Normal Conjunctival Epithelium Research Articles

Impression Cytology in the Evaluation of Ocular Surface Squamous Neoplasia- A Retrospective Cohort Study

Introduction: Ocular Surface Squamous Neoplasia (OSSN) are a diverse group of lesions varying from Conjunctival Intraepithelial Neoplasia (CIN) to invasive Squamous Cell Carcinomas (SCC). Histopathological examination is the gold standard technique in diagnosing them. However, it is invasive and may not be suitable in recurrent cases. Numerous minimally invasive techniques are available to diagnose OSSN cytologically. This study employs Impression Cytology (IC) to diagnose ocular squamous neoplastic lesions and compare it with the histological diagnosis. Aim: To utilise IC in the diagnosis of OSSN and to compare its findings with histopathological diagnosis. Materials and Methods: This was a retrospective cohort study, conducted in a Tertiary Eye care Hospital, Hubli, Karnataka, India during January 2023. The data included was over a period of three years (from July 2016 to June 2019). A total of 44 eyes (34 patients), in which both biopsy and IC done, were included. The patients’ age, sex and ocular examination findings were recorded. The cytological and histopathological slides were retrieved from the archives and examined morphologically. The cytological findings were compared with histopathological diagnosis and the sensitivity and specificity of the IC technique were calculated. The data was analysed using Microsoft excel sheets. Results: A total of 34 patients were studied ranging from 19 to 85 years. The mean age was 49 years, 21 (61.76%) of them were males and 13 (38.24%) were females. In 24 patients, single eye was affected and in 10 patients bilateral eye involvement was seen. Histology revealed that there were 18 (40.90%) CIN I cases, 4 (9.09%) CIN II, 5 (11.36%) CIN III and 17 (38.65%) SCC. On cytology, 3 (6.82 %) were normal conjunctival epithelium, 30 (68.18 %) were dysplasia and 11 (25.00 %) were SCC. On cyto- histomorphological analysis, 35 (79.55 %) were concordant, out of 44 diagnosis. IC has a sensitivity of 88.89% and specificity of 64.71% to detect dysplasia. To diagnose SCC, it has sensitivity of 64.71% and 100% specificity. Conclusion: According to the present study observations, IC is an excellent preliminary tool to investigate suspected neoplastic lesions of ocular surface. It has a very good concordance with histopathological diagnosis. IC is highly sensitive to detect dysplasia (CIN) and is very specific to diagnose SCC.

Identification of the circRNA-miRNA-mRNA Regulatory Network in Pterygium-Associated Conjunctival Epithelium.

To investigate the regulatory mechanism of pterygium formation, we detected differentially expressed messenger RNAs (DE-mRNAs) and differentially expressed circular RNAs (DE-circRNAs) in pterygium-associated conjunctival epithelium (PCE) and normal conjunctival epithelium (NCE). Genome-wide mRNA and circRNA expression profiles of PCE and NCE were determined using high-throughput sequencing. Bioinformatics analyses, including Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) analysis, were conducted. The microRNAs (miRNAs) interacting with the hub DE-mRNAs and DE-circRNAs were predicted and verified using real-time quantitative PCR (RT-qPCR). The data showed that there were 536 DE-mRNAs (280 upregulated and 256 downregulated mRNAs) and 78 DE-circRNAs (20 upregulated and 58 downregulated circRNAs) in PCE. KEGG enrichment analysis indicated that the DE-mRNAs were mainly involved in the following biological processes: IL-17 signalling pathway, viral protein interaction with cytokine and cytokine receptor, cytokine-cytokine receptor interaction, ECM-receptor interaction, and focal adhesion. The GSEA results revealed that the epithelial mesenchymal transition (EMT) process was significantly enriched in upregulated mRNAs. The pterygium-associated circRNA-miRNA-mRNA network was established based on the top 10 DE-circRNAs, 4 validated miRNAs (upregulated miR-376a-5p and miR-208a-5p,downregulated miR-203a-3p and miR-200b-3p), and 31 DE-mRNAs. We found that miR-200b-3p, as a regulator of FN1, SDC2, and MEX3D, could be regulated by 5 upregulated circRNAs. In addition, we screened out EMT-related DE-mRNAs, including 6 upregulated DE-mRNAs and 6 downregulated DE-mRNAs. The EMT-related circRNA-miRNA-mRNA network was established with the top 10 circRNAs, 8 validated miRNAs (upregulated miR-17-5p, miR-181a-5p, and miR-106a-5p, downregulated miR-124-3p, miR-9-5p, miR-130b-5p, miR-1-3p, and miR-26b-5P), and 12 EMT-related DE-mRNAs. We found that hsa_circ_0002406 might upregulate FN1 and ADAM12 by sponging miR-26b-5p and miR-1-3p, respectively, thus promoting EMT in pterygium. Briefly, the study provides a novel viewpoint on the molecular pathological mechanisms in pterygium formation. CircRNA-miRNA-mRNA regulatory networks participate in the pathogenesis of pterygium and might become promising targets for pterygium prevention and treatment.

