GNAQ and GNA11 mutations and downstream YAP activation in choroidal nevi.

BACKGROUND: Mutations in GNAQ/11 genes are considered an early event in the development of uveal melanoma that may derive from a pre-existing nevus. The Hippo pathway, by way of YAP activation, rather than MAP kinase, has a role in the oncogenic capacity of GNAQ/11 mutations. METHODS: We investigated 16 nevi from 13 human eyes for driver GNAQ/11 mutations using droplet digital PCR and determined whether nevi are clonal by quantifying mutant nevus cell fractions. Immunohistochemistry was performed on 15 nevi to analyse YAP activation. RESULTS: For 15 out of 16 nevi, a GNAQ/11 mutation was detected in the nevus cells albeit at a low frequency with a median of 13%. Nuclear YAP, a transcriptional co-activator in the Hippo tumour-suppressor pathway, was detected in 14/15 nevi. CONCLUSIONS: Our analysis suggests that a mutation in GNAQ/11 occurs in a subset of choroidal nevus cells. We hypothesise that GNAQ/11 mutant-driven extracellular mitogenic signalling involving YAP activation leads to accumulation of wild-type nevus cells.

Expression of cancer-testis antigens (MAGE-A1, MAGE-A3/6, MAGE-A4, MAGE-C1 and NY-ESO-1) in primary human uveal and conjunctival melanoma.

AIM: Metastatic disease in ocular melanoma remains untreatable, is associated with late detection and is resistant to conventional systemic therapies. Many tumours including cutaneous melanoma express specific cancer-testis (CT) antigens and vaccines targeting these antigens can induce T-cell-mediated and humoural immune responses. The authors examined primary uveal and conjunctival melanomas for expression of CT antigens to assess their potential as targets for ocular melanoma immunotherapy. METHODS: Paraffin-embedded uveal (n=32) and conjunctival (n=15) melanomas were assessed by immunohistochemistry for melanocyte differentiation antigens (gp100, Melan-A/MART-1 and tyrosinase), and CT antigens (MAGE-A1, MAGE-A3/6, MAGE-A4, MAGE-C1 and NY-ESO-1). RESULTS: Melanoma differentiation antigens, gp100, Melan-A/MART1 and tyrosinase, were expressed in >75% of tumour cells in all uveal and conjunctival melanomas tested. Expression of all five CT antigens tested was low in uveal melanomas, and when present, stained <25% of the tumour cells. MAGE-A1, MAGE-A4 and NY-ESO-1 were expressed in <10% of tumour cells in conjunctival melanomas, while MAGE-C1 and MAGE-A3/6 were expressed in ∼20% and ∼35% of tumour cells in this malignancy, respectively, with variable expression levels. CONCLUSIONS: Uveal and conjunctival melanomas consistently expressed high levels of the differentiation antigens (gp100, Melan-A/MART1 and tyrosinase). However, compared with other tumours, including cutaneous melanoma, only low levels of CT antigens were found in ocular melanomas. These observations suggest that immunotherapy directly targeting the CT antigens studied may not be effective for ocular melanoma.

Co-expression of CD147 (EMMPRIN), CD44v3-10, MDR1 and monocarboxylate transporters is associated with prostate cancer drug resistance and progression.

BACKGROUND: The aim of this study is to seek an association between markers of metastatic potential, drug resistance-related protein and monocarboxylate transporters in prostate cancer (CaP). METHODS: We evaluated the expression of invasive markers (CD147, CD44v3-10), drug-resistance protein (MDR1) and monocarboxylate transporters (MCT1 and MCT4) in CaP metastatic cell lines and CaP tissue microarrays (n=140) by immunostaining. The co-expression of CD147 and CD44v3-10 with that of MDR1, MCT1 and MCT4 in CaP cell lines was evaluated using confocal microscopy. The relationship between the expression of CD147 and CD44v3-10 and the sensitivity (IC(50)) to docetaxel in CaP cell lines was assessed using MTT assay. The relationship between expression of CD44v3-10, MDR1 and MCT4 and various clinicopathological CaP progression parameters was examined. RESULTS: CD147 and CD44v3-10 were co-expressed with MDR1, MCT1 and MCT4 in primary and metastatic CaP cells. Both CD147 and CD44v3-10 expression levels were inversely related to docetaxel sensitivity (IC(50)) in metastatic CaP cell lines. Overexpression of CD44v3-10, MDR1 and MCT4 was found in most primary CaP tissues, and was significantly associated with CaP progression. CONCLUSIONS: Our results suggest that the overexpression of CD147, CD44v3-10, MDR1 and MCT4 is associated with CaP progression. Expression of both CD147 and CD44v3-10 is correlated with drug resistance during CaP metastasis and could be a useful potential therapeutic target in advanced disease.

