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Purpose: To assess the efficacy and safety of human leukocyte antigen-matched allogeneic cultivated limbal epithelial stem cell grafts in the treatment of aniridia-associated keratopathy (AAK)., Methods: Six eyes of 6 patients with severe AAK received an allogeneic stem cell graft between January 2010 and March 2017. Anatomical and functional results were assessed at 6 months, 1 year, 2 years, and the final follow-up visit available. Safety analysis was performed by considering all perioperative and postoperative adverse events and additional surgeries required during the follow-up period., Results: The mean follow-up was 53.6 months (range 24-104 months). In most patients (80%), there was an early improvement of the keratopathy postoperatively, which slowly regressed during longer follow-up. At the final follow-up, 4 of the eyes were graded as failure and 1 eye was graded as partial success. Grading the sixth eye was not possible because of an adverse event. None of the patients maintained a total anatomical success in the long-term. Only 1 patient maintained a modest improvement in best-corrected visual acuity from hand motion to counting fingers. Four serious adverse events were recorded in 2 patients., Conclusions: Severe AAK remains a challenging condition to manage. Transplantation of allogenic ex vivo cultivated limbal stem cells may provide a temporary improvement in ocular surface stability, but anatomical and functional results are poor in the long-term. The eyes are prone to adverse events, and any surgical treatment should take this into consideration., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Limbal stem cell (LSC) transplantation is the only efficient treatment for patients affected by LSC deficiency (LSCD). Allogeneic LSC transplantation is one of the most successful alternative for patients with bilateral LSCD. Nevertheless, the high variability of the human leukocyte antigens (HLA) remains a relevant obstacle to long-term allogeneic graft survival. This study characterized the immunologic properties of LSCs and proposed a genetic engineering strategy to reduce the immunogenicity of LSCs and of their derivatives. Hence, LSC HLA expression was silenced using lentiviral vectors encoding for short hairpin (sh) RNAs targeting β2-microglobulin (β2M) or class II major histocompatibility complex transactivator (CIITA) to silence HLA class I and II respectively. Beside the constitutive expression of HLA class I, LSCs showed the capability to upregulate HLA class II expression under inflammatory conditions. Furthermore, LSCs demonstrated the capability to induce T-cell mediated immune responses. LSCs phenotypical and functional characteristics are not disturbed after genetic modification. However, HLA silenced LSC showed to prevent T cell activation, proliferation and cytotoxicity in comparison to fully HLA-expressing LSCs. Additionally; HLA-silenced LSCs were protected against antibody-mediated cellular-dependent cytotoxicity. Our data is a proof-of-concept of the feasibility to generate low immunogenic human LSCs without affecting their typical features. The use of low immunogenic LSCs may support for long-term survival of LSCs and their derivatives after allogeneic transplantation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Valdivia, Bertolin, Breda, Carvalho Oliveira, Salz, Hofmann, Börgel, Blasczyk, Ferrari and Figueiredo.)

Dry eye disease (DED) is a multifactorial disease of the ocular surface, characterized by loss of tear film homeostasis and ocular symptoms, in which neurosensory abnormalities have recently been shown to play an etiological role. Although the role of inflammation has been widely studied in DED, the kinetics of immune cells of the ocular surface in this complex disease are hereto unclear. Herein, we utilized intravital multiphoton imaging on transgenic mice to investigate the 3D morphology and kinetics of conventional dendritic cells (cDCs) and the role of ocular surface sensory nerves in regulating them in both the naïve state and experimental DED. Mice with DED had significantly lower tear secretion ( p < 0.01), greater corneal fluorescein staining ( p < 0.001), and higher cDC density in the ocular surface ( p < 0.05), compared to naïve mice. cDCs in DED mice showed morphological alterations in the limbus, exhibiting smaller surface area ( p < 0.001) and volume ( p < 0.001) compared to naïve mice. Furthermore, corneal cDCs showed greater sphericity in DED mice compared to naïve mice ( p < 0.01). In addition, limbal cDCs displayed significantly increased migratory kinetics in DED, including mean track speed, 3D instantaneous velocity, track length, and displacement, compared to naïve mice (all p < 0.05). In mice with DED, cDCs showed a higher meandering index in the limbus compared to central cornea ( p < 0.05). In DED, cDCs were less frequently found in contact with nerves in the limbus, peripheral, and central cornea ( p < 0.05). cDCs in contact with nerves demonstrated a larger surface area ( p < 0.001) and volume ( p < 0.001), however, they exhibited less sphericity ( p < 0.05) as compared to cDCs not in contact with nerves in naïve mice. Importantly, cDCs in contact with nerves during DED had a decreased track length, displacement, mean track speed, and 3D instantaneous velocity compared to those not in contact with nerves (all p < 0.05). Taken together, we present in vivo evidence of altered cDC kinetics and 3D morphology in DED. Furthermore, apparent neuronal contact significantly alters cDC kinetics and morphological characteristics, suggesting that ocular surface nerves may play a direct role in mediating immune responses in DED., (Copyright © 2020 Jamali, Seyed-Razavi, Chao, Ortiz, Kenyon, Blanco, Harris and Hamrah.)

Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new therapies to promptly restore vision. Innate lymphoid cells (ILCs) are a recently identified heterogeneous cell population that has been reported to orchestrate immunity and promote tissue repair in the lungs and skin after injury. However, whether ILCs can modulate the repair process in the cornea remains poorly understood. We identified a population of cornea-resident group 2 ILCs (ILC2s) in mice that express CD127, T1/ST2, CD90, and cKit. This cell population was relatively rare in corneas at a steady state but increased after corneal epithelial abrasion. Moreover, ILC2s were maintained and expanded locally at a steady state and after wounding. Depletion of this cell population caused a delay in corneal wound healing, whereas supplementation of ILC2s through adoptive transfer partially restored the healing process. Further investigation revealed that IL-25, IL-33, and thymic stromal lymphopoietin had critical roles in corneal ILC2 responses and that CCR2 - corneal macrophages were an important producer of IL-33 in the cornea. Together, these results reveal the critical role of cornea-resident ILC2s in the restoration of corneal epithelial integrity after acute injury and suggest that ILC2 responses depend on local induction of IL-25, IL-33, and thymic stromal lymphopoietin., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Stem Cells Translational Medicine 2017;6:761-766., (© 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Purpose of Review: Vernal keratoconjunctivitis (VKC) is a severe bilateral chronic allergic inflammatory disease of the ocular surface. In most of the cases, the disease is limited to the tarsal conjunctiva and to the limbus. However, in the more severe cases, the cornea may be involved, leading to potentially sight threatening complications. Prompt recognition of these complications is crucial in the management of VKC, which is one of the most severe ocular allergic diseases., Recent Findings: A vicious cycle of inflammation occurs as a result of a set of reciprocal interactions between the conjunctiva and the cornea, which results in damage to the corneal epithelium and corneal stoma, and to the formation of shield ulcers and plaques, infectious keratitis, keratoconus, scarring, and limbal stem cell deficiency. These corneal complications can cause permanent decrease or loss of vision in children suffering from VKC., Summary: Corneal complications in VKC are the result of an on-going process of uncontrolled inflammation. Proper recognition of the corneal complications in VKC is crucial, as most of these can be managed or prevented by a combination of medical and surgical measures.

Background: CD4+ T helper type 2 cells play a central role in the pathogenesis of vernal keratoconjunctivitis (VKC), and antigen-presenting cells are required for the cell activation. In this study, we aimed to survey the density, distribution, and morphology of dendritic cells (DCs) in patients with VKC by in vivo confocal microscopy., Methods: Thirty-five patients (mean, 12.4 ± 5.3 years) affected by VKC were included. All patients were treated with 0.1% fluorometholone eye drops and 0.5% cyclosporine A eye drops. The density and morphological and distributional characteristics of DCs in each right eye were evaluated by in vivo confocal microscopy before treatment and at 1, 3, and 6 months after treatment. Thirty-five age-matched normal subjects (mean, 16.5 ± 1.8 years) were studied as controls., Results: There was significant difference in age between the VKC group and the control group (F = 18.17, P < 0.05). Compared with normal eyes, increased numbers of DCs were found in patients with VKC, with mean cell densities of 244.09 ± 59.76 cells/mm(2) at the bulbar conjunctiva, 574.53 ± 87.34 cells/mm(2) at the limbus, and 403.32 ± 106.59 cells/mm(2) at the peripheral cornea before treatment. These DCs exhibited a typical dendritic shape. At 3 months after treatment, the DC density at the conjunctiva decreased significantly (P < 0.05), approximating that in the controls. At 3 and 6 months, the DC densities at the limbus and peripheral cornea also decreased significantly (P < 0.05), but were still statistically higher than those in the controls. These DCs, with small dendritic processes or irregular shapes, were observed to gradually locate at the epithelial basal membrane and subbasal nerve plexus., Conclusions: In vivo confocal microscopy appears to be a valuable tool in evaluating the dynamic change of DCs at the conjunctiva and cornea. DCs play an essential role in VKC and therefore may constitute a target for therapeutic intervention for VKC., (© 2013 John Wiley & Sons Ltd.)

Background Aims: Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation., Methods: MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells., Results: Human L-MSC cultures were typically CD34⁻, CD45⁻ and HLA-DR⁻ and CD73⁺, CD90⁺, CD105⁺ and HLA-ABC⁺. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation., Conclusions: L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Chronic inflammation in pterygium occurrence has not been explained. Whether damaged limbal basal epithelial cells are associated with pterygium occurrence in black Africans is not clear., Objective: To explain chronic inflammation in pterygium, and to clarify whether damaged limbal basal epithelial cells were associated with pterygium occurrence in black Africans., Methods: Chronic inflammatory changes and damaged limbal basal epithelial cells were assessed in 59 samples., Results: Chronic inflammatory cells were present in 59 pterygia. Inflammatory cell count in 5 (27.8%) of 18 small pterygia was >200 (high) while in 22 (53.7%) of 41 large growths was <200 (low); p = 0.25. The proportion of pterygia with high counts tended to increase with pterygium extent. Twenty (33.9%) of 59 pterygia recurred after surgery. Ten (50%) of 20 samples had high cell counts and 10 (50%), low counts; p = 0.40. P53 expression was detected in 11 (18.6%) of 59 pterygium samples and 5 (71.4%) of 7 controls; p = 0.007. MMP 1 staining was present in 14 (23.7%) of 59 sections and 5 (71.4%) of 7 controls; p = 0.02. MMP2 in 16 (27.1%) cases and 5 (71.4%) controls; p = 0.03. MMP3 was overexpressed in 16 (27.1%) of 59 cases and 5 (71.4%) controls; p = 0.03., Conclusions: Mild chronic inflammation has a tendency to be more frequent than severe inflammation in pterygia. It is clear that damaged limbal basal epithelial cells are unlikely to be related to pterygium occurrence.

