Epithelial Basement Research Articles

Expression of HGF/c‐Met in control and keratoconus corneas

AbstractPurpose Keratoconus (KC) is the most common primary degenerative corneal disease with a prevalence of around 1 in 2000 worldwide. A recent genome‐wide association study on potential genes associated with KC identified a significant association between KC and two single nucleotide polymorphisms in the promoter region of the HGF gene. The HGF/c‐Met pathway protects cells from apoptosis and is important to maintain epithelial homeostasis. In this study, we investigated if the HGF/c‐Met pathway is involved in KC by comparing HGF/c‐Met protein expression in control and KC samples.Methods HGF and c‐Met immunoreactivities in control (n=5) and KC (n=10) corneas were imaged by confocal microscopy.Basal tears were collected by capillary micropipettes from control and KC patients (n=5 each), and HGF protein expression determined by Western blot. Intracellular c‐Met (the membrane bound receptor for HGF) expression was detected by extracting proteins from both control and KC corneal epithelium (n=5 each) followed by Western blot.Results HGF and c‐Met were expressed in control and KC corneas. However,in KC obvious cytoplasmic staining of basal epithelium was noted compared to the more diffuse cytoplasmic epithelial immunostaining in control corneas. Western blot results showed that lower levels of HGF in KC tears compared to control tears, and the intracellular c‐Met expression was also weaker in KC cell lysates compared to controls.Conclusion We find that HGF/c‐met expression patterns were altered between KC and control corneas, and their expression levels were reduced in KC tears and corneal epithelium cell lysates suggesting a role for this pathway in the pathogenesis of KC. Funding: Medical School of Sydney University, Sydney Eye Hospital Foundation, Lions NSW Eye Bank

Expression and distribution of the transient receptor potential cationic channel ankyrin 1 (TRPA1) in the human vagina.

The transient receptor potential cationic channel type A1 (TRPA1), belonging to a superfamily of cationic membrane channels, has been suggested to act as mechano- and pain sensor and, thus, to play a role in neurotransmission in the human body, including the urogenital tract. While the expression of TRPA1 has been investigated in a variety of tissues, up until today, no study has addressed the expression and distribution in the female genital tract. The present study aimed to investigate the expression and distribution of TRPA1 protein in human vaginal tissue. Reverse transcriptase PCR (RT-PCR) was applied in order to identify messenger ribonuleic acid specifically encoding for TRPA/A1. The distribution of TRPA1 in relation to the neuronal nitric oxide synthase (nNOS) and the signaling peptide calcitonin gene-related peptide (CGRP) was examined by means of immunohistochemical methods (double-antibody technique, laser fluorescence microscopy). RT-PCR analysis revealed the expression of mRNA encoding sequences specific for TRPA in the vaginal wall and epithelium. Immunostaining related to TRPA1 was observed in the basal epithelium and in slender varicose nerve fibers transversing the subepithelial and stromal space of the vaginal sections. In addition, these fibers presented immunoreactivity specific for nNOS or CGRP. The smooth musculature of the vaginal wall and small vessels interspersing the tissue did not present signals related to TRPA1. The findings indicate that TRPA1 might be involved in afferent neurotransmission in the vagina and work synergistically together with the nitric oxide/cyclic guanosine monophosphate pathway.

Temporal changes in neutral endopeptidase/CD10 immunoexpression in the cyclic and early pregnant canine endometrium

CD10 is a multifunctional transmembrane neutral endopeptidase (NEP) that is considered to be a reliable marker of ectopic human endometrial stroma. Available information on NEP/CD10 protein expression in animal endometria is scarce. This study focused on the immunolocalization of NEP/CD10 in the canine uterus and on its temporal changes during the estrous cycle and early pregnancy (Days 11 to 23 post-LH surge) in healthy females. NEP/CD10 expression was found in the canine endometrial stroma in all stages of the estrous cycle, showing cyclic differences both in intensity and in distribution pattern. A small population of negative stromal cells in subsurface position was also observed. This population shared some morphological characteristics with the human predecidual cells, which became positive in progesterone-associated stages of the cycle. In addition, positive immunolabeling was also observed in canine myometrial stroma. In early pregnancy, the basal glandular epithelia and the syncytium cords remained negative to this marker contrasting with the trophoblast and the lacunar epithelium. A weak to moderate intensity of immunolabeling was observed in the decidual cells, whereas stromal immunolabeling was more intense at the delimitation of the syncytium cords. In conclusion, CD10 is consistently expressed in the canine endometrial stroma and myometrium but not in the endometrial epithelia. The characteristic pattern seen in early pregnancy also suggests a role for this molecule in the process of embryo invasion at implantation.