Expression and distribution of MMPs and TIMPs in human uveal melanoma.

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas (n=18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0-3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was > or =Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).

Beta-lapachone inhibits proliferation and induces apoptosis in retinoblastoma cell lines.

AIMS: To investigate the cytotoxicity of beta-lapachone, a potent agent that may selectively target tumour cells, in retinoblastoma (RB) cell lines. METHODS: Growth inhibitory effects of beta-lapachone were evaluated in Y79, WERI-RB1, and RBM human retinoblastoma cell lines. Pro-apoptotic effects of beta-lapachone were evaluated in Y79 cells by detection of caspase 3/7 activity, by enzyme-linked immunosorbent assay for nucleosome fragments, and by cellular morphological analysis. RESULTS: Beta-lapachone induced significant dose-dependent growth inhibitory effects in all three retinoblastoma cell lines. The 50% growth inhibitory concentration (IC(50)) of this agent was 1.9 microM in Y79 cells, 1.3 microM in WERI-RB1 cells, and 0.9 microM in RBM cells. Beta-lapachone also induced proapoptotic effects in RB cells. Treatment of Y79 cells with 1.9 microM beta-lapachone (IC(50)) resulted in a peak, fourfold induction of caspase 3/7 activity at 72 h post-treatment; a peak, 5.6-fold increase in nucleosome fragments at 96 h post-treatment; and a peak, 1.7-fold increase in the frequency of apoptotic cells at 48 h post-treatment, relative to vehicle-treated controls. CONCLUSION: Beta-lapachone induced potent cytotoxic effects in RB cell lines at low micromolar concentrations, suggesting this agent could be useful in the clinical management of RB.

Endothelial cell proliferation in the choriocapillaris during human retinal differentiation.

BACKGROUND: Differentiation patterns of the neural retina and its retinal vasculature are not well matched. The foveal region differentiates first, however the central retina is not vascularised until late in gestation. The authors explored the hypothesis that higher rates of endothelial cell proliferation in the choriocapillaris of the central retina might compensate for the slow growth of central retinal vessels, providing supplementary nutrients to the region during the early stages of neuronal maturation. METHODS: Frozen sections of five human fetal eyes (14-18.5 weeks' gestation), were examined for Ki-67 and CD34 immunoreactivity using confocal microscopy. Measurements of choriocapillaris area and the number of proliferating choroidal endothelial cells were used to calculate the rate of choroidal endothelial proliferation at five different chorioretinal locations. RESULTS: The choriocapillaris area is consistently greater in the foveal region than at other locations and increases progressively with age. A higher rate of endothelial cell proliferation was found in parts of the choriocapillaris associated with the undifferentiated (proliferating) neural retina, compared with the differentiated, central region. CONCLUSION: The findings suggest that mechanisms regulating proliferation and growth of the choroidal vasculature are independent of differentiation in the neural retina, and are thus profoundly different from mechanisms that regulate formation of the retinal vasculature.

Muller cell expression of glutamate cycle related proteins and anti-apoptotic proteins in early human retinal development.

