Acute Corneal Epithelial Rejection of LR-CLAL After SARS-CoV-2 Vaccination.

PURPOSE: The purpose of this study was to report a case of acute corneal epithelial rejection of living-related conjunctival limbal allograft (LR-CLAL) after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. OBSERVATIONS: A 27-year-old woman developed acute epithelial rejection of LR-CLAL 2 weeks after receiving the SARS-CoV-2 vaccine. She received the LR-CLAL transplant 4 years and 7 months previously and had a stable clinical course with no history of rejection. She had an ABO blood group and human leukocyte antigen compatible donor, no systemic comorbidities, and no rejection risk factors. CONCLUSIONS: The novel SARS-CoV-2 vaccine upregulates the immune system to produce an adaptive immune response. The SARS-CoV-2 vaccine may potentially be associated with increased risk of rejection in those with ocular surface transplants.

[The in vitro effect of antiseptics on epithelial cells of human cornea and conjunctiva]. / Vozdeistvie antiseptikov na epitelial'nye kletki rogovitsy i kon"yunktivy cheloveka in vitro.

Effective and safe antiseptic eye preparations are necessary for prevention and treatment of infectious and inflammatory eye diseases. PURPOSE: in vitro evaluation of the effect of antiseptic eye drops on corneal and conjunctival epithelial cells. MATERIAL AND METHODS: Antiseptic eye drops «Bactavit¼, «Vitabact¼ and «Ocomistin¼ were the object of the study. Immortalized human corneal epithelial cell lines (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) were used as the test systems. The viability of the cells was assessed by their metabolic activity and morphology using the MTT test and phase-contrast microscopy. RESULTS: Antiseptic eye drops belonging to different groups of chemical compounds induced cytotoxic effects on the cells of corneal epithelium (HCE) and human conjunctiva (Chang Conjunctiva, Clone 1-5c-4) of varying degrees, leading to morphological and functional changes in those cells. CONCLUSION: The study confirms the possibility of using cultured cells for the in vitro comparative assessment of the cytotoxic effect of antiseptic ophthalmic agents.

Therapeutic potentials of human microfluidic encapsulated conjunctival mesenchymal stem cells on the rat model of Parkinson's disease.

BACKGROUND AND AIM: Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the destruction of the dopaminergic neurons in the nigrostriatal pathway, leading to motor-behavioral complications. Cell therapy has been proposed as a promising approach for PD treatment using various cellular sources. Despite a few disadvantages mesenchymal stem cells (MSCs) represent, they have more auspicious effects for PD cell therapy. The present study aimed to evaluate a new source of MSCs isolated from human Conjunctiva (CJ-MSCs) impact on PD complications for the first time. MATERIALS AND METHODS: Parkinson's was induced by stereotactic injection of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB). An apomorphine-induced rotation test was used to confirm the model establishment. After PD model confirmation, green fluorescent protein (GFP) labeled CJ-MSCs and induced CJ-MSCs (microfluidic encapsulated and non-capsulated) were transplanted into the rats' right striatum. Then Rotation, Rotarod, and Open-field tests were performed to evaluate the behavioral assessment. Additionally, the immunohistochemistry technique was used for identifying tyrosine hydroxylase (TH). RESULTS: According to the obtained data, the cell transplantation caused a reduction in the rats' rotation number and improved locomotion compared to the control group. The previous results were also more pronounced in induced and microfluidic encapsulated cells compared to other cells. Rats recipient CJ-MSCs also have represented more TH-expressed GFP-labeled cell numbers in the striatum than the control group. CONCLUSION: It can be concluded that CJ-MSCs therapy can have protective effects against PD complications and nerve induction of cells due to their ability to express dopamine. On the other hand, CJ-MSCs microencapsulating leads to enhance even more protective effect of CJ-MSCs. However, confirmation of this hypothesis requires further studies and investigation of these cells' possible mechanisms of action.