Autologous simple conjunctival epithelial transplantation for primary pterygium

To evaluate the feasibility of a new method of conjunctival transplantation to achieve recovery of the normal conjunctival epithelium over the bare sclera after pterygium excision and prevent its recurrence. After excision of the primary pterygium, we performed simple conjunctival epithelial transplantation (SCET) in which we glued an amniotic membrane patch pre-loaded with tiny autologous conjunctival tissue fragments over the scleral defect. Slit-lamp evaluation was performed at 2 and 7-10days, and then at 1, 3, 6, and 12months after surgery, together with confocal microscopy at 3, 6, and 12months. Surgical excision and SCET for nasal primary pterygium were performed in 6 eyes (6 patients). No graft detachment occurred. An inflammatory granuloma was excised without sequelae in one patient 2months after surgery. No signs of recurrence or sight-threatening complications were recorded at 12months, and in vivo confocal microscopy showed progressive expansion of the conjunctival cell population and formation of a clear corneal-conjunctival transition. SCET takes advantage of the ability of the amniotic membrane and conjunctival cells to renew. Outcomes after SCET are comparable to conventional conjunctival flap surgery and can be achieved in less surgical time and with less donor tissue to be removed.

Conjunctival Myxoma: High-Resolution Optical Coherence Tomography Findings of a Rare Tumor

The purpose of this study was to describe 2 cases of biopsy-proven conjunctival myxomas and present their optical signs on high-resolution optical coherence tomography (HR-OCT) with clinical and histopathological correlations. Two middle-aged female patients with a clinical diagnosis of conjunctival cysts were referred for surgical treatment. Clinical assessment, photographs, ultrasound biomicroscopy, and HR-OCT images were obtained. Excisional biopsies were performed, and specimens were sent for histopathological and immunohistochemical analyses. Clinically, these patients presented with a well-circumscribed, semitranslucent, yellow-pinkish mass. Ultrasound biomicroscopy showed a dome-shaped epibulbar mass with medium-to-high internal reflectivity. No compromise of the underlying sclera was noted. HR-OCT showed a normal conjunctival epithelium, a subepithelial nonhomogeneous mass with hyperreflective and hyporeflective areas lined by a highly hyperreflective band, and mild posterior shadowing. Histopathological findings and immunoreactivity for CD34 and vimentin confirmed the diagnosis of conjunctival myxoma. The HR-OCT optical signs found in our 2 cases strongly correlated with the microscopic findings. Disclosing the optical signs observed on HR-OCT can help clinicians diagnose and differentiate this lesion, guiding its management. However, more studies with a larger number of patients comparing conjunctival myxoma and other ocular surface tumors are needed to enlighten readers about the unique pattern observed by HR-OCT.

Conjunctival Expression of Toll-Like Receptor 3 Plays a Pathogenic Role in the Formation of Ultraviolet Light-Induced Pterygium.

