Normal Skin Keratinocytes Research Articles

Gallium octacarboxyphthalocyanine hydroxide as a potential pro-apoptotic drug against cancer skin cells

Novel anticancer strategies reduce side effects on healthy tissues by elevating the lethal abilities of cancer cells. The development of effective particles with good bioavailability and selectivity remains problematic. For undesirable features, green chemistry is used to synthesize the best compounds, or natural-based particles are improved. Photodynamic therapy (PDT), modelled on phthalocyanines (Pcs), still delivers second-generation sensitizers which are complemented with metal ions, such as Zn2+, Al3+, or Ga3+. Gallium octacarboxyphthalocyanine hydroxide (Ga(OH)PcOC), was designed for skin cancer treatment, and was used as a pro-apoptotic and pro-oxidative agent on normal skin cell lines, fibroblasts (NHDF), and keratinocytes (HaCaT), with promising selectivity against melanoma cancer cells (Me45) in vitro. Compared to the previous reported findings, where the ZnPcOC acted on the skin cell lines at higher doses, the sensitivities to the Ga(OH)PcOC allows for an effective reduction of the sensitizer dose. The effective dose, for a novel Ga(OH)PcOC particle, was significantly reduced from 30 µM to 6 µM on Me45 cancer cells, tested using 24 h MTT viability, as well as cytometric pro-oxidative and pro-apoptotic assays. The promising photosensitizer did not reduce viability in normal fibroblasts and keratinocytes without reactive oxygen species (ROS) elevation or apoptosis induction. The improvement to the previous findings is better Ga-based photosensitizer selectivity against the cancer Me45 cells, then observed in Zn-based compounds.

Genetic evolution of keratinocytes to cutaneous squamous cell carcinoma.

We performed multi-omic profiling of epidermal keratinocytes, precancerous actinic keratoses, and squamous cell carcinomas to understand the molecular transitions during skin carcinogenesis. Single-cell mutational analyses showed that most keratinocytes in normal skin had lower mutation burdens than melanocytes and fibroblasts, however keratinocytes with TP53 or NOTCH1 mutations had substantially higher mutation burdens, suggesting that these mutations prime keratinocytes for transformation by increasing their mutation rate. Mutational profiling and spatial transcriptomics on squamous cell carcinomas adjacent to actinic keratoses revealed TERT promoter and CDKN2A mutations emerging in actinic keratoses, whereas additional mutations inactivating ARID2 and activating the MAPK-pathway delineated the transition to squamous cell carcinomas. Spatial variation in gene expression patterns was common in both tumor and immune cells, with high expression of checkpoint molecules at the invasive front of tumors. In conclusion, this study documents key events during the evolution of cutaneous squamous cell carcinoma.

Gram-negative anaerobes elicit a robust keratinocytes immune response with potential insights into HS pathogenesis.