Molecular characteristics and spatial distribution of adult human corneal cell subtypes.

Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.

Human Papillomavirus Related Neoplasia of the Ocular Adnexa.

Human papillomaviruses (HPV) are a large group of DNA viruses that infect the basal cells of the stratified epithelium at different anatomic locations. In the ocular adnexal region, the mucosa of the conjunctiva and the lacrimal drainage system, as well as the eyelid skin, are potential locations for HPV-related neoplasia. The role of HPV in squamous cell neoplasia of the ocular adnexa has been debated for several decades. Due to the rarity of all these tumors, large studies are not available in the scientific literature, thereby hampering the precision of the HPV prevalence estimates and the ability to conclude. Nevertheless, increasing evidence supports that defined subsets of conjunctival papillomas, intraepithelial neoplasia, and carcinomas develop in an HPV-dependent pathway. The role of HPV in squamous cell tumors arising in the lacrimal drainage system and the eyelid is still uncertain. Further, the potential of HPV status as a diagnostic, prognostic, or predictive biomarker in these diseases is a topic for future research.

Effect of processing methods on the cytotoxicity of methyl methacrylate-based ocular prostheses: An in vitro study.

The study evaluated the influence of cycles and methods of an ocular prosthesis resin on cytotoxicity toward human conjunctival cells. Resins were polymerized by water bath (WB, 74 °C or 100 °C for 30 min to 9 h), microwave (MW, 1200 W, 3 to 14 min and 30 s at 0 to 720 W), or autopolymerization (AP, room temperature for 20 min ± 60 °C for 30 min). Degree of conversion (DC), cytotoxicity, level of inflammatory mediators, gene expression of different markers, and apoptosis were evaluated. Data were submitted to ANOVA and Tukey test (p < 0.05). WB with longer processing time at higher temperature had highest DC (85.6%) and higher TGF ß1-gene expression (1.39); long cycle low power MW showed lowest DC (69.6%), lower cell proliferation (85.4%, MTT), and large IL-2 release (39,297 ng/mL). AP with additional processing time showed lower cell proliferation (75.3%, Alamar Blue), and AP polymerized at room temperature showed higher CASP 9-gene expression (1.21). AP methods showed higher IL-6 release (>277 pg/mL). Short cycle medium power MW had higher IL-23 release (534.2 pg/mL). MW (long and short cycles) and AP polymerizations have triggered a more intense inflammatory response. Among methods recommended by the manufacturer, WB showed high DC and less cytotoxicity.

Differential Effect of Proinflammatory Cytokines on Corneal and Conjunctival Epithelial Cell Mucins and Glycocalyx.

Purpose: Ocular surface mucins and glycocalyx are critical for providing ocular hydration as well lubrication and repelling pathogens or allergens. Elevated levels of tear proinflammatory cytokines in dry eye may have detrimental effect on mucins and glycocalyx. The present study tested the effect of proinflammatory cytokines IL-6, TNF-α, and IFN-γ on membrane-tethered mucins expression, glycocalyx, and viability of ocular surface epithelial cells. Methods: Stratified cultures of human corneal and conjunctival epithelial cells were exposed to different concentrations of IL-6, TNF-α, and IFN-γ for 24 hours. The mucins gene and protein expressions were quantified by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The glycocalyx was imaged using confocal microscopy after staining with Alexa 488-conjugated wheat germ agglutinin lectin. Apoptotic and necrotic cell death was quantified using flow cytometry. Results: IL-6, TNF-α, and IFN-γ treatment resulted in a significant increase in mucins (MUC)1 and MUC4 gene and protein expression in human corneal epithelial cells but caused no significant changes in the levels of these mucins in conjunctival epithelial cells. Further, these cytokines decreased MUC16 expression in both corneal and conjunctival epithelial cells. Moreover, no notable change in glycocalyx or apoptotic cell death in corneal and conjunctival epithelial cells was noted with any of the tested cytokines, but IL-6 and TNF-α exposure increased necrotic cell death in corneal and conjunctival epithelial cells, respectively. Conclusions: Our results demonstrate that proinflammatory cytokines have differential effects on human corneal and conjunctival epithelial cell mucins expression, but do not cause any damage to ocular surface epithelial cell glycocalyx.