PurposeToll-like receptor 3 (TLR3), as a damage-associated molecular pattern sensor, can detect self-RNA released from necrotic cells induced by ultraviolet B (UVB) radiation exposure. Pterygium formation is believed to be a tumorigenesis-like process induced by UVB exposure. In this study, we aimed to investigate the expression pattern of TLR3 in pterygium specimens and cultured pterygial epithelial cells (PECs).MethodsHuman pterygium and ipsilateral pterygium-free conjunctiva from the same patients were used in this study. The expression of TLR3 and nuclear factor-kappa B (NF-κB) was investigated in these specimens. PECs were exposed to UVB radiation to determine the effect of UVB on the expression of TLR3 and the activation of NF-κB.ResultsThe immunofluorescence study showed stronger TLR3 expression in superficial epithelial cells in the pterygial epithelium in comparison with the normal conjunctival epithelium. The expression of TLR3 decreased in intensity from the superficial epithelium toward the basal cell layer, implying a correlation between UVB exposure and TLR3 expression. Differential TLR3 expression patterns in pterygial and conjunctival tissues were also found in quantitative PCR analyses. PECs after UVB irradiation had higher protein levels of TLR3 and phospho-NF-κB than those of the PECs without irradiation. Immunofluorescence studies showed that UVB irradiation induced the nuclear translocation of NF-κB in the PECs. In PECs with the targeted TLR3 gene silencing, the expression of phospho-NF-κB was not induced by UVB irradiation.ConclusionsOur results indicate that UVB exposure, TLR3 expression, and NF-κB activation may be a critical sequence that leads to the formation of pterygium.

Tryptophan as a Safe Compound in Topical Ophthalmic Medications: In Vitro and In Vivo Studies

ABSTRACT Background To evaluate the effects of tryptophan (TRP) on normal human corneal and conjunctival epithelium in vitro and the re-epithelization of corneal erosion in rabbits. Materials and methods Corneal epithelial cell (10.014 pRSV-T) and conjunctival epithelial cell (HC0597) cultures were used. The cellular metabolism, viability, secretion of IL-1β, IL-6, IL-10, cytoskeleton organization, transwell migration were determined. Cells were incubated in the presence of TRP at 1–100 μM. After corneal de-epithelization rabbits received TRP drops (100 μM), 5 times a day. Results TRP increased conjunctival epithelium metabolism at 50 μM and increased the viability of corneal epithelium at 100 μM. TRP (10 μM) enhanced the production of IL-6 by the corneal epithelium and had no effect on IL-1β and IL-10. Conclusions TRP had no influence on the cellular cytoskeleton but induced a significant pseudopodia projection in both epithelia. TRP did not influence corneal re-epithelization in vivo. TRP was not toxic for corneal and conjunctival epithelia.

Advance in research of beta coronavirus receptors on ocular surface

2019-Novel coronavirus (2019-nCoV) caused an outbreak of corona virus disease 2019 (COVID-19) from December 2019 in China.2019-nCoV which was identified as a kind of beta coronavirus belongs to one of four coronavirus genera.Except 2019-nCoV, two other beta coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also quite harmful to human beings.2019-nCoV uses the same cell entry receptor, angiotensin-converting enzyme 2 (ACE2), as SARS-CoV.And dipeptidyl peptidase 4 (DPP4) or CD26 is the cell receptor for MERS-CoV.The expression of ACE2 was found to have obvious positive expression in human corneal and conjunctival epithelium, and corneal endothelium.DPP4 activity was presented in normal animal conjunctival epithelium and fibroblasts of the subjacent connective tissue.It was also presented in the whole corneal epithelium and tear fluid of animal with severe injured corneas.The two receptors, ACE2 and DPP4, are involved in many cellular signaling pathways and pathophysiological processes.Their expression in the cells of ocular surface may be an access route of corona virus in eye, which provides clues to elucidating the pathogenesis of corona virus in the eyeballs. Key words: Coronavirus; Receptor; Angiotensin-converting enzyme 2; Dipeptidyl peptidase 4; Ocular surface

Alteration of expression pattern of transient receptor potential vanilloid 2 and transient receptor potential vanilloid 3 in ocular surface neoplasm.

