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Previously, we reported a collagenase-based, animal product-free protocol for cultivated oral mucosal epithelial cell sheets for transplantation (COMET). Here, we reported the long-term outcomes of first 2 clinical cases. A 27-year-old man suffered from thermal burn, which resulted in symblepharon of lower fornix OD. COMET was performed, and the cornea remained clear with few peripheral NV and no more symblepharon 34 months postoperatively. Another 42-year-old man suffered from severe alkaline burn OD. He underwent COMET, followed by corneal transplantation half a year later. A biopsy taken two years after COMET showed stratified epithelium positive for keratin 4, 13, and 3 in the suprabasal layer. Staining for p63 and p75NTR was both positive in the basal layer. The graft remained clear up to post-OP 4 years. Our study confirmed the long-term survival of the transplanted OMECs, suggesting that collagenase-based spheroidal suspension culture is a promising technique for COMET.Trial registration ClinicalTrials.gov, ClinicalTrials.gov ID: NCT03943797 Registered 9 May 2019-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03943797.

White sponge nevus (WSN) is a rare autosomal dominant disorder, which is characterized by soft white mucosal plaques in the oral cavity and other regions. The white plaques exhibit surface irregularities, involving a wrinkly or spongy pattern. Its histopathological findings included a thick stratified squamous epithelium, which displays parakeratosis; cytoplasmic clarification and vacuolization in the stratum spinous layer cell, which were caused by swelling of the cytoplasm; and nuclear condensation. The widely accepted theory for the cause of the onset of WSN is mutation of the keratin 4 and/or 13 genes. We present a case of WSN, in which the patient, a Japanese female, exhibited white plaques on her bilateral buccal mucosae. A 56-year-old female visited our hospital, and her chief complaint was a white lesion on the buccal mucosa. White lesions were observed on her bilateral buccal mucosae. The lesions were widespread and spongy and had irregular surfaces. Tissue samples were obtained during biopsies performed at our hospital. The lesions’ histological findings included epithelial hyperparakeratosis with vacuolated cytoplasm in the spinous layer, but there was no evidence of epithelial dysplasia. In addition, spinous layer cells were nuclear condensation and perinuclear acidification. The final histological diagnosis was WSN; thus, we decided to follow-up the patient’s progress. Recently, a few studies have reported on the malignant transformation of WSN. Therefore, it is necessary to monitor patients with WSN regularly, and to investigate more cases of WSN to elucidate the cause of such transformation.

Background Cashmere goat is known for its precious undercoat. Being photoperiod-dictated, cashmere growth has been studied focusing mainly on hair follicle cycle phases (anagen, catagen and telogen). An accurate molecular knowledge of the goat hair follicle cycle, disentangling gene expression changes during phases and recognizing timing boundaries, could be useful to improve cashmere goat management and ultimately cashmere production. Results To better describe goat’s hair follicle transcriptome we applied RNA-sequencing to isolated hair follicles from five Italian cashmere goats, during the anagen and catagen phase, identifying total of 214 differentially expressed genes (DEGs): 97 were up-regulated while 117 were down-regulated in catagen with respect to anagen. Gene Ontology and pathway analysis were performed. We detected 144 significant pathways spanning from estrogen, pluripotency of stem cells, thermogenesis and fatty acid metabolism that were strongly expressed during the hair follicle phases analysed. Finally, we validated promising DEGs by RT-qPCR in the same set of samples as well as in hair follicles and entire skin biopsies of another cashmere goats cohort accounting for early anagen, anagen, early catagen, and catagen phases. Conclusions As in the isolated hair follicles, some target genes were homogenously modulated during the four hair follicle phases. Ceruloplasmin (CP) and Keratin 4 (K4), confirmed their clear cut expression between growing and resting phase. In fact, K4 was almost absent in catagen phases while CP was barely expressed in anagen phases. In particular, the strong expression of K4 in early anagen makes it an eligible marker to track the beginning of a new hair cycle, and therefore defining the optimum time for cashmere harvesting.

Background Oral white sponge nevus (WSN) is a rare autosomal dominant benign condition, characterized by asymptomatic spongy white plaques. Mutations in Keratin 4 (KRT4) and 13 (KRT13) have been shown to cause WSN. Familial cases are uncommon due to irregular penetrance. Thus, the aim of the study was: a) to demonstrate the clinical and histopathological features of a three-generation Turkish family with oral WSN b) to determine whether KRT4 or KRT13 gene mutation was the molecular basis of WSN. Material and Methods Out of twenty members of the family ten were available for assessment. Venous blood samples from six affected and five unaffected members and 48 healthy controls were obtained for genetic mutational analysis. Polymerase chain reaction was used to amplify all exons within KRT4 and KRT13 genes. These products were sequenced and the data was examined for mutations and polymorphisms. Results Varying presentation and severity of clinical features were observed. Analysis of the KRT13 gene revealed the sequence variant Y118D as the disease-causing mutation. One patient revealed several previously unreported polymorphisms including a novel mutation in exon 1 of the KRT13 gene and a heterozygous deletion in exon 1 of KRT4. This deletion in the KRT4 gene was found to be a common polymorphism reflecting a high allele frequency of 31.25% in the Turkish population. Conclusions Oral WSN may manifest variable clinical features. The novel mutation found in the KRT13 gene is believed to add evidence for a mutational hotspot in the mucosal keratins. Molecular genetic analysis is required to establish correct diagnosis and appropriate genetic consultation. Key words:White sponge nevus, leukokeratosis, oral mucosa, keratins, mutation.