AIMS: The distribution of glutamate cycle related proteins (glutamine synthetase (GS) and GLAST) and anti-apoptotic proteins (Bcl-2 and Bcl-X) was investigated in Müller cells during early human retinal development, relative to the onset of expression of synaptophysin, a presynaptic vesicle protein. METHODS: Using frozen sections of human fetal eyes (13-22 weeks gestation) (n = 10), Bcl-2, Bcl-X, GS, GLAST, and synaptophysin immunoreactivities (IR) were imaged using fluorescence microscopy and plotted as a function of eccentricity from the incipient fovea. Frozen sections of adult human retina (n = 4) were immunolabelled with antibodies to Bcl-2 and Bcl-X. RESULTS: Müller cell immunoreactivity for GS, GLAST, and Bcl-2 was initially detected in the incipient fovea, and then at more peripheral locations with increasing age. Synaptophysin-IR appeared earlier than all other target proteins. Within the synaptophysin-IR region, mature (differentiated) Müller cells expressed both Bcl-2 and Bcl-X-IR from 13 weeks gestation, ahead of GS-IR and GLAST-IR that were first seen at 14 weeks gestation. Additionally, from as early as 13 weeks gestation, ganglion cells and immature neuronal progenitor cells across the entire retina expressed Bcl-2-IR and Bcl-X-IR, respectively. In adult retina, ganglion cells and some bipolar cells expressed Bcl-X but not Bcl-2. CONCLUSION: Müller cells express Bcl-2 and Bcl-X after synaptogenesis has commenced, but before the onset of GS and GLAST expression, suggesting a protective role for these proteins in Müller cells during the onset of glutamatergic transmission in early human retinal development.

Human retinal microglia express candidate receptors for HIV-1 infection.

BACKGROUND/AIMS: Microglia are the primary antigen presenting cells in the central nervous system and the retina, and can harbour viral antigens that may damage neural tissue via the release of neurotoxins. All cells bearing CD4 molecules and co-receptors (members of the chemokine receptor and Fcgamma receptor families) are potential targets for the human immunodeficiency virus (HIV). In this study, retinal microglia (in vitro and in situ) were investigated for the expression of candidate HIV-1 binding receptors. METHODS: Cultured human retinal microglia and frozen sections of human retinas were used. Immunohistochemistry was used to investigate expression of cell surface receptors necessary for HIV-1 infection: CD4, CC chemokine receptor 5 (CCR5), and Fcgamma receptors. RESULTS: Human retinal microglia expressed detectable levels of CD4, CD16, CD64, and CCR5 in vitro and Fcgamma receptor I (CD64) in situ. CONCLUSIONS: Human retinal microglia express several candidate receptors required for viral binding and as such may be a potential reservoir for HIV-1 infection.

Conditioned medium from mixed retinal pigmented epithelium and Müller cell cultures reduces in vitro permeability of retinal vascular endothelial cells.

AIM: To investigate the in vitro effect of laser photocoagulation on blood-retinal barrier permeability. METHODS: Retinal capillary endothelial cells were exposed to supernatants from long term co-cultured cells that were argon laser treated. Endothelial cell permeability was analysed by (1) measurement of transendothelial electrical resistance and (2) equilibration of [(3)H] inulin and [(14)C] albumin across the cell monolayer. RESULTS: Laser photocoagulation of various retinal cells and control ECV304 cells in the lower chamber did not appreciably improve permeability of the endothelial cell monolayer compared with that of unlasered cells. However, medium that was conditioned by mixed retinal pigmented epithelium and Müller cells significantly reduced both inulin (43.2% (SD 6.5%) equilibration in mixed cultures v 59.8% (SD 7.0%) control cells, p<0.05) and albumin (15.1% (SD 3.8%) v 31.1% (SD 6.7%), p<0.05) permeability of the endothelial cell monolayers. A fourfold increase in transendothelial electrical resistance was also seen. CONCLUSIONS: These results are consistent with the hypothesis that interaction of Müller cells with retinal pigmented epithelium induced by laser treatment results in secretion of soluble factor(s), which reduces permeability of retinal vascular endothelium. Identification of these factor(s) may have implications for the clinical treatment of macular oedema secondary to diabetic retinopathy and other diseases.

Differential expression of GFAP in early v late AMD: a quantitative analysis.

