Epithelial Basement Research Articles

Depigmentation procedure by scrapping technique for management of gingival hyperpigmentation: Case report

Background: Currently, periodontal treatments for esthetic purposes are increasing. This treatment could be done by periodontal plastic surgery. One of the procedures of periodontal plastic surgery is gingival depigmentation. Gingival depigmentation is the treatment for gingival discoloration. Gingival discoloration occurs due to the hyper-deposition of melanin by melanocytes on the basal gingival epithelium. This condition causes a dark color of the gingiva. The purpose of this case report is to explain the management of gingival discoloration. Cases: A 22-year-old male patient with chief complaints of darkish gingival color and less confidence in smiling. The patient is not a smoker. Case management: Anterior maxilla gingival depigmentation was performed using scalpel #15. An Orban knife was used to shape the interdental area, followed by saline irrigation. A periodontal pack is placed on the surgical area. Two weeks of evaluation found that wound healing is fine, as painless and no infection occurred. Conclusion: Management of gingival discoloration by melanin with gingival depigmentation technique provides effective results.

Assessing the Impact of Ghee, Olive Oil and Margarine on Male Rabbit Fertility and Reproductive Hormones.

In the present investigation, the impact of natural ghee, olive oil and synthetic margarine on the fertility parameters of male rabbits was evaluated by examining semen quality, fertility hormones, antioxidant markers, lipid profile, and liver and kidney functions. Eighty male rabbits were randomly allotted into four groups (20 rabbits each, four replicates/group). The basal diet supplemented the control group; the margarine group was fed a 10% margarine diet, the ghee group was fed a 10% ghee diet, and the olive oil group was fed a 10% olive oil diet. In the margarine group, the semen quality parameters, total testosterone levels, free testosterone, luteinizing hormone (LH) and antioxidant enzyme levels as catalase showed a significant reduction compared to other groups. At the same time, they were enhanced in ghee and olive oil groups. A substantial increase of triglyceride (TAG), low-density lipoprotein (LDL) and cholesterol, with a decrease of high-density lipoprotein (HDL) levels, were observed in the margarine group contrasted to ghee and olive oil groups. The ghee and the olive oil-treated group showed strong immunoreactions of androgen, FSH, LH receptors and mild caspase 3 in testicular tissue compared to the margarine-treated group. Finally, histopathological examination of rabbit testicular tissue showed proliferation of basal spermatogenic cells, increased luminal spermatid of seminiferous epithelium, and proliferation of interstitial cells in normal interstitial tissue in the ghee and olive oil treated group. Still, it showed severe vacuolation and necrosis in the basal luminal seminiferous epithelium and congestion of blood vessels in the margarine group. This present study revealed that the health influence of olive oil and ghee is better than margarine on male fertility parameters.

Deletion of Fgfr2 in Ductal Basal Epithelium With Tamoxifen Induces Obstructive Meibomian Gland Dysfunction.

Fibroblast growth factor receptor 2 (Fgfr2) is crucial for the homeostasis of meibomian gland (MG). However, the role of Fgfr2 in MG ductal epithelial progenitors remains to be delineated. Herein, we created a new transgenic mouse model with conditional deletion of Fgfr2 from MG ductal progenitors and investigated the cell-specific role in the pathogenesis of obstructive meibomian gland dysfunction. Peritoneal injection of tamoxifen (TAM) at 50 µg/gm for three consecutive days was performed to induce conditional deletion of Fgfr2 in two-month-old Krt5Fgfr2CKO or Krt5Fgfr2CKO-mTmG mice. Phenotypes of MG after Fgfr2 deletion were monitored by meibography, lipid staining, and immunostaining against keratin-6a in MG whole mounts. Lineage tracing of the Krt5+ progenitors of MG and biomarkers for ductal differentiation and proliferation were also examined by immunostainings. The Krt5Fgfr2CKO mice developed extensive ductal occlusion and acinar atrophy at day 10 after TAM administration. Robust thickening of ductal epithelium with abnormal differentiation and proliferation of ductal basal meibocytes were observed in the MGs of Krt5Fgfr2CKO mice. In Krt5Fgfr2CKO-mTmG mice, the Krt5+ progenitors and its progeny were labeled by EGFP after Fgfr2 depletion by TAM with evident expansion of the suprabasal and superficial layers of MG ductal epithelium when compared with the controls. Our results substantiated the crucial role of Fgfr2 in homeostasis of the MG ductal epithelium. Deletion of Fgfr2 affects the MG ductal basal progenitors by impacting the differentiation of ductal meibocytes and the maintenance of acinar meibocytes, which are likely the underlying pathogenesis of obstructive MGD.