Development and optimization of a personalized fibrin membrane derived from the plasma rich in growth factors technology.

PURPOSE: To develop and characterize a new type of plasma rich in growth factors (PRGF) membrane for patients in which immune system is involved in the disease etiology. METHODS: Blood from three healthy donors was collected to obtain the different fibrin membranes by PRGF technology. PRGF obtained volumes were activated and divided into two groups: PRGF membrane (mPRGF) obtained after incubation at 37 °C for 30 min (control); and is-mPRGF: mPRGF obtained after incubation for 30 min at 56 °C. The concentration of several growth factors, proteins, immunoglobulin E and the complement activity was determined in the different mPRGF. The proliferative potential of heat-inactivated mPRGF were assayed on keratocytes (HK) and conjunctival fibroblasts (HConF). In addition, morphological and physical features of the inactivated mPRGF were evaluated in contrast to the control mPRGF. RESULTS: Heat-inactivation of the mPRGF preserves the content of most of the growth factors involved in the ocular wound healing while reducing drastically the content of IgE and the complement activity. The heat-inactivated mPRGF conserve the morphological and physical characteristics of the fibrin meshwork in comparison with the control mPRGF. Furthermore, no significant differences were found in the biological activity of the control mPRGF regarding the heat-inactivated mPRGF (is-mPRGF) in any of both ocular cell types evaluated. CONCLUSIONS: The heat-inactivation of the PRGF membranes (is-mPRGF) reduces drastically the content of IgE and complement activity while preserving the content of most of the proteins and morphogens involved in ocular wound healing. Furthermore, the morphological and physical features of the immunosafe mPRGF were also preserved after heat-inactivation.

Autologous serum eye drops improve tear production, both lachrymal flow and stability tests and conjunctival impression cytology with transfer in dry eye disease.

BACKGROUND: Autologous serum eye drops, produced by separation of liquid and cellular components of the patient's blood, contain biological nutrients present in natural tears. The aim of this study was to analyse changes in conjunctival impression cytology with transfer and both lachrymal stability and flow tests in patients with dry eye disease after treatment with autologous serum eye drops. MATERIALS AND METHODS: Conjunctival impression cytology and lachrymal flow and stability tests, namely Schirmer's and tear break-up time, were prospectively studied in patients with dry eye disease before and 1 month after treatment with autologous serum eye drops. RESULTS: Twenty-four patients (23 women, mean age 53.8±12.6 years) were included in the study. Ten patients (41.7%) had moderate and six (25.0%) had severe dry eye disease. Five patients had rheumatoid arthritis. After treatment, the number and density of conjunctival goblet cells, their size, the size of their nuclei and the nucleus/cytoplasm ratio increased significantly (202.3±107.5 vs 210.1±100.9 cells/mm2, p<0.01). Seven of ten patients with grade 3 or 4 metaplasia had an improvement in the degree of metaplasia. Both Schirmer's test and tear break-up time improved significantly in this subgroup of patients. In the multivariate study, the increase in conjunctival goblet cells was associated with the number of goblet cells and the size of the cytoplasm at baseline. No adverse reactions were noted. DISCUSSION: Treatment with autologous serum eye drops for 1 month was well tolerated and improved tear production, lachrymal flow and stability tests and conjunctival impression cytology with transfer, increasing the density of the goblet cells.

The Effect of Androgens on Proinflammatory Cytokine Secretion from Human Ocular Surface Epithelial Cells.

