Three stepwise pH progressions in stratum corneum for homeostatic maintenance of the skin.

The stratum corneum is the outermost skin layer with a vital role in skin barrier function. It is comprised of dead keratinocytes (corneocytes) and is known to maintain its thickness by shedding cells, although, the precise mechanisms that safeguard stratum corneum maturation and homeostasis remain unclear. Previous ex vivo studies have suggested a neutral-to-acidic pH gradient in the stratum corneum. Here, we use intravital pH imaging at single-corneocyte resolution to demonstrate that corneocytes actually undergo differentiation to develop three distinct zones in the stratum corneum, each with a distinct pH value. We identified a moderately acidic lower, an acidic middle, and a pH-neutral upper layer in the stratum corneum, with tight junctions playing a key role in their development. The upper pH neutral zone can adjust its pH according to the external environment and has a neutral pH under steady-state conditions owing to the influence of skin microbiota. The middle acidic pH zone provides a defensive barrier against pathogens. With mathematical modeling, we demonstrate the controlled protease activation of kallikrein-related peptidases on the stratum corneum surface that results in proper corneocyte shedding in desquamation. This work adds crucial information to our understanding of how stratum corneum homeostasis is maintained.

Visualization of intradermal blood vessel structures by dual-wavelength photoacoustic microscopy and characterization of three-dimensional construction of livedo-racemosa in cutaneous polyarteritis nodosa.

BACKGROUND: Photoacoustic microscopy is expected to have clinical applications as a noninvasive and three-dimensional (3D) method of observing intradermal structures. OBJECTIVE: Investigate the applicability of a photoacoustic microscope equipped with two types of pulsed lasers that can simultaneously recognize hemoglobin and melanin. METHODS: 16 skin lesions including erythema, pigmented lesions, vitiligo and purpura, were analyzed to visualize 3D structure of melanin granule distribution and dermal blood vessels. 13 cases of livedo racemosa in cutaneous polyarteritis nodosa (cPN) were further analyzed to visualize the 3D structure of dermal blood vessels in detail. Vascular structure was also analyzed in the biopsy specimens obtained from tender indurated erythema of cPN by CD34 immunostaining. RESULTS: Hemoglobin-recognition signal clearly visualized the 3D structure of dermal blood vessels and melanin-recognition signal was consistently reduced in vitiligo. In livedo racemosa, the hemoglobin-recognition signal revealed a relatively thick and large reticular structure in the deeper layers that became denser and finer toward the upper layers. The numerical analysis revealed that the number of dermal blood vessels was 1.29-fold higher (p<0.05) in the deeper region of the lesion than that of normal skin. The CD34 immunohistochemical analysis in tender indurated erythema revealed an increased number of dermal vessels compared with normal skin in 88.9% (8/9) of the cases, suggesting that vascular network remodeling had occurred in cPN. CONCLUSION: The photoacoustic system has an advantage in noninvasively detecting dermal blood vessel structures that are difficult to recognize by two-dimensional histopathology specimen examination and is worth evaluating in various skin diseases.

Calcium channel blockers may reduce the development of long COVID in females.

With the rising numbers of patients infected with severe acute respiratory syndrome coronavirus 2, long coronavirus disease 2019 (COVID-19)-a sequelae of COVID-19-has become a major problem. Different sexes and age groups develop different long COVID symptoms, and the risk factors for long COVID remain unclear. Therefore, we performed subgroup analyses of patients with COVID-19, classifying them into different groups. In this multicenter cohort study, using an original questionnaire, we examined patients (≥18 years old) diagnosed with COVID-19 from November 2020 to March 2022 and hospitalized at participating medical facilities. In total, 1066 patients were registered (361 female, 620 male). Hypertension was the most common comorbidity (n = 344; 32.5%). Females with hypertension were significantly less likely to develop long COVID symptoms than those without hypertension (odds ratio [OR] 0.51, 95% confidence interval [CI] 0.27-0.98; p = 0.043). In females, Ca channel blocker administration, rather than having hypertension, was significantly associated with reductions in the frequency of alopecia (OR 0.14, 95% CI 0.03-0.67, p = 0.015), memory impairment (OR 0.14, 95% CI 0.02-0.82, p = 0.029), sleeping disorders (OR 0.17, 95% CI 0.04-0.67, p = 0.012), tinnitus (OR 0.23, 95% CI 0.05-0.98, p = 0.047), sputum (OR 0.31, 95% CI 0.10-0.92, p = 0.035), and fever (OR 0.33, 95% CI 0.12-0.93, p = 0.036). Several long COVID symptoms, including alopecia, were significantly negatively associated with Ca channel-blocker administration in female patients with long COVID. Calcium channel blockers may reduce the development of long COVID in females.