Characterization of E-Cadherin, SSEA-1, MSI-1, and SOX-2 Expression and Their Association with Pale Cells in Adenomyosis

Adenomyosis (AM) is a gynecological disease characterized by the invasion of endometrial glands and stroma within the myometrium. The etiology and pathogenesis of AM remain inadequately understood. Pale cells were identified as a novel cell type characterized by the absence of desmosomal contacts and light-colored cytoplasm. These cells were observed to migrate individually through ultra-micro ruptures in the basal membrane of the endometrial glands, translocating into the stroma and then further into the myometrium. Our study aimed to explore the possible stem cell properties of these pale cells. Forty hysterectomy specimens were analyzed using immunohistochemistry and immunofluorescence to assess negative E-cadherin expression and the positive expression of stem cell markers SSEA-1, MSI-1, and SOX-2. Immunohistochemical analysis revealed the presence of pale cells and occasionally rounded, enlarged E-cadherin-negative cells predominantly in the basal endometrial epithelium. The stem cell marker SSEA-1 was significantly elevated in the basalis epithelium, as well as in the ectopic epithelium. SSEA-1 positive cells were also identified in the stroma and myometrium. Sporadic colocalization of SSEA-1+/E-cadherin– cells was confirmed through immunofluorescence. The positive staining of pale cells for SSEA-1 and MSI-1 was also confirmed at the ultrastructural level by immunoelectron microscopy. These findings indicate that pale cells may possess stem cell characteristics, particularly a positive SSEA-1 profile, warranting further in vitro investigation into their role in the pathogenesis of adenomyosis.

The Diagnostic Histopathological Techniques of Prostate Cancer

The histology of the prostate is critical for understanding both normal prostate function and the pathogenesis of prostate cancer. The prostate gland comprises a complex architecture that includes a functional secretory epithelium, a basal epithelium, and a supporting stroma with a variety of cell types. Prostate adenocarcinoma is the most common form of prostate cancer, characterized by acinar-type architecture. The consistent ranking of prostate cancer as a leading cause of cancer diagnosis and mortality reflects an urgent need for ongoing research into its etiology, risk factors, and effective treatment modalities. This review article cast a light on the diagnostic histopathological techniques of prostate cancer.

Switch-like Gene Expression Modulates Disease Susceptibility.

A fundamental challenge in biomedicine is understanding the mechanisms predisposing individuals to disease. While previous research has suggested that switch-like gene expression is crucial in driving biological variation and disease susceptibility, a systematic analysis across multiple tissues is still lacking. By analyzing transcriptomes from 943 individuals across 27 tissues, we identified 1,013 switch-like genes. We found that only 31 (3.1%) of these genes exhibit switch-like behavior across all tissues. These universally switch-like genes appear to be genetically driven, with large exonic genomic structural variants explaining five (~18%) of them. The remaining switch-like genes exhibit tissue-specific expression patterns. Notably, tissue-specific switch-like genes tend to be switched on or off in unison within individuals, likely under the influence of tissue-specific master regulators, including hormonal signals. Among our most significant findings, we identified hundreds of concordantly switched-off genes in the stomach and vagina that are linked to gastric cancer (41-fold, p<10-4) and vaginal atrophy (44-fold, p<10-4), respectively. Experimental analysis of vaginal tissues revealed that low systemic levels of estrogen lead to a significant reduction in both the epithelial thickness and the expression of the switch-like gene ALOX12. We propose a model wherein the switching off of driver genes in basal and parabasal epithelium suppresses cell proliferation therein, leading to epithelial thinning and, therefore, vaginal atrophy. Our findings underscore the significant biomedical implications of switch-like gene expression and lay the groundwork for potential diagnostic and therapeutic applications.