BACKGROUND/AIMS: Glial fibrillary acidic protein (GFAP) is an established indicator of retinal stress; its expression in retinal astrocytes and Müller cells has been demonstrated to be modulated by cytokines and retinal pathology, including age related macular degeneration (AMD). This study aims to quantify the modulation of GFAP expression in retinas with drusen and atrophic AMD versus normal age matched controls. METHODS: Following a histopathological survey, 17 donor retinas were classified into four groups: drusen (n=5), geographic atrophy (GA) (n=6), aged normal (n=3), and young normal (n=3). Paramacular cryosections were immunolabelled with GFAP antibody, examined by confocal microscopy, and quantified by NIH digital image analysis. Groups were matched for potential confounding factors including age, sex, and postmortem delay. RESULTS: A significant increase in GFAP immunolabelling of macroglia was noted in aged normal compared with young normal retinas (p<0.04). Upregulation of GFAP immunoreactivity involving astrocytes was observed in drusen retinas compared with control retinas (p<0.03). GFAP was also upregulated in retinas with GA compared with controls (p<0.05) and in retinas with GA compared with drusen (p<0.04), both involving Müller cells. Discrete regions of GFAP upregulation in Müller cells were associated with drusen formation. In GA specimens atrophied retinal pigment epithelium (RPE) was substituted by GFAP immunoreactive Müller cell processes (gliosis). CONCLUSION: This study provides a quantitative assessment of GFAP modulation in ageing and AMD affected retinas. Morphological observations were consistent with quantitative analyses indicating differential modulation of GFAP immunoreactivity in inner and outer retina. Upmodulation of GFAP in inner retina and astroglial processes was predominantly associated with drusen, while in outer retina Müller glia upmodulation of GFAP was associated with disruption of the RPE and blood-retinal barrier.

Human uveal melanoma expresses NG2 immunoreactivity.

BACKGROUND/AIMS: NG2 is the rat homologue of the human melanoma proteoglycan (HMP), also known as the high molecular weight melanoma associated antigen. Most cutaneous melanomas, as well as glioblastomas, chondrosarcomas, and some leukaemias express NG2 immunoreactivity, recognised using monoclonal antibody (mAb) 9.2.27. This antibody has also been used for molecular targeting in targeted alpha therapy for melanoma. The purpose of this study was to evaluate the expression of NG2 immunoreactivity in human uveal melanoma and normal ocular tissue using mAb 9.2.27. METHODS: Enucleated eyes from 26 patients with choroidal or ciliary body melanoma (n=26) were available as paraffin sections, and stained with haematoxylin and eosin to assess for tumour cell type and histopathology. Additional slides were investigated for NG2 immunoreactivity using mAb 9.2.27 and alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostaining. Two independent observers graded immunostaining using a semiquantitative scale from 0 (negative) to 3 (strong). RESULTS: Immunostaining for mAb 9.2.27 could not be graded in 7/26 cases with dense pigmentation of the tumour. For the remaining cases, grade 2 (moderate) or more immunostaining was seen in 18/19 tumours (95%). The retina, retinal pigment epithelium (RPE), and choroid displayed weak immunostaining (grade 0.5-1.5) in the majority of melanoma affected eyes. Normal retina and choroid (n=5) appeared negative for mAb 9.2.27. Optic nerve axon bundles in both control and melanoma affected eyes displayed moderate immunostaining. CONCLUSION: In the present study, the majority of human uveal melanomas expressed NG2 immunoreactivity, as detected using mAb 9.2.27. This antibody may be a suitable candidate for radioimmunotherapy to target ocular melanoma.

Immunological and aetiological aspects of macular degeneration.

Aetiological and immunological aspects of AMD, a leading cause of blindness in Western countries, have been reviewed. Developmental studies suggest that anatomical features unique to the fovea result in a critical relationship between metabolic demand and blood supply at the macula, which is maintained throughout life. Recent studies show a sufficient degree of consistency in the link between smoking and both dry and wet AMD to regard it as causative. Dry AMD is considered to be the natural endstage of the disease; epidemiological and morphological studies point to choroidal vascular atrophy as the causative event and it is suggested that signals associated with acute vascular compromise lead to the development of subretinal neovascularisation. The relationship between sub-pigment epithelial deposits, including basal laminar deposit, and the pathogenesis of AMD is examined. Much of the literature is consistent with a choroidal origin for the constituents of drusen. The blood-retinal barrier preserves the physiological environment of the neural retina and limits inflammatory responses. The factors, including cytokines, adhesion molecules and the presence of resident immunocompetent cells (microglia), which determine the immune status of the retina are considered. Historical descriptions of the involvement of inflammatory cells are provided, evidence implicating inflammation in the pathogenesis of AMD involving macrophages, giant cells and microglia has been derived from observations of human and animal subretinal neovascular lesions. The role of humoral factors such as anti-retinal autoantibodies and acute phase proteins together with clinical observations has been surveyed. Taken together these data demonstrate the involvement of both cellular and humoral immunity in the pathogenesis of AMD. It remains to be determined to what degree the influence of immunity is causative or contributory in both wet and dry AMD, however, the use of anti-inflammatory agents to ameliorate the condition further indicates the existence of an inflammatory component.

