Disorganisation of basement membrane zone architecture impairs melanocyte residence in vitiligo.

The basement membrane zone is the interface between the epidermis and dermis, and it is disrupted in several skin conditions. Here, we report the results of a comprehensive investigation into the structural and molecular factors of the basement membrane zone in vitiligo, a dermatological disorder characterised by depigmented patches on the skin. Using electron microscopy and immunofluorescence staining, we confirmed abnormal basement membrane zone morphology and disrupted basement membrane zone architecture in human vitiliginous skin. Furthermore, we identified elevated expression of matrix metalloproteinase 2 (MMP2) in human dermal fibroblasts as a key factor responsible for basement membrane zone matrix degradation. In our in vitro and ex vivo models, overexpression of MMP2 in fibroblasts led to basement membrane zone disruption and melanocyte disappearance. Importantly, we reveal that the loss of melanocytes in vitiligo is primarily linked to their weakened adhesion to the basement membrane, mediated by binding between integrin ß1 and laminin and discoidin domain receptor 1 and collagen IV. Finally, inhibition of matrix metalloproteinase 2 expression reversed depigmentation in a mouse model of vitiligo. In conclusion, our research shows the importance of basement membrane zone integrity in melanocyte residence and offers new avenues for therapeutic interventions to address this challenging skin condition. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Naked mole-rat TMEM2 lacks physiological hyaluronan-degrading activity.

Mouse transmembrane protein 2 (mTMEM2) has been identified as a hyaluronidase, which has extracellularly G8 and GG domains and PbH1 repeats; however, our previously study showed that human TMEM2 (hTMEM2) is not a catalytic hyaluronidase due to the absence of the critical amino acid residues (His248/Ala303) in the GG domain. Naked mole-rats (NMRs) accumulate abundant high-molecular weight hyaluronan (HA) in their tissues, suggesting decreased HA degradation. Therefore, we aimed to evaluate the HA-degrading activity of NMR TMEM2 (nmrTMEM2) and compare it with those of mTMEM2 and hTMEM2. The amino acid residues of nmrTMEM2 (Asn247/Val302) are similar to Asn248/Phe303 of hTMEM2, and nmrTMEM2-expressing HEK293T cells showed negligible activity. We confirmed the significance of these amino acid residues using an inactive chimeric TMEM2 with the human GG domain, which acquired catalytic activity when Asn248/Phe303 was substituted with His248/Ala303. Semi-quantitative comparison of the activities of the membrane-fractions derived from m/h/nmrTMEM2-expressing HEK293T cells revealed that at least 20- and 14-fold higher amounts of nmr/hTMEM2 were required to degrade HA to the same extent as by mTMEM2. Thus, unlike mTMEM2, nmrTMEM2 is not a physiological hyaluronidase. The inability of nmrTMEM2 to degrade HA might partially account for the high-molecular-weight HA accumulation in NMR tissues.

Epidermal keratinocytes regulate hyaluronan metabolism via extracellularly secreted hyaluronidase 1 and hyaluronan synthase 3.

Hyaluronan (HA) is a high-molecular-weight (HMW) glycosaminoglycan, which is a fundamental component of the extracellular matrix that is involved in a variety of biological processes. We previously showed that the HYBID/KIAA1199/CEMIP axis plays a key role in the depolymerization of HMW-HA in normal human dermal fibroblasts (NHDFs). However, its roles in normal human epidermal keratinocytes (NHEKs) remained unclear. HYBID mRNA expression in NHEKs was lower than that in NHDFs, and NHEKs showed no depolymerization of extracellular HMW-HA in culture, indicating that HYBID does not contribute to extracellular HA degradation. In this study, we found that the cell-free conditioned medium of NHEKs degraded HMW-HA under weakly acidic conditions (pH 4.8). This degrading activity was abolished by hyaluronidase 1 (HYAL1) knockdown but not by HYAL2 knockdown. Newly synthesized HYAL1 was mainly secreted extracellularly, and the secretion of HYAL1 was increased during differentiation, suggesting that epidermal interspace HA is physiologically degraded by HYAL1 according to pH decrease during stratum corneum formation. In HA synthesis, hyaluronan synthase 3 (HAS3) knockdown reduced HA production by NHEKs, and interferon-γ-dependent HA synthesis was correlated with increased HAS3 expression. Furthermore, HA production was increased by TMEM2 knockdown through enhanced HAS3 expression. These results indicate that NHEKs regulate HA metabolism via HYAL1 and HAS3, and TMEM2 is a regulator of HAS3-dependent HA production.

