Suprabasal Layers Research Articles

PAX6-WNK2 Axis Governs Corneal Epithelial Homeostasis.

Limbal stem/progenitor cells (LSCs) continuously proliferate and differentiate to replenish the corneal epithelium and play a vital role in corneal function and normal vision. A previous study revealed that paired box 6 (PAX6) is a master transcription factor involved in determining the fate of corneal epithelial cells (CECs). However, the molecular events downstream of PAX6 remain largely unknown. In this study, we aimed to clarify the regulation network of PAX6 in driving CEC differentiation. An air-liquid culture system was used to differentiate LSCs into mature CECs. Specific targeting PAX6 short-hairpin RNAs were used to knock down PAX6 in LSC. RNA sequencing (RNA-seq) was used to analyze shPAX6-transfected CECs and CEC differentiation-associated genes to identify the potential downstream targets of PAX6. RNA-seq analysis, quantitative real-time PCR, and immunofluorescence staining were performed to clarify the function of WNK lysine deficient protein kinase 2 (WNK2), a downstream target of PAX6, and its relationship with corneal diseases. WNK2 expression increased during CEC differentiation and decreased upon PAX6 depletion. The distribution of WNK2 was specifically limited to the central corneal epithelium and suprabasal layer of the limbus. Knockdown of WNK2 impaired the expression of CEC-specific markers (KRT12, ALDH3A1, and CLU), disrupted the corneal differentiation process, and activated the terms of keratinization, inflammation, and cell proliferation, consistent with PAX6-depleted CEC and published microbial keratitis. Thus, aberrant expression of WNK2 was linked to corneal ulcers. As a downstream target of PAX6, WNK2 plays an essential role in corneal epithelial cell differentiation and maintenance of corneal homeostasis.

Immunoexpression of metallothionein, Ki‐67 and PCNA in dermoid and epidermoid cysts of the oral cavity

AbstractObjectiveTo assess the immunoexpression of metallothionein and cell proliferation markers (Ki‐67 and PCNA) in a series of cases comprising four dermoid cysts and six epidermoid cysts of the oral cavity.Materials and MethodsThe percentages of nuclear and/or cytoplasmic staining in the basal and suprabasal layers of the cysts' epithelial lining were assessed.ResultsThis study included six males and four females, with a mean age of 33.1 years. The floor of the mouth was affected in 40% of the cases. The expression of metallothionein, Ki‐67, and PCNA was observed in all cases examined. For both lesions, the basal layer exhibited higher cytoplasmic and nuclear/cytoplasmic expression of metallothionein. Dermoid cysts exhibited higher expression of Ki‐67 and PCNA compared to epidermoid cysts in both basal and suprabasal layers, with a more pronounced expression in the suprabasal layers.ConclusionsOur results indicate that metallothionein is present in the epithelial lining of both dermoid and epidermoid cysts of the oral cavity. The cell proliferation indices observed in both conditions reflect their benign nature and slow growth.

Epidermal cell proliferation and differentiation in the beak of tadpoles of Rana dalmatina

AbstractEpidermal cell proliferation and differentiation during formation of the beak in tadpoles of Rana dalmatina. Acta Zoologica (Stockolm). The beak utilized in tadpoles of R. dalmatina for crushing vegetables is formed of cornified cell layers. A row of large wedge‐like cells forms a central pile that produces the sharp corneous lamina of the beak. The labial and oral surfaces of the beaks also accumulate corneous material during proliferation and differentiation of keratinocytes from the epidermis to form a sheath around the central corneous cells. Corneocytes are shed during the growth of tadpoles. Electron microscopy reveals that keratin and mucus granules are main components of these keratinocytes. Electron‐dense organelles of unknown composition and with inside lamellae are also present. Using immunohistochemistry after post‐injection of 5BrdU, a marker of cell proliferation, the main timing of formation of the beak has been determined. After 2–5 h from injection, labelled nuclei of keratinocytes are seen in the basal layer, and sparse suprabasal labelled nuclei are detected after 5 h. Labelled keratinocytes are seen in suprabasal layers at 2 days post‐injection. At 5 and 6 days labelled keratinocytes are present in central corneous cells and in the lateral shell of corneocytes forming the labial and oral beak sides. Shedding a complete stratum corneum likely takes more days, but this depends on feeding usage.

Myosin II mediates Shh signals to shape dental epithelia via control of cell adhesion and movement.