Purpose: The purpose of this study is to explore the effects of dihydrotestosterone (DHT) on lipopolysaccharide (LPS)-induced proinflammatory cytokine release in human ocular surface epithelial cells exposed to LPS and LPS-binding protein (LBP).Methods: Immortalized human corneal, conjunctival, and meibomian gland epithelial cells were cultured in keratinocyte-free medium. After confluency, they were exposed to a stratification medium Dulbecco's modified Eagle medium (DMEM)/F12 in the presence of fetal bovine serum and were exposed to vehicle, LPS + LBP, or DHT. Culture media were processed for multiplex-bead analysis of specific proinflammatory cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-8, IL-6, IL-10, IL-1ß, vascular endothelial growth factor (VEGF)-A. Cytokine concentrations were compared by analysis of variance with Tukey post hoc testing. p < 0.05 was considered statistically significant.Results: The results are LPS + LBP-induced the secretion of IFN-γ, IL-6, IL-10, IL-1ß, VEGF-A cytokines in corneal epithelial cells; TNF-α, IL-2, IL-8, IL-6, IL-1ß, VEGF-A cytokines in conjunctival epithelial cells; and IL-8, IL-6, IL-1ß, VEGF-A cytokines in meibomian gland epithelial cells. When these LPS + LBP-stimulated cells were exposed to DHT for 2 days, it was found that DHT suppressed the secretion of IL-6, IL-10, IL-1ß, VEGF-A cytokines in corneal epithelial cells; TNF-α, IL-6, IL-1ß, VEGF-A cytokines in conjunctival epithelial cells; and IL-6, IL-1ß, VEGF-A cytokines in meibomian gland epithelial cells.Conclusion: LPS + LBP is shown to induce the secretion of certain proinflammatory cytokines from ocular surface and adnexal epithelial cells. DHT showed anti-inflammatory activity by suppressing some of those cytokines in these cell lines.

Genistein-Calcitriol Mitigates Hyperosmotic Stress-Induced TonEBP, CFTR Dysfunction, VDR Degradation and Inflammation in Dry Eye Disease.

Dry eye disease (DED) signs and symptoms are causally associated with increased ocular surface (OS) inflammation. Modulation of key regulators of aberrant OS inflammation is of interest for clinical management. We investigated the status and the potential to harness key endogenous protective factors, such as cystic fibrosis transmembrane conductance regulator (CFTR) and vitamin D receptor (VDR) in hyperosmotic stress-associated inflammation in patients with DED and in vitro. Conjunctival impression cytology samples from control subjects (n = 11) and patients with DED (n = 15) were used to determine the status of hyperosmotic stress (TonEBP/NFAT5), inflammation (IL-6, IL-8, IL-17A/F, TNFα, MMP9, and MCP1), VDR, and intracellular chloride ion (GLRX5) by quantitative polymerase chain reaction and/or immunofluorescence. Human corneal epithelial cells (HCECs) were used to study the effect of CFTR activator (genistein) and vitamin D (calcitriol) in hyperosmotic stress (HOs)-induced response in vitro. Western blotting was used to determine the expression of these proteins, along with p-p38. Significantly, higher expression of inflammatory factors, TonEBP, GLRX5, and reduced VDR were observed in patients with DED and in HOs-induced HCECs in vitro. Expression of TonEBP positively correlated with expression of inflammatory genes in DED. Increased TonEBP and GLRX5 provides confirmation of osmotic stress and chloride ion imbalance in OS epithelium in DED. These along with reduced VDR suggests dysregulated OS homeostasis in DED. Combination of genistein and calcitriol reduced HOs-induced TonEBP, inflammatory gene expression, and p-p38, and abated VDR degradation in HCECs. Henceforth, this combination should be further explored for its relevance in the management of DED.

Differential Effects of TGF-ß2 on the Low-Density Lipoprotein Receptor Expression in Three Types of Human Subconjunctival Fibroblasts.