JAK inhibitors for the treatment of vitiligo.

Vitiligo is an autoimmune disease involving melanocyte-targeting T cells initiated by environmental and genetic factors. Steroids and tacrolimus have been used as topical treatments. Recently, novel topical agents targeting Janus kinase (JAK), a family of tyrosine kinases that regulates cytokine signaling, have emerged. Ruxolitinib is the first approved in vitiligo therapy. Furthermore, ritlecitinib is currently under clinical trials for oral treatment of active vitiligo. In this review, we discuss the possibility of topical JAK inhibitors as promising options for the treatment of vitiligo with regard to their mechanism of action, efficacy and safety.

Human TMEM2 is not a catalytic hyaluronidase, but a regulator of hyaluronan metabolism via HYBID (KIAA1199/CEMIP) and HAS2 expression.

Cutaneous hyaluronan (HA) is depolymerized to intermediate sizes in the extracellular matrix, and further fragmented in the regional lymph nodes. Previously, we showed that the HA-binding protein involved in HA depolymerization (HYBID), also known as KIAA1199/CEMIP, is responsible for the first step of HA depolymerization. Recently, mouse transmembrane 2 (mTMEM2) with high structural similarity to HYBID was proposed to be a membrane-bound hyaluronidase. However, we showed that the knockdown of human TMEM2 (hTMEM2) conversely promoted HA depolymerization in normal human dermal fibroblasts (NHDFs). Therefore, we examined the HA-degrading activity and function of hTMEM2 using HEK293T cells. We found that human HYBID and mTMEM2, but not hTMEM2, degraded extracellular HA, indicating that hTMEM2 does not function as a catalytic hyaluronidase. Analysis of the HA-degrading activity of chimeric TMEM2 in HEK293T cells suggested the importance of the mouse GG domain. Therefore, we focused on the amino acid residues that are conserved in active mouse and human HYBID and mTMEM2 but are substituted in hTMEM2. The HA-degrading activity of mTMEM2 was abolished when its His248 and Ala303 were simultaneously replaced by the corresponding residues of inactive hTMEM2 (Asn248 and Phe303). In NHDFs, enhancement of hTMEM2 expression by proinflammatory cytokines decreased HYBID expression and increased hyaluronan synthase 2-dependent HA production. The effects of proinflammatory cytokines were abrogated by hTMEM2 knockdown. A decreased HYBID expression by interleukin-1ß and transforming growth factor-ß was canceled by hTMEM2 knockdown. In conclusion, these results indicate that hTMEM2 is not a catalytic hyaluronidase, but a regulator of HA metabolism.

Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus.

Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-ß-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.

GPNMB Extracellular Fragment Protects Melanocytes from Oxidative Stress by Inhibiting AKT Phosphorylation Independent of CD44.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that plays an important role in cancer metastasis and osteoblast differentiation. In the skin epidermis, GPNMB is mainly expressed in melanocytes and plays a critical role in melanosome formation. In our previous study, GPNMB was also found to be expressed in skin epidermal keratinocytes. In addition, decreased GPNMB expression was observed in the epidermis of lesional skin of patients with vitiligo. However, the exact role of keratinocyte-derived GPNMB and its effect on vitiligo is still unknown. In this study, we demonstrated that GPNMB expression was also decreased in rhododendrol-induced leukoderma, as seen in vitiligo. The extracellular soluble form of GPNMB (sGPNMB) was found to protect melanocytes from cytotoxicity and the impairment of melanogenesis induced by oxidative stress. Furthermore, the effect of rGPNMB was not altered by the knockdown of CD44, which is a well-known receptor of GPNMB, but accompanied by the suppressed phosphorylation of AKT but not ERK, p38, or JNK. In addition, we found that oxidative stress decreased both transcriptional GPNMB expression and sGPNMB protein expression in human keratinocytes. Our results suggest that GPNMB might provide novel insights into the mechanisms related to the pathogenesis of vitiligo and leukoderma.

