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Background: Our laboratory previously reported that imatinib mesylate (IM) has an inhibitory effect on two retinoblastoma (Rb) cell lines in vitro.Aims: The purpose of this project was to determine the immunoexpression of platelet-derived growth factor receptor (PDGFR)-α, PDGFR-β and c-Abl in 61 human samples of Rb to determine if IM-sensitive receptors are present. Additionally, this paper seeks to establish a correlation between the expression of PDGFR, c-Abl and the histopathological prognosis. Methods: Sixty-one paraffin-embedded Rbs were collected from the Henry C. Witelson Ocular Pathology Registry. PDGFR-α, PDGFR-β and c-Abl immunostaining was performed according to the protocol provided by Ventana Medical System Inc. Immunoreactivity was correlated with the presence or absence of invasion into the choroid and optic nerve. Results: Overall, c-Abl expression was identified in 50 out of 61 specimens (81.97%), PDGFR-α was identified in 20 out of 60 specimens (33.33%) and PDGFR-β expression was identified in 57 out of 61 specimens (93.44%). Histopathological prognosis was not correlated with immunoreactivity except in the case of PDGFR-β. Conclusions: Rb is a cancer that expresses PDGFR-α, PDGFR-β and c-Abl, which are known targets of IM. These markers may be responsible for the documented therapeutic effect of IM on Rb cell lines.

Introduction Focal adhesion kinase (FAK) is implicated in tumor progression and metastatic cascade, and has been shown to be overexpressed in a variety of human cancers. However, the role of FAK in human uveal melanoma (UM) is not well defined. The purpose of this study was to evaluate the expression of FAK in UM tumors and normal eyes, and to determine the effect of Hsp90 inhibition on FAK expression in UM cells. Methods FAK expression was assessed in 39 UM specimens, FAK[pY397] expression was assessed in 51 UM specimens, and both FAK and FAK[pY397] expression were assessed in 20 normal eyes. The expression of FAK and FAK[pY397] was detected by Western blot in five UM cell lines after treatment with 10 μmol/L of 17-AAG. Results FAK was positive in 87.2% and FAK[pY397] in 90% of UM specimens. Low FAK expression was detected in non-tumor structures and in normal eyes. The cell lines with the most proliferative, invasive phenotype (92.1, SP6.5 and MKT-BR) displayed high expression of FAK[pY397], and the levels of FAK and FAK[pY397] were decreased in the presence of 17-AAG starting with 24 h of exposure. Conclusion FAK and FAK[pY397] were overexpressed in human UM tumors compared to normal ocular tissue and high levels of FAK[pY397] were seen in the most aggressive UM cell lines. Hsp90 inhibition led to downregulation of FAK expression. We propose a role for FAK in the pathogenesis of UM. Future studies are needed to explore the use of Hsp90 inhibitors as a feasible approach for modulating FAK in UM.

SIRT1 is a deacetylase that has been shown to be instrumental in embryonic and pathologic vascular formation. The purpose of this study was to evaluate the potential role of SIRT1 in the pathogenesis of choroidal neovascularization in age-related macular degeneration.The expression of SIRT1 was assessed via immunohistochemistry in nine excised human choroidal neovascularization membranes and seven non-age-related macular degeneration donor eyes. Enzyme-linked immunosorbent assay-based angiogenesis arrays were used to assess the potential of an SIRT1 inhibitor, nicotinamide, to reduce secretion of 10 unique proangiogenic cytokines from retinal pigment epithelial cells.SIRT1 was expressed more frequently in choroidal neovascularization membranes than donor eyes about vascular endothelial cells (78 vs. 29% positive cases) and retinal pigment epithelial cells (57 vs. 14% positive cases). SIRT1 inhibition in retinal pigment epithelial cells correlated with significantly decreased secretion of three potent proangiogenic cytokines: angiogenin, platelet-derived growth factor BB, and vascular endothelial growth factor A.SIRT1 levels appear elevated in human choroidal neovascularization membranes compared with control eyes. Moreover, inhibition of SIRT1 activity is correlated with decreased secretion of potent proangiogenic cytokines. Collectively, these data support a potential role for SIRT1 in the pathogenesis of neovascular age-related macular degeneration.

Background Age-related macular degeneration (AMD) is currently the leading cause of blindness in developed countries. Bevacizumab is a widely used anti-VEGF agent that is a commonly applied therapy for neovascular AMD; however, a consequence of bevacizumab therapy may be the activation of compensatory angiogenic signalling. Combination of bevacizumab with 3,4 dihydroxyphenyl ethanol (DPE) may attenuate this compensatory signalling. The goal of the study was to investigate this therapeutic option in a human retinal pigment epithelial cell line (ARPE-19). Methods ARPE-19 cells were incubated under both normoxic and hypoxic conditions. The cells were treated as follows: control, 100 µM DPE, 0.25 mg/ml bevacizumab, the combination of DPE and bevacizumab. Media was harvested after 24 h for sandwich ELISA-based angiogenesis assays. The secretion of the following 10 pro-angiogenic cytokines was measured: angiogenin, ANG2, EGF, bFGF, HB-EGF, PDGF-BB, Leptin, PIGF, HGF, and VEGF-A. Results Treatment of ARPE-19 cells with bevacizumab significantly increased the secretion of angiogenin. Secretion of angiogenin and VEGF-A were significantly reduced following treatment with DPE under both normoxia and hypoxia. In addition, angiogenin secretion was significantly reduced following treatment with the combination of DPE and bevacizumab compared to bevacizumab alone. Conclusions Compensatory angiogenic signalling may occur in neovascular AMD following treatment with bevacizumab. Here we show that DPE, both alone and in combination with bevacizumab, can reduce the secretion of angiogenin, a cytokine that has been upregulated following treatment with bevacizumab in RPE cells. Therefore, DPE may represent a possible therapeutic agent to be used in combination with bevacizumab for the treatment of neovascular AMD.