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.

Endoscopic diagnosis and treatment of lichen planus of the esophagus

Purpose of the study: to attract the attention of endoscopists and doctors of other specialties to the problem of diagnosis and treatment of lichen planus of the esophagus. Materials and methods. From January 2010 to December 2023, lichen planus was the cause of dysphagia in 7 of 17 patients with unexplained cicatricial strictures of the esophagus. Our own experience and literature data on endoscopic semiotics and treatment of lichen planus of the esophagus are presented. Results of the study. Lichen planus is most often localized in the upper third of the esophagus and is accompanied by a narrowing of the esophageal lumen. The mucosa is hyperemic, dull, with areas of epithelial detachment, erosions, and fibrin deposits. All 7 patients had grade 2-3 esophageal strictures with isolated esophageal involvement (3) or oral involvement (4). Morphological changes were nonspecific: ulcerations, granulation tissue and fibrosis of the underlying layers, atrophy and thinning of the epidermis, acanthosis. Only in 2 cases apoptotic Civatte bodies were detected in the suprabasal layer, which is a characteristic feature of lichen planus of the esophagus. All patients underwent courses of endoscopic bougienage, supplemented by intramural injections of triamcinolone. This manipulations led to stabilization of the esophageal lumen at 10-15 mm without a tendency to restenosis. Conclusion. Further experience is needed to determine optimal treatment strategies, but it is critical to pay particular attention to symptom assessment in patients with skin disorders and odynophagia or dysphagia. These actions will facilitate an earlier diagnosis of lichen planus of the esophagus and increase the effectiveness of endoscopic treatment.

Influence of peripheral nerve system on proliferation and migration of keratinocytes on site of the wound edges

AIM: to assess proliferation and migration of keratinocytes at the nonhealing edges of neuropathic wounds.MATERIALS AND METHODS: 25 patients with neuropathic ulcers and 5 patients without diabetes with decubitus were enrolled. Diabetic foot (DF) patients were underwent to standard treatment including debridement, atraumatic dressing, offloading, antibacterial therapy if it needs. Severity of peripheral neuropathy was assessed according to the NDS scale. Histo­logical (hematoxylin and eosin) and immunohistochemical (Ki-67 , α7nAChR markers) examination of wound edge were done during treatment (0, 10, 24 days).RESULTS: All patients have severe neuropathy according to NDSm (>8). The average size of DF ulcers before and on 10th day of treatment was of 4 cm2 and 2,5 cm2, respectively (p<0,004). Neuropathic ulcers were characterized by hyperproliferative epidermis. Mitotically active keratinocytes reside throughout the suprabasal layers. Ki-67 expressed all layers of the epidermis, but a greater staining density was detected in the basal layer. The density of a7nAChR-positive cells increased from 0 to 24 days (p=0,031).THE CONCLUSION: The data shows that neuropathy is one of the possible mechanisms of keratinocyte cell cycle disruption: proliferative activity and ability to migrate. Identification of new signaling pathways regulating the physiological repair of tissues and the study of their disorders in diabetes mellitus opens the prospect of developing an optimal therapeutic strategy.

ASH2L Mediates Epidermal Differentiation and Hair Follicle Morphogenesis through H3K4me3 Modification

The processes of epidermal development in mammals are regulated by complex molecular mechanisms, such as histone modifications. Histone H3 lysine K4 methylation mediated by COMPASS (complex of proteins associated with Set1) methyltransferase is associated with gene activation, but its effect on epidermal lineage development remains unclear. Therefore, we constructed a mouse model of specific ASH2L (COMPASS methyltransferase core subunit) deletion in epidermal progenitor cells and investigated its effect on the development of mouse epidermal lineage. Furthermore, downstream target genes regulated by H3K4me3 were screened using RNA sequencing combined with Cleavage Under Targets and Tagmentation sequencing. Deletion of ASH2L in epidermal progenitor cells caused thinning of the suprabasal layer of the epidermis and delayed hair follicle morphogenesis in newborn mice. These phenotypes may be related to the reduced proliferative capacity of epidermal and hair follicle progenitor cells. ASH2L depletion may also lead to depletion of the epidermal stem cell pools in late mouse development. Finally, genes related to hair follicle development (Shh, Edar, and Fzd6), Notch signaling pathway (Notch2, Notch3, Hes5, and Nrarp), and ΔNp63 were identified as downstream target genes regulated by H3K4me3. Collectively, ASH2L-dependent H3K4me3 modification served as an upstream epigenetic regulator in epidermal differentiation and hair follicle morphogenesis in mice.

