[Current Therapies in Superficial Malignant Tumors]. / Aktuelle Therapiekonzepte bei oberflächlichen malignen Tumoren.

This article is a review of diagnostic and therapeutic possibilities in common epibulbar malignant tumors, such as basal cell carcinoma, conjunctival lymphoma, squamous cell carcinoma and conjunctival melanoma. Most importantly, for every tumor patient there is a detailed anamnesis, split lamp examination and photo documentation. Further regular controls after therapy are required due to the risk of recurrences. Basal cell carcinoma is the most common tumor of the periocular skin, divided into three subtypes (nodular, superficial and morphea). The treatment consists of total excision with a sufficient safety margin.The most common tumor of the orbital and ocular adnexa are lymphoma. They can appear primary or secondary, as lymphatic system disease. The gold standard therapy is a percutaneous irradiation.Squamous cell carcinoma and its precancerous stages (CIN I-III) are frequently diagnosed on the conjunctiva. A complete tumor excision is required, but sometimes it is not possible due to large tumor growth. Therefore, an adjuvant therapy with mitomycin C eyedrops, brachytherapy with ruthenium plaque or proton radiotherapy is required.Conjunctival melanoma is a rare, but life-threatening disease. Melanosis with atypia has a high risk for degeneration and requires further treatment with mitomycin C eyedrops. In cases of malignant tumor growth, an excision with a non-touch technique and adjuvant therapy with brachy or proton irradiation should be applied.

[Current Therapies in Superficial Malignant Tumors]. / Aktuelle Therapiekonzepte bei oberflächlichen malignen Tumoren.

This article is a review of diagnostic and therapeutic possibilities in common epibulbar malignant tumors, such as basal cell carcinoma, conjunctival lymphoma, squamous cell carcinoma and conjunctival melanoma. Most importantly, for every tumor patient there is a detailed anamnesis, split lamp examination and photo documentation. Further regular controls after therapy are required due to the risk of recurrences.Basal cell carcinoma is the most common tumor of the periocular skin, divided into three subtypes (nodular, superficial and morphea). The treatment consists of total excision with a sufficient safety margin.The most common tumor of the orbital and ocular adnexa are lymphoma. They can appear primary or secondary, as lymphatic system disease. The gold standard therapy is a percutaneous irradiation.Squamous cell carcinoma and its precancerous stages (CIN I - III) are frequently diagnosed on the conjunctiva. A complete tumor excision is required, but sometimes it is not possible due to large tumor growth. Therefore, an adjuvant therapy with mitomycin C eyedrops, brachytherapy with ruthenium plaque or proton radiotherapy is required.Conjunctival melanoma is a rare, but life-threatening disease. Melanosis with atypia has a high risk for degeneration and requires further treatment with mitomycin C eyedrops. In cases of malignant tumor growth, an excision with a non-touch technique and adjuvant therapy with brachy or proton irradiation should be applied.

[Rehabilitation Following Limbal Stem Cell Deficiency: Current Treatment Options and Future Developments]. / Optische Rehabilitation nach Limbusstammzellinsuffizienz: aktuelle Behandlungsmöglichkeiten und Entwicklungen.

Limbal stem cell deficiency (LSCD) is a condition caused by the loss of corneal epithelial regenerative potential. The treatment of this condition is still a challenge. It results from various conditions both intrinsic as well as extrinsic. LSCD can be either uni- or bilateral and either partial or total. Today treatment options include a variety of techniques including transplantation of amniotic membrane and limbal tissue or tissue engineered cell sheets. This article summarizes the current techniques to treat LSCD and upcoming developments.

Expression of pluripotency and multipotency factors in human ocular surface tissues.

AIM: Mechanisms that control ocular surface stem cells (SCs) are unclear. Recent studies have shown that several adult SCs express pluripotency markers. Our objective was to analyze the expression of key molecules of pluripotency in human ocular surface tissues as well as in cultivated limbal epithelium. METHODS: Four samples of human corneal, limbal and on amniotic membrane cultivated limbal epithelium (HLEC-AM), as well as bulbar and fornical conjunctiva were analyzed. Human embryonic stem (ES) cells and human umbilical vein endothelial cells served as controls. Expression of corneal epithelial differentiation markers (K3, K12, Cx43), putative limbal SC markers (ABCG2, p63, K15), and molecules associated with pluripotency/multipotency (NANOG, OCT4, SOX2, KLF4, KIT, NESTIN, PAX6, NOTCH1) was examined using real-time polymerase chain reaction (PCR) and immunohistochemical staining. RESULTS: Limbal epithelium showed a significantly (p < 0.05) higher expression of K15, ABCG2, OCT4, SOX2, NESTIN and NOTCH1, but a lower expression of K3 than corneal epithelium. Besides a higher expression of ABCG2 in fornix, the expression of pluripotency markers was similar in both conjunctival regions, although lower than in limbal epithelium. Expression of pluripotency factors in ES cells was significantly higher than in ocular surface SCs, whereas the expression in limbal epithelium was the closest to ES cells. HLEC-AM in comparison to limbal epithelium showed a lower expression of differentiation markers, a similar expression of ABCG2 but a significantly lower expression of pluripotency factors. CONCLUSION: Human ocular surface epithelial cells and especially limbal epithelial cell express genes are important for pluripotency and may have preserved some common mechanisms with pluripotent SCs.

Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency.

BACKGROUND: Limbal stem cell deficiency (LSCD) leads to growth of abnormal fibro-vascular pannus tissue onto the corneal surface as well as chronic inflammation and impaired vision. Our aim was to investigate the clinical outcome of ocular surface reconstruction in LSCD using limbal epithelial cells expanded on amniotic membrane (AM). METHODS: Forty-four eyes of 38 patients (27 male, 11 female) with total (n = 32) or partial (n = 12) LSCD were treated by transplantation of autologous (n = 30) or allogeneic (n = 14) limbal epithelial cells expanded on intact AM. LSCD was caused by chemical and thermal burns (n = 22), pterygium (n = 9), congenital aniridia (n = 6), tumor excision (n = 2), perforating eye injury, mitomycin C, epidermolysis bullosa, bilateral graft-versus-host disease and chlamydial conjunctivitis (each n = 1). RESULTS: Mean follow-up time was 28.5 +/- 14.9 months. The corneal surface could be reconstructed to full stability in 30 (68%), and clear central cornea was achieved in 37 (84%) eyes. Grafting was significantly more successful in eyes treated by autologous than by allogeneic transplantation (76.7 vs. 50%, p < 0.05). The corneal surface could be successfully restored in 10 (83.3%) eyes with partial LSCD and in 20 (63.3%) eyes with total LSCD. Visual acuity (VA) increased significantly in 32 (73%) eyes, was stable in 10 (23%) eyes and decreased in 2 (4%) eyes. Mean VA increased significantly (p < 0.0001), from preoperative 1.7 +/- 0.9 log MAR (20/1,000) to 0.9 +/- 0.7 log-MAR (20/160). VA increased significantly after both autologous (p < 0.0001) and allogeneic transplantation (p < 0.005). CONCLUSIONS: In most patients with LSCD, transplantation of limbal epithelium cultivated on intact AM restores the corneal surface and results in significantly increased VA.

Ocular surface reconstruction with cultivated limbal epithelium in a patient with unilateral stem cell deficiency caused by Epidermolysis bullosa dystrophica hallopeau-Siemens.

PURPOSE: To report a case of partial limbal stem cell deficiency (LSCD) caused by epidermolysis bullosa dystrophica mutilans Hallopeau-Siemens treated by transplantation of autologous ex vivo expanded limbal epithelium. METHODS: Review of the clinical findings of an 11.5-year-old boy with unilateral LSCD and epidermolysis bullosa dystrophica who underwent ocular surface reconstruction in the right eye with autologous on intact human amniotic membrane cultivated limbal epithelial cells. RESULTS: Twenty-eight months after reconstruction, the corneal surface is clear, smooth, and stable showing no signs of LSCD recurrence. Three subconjunctival bevacizumab (Avastin) injections reduced the recurrent growth of symblepharon and corneal vascularization. The visual acuity has increased from hand motion to 20/50. CONCLUSION: Autologous transplantation of cultivated human limbal epithelial cells on intact human amniotic membrane can be a safe and effective method for corneal surface reconstruction in LSCD caused by recessive epidermolysis bullosa dystrophica.

Characterization of the corneal surface in limbal stem cell deficiency and after transplantation of cultivated limbal epithelium.