Lineage-restricted function of the pluripotency factor NANOG in stratified epithelia.

NANOG is a pluripotency transcription factor in embryonic stem cells; however, its role in adult tissues remains largely unexplored. Here we show that mouse NANOG is selectively expressed in stratified epithelia, most notably in the oesophagus where the Nanog promoter is hypomethylated. Interestingly, inducible ubiquitous overexpression of NANOG in mice causes hyperplasia selectively in the oesophagus, in association with increased cell proliferation. NANOG transcriptionally activates the mitotic programme, including Aurora A kinase (Aurka), in stratified epithelia, and endogenous NANOG directly binds to the Aurka promoter in primary keratinocytes. Interestingly, overexpression of Nanog or Aurka in mice increased proliferation and aneuploidy in the oesophageal basal epithelium. Finally, inactivation of NANOG in cell lines from oesophageal or head and neck squamous cell carcinomas (ESCCs or HNSCCs, respectively) results in lower levels of AURKA and decreased proliferation, and NANOG and AURKA expression are positively correlated in HNSCCs. Together, these results indicate that NANOG has a lineage-restricted mitogenic function in stratified epithelia.

Conditional Knockout of CTGF Affects Corneal Wound Healing

This study aimed to elucidate the role of connective tissue growth factor (CTGF) in healthy eyes and wounded corneas of mice and rabbits. Conditional knockout mice were used to determine the role of CTGF in corneal healing. CTGF expression was determined using transgenic mice carrying CTGF promoter driven-eGFP, quantitative RT-PCR, and immunofluorescent staining. Mice that carried two floxed CTGF alleles and a Cre/ERT2 transgene under the control of human ubiquitin C (ubc) promoter were used to conditionally delete CTGF gene in a tamoxifen-inducible manner. Phototherapeutic keratectomy (PTK) was used to generate an acute corneal wound and corneal re-epithelialization was assessed by fluorescein staining. Connective tissue growth factor expression was found in multiple ocular tissues with relatively high levels in the corneal endothelium, lens subcapsular epithelium, and in the vasculature of the iris and retina. Wounded corneas responded with an immediate upregulation of CTGF in the epithelium at the wound margin and a sustained CTGF induction during re-epithelialization. At the onset of haze formation, CTGF protein becomes more focused in the basal epithelium. Deletion of the CTGF gene caused a 40% reduction (P < 0.01) in the cornea re-epithelialization rate in knockout mice compared with wild-type mice. Connective tissue growth factor is expressed in the naïve cornea, lens, iris, and retina, and is expressed immediately after epithelial injury. Loss of CTGF impairs efficient re-epithelialization of corneal wounds.