Abstract 761: The relationship between ANGPTL4 and epidermal malignant transformation

Abstract High body mass index (BMI) is a risk factor for several types of cancers. Although epidemiology suggests obesity is negatively associated with non-melanoma skin cancer (NMSC), bariatric surgery has been shown to reduce the risk, suggesting a possible role of adipose tissue in the initiation and promotion of skin cancer. To gain mechanistic insight into how adipose tissue drives cutaneous carcinogenesis, we cultured non-tumorigenic mouse keratinocytes (JB6 P+ cells) with or without factors released from murine visceral adipose tissue (mFTF) and performed RNA-Sequencing. Published work demonstrated mFTF induces anchorage-independent cell growth, a surrogate marker for malignant transformation. Transcriptomic analysis demonstrated a significant induction of Angptl4 mRNA in JB6 P+ cells cultured with mFTF for 3 and 8 hours compared to the vehicle-treated cells. ANGPTL4 is an endogenous inhibitor of lipoprotein lipase that modulates free fatty acid delivery to adipose tissue and oxidative tissues such as muscle and liver. The C-terminal protein demonstrates tumor-associated activities such as angiogenesis, metastasis, protection against anoikis, and enhancement of cell survival. Therefore, we hypothesized that the induction of this gene promotes malignant transformation. RNA-seq data was validated in multiple cell models, including primary keratinocytes with human adipose tissue. Immunohistochemistry was used to assess ANGPTL4 expression in 37 actinic keratosis (pre-malignant) and cutaneous squamous cell carcinoma (cSCC) from patients with different BMIs (18.6 - 49.5). Notably, ANGPTL4 was only observed in the suprabasal epidermal layers in normal skin. For samples containing both lesional and perilesional skin, there was an increase in ANGPTL4 expression in the lesional epidermis relative to the normal perilesional skin. Furthermore, ANGPTL4 expression increased from transition to actinic keratosis to cSCC in situ, suggesting that ANGTL4 is induced during malignant transformation as observed in our pre-clinical models. Interestingly, once cSCC developed an invasive component, ANGTPL4 expression was lost in the invasive keratinocytes but persisted in the in situ component. There was no association between ANGPTL4 expression and BMI in lesional tissue. These data suggest that targeting ANTPLT4 may be a novel therapy for the prevention of skin cancer and that ANGPTL4-high patients or cancer patients with obesity may be a critical demographic for potential risk assessment or therapeutic intervention. Future studies will determine the impact of knocking out ANGTPL4 in our models of malignant transformation. Citation Format: Nat Ato Yawson, Jonathan D. Diedrich, Karen T. Liby, Jesse Veenstra, Jamie Jenna Bernard. The relationship between ANGPTL4 and epidermal malignant transformation [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 761.

In situ distribution of markers for stemness, quiescence, proliferation and differentiation in the human cornea and limbus

Aims/Purpose: Identify the distribution of the most commonly used markers for stemness, quiescence, proliferation, and differentiation in limbal compartments such as human limbal epithelial crypts, posterior and anterior limbus, as well as the central and posterior cornea.Methods: Corneal‐limbal biopsies from cadaver human donors (n = 3) embedded in paraffin were proceeded for immunohistochemistry fluorescence and examined for stemness/progenitor markers (p63α, SOX9, ABCG2, N‐cadherin), proliferation markers (ki‐67), quiescence marker (CEBPD), differentiation markers (CX43, KRT3, KRT12, KRT13), and epithelial marker (E‐cadherin). Images were obtained by Zeiss Axio Observer. Fluorescent images were obtained using the Zeiss Axio Imager M1 fluorescence microscope (ZEISS, Oberkochen, Germany).Results: The stemness/progenitor markers p63α and SOX9 were found in basal and suprabasal cells of limbal epithelial crypts and limbal epithelium, whereas the ABCG2 and N‐cadherin were exclusive for limbal basal epithelium in all limbal structures. Proliferation marker Ki‐67 was most abundant in suprabasal layers of the limbus and rarely found in the posterior cornea, while absent in anterior cornea. Quiescence marker CEBPD was positive in the basal cells of both, limbus and cornea. Differentiation markers KRT3 and KRT12 were positive in the whole cornea and intermediate and superficial layers in anterior and posterior limbus, while rarely present in limbal epithelial crypts. However, CX43 was staining almost all suprabasal layers in the limbus as well as basal and suprabasal layers in the cornea. KRT13 was exclusive for superficial layer of the anterior and posterior limbus layers staining conjunctival cells.Conclusions: Here we identify the localization of the most common markers used to determine the stemness, quiescence, proliferation, and differentiation status to unravel the in situ distribution of these markers in the limbal epithelial crypts, posterior and anterior limbus, as well as the central and posterior cornea. The study accumulated new knowledge about the distribution of these markers and the corneal‐limbal organization.

