Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis.

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

Whole-Mount Preparation and Microscopic Analysis of Epidermis.

The epidermis is a stratified epithelium. Compared to that for monolayered epithelia, understanding of the cell biology of stratified epithelia lags far behind. The major reason for this is the limitation of methods to reproduce the epidermis in vitro using cultured keratinocytes: for example, cultured keratinocyte cell sheets lack Langerhans cells, melanocytes, nerves, sweat ducts, and hair follicles. One current way to overcome this limitation is to observe the epidermis in vivo via whole-mount staining and three-dimensional imaging. Here, we describe how to prepare epidermal sheets from skin and how to immunostain and observe them in whole mount. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of mouse epidermal sheets by the ammonium thiocyanate method Alternate Protocol: Preparation of mouse epidermal sheets by the dispase method Basic Protocol 2: Preparation of human epidermal sheets by the dispase method Basic Protocol 3: Whole-mount immunostaining of epidermis.

CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury.

Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling between 2 unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL-21 and IFN-γ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis, and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153/CD30 signaling in TLT formation and propose targeting the CD153/CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.

Syntheses of Water-Soluble Silver(II)-Phthalocyanines toward Optical Sensing for Thiol Detection.

Water-soluble silver(II)-phthalocyanine complexes (AgPcs), tetrakis{4-(N-alkylpyridinium)thio}phthalocyaninato silver(II) tetrafluoroborate, [Ag(tRpySpc)](BF4)4, (R = Me and Et), have been synthesized for the first time by quaternization of pyridyl groups of tetrakis(4-pyridylthio)phthalocyaninato silver(II) by using Meerwein reagents and characterized by ESI-MS, elemental analyses, and optical absorption spectroscopy. Although they strongly aggregate in water, the presence of appropriate surfactants, such as polyethyleneglycol-monooleyl ether (n = approximately 50; PEG50) and sodium dodecyl sulfate, effectively disaggregates them to monomeric species. The spectral properties of the AgPcs and their aggregates in aqueous and nonaqueous solutions have been investigated by optical absorption, emission, and magnetic circular dichroism spectroscopy. These AgPcs rapidly react with thiols such as cysteine, glutathione, homocysteine, and sodium 2-sulfanylethanesulfonate (even on the order of 0.01 mM) in aqueous PEG50 solutions at room temperature to liberate the corresponding macrocyclic ligand, H2Pc, but not with the other amino-acid analogs without sulfhydryl groups. The molar ratio of thiol to AgPc has been determined to be 1:1. Since AgPcs are essentially nonfluorescent at room temperature, while H2Pcs emit intense red fluorescence, AgPcs can be a potent thiol-sensor toward bioimaging.

Spectral investigation of phthalocyanine complexes of high-valence silver and their aggregates.

Several novel silver(II) complexes ligating a tetra-substituted phthalocyaninate, [Ag(tbpc)] (where tbpc denotes tetra-tert-butylphthalocyaninate), [Ag(tppc)] (tppc = tetrakis(2,6-dimethylphenoxy)phthalocyaninate), [Ag(tObpc)] (tObpc = tetra-n-butoxyphthalocyaninate), and [Ag(tpySpc)] (tpySpc = tetrakis(4-pyridylthio)phthalocyaninate) have been synthesized and characterized by elemental analyses, MALDI-TOF MS, optical absorption, and magnetic circular dichroism (MCD) spectroscopy. Although all the compounds are well soluble in common organic solvents, concentration studies on their optical spectra in solutions have found that they are prone to strongly aggregate in a cofacial manner (i.e., H-aggregate). Silver(II) complexes, which are essentially non-fluorescent, are readily demetallated in the presence of appropriate reductant (e.g., I- or BH4-) to liberate the corresponding macrocyclic ligand, which emits intense red fluorescence. Chemical oxidation by using NOBF4 generates the corresponding silver(III) species.

Premature aging syndrome showing random chromosome number instabilities with CDC20 mutation.