In vitro effects of radiation on human retinoblastoma cells.

Retinoblastoma (Rb) is the most common intraocular tumor of childhood and has been demonstrated clinically to be very sensitive to external beam radiation (EBR). The purpose of this study was to determine the survival of Rb cell lines at different endpoints following irradiation. We also studied Rb-reconstituted cell lines postirradiation to gain insight into the role of Rb following DNA damage. Suspension cultures were exposed to single doses of 1, 2, 5, and 10 Gy. Following irradiation, cell viability and cell death was assessed for up to 72 hr using Trypan blue exclusion and acridine orange/ethidium bromide (AO/EB) staining, respectively. Morphological features were examined with light and electron microscopy (LM and EM). Clonogenic survival was assessed 2 weeks postirradiation. Cell cycle distribution was analyzed by flow cytometry. A time- and dose-dependent decrease in cell viability was induced in Y79 and WERI-Rb1 cells following irradiation, although not below approximately 45% for the times and doses used. A similar but much-reduced effect on viability was observed in Rb-reconstituted cells. AO/EB staining, LM, and EM revealed features characteristic of apoptosis following irradiation and a time- and dose-dependent increase in apoptosis was observed. For Y79 and WERI-Rb1 cells, 30-40% apoptosis was observed 72 hr after 10 Gy irradiation; however, apoptosis was much reduced in Rb-reconstituted cells (approximately 8% Y79LxRb28; approximately 18% WERILxRb8). Rb cell lines were extremely sensitive to radiation, although less so in Rb-reconstituted lines, with clonogenic survivals after 2 Gy (SF2) of 0.14 for Y79, 0.06 for WERI-Rb1, 0.36 for Y79LxRb28, and 0.19 for WERILxRb8. Postirradiation, a sub-G1 population was observed, consistent with radiation-induced apoptosis, and Rb-reconstituted cells displayed a prolonged G2 phase. The clonogenic survival parameters of Rb cell lines are consistent with clinical observations, where extreme sensitivity to irradiation has been reported. Additionally, Rb protein protected cells from DNA damage and may also play a role in radiation-induced G2 delay. This in vitro approach provides a useful model for further radiobiological studies of Rb.

Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration.

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age-related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood-retinal barrier (BRB). The influence of TA on the expression of ICAM-1 and MHC-I was studied on resting and phorbol myristate acetate (PMA)- or interferon-gamma (IFN-gamma)- and/or tumour necrosis factor-alpha (TNF-alpha)-activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA-activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down-regulated ICAM-1 expression, consistent with immunocytochemical observations. PMA-induced permeability changes were dose-dependent and TA decreased permeability of both resting and PMA-activated monolayers. MHC-I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM-1 expression in vitro correlate with clinical observations, suggesting re-establishment of the BRB and down-regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD-affected retinae.

The human hyaloid system: cell death and vascular regression.

The present study had investigated the roles of apoptosis and necrosis in the regression of the human fetal hyaloid vasculature. Normal human fetal hyaloid specimens (n = 67) ranging from 10 to 20 weeks' gestation were studied. Specimens were either immunolabeled with anti-von Willebrand factor and major histocompatibility complex class I antibodies or investigated using the terminal-deoxyribonucleotidyl transferase-mediated dUTP-biotin DNA nick-end labeling technique. A fluorescent DNA-binding dye acridine orange/ethidium bromide mixture was also applied to unfixed flat mounts of hyaloid vasculature and some specimens were processed for transmission electron microscopy. Vascular regression including cell loss in the connecting vessels, stretching and thinning of the vasa hyaloidea propria, tunica vasculosa lentis and the pupillary membrane was clearly evident after 13 weeks' gestation. Cresyl violet staining revealed condensed cells and pyknotic bodies throughout the hyaloid system; cell death occurred either in single cells or along small capillary segments associated with vascular regression. Acridine orange/ethidium bromide staining showed DNA condensation at early and late stages of cell death. Similarly, DNA nick-end labeling was positive in endothelial cells, pericytes and vessel and non-vessel associated hyalocytes. The observation of hyalocytes juxtaposed to cytolysed endothelial cells may indicate a role for these cells in vascular regression. Features of apoptosis were more evident during early vascular regression whilst necrosis was increasingly evident at later stages.