Background White sponge nevus is a rare autosomal dominant disorder that affects the non-keratinised stratified squamous epithelium. Mutations in the genes that encode mucosa-specific keratins 4 and 13 are strongly linked to the manifestation of white sponge nevus. This study involved mutational analysis of the genes encoding keratin-4 and keratin-13 in two Swedish families with white sponge nevus. Methods The diagnosis of white sponge nevus was based on disease history, clinical characteristics of the lesions and, in the majority of the cases, histopathological examination. Samples were collected from the affected buccal mucosa using buccal swabs. DNA was subsequently extracted and amplified using Touch-Down-PCR. The keratin-4 and -13 genes were sequenced and a genetic analysis was performed. Results A novel heterozygous missense mutation was identified in exon 1A of the keratin-4 gene in Family 2. In addition, previously reported heterozygous missense mutations were identified in the keratin-4 (E449K, A72V, Q156R, R208H) and keratin-13 (L115P) genes in both families. Conclusion We describe a novel heterozygous missense mutation in the keratin-4 gene of a Swedish family with white sponge nevus. Our results support the notion that mutations in keratin-4 and -13 are the underlying cause of white sponge nevus. This article is protected by copyright. All rights reserved.

// Tomohiro Enokida 1, 2 , Satoshi Fujii 3 , Mari Takahashi 4 , Youichi Higuchi 3 , Shogo Nomura 5 , Tetsuro Wakasugi 1 , Tomoko Yamazaki 1 , Ryuichi Hayashi 6 , Atsushi Ohtsu 2, 7 and Makoto Tahara 1 1 Department of Head and Neck Medical Oncology, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan 2 Advanced Clinical Research of Cancer, Juntendo University Graduate School of Medicine, Bunkyo-Ku, Tokyo 113-8421, Japan 3 Division of Pathology, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Kashiwa, Chiba 277-8577, Japan 4 Department of Digestive Endoscopy, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan 5 Biostatistics Division, Center for Research Administration and Support, National Cancer Center, Kashiwa, Chiba 277-8577, Japan 6 Head and Neck Surgery Division, National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan 7 National Cancer Center Hospital East, Kashiwa, Chiba 277-8577, Japan Correspondence to: Makoto Tahara, email: matahara@east.ncc.go.jp Satoshi Fujii, email: sfujii@east.ncc.go.jp Keywords: tongue squamous cell carcinoma, oral squamous cell carcinoma, gene expression profile, keratin 4, cytokeratin 4 Received: January 10, 2017 Accepted: May 23, 2017 Published: June 27, 2017 ABSTRACT Objectives: To establish a prognostic signature for locally advanced tongue squamous cell carcinoma (TSCC) patients treated with surgery. Results: In the discovery study, unsupervised hierarchical clustering analysis identified two clusters which differentiated the Kaplan-Meier curves of RFS [median RFS, 111 days vs. not reached; log-rank test, P = 0.023]. The 30 genes identified were combined into a dichotomous PI. In the validation cohort, classification according to the PI was associated with RFS [median RFS, 754 days vs. not reached; log-rank test, P = 0.026 in GSE31056] and DSS [median DSS, 540 days vs. not reached; log-rank test, P = 0.046 in GSE42743 and 443 days vs. not reached; P < 0.001 in GSE41613]. Among genes, positive immunohistochemical staining of cytokeratin 4 was associated with favorable prognostic values for RFS (hazard ratio (HR), 0.591, P = 0.045) and DSS (HR, 0.333, P = 0.004). Materials and methods: We conducted gene expression profiling of 26 clinicopathologically homogeneous advanced TSCC tissue samples using cDNA microarray as a discovery study. Candidate genes were screened using clustering analysis and univariate Cox regression analysis for relapse-free survival (RFS). These were combined into a prognostic index (PI), which was validated using three public microarray datasets of tongue and oral cancer (123 patients). Some genes identified in discovery were immunohistochemically examined for protein expression in another 127 TSCC patients. Conclusion: We identified robust molecular markers that showed significant associations with prognosis in TSCC patients. Gene expression profiling data were successfully converted to protein expression profiling data.

Herein, we present a sporadic case of white sponge nevus (WSN) on the cheek mucosa in a 27-year-old Japanese man. A definite diagnosis of WSN was obtained using the typical clinical appearance and microscopic features of the lesions. Histopathological and cytological studies should be conducted to differentiate this condition from other oral premalignant white lesions. A molecular mutation analysis revealed a missense mutation in exon 1A of the keratin 4 gene, which is correlated with the development of WSN. Hence, genetic analysis must be performed to obtain an accurate diagnosis in sporadic cases. The temporary regression of the lesion after treatment with oral amoxicillin hydrate (AMPC) 750 mg/day indicates that antibiotics are effective in symptomatic cases.

BACKGROUND The aim of this study was to investigate the roles of keratin 4 (KRT4) gene in the development of human white sponge nevus (WSN). METHODS Transgenic mice were created using the microinjection method with pcDNA3.1 vectors expressing KRT4 wild-type (WT) gene and E520K mutation. Polymerase chain reaction (PCR) and Western blotting were used to identify the genotype of transgenic founders and their filial generations. Expression of KRT4 in mouse oral mucosa was characterized by immunohistochemistry (IHC), and the whole epithelium layer of transgenic mice was observed using transmission electron microscope (TEM). RESULTS The positive rate of KRT4 transgenic mice in F1 generation was 45.5%. Expression level of KRT4 protein was significantly higher in 2-month-old transgenic mice than WT mice. Furthermore, all the epithelial lamina of 3-month-old transgenic mice showed reduced staining of KRT4. The surface and spinous layers were full of hyalocytes and bubble cells, which are similar to the clinical symptoms of WSN. For the ultrastructure, both tonofilaments and Odland bodies increased. CONCLUSIONS Our study indicated the mutated KRT4 gene may play important roles in the pathogenesis of WSN.