Hidradenitis Suppurativa (HS) is a chronic autoinflammatory skin disease with activated keratinocytes, tunnel formation and a complex immune infiltrate in tissue. The HS microbiome is polymicrobial with an abundance of commensal gram-positive facultative (GPs) Staphylococcus species and gram-negative anaerobic (GNA) bacteria like Prevotella, Fusobacterium and Porphyromonas with increasing predominance of GNAs with disease severity. We sought to define the keratinocyte response to bacteria commonly isolated from HS lesions to probe pathogenic relationships between HS and the microbiome. Type strains of Prevotella nigrescens, Prevotella melaninogenica, Prevotella intermedia, Prevotella asaccharolytica, Fusobacterium nucleatum, as well as Staphylococcus aureus and the normal skin commensal Staphylococcus epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was collected and analysed using RNAseq and RT-qPCR. The supernatant was collected from cell culture for protein quantification. Transcriptomic profiles between HS clinical samples and stimulated keratinocytes were compared. Co-staining of patient HS frozen sections was used to localize bacteria in lesions. A mouse intradermal injection model was used to investigate early immune recruitment. TLR4 and JAK inhibitors were used to investigate mechanistic avenues of bacterial response inhibition. GNAs, especially F. nucleatum, stimulated vastly higher CXCL8, IL17C, CCL20, IL6, TNF and IL36γ transcription in normal skin keratinocytes than the GPs S. epidermidis and S. aureus. Using RNAseq, we found that F. nucleatum (and Prevotella) strongly induced the IL-17 pathway in keratinocytes and overlapped with transcriptome profiles of HS patient clinical samples. Bacteria were juxtaposed to activated keratinocytes invivo, and F. nucleatum strongly recruited murine neutrophil and macrophage migration. Both the TLR4 and pan-JAK inhibitors reduced cytokine production. Detailed transcriptomic profiling of healthy skin keratinocytes exposed to GNAs prevalent in HS revealed a potent, extensive inflammatory response vastly stronger than GPs. GNAs stimulated HS-relevant genes, including many genes in the IL-17 response pathway, and were significantly associated with HS tissue transcriptomes. The close association of activated keratinocytes with bacteria in HS lesions and innate infiltration in murine skin cemented GNA pathogenic potential. These novel mechanistic insights could drive future targeted therapies.

Detecting normal and cancer skin cells via glycosylation and adhesion signatures: A path to enhanced microfluidic phenotyping

Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.

Abstract 4009: Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy

Abstract Century Therapeutics is developing NECTIN4 targeted induced Pluripotent Stem Cell (iPSC) derived Chimeric Antigen Receptor (CAR) T cell therapy. NECTIN4 targeted antibody drug conjugate enfortumab vedotin, while generally well tolerated, causes severe skin adverse events in some patients, thought to be driven by on-target off-tumor toxicity against NECTIN4 displaying skin keratinocytes. We include a safety switch in our first clinical-stage iPSC-derived CAR-NK cell therapy, CNTY-101, which provides an antibody target for elimination of the engineered cells should that be necessary. An additional method for reducing the incidence and severity of this type of adverse event is through the incorporation of an inhibitory CAR, also known as a NOT-gate, directed against antigen expressed on normal skin keratinocytes, but the optimal surface protein to target with such an inhibitory CAR for a NECTIN4 targeted therapy is unknown. Here, we identify DSG1 (Desmoglein-1) as a top inhibitory CAR target for NECTIN4 targeted CAR-T cell therapy by conducting a multi-omics analysis of public single-cell RNAseq, bulk RNAseq, and protein microarray immunohistochemistry datasets. We find that DSG1 is constitutively and uniformly displayed on the surface of skin keratinocytes in the layers of epidermis where they display NECTIN4, and DSG1 gene expression in normal skin is uniformly high across individuals. Conversely, DSG1 mRNA and protein are rarely expressed in NECTIN4 expressing cancer indications including bladder, breast, non-small cell lung, ovarian, and pancreatic. Further, autoimmune antibodies specific to DSG1 have been described, suggesting that DSG1 is targetable by a CAR in situ. Finally, DSG1 is not expressed by our iPSC-derived CAR-T cells. While NECTIN4 expression is greatest in skin keratinocytes, epithelial cells in other tissues express NECTIN4 at lower levels. In this study, we compare the expression levels of NECTIN4 to targets associated with tissue and cell-type specific on-target off-tumor toxicity in primary CAR-T cell clinical trials. Our findings support the strategy of DSG1-directed inhibitory CAR incorporation into NECTIN4 specific CAR-T cell therapeutic candidates. Citation Format: Matthew S. Hall, Christopher M. Dower, Michael Miller, John Wheeler, Jill Carton, Ohad Manor, Hyam Levitsky. Discovery of inhibitory CAR target DSG1 for damping NECTIN4 on-target off-tumor toxicity in iPSC-derived CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4009.