MP19-17 SURFACE CD24 DISTINGUISHES BETWEEN LOW DIFFERENTIATED AND TRANSIT-AMPLIFYING CELLS IN THE BASAL LAYER OF THE HUMAN PROSTATE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 2014MP19-17 SURFACE CD24 DISTINGUISHES BETWEEN LOW DIFFERENTIATED AND TRANSIT-AMPLIFYING CELLS IN THE BASAL LAYER OF THE HUMAN PROSTATE Christian Schwentner, Neli Petkova, Jörg Hennenlotter, Tilman Todenhöfer, Hans-Joerg Buehring, Stefan Aufderklamm, and Arnuf Stenzl Christian SchwentnerChristian Schwentner More articles by this author , Neli PetkovaNeli Petkova More articles by this author , Jörg HennenlotterJörg Hennenlotter More articles by this author , Tilman TodenhöferTilman Todenhöfer More articles by this author , Hans-Joerg BuehringHans-Joerg Buehring More articles by this author , Stefan AufderklammStefan Aufderklamm More articles by this author , and Arnuf StenzlArnuf Stenzl More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.718AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are common abnormalities in elderly men. It is considered that epithelial stem cells are involved in the etiology and development of both diseases. To distinguish aberrant from normal cells, the knowledge about primary epithelial stem/progenitor cells (ES/P) is essential. The aim of this study was to examine the role of surface markers to distinguish between different subsets of prostate basal epithelium. METHODS The expression pattern of prostate tissue single cell suspensions was analyzed by flow cytometry using different markers. Sorted cell populations were examined for their clonogenic capacity and the resulted colonies were analyzed with flow cytometry, Western blot, and qPCR for stem cell, basal, and luminal epithelium markers. Additionally, the histological localization of the examined markers was determined using immunofluorescence. RESULTS Using the combination of CD49f, Trop-2, and surface CD24, basal cell subsets with distinct differentiation capacities were dissected (CD49f(+) Trop-2(+) CD24(-) and CD49f(+) Trop-2(+) CD24(+) ). Although cells from the two subsets gave rise to similar basal colonies, qPCR of primary tissue revealed that higher levels of basal marker expression were detected in the CD49f(+) Trop-2(+) CD24(-) subset. Immunofluorescence analysis showed a prominent expression of CD24 by luminal and basal cells. CONCLUSIONS Subsets with distinct differentiation capacities within the basal epithelium (CD49f(+) Trop-2(+) CD24(-) and CD49f(+) Trop-2(+) CD24(+) ) can be distinguished in human prostate. CD24 is a marker expressed on the basal transit-amplifying cells (transition cells) and may play a role in the differentiation and migration of ES/P cells to the luminal layer. The knowledge of this mechanism is of relevance for treatment of both diseases. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e195 Peer Review Report Advertisement Copyright & Permissions© 2014MetricsAuthor Information Christian Schwentner More articles by this author Neli Petkova More articles by this author Jörg Hennenlotter More articles by this author Tilman Todenhöfer More articles by this author Hans-Joerg Buehring More articles by this author Stefan Aufderklamm More articles by this author Arnuf Stenzl More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Invasive leader cells: metastatic oncotarget.

Invasion into surrounding tissues is the first step of metastasis, and an understanding of the molecular basis of the invasive process may suggest novel strategies for anti-metastatic therapy. Breast tumors typically invade as adherent groups of cells, a process known as collective invasion. In vivo, collective invasion occurs within a complex tissue environment and involves interactions among the cancer cells, stromal cells, and the extracellular matrix (ECM). An additional challenge to understanding cancer invasion is that primary breast tumors contain diverse cancer cells with varied molecular phenotypes. It is difficult to determine the relative contribution of these subpopulations to invasion from analysis of histologic sections. We sought to reduce this complexity and to identify the most invasive cancer cells in the primary tumor in an unbiased fashion [1]. To accomplish this goal, we developed a three dimensional (3D) organoid invasion assay (Figure ​(Figure1A).1A). Briefly, we use enzymes and mechanical disruption to isolate organoids from primary breast tumors from mouse models and human patients. Each organoid is composed of several hundred cancer cells and is embedded in an invasion promoting ECM [2] (collagen I). Tumor organoids invade collectively and the cells at the tips of invasion strands are protrusive and highly interactive with the ECM. We refer to the cells at the tips of these strands as invasive leader cells (Figure ​(Figure1B1B). Figure 1 A. Tumor organoids are isolated from fresh primary tumors through a combination of mechanical disruption and enzymatic digestion. Tumor organoids are then embedded in 3D collagen I and become invasive. B. Organoids extend multiple invasive strands into ... We found that the molecular phenotype of invasive leader cells was distinct from the bulk tumor cells and conserved across breast cancer subtypes [1]. Leader cells expressed markers of basal epithelium, which are the cells in normal stratified tissues that interact with the ECM (Figure ​(Figure1C).1C). The basal cytokeratin, keratin-14 (K14), was expressed in ~90% of leader cells. Importantly, K14+ cells led collective invasion in multiple distinct mouse models and in diverse primary human breast tumors. We next used molecular biosensors to demonstrate that K14+ invasive leader cells were generated from non-invasive K14- bulk tumor cells. Importantly, knockdown of either K14 or p63, another leader cell marker, was sufficient to inhibit collective invasion. Our data establish three important concepts. First, we have demonstrated that the invasive behavior of the primary tumor is determined by a subset of specialized cancer cells1. Second, our data suggest a common molecular biology underlying breast cancer metastasis, because K14+ cells led collective invasion in 3D organoid invasion assays from patients with different breast cancer subtypes. In support of this concept, multiple groups have observed that K14 expression in breast tumors correlates with poor patient outcomes, independent of breast cancer subtype [3-5]. Third, since bulk tumor cells can convert into K14+ invasive leaders, we hypothesize that inhibition of the basal molecular program could control progression in patients with metastatic disease. Our 3D organoid invasion assay can also be utilized to identify invasive leader cells in other epithelial cancers. Because basal cells are found in many stratified epithelial tissues, the basal invasive program we identified may be utilized across diverse cancers. In support of this concept, basal cytokeratins are associated with invasion in both lung adenocarcinoma and hepatocellular carcinoma [6,7]. It is now critical to develop the translational framework to leverage our knowledge of invasive leader cells to improve patient outcomes. We are specifically interested to determine whether the abundance of invasive leader cells in the primary tumor will provide independent information about the prognosis of individual patients. Our invasion assays also provide an ideal platform to identify anti-invasive therapeutic compounds. Now that we have the invasive leader cell in our sights, we are working to defeat this new adversary.