Switch-like Gene Expression Modulates Disease Susceptibility.

A fundamental challenge in biomedicine is understanding the mechanisms predisposing individuals to disease. While previous research has suggested that switch-like gene expression is crucial in driving biological variation and disease susceptibility, a systematic analysis across multiple tissues is still lacking. By analyzing transcriptomes from 943 individuals across 27 tissues, we identified 1,013 switch-like genes. We found that only 31 (3.1%) of these genes exhibit switch-like behavior across all tissues. These universally switch-like genes appear to be genetically driven, with large exonic genomic structural variants explaining five (~18%) of them. The remaining switch-like genes exhibit tissue-specific expression patterns. Notably, tissue-specific switch-like genes tend to be switched on or off in unison within individuals, likely under the influence of tissue-specific master regulators, including hormonal signals. Among our most significant findings, we identified hundreds of concordantly switched-off genes in the stomach and vagina that are linked to gastric cancer (41-fold, p<10-4) and vaginal atrophy (44-fold, p<10-4), respectively. Experimental analysis of vaginal tissues revealed that low systemic levels of estrogen lead to a significant reduction in both the epithelial thickness and the expression of the switch-like gene ALOX12. We propose a model wherein the switching off of driver genes in basal and parabasal epithelium suppresses cell proliferation therein, leading to epithelial thinning and, therefore, vaginal atrophy. Our findings underscore the significant biomedical implications of switch-like gene expression and lay the groundwork for potential diagnostic and therapeutic applications.

Candidate proteins interacting with cytoskeleton in cells from the basal airway epithelium in vitro.

Introduction: The cytoskeleton consists of actin, microtubules, septins, and intermediate filaments and, in most cells, is anchored to an extracellular matrix. Each cell has a unique arrangement of this network and readjusts it from time to time. To investigate the regulation of these reorganizations, we identified interactors from extracts of four cultured lines representing basal cells from the airway epithelium. Methods: After immunoprecipitation with an antibody against keratin 17, samples were processed by liquid chromatography and tandem mass spectrometry. Samples not undergoing antibody-mediated capture were processed in parallel. Results: The main keratins of basal cells, namely, Krt14 (type I) and Krt5 (type II), constituted 67% of the total keratin recovered. Several other intermediate filament proteins, nestin, lamin-B1, and prelamin A/C, were present but not enriched upon immunoprecipitation. Although the class of armadillo-repeat proteins was represented by beta-catenin1 and plakoglobin, other desmosome plaque constituents were absent. Large cytolinkers were represented by the spectraplakin, microtubule-actin cross-linking factor (Macf1), which was enriched by immunoprecipitation, and the plakin, plectin, which was not enriched. Subunits of actin filaments and microtubules, along with numerous proteins associated with them, were recovered in both immunoprecipitated samples and those lacking the capture step. Coefficients of determination were computed based on abundance. The actin-associated proteins, alpha-spectrin and brain-specific angiogenesis inhibitor (Baiaip2l), were modestly correlated with keratin abundance but highly correlated with one another and with the keratin-binding protein, annexin A2. This interaction network resembled the pedestal formed by pathogenic Escherichia coli. Microtubule-associated proteins, dynamin 1-like protein and cytoplasmic dynein 1 heavy chain (Dync1h1), were enriched by immunoprecipitation, suggesting association with keratins, whereas kinesin-1 heavy chain and microtubule-associated protein retinitis pigmentosa 1 (EB1), were not enriched. Dync1h1 abundance was negatively correlated with that of all the septins, suggesting resemblance to a known antagonistic septin-dynein 1 relationship on microtubules. Conclusion: The cell lines showed remarkable uniformity with respect to the candidates interacting with cytoskeleton. The alpha-spectrin-Baiap2l network may link actin filaments to keratin precursor particles. A smaller interaction network centered on Dync1h1 was negatively correlated with all spectrin-Baiap2l constituents, suggesting that it and its binding partners are excluded from the pedestal-like domain.