Keratins are the major structural fibrous proteins of hair, feathers, wool, and nail. Because keratin is protein based, cheap, and biocompatible, it has found applications from tissue engineering to textile industry. Simultaneous UV-reactive electrospinning technique is used to fabricate nanofiber scaffolds with 4-vinyl benzene boronic acid–hydroxyapatite/poly(vinyl alcohol) composite containing different amounts of keratin. Human hair as keratin supports the scaffolds for cell culture applications in our study. Our aim was to obtain nanofiber scaffolds which were designed to be nontoxic. The structure and the morphology of electrospun membranes were investigated by scanning electron microscopy and Fourier transform infrared spectroscopy technique. For the cell culture applications, endothelium (ECV 304) and sarcoma osteogenic (SAOS) cells were seeded on the electrospun fibrous scaffolds. Nanofiber scaffolds were found to have an average diameter of 350 ± 20 nm. These scaffolds provided a medium for cells to grow. POLYM. COMPOS., 2015. © 2015 Society of Plastics Engineers

The molecular and cellular etiology of eosinophilic esophagitis (EoE), an emerging tissue-specific allergic disease, involves dysregulated gene expression in esophageal epithelial cells. Herein, we assessed the esophageal expression of IL-33, an epithelium-derived alarmin cytokine, in patients with EoE. IL-33 protein was markedly overexpressed within the nuclei of a subpopulation of basal layer esophageal epithelial cells in patients with active EoE compared to control individuals. IL-33 exhibited dynamic expression as levels normalized upon EoE remission. IL-33–positive basal epithelial cells expressed E-cadherin and the undifferentiated epithelial cell markers keratin 5 and 14 but not the differentiation marker keratin 4. Moreover, the IL-33–positive epithelial cells expressed the epithelial progenitor markers p75 and p63 and lacked the proliferation markers Ki67 and phospho-histone H3. Additionally, the IL-33–positive cells had low expression of PCNA. IL-33 expression was detected in ex vivo–cultured primary esophageal epithelial cells in a subpopulation of cells lacking expression of proliferation markers. Collectively, we report that IL-33 expression is induced in an undifferentiated, non-dividing esophageal epithelial cell population in patients with active EoE.

White sponge nevus (WSN) is an autosomal dominant hereditary disease. Keratin 4 (KRT4) and Keratin 13 (KRT13) gene mutations were involved in the WSN. We recruited two WSN Chinese families, and oral lesion biopsy with hematoxylin and eosin staining showed that patients had significant pathological characteristics. The mutations of KRT4 and KRT13 gene were detected by PCR and direct sequencing. The multiple alignments of KRT13 from 23 diverse species homology analyses were performed by the ClustalW program. The KRT13 expression was measured by Real-Time RT-PCR and Western blot analysis. Sequencing analysis revealed two mutations of KRT13 gene: one mutation was 332T>C and amino acid change was Leu111Pro. Another mutation was 340C>T and amino acid change was Arg114Cys. The sequence of KRT13 was highly conserved. Real-Time RT-PCR and Western blot analysis results show that KRT13 expression level is lower in patient but keep almost no change in mRNA level. When cells were treated with MG132, KRT13 protein level was increased and kept almost the same in normal and patient cells. We identified two heritable mutations in the KRT13 gene, which were associated with the development of WSN. The abnormal degradation of KRT13 protein of WSN may probably associate with the abnormal ubiquitination process.

We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33°C or an intermediate temperature of 37°C. At the nonpermissive temperature of 39°C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochemical analyses revealed that ROE2 cells at 37°C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive microarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells.

Objective To explore if climbazole enhances retinoid-associated biological activities in vitro and in vivo. Methods Primary human dermal fibroblasts (HDFs) were treated from six to 48 hours with either retinoids (retinol, retinyl propionate, retinyl palmitate) alone or in combination with climbazole, then assessed for cellular retinoic acid-binding protein 2 (CRABP2) mRNA expression by RT-qPCR. Next, skin equivalent (SE) cultures were topically treated with a retinol or retinyl propionate, and with or without climbazole, then measured for biological changes in retinoid biomarkers. Lastly, an IRB-approved clinical study was conducted on the outer forearm of 16 subjects to ascertain the effects of low (0.02%) or high (0.1%) levels of retinol, retinyl propionate (0.5%), climbazole (0.5%) or a combination of retinol (0.02%) /climbazole (0.5%). Indicators of retinoid activities were measured after 3 weeks. Results Treatment of HDFs with retinol or retinyl propionate were unaffected by climbazole but alone, resulted in a significantly (p

Background A key question in the allergy field is to understand how tissue-specific disease is manifested. Eosinophilic esophagitis (EoE) is an emerging tissue-specific allergic disease with an unclear pathogenesis. Objective Herein we tested the hypothesis that a defect in tissue-specific esophageal genes is an integral part of EoE pathogenesis. Methods We interrogated the pattern of expression of esophagus-specific signature genes derived from the Human Protein Atlas in the EoE transcriptome and in EPC2 esophageal epithelial cells. Western blotting and immunofluorescence were used for evaluating expression of esophageal proteins in biopsy specimens from control subjects and patients with active EoE. Whole-exome sequencing was performed to identify mutations in esophagus-specific genes. Results We found that approximately 39% of the esophagus-specific transcripts were altered in patients with EoE, with approximately 90% being downregulated. The majority of transcriptional changes observed in esophagus-specific genes were reproduced in vitro in esophageal epithelial cells differentiated in the presence of IL-13. Functional enrichment analysis revealed keratinization and differentiation as the most affected biological processes and identified IL-1 cytokines and serine peptidase inhibitors as the most dysregulated esophagus-specific protein families in patients with EoE. Accordingly, biopsy specimens from patients with EoE evidenced a profound loss of tissue differentiation, decreased expression of keratin 4 (KRT4) and cornulin (CRNN) , and increased expression of KRT5 and KRT14 . Whole-exome sequencing of 33 unrelated patients with EoE revealed 39 rare mutations in 18 esophagus-specific differentially expressed genes. Conclusions A tissue-centered analysis has revealed a profound loss of esophageal tissue differentiation (identity) as an integral and specific part of the pathophysiology of EoE and implicated protease- and IL-1–related activities as putative central pathways in disease pathogenesis.