Frizzled 7 Maintains the Undifferentiated State of Human Limbal Stem/Progenitor Cells

Wnt signaling pathway plays an important role in the regulation of human limbal stem/progenitor cells (LSCs). To examine the possible function of Frizzled (Fz) receptors in LSCs, the expression of 10 Fz receptors was profiled in the limbus and cornea. Only Fz7 had preferential expression in the basal limbal epithelium which contains the LSCs. The expression of Fz7 was colocalized with the putative LSC markers including p63α, N-cadherin and keratin (K) 14, and was minimum in cells expressing the corneal maturation marker K12. The expression of Fz7 was higher in the enriched LSCs population and decreased in cultured LSCs when there was a loss of progenitor phenotype. When the Fz7 was knocked down (Fz(KD)) using shRNA in primary LSCs, the expression of putative LSC markers ABCG2, ΔNp63α, and K14 was decreased significantly. The colony forming efficiency of the Fz7(KD) LSCs was significantly decreased in the subsequent passage 1 and 2 compared to the control. Our finding suggests that Wnt signaling is one of the factors of LSC niche, and Fz7 helps to maintain the undifferentiated state of LSCs.

Antigen-Presenting Cells Are Stratified Within Normal Human Corneas and Are Rapidly Mobilized During Ex Vivo Viral Infection

To define the phenotype and location of antigen-presenting cells (APCs) in normal donor human corneas, and to assess the response of APC to herpes simplex virus type 1 (HSV-1) infection. Donor human corneal tissue was analyzed by fluorescence confocal microscopy and flow cytometry to determine the phenotype and location of tissue-resident APCs. Confocal fluorescence microscopy was also utilized to investigate the response of corneal resident APCs to ex vivo infection with HSV-1. CD11c(+) dendritic cells (DCs) and CD207(+) Langerhans cells (LCs) were situated predominantly in the basal epithelium and CD68(+) macrophages in the anterior stroma of human corneas. The majority of DCs expressed major histocompatibility complex class II. Corneal resident APCs colocalized with HSV-1-infected corneal cells within 8 to 16 hours of ex vivo infection. The stratification of APCs found in human corneas is very similar to that previously reported in mice, confirming the relevance of murine models for the study of corneal APCs. Furthermore, corneal resident APCs are capable of rapidly mobilizing to the site of trauma and HSV-1 infection within the cornea.

Epitheliotrophic Cutaneous T Cell Lymphoma (CETL) in a Labrador Retriever Dog. A Case Report

A female 7 years old Labrador retriever was attended with a severe dermatological disease. The dog was treated with cephalexin (25 mg/kg/12 h/10 days), chlorhexidine shampoo and nutraceuticals (Omega 3, vitamins and minerals), with no clinical improvement. The clinical and histopathology results suggested a diagnosis of mycosis fungoides. Accordingly, treatment schedule was changed, but the dog did not show any clinical remission after several days under treatment and died. Immunohistochemistry evidenced a severe infiltration of CD3 T-cells collected around the basal squamous epithelia. Immunohistochemistry results confirmed the first mycosis fungoides case report in Colombia. Clinical findings and the most relevant treatment schedules are discussed.

