A method for measuring serum levels of melanin-associated indole metabolites using LC-MS/MS and its application to malignant melanoma.

BACKGROUND AND AIMS: With the development of novel therapies for advanced malignant melanoma (MM), biomarkers that can accurately reflect the progression of MM are needed. Serum levels of melanin-related indole metabolites such as 5-hydroxy-6-methoxyindole-2-carboxylic acid (5H6MI2C) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) are potential biomarkers for MM. Here, we describe the development of a mass spectrometry (MS)-based assay to determine serum levels of 5H6MI2C and 6H5MI2C. MATERIALS AND METHODS: We developed a stable isotope dilution-selective reaction monitoring-MS protocol using liquid chromatography tandem mass spectrometry (LC-MS/MS) to measure human serum 5H6MI2C and 6H5MI2C levels. Analytical evaluations of the method were performed and the method was applied to serum samples from MM patients (n = 81). RESULTS: The method established in this study showed high reproducibility and linearity. This novel method also found that serum 6H5MI2C levels were significantly elevated in patients with metastatic MM compared to those with non-metastatic MM. Unfortunately, 5H6MI2C did not show a comparable significant difference. CONCLUSION: We successfully established measurement methods for serum 5H6MI2C and 6H5MI2C levels using LC-MS/MS. Serum 6H5MI2C levels offer a potential marker for MM.

Analyzing the relationship between the efficacy of first-line immune checkpoint inhibitors and cumulative sun damage in Japanese patients with advanced BRAF wild-type nonacral cutaneous melanoma: A retrospective real-world, multicenter study.

BACKGROUND: Efficacy of anti-PD-1 antibody monotherapy (PD1) or anti-PD-1 plus anti-CTLA-4 combination therapy (PD1 +CTLA4) for melanoma is affected by its clinical subtype. The amount of tumor mutation burden (TMB) caused by cumulative sun damage (CSD) is occasionally used to explain this; however, their relationship in Japanese nonacral cutaneous melanoma (NACM) is still unclear. OBJECTIVE: To analyze the ICI efficacy and its relationship with CSD of the primary lesion in Japanese patients with NACM. METHODS: Japanese patients with advanced BRAF wild-type NACM who received first-line ICIs were recruited. Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS), and the degree of solar elastosis (SE) were evaluated. RESULTS: A total of 146 patients (PD1 group 113 and PD1 +CTLA4 group 33) were included. No significant differences in ORR were observed between the PD1 and PD1 +CTLA4 groups (35 % vs. 36 %; P = 0.67) or PFS and OS (median PFS 6.1 months vs. 8.5 months; P = 0.46, median OS 28.1 months vs. not reached; P = 0.59). Multivariate survival analysis revealed that PD1 +CTLA4 did not prolong the PFS and OS. The SE score had no effect on either PFS or OS. CONCLUSIONS: ICI efficacy was not as high as those reported in Western countries, and PD1 +CTLA4 did not present better clinical efficacy compared to PD1. Indicators of CSD did not serve as a predictor for clinical advantage. These findings may partially support the theory that ICI efficacy is affected by CSD; however, other unrecognized factors may also exist.

Characteristics of hair loss after COVID-19: A systematic scoping review.

OBJECTIVE: While there are literature reporting increased incidence of hair loss in COVID-19 patients, insufficient evidence exists on the topic to date. This review aims to identify the existing evidence and clinical characteristics of hair loss with COVID-19 infection. METHODS: Following the PRISMA Extension for Scoping Reviews, MEDLINE and EMBASE were searched for all peer-reviewed articles with relevant keywords including "Alopecia," "Telogen Effluvium (TE)," and "COVID-19" from their inception to November 20, 2021. RESULTS: A total of 26 articles, with 9 observational studies and 17 case reports or series (a total of 58 cases), were included. Most studies dealt with TE. There were no clear trends between COVID-19 severity and the extent of hair loss. Analysis of the 58 cases also found similar results with most of the cases being female (82.8%), the median onset of hair loss of 2.0 months, and the median time to recovery of hair loss of 5.0 months with a resolution rate of 95%. CONCLUSION: While this systematic review revealed uncertainty and a lack of strong evidence regarding the association of COVID-19 and hair loss, hair loss in COVID-19 may mainly include TE and be reversible in nature. Future studies are warranted to determine the detailed pathophysiology and risk factors of hair loss in COVID-19, including possible roles of estrogen, progesterone, and pro-inflammatory cytokines.