PURPOSE:We determined if the immunohistochemical expression pattern of transient receptor potential vanilloid (TRPV) family members and TRP ankyrin 1 (TRPA1) differs among a healthy conjunctival epithelium and diseased epithelia.MATERIALS AND METHODS:Subjects include a normal conjunctival epithelium, pterygium epithelium, epithelial dysplasia or carcinoma in situ.RESULTS:TRPV1, TRPV4 or TRPA1 was detected in both the cytoplasm and nuclei, or in either the nuclei or cytoplasm, of these different epithelial layers, respectively. There was no difference in the expression pattern of these three TRP isoforms. On the other hand, the expression patterns of TRPV2 and TRPV3 differed dramatically among these different subjects. TRPV2 was observed in the basal layer epithelium of a normal conjunctiva and pterygium. Its pattern was scattered in this region, although TRPV2 was absent throughout most of the dysplastic epithelium. TRPV2 was detected only in some of the suprabasal epithelial cells of a carcinoma in situ. TRPV3 was faintly detected in the cytoplasm of all the cell layers and also in the nuclei of some of the basal cells in a normal conjunctiva and in the pterygia epithelium, while in situ it was uniformly expressed in all of the dysplasia and carcinoma nuclei in all epithelial cell layers.CONCLUSION:These results suggest that TRPV2 and TRPV3 expression pattern analysis might be potential diagnostic markers of ocular surface epithelial disorders.

Insulin-like growth factor binding protein-7: A marker of conjunctivalization in an animal model of limbal stem cell deficiency

PurposeLimbal stem cell deficiency (LSCD) is characterized by the loss of limbal epithelial stem cells, resulting in a pathological process termed ‘conjunctivalization’ which compromises corneal transparency, leading to blindness. Current diagnosis for LSCD is limited because reliable conjunctiva-specific biomarkers are lacking. This study sought to address this shortcoming through the serendipitous discovery of insulin-like growth factor binding protein (IGFBP)-7. MethodsIGFBP-7 expression was determined in normal (n=83) and conjunctivalized (n=52) mouse corneas with experimentally-induced LSCD, and in cadaveric normal human corneas (n=7) and human pterygia (n=15); a disease characterized by the invasion of a conjunctivalized, fibrovascular pannus. Clinical assessments including slit-lamp microscopy, fluorescein staining and impression cytology, and biochemical, molecular and immunological assays were also conducted. ResultsMass spectrometry of conditioned media from mouse limbal explant-derived cells revealed the presence of IGFBP-7. This factor was expressed in normal limbal and conjunctival epithelium and conjunctivalized corneas from mice with LSCD, and in human pterygium epithelium but not in normal mouse or human corneal epithelium. Four weeks after inducing LSCD, IGFBP-7 staining was increased by 2.9-fold in mouse corneas compared to steady-state, and by 1.6-fold in impression cytology specimens derived from the same mice. Notably, IGFBP-7 was detected approximately 2-weeks earlier than Muc5AC. ConclusionsThis study provides novel insights into the specificity of IGFBP-7 for the mammalian conjunctival epithelium in health and disease. A point-of-care test for IGFBP-7 could be developed to assist clinicians in early diagnosis, and in monitoring disease progression, severity and therapeutic outcomes in patients with LSCD.

Pterygial body epithelium domination of pterygial proliferation with TCF4 as a potential key factor

To characterize the proliferative capacity of pterygial epithelium in different regions (head, neck and body) of pterygium and explore the function of transcription factor 4 (TCF4) in pterygium proliferation. Thirty pterygium tissues and 10 normal conjunctival tissues were obtained from Zhongshan Ophthalmic Center (ZOC) and Guangdong Eye Bank, respectively. Proliferative capacity of head, neck and body in pterygial epithelium was measured using clonal analysis, fold growth analysis and expression profile of proliferative markers revealed by immunofluorescent staining and real-time PCR. The expression of TCF4 was highlighted by double immunofluorescent staining with other proliferation related markers such as proliferating cell nuclear antigen (PCNA) and ATP-binding cassette sub-family G member 2 (ABCG2). The proliferative potential of pterygial epithelium was higher than that of normal conjunctival epithelium. High expression levels of proliferative markers (P63α, PCNA and ABCG2) in pterygial body epithelium were observed in immunofluorescent staining and real-time PCR (P<0.05). Also, epithelial cells isolated from pterygial body demonstrated higher proliferative capacity in clonal analysis and fold growth analysis, than those isolated from the head and neck regions. The TCF4 expression in pterygial epithelium was similar to other proliferative markers (P63α, PCNA and ABCG2), as higher in pterygial body than head and neck. Moreover, TCF4 showed coexpression with other proliferation-related markers (PCNA and ABCG2) in the double immunofluorescent staining experiment. The proliferative capacity in pterygial body epithelium is prominent than the head and neck regions, and upregulated TCF4 may be associated with enhanced proliferation in the pterygium.