Analysis of the Neoplastic Nature of Odontogenic Keratocyst and its Comparison with other Selected Benign Odontogenic Tumours: An Immunohistochemical Analysis

Odontogenic keratocyst (OKC) first described by Philipsen in 1956 constitutes approximately 11% of all cysts of the jaws. Adenomatoid odontogenic tumour (AOT) is an uncommon, benign epithelial lesion of odontogenic origin. The aim of this study was to analyse the expression of Bcl-2 in OKC and its comparison with other selected benign odontogenic tumours (OTs). Ten formalin fixed paraffin embedded blocks of OKCs, five each of AOT and unicystic ameloblastoma Bcl-2 protein is characterized by its ability to inhibit apoptosis. OKC were characterized by higher expression of Bcl-2 in basal cell epithelium. AOT and unicystic ameloblastoma differed from OKC in a wide spectrum of apoptosis and/or cell cycle-related protein expressions, higher proliferation in the basal cell layer, and vice versa, lower proliferation in the suprabasal cell layer. The solitary OKC seems to be less biologically aggressive and should be classified as a cyst rather than a tumour, means that at least few of OKCs manifests as ordinary cysts. Some of the present study findings could support the theory that OKCs are with high proliferative, probably that these lesions are developmental cysts with some neoplastic properties because of the high intrinsic growth potential. WHO recommends the term KCOT as it better reflects the neoplastic nature of the lesion; however, this reclassification has not yet been universally accepted.

Basal Cell Signaling by p63 Controls Luminal Progenitor Function and Lactation via NRG1

The mammary epithelium is organized as a bilayer ofluminal and basal/myoepithelial cells. During pregnancy, the luminal compartment expands for milk production, while basal cells are thought to provide structural and contractile support. Here, we reveal a pregnancy-specific role of basal epithelia as a central coordinator of lactogenesis. We demonstrate that genetic deletion of the transcription factor p63 (Trp63) gene exclusively within basal cells of the adult gland during pregnancy leads to dramatic defects in luminal cell proliferation and differentiation, resulting in lactation failure. This phenotype is explained by direct transcriptional activation of the epidermal growth factor family ligand gene Nrg1 by p63 selectively in basal cells, which is required for luminal ERBB4/STAT5A activation and consequent luminal progenitor cell maturation. Thus, paracrine basal-to-luminal cell signaling, controlled by p63 via NRG1, orchestrates the entire lactation program. Collectively, these findings redefine the paradigm for cellular interactions specifying the functional maturation of the mammary gland.

Retinoic acid regulation by CYP26 in vertebrate lens regeneration

Xenopus laevis is among the few species that are capable of fully regenerating a lost lens de novo. This occurs upon removal of the lens, when secreted factors from the retina are permitted to reach the cornea epithelium and trigger it to form a new lens. Although many studies have investigated the retinal factors that initiate lens regeneration, relatively little is known about what factors support this process and make the cornea competent to form a lens. We presently investigate the role of Retinoic acid (RA) signaling in lens regeneration in Xenopus. RA is a highly important morphogen during vertebrate development, including the development of various eye tissues, and has been previously implicated in several regenerative processes as well. For instance, Wolffian lens regeneration in the newt requires active RA signaling. In contrast, we provide evidence here that lens regeneration in Xenopus actually depends on the attenuation of RA signaling, which is regulated by the RA-degrading enzyme CYP26. Using RT-PCR we examined the expression of RA synthesis and metabolism related genes within ocular tissues. We found expression of aldh1a1, aldh1a2, and aldh1a3, as well as cyp26a1 and cyp26b1 in both normal and regenerating corneal tissue. On the other hand, cyp26c1 does not appear to be expressed in either control or regenerating corneas, but it is expressed in the lens. Additionally in the lens, we found expression of aldh1a1 and aldh1a2, but not aldh1a3. Using an inhibitor of CYP26, and separately using exogenous retinoids, as well as RA signaling inhibitors, we demonstrate that CYP26 activity is necessary for lens regeneration to occur. We also find using phosphorylated Histone H3 labeling that CYP26 antagonism reduces cell proliferation in the cornea, and using qPCR we find that exogenous retinoids alter the expression of putative corneal stem cell markers. Furthermore, the Xenopus cornea is composed of an outer layer and inner basal epithelium, as well as a deeper fibrillar layer sparsely populated with cells. We employed antibody staining to visualize the localization of CYP26A, CYP26B, and RALDH1 within these corneal layers. Immunohistochemical staining of these enzymes revealed that all 3 proteins are expressed in both the outer and basal layers. CYP26A appears to be unique in also being present in the deeper fibrillar layer, which may contain cornea stem cells. This study reveals a clear molecular difference between newt and Xenopus lens regeneration, and it implicates CYP26 in the latter regenerative process.