Hypericin-Based Photodynamic Therapy Displays Higher Selectivity and Phototoxicity towards Melanoma and Squamous Cell Cancer Compared to Normal Keratinocytes In Vitro

The aim of this study was to explore the potential of hypericin, a naturally occurring photosensi-tizer, for photodynamic therapy (PDT) in skin cancer, investigating its phototoxic effects and mechanisms of action in cancer cells compared to normal skin keratinocytes, squamous cell cancer (SCC-25) cells and melanoma (MUG-Mel2) cells. Hypericin was applied at concentrations ranging from 0.1–40 μM to HaCaT, SCC-25, and MUG-Mel2 cells. After 24 h of incubation, the cells were exposed to orange light at 3.6 J/cm2 or 7.2 J/cm2. Phototoxicity was assessed using MTT and SRB tests. Cellular uptake was measured by flow cytometry. Apoptosis-positive cells were estimated through TUNEL for apoptotic bodies’ visualization. Hypericin exhibited a higher phototoxic reaction in cancer cells compared to normal keratinocytes after irradiation. Cancer cells demonstrated increased and selective uptake of hypericin. Apoptosis was observed in SCC-25 and MUG-Mel2 cells following PDT. Our findings suggest that hypericin-based PDT is a promising and less invasive approach for treating skin cancer. The higher phototoxic reaction, selective uptake by cancer cells, and observed proapoptotic properties support the promising role of hypericin-based PDT in skin cancer treatment.

Abstract 4780: Comparison of safety and efficacy outcomes with nonthermal plasma and tirapazamine in porcine skin and mouse melanoma models

Abstract A novel preclinical experimental therapy for various cancer types including melanoma is the combination of nonthermal plasma (NTP) and tirapazamine (TPZ) delivered in situ. This patented combination therapy delivers reactive oxygen species (ROS) and reactive nitrogen species (RNS) from the atmosphere that are activated and ionized with helium gas to create the state of matter known as plasma. The combination with the prodrug, TPZ, that only activates to become a ROS in hypoxic environments, shows synergistic targeting of cancer cells. In this study, we assayed the effects of the dual therapy on porcine skin, which is the closest model to represent human skin and is commonly used for testing drug therapy. The results demonstrated that porcine skin showed no significant cell killing with the therapy. We also compared the combination therapy in normal skin keratinocytes and 1205Lu cells derived from a melanoma metastasis to the lung. The cells were treated with NTP and TPZ and maintained in a hypoxic chamber. Four days following therapy the keratinocytes continued to grow in culture, whereas the melanoma cells expressing endogenous gap junctions, or overexpressed Cx43 gap junctions showed decreased viability. However, the melanoma cells with a dominant negative Cx43 mutant did not show this loss in viability. This confirms our previous report that gap junctions mediate the passage of derivatives of ROS to increase the extent of cell death. To determine the effects of NTP on regulating the lethal dose of TPZ, we performed IC50 curves for TPZ in the presence or absence of NTP in B16-F10 murine metastatic melanoma cells. We demonstrated a decrease in the IC50 from 28 μM without NTP to 17.4 μM with NTP. In further analysis, the effects of treating with NTP in the presence or absence of tissue culture media were compared. The results showed a highly significant increased efficacy of NTP in the absence of media. The combination therapy was more effective than each independent treatment. To further confirm the experimental analysis, we induced tumor formation with the B16-F10 cells in a syngeneic C57BL/6 mouse model. The tumors from mice that were untreated or treated with NTP or TPZ alone, showed a significant increase in tumor volume as compared to the tumors from mice receiving the combination treatment in situ. In comparison of the tumor growth rates, the control, NTP-, or TPZ-treated tumors had increased by approximately 350% by day 10, whereas the tumors in the mice that received the dual therapy only increased by an average of 70%. The results from these studies collectively implicate the combination of NTP and TPZ as a potential synergistic treatment for in situ melanomas that offer selectivity as compared to normal tissue. Citation Format: Matthew Yehl, Dominik Kucharski, Michelle Eubank, Xavier Ramadan, Ahmad Dianat, Bianca Pham, Timothy C. Hutcherson, Sandra Sexton, Shoshanna N. Zucker. Comparison of safety and efficacy outcomes with nonthermal plasma and tirapazamine in porcine skin and mouse melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4780.