PURPOSE: Transplantation of in vitro-cultivated limbal epithelium (TCLE) recently was developed to treat limbal stem cell deficiency (LSCD). The objective of this study was to characterize changes in the cornea during LSCD and on the corneal surface after TCLE. DESIGN: Experimental study. PARTICIPANTS AND CONTROLS: The pannus tissue excised from the corneas of 17 LSCD patients was analyzed to characterize the changes in the cornea during LSCD. Five corneal buttons obtained during perforating keratoplasty (pKP) from patients who had undergone TCLE at least 6 months before pKP were examined to assess the effect of TCLE. Six samples of healthy central cornea and 6 of bulbar conjunctiva served as control tissue. METHODS: The expression of epithelial lineage markers (keratin [K] 3, K12, K19, and mucin 5AC) and inflammatory markers (interleukin-1alpha [IL-1alpha], IL-1beta, intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1], and vascular endothelial growth factor [VEGF]) were analyzed using real-time polymerase chain reaction, Western blotting, and immunofluorescence in the tissue samples. MAIN OUTCOME MEASURES: Comparison of the markers' expression patterns. RESULTS: The expression of all markers differed in healthy cornea and conjunctiva. Expression of lineage markers was similar in pannus to conjunctiva, but not to cornea. Interleukin-1beta, ICAM-1, VCAM-1, and VEGF were increased significantly in pannus compared with the levels in healthy cornea. Interleukin-1alpha, IL-1beta, and ICAM-1 were increased compared with healthy conjunctiva. The TCLE improved vision and reduced inflammation, vascularization, and discomfort. After TCLE, the lineage markers in the excised corneal buttons showed a corneal phenotype and a significant reduction in inflammatory markers in 4 of 5 cases. CONCLUSIONS: Limbal stem cell deficiency is characterized by ingrowth of abnormal inflamed tissue with a conjunctival phenotype. Transplantation of limbal epithelium cultivated in vitro on intact amniotic membrane restored a noninflamed ocular surface and a corneal phenotype. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Expression of membrane-associated mucins in limbal stem cell deficiency and after transplantation of cultivated limbal epithelium.

PURPOSE: Membrane-associated mucins are important components of the ocular tear film. Our objective was to characterize the expression of membrane-associated mucins MUC1, MUC4, and MUC16 in healthy cornea, healthy conjunctiva, fibrovascular tissue (pannus) covering the corneal surface in limbal stem cell deficiency (LSCD), human limbal epithelial cells cultivated on intact amniotic membrane (HLEC-AM), and corneal epithelium after transplantation of cultivated limbal epithelium (TCLE). METHODS: Total RNA was isolated from six samples of healthy human cornea, healthy conjunctiva, pannus, limbal epithelial cell cultures, and four corneal samples after TCLE. The expression of MUC1, MUC4, and MUC16 was evaluated by real-time polymerase chain reaction (PCR), Western blotting, and immunofluorescence. RESULTS: Conjunctiva showed a significantly higher expression of MUC1 and MUC4 than cornea, but the expression of MUC16 was similar in both tissues. The expression of MUC1 in HLEC-AM increased, MUC16 decreased, and MUC4 showed no significant difference when compared to cornea. The expression of all studied mucins in conjunctiva was significantly higher than in HLEC-AM. Pannus revealed a similar expression pattern of membrane-associated mucins to conjunctiva. The expression of all studied mucins in pannus was significantly higher than in cornea or HLEC-AM. A corneal expression pattern of membrane-associated mucins was found in all four samples of cornea after TCLE. CONCLUSION: A tissue-specific expression of MUC1 and MUC4 but not MUC16 was found in human cornea and conjunctiva. LSCD led to a conjunctival expression pattern of mucins on the corneal surface. A corneal phenotype could be restored after TCLE, confirming the potential of this method to reconstruct the corneal surface.

Correlation of cell cycle kinetics with p63 expression in human limbal epithelial cells expanded on intact human amniotic membrane.

AIM: To analyse the correlation of p63 expression and cell cycle kinetics of human limbal epithelial cells (HLECs) expanded on amniotic membrane (AM) or plastic. METHODS: Primary HLECs were cultured either on cryopreserved intact AM or plastic dishes for 2 weeks. Cells were labelled with 5 microM 5-bromo-2'-deoxyuridine (BrdU) for 3 days, followed by an interval of either 7 or 14 days in BrdU-free medium. The expression of p63 and BrdU labelling was detected by immunocytochemistry. RESULTS: More cells on AM (56%) than on plastic (24%) retained their BrdU label after the 7-day interval (p < 0.001). The difference was even more pronounced after 14 days (20 and 2%, p < 0.001). All BrdU-labelled cells were also p63 positive. 2.5-fold more cells on AM (54%) than on plastic (21%) were BrdU positive/p63 positive after 7 days (p < 0.001). It increased to a 9-fold difference after 14 days (p < 0.001). The BrdU label was lost more quickly than the p63 expression during the observation period, indicating that p63 expression was not confined to stem cells but existed also in transient amplifying cells. CONCLUSIONS: The combination of p63 expression and BrdU label retention is a better criterion to characterize stemness than either marker alone. AM as a culture substrate preserves stemness better than plastic.