Epithelialization of a synthetic polymer in the feline cornea: a preliminary study.

PURPOSE: This study examined the potential of a synthetic polymer to support stable epithelial growth when implanted in the feline cornea. METHODS: A perfluoropolyether-based polymer was cast into lenticules that were coated with collagen I and implanted in four feline corneas. Epithelial growth onto the lenticules was monitored clinically for 6 weeks, after which time the animals were killed, and three corneas were evaluated histologically. Immunohistochemistry was used to identify proteins associated with the formation of a basement membrane (laminin) and adhesion complexes (bullous pemphigoid antigen and collagen VII). Electron microscopy was used to examine the tissue-polymer interface for evidence of the assembly of these adhesive structures. RESULTS: Postoperative epithelial growth began on days 2 to 3, and lenticules were fully epithelialized by days 5 to 9. Lenticules were clinically well tolerated and histology showed epithelium consisting of multiple layers adherent to the lenticule's surface. Laminin, bullous pemphigoid antigen and collagen VII were identified at the tissue-polymer interface using immunohistochemistry. Ultrastructural examination showed evidence of assembly of these proteins into a recognizable basement membrane and hemidesmosomal plaques. CONCLUSIONS: A perfluoropolyether-based polymer coated with collagen I was implanted in the feline cornea and supported epithelial growth that showed signs of persistent adhesion, both clinically and histologically. This polymer shows potential for ophthalmic applications that require sustained epithelialization.

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas.

PURPOSE: To determine whether MT1-MMP and MMP-2 are expressed in normal and keratoconic corneas, and to investigate the ability of MT1-MMP, expressed on cultured keratocytes after stimulation with concanavalin A, to activate pro-gelatinase A (pro-MMP 2). METHODS: Specimens of keratoconus corneas (n = 20), removed at corneal transplantation, were obtained from pathology archives, sections were cut, and were stained with an antibody to MT1-MMP, using peroxidase immunohistochemistry. Eye banked corneas served as controls (n = 14). Normal human keratocyte cultures were initiated from eye bank corneas, and after stimulation with con A, MMPs in the media were examined using gelatin zymography and immunoblotting, and MT1-MMP expression was analysed by flow cytometry and immunoblotting. RESULTS: All corneas showed some expression of MT1-MMP and MMP-2, although the degree of staining varied greatly. The MMPs were present in the epithelium, endothelium and stroma. Expression of MT1-MMP, but not MMP-2, in the epithelium and stroma, was significantly elevated in keratoconus, compared to normal corneas. In vitro, keratocytes stimulated with con A expressed MT1-MMP and produced active MMP-2, detected by zymography. These responses to con A were concentration-dependent and MT1-MMP expression and MMP-2 activation correlated significantly (p = 0.0003) In addition, MMP inhibitors abolished MMP-2 activation, providing further evidence that MT1-MMP activated MMP-2. CONCLUSION: The observation that MT1-MMP expression may be up-regulated in keratoconus corneas, taken together with the demonstration that human corneal cells can express this enzyme, which in turn can activate latent MMP-2, provide evidence for a possible role for MT1-MMP in the pathogenesis of keratoconus.

Soluble TNF-alpha receptors bind and neutralize over-expressed transmembrane TNF-alpha on macrophages, but do not inhibit its processing.