Exogenous Tissue Inhibitor of Metalloproteinase-2 Affects Matrix Metalloproteinase-2 Expression in Conjunctival Filtering Blebs and Bleb Scarring in Rats.

Objective To examine the effect of tissue inhibitor of metalloproteinase-2 (TIMP-2) on conjunctival filtering bleb scarring. Methods A model of conjunctival filtering bleb was established whereby rats were injected with saline, blank adenoviral vector, or adenoviral vector carrying TIMP-2 into the bleb. Filtration bleb formation and matrix metalloproteinase-2 (MMP-2) expression were examined. Results All operated eyes formed obvious elevated blebs on day 1. In the normal saline group, empty plasmid group, and gene transfection group maintenance time of filtrating blebs was 5–14, 5–14, and 6–16 days, and average survival time was 8.24, 8.16, and 9.44 days, respectively. MMP-2 expression increased slightly in the gene transfection group at 3 and 5 days after surgery, reached a peak after 14 days, and then gradually decreased. MMP-2 expression was weakly positive in the normal conjunctival epithelium, but was hardly detected in the lamina propria. Seven days after surgery, the epithelium and lamina propria of the conjunctival filtering bleb exhibited strong positive expression in the empty plasmid group but only weak expression in the adenovirus group. Conclusion Exogenous TIMP-2 interfered with local MMP-2 expression, delaying peak expression of MMP-2 and slowing the scarring of filtering blebs during wound healing of subconjunctival tissue.

Research on the expression of TSG-6 and PTX3 and on apoptosis in conjunctival resection tissue in conjunctivochalasis

Objective To investigate the expression of TSG-6 and PTX3 as well as the apoptosis in conjunctival tissue of conjunctivochalasis (CCh). Methods Conjunctival tissue from the conjunctival resection tissue of CCh patients and simple cataract patients were collected and then the frozen sections of conjunctival tissue were prepared. The expressions of TSG-6 and PTX3 were detected by immunofluorescence and the apoptosis was detected by Tunel assay. Results The expression of TSG-6 was strongly positive in conjunctival epithelium of the CCh group. The average fluorescent intensity (AFI) was (76.67±26.87)which was significantly higher than that in normal control group (22.53±7.21)and the difference was statistically significant (t=8.460, P<0.001). TSG-6 was expressed in the conjunctival stroma and Tenon capsule of the CCh group (AFI 39.25±13.24) which was higher than that in normal control group (AFI 13.81±9.60) and the difference was statistically significant (t=7.440, P<0.001). PTX3 in conjunctival epithelium (AFI 52.41±11.61), matrix and Tenon's capsule (AFI 35.96±10.91) of the CCh group were both positive which were significantly higher than those (AFI 13.88±5.42 and 4.47±2.49) in normal control group (t=13.887, P<0.001; t=12.658, P<0.001 ). No statistically significant difference was found in the apoptosis rate between CCh (8.64±0.13%) and normal (8.33±0.11%) conjunctival epithelium (t=1.810, P=0.072). The apoptosis rate was significantly increased in CCh stroma and Tenon's capsule (40.42±0.24%) compared with that of normal control group (10.87±0.25%) (t=90.171, P<0.001). Conclusion The expressions of TSG-6 and PTX3, as well as cell apoptosis, increase significantly in conjunctival tissue of conjunctivohalsis, which indicates that inflammation and apoptosis involve in the pathogenesis of conjunctivochalasis. Key words: Conjunctivochalasis; Conjunctival resection tissue; TSG-6; PTX3; Apoptosis

Lymphatic and Blood Vessel Density in Human Conjunctiva After Glaucoma Filtration Surgery.

