Suppressive effect of Yokukansan on glutamate released from canine keratinocytes.

Background: Canine atopic dermatitis (CAD) is caused by skin barrier dysfunction due to allergen exposure. Excessive glutamate release in the skin is associated with delayed skin barrier function recovery and epidermal thickening and lichenification. Treatment with Yokukansan (YKS), a traditional Japanese medicine, reduces dermatitis severity and scratching behavior in NC/Nga mice by decreasing epidermal glutamate levels. However, the association between canine keratinocytes and glutamate and the mechanism by which YKS inhibits glutamate release from keratinocytes remains unknown. Aim: We aimed to investigate glutamate release from canine progenitor epidermal keratinocytes (CPEKs) and the inhibitory effect of YKS on this release. We also explored the underlying mechanism of YKS to enable its application in CAD treatment. Methods: Glutamate produced from CPEKs in the medium at 24 hours was measured. The measurement conditions varied in terms of cell density and YKS concentration. CPEKs were treated with a glutamate receptor antagonist (MK-801), a glutamate transporter antagonist (THA), and a glutamate dehydrogenase inhibitor (epigallocatechin gallate; EGCG), and the inhibitory effect of YKS, YKS + THA, MK-801, and EGCG on this release was determined. MK-801 and glutamate dehydrogenase inhibitor were tested alone, and THA was tested in combination with YKS. Finally, glutamine incorporated into CPEKs at 24 hours was measured using radioisotope labeling. Results: CPEKs released glutamate in a cell density-dependent manner, inhibited by YKS in a concentration-dependent manner. Moreover, YKS reduced the intracellular uptake of radioisotope-labeled glutamine in a concentration-dependent manner. No involvement of glutamate receptor antagonism or activation of glutamate transporters was found, as suggested by previous studies. In addition, EGCG could inhibit glutamate release from CPEKs. Conclusion: Our findings indicated that glutamate release from CPEKs could be effectively inhibited by YKS, suggesting the utility of YKS in maintaining skin barrier function during CAD. In addition, CPEKs are appropriate for analyzing the mechanism of YKS. However, we found that the mechanism of action of YKS differs from that reported in previous studies, suggesting that it may have had a similar effect to EGCG in this study. Further research is warranted to understand the exact mechanism and clinical efficacy in treating CAD.

Calcitriol modulates epidermal tight junction barrier function in human keratinocytes.

BACKGROUND: The aberrant expression of tight junction (TJ) proteins play an important role in several diseases with impaired skin barriers, including atopic dermatitis, psoriasis, and chronic wounds. The evidence provided thus far suggests an important role of calcitriol in skin homeostasis. However, it is not known whether calcitriol improves the impaired skin barrier. OBJECTIVE: To investigate the effect of calcitriol on TJ barrier function in human primary keratinocytes. METHODS: Normal human primary keratinocytes were stimulated with calcitriol, and the expression of TJ-related proteins was measured by real-time PCR and Western blotting. Immunofluorescence was used to examine the intercellular distribution of TJ-related proteins. TJ barrier function was assessed by the transepithelial electrical resistance (TER) assay. RESULTS: We demonstrated that calcitriol increased the expression levels of TJ-related proteins, including claudin-4, claudin-7, occludin, and zonula occludens (ZO)- 1. Calcitriol enhanced the distribution of TJ-related proteins at cellcell borders and induced the phosphorylation of pathways involved in the regulation of TJ barrier function, such as atypical protein kinase C (aPKC), Ras-related C3 botulinum toxin substrate 1 (Rac1), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt), as evidenced by the effects of specific inhibitors on the above pathways. Indeed, we confirmed that calcitriol enhanced TER in keratinocyte monolayers. CONCLUSION: These findings showed that calcitriol could modify the expression of keratinocyte TJ proteins, contributing to the maintenance of homeostatic barrier function.

Antigen Protease Activity on Intact or Tape-Stripped Skin Induces Acute Itch and T Helper Sensitization Leading to Airway Eosinophilia in Mice.