Expression and Activity of Collagenases in the Digital Laminae of Horses with Carbohydrate Overload‐Induced Acute Laminitis

BackgroundMatrix metalloproteinases (MMP) are hypothesized to degrade structurally important components of the laminar extracellular matrix (ECM) in horses with laminitis.ObjectiveTo compare levels of expression of stromelysin‐1 (MMP‐3), collagenases (MMP‐1, ‐13), and membrane type‐MMPs (MMP‐14, ‐15, ‐16), and the distribution of their ECM substrates, in laminae of healthy horses and horses with carbohydrate overload laminitis.AnimalsTwenty‐five adult horses.MethodsGene and protein expression were determined in extracts of laminae using real‐time quantitative polymerase chain reaction and Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis. Distribution of MMP‐13 and ECM components was determined using indirect immunofluorescent microscopy of nonfixed frozen sections. ECM morphology was assessed by hematoxylin and eosin staining.ResultsOf the genes studied, only those encoding MMP‐1 and ‐13 were upregulated in CHO‐induced laminitis; MMP‐1 at Obel grade (OG)1 lameness and MMP‐13 at OG3 lameness. Laminar MMP‐1 was present as 52 kDa proenzyme only. MMP‐13 was present as pro‐ (61 kDa) and processed (48 kDa) enzyme. MMP‐13 localized to the basal epithelium of the secondary epidermal laminae and its increased expression were accompanied by the appearance in secondary dermal laminae (SDL) of multiple foci that were devoid of collagen I, fibronectin, chondroitin and keratan sulfate glycosaminoglycans, and eosin‐staining material.Conclusions and Clinical Relevance MMP‐13 is upregulated in laminae of horses with CHO‐induced OG3 lameness and, by degrading components of the ECM, may contribute to the formation of ECM‐free lesions (gaps or tears) that appear in the SDL with OG3 lameness.

Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing

We reported the expression of phosphorylated HSP27 during epithelial wound healing in murine corneas (Jain et al., 2012) in July of 2012. This in vivo investigation demonstrated that the expression levels of phosphorylated HSP27 were greater in wounded corneal epithelial cells than in unwounded controls and that the localization of phosphorylated HSP27 was in the basal and superficial epithelia three days following corneal epithelial wounding. We suggested that phosphorylated HSP27 had a role in the early phase of corneal epithelial wound healing. The purpose of this study was to investigate the exact role of heat shock protein 27 (HSP27) phosphorylation for the wound healing of cultured human corneal epithelial cells (HCECs). HSP27-specific siRNAs and control-siRNAs, with no known homologous targets in HCECs, were created. The cultured HCECs were divided into two groups: Scrambled control-siRNA-transfected group vs. HSP27-specific siRNA-transfected group. The scratch-induced directional wounding assay, Western blotting, using antibodies against non-phosphorylated and phosphorylated HSP27, non-phosphorylated and phosphorylated Akt, and Bcl-2–associated X protein (Bax), immunofluorescence staining to determine the filament actin, flow cytometry to measure apoptosis, and proliferation assay were performed to determine the role of HSP27. Western blot assay showed that the expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding, compared with those in unwounded HCECs (all p < 0.05). Western blot assay also showed HSP27-specific siRNAs effectively blocked the expression of non-phosphorylated HSP27. The HSP27-specific siRNA-transfected group had more Bax expression, less phosphorylated Akt expression, and less non-phosphorylated and phosphorylated HSP27 expression (all p < 0.05). The scratch-induced directional wounding assay showed the HSP27-specific siRNA-transfected group with a less migrating cell number than the control-siRNA-transfected group (p < 0.05). Immunofluorescence staining showed that reorganization of actin cytoskeleton prominently decreased in the HSP27-specific siRNA-transfected group, compared with the control siRNA-tranfected group. Flow cytometry revealed that the HSP27-specific siRNA-transfected group had more HCEC apoptosis. Proliferation assay showed no difference between the two groups. In conclusion, the role of HSP27 in corneal epithelial wound healing can be epithelial cell apoptosis, as well as epithelial migration. HSP27 is involved in HCEC migration by the reorganization of actin cytoskeleton.