Cornea transplantation has been considered to be different from other solid organ transplantation because of the assumed immune-privileged state of the anterior chamber of the eye. Three major lines of thought regarding the molecular mechanisms of immune privilege in the eye are as follows: (a) anatomical, cellular, and molecular barriers in the eye; (b) anterior chamber-associated immune deviation; and (c) immunosuppressive microenvironment in the eye. However, cornea transplants suffer allograft rejection when breached by vascularization. In recent developments, cellular corneal transplantation from cultivated limbal epithelial cells has shown impressive advances as a future therapy. The limbal stem cell niche contains stem cells that promote proliferation and migration and have immunosuppressive mechanisms to protect them from immunological reactions. Limbal stem cells are also noted to display an enhanced expression of genes for the antiapoptotic proteins, a property that is imperative for the survival of transplanted tissues. Further investigation of the molecular mechanisms regulating the immune regulation of limbal stem cells is relevant in the clinical setting to promote the survival of whole corneal and limbal stem cell transplantation.

Purpose: We determined the plausible functional role of angiopoietin-like protein 2 (Angptl2) in inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo., Methods: Corneal hemangiogenesis and lymphangiogenesis were induced by suturing 10-0 nylon 1 mm away from the limbal vessel in Angptl2 knockout and K14-Angptl2 transgenic mice. We analyzed Angptl2 and interleukin 1β (IL-1β) expressions in normal and vascularized corneas by real-time RT-PCR and immunohistochemistry. Corneal hemangiogenic and lymphangiogenic responses, and macrophage infiltration were assessed by immunofluorescent microscopic studies using specific antibodies against CD31, LYVE-1, and F4/80, and compared to their corresponding background. Subconjunctival injection of Angptl2 siRNA to the sutured corneas was also performed., Results: Angptl2 mRNA expression increased markedly in the neovascularized corneas compared to the normal cornea. Angptl2 protein was expressed strongly in the corneal epithelium and stroma of the vascularized cornea. The regions showing hemangiogenesis and lymphangiogenesis were increased significantly in K14-Angptl2 mice and reduced in Angptl2(-/-) mice compared to their corresponding background strains. In contrast to control mice, the number of F4/80-positive cells, as well as the expressions of F4/80 and IL-1β were found to be higher in K14-Angptl2 mice and lower in Angptl2(-/-) mice. Subconjunctival injection of Angptl2 siRNA significantly inhibited hemangiogenesis and lymphangiogenesis in the sutured corneas., Conclusions: Our findings demonstrated Angptl2 to be upregulated in corneal inflammation, and highlight that corneal hemangiogenesis and lymphangiogenesis may be driven by Angptk2 overexpression via macrophage infiltration and IL-1β expression. Angptl2 may be a novel therapeutic target for preventing blindness.

Background: The purpose of this study is to conduct a histopathological research of the conjunctival findings and eosinophilic inflammation of novel atopic keratoconjunctivitis in a NC/Nga mouse model using crude Dermatophagoides farina., Methods: NC/Nga mice were sensitized by repeated topical applications of an ointment containing Dermatophagoides farinae body (Dfb). They were then divided into 4 groups depending on the following topical ophthalmic treatment: DFb group undergoing topical ophthalmic ointment containing Dfb; DFco group undergoing topical instillation of allergen extracts of Dermatophagoides farinae; Ba group undergoing topical ointment with substrate alone and NT group without after-topical ophthalmic treatment. At 24 hours after the last ophthalmic treatment, histopathological examination was performed. The density of the subepithelial infiltration of the eosinophils was determined. Serum total IgE and house-dust-mite (HDM)-specific IgE antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA)., Results: In the DFb group, the conjunctiva showed similar findings to those of atopic keratoconjunctivitis, i.e. intraepithelial pseudotubular formation, Torus-form infiltration due to massive lymphocytes in the palpebral conjunctiva and gelatinous hyperplasia in the limbus with subepithelial granuloma composed of lymphocytes and eosinophils. Subepithelial infiltration of eosinophil density in the DFb group [878.4 ± 399.7cells/mm2 (mean ± SD)] was significantly higher than in the other 2 groups (DFco 85.6 ± 40.1 Ba 49.2 ± 32.3) (P < 0.001). Total serum IgE concentration and HDM-specific serum IgE antibody concentration in the DFb group and the DFco group were significantly higher compared with those in the NT group., Conclusions: Topical application of an ointment containing DFb to both the skin and eyes of NC/Nga mice can induce an atopic keratoconjunctivitis (AKC) model in these mice.