BRAF Immunoexpression Can Be Intralesionally Heterogeneous but BRAF V600E Mutation Status Is Intralesionally Homogeneous and Interlesionally Concordant in Melanoma: A Study of 140 Lesions From 98 Patients.

ABSTRACT: This study sought to confirm the homogeneity of BRAF V600E mutation status in melanoma. BRAF immunohistochemistry was performed on 102 lesions from 60 patients of melanoma with BRAF V600E mutation and 38 negative-control melanoma lesions from 38 patients, both of which were confirmed by real-time PCR or the MassARRAY System. In the positive-control lesions, 9 lesions from 7 patients with preceding BRAF-inhibitor therapy were included. Of the 102 BRAF-mutant lesions, 101 (99.0%) showed diffuse BRAF immunoexpression, but 39 (38.2%) of them showed various heterogeneous intensities. The heterogeneous intensity of immunostaining was due to necrosis (n = 10), minimal or clear cytoplasm (n = 5), tissue crush (n = 8), insufficient fixation (n = 24), or technical error (n = 4). Only 1 lesion (1.0%) with nondiffuse immunoexpression harbored 80% weakly BRAF-positive tumor area and 20% BRAF-negative area with tissue damage. Sanger sequencing performed on the weak or negative regions in 7 lesions revealed BRAF V600E mutation in all the tested lesions. By contrast, all 38 negative-control lesions demonstrated no BRAF immunoexpression. This study demonstrated intralesional homogeneity and interlesional concordance for BRAF V600E mutation status and intralesional frequent heterogeneity for BRAF immunoexpression. The abovementioned 5 phenomena caused substantial reduction in BRAF immunostaining intensity. In 9 lesions within this study, BRAF immunoexpression and BRAF V600E point mutation status were not affected by preceding BRAF inhibitor therapy. Our data would also support the position that it does not matter whether we select primary or metastatic samples for BRAF mutation analysis.

Multifaceted Analysis of IL-23A- and/or EBI3-Including Cytokines Produced by Psoriatic Keratinocytes.

Interleukin (IL) 23 (p19/p40) plays a critical role in the pathogenesis of psoriasis and is upregulated in psoriasis skin lesions. In clinical practice, anti-IL-23Ap19 antibodies are highly effective against psoriasis. IL-39 (p19/ Epstein-Barr virus-induced (EBI) 3), a newly discovered cytokine in 2015, shares the p19 subunit with IL-23. Anti-IL-23Ap19 antibodies may bind to IL-39; also, the cytokine may contribute to the pathogenesis of psoriasis. To investigate IL23Ap19- and/or EBI3-including cytokines in psoriatic keratinocytes, we analyzed IL-23Ap19 and EBI3 expressions in psoriasis skin lesions, using immunohistochemistry and normal human epidermal keratinocytes (NHEKs) stimulated with inflammatory cytokines, using quantitative real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-electrospray tandem mass spectrometry (LC-Ms/Ms). Immunohistochemical analysis showed that IL-23Ap19 and EBI3 expressions were upregulated in the psoriasis skin lesions. In vitro, these expressions were synergistically induced by the triple combination of tumor necrosis factor (TNF)-α, IL-17A, and interferon (IFN)-γ, and suppressed by dexamethasone, vitamin D3, and acitretin. In ELISA and LC-Ms/Ms analyses, keratinocyte-derived IL-23Ap19 and EBI3, but not heterodimeric forms, were detected with humanized anti-IL-23Ap19 monoclonal antibodies, tildrakizumab, and anti-EBI3 antibodies, respectively. Psoriatic keratinocytes may express IL-23Ap19 and EBI3 proteins in a monomer or homopolymer, such as homodimer or homotrimer.

Staphylococcal enterotoxin B- and lipopolysaccharide-induced toxic shock syndrome in a burn patient.

Toxic shock syndrome (TSS) is caused by toxic shock syndrome toxin 1 or enterotoxins secreted by Staphylococcus aureus. Lipopolysaccharide (LPS) has also been shown to play a major role in the development of sepsis. Staphylococcal superantigens and LPS operate synergistically in conditioning cytokine release and lethal shock in mice. An 80-year-old woman was admitted because of a 20% mixed-depth flame burn. Despite two excisions and grafts, necrotic ulcers with methicillin-resistant Staphylococcus aureus (MRSA) colonization remained. On the 7th day after the operation, she developed shock with an erythematous rash. Blood examination revealed evidence of disseminated intravascular coagulation, and liver and renal dysfunction. A blood culture revealed a staphylococcal enterotoxin B (SEB)-producing strain of MRSA and Klebsiella pneumoniae. The septic symptoms were prolonged, but the condition gradually improved with extensive treatment. T-cell receptor analysis demonstrated a marked accumulation of SEB-mediated Vß T cells. Stimulation of peripheral blood mononuclear cells in the recovery phase with SEB and LPS induced additive effects on tumor necrosis factor-α, interferon-γ, and interleukin-6 production. Although the present case did not fulfill the clinical criteria for TSS, the additive effects of SEB and LPS might have caused the severe septic shock.