A context-aware driver model for determining recommended speed in blind intersection situations.

The near-miss events involving vulnerable road users can lead to serious accidents. Safe and careful expert drivers perform a hazard-anticipatory driving and they will naturally seek to reduce the uncertainty by attempting to fit their current driving context into a pre-existing category they have already developed, that is, predicting what can happen. In this study, our target situation consists of a cyclist attempting a road crossing at a blind spot. This study aims at developing a context-aware driver model for determining the recommended driving speed at blind intersections based on the analysis of near-miss-incidence database, which includes the data on driver behavior and road environmental factors just before the near-miss. First, we extracted the drive-recorder data using the management tool provided in the database. Second, risk, which is defined as the time margin for drivers to perform evasive actions to avoid a crash, was quantified for the extracted data using the safety-cushion time. The safety-cushion time can be observed as a result of the driver's adjustment to the vehicle velocity depending on the given road environment. One of the key aspects in developing the context-aware driver model is to categorize the extracted near-miss data into two levels based on the risk quantifications: low- and high-risk events. The low- and high-risk events were regarded as a result of the driver's appropriate adjustment of, and inability or failure to adjust the vehicle velocity depending on the given road environment, respectively. Third, based on a multiple linear regression analysis with low-risk event dataset, we constructed a context-aware driver model to produce the recommended vehicle speed depending on the given road environment. The road environment variables, determined by stepwise regression, were identified as factors that reduced or increased the vehicle velocity at blind intersections, and were incorporated into the model as predictors. Furthermore, we quantitatively visualized drivers setting the baseline for speed adjustment and increasing or decreasing the speed according to the given road environment context. Fourth, the model validation demonstrated a coefficient of determination (R2) of 0.20, and a mean absolute error (MAE) of 6.54 km/h on average in the 5-fold cross-validation. Finally, to investigate the effectiveness of the constructed driver model on safety performance, we used the dataset of high-risk events as test data. Theoretically, the constructed driver model guided the drivers to drive the vehicle at the recommended speed, and thus convert more than half of the high-risk events into low-risk events. These results indicate that the context-aware driver model is feasible to be used to adjust the approaching speed at blind intersections in accordance with the road environment factors.

Fish allergy tolerance 16 months after diagnosis.

Fish allergy is generally thought to be persistent, and approximately 80% of patients with fish allergies do not develop tolerance even 10 years after diagnosis. There have been no reports of rapid tolerance development in patients with severe fish allergies. We report the development of tolerance 16 months after the diagnosis of fish allergies. A 13-month-old boy was diagnosed with rosefish allergy (Sebastes matsubarae) and Japanese jack mackerel allergy (Trachurus japonicus). To find out which species of fish he could consume safely, he underwent several oral food challenge (OFC) tests. It was determined that he could consume tuna, salmon, cod, sardine, chub mackerel (Scomber japonicus), and Japanese amberjack (Seriola quinqueradiata) without eliciting signs of allergy. He continued to eat the fish that did not produce allergic reactions three to four times a week. The titer of serum allergen-specific immunoglobulin E (IgE) to fish had decreased in a subsequent ImmunoCAP®-specific IgE blood test performed 16 months after the diagnosis of the rosefish allergy. Following this test result, he underwent OFCs with rosefish and Japanese jack mackerel, both of which turned out to be negative, and it was determined that he had developed tolerance to fish. In this case, the repeated OFCs were useful in identifying fish species that were safe for consumption. In addition, the decrease in allergen-specific IgE was useful in predicting the development of tolerance. We hypothesize that proactive consumption of available fish species may lead to this rapid induction of tolerance to fish allergens.