Effect of Hypertrophic Scar Fibroblast-Derived Exosomes on Keratinocytes of Normal Human Skin

Epidermal keratinocytes are highly activated, hyper-proliferated, and abnormally differentiated in the post-burn hypertrophic scar (HTS); however, the effects of scar fibroblasts (SFs) on keratinocytes through cell-cell interaction in HTS remain unknown. Here, we investigated the effects of HTSF-derived exosomes on the proliferation and differentiation of normal human keratinocytes (NHKs) compared with normal fibroblasts (NFs) and their possible mechanism to provide a reference for clinical intervention of HTS. Fibroblasts were isolated and cultured from HTS and normal skin. Both HTSF-exosomes and NF-exosomes were extracted via a column-based method from the cell culture supernatant. NHKs were treated for 24 or 48 h with 100 μg/mL of cell-derived exosomes. The expression of proliferation markers (Ki-67 and keratin 14), activation markers (keratins 6, 16, and 17), differentiation markers (keratins 1 and 10), apoptosis factors (Bax, Bcl2, caspase 14, and ASK1), proliferation/differentiation regulators (p21 and p27), and epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and vimentin) was investigated. Compared with NF-exosomes, HTSF-exosomes altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, proliferation/differentiation regulators of NHKs, and EMT markers differently. In conclusion, our findings indicate that HTSF-derived exosomes may play a role in the epidermal pathological development of HTS.

Phosphorylation of Janus kinase 1 and signal transducer and activator of transcription 3 and 6 in keratinocytes of canine atopic dermatitis.

Canine atopic dermatitis (cAD) is a disease associated with Type 2 helper T (Th2) immune responses in the acute phase of the disease. In humans, keratinocytes are activated by Th2 cytokines via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. However, the activation of keratinocytes by Th2 cytokines in cAD has not yet been demonstrated. To evaluate keratinocyte activation based on the phosphorylation (p) of JAK1, STAT3 and STAT6. Seven dogs with cAD and three healthy dogs. Immunohistochemical analysis was performed to detect pJAK1, pSTAT3 and pSTAT6 in keratinocytes in normal canine skin, and the skin of atopic dogs. In the latter group samples were collected from both primary and secondary lesions, and nonaffected skin. The percentage of pJAK1-positive keratinocytes was significantly higher in primary cAD lesions than in healthy skin (p < 0.05). No significant differences were observed in pSTAT3-positive keratinocytes among the groups. The percentage of pSTAT6-positive keratinocytes was significantly higher in primary and secondary lesions than in healthy skin (p < 0.05, respectively). The novel finding in this study was the activation of keratinocytes as demonstrated by the phosphorylation of JAK1/STATs in lesional and nonlesional cAD skin. These results suggest the potential of not only JAK1, butalso of STAT6 as therapeutic targets for cAD.

Thioredoxin domain-containing protein 9 protects cells against UV-B-provoked apoptosis via NF-κB/p65 activation in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC), a type of non-melanoma skin cancer (NMSC), is the most common malignancy worldwide. Thioredoxin (TXN) domain-containing protein 9 (TXNDC9) is a member of the TXN family that is important in cell differentiation. However, the biological function of this protein in cancer, particularly cSCC, is still unknown. In the present study, our experiments revealed the protective effects of TXNDC9 on UV-B-irritated cSCC cells. The initial findings showed that TXNDC9 is significantly upregulated in cSCC tissue and cells compared to normal skin tissue and keratinocytes. UV-B radiation robustly induces the expression of TXNDC9, and UV-B-induced cSCC cell death is boosted by TXNDC9 deficiency. Moreover, cSCC cells lacking TXNDC9 displayed attenuated activation of the NF-κB pathway. Additional studies by inhibiting TXNDC9 confirmed this finding, as TXNDC9 deficiency attenuated UV-B radiation-induced translocation of NF-κB p65 from the cytoplasm to the nucleus of cSCC. In conclusion, our work demonstrates the biological roles of TXNDC9 in cSCC progression and may provide a novel therapeutic target to treat cSCC in the future.

