequencing covering common cancer-related mutations. Both tumors possessed TP53 Q192X mutation, whereas SMAD4 S517T was found only in SCC, suggesting that both tumor components originated from a founder clone with a stop-gain mutation in TP53. The somatic mutation profile of the tumors indicated that that loss of heterozygosity (LOH) at the TP53 gene might have occurred during the differentiation of the founder clone into NEC, while a SMAD4 mutation might have contributed to SCC development, indicating branching and subclonal evolution from common founder clone to both NEC and SCC. The mutation assessments provided valuable information to better understand the clonal evolution of metachronous cancers.