Fish allergy tolerance 16 months after diagnosis

Fish allergy is generally thought to be persistent, and approximately 80% of patients with fish allergies do not develop tolerance even 10 years after diagnosis. There have been no reports of rapid tolerance development in patients with severe fish allergies. We report the development of tolerance 16 months after the diagnosis of fish allergies. A 13-month-old boy was diagnosed with rosefish allergy (Sebastes matsubarae) and Japanese jack mackerel allergy (Trachurus japonicus). To find out which species of fish he could consume safely, he underwent several oral food challenge (OFC) tests. It was determined that he could consume tuna, salmon, cod, sardine, chub mackerel (Scomber japonicus), and Japanese amberjack (Seriola quinqueradiata) without eliciting signs of allergy. He continued to eat the fish that did not produce allergic reactions three to four times a week. The titer of serum allergen-specific immunoglobulin E (IgE) to fish had decreased in a subsequent ImmunoCAP®-specific IgE blood test performed 16 months after the diagnosis of the rosefish allergy. Following this test result, he underwent OFCs with rosefish and Japanese jack mackerel, both of which turned out to be negative, and it was determined that he had developed tolerance to fish. In this case, the repeated OFCs were useful in identifying fish species that were safe for consumption. In addition, the decrease in allergen-specific IgE was useful in predicting the development of tolerance. We hypothesize that proactive consumption of available fish species may lead to this rapid induction of tolerance to fish allergens (AU)

Markedly Elevated Procalcitonin in Food Protein Induced Enterocolitis Syndrome.

Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated gastrointestinal food allergy. Some studies have reported that FPIES was associated with elevated C-reactive protein (CRP). However, the number of reports on the relationship between FPIES and procalcitonin (PCT) is limited. This case report highlights the fact that PCT levels can be markedly elevated in patients with acute FPIES. An 11-month-old girl previously diagnosed with FPIES underwent an oral food challenge test (OFC). Her serum PCT levels were measured after she developed severe symptoms including fever and shock following administration of 100mL of formula milk. The PCT levels were extremely elevated but improved without antibiotics the next day. The fact that serum PCT levels may be significantly elevated in FPIES means that differentiating severe FPIES from sepsis could be more challenging than was previously thought.

Interferon-γ downregulates tight junction function, which is rescued by interleukin-17A.

Although atopic dermatitis (AD) has been reported to be a typical type 2 immune response disease, it is also an inflammatory skin disease that involves cytokines, such as Th1, Th17 and Th22. However, little is known about the mechanism by which the candidate cytokines, alone or in combination, are involved in AD pathology. Differences in cytokine balance, which contribute to the complexity of AD pathology, may influence the stratum corneum barrier function through tight junction (TJ) functional stability and contribute to disease severity. To confirm the regulatory mechanism of TJ protein expression in AD, we investigated the Th1 and Th17 pathways, which are the initiation factors of chronic AD pathology. We examined the effects of these cytokines on TJ protein expression in normal human epidermal keratinocytes in vitro, and also examined their function in a human skin equivalent model. We observed a time- and dose-dependent inhibitory effect of IFN-γ on claudin-1 expression via the IFN-γ receptor/JAK/STAT signalling pathway. IFN-γ impaired TJ function in a human skin equivalent model. Moreover, we investigated co-stimulation with IL-17A, which is highly expressed in AD skin lesions and found that IL-17A restores IFN-γ-induced TJ dysfunction. This restoration of TJ function was mediated by atypical protein kinase C zeta activation without recovery of TJ protein expression. These results are informative for personalized AD treatment via systemic therapies using anti-cytokine antibodies and/or JAK inhibitors.