Keratinocytes of the Upper Epidermis and Isthmus of Hair Follicles Express Hemoglobin mRNA and Protein.

The epidermis, the keratinized stratified squamous epithelium surrounding the body surface, offers a valuable framework to investigate how terrestrial animals overcome environmental stresses. However, the mechanisms underlying epidermal barrier function remain nebulous. In this study, we examined genes highly expressed in the human and mouse upper epidermis, the outer frontier that induces various barrier-related genes. Transcriptome analysis revealed that the messenger RNA level of hemoglobin α (HBA), an oxygen carrier in erythroid cells, was enriched in the upper epidermis compared with that in the whole epidermis. Immunostaining analysis confirmed HBA protein expression in human and mouse keratinocytes (KCs) of the stratum spinosum and stratum granulosum. HBA was also expressed in hair follicle KCs in the isthmus region; its expression levels were more prominent than those in interfollicular KCs. HBA expression was not observed in noncutaneous keratinized stratified squamous epithelia of mice, for example, the vagina, esophagus, and forestomach. HBA expression was upregulated in human epidermal KC cultures after UV irradiation, a major cause of skin-specific oxidative stress. Furthermore, HBA knockdown increased UV-induced production of ROS in primary KCs. Our findings suggest that epidermal HBA expression is induced by oxidative stress and acts as an antioxidant, contributing to skin barrier function.

Immune Regulation by Cytosolic DNA Sensors in the Tumor Microenvironment.

cGAS and AIM2 are CDSs that are activated in the presence of cytosolic dsDNA and are expressed in various cell types, including immune and tumor cells. The recognition of tumor-derived dsDNA by CDSs in the cytosol of tumor-infiltrating dendritic cells (TIDCs) activates the innate and acquired immunity, thereby enhancing anti-tumor immune responses. STING is the downstream signaling effector of cGAS that induces type I interferon (IFN) signaling. Owing to their ability to activate TIDCs, STING agonists have been intratumorally injected in several clinical trials to enhance the anti-tumor immune response elicited by immune checkpoint antibodies. However, they have shown minimal effect, suggesting the importance of optimizing the dose and route of administration for STING agonists and deciphering other immune pathways that contribute to anti-tumor immune responses. Recent studies have revealed that AIM2 activity induces pro-tumor growth through multiple parallel pathways, including inhibition of STING-type I IFN signaling. Thus, AIM2 could be a potential molecular target for cancer immunotherapies. This review summarizes the current research on the roles of cGAS, STING, and AIM2 in immune cells and tumor cells in the tumor microenvironment and discusses the future prospects of anti-tumor treatment approaches based on these molecules.

IFNγ score-based neoadjuvant immunotherapy for stage III melanoma.

In this issue of JEM, Reijers et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20221952) demonstrate that pre- and post-treatment IFNγ-related gene expression scores are promising markers for choosing neoadjuvant immunotherapy for stage III melanoma.

Cutaneous eyelid melanoma with lacrimal sac metastasis: the potential role of lacrimal fluid as a metastatic pathway.

Here we present a patient with cutaneous eyelid melanoma patient with lacrimal sac metastasis. Clinicopathological findings in this case support the theory that lacrimal fluid can be a metastatic pathway for tumour cells. Dermatologists should be aware of the possibility that cutaneous eyelid melanoma may involve the nasolacrimal system and should examine it during the perioperative period and in postoperative follow-up.

Microbiota-Independent Spontaneous Dermatitis Associated with Increased Sebaceous Lipid Production in Tmem79-Deficient Mice.