Damage to the genome can accelerate aging. The percentage of aneuploid cells, that is, cells with an abnormal number of chromosomes, increases during aging; however, it is not clear whether increased aneuploidy accelerates aging. Here, we report an individual showing premature aging phenotypes of various organs including early hair loss, atrophic skin, and loss of hematopoietic stem cells; instability of chromosome numbers known as mosaic variegated aneuploidy (MVA); and spindle assembly checkpoint (SAC) failure. Exome sequencing identified a de novo heterozygous germline missense mutation of c.856C>A (p.R286S) in the mitotic activator CDC20. The mutant CDC20 showed lower binding affinity to BUBR1 during the formation of the mitotic checkpoint complex (MCC), but not during the interaction between MCC and the anaphase-promoting complex/cyclosome (APC/C)-CDC20 complex. While heterozygous knockout of CDC20 did not induce SAC failure, knock-in of the mutant CDC20 induced SAC failure and random aneuploidy in cultured cells, indicating that the particular missense mutation is pathogenic probably via the resultant imbalance between MCC and APC/C-CDC20 complex. We postulate that accelerated chromosome number instability induces premature aging in humans, which may be associated with early loss of stem cells. These findings could form the basis of a novel disease model of the aging of the body and organs.

Monitoring the roles of prothrombin activation fragment 1 and 2 (F1 + 2) in patients with atrial fibrillation receiving rivaroxaban and apixaban.

Factor Xa (FXa) inhibitors are recommended for use in fixed doses without laboratory monitoring. However, prior studies reported the importance of establishing biomarkers representing anticoagulation intensity related to bleeding or thrombotic events. To test the hypothesis that prothrombin activation fragment 1 and 2 (F1 + 2), a non-specific marker of thrombin generation, could be altered during FXa inhibitor treatment in patients with atrial fibrillation. We conducted the study in two different clinical settings. First, the interrelations among biomarkers representing coagulation/fibrinolysis were investigated in 80 patients in an outpatient clinic. Second, these biomarkers were evaluated in 75 patients who underwent radiofrequency catheter ablation. Plasma concentration of FXa inhibitors was evaluated using an anti-FXa chromogenic assay (C-Xa). In the outpatient study, only F1 + 2 exhibited a significant and negative association with C-Xa (rS = - 0.315, p = 0.026), and 37% of the variance could be explained by C-Xa levels. F1 + 2 levels above the reference range (> 229 pmol/L) could be considered as a cut-off to identify poor patient compliance or under-dosing. In the peri-ablation study, increased F1 + 2 levels were associated with decline of C-Xa levels after periprocedural discontinuation of FXa inhibitors, which was greater in the rivaroxaban group than in the apixaban group. F1 + 2 showed modest and inverse association with plasma concentration of rivaroxaban and apixaban in patients with atrial fibrillation. Larger study to test the hypothesis that continued thrombin generation despite anticoagulation is associated with a heightened risk of clinical events is required.

Clonal Expansion of Second-Hit Cells with Somatic Recombinations or C>T Transitions Form Porokeratosis in MVD or MVK Mutant Heterozygotes.

Patients with disseminated superficial actinic porokeratosis (DSAP) and linear porokeratosis (LP) exhibit monoallelic germline mutations in genes encoding mevalonate pathway enzymes, such as MVD or MVK. Here, we showed that each skin lesion of DSAP exhibited an individual second hit genetic change in the wild-type allele of the corresponding gene specifically in the epidermis, indicating that a postnatal second hit triggering biallelic deficiency of the gene is required for porokeratosis to develop. Most skin lesions exhibited one of two principal second hits, either somatic homologous recombinations rendering the monoallelic mutation biallelic or C>T transition mutations in the wild-type allele. The second hits differed among DSAP lesions but were identical in those of congenital LP, suggesting that DSAP is attributable to sporadic postnatal second hits and congenital LP to a single second hit in the embryonic period. In the characteristic annular skin lesions of DSAP, the central epidermis featured mostly second hit keratinocytes, and that of the annular ring featured a mixture of such cells and naïve keratinocytes, implying that each lesion reflects the clonal expansion of single second hit keratinocytes. DSAP is therefore a benign intraepidermal neoplasia, which can be included in the genetic tumor disorders explicable by Knudson's two-hit hypothesis.

Characterization of centriole duplication in human epidermis, Bowen's disease, and squamous cell carcinoma.