Purpose: To investigate whether TGF-ß2 had a different effect on the expression levels of low-density lipoprotein receptor (LDLr) in the subconjunctival fibroblasts from glaucoma patients who underwent a reoperation (RGSFs) compared with those from glaucoma patients who underwent first filtering surgery (GSFs) and control patients with cataracts (HSFs). Methods: Human subconjunctival fibroblasts were obtained from the three groups of patients. Different concentrations of TGF-ß2 were added to the fibroblasts for 1, 3, and 5 days. The proliferation of the fibroblasts was determined by CCK-8 assays. Real-time PCR and western blotting were performed to analyze the mRNA and protein levels of LDLr. The uptake of DiI-labeled LDL was determined by confocal microscopy. Results: The results revealed that under TGF-ß2 exposure, fibroblast proliferation was positively correlated with LDLr expression (all p < .001). The LDLr mRNA and protein levels were affected by TGF-ß2 in a concentration-dependent and time-dependent manner in the RGSFs, GSFs and HSFs. The maximal expression of LDLr after TGF-ß2 stimulation was consistent with the peak uptake of DiI-LDL, which was obviously highest in the RGSFs, followed by the GSFs, and then the HSFs (all p < .05). All 3 groups took up DiI-LDL in a similar time-dependent manner, with maximal uptake at 6 h following DiI-LDL incubation (all p < .05). In addition, there were significant differences in the LDLr protein levels in the subconjunctival tissues isolated from the glaucoma patients during reoperation, the glaucoma patients during first filtering surgery and the control patients at day 3 (p < .05). The highest protein expression of LDLr was observed in the RG group. Conclusion: These data suggested that the RGSFs had the highest LDLr expression and the highest peak uptake of LDL among three groups. The LDLr-drug-LDL delivery system could potentially be used for targeted delivery of antimetabolite agents in anti-scarring therapy for glaucoma patients after filtering surgery.

Ocular surface findings in impression cytology after interferon a2b or mitomycin C in rabbits / Achados em citologia de impressão da superfície ocular após uso de interferon alfa-2b ou mitomicina C: estudo em coelho

ABSTRACT Objective: To describe ocular surface findings in impression cytology obtained from healthy rabbit conjunctiva treated with interferon alpha-2b eyedrop, and compare them to findings after use of mitomycin C 0.02%. Methods: An experimental study using a rabbit model was performed between September 2013 and October 2014 at the Faculdade de Medicina de Marília, Universidade Federal de São Paulo, Clínica de Olhos Moacir Cunha. Thirty New Zealand white rabbits were divided into 6 groups and received interferon alpha-2b or mitomycin C 0.02%. Impression cytology (IC) was performed prior to topical applications and at15, 30 and 60 days of use. The following variables were analyzed in impression cytology: goblet cells, cellularity, cell-to-cell adhesion, nucleus/cytoplasm ratio, nuclear chromatin, inflammatory cells keratinization, and cytomegaly. Results: The major findings in impression cytology after us of interferon alpha-2b included loss of goblet cells (50.8%), reduced cell-to-cell adhesion (26.2%), abnormal nucleus/cytoplasm ratio (20%) and reduced cellularity (15.4%). After use of mitomycin C 0.02%, the most common changes included loss of goblet cells (46.2%), abnormal nucleus/cytoplasm ratio (25.6%), less cell-to-cell adhesion (23.1%), and reduced cellularity (20.5%). There were no significant differences in any variable when comparing impression cytology after interferon alpha-2b and after mitomycin C 0.02%. Goblet cell loss was more pronounced at days 30 and 60, as compared to impression cytology at day 15 for both drugs. Conclusion: The loss of goblet cells, reduced cell-to-cell adhesion and cellularity, along with abnormal nucleus/cytoplasm ratio were the most common findings in impression cytology after use of interferon alpha-2b. These findings are similar to those described for use of mitomycin C 0.02%. ..

