Human Conjunctival Stem Cells are Predominantly Located in the Medial Canthal and Inferior Forniceal Areas.

PURPOSE: The conjunctiva plays a key role in ocular surface defence and maintenance of the tear film. Ex vivo expansion of conjunctival epithelial cells offers potential to reconstruct the ocular surface in cases of severe cicatrising disease, but requires initial biopsies rich in stem cells to ensure long-term success. The distribution of human conjunctival stem cells, however, has not been clearly elucidated. METHODS: Whole human cadaveric conjunctiva was retrieved and divided into specific areas for comparison. From each donor, all areas from one specimen were cultured for colony-forming efficiency assays and immunocytochemical studies; all areas from the other specimen were fixed and paraffin embedded for immunohistochemical studies. Expression of CK19, p63, and stem cell markers ABCG2, ΔNp63, and Hsp70 were analyzed. Results were correlated to donor age and postmortem retrieval time. RESULTS: Conjunctiva was retrieved from 13 donors (26 specimens). Colony-forming efficiency and expression of stem cell markers ABCG2, ΔNp63, and Hsp70 in cultures and ABCG2 in fixed tissue were all consistently demonstrated throughout the tissue but with highest levels in the medial canthal and inferior forniceal areas (P < 0.01 for each). Both increasing donor age and longer postmortem retrieval times were associated with significantly lower colony-forming efficiency, stem cell marker expression in cell cultures and ABCG2 expression in fixed tissue. CONCLUSIONS: Biopsies from the medial canthus and inferior forniceal areas, from younger donors, and with short postmortem retrieval times offer the greatest potential to developing conjunctival stem cell-rich epithelial constructs for transplantation.

Does the presence of an epiretinal membrane alter the cleavage plane during internal limiting membrane peeling?

PURPOSE: To determine whether the presence of a clinically and/or microscopically detectable epiretinal membrane (ERM) alters the cleavage plane during internal limiting membrane (ILM) peeling. DESIGN: Retrospective, observational, immunohistochemical study of ILM specimens using archival formalin-fixed, paraffin-embedded tissue. PARTICIPANTS: Fifty-one patients who had had ILM excision. METHODS: Fifty-one ILM specimens peeled during vitrectomy for various etiologies were examined by light microscopy. The removal of ILM was assisted using Trypan blue (n = 30), indocyanine green (n = 7), or brilliant blue G (n = 14). Monoclonal antibodies to glial fibrillary acidic protein and to neurofilament protein were used to detect glial or neuronal cells respectively on the vitreous or retinal surfaces of the ILM. Specimens were divided into 2 groups: ILM peeled for full-thickness macular hole (MH; n = 31) and ILM peeled after removal of clinically detectable ERM (n = 20). MAIN OUTCOME MEASURES: Primary outcome measure was the localization of immunohistochemical markers to neuronal or glial cells on the vitreous or retinal surfaces of ILM. The secondary outcome measure was the correlation of the results of the primary measure with the dyes used to facilitate ILM peeling. RESULTS: Glial and/or neuronal cells were detected on the retinal surface of the ILM in 10 of 31 (32%) of the MH ILM specimens and in 13 of 20 (65%) of the ILM peeled after ERM excision; the difference was significant (P = 0.02). There was no association between the presence of neuronal and glial cells with the type of dye used (P = 0.2). Of the 23 ILM specimens with cells attached to the retinal surface, 21 (91%) were associated with clinical and/or histologic evidence of ERM and 2 (9%) were not. The correlation between the presence of cells on the vitreous and the retinal surfaces of ILM was high (P<0.0001). CONCLUSIONS: The findings suggest that ERM may be associated with sub-ILM changes that alter the plane of separation during ILM peeling. This study does not confirm any influence of dyes on the cleavage plane during surgery.

Labeling of stem cells with monocrystalline iron oxide for tracking and localization by magnetic resonance imaging.

Precise localization of exogenously delivered stem cells is critical to our understanding of their reparative response. Our current inability to determine the exact location of small numbers of cells may hinder optimal development of these cells for clinical use. We describe a method using magnetic resonance imaging to track and localize small numbers of stem cells following transplantation. Endothelial progenitor cells (EPC) were labeled with monocrystalline iron oxide nanoparticles (MIONs) which neither adversely altered their viability nor their ability to migrate in vitro and allowed successful detection of limited numbers of these cells in muscle. MION-labeled stem cells were also injected into the vitreous cavity of mice undergoing the model of choroidal neovascularization, laser rupture of Bruch's membrane. Migration of the MION-labeled cells from the injection site towards the laser burns was visualized by MRI. In conclusion, MION labeling of EPC provides a non-invasive means to define the location of small numbers of these cells. Localization of these cells following injection is critical to their optimization for therapy.

Neoplastic transformation of ciliary body epithelium is associated with loss of opticin expression.