Rhododendrol-induced leukoderma update II: Pathophysiology, mechanisms, risk evaluation, and possible mechanism-based treatments in comparison with vitiligo.

A small proportion of individuals utilizing cosmetics containing rhododendrol developed leukoderma with various pathological conditions, in some cases indistinguishable from vitiligo. In this review, we investigate and evaluate the major considerations for developing rhododendrol-induced leukoderma based on data from original or review articles published in the literature to provide a wide range of information regarding the pathophysiology, mechanisms, risk evaluation, and possible mechanism-based treatments. We compile and discuss the latest information, including data related to the cytotoxicity of rhododendrol, cytoprotective functions, and involvement of the immune system, and consider the possibility of novel treatments based on the differences between individual patients and on the mechanism underlying the onset of the condition. Understanding the pathophysiology of rhododendrol-induced leukoderma helps not only elucidate the mechanisms of non-segmental vitiligo onset and progression, but also suggests prevention and treatment.

HYBID derived from tumor cells and tumor-associated macrophages contribute to the glioblastoma growth.

Glioblastoma is the most malignant tumor of the brain associated with poor prognosis and outcome, and hence there is an urgent need to develop novel treatments for glioblastoma. In this study, we focused on hyaluronan binding protein (HYBID, as known as CEMIP/KIAA1199), a protein involved in hyaluronan depolymerization in chondrocytes and synoviocytes. We previously reported that Hybid-deficient (KO) mice show accumulation of hyaluronan in the brain, and memory impairment. To elucidate the role of HYBID in glioblastoma pathogenesis, we knocked down HYBID in human glioblastoma cells using siRNAs and developed a murine orthotopic xenograft model in the Hybid KO mice. Downregulation of HYBID in glioblastoma cells resulted in inhibition of cell proliferation and migration, and increased cell death. The growth of glioblastoma cells implanted in the mouse brain was suppressed in Hybid KO mice compared to that in the wild-type mice. Interestingly, infiltration of macrophages in the glioblastoma tissue was decreased in Hybid KO mice. Using intraperitoneal macrophages derived from Hybid KO mice and glioma cell supernatants, we examined the role of HYBID in macrophages in the tumor environment. We showed that HYBID contributes to macrophage migration and the release of pro-tumor factors. Moreover, we revealed that HYBID can be a poor prognostic factor in glioma patients by bioinformatics approaches. Our study provides data to support that HYBID expressed by both glioblastoma cells and tumor-associated macrophages may contribute to glioblastoma progression and suggests that HYBID may be a potential target for therapy that focuses on the tumor microenvironment of glioblastoma.

Rhododendrol-induced leukoderma update I: Clinical findings and treatment.

Individuals who used skin-whitening cosmetics (quasi-drugs) containing 2% rhododendrol-containing agents, developed leukoderma at a higher frequency than those who have used other skin-whitening cosmetics. The Rhododenol Research Team (RD-Team) was formed and commissioned by Kanebo Cosmetics Inc. to conduct research in treatments of rhododendrol-induced leukoderma (RDL), to evaluate effective treatment options from a medical standpoint, and provide information to a wide range of people. In this study, we evaluated the efficacy of various treatments for RDL from a medical perspective, based on the information published in the literature as original or review articles. We searched the PubMed (international) and the Igaku Chuo Zasshi (ICHUSHI) (Japanese) databases using the keywords "Rhododenol" and "rhododendrol", for articles published between July 2013 and November 2020. We discuss the main clinical findings and treatments (topical, oral, phototherapy, and surgical) of this condition based on the literature review. We found that ultraviolet light therapy is the most effective treatment for RDL. We have also summarized reports of the efficacy of oral vitamin D3 in RDL. A topical prostaglandin derivative has been reported in a new study to be effective. We have provided guidance for patients using self-tanning and skin-whitening agents to improve their quality of life. Finally, we have highlighted the importance of providing patients with information on contact dermatitis and instructing them to discontinue product use immediately if they develop any symptoms of contact dermatitis while using skin-whitening agents.