BACKGROUND: Centrosomes contain two centrioles: a pre-existing mature centriole and a newly formed immature centriole. Each centriole is duplicated once within a cell cycle, which is crucial for proper centrosome duplication and cell division. OBJECTIVE: To describe the centrosome duplication cycle in human epidermis, Bowen's disease (BD), and squamous cell carcinoma (SCC). METHODS: Immunofluorescent staining of centriolar proteins and Ki-67 was used to evaluate cell cycles and the number of centrioles. Centrobin and Outer dense fiber of sperm tails 2 (ODF2) were used as markers for immature and mature centrioles, respectively. RESULTS: Normal human primary epidermal keratinocytes in a monolayered culture have one centrobin+ centriole (CTRB1+ cells) supposed in G0/G1 phases or have two centrobin+ centrioles (CTRB2+ cells) supposed in S-G2 phase. In a three-dimensional culture and in vivo human epidermis, the majority of suprabasal cells were CTRB2+ cells, in spite of their non-proliferative Ki-67- nature. The tumor mass of BD and SCC contained CTRB1+ cells and Ki-67+ proliferating and Ki-67- non-proliferative CTRB2+ cells. Clumping cells in BD had increased numbers of centrioles, with an approximate 1:1 to 2:1 ratio of centrobin+ to ODF2+ centrioles. CONCLUSIONS: The cell cycle arrest of suprabasal cells is distinct from the G0 arrest of monolayered epithelial cells. Tumor mass of BD and SCC contained non-proliferative cells with the characteristics of the suprabasal cells of normal epidermis. A constant ratio of the number of centrobin+ to ODF2+ centrioles indicates that multiple centrioles were induced by cell division failure rather than centriole overduplication in clumping cells.

Amphoteric phosphorous(V)-phthalocyanines as proton-driven switchable fluorescers toward deep-tissue bio-imaging.

Spectral (optical absorption and emission) properties of three amphoteric phosphorous(V)-phthalocyanine derivatives, [P(Pc)(O)OH], where Pc=tetra(tert-butyl)phthalocyaninate (tbpc), tetrakis(2',6'-dimethylphenoxy)phthalocyaninate (tppc), and octakis(4'-tert-butylphenoxy)phthalocyaninate (obppc), have been investigated in ethanolic solutions. Spectral changes upon protonation/deprotonation (the reaction sites have been determined to be their axial ligands by magnetic circular dichroism study) are drastic and rapid. All the initial ([P(Pc)(O)OH]), protonated ([P(Pc)(OH)2]+), and deprotonated ([P(Pc)(O)2]-) species are possessed with sufficient brightness (defined as the product of their molar extinction coefficient, ε (inM-1cm-1), and fluorescence quantum yield, ΦF) in bio-imaging window (650-900nm). For example, spectral characteristics of the tbpc derivatives have been determined as follows: ε=1.65×105 (absorption maximum 676nm) and ΦF=0.80 (emission maximum 686nm) for [P(tbpc)(O)(OH)] while ε=1.45×105 (697nm) and ΦF=0.27 (714nm) for [P(tbpc)(OH)2]+, and ε=2.25×105 (662nm) and ΦF=0.90 (667nm) for [P(tbpc)(O)2]-. Emission of tppc and obppc derivatives behave in essentially the same manner irrespective of nature of the peripheral substituents and hence ΦF values are greater with increasing emission peak wavenumbers in line with the "energy gap law". These characteristics make these compounds promising candidates as chemical probes for deep-tissue bio-imaging.

A familial case of nail patella syndrome with a heterozygous in-frame indel mutation in the LIM domain of LMX1B.

Nail patella syndrome is a autosomal dominant disorder caused by a genetic alteration in LMX1B. We identified a novel heterozygous in-frame indel mutation of LMX1B in a family of Nail patella syndrome. Impaired transcriptional activity but not dominant negative effect of mutant LMX1B were revealed using a transcriptional reporter assay, indicating that the mutation caused nail patella syndrome in this family via haploinsufficiency of the transcriptional activity of LMX1B.

Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney.

Acute kidney injury (AKI) is a common clinical condition defined as a rapid decline in kidney function. AKI is a global health burden, estimated to cause 2 million deaths annually worldwide. Unlike AKI in the young, which is reversible, AKI in the elderly often leads to end-stage renal disease, and the mechanism that prevents kidney repair in the elderly is unclear. Here we demonstrate that aged but not young mice developed multiple tertiary lymphoid tissues (TLTs) in the kidney after AKI. TLT size was associated with impaired renal function and increased expression of proinflammatory cytokines and homeostatic chemokines, indicating a possible contribution of TLTs to sustained inflammation after injury. Notably, resident fibroblasts from a single lineage diversified into p75 neurotrophin receptor+ (p75NTR+) fibroblasts and homeostatic chemokine-producing fibroblasts inside TLTs, and retinoic acid-producing fibroblasts around TLTs. Deletion of CD4+ cells as well as late administration of dexamethasone abolished TLTs and improved renal outcomes. Importantly, aged but not young human kidneys also formed TLTs that had cellular and molecular components similar to those of mouse TLTs. Therefore, the inhibition of TLT formation may offer a novel therapeutic strategy for AKI in the elderly.