Biallelic TUFT1 variants cause woolly hair, superficial skin fragility and desmosomal defects.

Desmosomes are complex cell junction structures that connect intermediate filaments providing strong cell-to-cell adhesion in tissues exposed to mechanical stress. To identify causal variants in individuals with woolly hair and skin fragility of unknown genetic cause. This research was conducted using whole-genome sequencing, whole-exome sequencing, clinical phenotyping, haplotype analysis, single-cell RNA sequencing data analysis, immunofluorescence microscopy and transmission electron microscopy. We identified homozygous predicted loss-of-function tuftelin-1 (TUFT1) variants in nine individuals, from three families, with woolly hair and skin fragility. One donor splice-site variant, c.60+1G>A, was present in two families, while a frameshift variant, p.Gln189Asnfs*49, was found in the third family. Haplotype analysis showed the c.60+1G>A substitution to be a founder variant in the Irish population that likely arose approximately 20 generations ago. Human and mouse single-cell RNA sequencing data showed TUFT1 expression to be enriched in the hair dermal sheath and keratinocytes. TUFT1 expression was highly correlated with genes encoding desmosomal components implicated in diseases with phenotypes that overlap with the cohort presented here. Immunofluorescence showed tuftelin-1 to be mainly localized to the peripheral cell membranes of keratinocytes in normal skin. Skin samples from individuals with TUFT1 variants showed markedly reduced immunoreactivity for tuftelin-1, with a loss of the keratinocyte cell membrane labelling. Light microscopy revealed keratinocyte adhesion, mild hyperkeratosis and areas of superficial peeling. Transmission electron microscopy showed panepidermal acantholysis with widening of intercellular spaces throughout the epidermis and desmosomal detachment through the inner plaques. Biallelic loss-of-function TUFT1 variants cause a new autosomal recessive skin/hair disorder characterized by woolly hair texture and early-onset skin fragility. Tuftelin-1 has a role in desmosomal integrity and function.

New Polyesterified Ursane Derivatives from Leaves of Maesa membranacea and Their Cytotoxic Activity.

Maesa membranacea A. DC. (Primulaceae) is a plant species that has been frequently used by practitioners of the traditional ethnobotany knowledge from northern and central Vietnam. However, the chemical constituents of the plant remained unknown until recently. Chromatographic separation of a chloroform-soluble fraction of extract from leaves of M. membranacea led to the isolation of two new polyesterified ursane triterpenes (1–2) and two known apocarotenoids: (+)-dehydrovomifoliol (3) and (+)-vomifoliol (4). The chemical structures of the undescribed triterpenoids were elucidated using 1D and 2D MNR and HRESIMS spectral data as 2α,6β,22α-triacetoxy-11α-(2-methylbutyryloxy)-urs-12-ene-3α,20β-diol (1) and 2α,6β,22α-triacetoxy-urs-12-ene-3α,11α,20β-triol (2). The newly isolated triterpenoids were tested for their cytotoxic activity in vitro against two melanoma cell lines (HTB140 and A375), normal skin keratinocytes (HaCaT), two colon cancer cell lines (HT29 and Caco-2), two prostate cancer cell lines (DU145 and PC3) and normal prostate epithelial cells (PNT-2). Doxorubicin was used as a reference cytostatic drug. The 2α,6β,22α-triacetoxy-11α-(2-methylbutyryloxy)-urs-12-ene-3α,20β-diol demonstrated cytotoxic activity against prostate cancer cell lines (Du145—IC50 = 35.8 µg/mL, PC3—IC50 = 41.6 µg/mL), and at a concentration of 100 µg/mL reduced viability of normal prostate epithelium (PNT-2) cells by 41%.