TMEM79 is a predisposing gene for atopic dermatitis. Tmem79-deficient mice develop spontaneous dermatitis in a biphasic pattern. The first-phase dermatitis is unique because it occurs independent of microbiota status, whereas the second-phase dermatitis is microbiota dependent. In this study, we sought to identify the key factors mediating the development of first-phase dermatitis. Structural analysis showed that sebaceous gland hyperplasia started from first-phase dermatitis. Longitudinal RNA sequencing analysis revealed significant activation of fatty acid lipid metabolism pathways in first-phase dermatitis, whereas T helper 17‒based immune response genes were highly expressed in second-phase dermatitis. Quantitative RT-PCR analysis revealed that genes involved in fatty acid elongation and sebocyte differentiation were upregulated in first-phase dermatitis. The results of thin-layer chromatography supported these findings with an increased abundance of wax esters, cholesterol esters, and fatty alcohols in hair lipids. Further gas chromatography-tandem mass spectrometry analysis showed an increase in total fatty acid production, including that of elongated C20-24 saturated and C18-24 monounsaturated fatty acids. Collectively, these results suggest that aberrant production of sebaceous long-chain fatty acids is associated with microbiota-independent dermatitis. Further investigation of Tmem79-deficient mice may clarify the role of certain fatty acids in dermatitis.

Networks of CD8+ T Cell Response Activation in Melanoma and Vitiligo.

Melanoma is an aggressive skin cancer derived from melanocyte, which shows high response rate to cancer immunotherapy, such as immune checkpoint inhibitors (ICIs). Vitiligo is an autoimmune skin disease resulting from the destruction of melanocytes by autoreactive CD8+ T cells. Vitiligo induced by cancer immunotherapy is a favorable prognostic factor in patients with melanoma, and growing evidence supports the fact that melanocyte/melanoma-shared antigen (MSA)-specific CD8+ T cells infiltrated in the tumor (melanoma) and skin (vitiligo) microenvironment play pivotal roles in the prognosis of both diseases. Thus, cellular communications that promote MSA-specific CD8+ T cells recruitment, proliferation, and effector functions are now seen as key targets to enhance the efficacy of current therapies for both diseases. Here, we discussed recent advancements in illustrating immune signaling pathways and immune cell types that regulate migration, proliferation, and function of MSA-specific CD8+ T cells in melanoma and vitiligo; and future immunotherapeutic approaches that may enhance clinical outcomes of both diseases.

Management and outcomes of hydronephrosis in patients with metastatic extramammary Paget's disease: A retrospective analysis.

Hydronephrosis in extramammary Paget's disease (EMPD) with distant metastasis (metastatic EMPD) has been observed in medical practice; however, its prognosis remains unclear. Retrospective analyses were performed to assess the management and outcomes of hydronephrosis in metastatic EMPD. During a follow-up of 44 patients with metastatic EMPD, 13 (30%) developed hydronephrosis. Ten (77%) of the 13 patients with hydronephrosis had impaired renal function (estimated glomerular filtration rate: <60 ml/min/1.73 m2 ), and ureteral stents were placed in every patient with impaired renal function. The stent was placed successfully in all 10 patients, and their renal function recovered within a median period of 7 days. Importantly, each of these patients continued chemotherapy, and none of them experienced stent failure. The median overall survival time (OS) in patients with metastatic EMPD and hydronephrosis (n = 13) was 7.8 months. Treatment for hydronephrosis was not a significant factor for OS, and median OS in patients who underwent ureteral stent replacement (n = 10) was 14.7 months. Collectively, our results indicate that hydronephrosis is relatively common, and ureteral stent placement should be considered in cases of metastatic EMPD with hydronephrosis to maintain renal function and continue chemotherapy toward a better prognosis.

Imatinib mesylate in combination with pembrolizumab in patients with advanced KIT-mutant melanoma following progression on standard therapy: A phase I/II trial and study protocol.