BACKGROUND: Opticin is a recently discovered glycoprotein present predominantly in the vitreous humour. It is synthesised and secreted by the ciliary body epithelium (CBE) from the initiation of CBE development in the embryo, and production continues throughout life. AIM: To determine whether a variety of ciliary body tumours synthesise opticin to characterise further its role in ciliary body health and disease. METHODS: Immunohistochemistry was used to determine the distribution of opticin in normal human CBE, and in hyperplastic and neoplastic CBE lesions. RESULTS: Opticin was immunolocalised to the basal cell surface and basement membrane material of the non-pigmented CBE in nine donor eyes as well as four hyperplastic lesions of the CBE (Fuchs's adenoma). By contrast, none of eight neoplastic lesions (two adenoma and six adenocarcinoma) of CBE stained for opticin. CONCLUSION: The present series supports the theory that opticin is produced by the non-pigmented CBE throughout adult life. Loss of opticin expression by this tissue is associated with and could contribute towards neoplastic transformation.

Transretinal choroidal tumor biopsy with a 25-gauge vitrector.

OBJECTIVE: To describe and evaluate transretinal biopsy of choroidal tumors using 25-gauge vitrectomy instrumentation. DESIGN: Retrospective, consecutive, noncomparative case series. PARTICIPANTS: Fourteen patients undergoing choroidal tumor biopsy at an ocular oncology center. METHODS: The biopsies were performed under local or general anesthesia, alone or in combination with ruthenium plaque or tantalum marker insertion. Immunohistochemistry was performed on all samples, and some melanomas were also analyzed cytogenetically. RESULTS: Surgery was uneventful in all cases. A positive tissue diagnosis was made in 13 of 14 patients, albeit at the second attempt in 1 patient. The only failure occurred because the tumor was calcified. CONCLUSION: Transretinal choroidal biopsy with 25-gauge instrumentation yields a larger sample than fine-needle aspiration biopsy, usually producing sufficient tissue for cytogenetic studies. We did not identify safety concerns in this series of patients. Insufficient samples can occur in some patients, and further studies are needed to understand the reason for such failure.

Presymptomatic diagnosis of bronchogenic carcinoma associated with bilateral diffuse uveal melanocytic proliferation.

A 62-year-old man presented with bilateral diffuse uveal melanocytic proliferations (BDUMP) and painful flexor contractures of the fingers of both hands. All these features were considered paraneoplastic but extensive and repeated investigations revealed no underlying malignancy. Oral steroids and orbital radiotherapy were ineffective. The diagnosis was confirmed by trans-scleral biopsy of the right choroid. Rapidly progressive cataracts were treated by phacoemulsification. Severe exudative retinal detachment with rubeosis and neovascular glaucoma in the left eye were treated successfully by partial choroidectomy. Fifteen months after presentation, investigations detected a 22 mm, poorly differentiated adenocarcinoma, which was resected without complication. The ocular tumours in both eyes regressed, without improvement in vision of Light Perception, and the palmar fasciitis also improved. The patient remained free of tumour recurrence until sudden death from myocardial infarction five years after he first presented.

The presence of AC133-positive cells suggests a possible role of endothelial progenitor cells in the formation of choroidal neovascularization.

PURPOSE: Recent evidence suggests that vasculogenesis as well as angiogenesis occurs throughout the body during neovascularization. The recruitment of circulating stem cells is a key feature of vasculogenesis. The purpose of the present study was to determine whether markers of endothelial progenitor cells (EPCs) are present in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). METHODS: Surgically excised CNV (n = 9) membranes from patients with AMD were probed with immunohistochemical techniques using the following monoclonal antibodies: AC133 a putative marker of EPCs and hematopoietic stem cells (HSCs); the endothelial cells markers CD31, CD34, and von Willebrand factor (vWF); and cytokeratins and CD68, markers for retinal pigment epithelium (RPE) and macrophages, respectively. After secondary antibody amplification, reactions were visualized with fast red substrate. RESULTS: Six of nine specimens demonstrated cells positive for AC133 that were all found within predominantly cellular regions of the specimens. In the avascular fibrous stromal core of all specimens, the predominant cells were RPE cells and macrophages. The peripheral component of all CNV membranes was highly vascular and showed varying immunoreactivity for all endothelial markers. The greatest immunoreactivity for endothelial markers was observed with CD34 and vWF and least for CD31. CONCLUSIONS: These findings support animal studies that vasculogenesis, in addition to angiogenesis, may contribute to the neovascularization that occurs in AMD.

BRAF mutations are detectable in conjunctival but not uveal melanomas.

Activating mutations in exon 15 of BRAF have been detected in a high proportion of cutaneous melanomas. To determine whether such mutations are a feature of conjunctival or uveal melanomas, we screened DNA from these tumours. Twenty-one conjunctival and 88 uveal tumours were included in the study. Mutation analysis of BRAF exons 11 and 15 was undertaken using a combination of conformationally sensitive gel electrophoresis and direct sequencing. Mutations in exon 15 were detected in three of the conjunctival tumours (two V599E and one E585 K). None of the uveal tumours possessed a BRAF mutation in either exon 15 or 11. We conclude that uveal melanomas arise independently of oncogenic BRAF mutations, but the development of a proportion of conjunctival tumours involves mutation of this gene.