The structure and characterization of the oligopeptide crystals formed from the feather keratin solution obtained by superheated water treatment are reported. The FTIR spectra and 1H and 13C solid-state NMR results indicate that the peptides, arranged mostly in a beta-sheet structure in feather, reorganize into a mainly alpha-helix and less beta-sheet mixed secondary structure, when self-assemble from the solution at room temperature. MALDI-ToF-ToF spectra confirm that the most primary sequence with the mass 1884 come from the feather keratin 4, KRFA_CHICK of Gallus gallus. The largely preservation of all but cystine amino acid species and the increase of hydrophobic amino acids content in the oligopeptide crystals are proved by the amino acid analysis.

Serine-rich repeat protein 1 (Srr-1) is a surface protein from Streptococcus agalactiae. A 17 kDa region of this protein has been identified to bind to human keratin 4 (K4) and is termed the Srr-1 K4-binding domain (Srr-1-K4BD). Recombinant Srr-1-K4BD was overexpressed in Escherichia coli BL21 (DE3) cells. Native and selenomethionine-substituted proteins were prepared using Luria–Bertani (LB) and M9 minimal media, respectively. A two-step purification protocol was carried out to obtain a final homogenous sample of Srr-1-K4BD. Crystals of native Srr-1-K4BD were obtained using PEG 3350 as a precipitant. The crystals diffracted to 3.8 Å resolution using synchrotron radiation and belonged to space group P21, with unit-cell parameters a = 47.56, b = 59.48, c = 94.71 Å, β = 93.95°.

Disorders of keratinization are often treated with vitamin A derivatives (retinoids) which affect keratinocyte differentiation, including keratin (KRT) gene expression. In vivo, suprabasal keratinocytes normally express only keratin (K) 1, K2 and K10, but after topical application of all-trans retinoic acid (ATRA), the granular cells will additionally express K4 and K13, i.e. keratins normally present in oral mucosa and in cultured epidermal keratinocytes. To learn more about the retinoid regulation of keratin expression under in vivo-like conditions, we cultured keratinocytes on de-epidermized dermis in only 0.5% serum. These cells produce a normal-looking epidermis that expresses high mRNA levels of KRT1, KRT2 and KRT10, but minimal amounts of KRT4 and KRT13. Addition of ATRA to the medium for 48 h caused a dose-dependent increase in KRT4/KRT13 and a down-regulation of KRT2 mRNA. An increase in K4 protein was also found. The response was greater than the up-regulation of another retinoid-regulated gene, CRABPII. By studying 10 retinoids with different affinities for the retinoic acid receptors (RAR) and retinoid X receptors (RXR) isoforms, the reciprocal expression of KRT2 and KRT4/KRT13 could be connected with agonists for RARalpha. Two of these agonists, CD336/Am580 and CD2081, altered the expression profile with similar potency as the pan-RAR agonists ATRA and CD367. Co-addition of a pan-RAR antagonist (CD3106/AGN193109) markedly inhibited the induction of KRT4/KRT13 expression, whereas the down-regulation of KRT2 was less affected. In conclusion, RARalpha agonists elicit a reciprocal modulation of KRT2 and KRT4/KRT13 expression in human epidermis, but whether or not the keratin genes also possess RARalpha-specific regulatory elements is still unclear.

White sponge nevus (WSN) in the oral mucosa is a rare autosomal dominant genetic disease. The involved mucosa is white or greyish, thickened, folded and spongy. The genes associated with WSN include mutant cytokeratin keratin 4 (KRT4) and keratin 13 (KRT13). In recent years, new cases of WSN and associated mutations have been reported. Here, we summarise the recent progress in our understanding of WSN, including clinical reports, genetics, animal models, treatment, pathogenic mechanisms and future directions. Gene-based diagnosis and gene therapy for WSN may become available in the near future and could provide a reference and instruction for treating other KRT-associated diseases.

To identify differentially expressed genes during the development of oral malignancy, differential display, northern blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses were undertaken using oral squamous cell carcinoma (OSCC) and leukoplakia tissues. Significantly higher levels of keratin (Ker)-14 and -17 mRNAs, combined with lower levels of Ker-4, Ker-13 and transglutaminase 3 (TG-3) transcripts, were observed in OSCC and severely dysplastic tissues, whereas this expression profile was reversed in hyperplasia and in mild to moderate dysplasia. The expression of Ker-4 and Ker-13 was elevated in density-arrested OSCC cell lines (Ca9-22, HSC-2, -3 and -4) but the expression of Ker-17 mRNA was elevated in these cells, regardless of the growth conditions. In addition, Ker-4 and Ker-13 proteins were predominantly expressed in moderate dysplasia and hyperplasia, whereas Ker-17 was markedly expressed in OSCC tissues. The expression patterns of these genes could therefore be an important determinant of the manifestation of oral malignancy.