LYVE-1(+) corneal lymphatics contribute to drainage and immunity. LYVE-1 is widely accepted as the most reliable lymphatic marker because of its continuous expression in lymphatic endothelium. LYVE-1 expression in corneal lymphatics has not been examined. In this study, we report intact CD31(+) corneal lymphatic capillary endothelial cells that do not express LYVE-1. The number of LYVE-1(-) gaps initially increased until 8 wk of age but was significantly reduced in aged mice. C57BL/6 mice showed a notably higher number of the LYVE-1(-)/CD31(+) lymphatic regions than BALB/c mice, which suggests a genetic predisposition for this histological feature. The LYVE-1(-) lymphatic gaps expressed podoplanin and VE-cadherin but not αSMA or FOXC2. Interestingly, the number of LYVE-1(-) gaps in FGF-2, but not VEGF-A, implanted corneas was significantly lower than in untreated corneas. Over 70% of the CD45(+) leukocytes were found in the proximity of the LYVE-1(-) gaps. Using a novel in vivo imaging technique for visualization of leukocyte migration into and out of corneal stroma, we showed reentry of extravasated leukocytes from angiogenic vessels into newly grown corneal lymphatics. This process was inhibited by VE-cadherin blockade. To date, existence of lymphatic valves in cornea is unknown. Electron microscopy showed overlapping lymphatic endothelial ends, reminiscent of microvalves in corneal lymphatics. This work introduces a novel corneal endothelial lymphatic phenotype that lacks LYVE-1. LYVE-1(-) lymphatic endothelium could serve as microvalves, supporting unidirectional flow, as well as immunological hot spots that facilitate reentry of stromal macropahges.

Purpose: Mesenchymal stromal stem cells (MSC) are non-hemopoietic cells with the capacity to self-renewal and to differentiate into various cell lineages of mesenchymal origin. More recently, the immune regulatory potential of MSC has been focused on. Furthermore, mesenchymal stem cells obtained from diverse tissues possess immunomodulatory properties and inhibit proinflammatory immune reactions. The aim of this study was to determine the immunosuppressive characteristics of mesenchymal stem cells isolated from human limbal (L-MSC) tissue., Methods: L-MSC were enzymatically obtained from cadaveric sclero-corneal rims and expanded in vitro. The cells were characterized by flow cytometry using specific antibodies to mesenchymal stem cells markers. Clonogenic and tissue transdifferentiation in vitro assays were performed. The effect of L-MSC soluble factors on T cell proliferation was determined by flow cytometry. Cytokines such as transforming growth factor-b1 (TGF-β1) and Interleukin-10 (IL-10) on supernatants from L-MSC were identified by enzyme-linked immunosorbent assay (ELISA)., Results: Herein, we described that L-MSC cells in vitro-expanded were positive for the expression of vimentin, CD29, CD34, CD39, CD73 and CD105 mesenchymal stem cells markers; meanwhile, this cell population was negative to CD45 and HLA-DR hemopoietic markers as well as to cytokeratin expression. Clonogenic assays showed that these cells were able to form colonies. In addition, this L-MSC population had the ability to transdifferentiate into neurons and chondrocytes and to form tubular networks on matrigel in the presence of vascular endothelial growth factor (VEGF). These results indicated that these cells were stem cells. Additionally, soluble factors secreted by L-MSC were capable of mediating the suppression of T-cell receptor (TCR)-engagement lymphocyte proliferation. In an attempt to identify the possible immunosuppressive factors secreted by L-MSC, TGFβ1 and IL-10 cytokines were determined in the L-MSC supernatants by ELISA; interestingly, TGFβ1 was constitutively secreted by this cell population; in contrast, IL-10 was not detectable. Moreover, TGFβRII neutralizing antibodies were able to revert the TCR-engagement lymphocyte proliferation inhibition mediated by L-MSC. Thus, TGFβ1 secreted by L-MSC was able to suppress T cell proliferation., Conclusions: Taken together these results, explain in part the immunosuppressive features of this cell population obtained from the human limbus. All these characteristics make this cell population an excellent source to be used in the regenerative medicine.

Purpose: To investigate the effect of ex vivo culture on the expression of human leukocyte antigen (HLA) in human corneal limbal epithelial cells (LECs), and to evaluate the expression of HLA and costimulatory molecules on human corneal limbal stromal cells (LSCs)., Methods: The expression of HLA-ABC and HLA-DR were evaluated using flow cytometry on cultured human LECs and LSCs after serial passage and compared with that on freshly isolated cells. Additionally, the expression of costimulatory molecules CD80, CD86, CD40, and immunoregulatory molecule HLA-G were investigated on LSCs with or without an addition of interferon (IFN) γ (250 U/mL, 4 days)., Results: HLA-ABC and HLA-DR were expressed in 99.12 ± 1.02% and 25.86 ± 4.34% of freshly isolated LECs, respectively. After culture, the HLA-DR-expressing cells significantly decreased with serial passage, whereas the percentage of HLA-ABC-expressing cells did not change. More than 90% of LSCs expressed HLA-ABC and HLA-G, whereas the expression of HLA-DR, CD80, CD86, and CD40 was negligible. This expression profile did not change after serial passage. However, the treatment with IFN-γ induced a significant increase in HLA-DR (0.17% vs. 17.68%) and CD40 (0.19% vs. 13.19%) expression on LSCs., Conclusions: The immunogenicity of LECs can be lowered after ex vivo cultivation, whereas LSCs can be immunogenic in the presence of IFN-γ.