Multifaceted Analyses of Epidermal Serine Protease Activity in Patients with Atopic Dermatitis.

The serine proteases kallikrein-related peptidase (KLK) 5 and KLK7 cleave cell adhesion molecules in the epidermis. Aberrant epidermal serine protease activity is thought to play an important role in the pathogenesis of atopic dermatitis (AD). We collected the stratum corneum (SC) from healthy individuals (n = 46) and AD patients (n = 63) by tape stripping and then measuring the trypsin- and chymotrypsin-like serine protease activity. We also analyzed the p.D386N and p.E420K of SPINK5 variants and loss-of-function mutations of FLG in the AD patients. The serine protease activity in the SC was increased not only in AD lesions but also in non-lesions of AD patients. We found, generally, that there was a positive correlation between the serine protease activity in the SC and the total serum immunoglobulin E (IgE) levels, serum thymus and activation-regulated chemokine (TARC) levels, and peripheral blood eosinophil counts. Moreover, the p.D386N or p.E420K in SPINK5 and FLG mutations were not significantly associated with the SC's serine protease activity. Epidermal serine protease activity was increased even in non-lesions of AD patients. Such activity was found to correlate with a number of biomarkers of AD. Further investigations of serine proteases might provide new treatments and prophylaxis for AD.

Immune-related adverse events correlate with improved survival in patients with advanced mucosal melanoma treated with nivolumab: A single-center retrospective study in Japan.

Immune-related adverse events (irAE) were reported to be associated with better outcomes in various cancers treated with the immune checkpoint inhibitor nivolumab. Considering that their development depends on host immune activation, irAE may reflect antitumor response in mucosal melanoma (MM). This single-center retrospective study including patients with advanced MM receiving nivolumab monotherapy between August 2014 and September 2018 investigated whether the development of irAE was associated with clinical efficacy. The study patients were divided into those with and without irAE, and treatment efficacy and safety were evaluated. The study cohort of 27 patients included 20 (74%), six (22%) and one (4%) patient with primary MM in the head and neck, genitourinary and anorectal regions, respectively. The irAE onset was not significantly associated with the objective response rate in patients while it was significantly associated with the disease control rate. The median progression-free survival in patients with and without irAE was 301 and 63 days, respectively. The median overall survival (OS) in patients with and without irAE was 723 and 199 days, respectively. According to the timing of irAE onset, the OS was better in seven patients who developed irAE after 180 days than in nine patients who developed irAE within 180 days. Although 16 patients (59%) experienced any grade irAE, including three (11%) with grade 3 or more irAE, there were no treatment-related deaths. These results indicated that the development of irAE may correlate with improved survival in patients with MM treated with nivolumab monotherapy. Further studies are necessary to confirm these findings.

TNF-α and IL-17A induce the expression of lympho-epithelial Kazal-type inhibitor in epidermal keratinocytes.

BACKGROUND: Serine proteases have important roles in skin barrier function and desquamation, and the aberrant expression or the dysfunction of serine proteases is associated with the pathogenesis of skin diseases. Serine protease activities are tightly regulated by serine proteases such as kallikrein-related peptidases (KLKs) and serine protease inhibitors such as lympho-epithelial Kazal-type related inhibitor (LEKTI). For a better understating of diseases' pathogenesis, the regulation mechanism of serine proteases and the inhibitors' expression in epidermal keratinocytes must be clarified. OBJECTIVES: To investigate the effects of the cytokines on the expression of LEKTI in epidermal keratinocytes. METHODS: Normal human epidermal keratinocytes (NHEKs) were stimulated with panels of inflammatory cytokines. The expression of serine protease inhibitors was analyzed using quantitative real-time PCR and ELISA. LEKTI expression in normal human skin and lesions from psoriasis or atopic dermatitis (AD) were analyzed by immunohistochemically and tape-stripping. Trypsin- and chymotrypsin-like serine protease activities in culture supernatants were measured by using specific substrates. RESULTS: TNF-α and IL-17A significantly induced the expression of LEKTI in NHEKs. The immunohistochemical and tape-stripping analysis revealed that psoriatic skin lesions had higher LEKTI expression compared to normal skin and AD lesions. Trypsin- and chymotrypsin-like protease activities in the culture media were upregulated 3-5 days later but attenuated 6-7 days later period by these cytokines. CONCLUSIONS: In epidermal keratinocytes, the Th1&Th17 cytokines TNF-α and IL-17A induce the expression of serine protease inhibitor LEKTI, and it might occur to suppress the increase in the serine protease activities under inflammation.