Objectives. Microarray expression analysis of cervical tumors has revealed differential expression of genes that may be useful as markers or targets for treatment. We question the application of array findings across the major categories of cervical cancer. We sought to identify differences between normal squamous epithelium (NSQ) and glandular epithelium (NGL) of the uterine cervix and their malignant variants: squamous cell cancer (SCC) and adenocarcinoma (ACA). Methods. Eight genes were selected: 12-lipoxygenase (12-LOX), keratin 4, trypsinogen 2 (TRY2), Rh glycoprotein C (RhGC), collagen type V alpha 2, integrin alpha 5, integrin alpha 6, and c- myc . Ten cases each of SCC and ACA of the cervix were selected from our tumor bank. NSQ and NGL epithelia were obtained from consecutive patients undergoing surgery for benign disease. RNA extraction, cDNA synthesis, and DNA amplification of all samples were performed according to an established protocol. Electrophoresis of the multiplexed polymerase chain reaction (PCR) products was performed under standard conditions, followed by digital image capture. A ratio of target to control gene (β-actin) was obtained for each sample. Analysis of variance was applied to the mean ratios for each tissue to establish significant differences. Individual pairwise comparisons were made by Student t tests and verified with the Tukey-Kramer test. Results. Clinically valid comparisons are NSQ to NGL, NSQ to SCC, NGL to ACA, and SCC to ACA. Various expression patterns were observed between the epithelia and their malignant phenotypes. Significant differences in gene expression were observed between benign squamous and glandular epithelium in four of the eight genes and between malignant squamous and glandular epithelium in three of the eight genes. Significant differences in gene expression between benign and malignant tissues were demonstrated in four of the eight genes. Conclusions. We have defined significant differential expression changes between the two principal cervical tumor types. Differences in genes are demonstrated and must be considered if array technology is applied to the study of the biologic behavior of these tumors as well as their screening and management. The observed differential expression should be a compelling argument to perform type-specific expression analysis for other tumors with histological variants.

The leucine rich repeats and immunoglobulin-like protein 1 (LRIG1) is a newly discovered negative regulator of epidermal growth factor receptor (EGFR) and a proposed tumor suppressor. It is not universally downregulated in human cancers, and its role in neoplastic transformation and tumorigenesis is not well-documented. In this study, we show the expression of LRIG1 as a novel potential marker for neoplastic transformation in ocular-surface squamous neoplasia (OSSN). The following two groups were included in this study: 1) benign group (3 cases; 1 with papilloma and 2 with dysplasia) and 2) malignant group (3 cases with squamous cell carcinoma (SCC)). In both groups, immunofluorescence analysis was firstly performed for keratins 4, 12, 13, and 15 to characterize the state of differentiation, and for Ki67 to evaluate the proliferation activity. Subsequently, LRIG1 and EGFR expression was analyzed. Either keratin 4 and/or 13, both non-keratinized epithelial cell markers, were generally expressed in both groups, except for 1 severe SCC case. Keratin 15, an undifferentiated basal cell marker, was more strongly expressed in the malignant cases than in the benign cases. The Ki67 index was significantly higher (P

We established two gingival epithelial cell lines (GE1 and GE6), originating from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. GE1 and GE6 grew at a permissive temperature (33 degrees C) in a pavement arrangement and solely formed multilayers that exhibited morphological features similar to those of the stratified oral epithelium, with neither the use of stromal equivalents nor feeder layers. Both GE cells underwent apoptosis at a non-permissive temperature (39 degrees C). Characteristic keratin peptides, keratin 4 and 13, for mucosal epithelium were obviously expressed in the suprabasal cells, and keratohyalin granules and involucrin were present in the surface flat cells in the multilayered culture. Keratin 10 (one of the markers for higher keratinized gingival epithelium) was rarely found in some uppermost cells, and filaggrin (a component of keratohyalin granules) appeared sparsely in uppermost desquamating cells in the older cultures. These observations indicated that GE1 and GE6 cells exhibited the phenotype characterizing nonkeratinized sulcular epithelium, which possessed the potency undergoing keratinization in such highly stratified cultures as oral gingival epithelium. GE cells increased the expression levels of mRNA of interleukin-1beta and tumor necrosis factor alpha by the stimulation of lipopolysaccharide and extracellular substances of oral streptococci. The GE cell lines thus could serve as an excellent experimental system for further studies on the physiology of gingival epithelium and corresponding diseases, such as periodontal disease, epithelial hyperplasia, and gingival tumors.

Zf9/CPBP/KLF6 is a widely expressed member of the Krüppel-like family of transcriptional factors which regulates gene expression in hepatic stellate cells. Because of its ubiquitous expression including in the esophagus, we have explored its function in the esophageal squamous epithelium, a model system to study cellular proliferation and differentiation. Reverse transcription-PCR (RT-PCR) and Western blot analyses revealed that Zf9 was highly expressed in human esophageal squamous cancer cell lines. Additionally, Zf9 localizes to the esophageal squamous epithelium by immunohistochemistry. Using transient transfection, Zf9 transactivates the human keratin 4 (K4) promoter reporter gene construct in a subset of the esophageal cancer cell lines through indirect mechanisms. Co-transfection of Zf9 and GKLF/KLF4, which is also a member of the Krüppel-like factors and expressed in the esophageal squamous epithelium, leads to coactivation in an additive fashion. Furthermore, we demonstrate that there is a physical interaction between GKLF and Zf9, a novel finding for Krüppel-like family members.

The ets family of transcription factors comprises many members which contribute to diverse cellular functions that vary depending upon the cell- and tissue-type context. Recently, different groups have identified a novel member of the ets family that is epithelial-specific. Variably called ESE-1, ERT, jen, ESX, this gene is designated currently as ELF3. In order to understand transcriptional regulatory mechanisms mediated by ELF3, we investigated its effect on the human keratin 4 gene promoter based upon the role of keratin 4 in early differentiation of the esophageal squamous epithelium. Interestingly, ELF3 suppressed basal keratin 4 promoter activity in both esophageal and cervical epithelial cancer cell lines, a novel result, while simultaneously activating the late-differentiation linked SPRR2A promoter. Furthermore, serial deletion constructs of the keratin 4 promoter continued to be suppressed by ELF3, a phenomenon that was only partially rescued by ELF3 ets domain mutants, but completely abrogated by deletion of the ELF3 pointed domain. These results suggest that ELF3 may have dual functions in the transcriptional regulation of genes involved in squamous epithelial differentiation. One of these functions may not be exclusively mediated through DNA binding in the context of transcriptional suppression of the keratin 4 promoter.