Limbal stem cell deficiency (LSCD) is clinically characterized by growth of fibrovascular pannus onto the corneal surface, chronic inflammation and impaired visual acuity. The aim of this study was to characterize the pannus tissue. Total RNA was isolated from six pannus samples and protein from three pannus samples from patients with LSCD caused by chemical burns. Normal human corneal tissue (n=6) and conjunctiva (n=6) were used as control tissues. The expression of the epithelial lineage markers keratin 3 (K3), K19 and MUC5AC, the inflammatory markers IL-1beta, ICAM-1 and VEGF was analyzed by Western Blotting and/or real-time PCR. Normal corneal tissue had a higher expression of K3 and a lower expression of K19 and MUC5AC in comparison to normal conjunctiva. A higher expression of inflammatory markers was seen in conjunctiva compared to corneal tissue. The pannus tissue showed a similar expression of lineage markers to conjunctiva but a higher expression of most inflammatory markers as compared to both control tissues. The analyzed samples of pannus in LSCD resembled conjunctiva and not cornea and showed a high expression of inflammatory markers indicating chronic inflammation.

Purpose: Squamous metaplasia of the ocular surface epithelium in severe Sjögren syndrome (SS) dry eye has been implicated to be associated with chronic engagement of immune-mediated inflammation. While the detailed immunopathological mechanism underlying keratinization of the ocular muco-epithelium in this setting remains unclear, mice deficient in the autoimmune regulator gene (Aire) demonstrate SS-like pathological changes in the exocrine organs and ocular surface including squamous metaplasia. Using this murine model, we sought to determine the specific immune events that predict squamous metaplasia of the cornea in Aire deficiency., Methods: Lissamine green staining, goblet cell density, and corneal small proline-rich protein 1B (SPRR1B) were compared in Aire-sufficient and -deficient mice at 4, 8, and 16 weeks of age. Corneal, limbal and conjunctival infiltration of CD4(+) and CD8(+) T cells as well as CD11c(+) and MHC class II (I-A(d+)) dendritic cells (DCs) were examined at the same time points. Ordinary least squares regression was used to model SPRR1B's relationship with lissamine green staining, goblet cell density, and immune cell infiltration., Results: Lissamine green staining was present in Aire-deficient mice by four weeks of age and increased over time. Compared to Aire-sufficient controls, conjunctival goblet cell density (GCD) decreased and corneal SPRR1B increased in Aire-deficient mice with significant differences noted at both 8 and 16 weeks. Immune-mediated CD4(+) T cell infiltration of the conjunctiva and limbus peaked at eight weeks and then decreased. In contrast, corneal T cell infiltration continued to increase over time, reaching a maximum cell number at 16 weeks. CD11c(+) myeloid-derived DCs were found in the conjunctiva and limbus at all time points. As the mice aged, there was a notable increase in corneal CD11c(+) cell counts. Interestingly, the dynamic of activated MHC class II(+) DCs was nearly identical to that of CD4(+) T cells, peaking first in the limbus at eight weeks with maximum infiltration of the cornea by 16 weeks. Regression analysis showed that squamous metaplasia biomarker, SPRR1B, is strongly related to the lissamine green staining of the ocular surface. Corneal infiltration of activated DCs was most prognostic of corneal SPRR1B expression while the presence of precursor DCs, activated DCs, and CD4(+) T cells in the limbus were also significant predictors of SPRR1B., Conclusions: Aire-deficient mice represent a useful model to study Sjögren-like autoimmune-mediated ocular surface disease. Results of the current study suggest that squamous cell precursor protein, SPRR1B, provides an important readout to evaluate ocular surface damage and specific events related to immune-mediated inflammation. Results also define an appropriate time frame for interventional studies to develop more effective therapies for keratinizing ocular surface disease.

Purpose: To evaluate the long-term outcome of HLA-matched lr-CLAL for bilateral ocular surface disorders., Methods: A retrospective, non-comparative interventional case series study of 39 eyes of 32 patients with bilateral surface disorders and clinical diagnosis of limbal stem cell deficiency who underwent HLA-matched lr-CLAL. Visual acuity (VA), ambulatory vision (> or = 20/200) and ocular surface stability were evaluated as main outcomes. Donor limbus was obtained from a sibling or a parent of the patient, after an appropriate Class I and II HLA match., Results: One year after surgery, VA improved in 46.2%, ambulatory vision was achieved in 48.7% and a stable corneal surface was achieved in 84.6% of the eyes. At the final follow-up (mean, 48.7 +/- 30.6 months), 66.6% of the eyes that had gained VA one year after surgery maintained an improved VA (p=0.28), 94.7% of eyes that had achieved ambulatory vision one year after surgery maintained 20/200 or better (p<0.001) and 93.9% still had a stable corneal surface (p=0.043) at the final follow-up., Conclusions: HLA-matched lr-CLAL can be an adequate method of treatment for bilateral ocular surface disorders, with a reasonable percentage of success of long-term results.