Respiratory allergen sources such as house dust mites frequently contain proteases. In this study, we demonstrated that the epicutaneous application of a model protease antigen, papain, onto intact or tape-stripped ear skin of mice induced acute scratching behaviors and T helper (Th)2, Th9, Th17/Th22, and/or Th1 sensitization in a protease activity-dependent manner. The protease activity of papain applied onto the skin was also essential for subsequent airway eosinophilia induced by an intranasal challenge with low-dose papain. With tape stripping, papain-treated mice showed barrier dysfunction, the accelerated onset of acute scratching behaviors, and attenuated Th17/Th22 sensitization. In contrast, the protease activity of inhaled papain partially or critically contributed to airway atopic march responses in mice sensitized through intact or tape-stripped skin, respectively. These results indicated that papain protease activity on epicutaneous application through intact skin or skin with mechanical barrier damage is critical to the sensitization phase responses, including acute itch and Th sensitization and progression to the airway atopic march, whereas dependency on the protease activity of inhaled papain in the atopic march differs by the condition of the sensitized skin area. This study suggests that exogenous protease-dependent epicutaneous mechanisms are a target for controlling allergic sensitization and progression to the atopic march.

A Systemic Lupus Erythematosus Patient with Cutaneous Mycobacterium haemophilum Infection under Belimumab Treatment: A Case Report.

A 38-year-old female with systemic lupus erythematosus (SLE) initiated belimumab treatment. One month later, she presented with a reddish painful swelling on her right lower leg. She was treated with ceftriaxone and vancomycin. However, novel erythematous papules and indurated nodules appeared on both her lower legs. Skin biopsy revealed microabscess formation with mixed cell granuloma surrounded by inflammatory cell infiltration within the dermis with subcutaneous fat tissue. A large number of acid-fast bacilli were observed with Ziehl-Neelsen staining. DNA sequencing of both the hsp65 and the 16S rRNA sequences showed a 100% match with the corresponding region of Mycobacterium haemophilum. Mycobacterial culture revealed satellite growth enhancement on Middlebrook 7H11 agar plates around a paper strip containing hemin. She was treated with levofloxacin, rifabutin, and ethambutol. Within 13 months, her cutaneous lesions improved markedly without any side effects. The B cell-targeted biologic belimumab, a fully humanized IgG1γ monoclonal antibody that inactivates B lymphocyte stimulator, has been considered to be beneficial for active SLE. However, this therapy could increase the risk for the development of biologic therapy-associated mycobacterial infections, both tuberculosis and nontuberculous mycobacteria infections.

Clinical Findings, Response to Steroid Treatment, and Recurrence Rate in Alopecia Areata Patients with or without a Nonsynonymous Variant of Coiled-Coil Alpha-Helical Rod Protein 1.

BACKGROUND: Alopecia areata (AA) is considered complex genetic and tissue-specific autoimmune disease. We recently discovered a nonsynonymous variant in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene within the AA risk haplotype. And a water avoidance stress test on CCHCR1 knockout mice induced AA-like lesions. OBJECTIVE: To investigate the difference clinical findings of AA in patients with the CCHCR1 variant and without. METHODS: We conducted a retrospective analysis of the data from 142 AA patients. Among these patients, 20 (14.1%) had a variant of CCHCR1. We evaluated the sex distribution of the patients, age at onset, distribution of the clinical types, prevalence of a positive family history of AA, prevalence of association of AA with atopic dermatitis, response to steroid therapy, and recurrence rate. We used multivariate logistic regression analysis and Fisher's exact test for statistical analysis. We also investigate electron microscopic observations of hair samples with the CCHCR1 variant and without. RESULTS: The results showed a significant correlation between the CCHCR1 variant and the recurrence rate compared with the variant-negative group (p=0.0072). Electron microscopy revealed abnormalities in the hair shaft structure and hair cuticle in patients with the CCHCR1 variant (p=0.00174). CONCLUSION: Our results suggest that AA with CCHCR1 variant is clinically characterized by a high recurrence rate and hair morphological abnormality.

A Case of Kerion Celsi due to Trichophyton tonsurans.