INTRODUCTION: Genetic alterations of KIT gene are known to be one of the major causes of melanoma. Those are more common in the mucous and acral subtypes and KIT is regarded as major oncogene in Asian melanomas, where the prevalence of these subtypes is high. Up to date, several clinical trials have been conducted to target KIT gene alterations in melanoma with unsatisfied efficacies. Imatinib mesylate, a small-molecule inhibitor of the KIT tyrosine kinase, provides a rapid but not durable clinical response in KIT-mutant melanoma. Meanwhile, recent basic and clinical evidence have revealed another aspect of KIT-targeted therapy, namely the enhancement of antitumor activity of immune checkpoint inhibitors. Herein, we designed clinical trial of co-administrating imatinib mesylate and pembrolizumab (anti-PD-1 antibody) to evaluate its safety and efficacy. METHODS AND ANALYSIS: This is an open-label, single-arm, phase I/II clinical trial involving Japanese patients with metastatic KIT-mutant melanoma that are refractory to standard therapy including anti-PD-1 therapy. Phase I study is a dose-escalation study comprising two dose levels of imatinib mesylate (200 and 400 mg/day, respectively) with fixed dose of pembrolizumab (200 mg every 3 weeks) to evaluate safety and tolerability and determine recommended phase II dose. The primary endpoint of the phase II study is the objective response rate after 4 cycles (3 weeks/cycle) of pembrolizumab and imatinib mesylate at the dose determined in phase I, based on RECIST version 1.1. A Simon's minimax two-stage design is employed to test the null hypothesis of a 5% response rate vs 30% alternative, which will be rejected when a lower confidence limit of two-sided 90% confidence interval of true response rate is over than threshold response rate. The secondary endpoints include progression free survival, overall survival, best overall response and incidence of adverse events. Totally, a target size of 22 patients will be expected. DISCUSSION: If this study shows efficacy and acceptable safety profile, it will contribute to the development of novel treatment option for patients with KIT-mutant melanoma that are refractory to standard therapy. TRIAL REGISTRATION: NCT04546074. Date of Registration: September 11, 2020 (https://clinicaltrials.gov/ct2/show/NCT04546074). Date of First Participant Enrollment: December 23, 2020.

Comprehensive and long-term surveys of COVID-19 sequelae in Japan, an ambidirectional multicentre cohort study: study protocol.

INTRODUCTION: The rapid spread of COVID-19 posed a global burden. Substantial number of people died of the disease in the acute phase of infection. In addition, a significant proportion of patients have been reported to suffer from post-acute phase symptoms, sequelae of COVID-19, which may negatively influence the quality of daily living and/or socioeconomic circumstances of the patients. However, no previous study has comprehensively and objectively assessed the quality of life of patients by using existing international scales. Further, evidence of socioeconomic consequences among patients with COVID-19 is scarce. To address the multidimensional issues from sequelae of COVID-19, evidence from comprehensive surveys beyond clinical perspectives is critical that investigates health, and social determinants of disease progression as well as socioeconomic consequences at a large scale. METHODS AND ANALYSIS: In this study, we plan to conduct a nationwide and comprehensive survey for the sequelae of COVID-19 in a total of 1000 patients diagnosed at 27 hospitals throughout Japan. This study will evaluate not only the health-related status of patients from clinical perspectives but also the Health-related Quality of Life (HRQoL) scores, socioeconomic status and consequences to discuss the sequelae of the disease and the related risk factors. The primary endpoint is the frequency of long-term complications of COVID-19 infection. The secondary endpoints are risk factors for progression to sequelae of COVID-19 infection. The study will provide robust and important evidence as a resource to tackle the issues from the sequelae of COVID-19 from the multi-dimensional perspectives. ETHICS AND DISSEMINATION: This trial was approved by the Keio University School of Medicine Ethics Committee (20200243, UMIN000042299). The results of this study will be reported at a society meeting or published in a peer-reviewed journal.

AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma.

The STING and absent in melanoma 2 (AIM2) pathways are activated by the presence of cytosolic DNA, and STING agonists enhance immunotherapeutic responses. Here, we show that dendritic cell (DC) expression of AIM2 within human melanoma correlates with poor prognosis and, in contrast to STING, AIM2 exerts an immunosuppressive effect within the melanoma microenvironment. Vaccination with AIM2-deficient DCs improves the efficacy of both adoptive T cell therapy and anti-PD-1 immunotherapy for "cold tumors," which exhibit poor therapeutic responses. This effect did not depend on prolonged survival of vaccinated DCs, but on tumor-derived DNA that activates STING-dependent type I IFN secretion and subsequent production of CXCL10 to recruit CD8+ T cells. Additionally, loss of AIM2-dependent IL-1ß and IL-18 processing enhanced the treatment response further by limiting the recruitment of regulatory T cells. Finally, AIM2 siRNA-treated mouse DCs in vivo and human DCs in vitro enhanced similar anti-tumor immune responses. Thus, targeting AIM2 in tumor-infiltrating DCs is a promising new treatment strategy for melanoma.

Adoptive cell therapy using tumor-infiltrating lymphocytes for melanoma refractory to immune-checkpoint inhibitors.