To investigate the lymphatic vascular microvessel density (LVD) and the blood vascular microvessel density (MVD) and their distribution in excised leaking blebs after mitomycin C trabeculectomy and normal conjunctiva specimens. LVD and MVD in normal human conjunctiva (n=8) and excised blebs in the hypocellular stroma and peribleb tissue (conjunctiva adjacent to hypocellular bleb tissue) (n=8) were evaluated by immunohistochemistry using antibodies raised against Lymphatic Vessel Endothelial Receptor 1 (D2-40, lymphatic endothelium) and CD34 (vascular endothelium). LVD and MVD counts were performed by light microscopy in 5 fields at ×20 magnification by 3 observers. Differences were determined using Mann-Whitney U test (P<0.05 was considered significant). The leaking blebs showed typical epithelial-stromal domes with areas of acellular stroma covered by attenuated epithelium and surrounded by normal conjunctival epithelium and a dense scar-like matrix replacing the substantia propria. The LVD and MVD were significantly reduced to nil in the hypocellular conjunctival stroma of the excised blebs compared with normal conjunctiva (21.42 vs. 1.16, P<0.002 and 24.28 vs. 1, P<0.008, respectively). The LVD and MVD was also reduced (2- to 2.5-fold) in the peribleb stroma when compared with normal conjunctiva specimens. In this study we show reduced LCD and MVD in the hypocellular and peribleb stroma. These results may suggest a role of these vessels in an altered immune response in leaking blebs leading to an increased risk for blebitis.

Interleukin-17 and its correlation with vascular endothelial growth factor expression in ocular surface pathologies: a histologic study.

Interleukin-17 (IL-17), a cytokine that was recently associated with various inflammatory processes, was evaluated in ocular surface inflammatory pathologies, namely pterygium and inflamed juvenile conjunctival nevus (IJCN). Its expression was examined in correlation with vascular endothelial growth factor (VEGF) expression. Expression of IL-17 and VEGF was immunohistochemically evaluated in 50 pterygia and 9 IJCN specimens. The pattern of IL-17 and VEGF expression was studied as well as the correlation between the 2 cytokines. Interleukin-17 was expressed in all IJCN samples and in 84% of pterygia (Fisher exact test, p&lt;0.002). In pterygium, it was expressed mainly in the basal layer of the epithelial cells, in the perivascular tissues, and in the vascular endothelial cells. In IJCN, it was mainly expressed in the inflammatory infiltrate. In normal conjunctival epithelium, it was barely detected and was positive in only 11%. Vascular endothelial growth factor was expressed in all IJCN samples and in 92% of pterygia (Fisher exact test, p&lt;0.002). It was expressed in the surface epithelial layers of the pterygium and in the vascular endothelium. In IJCN, it was again seen in the vascular endothelial cells and in the inflammatory infiltrate. Vascular endothelial growth factor expression was noted in 67% of normal conjunctival sections. The present study describes the expression of IL-17 in pterygium and in IJCN. These findings provide new insights into pterygium and IJCN pathogenesis. Interleukin-17 blockade may have a potential role in management of pterygium and IJCN or preventing pterygium recurrence.

Healing indicators after pterygium excision by optical coherence tomography.