Anti-Malignant Effect of Tensile Loading to Adherens Junctions in Cutaneous Squamous Cell Carcinoma Cells.

Actomyosin contractility regulates various cellular processes including proliferation and differentiation while dysregulation of actomyosin activity contributes to cancer development and progression. Previously, we have reported that actomyosin-generated tension at adherens junctions is required for cell density-dependent inhibition of proliferation of normal skin keratinocytes. However, it remains unclear how actomyosin contractility affects the hyperproliferation ability of cutaneous squamous cell carcinoma (cSCC) cells. In this study, we find that actomyosin activity is impaired in cSCC cells both in vitro and in vivo. External application of tensile loads to adherens junctions by sustained mechanical stretch attenuates the proliferation of cSCC cells, which depends on intact adherens junctions. Forced activation of actomyosin of cSCC cells also inhibits their proliferation in a cell-cell contact-dependent manner. Furthermore, the cell cycle arrest induced by tensile loading to adherens junctions is accompanied by epidermal differentiation in cSCC cells. Our results show that the degree of malignant properties of cSCC cells can be reduced by applying tensile loads to adherens junctions, which implies that the mechanical status of adherens junctions may serve as a novel therapeutic target for cSCC.

Development of simultaneous quantitative analysis of tricarboxylic acid cycle metabolites to identify specific metabolites in cancer cells by targeted metabolomic approach

The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.

Immunohistological Analysis of CD34-Positive Dermal Dendritic Cells and Microvessel Density in the Genital and Extragenital Lichen Sclerosus

BackgroundLichen sclerosus (LiS) is a chronic scleroatrophic condition that usually affects the anogenital area and occasionally the extragenital sites. CD34-positive dermal dendritic cells (DDCs) contribute to the maintenance of the dermal microarchitecture and modulation of the immune response. p53 is a tumor suppressor gene important for the regulation of the cell cycle and apoptosis. Similar to morphea (a LiS-closely related scleroatrophic condition), dermal sclerosis, alterations of DDCs, and dermal microvasculature may be important underlying pathogenetic mechanisms in LiS. ObjectivesTo examine the profile of CD34-positive DDCs, microvessel density (MVD), and p53 protein in LiS. Materials and methodsThe immunohistological profiles of DDCs, MVD, and p53 were examined in 19 cases of LiS and their age- and sex-matched normal skin (10 specimens), using antibodies against CD34 and p53. ResultsThere was a markedly decreased counts (1.7 ± 0.5/mm2) or complete loss of CD34-positive DDCs in LiS against their abundance in the normal skin (23.4 ± 2.1/mm2, p = 0.000). MVD was markedly increased in LiS lesions (20 ± 0.47) as compared to normal skin (5.50 ± 0.20, p = 0.000). Discontinuous single-cell p53 weakly positive nuclear staining was seen in the epidermal basal cell keratinocytes in normal skin and LiS lesions. ConclusionsTo the best of this author's knowledge, this is the first study analyzing DDCs, MVD, and p53 profiles together in LiS. The findings suggest that alterations of DDCs and MVD have roles in the pathogenesis of LiS.