Ocular surface reconstruction in graft-versus-host disease with HLA-identical living-related allogeneic cultivated limbal epithelium after hematopoietic stem cell transplantation from the same donor.

PURPOSE: To report a case of HLA-identical allogeneic living-related ex vivo expanded limbal epithelium in ocular surface reconstruction for chronic graft-versus-host disease (cGVHD). METHODS: Review the clinical findings in a 58-year-old woman with bilateral limbal stem cell deficiency caused by cGVHD who underwent ocular surface reconstruction on the left eye with cultivated limbal epithelial cells (LECs) on intact human amniotic membrane combined with extracapsular cataract extraction and intraocular lens implantation. LECs were harvested from a small biopsy of the same HLA-identical living-related donor who already donated peripheral blood cells for hematopoietic stem cell transplantation. RESULTS: At the present state, after a follow-up of 31 months, the patient shows a successful ocular surface reconstruction with a clear, smooth, and stable corneal ocular surface without recurrence of limbal stem cell deficiency. Nine months postoperatively, patients' corneal thinning progressed to a small perforation, which was successfully treated with a tectonic perforating keratoplasty combined with the removal of irritating lid lashes. CONCLUSION: The technique of HLA-typed allogeneic ex vivo expansion of LECs harvested from the same living-related donors who already donated hematopoietic stem cells offers a possibility to reconstruct ocular surface in cGVHD.

Transplantation of amniotic membrane in murine herpes stromal keratitis modulates matrix metalloproteinases in the cornea.

PURPOSE: To study matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the corneas from mice with ulcerative herpes stromal keratitis (HSK) treated with amniotic membrane transplantation (AMT). METHODS: The corneas from BALB/c mice were infected with HSV-1. Mice with ulcerative HSK on postinfection (PI) day 14 were used for the experiments. In one group of mice, the corneas were treated with transplantation of amniotic membrane (AMT) that was secured with a tarsorrhaphy, and a control group underwent tarsorrhaphy alone. After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically. Corneal sections were studied by immunohistochemistry for the expression of MMP-2, -8, and -9 and TIMP-1 and -2. MMP activity in the corneas was investigated by zymography, and the expression of the enzymes was measured by the Western blot technique. RESULTS: At day 14 PI, the ulcers stained intensely positive for MMP-2, -8, and -9 and TIMP-1 and -2. Ulceration (P < 0.001), stromal inflammation (P < 0.01) and inflammatory cell infiltration (P < 0.001) markedly improved by day 2 after AMT. This was associated with reduced expression (P < 0.01) and activity of MMP-8, and -9 and increased localization of TIMP-1 (P < 0.01), whereas TIMP-2 was not affected. In contrast, high levels of expression of MMP-8 and -9 remained in the cornea after tarsorrhaphy, and the TIMP-1 expression was only slightly upregulated. CONCLUSIONS: Rapid improvement of HSV-1-induced ulcerative keratitis is noted after amniotic membrane transplantation. This may be caused by reduced expression and activity of MMP-8 and -9, increased expression of TIMP-1, and sustained expression of TIMP-2.

Clinical results of anti-inflammatory therapy in Graves' ophthalmopathy and association with thyroidal autoantibodies.

OBJECTIVE: Graves' ophthalmopathy (GO) is clinically associated with autoimmune thyroid disease, and autoantibodies to thyroidal antigens, especially to the TSH-receptor (TRAb), might be involved in the disease process. While there is mounting evidence that TRAb are associated with GO at the onset of the disease, so far no studies have looked at the association between thyroidal autoantibodies and the clinical outcome of GO therapy. The aim of this retrospective study was to evaluate whether TSH binding inhibitory immunoglobulins (TBII) and thyroid stimulating antibodies (TSAb) are still associated with the clinical activity and severity of GO after the completion of anti-inflammatory therapy. In addition, we wanted to elucidate whether thyroid peroxidase (TPO) or thyroglobulin (TG) autoantibodies (TPOAb and TGAb) are in any way related to GO. DESIGN PATIENTS AND MEASUREMENTS: Clinical activity score (CAS) and the severity of GO (modified NOSPECS score) were assessed in 108 patients with GO after steroid therapy and, if indicated, orbital irradiation. Patients were grouped according to their clinical presentation and autoantibody levels (TBII, TSAb, TPOAb and TGAb) were measured. After therapy for hyperthyroidism, all patients were clinically euthyroid but showed clear heterogeneity for GO 4-12 months after the end of anti-inflammatory therapy. Fifty-two patients had inactive GO, 41 had moderately active and 15 still had very active (non-responsive) GO. Concerning severity, 27 patients had mild GO, 64 moderately severe and 17 severe GO. RESULTS: TBII titres were still positive in 14 (93%) of 15 patients in the non-responsive group (CAS > 6) compared to 22 (42%) of 52 patients (P < 0.001) with post-therapeutic inactive GO (CAS

Expression of Delta Np63 in response to phorbol ester in human limbal epithelial cells expanded on intact human amniotic membrane.