Retinoids affect keratinocyte differentiation and modulate the expression of many epidermal proteins, among them cellular retinoic acid-binding protein II and the family of cytokeratins. The upregulation of the former protein is a well-known phenomenon, whereas the retinoid-induced regulation of epidermal keratin expression is more complex and only partially understood. We studied the effect of topical retinoids on the expression in healthy skin of cellular retinoic acid-binding protein II, tazarotene-induced genes 1 and 2, several epidermal keratins (K1, K2e, and K10), and two mucous keratins (K4 and K13) known to appear in epidermis under certain abnormal conditions. Reverse transcription–polymerase chain reaction experiments showed that the K4 expression was the one most overtly induced by 2 wk of open treatment with 0.05% of retinoic acid and tazarotene. Using real-time quantitative polymerase chain reaction (TaqMan) and normalization of the mRNA values to β-actin, the increase in K4 was found to be 100–1000-fold. In comparison, the expression of K13 and cellular retinoic acid-binding protein II was increased 10–50-fold, the K1 and K10 mRNA levels remained unchanged, and the K2e level decreased by a factor of 100–1000. In parallel biopsies, immunohistochemistry showed no change in K1, K2e, or K10 staining, but a strong de novo appearance of K4 in the granular layer after retinoid treatment. In a separate study, occlusive application of 0.025% retinoic acid in four healthy subjects produced a maximal K4 mRNA signal after 48 h and strong K4 staining after 80 h. Finally, a dose–response study showed that the de novo appearance of K4 can be utilized as a sensitive test for retinoid bioactivity in epidermis in vivo.

The stratified squamous epithelium comprises actively proliferating basal cells that undergo a program of differentiation accompanied by morphological, biochemical, and genetic changes. The transcriptional regulatory signals and the genes that orchestrate this switch from proliferation to differentiation can be studied through the keratin gene family. Given the localization of keratin 4 (K4) to the early differentiated suprabasal compartment and having previously demonstrated that targeted disruption of this gene in murine embryonic stem cells results in impairment of the normal differentiation program in esophageal and corneal epithelial cells, we studied the transcriptional regulation of the human K4 promoter. A panel of K4 promoter deletions were found in transient transfection assays to be predominantly active in esophageal and corneal cell lines. A critical cis-regulatory element resides between -163 and -140 bp and contains an inverted CACACCT motif. A site-directed mutated version of this motif within the K4 promoter renders it inactive, whereas the wild-type version is active in a heterologous promoter system. It specifically binds esophageal-specific zinc-dependent transcriptional factors. Our studies demonstrate that regulation of the human K4 promoter is in part mediated through tissue-specific transcriptional factors.

Keratins are intermediate filaments of epithelial cells. Mutations in keratin genes expressed in skin lead to human disorders, including epidermolysis bullosa simplex and epidermolytic hyperkeratosis. We examined the role of keratin 4 (K4) in maintaining the integrity of internal epithelial linings by using gene targeting to generate mice containing a null mutation in the epithelial K4 gene. Homozygous mice that do not express K4 develop a spectrum of phenotypes that affect several organs which express K4 including the esophagus, tongue, and cornea. The cellular phenotypes include basal hyperplasia, lack of maturation, hyperkeratosis, atypical nuclei, perinuclear clearing, and cell degeneration. These results are consistent with the notion that K4 is required for internal epithelial cell integrity. As mutations in K4 in humans lead to a disorder called white sponge nevus, the K4-deficient mice may serve as models for white sponge nevus and for understanding the role of K4 in cellular proliferation and differentiation.

The Kruppel-like family of transcription factors comprises genes that appear to have tissue-restricted functions. Expression of gut-enriched Kruppel-like factor (GKLF) may be important in the switch from proliferation to differentiation in the squamous epithelium. We sought to determine transcriptionally mediated effects of GKLF on two promoters active in the esophageal squamous epithelium, namely the Epstein-Barr virus ED-L2 and human keratin 4 promoters. Both promoters contain a CACCC-like motif previously shown to bind GKLF. To determine whether GKLF regulates genes containing this element, we first demonstrated expression and then cloned the full-length human GKLF from an esophageal squamous carcinoma cell line. In a transient transfection system, GKLF increased the activity of both promoters >25-fold, localized to regions containing the CACCC-like element. Recombinant GKLF specifically binds the CACCC-like motif in both promoters. GKLF epitope-tagged protein leads to the formation of two proteins of 65 and 34 kDa. The chromatographically purified 65-kDa protein binds the CACCC-like element from both Epstein-Barr virus ED-L2 and keratin 4 promoters, which is not attenuated by the 34-kDa protein. In summary, GKLF is expressed in esophageal squamous epithelial cells and transcriptionally activates two esophageal epithelial promoters important at the transition toward differentiation.

The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.

Complex nanomicelles were made from casein and keratin through electrostatic self-assembly and transglutaminase fixation that was proved to be harmless and green. The complex nanomicelles were characterized by dynamic light scattering, scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and steady-state florescence. The results show that the complex nanomcelles acquired at the neutral pH in the mass ratio of casein to keratin 4 : 1 exhibit an anomalous sphere shape with uniform size which the diameter is about 40–70 nm. The complex nanomicelles in solution possess excellent dilution and storage stability due to the fixation and their highζ-potential (22.8 mV). The complex nanomicelles are relatively hydrophilic and have a good potential for industrial application.