The patient was a 13-year-old boy who was a member of the judo club at his junior high school. Approximately 1 week prior to his presentation, he developed multiple erythematous pilaris papules on his occipital area and was treated by a local doctor. The erythematous lesions expanded to 10 × 10 cm, showing granulation with drainage and strong spontaneous pain. At this point, he visited our hospital. He was diagnosed with kerion celsi due to Trichophyton tonsurans by fungal examination. The patient was treated with terbinafine (125 mg/day) for 6 weeks, and a brush test at 6 weeks was negative. All 18 members of the judo club, including this patient, were investigated; brush tests were positive in 4 cases, and one was positive for tinea corporis alone. The patient's family members parents were both negative. When an athlete is diagnosed with ringworm, T. tonsurans infection should be considered, and testing and treatment of family members and fellow athletes should be carried out to prevent the spread of infection.

Recent Advances in Clinical Research on Rare Intractable Hereditary Skin Diseases in Japan.

Our Research Group for Rare and Intractable Skin Diseases operates within the Project for Research on Intractable Diseases of the Ministry of Health, Labour, and Welfare of Japan and is conducting research on eight rare intractable skin diseases. Five of these are monogenic disorders (epidermolysis bullosa, congenital ichthyoses, oculocutaneous albinism, pseudoxanthoma elasticum, and hereditary angioedema), and for a sixth [generalized pustular psoriasis (GPP)], genetic predisposing factors are important. This review introduces our activities for raising public awareness of these six intractable hereditary skin diseases and summarizes our recent achievements in clarifying the situation of medical treatments for these diseases in Japan. We note our current progress in elucidating the pathogeneses of these diseases and in developing new treatment methods, and we discuss our progress in establishing clinical practice guidelines. A nationwide survey on epidermolysis bullosa and a clinical survey on congenital ichthyoses are progressing. The Angioedema Activity Score and the Angioedema Quality-of-Life Questionnaire, the latter of which is a quality-of-life evaluation tool, have been established for hereditary angioedema. Registries of patients with oculocutaneous albinism and pseudoxanthoma elasticum have been created, and the registry for the latter has achieved its target of 170 cases. For GPP, the results of our survey on clinical practice were published in 2021. Information regarding all six of these hereditary skin diseases has been disseminated to academic societies, medical professionals, patients, and the general public.

Antifungal Susceptibility Testing for Microsporum canis from Cats in Japan.

Control of infection caused by Microsporum canis in pet animals are important for prevention of zoonosis. Treatments for animal dermatophytosis have generally consisted of itraconazole (ITZ) and terbinafine (TRF); however, a TRF-resistant M. canis strain from a case of feline dermatophytosis has been reported. In the present study, we examined the in vitro susceptibility of clinical isolates of M. canis to new antifungal drugs, such as ravuconazole (RVZ) and luliconazole (LCZ). The results indicated that RVZ and LCZ are more effective than ITZ and TRF. Therefore, oral administration of RVZ or topical application of LCZ may serve as new treatment options.

The Antimicrobial Peptide AMP-IBP5 Suppresses Dermatitis-like Lesions in a Mouse Model of Atopic Dermatitis through the Low-Density Lipoprotein Receptor-Related Protein-1 Receptor.

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) exhibits antimicrobial activities and immunomodulatory functions in keratinocytes and fibroblasts. However, its role in regulating skin barrier function remains unclear. Here, we investigated the effects of AMP-IBP5 on the skin barrier and its role in the pathogenesis of atopic dermatitis (AD). 2,4-Dinitrochlorobenzene was used to induce AD-like skin inflammation. Transepithelial electrical resistance and permeability assays were used to investigate tight junction (TJ) barrier function in normal human epidermal keratinocytes and mice. AMP-IBP5 increased the expression of TJ-related proteins and their distribution along the intercellular borders. AMP-IBP5 also improved TJ barrier function through activation of the atypical protein kinase C and Rac1 pathways. In AD mice, AMP-IBP5 ameliorated dermatitis-like symptoms restored the expression of TJ-related proteins, suppressed the expression of inflammatory and pruritic cytokines, and improved skin barrier function. Interestingly, the ability of AMP-IBP5 to alleviate inflammation and improve skin barrier function in AD mice was abolished in mice treated with an antagonist of the low-density lipoprotein receptor-related protein-1 (LRP1) receptor. Collectively, these findings indicate that AMP-IBP5 may ameliorate AD-like inflammation and enhance skin barrier function through LRP1, suggesting a possible role for AMP-IBP5 in the treatment of AD.

Examination of colorectal cancer cases with metal allergy.