Aim: In 2005 we reported the discovery of a novel anatomical structure at the limbus, which we termed the limbal epithelial crypt (LEC). The purpose of this study was to further evaluate the distribution, immunophenotypical, and ultra structural characteristics of the LEC as a putative niche of stem cells., Methods: Sequential histological sections of human corneo-scleral limbal rims were examined for the presence and distribution of the LEC. Immunophenotypical characterisation of the LEC cells using a panel of antibodies of interest was undertaken. Transmission electron microscopy of the LEC was used to examine the ultra structural and morphometric features of cells within the LEC and adjacent limbus., Results: A total of 74 LECs were identified in eight corneo-scleral rims. These varied in number, size and distribution within rims. Cells within the crypt demonstrated the following phenotype: CK3-/CK19+/CD 34-/Vimentin+/p63+/Connexin 43+/MIB1 (Ki67)-. Presence of Cx43 was also demonstrated in the rete pegs adjacent to the LEC. Basal cells of the LEC were significantly smaller than basal cells found in adjacent rete pegs and also smaller than suprabasal limbal and central corneal epithelial cells (p<0.05). Morphologically they had a high nuclear:cytoplasmic ratio and were adherent to the underlying basement membrane by means of complex convolutions of cytoplasmic processes., Conclusions: LECs are sparse but a consistent finding in the human corneo-scleral limbus. The LEC contains a unique sub-population of cells expressing several characteristics that are consistent with it representing a putative stem cell niche.

Limbal transplants in humans show a high rate of rejection even under local and systemic immunotherapy. In order to test immunomodulatory treatments a new limbal transplant model in the rat was developed using enhanced green fluorescent protein (E-GFP) as marker for follow-up. Sixty E-GFP-positive limbal transplants from Sprague-Dawley TgN(act-EGFP)Osb4 rats were transplanted onto 18 wild-type inbred Sprague-Dawley (isografts) rats, six wild-type litter mate Sprague-Dawley (sibling) rats, 18 Fischer 344 (allografts) rats, and 18 Fischer 344 rats depleted from monocytes and macrophages by subconjunctival treatment with clodronate liposomes. All rats were monitored three times a week with fluorescence microscopy, until fluorescence had disappeared. At postoperative days 6, 9, 12, and 15, three rats of all groups were killed for immunohistochemical analysis of infiltrating cells. Using a modified digital fluorescence microscope, we were able to monitor transplant behavior over time without disturbance of the ocular surface. The average days of rejection were 14 days in the isograft group, the sibling group, and the untreated allograft group. However, the average day of rejection in the allogeneic macrophage-depleted group was 27 days. Marked infiltration of macrophages and lymphocytes was seen in the untreated isografts and allografts. In the clodronate liposome-treated allografts infiltration was minor. A successful new limbal transplant model is described. The transplant can be accurately followed up in vivo by E-GFP labeling of the donor tissue without disturbing the corneal surface. Although E-GFP itself proved to be immunogenic, local clodronate liposome injections significantly increased graft survival. So the model seems to be useful for testing immunosuppressive or modulatory agents in limbal transplantation studies.

The aim of the present study was to determine the capacity of resident corneal and limbal dendritic cells (DC) and macrophages to capture antigen (Ag) in vivo and compare this to their capacity in vitro to take up Ag during organ culture conditions. To investigate Ag uptake in vivo 3 microl (30 microg) of fluorescently labelled Dextran, bovine serum albumin (BSA) or ovalbumin (OVA) were either placed on the intact ocular surface or injected into the anterior chamber (AC) or subconjunctival space of the Lewis rat eye. The presence of Ag+ cells in the cornea was assessed using intravital fluorescence video microscopy. Animals were sacrificed 24h after Ag injections or topical application and the distribution and phenotype of Ag+ cells were analysed ex vivo by fluorescence and confocal microscopic analysis of immunostained and unstained corneal tissue wholemounts or frozen sections. Corneal buttons and corneoscleral rims from naive Lewis rats were placed in organ culture conditions in the presence of LPS with or without FITC-Dextran for 48 h and 72 h. The explants were examined by epi-fluorescence microscopy and the phenotype of Ag+ cells in the supernatant from the organ cultures was analyzed by flow cytometry using a range of macrophage and DC markers. In vivo observations and microscopic examination of corneas 24h following Ag topical application failed to reveal evidence of Ag retention by ocular cells. Those in which Ag had been placed in the AC or subconjunctival space revealed Ag+ cells within the corneal stroma. The distribution of Ag+ cells displayed a centripetal gradient, the most marked uptake of Ag being by cells in the circumferential limbal zone. Immunophenotypic studies revealed that Ag uptake was predominantly performed by cells that were CD68+, CD172+ but rarely MHC class II+, a profile characteristic of macrophages. Occasional Ag+ keratocytes were noted. In vitro studies of corneal buttons placed in culture conditions revealed that cells from the limbal zone, but not the central cornea, were able to take up Ag from the supernatant. Significant numbers of the cells that had migrated from the corneal buttons and captured fluorescent labelled Ag in the presence of LPS were revealed by flow cytometry to consist of CD163+ and CD11b+ macrophages, but none expressed the DC markers CD11c or OX62 and they were also generally MHC class II(-). In conclusion the present study revealed that macrophages and keratocytes in the corneal stroma and limbal episcleral tissue have the capacity to internalise fluorescent mock Ag injected into the AC or subconjunctival space or in culture conditions. The failure to demonstrate significant Ag trapping ability by corneal or limbal stromal or epithelial DC may either be due to the rarity of such cells or their lack of Ag trapping ability.