Droplet-based proteomics reveals CD36 as a marker for progenitors in mammary basal epithelium

Deep proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present DROPPS (droplet-based one-pot preparation for proteomic samples), an accessible low-input platform that generates high-fidelity proteomic profiles of 100-2,500 cells. By applying DROPPS within the mammary epithelium, we elucidated the connection between mitochondrial activity and clonogenicity, identifying CD36 as a marker of progenitor capacity in the basal cell compartment. We anticipate that DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations.

Abstract 4771: Assessment of Ki67 proliferation index in pre-malignant colonic adenomas: Implication for new biomarker testing to predict metachronous disease (The INCISE project)

Abstract Approximately 40% of patients are found to have polyps at bowel screening colonoscopy, which are associated with a higher risk of developing future polyps or colorectal cancer. The British Society of Gastroenterology 2020 (BSG20) guidelines stratify patients into high/low risk of metachronous disease, using the size, dysplasia, and number of polyps found at index colonoscopy. This study aims to explore the prognostic significance of Ki67 proliferation percentages in adenomatous polyps, with metachronous disease. Tissue microarray (TMA) was constructed from 1201 index adenomatous polyps with representative cores of basal and luminal adenomatous epithelia. 67% patients were identified as high risk at index colonoscopy. The median time to detect metachronous polyps was 55 months (range 6-72 months) with 56% of patients developing metachronous polyps by the end of follow-up. TMA was stained for Ki67 using immunohistochemistry. Ki67 proliferation percentages in adenomatous epithelia was quantified using QuPath. Data was split into training (n=830) and test (n=371) datasets. The Ki67 proliferation cutpoint was made using Survmine R package using the training dataset. X2 test was used to study associations with clinicopathological criteria and Kaplan survival analysis was used to assess Ki67 association with metachronous lesions. Ki67 percentages were higher in lumina than in basal adenomatous epithelium (median Ki67 were 63% &amp; 48%; respectively). Total Ki67 percentages were computed by averaging luminal and basal values. Thresholds of 70%, 64% and 59% were employed to classify patients into low/high categories based on luminal, basal or total percentages; respectively. In the training cohort, high luminal, basal or total Ki67 were associated with metachronous lesions (p values 0.032, &amp;lt;0.001 and 0.006; respectively). Other associations were significant between luminal or total Ki67 and location of index polyps (p=0.017 &amp; p=0.044; respectively), histology (p=0.019) and advanced future lesion status (p=0.002 &amp; p&amp;lt;0.001; respectively). In multivariate cox regression analysis, high Ki67 was an independent prognostic factor for metachronous polyps (luminal Ki67 HR=1.28, CI: 1.052-1.560, p=0.014; basal Ki67 HR=1.639, CI: 1.313-2.046, p&amp;lt;0.001; total Ki67 HR=1.344, CI: 1.105-1.635, p=0.003) when adjusted to number of index polyps and BSG20 risk score. BSG20 risk score stratification of patients in terms of metachronous disease was improved when combined with luminal, basal or total Ki67 in the training datasets (all p&amp;lt;0.05). Prediction of metachronous disease by the combined groups was validated in the test cohort for luminal, basal and total Ki67 (all p&amp;lt;0.05). High Ki67 proliferation rates in adenomatous polyps are independently associated with the development of metachronous polyps and may help improve the BSG20 criteria. Citation Format: Gerard P. Lynch, Aula Ammar, Christopher Bigley, Mark Johnstone, Jakub Jawny, Hannah Morgan, Jennifer Hay, Noori Maka, Stephen McSorley, Joanne Edwards. Assessment of Ki67 proliferation index in pre-malignant colonic adenomas: Implication for new biomarker testing to predict metachronous disease (The INCISE project) [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 4771.

Single cell analysis of short-term dry eye induced changes in cornea immune cell populations.