PURPOSE: To report our initiatives and treatment results for patients with colorectal cancer with metal allergy. METHODS: A total of 27 patients (2.6%) with a history of metal contact dermatitis were identified among 1027 patients who underwent curative resection of colorectal cancer from 2014 to 2020. The results of the patch test, perioperative results, and postoperative colonoscopy findings were also investigated. RESULTS: The patch test for metal allergens and staples was performed in 21 patients (77.8%), and 13 of them (61.9%) tested positive for at least one metal allergen. Ni (38.1%), Co (28.6%), and Pd (19.0%) showed higher positive rates than other metals, and 1 patient (4.8%) tested positive for staples. Stapled anastomosis/suturing was performed as planned in 15 of 27 patients. In 10 patients, the anastomosis method was changed from stapled to hand-sewn according to the no-patch test results (60%), positivity for multiple metals (20%), positivity for staples (10%), and surgeon's judgment (10%). No complications and abnormal colonoscopy findings were found to be associated with stapled anastomosis/suturing. CONCLUSION: The patch test is useful for selecting an optimal anastomosis method for patients with suspected metal allergy.

Measurement of human skin moisture via high-frequency spectroscopy.

High-frequency spectroscopy (HFS) is an analytical method that is sensitive to slight changes in the dielectric properties of materials. Since water has high permittivity, HFS can be used to detect changes in water content in materials. In this study, we employed HFS to measure human skin moisture during a water sorption-desorption test. Skin without any treatment showed a resonance peak at approximately 1150 MHz. Furthermore, the peak shifted to lower frequency immediately after the application of water to the skin and gradually returned to its original frequency as time progressed. The resonance frequency obtained via least-squares fitting showed that the applied water remained in the skin after 240 s from the beginning of the measurement. These results illustrated that HFS measurements can monitor the progression of decreasing moisture content in human skin during a water sorption-desorption test.

Cathelicidin LL-37 Activates Human Keratinocyte Autophagy through the P2X7, Mechanistic Target of Rapamycin, and MAPK Pathways.

Human cathelicidin LL-37 is a multifunctional antimicrobial peptide that exhibits antimicrobial and immunomodulatory activities. LL-37 regulates skin barrier function and was recently reported to activate autophagy in macrophages. Because autophagy deficiency is associated with skin diseases characterized by a dysfunctional epidermal barrier, we hypothesized that LL-37 might regulate the skin barrier through autophagy modulation. We showed that LL-37 activated autophagy in human keratinocytes and three-dimensional skin equivalent models as indicated by increases in LC3 puncta formation, decreases in p62, and autophagosome and autolysosome formation. LL-37‒induced autophagy was suppressed by P2X7 receptor, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 inhibitors, suggesting that the P2X7, adenosine monophosphate‒activated protein kinase, and unc-51-like kinase 1 pathways are involved. Moreover, LL-37 enhanced the phosphorylation of adenosine monophosphate‒activated protein kinase and unc-51-like kinase 1. In addition, LL-37‒mediated autophagy involves the mechanistic target of rapamycin and MAPK pathways. Interestingly, the LL-37‒induced distribution of tight junction proteins and improvement in the tight junction barrier were inhibited in autophagy-deficient keratinocytes and keratinocytes and skin models treated with autophagy inhibitors, indicating that the LL-37‒mediated tight junction barrier is associated with autophagy activation. Collectively, these findings suggest that LL-37 is a potential therapeutic target for skin diseases characterized by dysfunctional autophagy and skin barriers.

Microsporum canis Infection in a Cat Breeder Family and an Investigation of Their Breeding Cats.

Two pediatric cases of Microsporum canis infection that occurred in a cat breeder family and the isolation of dermatophytes from their 166 breeding cats are reported. The patients were a 16-month-old girl and her 26-month-old sister who both had tinea capitis. Their family consisted of six members: the sisters, their great-grandmother, grandmother, grandfather, and mother. Except for the two sisters, the family had no history of skin lesions. The grandmother had been a cat breeder for 20 years. We tested the cats using the hairbrush technique, and 56 of the 158 cats (35%) tested were positive for M. canis. In particular, cultures performed from 4 cats developed M. canis colonies that grew densely from all spikes on the hairbrush. On the basis of observations of the cultures, cutaneous infection was suspected when five or more colonies grew on a single plate medium (9 cats), whereas growth of fewer colonies was thought to suggest saprophytic colonization on cat hair. M. canis is known to be highly transmittable among cats, but 65% of the cats investigated remained negative. It was thus considered possible to prevent further spread of infection by practicing basic infection control and improving the environment.