In a previous investigation into the fate of fluorescently labelled antigen (Ag) injected into the anterior chamber (AC) of the rat eye, a large number of Ag+ cells were noted in the conventional and non-conventional aqueous humour outflow pathways together with the external limbus. The aim of this study was to investigate the precise distribution and phenotype of these cells and compare their ability to capture fluorescent-labelled protein (bovine serum albumin, BSA, and ovalbumin, OVA) and polysaccharides (dextran, Dx) injected into the AC. The density of Ag+ cells in the iris and limbus was investigated using in vivo video fluorescence microscopy 24 hr post-injection. The distribution and phenotype of Ag+ cells in ocular tissues was analysed by confocal microscopy of frozen sections and in iris and corneoscleral/limbal wholemounts from animals sacrificed 24 hr post injection. The general distribution of labelled Ag was equivalent in OVA, BSA and Dx injected animals. Antigen-bearing cells were observed within the iris, iridocorneal angle, pre-equatorial choroid and around limbal/episcleral vessels. Localization of Ag+ cells and free Ag in the anterior segment suggests that substances of these molecular weights (40-70 kDa) leave the eye through the conventional and non-conventional aqueous outflow pathways. The cells that internalized BSA, OVA or Dx in ocular tissues were of a similar phenotype, namely, ED1+, ED2+, occasionally ED3+ and predominantly MHC class II-, thus suggesting that they are of the macrophage phenotype. However, a few Ag+ MHC class II+ dendriform cells (putative DC) were also observed in the iris, trabecular meshwork, choroid and episclera. In conclusion our data reveal that the majority of intracamerally injected soluble Ag retained in the eye is taken up by resident macrophages not only in the iris but in all tissues lining the AC of the eye.

Aim: To evaluate the incidence and clinical course of corneal complications in patients with severe dry eye syndrome after hematopoietic stem cell transplantation (HSCT)., Patients and Methods: 50 consecutive patients (aged 9-65 years; average=42+/-11 years) with Sjögren-like syndrome after hematopoietic stem cell transplantation were examined. In order to assess the severity of the dry eye syndrome, the frequency of blinking and break-up time were determined and Schirmer-I, fluorescein,and rose bengal tests were carried out. Biopsy and histological examination were performed in cases with suspected conjunctival graft-versus-host reaction., Results: Patients with Sjögren-like dry eye syndrome were referred within an average of 13 months after HSCT (SD+/-20 months).The follow-up was up to 83 months (mean: 10.2 months,SD+/-14.97). Of the 50 patients 15 developed severe corneal complications with significant loss of vision which resulted in enucleation of the eye in 1 patient. Of these 15 patients 4 had a viral and 2 a bacterial keratitis,7 had trophic corneal thinning and sterile ulcers. Two patients had limbal stem cell insufficiency or pseudomembranous conjunctivitis, both due to conjunctival graft-versus-host reaction. Another patient developed a toxic keratopathy, probably induced by cyclosporin A eye drops. Of 50 patients 35 presented with signs of an inactive or active conjunctival graft-versus-host reaction which could be proven histologically in 26 patients., Discussion: Patients with Sjögren-like dry eye syndrome after hematopoietic stem cell transplantation are at high risk to develop corneal complications.These complications may result from an aggressive extension of the graft-versus-host reaction towards the conjunctiva and/or the lacrimal gland and seem to occur more often during the period of reduction of systemic immunosuppressive therapy.Furthermore, infectious as well as trophic or toxic corneal complications may be supported by local immunosuppressive therapy. We suggest frequent ophthalmological checks of patients receiving hematopoietic stem cell transplantation who need local immunosuppressive therapy or are in the phase of reduction of systemic immunosuppressive therapy

Purpose: Bilateral stem cell deficiency can be overcome only by keratoplasty plus additional homologous limbal transplantation. We have four years' experience with a new surgical one-stage procedure, homologous penetrating central limbo-keratoplasty (HPCLK)., Methods: A clinical trial was performed in order to evaluate the effectiveness of this new method. The first 25 eyes after HPCLK for limbal stem cell deficiency have been followed for more than 12 months. The eccentrically trephined unmatched grafts contained 40% limbus and were transplanted centrally in the host. Systemic cyclosporin A (CSA) was administered for at least one year. Central clear graft survival was the main outcome criterion., Results: 18 grafts failed, mostly because of postoperative surface disorders. Seven grafts have remained centrally clear 12-41 months after HPCLK., Conclusions: In the majority of the grafts the transplanted limbal stem cells underwent immune destruction. The survival of seven grafts, however, shows that HPCLK is principally a promising new procedure. Further progress can be expected from the use of well HLA-matched grafts instead of unmatched grafts and from further improved systemic immune modulation.