Acorus calamus extract and its component α-asarone attenuate murine hippocampal neuronal cell death induced by l-glutamate and tunicamycin.

The Asian traditional medicinal plant Acorus calamus and its component α-asarone exhibited various biological activities, such as antiinflammation and antioxidant effects. In the present study, we investigated the in vitro effects of A. calamus extract and α-asarone on oxidative stress- and endoplasmic reticulum (ER) stress-induced cell death in hippocampal HT22 cells. A. calamus extract and α-asarone both significantly suppressed cell death induced by the oxidative stress inducer l-glutamate and ER stress inducer tunicamycin. A. calamus extract and α-asarone also significantly reduced reactive oxygen species (ROS) production induced by l-glutamate. Moreover, A. calamus extract and α-asarone suppressed the phosphorylation of protein kinase RNA-like ER kinase (PERK) induced by tunicamycin. These results suggest that A. calamus extract and α-asarone protect hippocampal cells from oxidative stress and ER stress by decreasing ROS production and suppressing PERK signaling, respectively. α-Asarone has potential as a potent therapeutic candidate for neurodegenerative diseases, including Alzheimer's disease.

Pro-inflammatory cytokines suppress HYBID (hyaluronan (HA) -binding protein involved in HA depolymerization/KIAA1199/CEMIP) -mediated HA metabolism in human skin fibroblasts.

In the skin, the metabolism of hyaluronan (HA) is highly regulated. Aging leads to chronic low-grade inflammation, which is characterized by elevated levels of pro-inflammatory cytokines; however, the relationship between inflammation and HA metabolism is not clear. Herein, we investigated the effects of a mixture of pro-inflammatory cytokines containing TNF-α, IL-1ß, and IL-6 on HA metabolism in human skin fibroblasts. Treatment with the cytokine mixture for 24 h suppressed HA depolymerization via downregulation of HYBID (HA-binding protein involved in HA depolymerization/KIAA1199/CEMIP) and promoted HA synthesis via upregulation of HAS2 in human skin fibroblasts. Moreover, HAS2-dependent HA synthesis was driven mainly by IL-1ß with partial contribution from TNF-α. Transmembrane protein 2 (TMEM2/CEMIP2), which was previously reported as a candidate hyaluronidase, was upregulated by the cytokine mixture, suggesting that TMEM2 might not function as a hyaluronidase in human skin fibroblasts. Furthermore, the effects of the cytokine mixture on HA metabolism were observed in fibroblasts after 8 days of treatment with cytokines during three passages. Thus, we have shown that HYBID-mediated HA metabolism is negatively regulated by the pro-inflammatory cytokine mixture, providing novel insights into the relationship between inflammation and HA metabolism in the skin.

An indigo-producing plant, Polygonum tinctorium, possesses a flavin-containing monooxygenase capable of oxidizing indole.

Polygonum tinctorium (P. tinctorium) is an indigo plant that is cultivated for a specific metabolite that it produces i.e., indoxyl ß-D-glucoside (indican). In this study, flavin-containing monooxygenase (PtFMO) from P. tinctorium was cloned. When recombinant PtFMO was expressed in E. coli in the presence of tryptophan, indigo production was observed. Furthermore, we measured the activity of PtFMO using the membrane fraction from E. coli and found that it could produce indigo using indole as a substrate. The co-expression of PtFMO with indoxyl ß-D-glucoside synthase (PtIGS), which catalyzes the glucosylation of indoxyl, brought about the formation of indican in E. coli. The results showed that indican was synthesized by sequential reactions of PtFMO and PtIGS. In three-week-old P. tinctorium specimens, the first leaves demonstrated higher levels of PtFMO expression than the subsequent leaves. This result coincided with that of our prior study on PtIGS expression level. Our study provides evidence that PtFMO might contribute to indican biosynthesis.