Immunohistochemical Expression of CK14 and Bcl-2 in Odontogenic Keratocyst and Its Variants.

Odontogenic keratocysts (OKCs) are aggressive cystic jaw lesions with a high epithelial turnover rate and increased propensity for recurrence. Sometimes, the characteristic histopathological features of OKCs are either completely lost or seen focally due to previous marsupialization or inflammation. This research aimed to determine whether specific patterns of CK14 and Bcl-2 staining could assist in diagnosing OKCs with altered epithelial features and provide clues in elucidating their aggressive nature. CK14 expression was restricted to basal and suprabasal layers near satellite cysts and in areas showing subepithelial split. The entire epithelial lining showed CK14 expression in areas of inflammation and after marsupialization. The typical basal/suprabasal staining of Bcl-2 was lost in areas of inflammation and intensity is decreased in OKCs after marsupialization. These new findings could offer a hint into the biological nature and pathogenesis of OKCs. Because of its therapeutic consequences and high recurrence rate, proper recognition and diagnosis are essential for treatment planning.

Three atypical/malignant onychopapillomas in a 52-case series with immunohistochemical study.

Onychopapilloma (OP) is a benign tumor of the nail. Haneke reported one case of malignant OP in 2021. No systematic immunohistochemistry study has been conducted on OP. The aim of our study was to identify possible malignant OP in our series of OP and to describe the immunohistochemical expression of p16, p53, and Ki67 in typical and atypical/malignant ones. Ninety-one cases were available for pathological review. Immunohistochemical analysis could be performed on 52 cases. In 88 of 91 cases, the diagnosis of OP was confirmed. Three atypical/malignant cases were observed. No OP expressed p16. A normal p53 expression was observed in two thirds of the cases, an abnormal increased p53 expression in one third, including the three atypical cases. A normal Ki67 expression was observed in 84% of the cases, an abnormal Ki67 expression with focal heterogeneous expression in the suprabasal layers in 6% and in all suprabasal cell layers in 10%, including the three atypical cases. The diagnosis of atypical/malignant OP may be underestimated. The expression of Ki67 and p53 in these tumors differs from the expression observed in conventional OP. The absence of p16 expression confirms that human papillomavirus does not play a role in the etiopathogenesis of OP.

Agent-based approach for elucidating the release from collective arrest of cell motion in corneal epithelial cell sheet

Changes in cell fluidity have been observed in various cellular tissues and are strongly linked to biological phenomena such as self-organization. Recent studies suggested variety of mechanisms and factors, which are still being investigated. This study aimed to investigate changes in cell fluidity in multi-layered cell sheets, by exploring the collective arrest of cell motion and its release in cultures of corneal epithelial cells. We constructed mathematical models to simulate the behaviors of individual cells, including cell differentiation and time-dependent changes in cell-cell connections, which are defined by stochastic or kinetic rules. Changes in cell fluidity and cell sheet structures were expressed by simulating autonomous cell behaviors and interactions in tissues using an agent-based model. A single-cell level spatiotemporal analysis of cell state transition between migratable and non-migratable states revealed that the release from collective arrest of cell motion was initially triggered by a decreased ability to form cell-cell connections in the suprabasal layers, and was propagated by chain migration. Notably, the disruption of cell-cell connections and stratification occurred in the region of migratable state cells. Hence, a modeling approach that considers time-dependent changes in cell properties and behavior, and spatiotemporal analysis at the single-cell level can effectively delineate emergent phenomena arising from the complex interplay of cells.

Expression profiles of glucocorticoid-inducible proteins in human papilloma virus-related oropharyngeal squamous cell carcinoma.