Immunological privilege operates within the normal eye by multiple passive and active mechanisms, including antigen sequestration, maintenance of an immunosuppressive local environment and induction of apoptotic death in infiltrating cells of the immune system. Ocular privilege might have developed to protect the eye from the collateral damage associated with an inflammatory response to invading pathogens. Nevertheless, corneal grafts do undergo irreversible immunological rejection and, furthermore, corneal graft rejection is very similar at a histological level to the rejection processes that operate in vascularized organ grafts. Ocular privilege is thus relative. The question arises as to how corneal grafts are rejected in the face of so many mechanisms designed to prevent immune responses from operating inside the eye--a question that is still essentially unanswered.

Failure of a specialized population of corneal epithelial stem cells found in the peripheral cornea and limbus results in ocular surface disease, which may be amenable to treatment by transplantation of limbal tissue. This study was designed to investigate donor limbal stem cell allograft survival in rabbits with ocular surface disease. Rabbits underwent corneal epithelial debridement and limbal ablation to induce ocular surface disease and were then treated by limbal stem cell allotransplantation, by allotransplantation plus topical steroid, or by topical steroid only (n = 7 for each group). Donors and recipients were sex mismatched. Recipients were followed for up to 5 months. Outcome was assessed by daily slit-lamp examination, weekly impression cytology and photographic record, end-point sex chromatin and fluorescent cell tracer analyses, histology, and immunohistochemistry. In no case was a completely normal ocular surface regained, but some animals that received grafts plus corticosteroids fared best by all criteria used. In the absence of immunosuppression, graft hemorrhagia (believed to be a manifestation of graft rejection) occurred within the first month, the cornea became resurfaced with conjunctiva-derived cells, and no donor cells survived centrally in the long term. Topical corticosteroids reduced the number and severity of these episodes significantly, and were associated with survival of some donor-derived cells in the central cornea of some grafted animals. Thus, rabbit limbal stem cell allografts appeared to undergo rejection, which could be modified by immunosuppression, but useful regeneration of the ocular surface occurred only where rejection was circumvented.

Background: Thymus-derived lymphocytes play a critical role in the development of herpes simplex keratitis (HSK). T-cell subsets defined by their expression of various T-cell receptor (TCR) V beta segments were studied following corneal HSV-1 infection (p.i.)., Methods: Conjunctiva, corneal limbus and corneal stroma of two inbred BALB/c congenic mouse strains which differ only in the gene products closely linked to the Igh-1 locus on chromosome 12 were analyzed., Results: While C.B-17 mice (Igh-1b) were resistant to HSK, C.AL-20 mice (Igh-1d) clinically developed severe necrotizing keratitis by day 11 p.i. The corneal stroma of C.B-17 mice remained clear, while it was increasingly infiltrated by mononuclear cells and neutrophils in C.AL-20 mice by day 11 p.i. In C.B-17 mice, Thy1.2+ cells were found in the conjunctiva between days 2 to 4 p.i., and subsequently decreased. Only a few Thy1.2+ cells were found in the limbus, and no such cells were found in the stroma. In contrast, in C.AL-20 mice the numbers of Thy1.2+ cells (activated CD4+, V beta 8+ T cells) profoundly increased in the conjunctiva by day 4 p.i. These cells infiltrated the limbus between days 7 and 11 p.i. and eventually entered the stromal tissue by day 11 p.i., Conclusions: Our data suggest that the HSV-1-induced corneal tissue destruction is mediated by mononuclear cells and neutrophils and that these cells are probably attracted into the cornea by cytokines elaborated by activated CD4+, V beta 8+ T cells.

Conjunctiva associated lymphoid tissue shows several similarities to mucosa associated lymphoid tissue of the gut and respiratory tract. These similarities have been described in relation to lymphocyte subpopulations and epithelial cell morphology. However, unlike the lymphoid tissue of the gut and respiratory tract, mucosa specific lymphocytes have not been described in the ocular mucosa. In this study we demonstrated the presence of mucosa specific lymphocytes bearing the Human Mucosal Lymphocyte-1 antigen (beta 7 integrin), in the human conjunctiva, limbus and lacrimal gland. The distribution of this subset of lymphocytes corresponded to the distribution of CD8+ T-cells and was found maximally in the epithelium of the epibulbar conjunctiva and in the lacrimal gland. The Human mucosal lymphocyte antigen may function to determine mucosal homing of this particular subset of CD8+ T-cells, which in turn, may have special function in immunological defense and tolerance mechanisms occurring at mucosal surfaces.