PURPOSE: To evaluate the effect of phorbol 12-myristate 13-acetate (PMA) on the expression of Delta Np63 in human limbal epithelial cells (HLECs) during ex vivo expansion on amniotic membrane (AM). METHODS: Primary HLECs were cultured either on AM or plastic surfaces and were treated with 1 micro g/mL PMA for 24 hours. Expression of Delta Np63 and the differentiation-associated gap junctional protein connexin 43 (Cx43) were studied by laser scanning microscopy. RESULTS: The labeling index (LI) of Delta Np63 was higher in HLECs cultured on AM than in HLECs grown on plastic (81.4% +/- 12.2% and 66.6% +/- 16.5%, respectively; P < 0.001). After PMA treatment, Delta Np63 expression in HLECs on plastic dramatically decreased to 20.4% +/- 11.4%. However, HLECs cultured on AM showed only a moderate decrease in Delta Np63 expression (56.4% +/- 10.9%, P < 0.001) after PMA treatment. It was also observed that 72.8% +/- 17.5% of the Delta Np63-positive cells in untreated HLECs cultured on plastic coexpressed Cx43, in contrast to only 21.9% +/- 3.7% of the Delta Np63-positive cells in HLECs cultured on AM (P < 0.001). The latter indicates that growth over AM preserves limbal phenotype, whereas growth over plastic surface induces or allows transition toward corneal peripheral phenotype. CONCLUSIONS: Delta Np63 protein is typically detected in human corneal epithelial cells with high proliferative capacity including, limbal epithelial stem cells (SCs) and probably also transient amplifying cells (TACs). AM supports Delta Np63 protein expression in HLECs and maintains a higher resistance against phorbol ester-induced differentiation, indicating that characteristic signs of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM.

Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane.

The aim of the study was to determine if human limbal epithelial cells (HLEC) do not form gap junctions (GJ) during ex vivo expansion on preserved and intact human amniotic membrane (AM). Thereby, we attempt to evaluate if characteristic features of the limbal epithelial progenitor cells are preserved on AM. Primary human limbal (HLEC) and peripheral corneal (HPCEC) epithelial cells from limbal and peripheral corneal explants were cultured with SHEM either on intact AM or plastic. After 3-4 weeks, cell cultures were terminated and processed for immunofluorescence. In all cell cultures, formations of GJs were analyzed with a mouse monoclonal antibody to connexin 43 (Cx43) and a rabbit affinity purified antibody against connexin 26 (Cx26). Sections of human limbus and cornea served as positive control. Lucifer yellow (LY) known to be a GJ permeant dye was used to analyse functionality of GJ. Microinjection of LY into single cells was performed with a pressure microinjection device under visual control and with the aid of phase contrast optics. Dye spread of LY into adjacent cells indicating intercellular communication was compared between HLEC and HPCEC cultured either on AM or plastic. In vivo, a punctate pattern of Cx43 was typically found in basal and suprabasal corneal epithelial cells and labeling for Cx26 was observed in all cell layers of the human corneal epithelium, however, subpopulations of limbal basal epithelial cells lacked detectable fluorescence signals for both connexins. In HLEC cultured on AM, a scanty immunolabeling for Cx43 (12.6%) was noted, but HPCEC cultured on AM as well as HLEC cultured on plastic showed a higher labeling index (LI) for Cx43 (42.7 and 52.3%, respectively). A significant lower immunostaining for Cx26 was observed in HLEC cultured on AM (LI: 35.16%) in comparison to HLEC cultured on plastic (68.4%), as well as, HPCEC cultured either on AM or plastic (61% and 79.3%, respectively; p<0.001). Gap junctional communication was evidenced more frequently in HLEC cultured on plastic (51%, p<0.05) in contrast to HLEC cultures on AM, which exhibited a limited dye spread in 29.7% of injected cells. A significant difference in dye coupling was also evidenced between HPCEC on AM (52.9%; p<0.05) and HLEC on AM. Subpopulations of HLEC cultured on AM remain Cx43 and Cx26 negative and without functional GPs indicating that characteristic features of limbal epithelial progenitor cells might be preserved during ex-vivo expansion on AM. These data provide support to the use of the ex-vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.