Análisis inmunohistológico de las células dendríticas dérmicas positivas para CD34 y de la densidad de microvasos en el liquen escleroso genital y extragenital

BackgroundLichen sclerosus (LiS) is a chronic scleroatrophic condition that usually affects the anogenital area and occasionally the extragenital sites. CD34-positive dermal dendritic cells (DDCs) contribute to the maintenance of the dermal microarchitecture and modulation of the immune response. p53 is a tumor suppressor gene important for the regulation of the cell cycle and apoptosis. Similar to morphea (a LiS-closely related scleroatrophic condition), dermal sclerosis, alterations of DDCs, and dermal microvasculature may be important underlying pathogenetic mechanisms in LiS. ObjectivesTo examine the profile of CD34-positive DDCs, microvessel density (MVD), and p53 protein in LiS. Materials and methodsThe immunohistological profiles of DDCs, MVD, and p53 were examined in 19 cases of LiS and their age- and sex-matched normal skin (10 specimens), using antibodies against CD34 and p53. ResultsThere was a markedly decreased counts (1.7±0.5/mm2) or complete loss of CD34-positive DDCs in LiS against their abundance in the normal skin (23.4±2.1/mm2, p=0.000). MVD was markedly increased in LiS lesions (20±0.47) as compared to normal skin (5.50±0.20, p=0.000). Discontinuous single-cell p53 weakly positive nuclear staining was seen in the epidermal basal cell keratinocytes in normal skin and LiS lesions. ConclusionsTo the best of this author's knowledge, this is the first study analyzing DDCs, MVD, and p53 profiles together in LiS. The findings suggest that alterations of DDCs and MVD have roles in the pathogenesis of LiS.

Effects of Regulating Smad7 Gene on Epithelial-Mesenchymal Transition in Keloid Keratinocyte

The effect of Smad7 on epithelial-Mesenchymal Transition (EMT) of keloid keratinocytes was studied. Culture formed keloid cutin cells (KK) and normal skin cutin cell (NK cells), built the Smad7 too slow virus slow virus vector and Smad7 interference expression vector, screening the best expression and interfering with the slow virus infection NK and KK cells respectively, and contrast carrier puro screening stable expression cell lines, stem cells can be divided into 8 groups: NK-Control (normal training of NK cells); NK-NC (NK cells screened against lentivirus); NK-shSmad7 (NK cells that interfere with lentivirus screening); NK-mSmad7 (NK cells screened for overexpression of lentivirus); KK-control (normal cultured KK cells); KK-NC (KK cells screened against lentivirus); KK-shSmad7 (KK cells that interfere with lentivirus screening); KK-mSmad7 (KK cells screened for overexpression of lentivirus). Cell proliferation was observed by the CCK-8 method, cell apoptosis was detected by flow cytometry, cell migration ability was detected by Transwell chamber, and expression of key proteins (N-cadherin and Occludin) in epithelium-interstitial transform was detected by Western blot. The Smad7 interfering lentivirus vector and Smad7 overexpressing lentivirus vector were successfully constructed. Interference with Smad7 can promote NK cell and KK cell proliferation and migration, and inhibit KK cell apoptosis, but it has no significant effect on NK cell apoptosis ( P>0.05). Overexpression of Smad7 inhibited the proliferation and migration of NK cells and KK cells, and promoted their apoptosis. After interfering with lentivirus infection, NK cells and KK cells showed decreased expression of Occludin protein compared with NC group ( P<0.01), increased N-cadherin protein expression in KK cells ( P<0.01), but there was no significant change in N-cadherin protein expression in NK cells ( P>0.05); After lentivirus overexpression, NK and KK cells showed increased expression of Occludin protein ( P<0.05), the expression of N-cadherin protein in NK cells decreased ( P<0.05), but there was no significant change in N-cadherin protein expression in KK cells ( P>0.05). The regulation of Smad7 gene can affect the EMT in normal skin keratinocytes and keloid keratinocytes, and further regulate the ability of cell proliferation, migration and apoptosis. The effect of Smad7 gene regulation on EMT in keloid keratinocytes was greater than that on normal skin keratinocytes.

Challenge of reflectance confocal microscopy in the diagnosis of genital warts.