In vivo confocal microscopy of bulbar conjunctiva in patients with Graves' ophthalmopathy

To investigate the bulbar conjunctival changes in patients with Graves' ophthalmopathy (GO) using in vivo confocal microscopy. A total of 15 GO patients and 15 control patients were recruited. Images of the superior site and temporal bulbar conjunctivas were taken by the Heidelberg Retina Tomograph/Rostock Corneal Module. The conjunctival thickness and cell density of superior epithelium, basal epithelium, Langerhans cells, and goblet cells were analyzed. Conjunctival impression cytology was performed to assess the grading of squamous metaplasia of the conjunctival epithelium. The superficial epithelial cell density in the superior bulbar conjunctiva of the GO group was 856.93 ± 461.68 cells/mm(2), which was significantly lower than that in the control group (1581.13 ± 556.34 cells/mm(2); p = 0.002). However, the difference in superficial epithelial cell density in the temporal bulbar conjunctiva between the two groups was not statistically significant. No significant differences in conjunctival thickness and the basal epithelial cell density were noted between the two groups. Increased Langerhans cell density and reduced goblet cell density were noted in the GO group. Impression cytology of the conjunctival epithelium revealed significantly higher degree of squamous metaplasia in the GO group. The superficial epithelial cell density in the upper bulbar conjunctiva showed negative correlation with marginal reflex distance in the GO group. GO patients suffered from more severe bulbar conjunctival damage and inflammation with the superior site than the temporal site. In vivo confocal microscopy can be a rapid and noninvasive tool for the quantitative evaluation of ocular surface changes in patients with GO.

The effects of ocular hypotensive drugs on the cornea: An in vivo analysis with confocal microscopy

PurposeTo evaluate the effects of anti-glaucoma treatments containing benzalconium chloride (BAC) on the human cornea. MethodsA prospective single masked cohort study was conducted on the 50 eyes of 50 patients. The inclusion criteria were recently diagnosed glaucoma or ocular hypertension with previous treatment, or ophthalmologist-prescribed anti-glaucoma therapy, and oral consent to participate in the study. The patients were not randomized, as the ophthalmologist decided the best therapy according to clinical criteria. The patients were divided in 2 cohorts: those exposed to BAC (23 patients), and those not exposed (27 patients). The mean follow-up period was 22 weeks (range 18–30). The change in cell density before and after therapy was measured in basal layer epithelium, basal layer of limbal epithelium and endothelium. The change in stromal reflectivity and the number of nerve branches in sub-basal nerve plexus was also measured. BAC exposure was blinded to the main researcher. ResultsA greater increase in basal layer epithelium cell density was observed in BAC exposed cohort (p<0.05). No significant differences were detected in the endothelium, limbal cell density, stromal reflectivity, or sub-basal nerve plexus. Age, sex, IOP, active ingredient or BAC concentration did not affect the direction or magnitude of the ocular surface alterations found. ConclusionChronic anti-glaucoma therapy induces changes in the corneal epithelium. Preservative free drops showed less disruption of the ocular surface by confocal microscopy analysis. Further studies should be conducted to evaluate the clinical impact of these histological findings.

The effect of prenatal exposure to 900-megahertz electromagnetic field on the newborn rat testicles

Intensive use in daily life of devices with an electromagnetic field (EMF), such as radios, televisions, computers and mobile phones, results in constant exposure. The fact that more than 80% of people in many countries use mobile phones has further increased concerns about the effect of EMFs. The aim of this study was to investigate the effect of exposure to a 900-megahertz (MHz) EMF in the prenatal term on the newborn rat testicles. Six pregnant, female Sprague Dawley rats were equally divided into control (CG) and EMF (EMFG) groups. No procedure was performed on CG. EMFG was exposed to a 900 MHz EMF for 1 h during days 13-21 of pregnancy at the same hour every day. No procedure was performed on newborn female rats after birth, and the study continued with newborn male rats. Newborn CG (NCG, n: 10) male rats were obtained from mothers in the CG, and newborn EMFG (NEMFG, n: 10) male rats from mothers in the EMFG. All animals were sacrificed and the testicles extracted at postnatal day 21. Testicular tissue samples were evaluated histopathologically under light microscope. The TUNEL technique was used to determine apoptosis in testicles. Lipid peroxidation and DNA oxidation levels were evaluated biochemically. Histopathologically, NEMFG male rats exhibited irregularities in the seminiferous tubule basal membrane and epithelium of seminiferous tubules, immature germ cells in the lumen, and a decreased diameter of seminiferous tubules and thickness of epithelium. Apoptotic index, lipid per oxidation and DNA oxidation were higher in NEMFG male rats than in NCG. Our results showed that the newborn rat testicles exposed to 900 MHz EMF in the prenatal period has been adversely affected.