A CD153+CD4+ T follicular cell population with cell-senescence features plays a crucial role in lupus pathogenesis via osteopontin production.

Immune aging results in diminished adaptive immunity and increased risk for autoimmunity. We previously reported a unique PD-1(+) CD44(high)CD4(+) T cell population that increases with age in normal mice. In this study, we indicate that the age-dependent PD-1(+) CD44(high)CD4(+) T cells develop as unique T follicular (TF) cells in a B cell-dependent manner and consist of two subpopulations, as follows: CD153(+) cells preferentially secreting abundant osteopontin on TCR stimulation and CD153(-) cells that are apparently TCR anergic. These unique TF cells with essentially similar features increase much earlier and are accumulated in the spontaneous germinal centers (GCs) in lupus-prone female BWF1 (f-BWF1) mice. These TF cells showed characteristic cell-senescence features and developed in association with extensive CD4(+) T cell proliferation in vivo, suggesting replicative senescence. Although the CD153(+) TF cells were defective in proliferation capacity, they were quite stable and specifically responded to self GC-B cells to secret abundant osteopontin, which inhibited B cell receptor-induced GC-B cell apoptosis in f-BWF1 mice. Transfer of CD153(+) PD-1(+) CD4(+) T cells promoted the growth of spontaneous GCs, whereas administration of anti-osteopontin Ab suppressed GC enlargement and anti-nuclear Ab production and ameliorated clinical lupus nephritis of f-BWF1 mice. Current results suggest that senescent CD153(+) TF cells generated as a consequence of extensive endogenous CD4(+) T cell proliferation play an essential, if not sufficient, role in lupus pathogenesis in lupus-prone genetic background and may also contribute to an increased autoimmunity risk with age.

Distinct behavior of human Langerhans cells and inflammatory dendritic epidermal cells at tight junctions in patients with atopic dermatitis.

BACKGROUND: The stratum corneum and tight junctions (TJs) form physical barriers in the epidermis. Dendrites of activated Langerhans cells (LCs) extend beyond the TJs to capture external antigens in mice. LCs and inflammatory dendritic epidermal cells (IDECs) are observed in the skin of patients with atopic dermatitis (AD). OBJECTIVE: We sought to investigate the characteristics of LCs and IDECs and the distribution of their antigen capture receptors in relation to TJs in normal and AD skin. METHODS: We characterized the interactions of LCs and IDECs with TJs and the expression patterns of langerin and FcεRI by using whole-mount epidermal sheets from healthy subjects and patients with AD, ichthyosis vulgaris, and psoriasis vulgaris. RESULTS: As in mouse skin, activated LCs penetrate TJs in human skin. The number of LCs with TJ penetration increased approximately 5-fold in erythematous lesional skin of patients with AD but not in nonlesional skin of patients with AD or lesions of patients with ichthyosis vulgaris or psoriasis. In contrast, IDECs localized in the lower part of the epidermis, and their dendrites extended horizontally without penetration through TJs. Although langerin accumulated on the tips of dendrites of activated LCs, FcεRI was expressed diffusely on the cell surfaces on LCs and IDECs in lesional skin from patients with AD. CONCLUSIONS: These findings highlight interesting differences between LCs and IDECs in epidermis of patients with AD, where LCs, but not IDECs, extend dendrites through the TJs, likely to capture antigens from outside the TJ barrier with a polarized distribution of langerin but not FcεRI. These behavioral differences between skin dendritic cells might reflect an important pathophysiology of AD.

Enteroendocrine cells are specifically marked by cell surface expression of claudin-4 in mouse small intestine.