RESUMO Objetivo: Descrever os achados em citologia de impressão de conjuntiva sadia de coelho submetida ao uso de colírio de interferon alfa-2b e compará-los ao que foi encontrado após uso da mitomicina C 0,02%. Métodos: Estudo experimental realizado em modelo animal no período entre setembro de 2013 e outubro de 2014 nas dependências da Faculdade de Medicina de Marília, da Universidade Federal de São Paulo e da Clínica de Olhos Moacir Cunha. Trinta coelhos albinos da raça Nova Zelândia foram divididos em seis grupos e receberam interferon alfa-2b ou mitomicina C. A citologia de impressão foi realizada antes do início dos colírios e após 15, 30, 60 dias de seu uso. As seguintes variáveis foram analisadas na citologia de impressão: células caliciformes, celularidade, adesão intercelular, razão núcleo/citoplasma, cromatina, células inflamatórias, queratinização e citomegalia. Resultados: Os principais achados na citologia de impressão após o uso do interferon alfa-2b foram a redução de células caliciformes (50,8%), a diminuição da adesão intercelular (26,2%), a alteração da razão N/C (20%) e a redução da celularidade (15,4%). Após o uso da mitomicina C 0,02%, foram mais frequentes a redução das células caliciformes (46,2%), a alteração da razão N/C (25,6%), a adesão intercelular (23,1%) e a redução da celularidade (20,5%). Não houve diferença estatisticamente significante para nenhuma das variáveis estudas quando se compararam as citologias de impressão após interferon alfa-2b com as citologias de impressão após mitomicina C 0,02%. Independentemente da substância utilizada, as citologias colhidas 30 e 60 dias após início das drogas apresentaram maior redução de células caliciformes quando comparadas com as citologias de impressão colhidas após 15 dias. Conclusão: A redução das células caliciformes, a diminuição da adesão intercelular, a alteração da razão N/C e a diminuição da celularidade foram as alterações mais frequentes na citologia de impressão colhida após o uso de interferon alfa-2b. Os achados em citologias de impressão após o uso de interferon alfa-2b são semelhantes àqueles encontrados após o uso da mitomicina C 0,02%.

Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy.

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10-20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

A Tenon's capsule/bulbar conjunctiva interface biomimetic to model fibrosis and local drug delivery.

Glaucoma filtration surgery is one of the most effective methods for lowering intraocular pressure in glaucoma. The surgery efficiently reduces intra-ocular pressure but the most common cause of failure is scarring at the incision site. This occurs in the conjunctiva/Tenon's capsule layer overlying the scleral coat of the eye. Currently used antimetabolite treatments to prevent post-surgical scarring are non-selective and are associated with potentially blinding side effects. Developing new treatments to target scarring requires both a better understanding of wound healing and scarring in the conjunctiva, and new means of delivering anti-scarring drugs locally and sustainably. By combining plastic compression of collagen gels with a soft collagen-based layer, we have developed a physiologically relevant model of the sub-epithelial bulbar conjunctiva/Tenon's capsule interface, which allows a more holistic approach to the understanding of subconjunctival tissue behaviour and local drug delivery. The biomimetic tissue hosts both primary human conjunctival fibroblasts and an immune component in the form of macrophages, morphologically and structurally mimicking the mechanical proprieties and contraction kinetics of ex vivo porcine conjunctiva. We show that our model is suitable for the screening of drugs targeting scarring and/or inflammation, and amenable to the study of local drug delivery devices that can be inserted in between the two layers of the biomimetic. We propose that this multicellular-bilayer engineered tissue will be useful to study complex biological aspects of scarring and fibrosis, including the role of inflammation, with potentially significant implications for the management of scarring following glaucoma filtration surgery and other anterior ocular segment scarring conditions. Crucially, it uniquely allows the evaluation of new means of local drug delivery within a physiologically relevant tissue mimetic, mimicking intraoperative drug delivery in vivo.

Goblet Cell Differentiation Potential in Human Corneal Limbal Epithelial Progenitor Cells In Vitro.