Human papillomavirus virus-related oropharyngeal squamous cell carcinoma (HPV-OPSCC) comprises a significant portion of head and neck cancers. Several glucocorticoid-inducible proteins play important roles in pathogenesis of some cancers but their status and roles in HPV-OPSCC remain elusive; these include the glucocorticoid-induced leucine zipper (GILZ), Annexin-A1 and serum glucocorticoid-regulated kinase-1 (SGK-1). We determined expression profiles of these proteins, using immunohistochemistry, in archived biopsy samples of patients diagnosed with HPV-OPSCC; samples of non-cancer oral lesions (e.g., hyperkeratosis) were used as controls. GILZ staining was primarily confined to nuclei of all tissues but, in HPV-OPSCC specimens, neoplastic cells exhibiting mitosis displayed prominent cytoplasmic GILZ expression. On the other hand, nuclear, cytoplasmic and membranous Annexin-A1 staining was observed in suprabasal cell layers of control specimens. A noted feature of the HPV-OPSCC specimens was few clusters of matured and differentiated nonbasaloid cells that showed prominent nuclear and cytoplasmic Annexin-A1 staining while the remainder of the tumor mass was devoid of staining. Cytoplasmic and nuclear staining for SGK-1 was prominent for control than PV-OPSCC specimens while staining for phosphorylated SGK-1 (pSGK-1; active) was prominent for cell membrane and cytoplasm of control specimens but HPV-OPSCC specimens showed mild and patchy nuclear and cytoplasmic staining. Semi-quantitative analysis of GILZ immunostaining indicated increased staining area but similar normalized staining for HPV-OPSCC compared to control specimens. By contrast, staining area and normalized staining were reduced for other proteins in HPV-OPSCC than control specimens. Our collective observations suggest differential cellular localization and expression of glucocorticoid-inducible proteins in HPV-OPSCC suggestive of different functional roles in pathogenesis of this condition.

Biomimetic human skin model patterned with rete ridges

Rete ridges consist of undulations between the epidermis and dermis that enhance the mechanical properties and biological function of human skin. However, most human skin models are fabricated with a flat interface between the epidermal and dermal layers. Here, we report a micro-stamping method for producing human skin models patterned with rete ridges of controlled geometry. To mitigate keratinocyte-induced matrix degradation, telocollagen–fibrin matrices with and without crosslinks enable these micropatterned features to persist during longitudinal culture. Our human skin model exhibits an epidermis that includes the following markers: cytokeratin 14, p63, and Ki67 in the basal layer, cytokeratin 10 in the suprabasal layer, and laminin and collagen IV in the basement membrane. We demonstrated that two keratinocyte cell lines, one from a neonatal donor and another from an adult diabetic donor, are compatible with this model. We tested this model using an irritation test and showed that the epidermis prevents rapid penetration of sodium dodecyl sulfate. Gene expression analysis revealed differences in keratinocytes obtained from the two donors as well as between 2D (control) and 3D culture conditions. Our human skin model may find potential application for drug and cosmetic testing, disease and wound healing modeling, and aging studies.

Mammalian esophageal stratified tissue homeostasis is maintained distinctively by the epithelial pluripotent p63+Sox2+ and p63-Sox2+ cell populations.

Self-renewing, damage-repair and differentiation of mammalian stratified squamous epithelia are subject to tissue homeostasis, but the regulation mechanisms remain elusive. Here, we investigate the esophageal squamous epithelial tissue homeostasis in vitro and in vivo. We establish a rat esophageal organoid (rEO) in vitro system and show that the landscapes of rEO formation, development and maturation trajectories can mimic those of rat esophageal epithelia in vivo. Single-cell RNA sequencing (scRNA-seq), snapshot immunostaining and functional analyses of stratified "matured" rEOs define that the epithelial pluripotent stem cell determinants, p63 and Sox2, play crucial but distinctive roles for regulating mammalian esophageal tissue homeostasis. We identify two cell populations, p63+Sox2+ and p63-Sox2+, of which the p63+Sox2+ population presented at the basal layer is the cells of origin required for esophageal epithelial stemness maintenance and proliferation, whereas the p63-Sox2+ population presented at the suprabasal layers is the cells of origin having a dual role for esophageal epithelial differentiation (differentiation-prone fate) and rapid tissue damage-repair responses (proliferation-prone fate). Given the fact that p63 and Sox2 are developmental lineage oncogenes and commonly overexpressed in ESCC tissues, p63-Sox2+ population could not be detected in organoids formed by esophageal squamous cell carcinoma (ESCC) cell lines. Taken together, these findings reveal that the tissue homeostasis is maintained distinctively by p63 and/or Sox2-dependent cell lineage populations required for the tissue renewing, damage-repair and protection of carcinogenesis in mammalian esophagi.