Toll-like receptor signaling induces the expression of lympho-epithelial Kazal-type inhibitor in epidermal keratinocytes.

BACKGROUND: Lympho-epithelial Kazal-type inhibitor (LEKTI) tightly controls the activities of serine proteases such as kallikrein-related peptidase (KLK) 5 and KLK7 in the epidermis. LEKTI is known to be an essential molecule for the epidermal skin barrier, as demonstrated by SPINK5 nonsense mutation, which results in Netherton syndrome. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns or damage-associated molecular patterns and produce inflammatory cytokines, chemokines, and antimicrobial peptides. However, the effect of TLR signaling on the expression of LEKTI is not clear. OBJECTIVE: To investigate whether TLR signaling can affect expression of LEKTI in epidermal keratinocytes. METHODS: We stimulated a panel of TLR ligands and investigated the expression of LEKTI in normal human epidermal keratinocytes (NHEKs). We further measured trypsin or chymotrypsin-like serine protease activity in NHEK cultured media under stimulation with TLR3 ligand, poly (I:C). Immunostaining for LEKTI was performed using skin samples from skin infectious diseases. RESULTS: TLR1/2, 3, 5, and 2/6 ligands induced the expression of LEKTI in NHEKs. The trypsin or chymotrypsin-like serine protease activity in NHEKs was up-regulated with the stimulation of poly (I:C). The gene expressions of KLK6, KLK10, KLK11, and KLK13 were also increased by poly (I:C). An immunohistochemical analysis demonstrated that the expression of LEKTI was up-regulated in the lesions of varicella, pyoderma, and rosacea. CONCLUSIONS: TLR signaling induces the expression of LEKTI in epidermal keratinocytes, which might contribute to the control of aberrant serine protease activities in inflammatory skin diseases.

Analysis of All 34 Exons of the SPINK5 Gene in Japanese Atopic Dermatitis Patients.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a large multidomain serine protease inhibitor that is expressed in epidermal keratinocytes. Nonsense mutations of the SPINK5 gene, which codes for LEKTI, cause Netherton syndrome, which is characterized by hair abnormality, ichthyosis, and atopy. A single nucleotide polymorphism (SNP) of SPINK5, p.K420E, is reported to be associated with the pathogenesis of atopic dermatitis (AD). We studied all 34 exons of the SPINK5 gene in Japanese 57 AD patients and 50 normal healthy controls. We detected nine nonsynonymous variants, including p.K420E; these variants had already been registered in the SNP database. Among them, p.R654H (n=1) was found as a heterozygous mutation in the AD patients, but not in the control. No new mutation was detected. We next compared the data of the AD patients with data from the Human Genetic Variation Database provided by Kyoto University; a significant difference was found in the frequency of the p.S368N genotype distribution. PolyPhen-2 and SIFT, two algorithms for predicting the functional effects of amino acid substitutions, showed significant scores for p.R654H. Therefore, R654H might be a risk factor for epidermal barrier dysfunction in some Japanese AD patients.

Composition of chemical species of selenium contained in selenium-enriched shiitake mushroom and vegetables determined by high performance liquid chromatography with inductively coupled plasma mass spectrometry.