To establish the sequence of tomographic changes in the tissue recovery process after pterygium excision and to propose healing indicators. Optical coherence tomography (OCT) images were taken at 1 week, 1, 3 and 6 months after lesion excision in 73 eyes of 73 patients (33 male, 40 female; mean age 50, S.D. 5.0, range 40-70 years) with primary nasal pterygium. Biomicroscopy was performed at each visit and at 12 months, to diagnose clinical healing or lesion recurrence. The presence of well demarcated corneal epithelium, conjunctival epithelium, limbal demarcation area, and graft thickening were analysed. Comparisons between tomographic data of both clinical situations were made at each time point using contingency tables. Eleven eyes displayed lesion recurrence (R group) and 62 eyes showed no recurrence (NR group). Normal anatomical structures, corneal and conjunctival epithelium and limbal demarcation area, were identified by OCT images in a higher percentage of NR cases over time, compared to the R group where most of the cases presented without these markers of tissue recovery. In contrast, the variable graft thickening, which analysed a pathological event, revealed similar results in both groups (p > 0.05; Fisher's exact statistic), with a clear decrease of cases which showed graft thickening over time. Differences between groups started at 1 month, when no eye had yet presented clinical recurrence, with greater identification of corneal epithelium in the NR group (p = 0.04; Fisher's exact statistic). At 3 months, corneal and conjunctival epithelium identification tended to be more frequent in the NR than in the R group (in both cases, p = 0.0001; Fisher's exact statistic). Finally, at 6 months these different patterns consolidated, with a significantly higher number of limbal demarcation areas being identified in the NR group (p = 0.001; Fisher's exact statistic). In fact, this landmark of a normally structured limbus was never found in the R group. The sequence of tissue restoration, according to OCT images, seems to start in the cornea and end in the limbal area, similar to the process of pterygium injury in reverse. Although the visualisation of corneal epithelium could be an early indicator of successful surgery, identification of the limbal demarcation area, as a normal limbal pattern in OCT images, seems to be a better positive predictive value in diagnosing healing.

Inductive differentiation of conjunctival goblet cells by γ-secretase inhibitor and construction of recombinant conjunctival epithelium

γ-secretase inhibitor has been shown to promote intestinal goblet cell differentiation. We now demonstrated that the in vitro addition of γ-secretase inhibitor in the culture of human conjunctival epithelial cells significantly promoted the differentiation of conjunctival goblet cells with typical droplet-like phenotype, positive periodic acid-Schiff and goblet cell-specific Muc5Ac, cytokeratin 7 and Helix pomatia agglutinin lectin staining. Moreover, topical application of γ-secretase inhibitor promoted the differentiation of mouse conjunctival goblet cells in vivo. Furthermore, the expression of Notch target gene HES-1 was down-regulated during the differentiation of conjunctival goblet cells. In addition, we found that the recombinant conjunctival epithelium on amniotic membrane showed less goblet cell density and abnormal location when compared with normal conjunctival epithelium, which were improved by the addition of γ-secretase inhibitor in the final induction.

Canaliculops: Clinicopathologic Features and Treatment With Marsupialization

To report the features of the rare and under-recognized condition of canaliculops (or canaliculocele) of the eyelid, which is a dilation of the canaliculus, and to evaluate treatment with marsupialization. Retrospective interventional case series. The records of 2 patients with canaliculops from the Massachusetts Eye and Ear Infirmary were reviewed. Data collected included clinical history, surgical technique, histopathologic analysis, and comparative immunohistochemical analysis of a range of cytokeratins in normal conjunctival epithelium, normal canalicular epithelium, and canaliculops epithelium. Two women, 53 and 66 years of age, experienced chronic, noninflammatory, painless medial eyelid and eyelid margin fluctuant swelling after earlier trauma or eyelid surgery. The external mass was accompanied by a whitish opalescent or bluish discoloration of a palpebral surface bulge. Biopsy revealed multilaminar (up to 12 cells thick), nonkeratinizing, tightly packed small squamous epithelial cells that surmounted a highly regimented basal layer with a picket fence arrangement. No goblet cells or subepithelial inflammation were present. Immunohistochemistry revealed only superficial CK7 immunostaining and positive patchy suprabasilar CK17 staining in the canaliculops epithelium, contrasting with their full-thickness positivity and negativity, respectively, in normal conjunctival epithelium. Marsupialization achieved resolution of the condition in each patient. An improved awareness of the normal canalicular epithelial structure and its immunohistochemical features can definitively separate canaliculops from conjunctival cysts. Previous treatment of canaliculops has involved complete excisions. Canaliculops may, however, be effectively treated with less invasive marsupialization while obtaining an adequate biopsy specimen for histopathologic diagnosis.