Immunolocalization of Some Epidermal Proteins and Glycoproteins in the Growing Skin of the Australian Lungfish (Neoceratodus forsteri).

Here we report the immunolocalization of mucin, nestin, elastin and three glycoproteins involved in tissue mineralization in small and large juveniles of Neoceratodus forsteri. Both small and larger juvenile epidermis are mucogenic and contain a diffuse immunolabeling for nestin. Sparse PCNA-labeled cells, indicating proliferation, are found in basal and suprabasal epidermal layers. No scales are formed in small juveniles but are present in a 5 cm long juvenile and in larger juveniles. Elastin and a mineralizing matrix are localized underneath the basement membrane of the tail epidermis where lepidotriches are forming. The latter appears as "circular bodies" in cross sections and are made of elongated cells surrounding a central amorphous area containing collagen and elastin-like proteins that undergo calcification as evidenced using the von Kossa staining. However, the first calcification sites are the coniform teeth of the small juveniles of 2-3 cm in length. In the superficial dermis of juveniles (16-26 cm in length) where scales are formed, the spinulated outer bony layer (squamulin) of the elasmoid scales contains osteonectin, alkaline phosphatase, osteopontin, and calcium deposits that are instead absent in the underlying layer of elasmodin. In particular, these glycoproteins are localized along the scale margin in juveniles where scales grow, as indicated by the presence of PCNA-labeled cells (proliferating). These observations suggest a continuous deposition of new bone during the growth of the scales, possibly under the action of these mineralizing glycoproteins, like in the endoskeleton of terrestrial vertebrates.

Two cases of cutaneous-type pemphigus vulgaris and a case of pemphigus foliaceus without mucosal involvement despite high anti-desmoglein 3 autoantibody levels.

Pemphigus is an autoimmune blistering disease with two major subtypes, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Although most patients with PV show oral lesions, cutaneous type PV (C-PV) is a rare subtype clinically characterized by predominant cutaneous involvement with no or subtle mucosal lesions. Patients with PF present with only skin involvement; they do not have mucosal lesions. Serologically, autoantibodies against desmoglein (Dsg) 3 and Dsg1 are observed in C-PV whereas PF is associated with anti-Dsg1 antibodies only. Herein, we describe three cases of pemphigus presenting with predominant skin lesions and no mucosal involvement despite high anti-Dsg 3 autoantibody levels in chemiluminescent enzyme immune assays (CLEIAs). In addition, anti-Dsg 1 autoantibodies were positive in patients 2 and 3, but negative in patient 1 based on CLEIAs. Histological examination of the skin showed suprabasal acantholysis in patients 1 and 2, and blister formation in the upper epidermis in patient 3. Histopathology of the oral membrane in patients 1 and 2 showed subtle acantholysis in the suprabasal layer. Thus, we diagnosed patients 1 and 2 as having cutaneous type PV and patient 3 as having PF. Ethylenediaminetetraacetic acid-treated enzyme-linked immunosorbent assay demonstrated a low proportion of anti-Dsg3 autoantibodies recognizing Ca2+ -dependent epitopes, antibodies against which are thought to be the main contributor to acantholysis. Thus, along with Dsg1 antibodies, weak anti-Dsg3 antibodies could induce acantholysis in the skin, but they are insufficient to induce mucosal lesions.

Expression of connexin43 in oral submucous fibrosis, oral epithelial dysplasia and oral squamous cell carcinoma: A cross-sectional immunohistochemical study