Lichen planus (LP) is a chronic mucocutaneous disease with unknown cause. Patients with LP often have both oral and genital lesions, but these conditions are often considered as separate diseases and treated accordingly. To find out which genes are differently expressed in mucosal LP compared to normal mucosa and establish whether oral and genital LP are in fact the same disease, whole genome expression analysis was performed on epithelium from 13 patients diagnosed with oral and/or genital LP and normal controls. For confirmation of keratin 4 and corneodesmosin expression, quantitative reverse-transcription PCR and immunohistochemistry were used. Many genes involved in epithelial development and differentiation are differently expressed in epithelium from LP compared to normal epithelium. Several of the differentially expressed genes are common for oral and genital LP and the same biological processes are altered which supports the fact that oral and genital LP are manifestations of the same disease. The change in gene expression indicates that differentiation is altered leading to changes in the epithelial barrier.

Liarozole, a novel imidazole derivative, inhibits the cytochrome P450-dependent 4-hydroxylation of retinoic acid, resulting in increased tissue levels of retinoic acid. Twelve male patients with ichthyosis were given oral liarozole, 150 mg twice daily, in an open study for 12 weeks. Immunohistochemical parameters of inflammation, epidermal proliferation and differentiation were assessed before and after treatment. Extent and severity of the skin lesions was markedly reduced in all patients. Clinical side-effects were reminiscent of those with synthetic retinoids. No relevant changes were found in the haematological, urinary and biochemical parameters. Immunohistochemical assessment showed a statistically significant induction of keratin 4 after liarozole treatment in 10 of 12 patients. In two of these patients keratin 13 was induced. This open study showed that oral liarozole treatment was efficacious and well tolerated in the treatment of different types of ichthyosis. The immunohistochemical results suggest a retinoid mechanism as the mode of action.

Most previously described immunohistochemical markers of cervical high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma may help to improve diagnostic accuracy but have a minimal prognostic value. The goals of the current study were to identify and validate novel candidate biomarkers that could potentially improve diagnostic and prognostic accuracy for cervical HSIL and squamous cell carcinoma. Microdissected tissue sections from formalin-fixed paraffin-embedded normal ectocervical squamous mucosa, low-grade squamous intraepithelial lesion (LSIL), HSIL and squamous cell carcinoma sections were analyzed by mass spectrometry-based shotgun proteomics for biomarker discovery. The diagnostic specificity of candidate biomarkers was subsequently evaluated by immunohistochemical analysis of tissue microarrays. Among 1750 proteins identified by proteomic analyses, keratin 4 (KRT4) and keratin 17 (KRT17) showed reciprocal patterns of expression in the spectrum of cases ranging from normal ectocervical squamous mucosa to squamous cell carcinoma. Immunohistochemical studies confirmed that KRT4 expression was significantly decreased in squamous cell carcinoma compared with the other diagnostic categories. By contrast, KRT17 expression was significantly increased in HSIL and squamous cell carcinoma compared with normal ectocervical squamous mucosa and LSIL. KRT17 was also highly expressed in immature squamous metaplasia and in endocervical reserve cells but was generally not detected in mature squamous metaplasia. Furthermore, high levels of KRT17 expression were significantly associated with poor survival of squamous cell carcinoma patients (Hazard ratio = 14.76, P = 0.01). In summary, both KRT4 and KRT17 expressions are related to the histopathology of the cervical squamous mucosa; KRT17 is highly overexpressed in immature squamous metaplasia, in HSIL, and in squamous cell carcinoma and the level of KRT17 in squamous cell carcinoma may help to identify patients who are at greatest risk for cervical cancer mortality.

The molecular basis for cross-reactive antibody binding to human T cell leukemia virus type I (HTLV-I) p19 core protein and human thymic epithelium has been defined with two monoclonal antibodies (mAbs), 12/1-2 and 13B12, raised to HTLV-I p19. The mAb 12/1-2 has previously been shown to react with HTLV-I p19, HTLV-II p22, and antigens of normal human thymic epithelium, placenta, and foreskin, whereas mAb 13B12 binds only to the carboxyl terminus of HTLV-I p19. In the present study, mAb 12/1-2 bound to a subset of Triton X-100-insoluble intermediate filaments in human thymic epithelium also recognized by antikeratin antibodies AE1 and AE3. The mAb 12/1-2 also reacted in Western blot assays with proteins of 54, 46, and 40 kDa present in extracts of human thymic epithelium and with hexameric peptides containing overlapping sequences of HTLV-I p19 with the amino acids IPP (amino acids 117-119). In contrast, the HTLV-I-specific mAb 13B12 did not bind to human thymic epithelium and reacted with a single hexameric peptide containing the carboxy-terminal HTLV-I p19 sequence IPPPYV (amino acids 117-122). Binding of mAb 12/1-2 to thymic epithelium could be inhibited by adsorption with peptide SP-79 containing a C-terminal sequence (amino acids 112-125) of p19. The crossreactive IPP site is within a region of p19 that has been previously shown to be highly immunogenic in HTLV-I-infected individuals and that is also encoded by genes or mRNA of human cytokeratin 17, keratin 4, epidermal cytokeratin 2, and 50-kDa type I epidermal keratin. Thus, our studies define the sequence of a cross-reactive antigen on HTLV-I p19 that is also associated with keratin intermediate filaments from human thymic epithelium and other normal human tissues and that could serve as a focus of an autoimmune response during HTLV-I infection.