Role of Ultrastructural Alterations of Intercellular Junction and Tight-junction Proteins in Patients With Gastroesophageal Reflux Disease

TO THE EDITOR: I read with great interest the study by Liu et al 1 regarding the significant correlation between presence of desmosomal junction alterations and the severity of gastroesophageal reflux disease (GERD). The authors reported that tight-junction proteins (TJP) such as claudin-1 and claudin-2 were abundant not only in asymptomatic controls but also in patients with gastroesophageal reflux disease and, a significant intercellular space (ICS) dilatation occurred only in upper stratified squamous epithelium of patients with moderate erosive esophagitis, while there was no difference in ICS dilatation of lower basal cuboidal epithelium among different groups of patients with GERD and controls. Furthermore, this study revealed that alterations in desmosomal junctions and their processes under transmission electron microscopy increased in parallel with the severity of GERD. Esohageal mucosal ICS were frequently seen with nonerosive reflux disease (NERD) and patients with esophagitis. 2 In addition, dilatation of intercellular spaces was an early marker of damage caused by gastroesophageal reflux, and treatment of proton pump inhibitor led to a complete recovery of dilated intercellular spaces in 97.4%. 3 Although the authors showed an important results of ultrastructural alterations of esophageal mucosa in patients with GERD, there were a few limitations most of which had been discussed in this paper. First, in contrast to this study, previous studies demonstrated that dilated ICS was associated with NERD and erosive GERD. For instance, Caviglia et al 4 reported that mean ICS diameter in NERD patients was 3 times greater than in controls. Moreover, Zhong et al 5 reported that reflux esophagitis (RE) patients showed wider ICS (1.29 μm [1.10-1.46 μm], n = 6) than NERD patients (1.10 μm [0.95-1.21 μm], n = 16) and controls (1.01 μm [0.94-1.17 μm], n = 12). Differences in the study population and definition of GERD severity may contribute to this discrepancy. In this study, authors defined mild RE in case of Los Angeles classification A (LA-A) esophagitis and moderate RE in case of LA-B esohagitis, but there were no patients with LA-C or LA-D eosphagitis. However, Zhong et al 5 classified LA-A/B into mild RE and LA-C/D into severe RE. I wonder whether the severe GERD patients including LA-C/D may have influence on the results or not, but there was no explanation regarding this matter in the discussion. Second, the authors showed that there were no differences in the presence of claudin-1 and claudin-2 expression among patients with GERD, NERD and controls. However, previous study revealed that claudin-1 expression in RE and NERD patients were significantly higher than in the control. Moreover, expression of occludin tended to be lower in the RE patients and the expression of claudin-3 tended to be higher in the RE and NERD patients, however, none of these were statistically significant. 3 These findings suggested the role of several other TJP including several types of claudins, occludins and junctional adhesion molecules, as authors have mentioned in the discussion. Despite these limitations of the present study, it provides us with the important information in the ultrastructural alterations of ICS and TJP in the inflamed esophageal mucosa. Further studies are needed to validate the results of the present study.

The effect of prenatal exposure to 900-MHz electromagnetic field on the 21-old-day rat testicle

The aim of this study was to investigate the effect of exposure to a 900-MHz electromagnetic field (EMF) in the prenatal term on the 21-old-day rat testicle. Pregnant rats were divided into control (CG) and EMF (EMFG) groups. EMFG was exposed to 900-MHz EMF during days 13–21 of pregnancy. Newborn CG rats were obtained from the CG and newborn EMFG (NEMFG) rats from the EMFG. Testicles were extracted at postnatal day 21. Lipid peroxidation and DNA oxidation levels, apoptotic index and histopathological damage scores were compared. NEMFG rats exhibited irregularities in seminiferous tubule basal membrane and epithelium, immature germ cells in the lumen, and a decreased diameter in seminiferous tubules and thickness of epithelium. Apoptotic index, lipid peroxidation and DNA oxidation were higher in NEMFG rats than in NCG. 21-day-old rat testicles exposed to 900-MHz EMF in the prenatal term may be adversely affected, and this effect persists after birth.