Dry eye causes corneal inflammation, epitheliopathy and sensorineural changes. This study evaluates the hypothesis that dry eye alters the percentages and transcriptional profiles of immune cell populations in the cornea. Desiccating stress (DS) induced dry eye was created by pharmacologic suppression of tear secretion and exposure to drafty low humidity environment. Expression profiling of corneal immune cells was performed by single-cell RNA sequencing (scRNA-seq). Cell differentiation trajectories and cell fate were modeled through RNA velocity analysis. Confocal microscopy was used to immunodetect corneal immune cells. Irritation response to topical neurostimulants was assessed. Twelve corneal immune cell populations based on their transcriptional profiles were identified at baseline and consist of monocytes, resident (rMP) and MMP12/13 high macrophages, dendritic cells (cDC2), neutrophils, mast cells, pre T/B cells, and innate (γDT, ILC2, NK) and conventional T and B lymphocytes. T cells and resident macrophages (rMP) were the largest populations in the normal cornea comprising 18.6 and 18.2 percent, respectively. rMP increased to 55.2% of cells after 5 days of DS. Significant changes in expression of 1,365 genes (adj p < 0.0001) were noted in rMP with increases in cytokines and chemokines (Tnf, Cxcl1, Ccl12, Il1rn), inflammatory markers (Vcam, Adam17, Junb), the TAM receptor (Mertk), and decreases in complement and MHCII genes. A differentiation trajectory from monocytes to terminal state rMP was found. Phagocytosis, C-type lectin receptor signaling, NF-kappa B signaling and Toll-like receptor signaling were among the pathways with enhanced activity in these cells. The percentage of MRC1+ rMPs increased in the cornea and they were observed in the basal epithelium adjacent to epithelial nerve plexus. Concentration of the chemokine CXCL1 increased in the cornea and it heightened irritation/pain responses to topically applied hypertonic saline. These findings indicate that DS recruits monocytes that differentiate to macrophages with increased expression of inflammation associated genes. The proximity of these macrophages to cornea nerves and their expression of neurosensitizers suggests they contribute to the corneal sensorineural changes in dry eye.

TP63–TRIM29 axis regulates enhancer methylation and chromosomal instability in prostate cancer

BackgroundProstate adenocarcinoma (PRAD) is the second leading cause of cancer-related deaths in men. High variability in DNA methylation and a high rate of large genomic rearrangements are often observed in PRAD.ResultsTo investigate the reasons for such high variance, we integrated DNA methylation, RNA-seq, and copy number alterations datasets from The Cancer Genome Atlas (TCGA), focusing on PRAD, and employed weighted gene co-expression network analysis (WGCNA). Our results show that only single cluster of co-expressed genes is associated with genomic and epigenomic instability. Within this cluster, TP63 and TRIM29 are key transcription regulators and are downregulated in PRAD. We discovered that TP63 regulates the level of enhancer methylation in prostate basal epithelial cells. TRIM29 forms a complex with TP63 and together regulates the expression of genes specific to the prostate basal epithelium. In addition, TRIM29 binds DNA repair proteins and prevents the formation of the TMPRSS2:ERG gene fusion typically observed in PRAD.ConclusionOur study demonstrates that TRIM29 and TP63 are important regulators in maintaining the identity of the basal epithelium under physiological conditions. Furthermore, we uncover the role of TRIM29 in PRAD development.

Isolation and characterization of rabbit limbal niche cells

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs.Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRβ) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRβ and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRβ in rabbit-LNCs P4.In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRβ+).

One therapy to protect them all: Controlling diabetic eye disease in anterior and posterior segment via mitophagy‐activating drugs