Purpose: The existence of goblet cells has been regarded as a critical differential point to distinguish conjunctival epithelium from corneal epithelium in vivo. We tested differentiation potential of single progenitor cells from corneal limbal epithelium with growth factors in vitro. Methods: Dissociated single cells from corneal limbal epithelium were cultured in the serum- and feeder cell-free medium containing B27 and various growth factors using nontissue culture dishes. Specific marker expression was examined in the colonies stimulated with growth factors. Differentiation of some mucosal epithelia was tested. Results: Adherent single cells from dissociated single cells in corneal limbal epithelium did not proliferate in the serum- and feeder cell-free medium containing B27 only and formed corneal epithelium with B27 plus epidermal growth factor, while they gave rise to goblet cell with periodic acid Schiff-positive mucin and cytokeratin-3 and-12 expressing corneal epithelium with fibroblast growth factor (FGF)2 stimulation. Colonies stimulated with FGF2 expressed goblet cell specific MUC5AC and cytokeratin-7 mRNA and protein. FGF receptor 1 was a functional receptor for the differentiation to goblet cells and corneal epithelium. Conclusions: Single corneal limbal progenitor cells give rise to goblet cells and corneal epithelium by FGF2 stimulation via FGF receptor 1 in vitro.

Immunohistochemical Study of SARS-CoV-2 Viral Entry Factors in the Cornea and Ocular Surface.

PURPOSE: To confirm the ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by evaluating the expression of viral entry factors in human ocular tissues using immunohistochemistry. METHODS: Fresh donor corneas and primary explant cultures of corneal, limbal, and conjunctival epithelial cells were evaluated for the expression of viral entry factors. Using immunohistochemistry, the samples were tested for the expression of angiotension-converting enzyme 2 (ACE2), dendritic cell-specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), DC-SIGN-related protein (DC-SIGNR), and transmembrane serine protease 2 (TMPRSS2). RESULTS: In total, 5 donor corneas were evaluated for the expression of viral entry factors. In all specimens, both ACE2 and TMPRSS2 were expressed throughout the surface epithelium (corneal, limbal, and conjunctival) and corneal endothelium. In corneal stromal cells, ACE2 was sporadically expressed, whereas TMPRSS2 was absent. DC-SIGN/DC-SIGNR expression varied between donor specimens. Four specimens expressed DC-SIGN/DC-SIGNR in a similar distribution to ACE2, but 1 specimen from a young donor showed no expression of DC-SIGN/DC-SIGNR. ACE2, TMPRSS2, and DC-SIGN/DC-SIGNR were all expressed in the cultured corneal, limbal, and conjunctival epithelial cells. CONCLUSIONS: Both corneal and conjunctival epithelia express ACE2, DC-SIGN/DC-SIGNR, and TMPRSS2, suggesting that the ocular surface is a potential route for the transmission of SARS-CoV-2. The risk of viral transmission with corneal transplantation cannot be ruled out, given the presence of ACE2 in corneal epithelium and endothelium. Cultured corneal, limbal, and conjunctival epithelial cells mimic the expression of viral entry factors in fresh donor tissue and may be useful for future in vitro SARS-CoV-2 infection studies.

The potency of hsa-miR-9-1 overexpression in photoreceptor differentiation of conjunctiva mesenchymal stem cells on a 3D nanofibrous scaffold.