Experimental and Clinical Evidence Suggests That Treatment with Betacellulin Can Alleviate Th2-Type Cytokine-Mediated Impairment of Skin Barrier Function.

Betacellulin (BTC) is a peptide ligand that belongs to the epidermal growth factor family, the members of which have been implicated in skin morphogenesis, homeostasis, repair, and angiogenesis; however, the role of BTC in the regulation of the skin barrier remains unknown. To examine the role of BTC in skin barrier function, we analyzed atopic dermatitis (AD) transcriptomic data from Gene Expression Omnibus (GEO) datasets, performed BTC immunohistochemistry using human skin tissues, and evaluated the effects of BTC on primary human keratinocytes by real-time PCR, Western blotting, and assay of the transepidermal electrical resistance (TER), a functional parameter to monitor the tight junction barrier. We found that the gene expression of BTC was downregulated in skin lesions from patients with AD, and this downregulated expression recovered following biological treatments. Consistently, the BTC protein levels were downregulated in the lesional skin of AD patients compared with the normal skin of healthy participants, suggesting that the BTC levels in skin might be a biomarker for the diagnosis and therapy of AD. Furthermore, in human keratinocytes, BTC knockdown reduced the levels of skin-derived antimicrobial peptides and skin barrier-related genes, whereas BTC addition enhanced their levels. Importantly, in human skin equivalents, BTC restored the increased tight junction permeability induced by Th2 cytokine IL-4/IL-13 treatment. In addition, specific inhibitors of epidermal growth factor receptor (EGFR) and protein kinase C (PKC) abolished the BTC-mediated improvement in skin barrier-related proteins in keratinocyte monolayers. Collectively, our findings suggest that treatment with BTC might improve the Th2-type cytokine-mediated impairment of skin barrier function through the EGFR/PKC axis and that BTC might be a novel potential biomarker and therapeutic target for the treatment of skin conditions characterized by the overproduction of Th2 cytokines and dysfunctional skin barriers, such as AD.

Skin Treatment with Detergent Induces Dermatitis with H1-Antihistamine-Refractory Itch and Upregulates IL-4 and Th17/Th22 Cytokine Gene Expression in C57BL/6 Mice.

INTRODUCTION: Repeated skin contact to detergents causes chronic irritant contact dermatitis (ICD) associated with itch sensation and eczema. However, the mechanisms of detergent-induced ICD are poorly understood. Here, we established a new murine model of detergent-induced ICD with H1-antihistamine-refractory itch. METHODS: Ear skin of wild-type and mast cell-deficient mice on the C57BL/6 genetic background was treated with a detergent, sodium dodecyl/lauryl sulfate (SDS), daily for approximately 2 weeks with or without administration of an H1-antihistamine, fexofenadine. Skin inflammation, barrier dysfunction, and itching were analyzed. Quantitative PCR for earlobe gene expression and flow cytometry analysis for draining lymph node cells were conducted. RESULTS: SDS treatment induced skin inflammation with ear swelling, increased transepidermal water loss, and hind-paw scratching behaviors in the wild-type and mast cell-deficient mice. The peak value of scratching bouts was retained for at least 48 h after the last SDS treatment. H1-antihistamine administration showed no or little reduction in the responses. SDS treatment upregulated gene expression for a Th2 cytokine IL-4 and Th17/Th22 cytokines, IL-17A, IL-17F, and IL-22, and increased cell numbers in draining lymph nodes of CD4+ T, CD8+ T, and γδT cells with enhanced expression of GATA3, RORγt, T-bet, or FOXP3 compared with untreated mice. CONCLUSIONS: The present study showed that SDS treatment of ear skin in C57BL/6 mice induces mast cell-independent skin inflammation with H1-antihistamine-refractory itch and suggested a possible Th cytokine- and/or lymphocyte-mediated regulation of the model. The model would be useful for elucidation of mechanisms for inflammation with H1-antihistamine-refractory itch in detergent-induced ICD.