Aims/Purpose: The accumulation of dysfunctional mitochondria has been implicated in the pathogenesis of diabetic eye disease through the impairment of mitochondrial quality control (MQC). This study aimed to investigate whether mitophagy‐activating drugs can alleviate pathology in the anterior and posterior ocular tissues of type‐1 diabetic mice.Methods: Mitophagy was evaluated in corneal and retinal tissues from diabetic mitophagy reporter mice (mitoQC‐Ins2Akita). The therapeutic potential of targeting mitophagy was evaluated by treating diabetic Ins2Akita mice with PIA‐01 (a PINK1‐independent mitophagy activator). Treatment was delivered orally between 4 and 8 months of diabetes. Ocular degenerative changes were evaluated by scotopic electroretinography (ERG), along with immunohistochemical analysis of retinal neurons and corneal cytoarchitecture.Results: Indicative of deteriorated MQC, mitophagy and mitochondrial transcription factor A (TFAM) positive mitochondrial nucleoids (which contain mtDNA) significantly decreased in the cornea and retina by 8 months of diabetes. Oral delivery of PIA‐01 restored TFAM+ mitochondrial nucleoids, leading to improved visual outcomes in Ins2Akita mice. In the retina, this was reflected by improved ERG responses (e.g. increased scotopic a‐wave and b‐wave amplitudes), while also protecting retinal neurons, including cone‐photoreceptors, synaptic structures, amacrine cells and retinal ganglion cells. This protection extended to the anterior segment, where PIA‐01 rescued corneal cytoarchitecture (basal epithelium and stroma) in diabetic mice.Conclusions: Diabetes progresses with deterioration of MQC in anterior and posterior ocular tissues. Rescuing MQC via mitophagy‐activating drugs may allow for multisystem therapies to control diabetic eye disease.

Nucleus-Mitochondria Contact Sites Are Associated With Asynthetic Fission in Zebrafish Skin.

Rapid increase in body surface area of growing zebrafish larvae (Danio rario) is partially accomplished by asynthetic fission of superficial epithelial cells (SECs) of the skin. There are two cycles of this atypical form of cell division which is unaccompanied by DNA replication; resulting in cells with a variable DNA content. Here, electron microscopy of basal epithelium cells that give rise to these SECs in zebrafish larvae shows aggregation of mitochondria around the nucleus and the formation of nucleus-mitochondria membrane contact sites. Membrane aggregates appear in the lumen of the nuclear envelope at these sites of membrane contact in some cells, suggesting lipid turnover in this vicinity. As the epithelial cells mature and stratify, the mitochondria are engulfed by extensions arising from the nuclear envelope. The mitochondrial outer membrane fragments and mitochondria fuse with the nuclear envelope and parts of the endoplasmic reticulum. Other organelles, including the Golgi apparatus, progressively localize to a central region of the cell and lose their integrity. Thus, asynthetic fission is accompanied by an atypical pattern of organelle destruction and a prelude to this is the formation of nucleus-mitochondria membrane contact sites.

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.

Allogeneic serum eye drops Sjogren syndrome‐related dry eye disease: Clinical evaluation of the ocular surface and in vivo confocal microscopy of the cornea

Aims/Purpose: To evaluate the symptomatic and clinical efficacy and in vivo confocal microscopy (IVCM) changes of the cornea after allogeneic serum eye drop therapy in patients with Sjögren's syndrome (SS)‐related Dry Eye Disease (DED).Methods: In this prospective clinical study 15 patients (30 eyes) with primary or secondary SS‐related DED were evaluated for Ocular Surface Disease Index (OSDI) questionnaire, ocular inflammation grade, Schirmer's test, tear film break‐up time (TBUT), corneal and conjunctival staining, and in vivo confocal microscopy (IVCM) analysis.Results: The average age of patients was 63.87 ± 9.18 years. The patients received treatment for a mean of 81.13 ± 56.84 days. The difference between before and after treatment with allogeneic serum eye drop therapy was statistically significant for OSDI parameters (p = 0.001), ocular inflammation (p = 0.004), TBUT (p = 0.007), Oxford Staining Scale (p = 0.007), Van Bijsterveld score (p = 0.006), basal epithelium density (p = 0.005), intermediate epithelium density (p = 0.041), Corneal Nerve Branch Density (CNBD) (p = 0.040), Beadings density (p = 0.047) and Fibre Tortuosity (p = 0.002).Conclusions: Allogeneic SEDs treatment in SS related DED patients, showed great results in terms of improvement in dry eye symptoms, clinical signs and IVCM changes. Further studies that aim at propagating and standardizing its production with clear international guidelines are needed.

Hypoxia-inducible microRNA-155 negatively regulates epithelial barrier in eosinophilic esophagitis by suppressing tight junction claudin-7.