Selenium (Se) species in Se-enriched shiitake mushroom (Lentinula edodes) were identified and quantified by high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICPMS). Two types of Se-enriched shiitake obtained from selenite- or selenate-fertilized mushroom beds were used. More than 80% of Se in both shiitake samples could not be extracted with 0.2 M HCl. Protease digestion released a large amount of selenomethionine from the shiitake enriched with selenite. However, most of the Se in the shiitake enriched with selenate was not released by protease but was released by a cell wall digestive enzyme and most of the Se released was identified as selenate. These results indicate that the main Se species in the shiitake enriched with selenite or selenate is selenomethionine bound to protein or selenate bound to polysaccharides in the cell wall, respectively. Several Se-enriched vegetables grown on a soil fertilized with selenate were also analyzed by HPLC-ICPMS. Four Se species, selenate, Se-methylselenocysteine, selenomethionine, gamma-glutamyl-Se-methylselenocysteine, and an unknown Se compound were detected in the vegetables. The composition of Se species varied with the kinds or parts of vegetables. The main Se species in bulbs, leaves or flowers of the Se-enriched garlic, onions, cabbage and ashitaba were selenate, Se-methylselenocysteine or gamma-glutamyl-Se-methylselenocysteine, while those in fruit bodies of the peppers and pumpkin were selenomethionine bound to protein. Bioavailabilities of Se in the shiitake mushroom enriched with selenite and the vegetables enriched with selenate are expected to be high, but that in shiitake enriched with selenate may be low.

Effect of intermittent supplementation with selenate on selenium status of rats fed selenium-deficient diet.

To examine the selenium (Se) status of rats intermittently supplemented with Se, we measured tissue Se contents and glutathione peroxidase (GPx) activities in rats fed a Se-deficient diet intermittently supplemented with selenate. In experiment 1, four groups of male 4-wk-old Wistar rats were fed a Torula yeast-based Se-deficient diet (Se content, < 0.01 microg/g) for 28 d. During the experimental period, the diet of each group was supplemented with sodium selenate (0.17 microg Se/g) for 0, 1, 2 or 7 d/wk. The tissue Se contents and GPx activities both increased gradually with an increase in frequency of the selenate supplementation, and significant linear regressions were observed between the frequency and these Se indices. In particular, the correlation coefficient in the liver and plasma indices was nearly equal to a value of 1.0. In experiment 2, three groups of rats were fed the Se-deficient basal diet for 28 d. Among these, one group was daily supplemented with sodium selenate to the Se-deficient diet at a level of 0.17 microg Se/g, and another group was intermittently supplemented with the selenate at a level of 1.19 microg Se/g for 1 d/wk. The tissue Se contents and GPx activities both were increased by the selenate supplementation and no significant difference was observed between daily and weekly supplementation in the Se indices except in erythrocyte Se. These results indicate that Se status in the growth period is dependent on total Se intake in this period and that weekly intermittent supplementation with Se can maintain adequate Se status.

Preparation of selenium-enriched sprouts and identification of their selenium species by high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

Sprouts of several plants (10 families and 28 species) were cultivated in a high selenium environment, and the chemical species of selenium in these selenium-enriched sprouts were identified by using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). Cultivation of sprouts of kaiware daikon (type of radish) with 5.0 microg/ml or 10.0 microg/ml of selenium as selenite inhibited the growth. However, no abnormalities in the shape or color were apparent even in the sprouts exposed to 10.0 microg/ml of selenium. The selenium concentration in the sprouts of most plants examined was higher than that from environmental exposure. Among the types of selenium that were accumulated, a large part (69-98%) was extractable in 0.2 M HCl. Chemical analysis of selenium in the HCl extract showed that the main selenium species in all the sprouts examined was Se-methylselenocysteine. In addition to Se-methylselenocysteine, selenomethionine, non-metabolized selenite, gamma-glutamyl-Se-methylselenocysteine and an unknown selenium compound were also detected in several high-selenium sprouts. Since higher anticarcinogenic activities of these monomethylated selenoamino acids have been observed, it is anticipated that such selenium-enriched sprouts will be used as a foodstuff for cancer prevention.

Digestibility and chemical species of selenium contained in high-selenium yeast.

To evaluate the bioavailability of selenium (Se) in high-Se yeast (SeY), the digestibility and chemical species of Se in SeY were investigated. Both Se and nitrogen in SeY were readily released into the soluble fraction through trypsin digestion. In a Sephadex G-25 gel chromatography of the trypsin digest of SeY, the range in which Se was eluted was coincident with the range in which peptide fragments were eluted. Se was distributed almost uniformly within the range and there was no fraction that contained Se in a specifically high amount. A proteolytic enzyme extract of SeY was found to contain Se as selenomethionine (74.8%), selenocystine (9.9%), selenite (5.1%) and as at least three unknown Se compounds (10.2%) when analyzed using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). These results indicate that Se in SeY is mainly present as selenomethionine non-specifically incorporated into peptide chains and is highly digestible. Accordingly, it is concluded that the bioavailability of Se in SeY is high.