TSG-6 controls transcription and activation of matrix metalloproteinase 1 in conjunctivochalasis.

To investigate the role of anti-inflammatory TSG-6 in controlling MMP-1 and MMP-3, which have been shown to be upregulated in conjunctivochalasis (CCh). Immunostaining of TSG-6 was compared between normal and CCh conjunctiva and Tenon's capsule. Second cultures of normal and CCh fibroblasts were transfected with or without TSG-6 siRNA and then with or without the addition of TNF-α or IL-1β. Cell lysates and culture media were collected to assess apoptosis with the use of ELISA and the expression of TSG-6, MMP-1, and MMP-3 transcripts and proteins with the use of qRT-PCR and Western blot analysis, respectively. TSG-6 expression was constitutive in the in vivo normal conjunctival epithelium. Significantly more TSG-6-positive cells than normal specimens were noted in CCh subconjunctival tissue and Tenon's capsule. TSG-6 was constitutively expressed intracellularly by both resting normal and CCh fibroblasts but was secreted extracellularly only by resting CCh fibroblasts. Intracellular and extracellular TSG-6 proteins were markedly upregulated by TNF-α or IL-1β in normal and CCh fibroblasts. Active MMP-1 was found in CCh fibroblasts intracellularly and extracellularly, whereas only proMMP-1 was found intracellularly in normal fibroblasts. Knockdown by TSG-6 siRNA upregulated more MMP-1 than MMP-3 transcripts in normal and CCh fibroblasts. TSG-6 siRNA led to extracellular MMP-1 expression by normal fibroblasts such as CCh fibroblasts. This activation of MMP-1 was further enhanced by IL-1β. Cell apoptosis was higher in CCh fibroblasts and further aggravated by TSG-6 siRNA knockdown. TSG-6 exerts an anti-inflammatory function by counteracting the transcription of MMP-1 and MMP-3 and the activation of MMP-1. Dysfunction of TSG-6 might play a role in the pathogenesis of CCh.

Conjunctival Reconstruction with Progenitor Cell-Derived Autologous Epidermal Sheets in Rhesus Monkey

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

Prostaglandin E Receptor Subtype EP3 Expression in Human Conjunctival Epithelium and Its Changes in Various Ocular Surface Disorders

BackgroundIn our earlier genome-wide association study on Stevens-Johnson Syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), we found that in Japanese patients with these severe ocular surface complications there was an association with prostaglandin E receptor 3 (EP3) gene (PTGER3) polymorphisms. We also reported that EP3 is dominantly expressed in the ocular surface-, especially the conjunctival epithelium, and suggested that EP3 in the conjunctival epithelium may down-regulate ocular surface inflammation. In the current study we investigated the expression of EP3 protein in the conjunctiva of patients with various ocular surface diseases such as SJS/TEN, chemical eye burns, Mooren’s ulcers, and ocular cicatricial pemphigoid (OCP).Methodology/Principal FindingsConjunctival tissues were obtained from patients undergoing surgical reconstruction of the ocular surface due to SJS/TEN, chemical eye burns, and OCP, and from patients with Mooren's ulcers treated by resection of the inflammatory conjunctiva. The controls were nearly normal human conjunctival tissues acquired at surgery for conjunctivochalasis. We performed immunohistological analysis of the EP3 protein and evaluated the immunohistological staining of EP3 protein in the conjunctival epithelium of patients with ocular surface diseases. EP3 was expressed in the conjunctival epithelium of patients with chemical eye burns and Mooren’s ulcer and in normal human conjunctival epithelium. However, it was markedly down-regulated in the conjunctival epithelium of SJS/TEN and OCP patients.ConclusionsWe posit an association between the down-regulation of EP3 in conjunctival epithelium and the pathogenesis and pathology of SJS/TEN and OCP, and suggest a common mechanism(s) in the pathology of these diseases. The examination of EP3 protein expression in conjunctival epithelium may aid in the differential diagnosis of various ocular surface diseases.