Introduction: Connexins are specialized gap junction (GJ) proteins that are necessary for the maintenance of cellular homeostasis. The loss of GJ leads to the loss of cellular cohesion, promoting tumor progression. Not much is known about the role of connexin43 (Cx43) in oral submucous fibrosis (OSF), oral epithelial dysplasia (OED), and in OSF that had transformed into oral squamous cell carcinoma (OSCC). Materials and methods: A total of 39 tissue samples was grouped into group I: Normal oral mucosa (NC) (n = 6), group II: OSF (n = 14), group III: OED (n = 12), and group IV: OSCC with a history of OSF (n = 7). Sections were stained with Cx43 antibody and analyzed for staining intensity in the basal, suprabasal cell layers, and connective tissue. The Chi-square test was used to assess significant association and Kappa statistics between the variable and interobserver variability. Results: Cx43 exhibited varying levels of expression in the cell membrane of suprabasal cell layer across all the groups. The difference in expression was statistically significant (P = 0.033). In OSF, there was a moderate presence of Cx43, whereas NC, dysplasia, and OSCC with OSF showed no detectable Cx43 expression. Notably, OSF displayed higher Cx43 expression compared to OED. Conversely, OSCC with OSF demonstrated reduced expression of Cx43 in epithelial tumor islands that had invaded the connective tissue (P = 0.033). Conclusion: Cx43 expression was significantly decreased as there was a progression in OED severity and reduced in OSCC with a history of OSF.

Development of a lip vermilion epithelium reconstruction model using keratinocytes from skin and oral mucosa.

Lip vermilion is unique and can be distinguished from the adjacent skin and oral mucosa. However, because of the lack of appropriate evaluation tools, skin and/or oral mucosa substitutes such as in vitro vermilion epithelial models have been used for lip product testing. We aimed to develop and characterize a lip vermilion epithelium reconstruction model (LVERM) using skin and oral keratinocytes. LVERM was manufactured by co-culturing primary skin and oral keratinocytes, using a device that allowed the separation of cell seeding, and created an intercalated cell-free zone, referred to as the vermilion part. After removing the device, LVERM construction was completed in 8 days, in a submerged condition. Subsequently, they were placed in an air-liquid interface for 7 days. To determine the epithelial characteristics of LVERM, keratin 2e (KRT2) and small proline-rich protein 3 (SPRR3) expression patterns were examined. The in vivo expression profiles of KRT2 and SPRR3 genes in vermilion were also examined. We found that a continuous multi-layered epithelium was generated in the LVERM that exhibited ortho- and para-keratinization in the skin and oral mucosa parts, respectively. Although an intermediate keratinization pattern was observed in the vermilion part, KRT2 and SPRR3 were co-expressed in the suprabasal layer, consistent with the expression pattern of a single vermilion epithelial model. Clustering analysis revealed that KRT2 and SPRR3 gene expression in vermilion was location-dependent within the sample. Therefore, LVERM can be used as an evaluation tool for lip products and has great importance in innovative approaches for cosmetic testing.

Ki-67 Expression as a Prognostic Marker: A Comparative Immunohistochemical Analysis of Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma.

Introduction Oral dysplasia is a frequent precancerous condition that may lead to oral cancer. The histopathologic abnormalities exhibited in a chronic, progressive, and premalignant condition of the oral mucosa are referred to as oral epithelial dysplasia (OED). It might show up as erythroplakia, leukoplakia, or leukoerythroplakia. OED is a premalignancy histologic marker that predicts a higher likelihood of squamous cell carcinoma development. Aims and objectives The aim of this study is to identify an association between Ki-67 protein expression and histological grading of OED and oral squamous cell carcinoma (OSCC) and to compare the expression of Ki-67 in different grades of OED and OSCC with the prognosis. Materials and methods The current retrospective research is focused on evaluating epithelial dysplasia and analyzing the function of Ki-67 as a prognostic marker after receiving institutional ethical approval. Group I - normal oral mucosa (NOM), Group II - OED, and Group III - OSCC were included in the study. For statistical analysis, SPSS Statistics version 21.0 (IBM Corp. Released 2021. IBM SPSS Statistics for Windows, Version 28.0. Armonk, NY: IBM Corp) was utilized. The Cox regression model was employed to look at interactions between various prognostic variables. At p<0.05, differences were deemed statistically significant. Results Ki-67 expression was confined to the basal layers in the normal oral epithelium and in the basal, suprabasal, and spinous layers in OED.Ki-67 positive cells were mostly found on the perimeter of well, moderate, and poorly differentiated OSCC tumor nests with Ki-67 positive cells scattered throughout OSCC.According to statistical analysis, there is a substantial difference in expression between OEDand NOM, OSCC and NOM, and OED and OSCC. Conclusion Our study showed that there is a progressive increase in Ki-67 expression across various grades of OED, and the highest expression was noted in OSCC. Early identification and prompt treatment will help in improving the quality of life of such patients.