White sponge nevus (WSN) is a rare autosomal dominant disorder characterized by white plaques of oral mucosa; it is benign condition with no effective treatment. The disorder usually manifests during early childhood or adolescence. Mutations of keratin 4 or 13 gene have been identified as causing WSN. The aim of this study is to determine whether keratin 4 or 13 gene mutation was the molecular basis of WSN in a Japanese family. The proband in this family was an 11-year-old boy, with three other people affected by WSN. Genomic DNA was extracted from two affected members and an unaffected member. Segments of keratin 4 and 13 genes were amplified by PCR, and direct DNA sequencing was carried out. Sequence analysis revealed a heterozygous 3bp deletion (N160Del) localized in the helix-initiation motif at the beginning of alpha-helical domain 1A of keratin 4 gene from affected members. One member lacking the phenotype was genetically tested normal. The authors identified a mutation of the keratin 4 gene recurrent in a family affected by WSN. Further investigation of the multifunctional role of keratin genes is warranted in the group of inherited epithelial disorders that may result in identification of effective treatment for this genetic disease.

The present study was designed to explore if *etinaldehyde, a natural metabolite of vitamin A, has any biologic activity and is tolerated by human skin. Biologic activity was shown by the induction of cellular retinoic acid-binding protein type 2 (CRABP 2) mRNA and protein; the rank order for CRABP-2 increase was retinoic acid > retinaldehyde > 9 cis retinoic acid > retinol > beta carotene. In volunteers treated 1-3 months with 0.5, 0.1, and 0.05% retinaldehyde, there was a dose-dependent and significant increase in epidermal thickness, keratin 14 immunoreactivity, and Ki67-positive cells. The area of distribution of involucrin, transglutaminase, and filaggrin immunoreactivity was also increased in a dose-dependent manner, and keratin 4 immunoreactivity was induced in the upper epidermis. In pilot clinical tolerance studies, 229 patients received topical retinaldehyde at different concentrations; the 1% preparation was tolerated by up to 70% of the treated subjects; tolerance of the 0.5% preparation was slightly better, whereas both 0.1 and 0.05% preparations applied on facial skin were well tolerated and allowed prolonged use (up to 3 years) in patients with inflammatory dermatoses. These findings indicate that topical retinaldehyde has biologic activity and is well tolerated on human skin.

Severe ocular surface diseases are some of the most challenging problems that the clinician faces today. Conventional management is generally unsatisfactory, and the long-term ocular consequences of these conditions are devastating. It is significantly important to find a substitute for conjunctival epithelial cells. This study was to explore the possibility of progenitor cell-derived epidermal sheets on denuded amniotic membrane to reconstruct ocular surface of conjunctiva damaged monkeys. We isolated epidermal progenitor cells of rhesus monkeys by type IV collagen adhesion, and then expanded progenitor cell-derived epidermal sheets on denuded amniotic membrane ex vivo. At 3 weeks after the conjunctiva injury, the damaged ocular surface of four monkeys was surgically reconstructed by transplanting the autologous cultivated epidermal progenitor cells. At 2 weeks after surgery, transplants were removed and examined with Hematoxylin-eosin staining, Periodic acid Schiff staining, immunofluorescent staining, scanning and transmission electron microscopy. Histological examination of transplanted sheets revealed that the cell sheets were healthy alive, adhered well to the denuded amniotic membrane, and had several layers of epithelial cells. Electron microscopy showed that the epithelial cells were very similar in appearance to those of normal conjunctival epithelium, even without goblet cell detected. Epithelial cells of transplants had numerous desmosomal junctions and were attached to the amniotic membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the conjunctival specific markers, mucin 4 and keratin 4, in the transplanted epidermal progenitor cells. In conclusion, our present study successfully reconstructed conjunctiva with autologous transplantation of progenitor cell-derived epidermal sheets on denuded AM in conjunctival damaged monkeys, which is the first step toward assessing the use of autologous transplantation of progenitor cells of nonocular surface origin. Epidermal progenitor cells could be provided as a new substitute for conjunctival epithelial cells to overcome the problems of autologous conjunctiva shortage.

Human sebocytes maintained in medium containing delipidized serum were studied for ultrastructural characteristics, cell proliferation, lipid synthesis, immunophenotype, and keratin expression before and after the addition of the synthetic retinoids isotretinoin and acitretin (10(-8)-10(-5) M). Compared to the properties of sebocytes cultured in normal sebocyte medium (1-2 x 10(-7) M vitamin A), the use of delipidized serum (undetectable amounts of vitamin A) resulted in prominent decrease of i) proliferation; ii) number of intracellular lipid droplets and synthesis of total lipids, especially triglycerides, squalene, and wax esters; and iii) labeling with monoclonal antibodies identifying progressive and late-stage sebocyte differentiation. Intercellular spaces narrowed and cell-to-cell contacts were established by abundant desmosomes. Lanosterol was induced. Keratins 14, 16, 17, and 18 were upregulated and the keratin 16: keratin 4 ratio, negatively correlating with sebocyte differentiation, increased. Addition of isotretinoin and acitretin exerted a biphasic effect. At concentrationsor = 10(-7) M, both compounds enhanced sebocyte proliferation and synthesis of total lipids, especially triglycerides and cholesterol, and decreased lanosterol, keratin 16, and the keratin 16:keratin 4 ratio. In contrast, retinoid concentrations10(-7) M inhibited sebocyte proliferation in a dose-dependent manner. Our findings indicate that vitamin A is essential for proliferation, synthetic activity, and differentiation of human sebocytes in vitro. Synthetic retinoids partially reinstate the altered functions of sebocytes maintained in medium containing delipidized serum. In contrast to the previously shown isotretinoin-specific response of cultured sebocytes in the presence of vitamin A, similar effects of isotretinoin and acitretin were obtained in its absence. This suggests different interactions of synthetic retinoids with vitamin A, possibly influencing their efficacy on the sebaceous gland.