MicroRNA (miRNA)-mediated mRNA regulation directs many homeostatic and pathological processes, but how miRNAs coordinate aberrant esophageal inflammation during eosinophilic esophagitis (EoE) is poorly understood. Here, we report a deregulatory axis where microRNA-155 (miR-155) regulates epithelial barrier dysfunction by selectively constraining tight junction CLDN7 (claudin-7). MiR-155 is elevated in the esophageal epithelium of biopsies from patients with active EoE and in cell culture models. MiR-155 localization using insitu hybridization (ISH) in patient biopsies and intra-epithelial compartmentalization of miR-155 show expression predominantly within the basal epithelia. Epithelial miR-155 activity was evident through diminished target gene expression in 3D organotypic cultures, particularly in relatively undifferentiated basal cell states. Mechanistically, generation of a novel cell line with enhanced epithelial miR-155 stable overexpression induced a functionally deficient epithelial barrier in 3D air-liquid interface epithelial cultures measured by transepithelial electrical resistance (TEER). Histological assessment of 3D esophageal organoid cultures overexpressing miR-155 showed notable dilated intra-epithelial spaces. Unbiased RNA-sequencing analysis and immunofluorescence determined a defect in epithelial barrier tight junctions and revealed a selective reduction in the expression of critical esophageal tight junction molecule, claudin-7. Together, our data reveal a previously unappreciated role for miR-155 in mediating epithelial barrier dysfunction in esophageal inflammation.

Immunohistochemical Study of MDM2 Expression in Odontogenic Keratocyst andDentigerous Cyst

Introduction: Dentigerous cyst and odontogenic keratocyst (OKC) are two of the most common developmental odontogenic cysts. Different developmental mechanism and biological behavior of OKC is due to unknown factors inherent in the epithelium or enzymatic activity in the wall. P53 plays a role in determining the pathways for DNA repair، apoptosis، angiogenesis and genomic stability and is negatively confronted with the MDM2 oncoprotein. Aim of this study was to evaluate the expression of MDM2 as an important indicator of proliferation and cell cycle regulation in OKC and dentigerous cyst.&#x0D; Methods: In this descriptive-cross-sectional study، MDM2 expression in the suprabasal and basal epithelium regions of 15 samples from each of the dentigerous cysts and OKC was assessed using immunohistochemistry. Finally، the data were entered into the SPSS16 software and analyzed using the t-test.&#x0D; Results: MDM2 expression in the suprabasal and basal epithelium regions in OKC was higher than dentigerous cyst، but there was not seen a statically significant difference (P: 0.551)، (P: 0.825). The expression level of this marker in both groups in the suprabasal region was significantly higher than the basal region (P: 0.005)، (P: 0.004).&#x0D; Conclusion: Higher expression of MDM2 in OKC، especially in the suprabasal areas، could indicate a secondary role of this protein in the pathogenesis، growth and spread of this cyst and the proliferation، apoptosis and differentiation processes confirm the neoplastic nature and specific biological behavior of this lesion.

Systemic Histology of the Marine Water-Strider Halobates hayanus (Heteropera, Gerridae)

The structure and cell types of the organs of Halobates hayanus from Libong Island, Thailand, were investigated using a histological method. The integumentary system of this species consists of three layers: epicuticle, exocuticle, and endocuticle. Throughout the body, the muscular system exclusively included skeletal muscle. In the excretory system, we found four fully formed Malpighian tubules, each one lined with a straightforward cuboidal epithelium harbouring secretory granules. The digestive system comprised a foregut, midgut, and hindgut. The midgut had three distinct cell types: basal cells, epithelium, and absorptive cells. The nervous system comprised two regions: A ventral nerve cord and a frontal ganglion, which is connected to the eye structure. The frontal ganglion was composed of two lobes each containing an outer cortex and inner medulla. The outer cortex presented neurosecretory cells, neuroglia cells, and neurons. The neurosecretory cells were large and contained secretory granules in their cytoplasm. This histological study also showed the reproductive system of this gerrid species, including the reproductive tract, and the accessory organ.