Tumor necrosis factor alpha (TNF-alpha) is initially synthesized as a type II integral membrane protein (transmembrane TNF-alpha) after macrophage activation. In this study we have investigated some aspects of the regulation of expression and biological activity of transmembrane TNF-alpha by both soluble TNF-alpha receptors (sTNF-alphaR) and inhibitors of TNF-alpha processing. We show, using the technique of receptor-mediated ligand precipitation (RMLP), that a dimeric construct of the type I sTNF-alphaR binds to transmembrane TNF-alpha, expressed on the mouse macrophage cell line RAW264.7, under cell culture conditions. This interaction between sTNF-alphaR and transmembrane TNF-alpha does not prevent processing and release of soluble TNF-alpha. A specific hydroxamic acid-based inhibitor of processing, BB1101 (British Biotech), was found to increase the total cellular levels of whole-cell, 26-kDa, precursor TNF-alpha by 2.2-fold. However, the inhibitor increased the levels of precursor TNF-alpha present solely on the cell surface (i.e., transmembrane TNF-alpha) by 5.1- to 7.5-fold. This increase in the levels of transmembrane TNF-alpha on the activated human monocytoid cell line mono mac 6 was associated with a similar (6.7-fold) increase in TNF-alpha-mediated cytotoxicity toward the human adenocarcinoma cell line Colo 205, which is sensitive only to the transmembrane form of TNF-alpha. Mono mac 6 cells, expressing transmembrane TNF-alpha, were found to be killing the Colo 205 target cells through apoptosis. This cytotoxicity could be neutralized by pre-incubating the mono mac 6 cells with either sTNF-alphaR or polyclonal anti-TNF-alpha serum.

Astrocyte proliferation during development of the human retinal vasculature.

Wholemounts of human fetal retinas were labeled with antibodies to Ki67 or proliferating cell nuclear antigen, to map the distribution of proliferating cells in the developing primary vasculature and neural retina. Double labeling was used to determine the relative proportions of endothelial cells (CD34), astrocytes (glial fibrillary acidic protein - GFAP) and microglia (major histocompatability complex class II) associated with the developing vessels. The differentiated region of neural retina (cold spot) was 3.5 mm(2)at 15 weeks gestation (WG), centred on the incipient fovea, and increased in size with age to 80.5 mm(2)by 23-24 WG. Ki67 immunoreactive cells were distributed throughout the developing vasculature at all ages. The mean density of dividing cells in the neural retina increased with gestational age from 146 mm(-2)at 15 WG, to 624 mm(-2)at 23-24 WG. By 20 WG proliferation in the vasculature overlapped the margins of the cold spot, which was almost completely vascularized by 23-24 WG, except for a narrow strip on the horizontal meridian, which included the incipient fovea. Counts of CD34/Ki67 immunoreactive cells indicated that 15-52% of proliferations in the developing vasculature at 18 WG are endothelial cells. In contrast, in the fellow retina 65-85% cells were Ki67/GFAP immunoreactive, indicating proliferation of astrocytes in situ. No dividing microglia were observed. The findings suggest that large numbers of proliferating astrocytes accompany the developing vessels as they migrate across the primate retina.

Apoptosis during development of the human retina: relationship to foveal development and retinal synaptogenesis.

Apoptosis in the ganglion cell (GCL) and inner nuclear (INL) layers of human fetal retinae aged 14-35 weeks of gestation (WG) was investigated in relation to synaptogenesis and foveal depression formation. Terminal transferase dUTP-biotin nick end labeling (TUNEL) was used to identify apoptosis, and synapse development was demonstrated by synaptophysin immunoreactivity (-IR). The distribution of apoptotic cells and synaptophysin-IR was studied as a function of eccentricity. Between 14 and 23-24 WG in the GCL, rates of apoptosis were relatively low in central retina. A shallow fovea was detected at 23-24 WG. In the central GCL, the rate of apoptosis was 0.21% of viable cells compared with a higher incidence of 0.79-1.64% peripherally. Apoptosis in the INL was 2-8 times greater than that in the GCL. At 14-15 WG, peak death occurred at the incipient fovea; however, by 20 WG the distribution was bimodal, with peaks at more eccentric locations on either side of the incipient fovea with increasing age. Approximately 90% of INL apoptotic cells were in the middle and outer regions, suggesting that bipolar cells formed the majority of dying neurons. Synaptophysin-IR was present in cones, bipolar cells, and processes in the inner and outer plexiform layers at the incipient fovea at 14 WG and spread peripherally with increasing age. The peripheral margin of synaptophysin-IR coincided with areas of peak INL apoptosis. This pattern suggests that bipolar cell elimination is associated with the onset of synaptogenesis. Apoptosis in the GCL and INL is not a significant factor in foveal depression morphogenesis.