Reflection confocal microscopy (RCM) is commonly used to assist in the diagnosis of skin diseases. RCM has been found to be useful in the diagnosis of genital warts. The characteristics of genital warts under RCM are not specific. This study aimed to face the challenge of diagnosing genital warts with RCM. We retrospectively studied 161 patients with clinical diagnosis of genital warts who underwent RCM examination. Histopathological examination was performed for 75 of these patients. The results of histological diagnosis and RCM diagnosis were compared and analyzed. According to the RCM images, 102 of the 161 (63.35%) patients were diagnosed as genital warts. By comparison with histological results, the misdiagnosis rate of the 75 biopsy cases was 35.48% and the missed diagnosis rate was 20.45%.The diseases of misdiagnosis and missed diagnosis mainly included bowenoid papulosis, pseudocondylona of vulvae, squamous cell carcinoma in situ, molluscum contagiosum, and lichen planus. We further found that the RCM characteristics of genital warts conformed by histology were koilocytes, which mainly appeared in the granular layer or the spinous layer, larger than normal skin keratinocytes, and the cytoplasm with low refractive index. Due to the commonality of the microscopic characteristics of genital warts and related diseases, some cases are easy to be misdiagnosed or missed diagnosed. Diagnostic koilocytes could be an important basis for the diagnosis of genital warts.

Targeting 14-3-3ε activates apoptotic signaling to prevent cutaneous squamous cell carcinoma.

More than a million cases of cutaneous squamous cell carcinoma are diagnosed in the USA each year, and its incidence is increasing. Most of these malignancies arise from premalignant lesions, providing an opportunity for intervention before malignant progression. We previously documented how cytoplasmic mislocalization of CDC25A in premalignant and malignant skin cancers confers resistance to apoptotic cell death via a mechanism that depends on its interaction with 14-3-3ε. From these data, we hypothesized that 14-3-3ε overexpression drives skin tumor development and progression, such that targeting 14-3-3ε may be a useful strategy for skin cancer treatment. Like CDC25A, 14-3-3ε was overexpressed and mislocalized to the cytoplasm of both benign and malignant human skin cancer. Skin-targeted deletion of the 14-3-3ε gene reduced skin tumor development by 75% and blocked malignant progression. 14-3-3ε suppressed apoptosis through activation of Akt, leading to inhibition of BCL2 associated agonist of cell death and upregulation of Survivin. Using virtual tetrapeptide libraries, we developed a novel peptide that specifically blocked 14-3-3ε heterodimerization and thereby prevented its interaction with CDC25A. The peptide reduced prosurvival signaling, killed skin cancer cells and reduced skin tumor growth in xenograft. Normal skin keratinocytes were unaffected by inhibition or deletion of 14-3-3ε. Thus, targeting of 14-3-3ε dimerization is a promising strategy for the treatment of premalignant skin lesions.

Endoplasmic reticulum stress links psoriasis vulgaris with keratinocyte inflammation.

IntroductionEndoplasmic reticulum stress (ERS) has been implicated in the pathogenesis of various inflammatory diseases. However, the role of ERS in psoriasis is still unclear.AimTo examine ERS in psoriasis keratinocytes and to assess the association of ERS with skin inflammation response.Material and methodsWe investigated ERS in keratinocytes of normal skin, lesional and perilesional psoriasis vulgaris (PV) skin tissues using transmission electron microscope (TEM) examination, Western blot and immunostaining analysis.ResultsBy TEM examination, we found that endoplasmic reticulum (ER) in psoriatic keratinocytes was ultrastructurally abnormal, with changes in ER morphology and the ER expansion. Using Western blot and immunostaining analysis, we showed that the expression of ERS-associated proteins, such as BiP, CHOP and XBP1, was enhanced in PV epidermis compared to the healthy skin. Moreover, abundant TNF-α protein was correlated to the increased BiP, CHOP and XBP1 expression in PV epidermis.ConclusionsOur findings demonstrate that PV keratinocytes have an increased ERS, which may contribute to the pathogenesis of PV.