To evaluate the feasibility of adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) in Japanese patients with melanoma who failed immune-checkpoint inhibitor therapy, an open-label, single-arm, pilot study was conducted. We investigated the immunological and genetic factors of the pretreatment tumor and expanded TILs that may be associated with the clinical response. The treatment protocol comprised preparation of TIL culture, lympho-depleting non-myeloablative preconditioning with cyclophosphamide and fludarabine, TIL infusion, and intravenous administration of low-dose IL-2. Three patients of clinical subtypes mucosal, superficial spreading, and acral melanoma underwent TIL-ACT. Most severe adverse events, including fever and leukopenia, were manageable with the supportive regimen specified in the protocol, suggesting that the TIL-ACT regimen is suitable for Japanese patients with melanoma. One patient showed a short-term partial response, one relatively long-stable disease, and one experienced disease progression. Whole-exome and transcriptional sequencing of isolated tumor cells and immunohistochemical analyses before TIL-ACT revealed various immunostimulatory factors, including a high tumor mutation burden and immune cell-recruiting chemokines, as well as various immunosuppressive factors including TGF-ß, VEGF, Wnt/ß-catenin, and MAPK signaling and epithelial-to-mesenchymal transition, which might influence the efficacy of TIL-ACT. Our results imply mechanisms for the antitumor effect of and resistance to TIL-ACT. Further studies of immune-resistant mechanisms of TIL-ACT are warranted. This study is registered with the UMIN Clinical Trial Registry (UMIN 000011431).

Type I interferon signaling limits viral vector priming of CD8+ T cells during initiation of vitiligo and melanoma immunotherapy.

Vitiligo is an autoimmune skin disease in which epidermal melanocytes are targeted for destruction by CD8+ T cells specific for melanocyte/melanoma-shared antigens. IFNγ is the central cytokine driving disease, but the role of type I IFN in vitiligo remains unclear. We investigated the functional role of type I IFN during vitiligo progression using two different mouse models: one induced with a vaccinia virus (VV) vaccine and one induced with dendritic cells to prime autoimmune T cells. Induction of vitiligo by VV in IFNaR-deficient mice led to the development of severe vitiligo compared with wild-type (WT) mice and was characterized by a significantly enhanced effector CD8+ T-cell response. Severe vitiligo in this model was a result of VV persistence, because exacerbation of disease in IFNaR-deficient mice was not observed when antigen-pulsed dendritic cells were used to induce vitiligo instead of virus. Treatment of B16F10 melanoma-inoculated mice with VV vaccine therapy also induced a significantly enhanced anti-tumor response in IFNaR-deficient mice compared with WT. These results not only help define the pathways responsible for vitiligo progression but also suggest that blockade of type I IFNs following administration of a VV vaccine may provide increased immunogenicity and efficacy for melanoma immunotherapy.

Anti-PD-1 antibody therapy for epithelial skin malignancies: An investigator-initiated, open-label, single-arm, multicenter, phase II clinical trial (NMSC-PD1 Study).

INTRODUCTION: Malignant cutaneous epithelial tumors comprise various skin malignancies originating from the cutaneous epithelium, including cutaneous squamous cell carcinoma, basal cell carcinoma, and malignant cutaneous adnexal tumors. Treatment options are limited, as the rarity of these tumors, especially among Asians, renders well-controlled clinical trials extremely challenging to conduct. Thus, we designed a clinical trial to evaluate the efficacy and safety of the anti-programmed cell death-1 (PD-1) monoclonal antibody nivolumab in patients with metastatic cutaneous squamous cell carcinomas and other rare metastatic cutaneous epithelial tumors. METHODS AND ANALYSIS: This is an open-label, single-arm, multicenter, phase 2 clinical trial involving patients with metastatic malignant cutaneous epithelial tumors. Nivolumab (480 mg) will be administered intravenously every 4 weeks for a maximum of 26 doses. The primary outcome of the study will be the response rate based on response evaluation criteria in solid tumors, version 1.1. Assuming a null hypothesis of a response rate ≤5% and an alternative hypothesis of a 25% response rate, a minimum of 26 patients are required to achieve a 5% two-sided type I error and 80% power based on the exact binomial distribution. Finally, a target cohort size of 30 patients was determined as some patient dropout will be expected. DISCUSSION: This is the first phase 2 clinical trial evaluating the efficacy and safety of the PD-1 inhibitor nivolumab in Asian patients with metastatic malignant cutaneous epithelial tumors. The findings of the study will contribute to the development of novel treatment approaches for patients with rare cutaneous malignancies, which remains an unmet clinical need. TRIAL REGISTRATION: Registry number: jRCT 2031190048.