Enteroendocrine cells are solitary epithelial cells scattered throughout the gastrointestinal tract and produce various types of hormones, constituting one of the largest endocrine systems in the body. The study of these rare epithelial cells has been hampered by the difficulty in isolating them because of the lack of specific cell surface markers. Here, we report that enteroendocrine cells selectively express a tight junction membrane protein, claudin-4 (Cld4), and are efficiently isolated with the use of an antibody specific for the Cld4 extracellular domain and flow cytometry. Sorted Cld4+ epithelial cells in the small intestine exclusively expressed a chromogranin A gene (Chga) and other enteroendocrine cell-related genes (Ffar1, Ffar4, Gpr119), and the population was divided into two subpopulations based on the activity of binding to Ulex europaeus agglutinin-1 (UEA-1). A Cld4+UEA-1- cell population almost exclusively expressed glucose-dependent insulinotropic polypeptide gene (Gip), thus representing K cells, whereas a Cld4+UEA-1+ cell population expressed other gut hormone genes, including glucagon-like peptide 1 (Gcg), pancreatic polypeptide-like peptide with N-terminal tyrosine amide (Pyy), cholecystokinin (Cck), secretin (Sct), and tryptophan hydroxylase 1 (Tph1). In addition, we found that orally administered luminal antigens were taken up by the solitary Cld4+ cells in the small intestinal villi, raising the possibility that enteroendocrine cells might also play a role in initiation of mucosal immunity. Our results provide a useful tool for the cellular and functional characterization of enteroendocrine cells.

The synthesis and spectral investigation of a novel highly water-soluble, aggregation-free antimony(V)-phthalocyanine absorbing light in optical therapeutical window.

A novel antimony-phthalocyanine, [Sb(H(3)tsppc)(OH)(2)], where H(3)tsppc denotes monodeprotonated tetrakis{(2',6'-dimethyl-4'-sulfonic acid)phenoxyl}phthalocyaninate, has been synthesized through sulfonation of [Sb(tppc)(OH)(2)](+) (tppc denotes tetrakis{(2',6'-dimethyl)phenoxyl}phthalocyaninate) in concentrated sulfuric acid. This compound is highly soluble in water (ca. 4 × 10(-2) M) without surfactant or alcohol. Moreover, it has been found free from aggregation in water up to almost 10(-4) M, unlike its copper and metal-free analogues, and show an intense optical absorption and emission band in optical therapeutical window (700-800 nm). The axial hydroxyl groups play a crucial role in disaggregation of the antimony derivative in water.

The syntheses of amphiphilic antimony(V)-phthalocyanines and spectral investigation on their aggregation behaviors in aqueous and non-aqueous solutions.

Three amphiphilic antimony(V)-phthalocyanines have been synthesized by treating [Sb(R(4)Pc)(OH)(2)](+) salts in concentrated H(2)SO(4) and isolated as zwitter ions, [Sb(R(4)Pc)(SO(4)H)(SO(4))], where R(4)Pc denotes tetra-substituted phthalocyaninate; R(4)Pc=pc (R=H), tbpc (R=(t)Bu), and tObpc (R=O(n)Bu). Their solubility (R=tbpc>pc >>tObpc in H(2)O (much improved by the presence of surfactant or alcohol) while tbpc>tObpc >>pc in CH(2)Cl(2)) and aggregation behaviors are highly sensitive to the nature of the peripheral substituents. The pc and tbpc derivatives form well-behaved J-aggregates in aqueous media in the presence of surfactant or alcohol.

Claudin-4 deficiency results in urothelial hyperplasia and lethal hydronephrosis.

Claudin (Cld)-4 is one of the dominant Clds expressed in the kidney and urinary tract, including selective segments of renal nephrons and the entire urothelium from the pelvis to the bladder. We generated Cldn4(-/-) mice and found that these mice had increased mortality due to hydronephrosis of relatively late onset. While the renal nephrons of Cldn4(-/-) mice showed a concomitant diminution of Cld8 expression at tight junction (TJ), accumulation of Cld3 at TJ was markedly enhanced in compensation and the overall TJ structure was unaffected. Nonetheless, Cldn4(-/-) mice showed slightly yet significantly increased fractional excretion of Ca(2+) and Cl(-), suggesting a role of Cld4 in the specific reabsorption of these ions via a paracellular route. Although the urine volume tended to be increased concordantly, Cldn4(-/-) mice were capable of concentrating urine normally on dehydration, with no evidence of diabetes insipidus. In the urothelium, the formation of TJs and uroplaques as well as the gross barrier function were also unaffected. However, intravenous pyelography analysis indicated retarded urine flow prior to hydronephrosis. Histological examination revealed diffuse hyperplasia and a thickening of pelvic and ureteral urothelial layers with markedly increased BrdU uptake in vivo. These results suggest that progressive hydronephrosis in Cldn4(-/-) mice arises from urinary tract obstruction due to urothelial hyperplasia, and that Cld4 plays an important role in maintaining the homeostatic integrity of normal urothelium.