Expression of serotonin receptor mRNAs in human ciliary body: a polymerase chain reaction study.

BACKGROUND: Previous research has indicated a role for serotonin (5-HT) in the anterior uvea of the eye. The purpose of this study was to examine whether mRNAs encoding particular 5-HT receptors are expressed in the ciliary body and iris of a number of human subjects. METHODS: The presence of mRNA encoding 5-HT receptors in four human ciliary body samples was determined by reverse transcription-polymerase chain reaction experiments using a standard methodology. RESULTS: Positive signals for 5-HT(1A), 5-HT(2A), 5-HT(2B), 5-HT(2C) and 5-HT(7) receptor mRNAs were detected in the samples prepared from various human ciliary body samples. CONCLUSION: The detection of certain 5-HT receptor mRNAs in the human ciliary body supports the hypothesis that serotonin is involved in the control of aqueous dynamics and indicates that ligands acting on these 5-HT receptors may have potential use as intraocular pressure-lowering agents.

Histopathology of eyes enucleated after endoresection of choroidal melanoma.

PURPOSE: To demonstrate the histopathologic features of eyes enucleated after endoresection for choroidal melanoma to assess the complications of this treatment and to determine indications for further treatment after endoresection in the setting of possible tumor recurrence. DESIGN: Retrospective, observational case series. PARTICIPANTS: Sixty-one consecutive patients who had undergone endoresection for uveal melanoma. METHODS: Eyes that had undergone enucleation after endoresection were identified, and their charts and histologic characteristics were reviewed. Pertinent features were described. One patient was excluded because enucleation was performed as a primary treatment when endoresection was abandoned at the time of his initial treatment. MAIN OUTCOME MEASURES: The outcome measures included: reasons for enucleation; tumor recurrence; and location, clinical, and histologic characteristics of each recurrence. RESULTS: Twelve eyes were identified that had undergone enucleation after endoresection. The reasons for enucleation were: (1) local tumor recurrence detected by ophthalmoscopy (2 patients) or echography (1 patient); (2) opaque media preventing adequate ophthalmoscopy (4 patients); (3) blind and painful eye of uncertain cause (1 patient); and (4) a combination of blind eye and limited fundus view (4 patients), which was the result of untreatable retinal detachment (3 patients) and endophthalmitis (1 patient). Eight of 12 patients had recurrent choroidal melanoma. Recurrences were all located adjacent to the resection site, although in 1 patient there was extensive diffuse recurrence throughout the eye. The recurrence was visible clinically in 3 patients and obscured because of opaque media (2 patients), a combination of inadequate echography and retinal detachment (1 patient), retinal detachment (1 patient), and endophthalmitis (1 patient). CONCLUSIONS: Recurrent disease occurred at the site of the primary tumor with no seeding except in 1 patient, whose marginal recurrence was not immediately detected and treated because of opaque media. As with other treatments conserving the eye, enucleation should be performed if adequate ocular examination is not possible, and follow-up should be lifelong.

The role of matricellular proteins thrombospondin-1 and osteonectin during RPE cell migration in proliferative vitreoretinopathy.

PURPOSE: To investigate the hypothesis that the Matricellular proteins thrombospondin 1 (TSP1), tenascin (TN) and Secreted Protein Acidic and Rich in Cysteine (SPARC) modulate the migration of RPE cells in the epiretinal membranes of proliferative vitreoretinopathy. METHODS: Ten PVR epiretinal membranes were studied by immunohistochemical methods in which aggregates of RPE cells were identified by their expression of a broad range of cytokeratins. RPE subsets containing migratory RPE cells were detected by immunoreactivity for the monoclonal antibody RGE53 (which detects an epitope on cytokeratin-18 on motile RPE cells). Co-localisation of the RPE subsets with the glycoproteins TSP-1, SPARC and TN was evaluated. RESULTS: Nineteen migratory RPE (RGE53 positive) subsets and 13 RPE (RGE53 negative) subsets were identified. All of the RGE53+ subsets colocalised with TSP1 and SPARC and 17 with TN. Ten of the RGE53- aggregates stained for TN, 6 for SPARC and 5 for TSP1. The association between the presence of RGE53+ cells in the RPE cell aggregates and TSP1 immunoreactivity in the aggregates was significant (p < 0.001), and there was a comparable significant association between RGE53+ cells and SPARC (p < 0.001). No such association was detected for RGE53+ cells and TN (p > 0.2). CONCLUSIONS: The findings support the concept that the migration of retinal pigment cells in epiretinal membranes is modulated by TSP1 and SPARC and thus that these two proteins ultimately may represent therapeutic targets in the management of the membranes.