MiRNAs are small non-coding RNAs that are ordinarily involved in modulating mRNAs and stem cell differentiation. 3D nanofibrous scaffolds have an important role in the differentiation of stem cells due to their similarity to the extracellular matrix (ECM). In the present study, we tried to introduce a new approach to guiding the differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells by hsa-miR-9-1 delivery on both 2D and 3D substrates. First, the CJMSCs were transduced by a lentiviral vector carrying miR-9 (pCDH + hsa-miR-9-1) and then cell transduction efficacy verified by using fluorescent microscopy, flow cytometry, and qPCR analyses. Silk Fibroin-poly-L-lactic acid (SF-PLLA) scaffold was fabricated by the electrospinning technique while the scaffold characteristics including morphology, chemical properties, and biocompatibility were evaluated by SEM, FTIR, and MTT assays, respectively. Then, the miR-9-CJMSCs were seeded on both TCPS and the scaffold; photoreceptor gene and protein expressions were evaluated by RT-qPCR and immunostaining after 14 and 21 days of transduction. More than 80% of CJMSCs were transduced and miR-9 expression was significantly higher in miR-9-CJMSCs compared with empty vector (EV)-CJMSCs. SEM and FTIR confirmed the fabrication of the SF/PLLA hybrid structure. RT-qPCR and immunostaining analyses showed that the specific photoreceptor genes and proteins were expressed in miR-9 transduced CJMSCs. Mir-9 induced CJMSCs into photoreceptor-like cells in a time-dependent manneron on both TCPS and nanofibrous scaffold.We have proved that hsa-miR-9-1 has the potency to guide the photoreceptor differentiation of mesenchymal stem cells and promote retinal regeneration.

Lymphatic lacunae of the human eye conjunctiva embedded within a stroma containing CD34+ telocytes.

An accurate identification of telocytes (TCs) was limited because of the heterogeneity of cell types expressing the markers attributed to TCs. Some endothelial lineage cells also could fit within the pattern of TCs. Such endothelial cells could line conjunctival lacunae previously assessed by laser confocal microscopy. We have been suggested that an accurate distinction of TCs from endothelial cells in the human eye conjunctiva could be achieved by use of CD31, CD34 and D2-40 (podoplanin); and that the conjunctival lacunae are in fact lymphatic. We aimed as testing the hypothesis by an immunohistochemical study on human eye conjunctiva biopsy samples. Samples of human eye conjunctiva from 30 patients were evaluated immunohistochemically by use of the primary antibodies: CD34, D2-40 and CD31. D2-40 was equally expressed within epithelia and laminae propria. Basal epithelial cells were D2-40 positive. Within the stromal compartment, the lymphatic marker D2-40 labelled several lymphatic vessels. CD31 labelled both vascular and lymphatic endothelial cells within the lamina propria. When capillary lymphatics were tangentially cut, they gave the false appearance of telocytes. Blood endothelial cells expressed CD34, whereas lymphatic endothelial cells did not. Stromal CD34-expressing cells/telocytes were found building a consistent pan-stromal network which was equally CD31-negative and D2-40-negative. The conjunctival lymphatic lacunae seem to represent a peculiar anatomic feature of eye conjunctiva. They are embedded within a CD34-expressing stromal network of TCs. The negative expression of CD31 and D2-40 should be tested when discriminating CD34-expressing TCs.

The TßR II-targeted aptamer S58 prevents fibrosis after glaucoma filtration surgery.

Glaucoma filtration surgery (GFS) is an effective clinical treatment for glaucoma when intraocular pressure (IOP) control is poor. However, the occurrence of conjunctival scarring at the surgical site is the main reason for failure of the surgery. In a previous study, we isolated and developed S58, a novel nucleic acid aptamer targeting TßR II, by systematic evolution of ligands by exponential enrichment (SELEX). Here, we show how S58 sterically inhibits the TßR II interaction with TGF-ß. The effects of topical S58 treatment were studied in a rabbit model of GFS. At 6 postoperative weeks, S58 reduced fibrosis and prolonged bleb survival in rabbits after GFS. Further in vitro tests showed that the levels of fibrosis in S58 treated-Human Conjunctival Fibroblasts (HConFs) were decreased and that antioxidant defense was increased. In addition, the loss of nuclear factor erythroid 2-related factor 2 (Nrf2) or the inhibition of phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) reversed the anti-fibrotic effects of S58. The present work suggests that S58 could effectively improve GFS surgical outcomes by activating the intracellular antioxidant defense PI3K/Akt/Nrf2 signaling pathway.