Purpose: The cellular origin of retinoblastoma is uncertain as constituent tumor cells heterogeneously express markers of both immature and mature retinal cells. An immunohistochemical analysis of cellular origin may yield valuable insights into disease progression and treatment options. This study aimed to determine the cellular origin of retinoblastoma in a large case series and correlate these findings with histopathological prognostic factors. Methods: Thirty-nine retinoblastoma cases were histopathologically diagnosed and analyzed by immunohistochemistry using monoclonal antibodies against the immature neural cell marker SRY-box containing gene 2 (SOX-2), the mature neuronal cell marker microtubule-associated protein 2 (MAP2), and the mature glial cell marker glial fibrillary acidic protein (GFAP). Histopathological features were also evaluated, including patterns of growth, differentiation, vitreous seeding, and choroidal/scleral, optic nerve, and anterior chamber invasion. Two retinoblastoma cell lines, WERI-1 and Y79, were studied by immunocytochemistry using the same antibodies. Results: Expression of SOX-2 was strong in 97.4% of retinoblastoma cases, while MAP-2 was expressed in 59% of cases. Immunostaining for GFAP was positive only in reactive stromal astrocytes interspersed amongst tumor cells and in peritumoral tissue. There was no correlation between histopathological prognostic factors and immunohistochemical markers. Retinoblastoma cell lines showed strong positivity for SOX2 (90% of WERI-1 cells and 70% of Y79 cells) and MAP2 (90% of cells in both lines). GFAP was completely negative in both cell lines. Conclusion: The majority of retinoblastomas and both RB cell lines expressed an immature neural and/or a mature neuronal cell marker, but not a glial marker. These results indicate a typical neuroblast or neuronal origin and eliminate astrocyte differentiation from neural stem cells as the source of retinoblastoma.

Purpose. To describe the histopathological features of vitreous samples obtained after vitrectomy surgery from diabetic and nondiabetic patients.Methods. Vitreous specimens from 137 patients who underwent vitrectomy for different clinical conditions were analysed. All samples were centrifuged and each resulting pellet was fixed and processed as part of routine paraffin section histopathology. The histopathological features were categorized in a semiquantitative fashion. The samples from diabetic and nondiabetic patients were compared.Results. The 125 included patients (58 diabetic, 60% males) were aged64.2±13.9years. The presence of hemorrhage, inflammatory cells, and histiocytes was significantly higher in the diabetic group (P<0.001,P=0.028, andP=0.016, resp.), showing more vessels (P<0.001) and ghost vessels (P=0.049). The presence of inflammatory cells was the feature with the highest sensitivity for detecting diabetes mellitus (98%) and also the highest negative predictive value (89%). In the multivariate analysis, three variables emerged as independent significant predictors of diabetes in vitrectomy samples: hemorrhage, endothelial-lined vessels, and age (P<0.001,P<0.001, andP=0.019, resp.).Conclusions. Different histopathological features can be found in vitreous samples from diabetic patients. Analysis of vitrectomy samples may serve as a tool for diabetes management.

Our aim was to evaluate the potential effect of imatinib mesylate (IM), a small molecule that specifically inhibits the tyrosine quinase receptors, on the proliferation and invasive abilities of two human retinoblastoma (Rb) cell lines. Furthermore, the ability of IM to radiosensitize Rb cells was evaluated. The potential targets of IM (C-kit, PDGRF-α and -β, and c-Abl) were also investigated in these cell lines.Two human Rb cell lines (WERI-RB-1 and Y79) were cultured under normal growth conditions. An MTT-based proliferation assay and a Matrigel invasion assay were performed with and without exposure to 10 μM of IM. The cells were also irradiated with graded dosages of 0, 2, 4, 6, 8, and 10 Gy with and without IM and their proliferations rates were analyzed. Western blot and immunocytochemical analysis of cytospins were performed to evaluate the expression of C-kit, PDGRF-α and -β, and c-Abl.When IM was added to both cell lines a statistically significant (P0.05) reduction in proliferation and invasive ability were observed. Exposure to IM also significantly increased the radiosensitivity of both Rb cell lines. The c-Abl expression was strongly positive, PDGRF-α and -β expression were also positive but the C-kit expression was negative in both cell lines.These results indicate that Gleevec may be useful as an adjuvant treatment in Rb patients, specially those considered for radiation therapy.

Background: Recent evidence has suggested a role for toll-like receptor 3 (TLR3) in experimental models of age-related macular degeneration (AMD). To date, however, few data exist about TLR3 in human AMD. The purpose of this study was to investigate the expression of TLR3 in human choroidal neovascular (CNV) membranes. Methods: Immunostaining for TLR3 was performed on sections of CNV membranes from 8 AMD patients and eyes from 4 donors without CNV. Results: All CNV membranes expressed TLR3 in retinal pigment epithelial (RPE) cells. One was classified as having strong intensity, 5 as having moderate intensity and 2 as having weak intensity. All cases had ≧30% of the RPE cells staining for TLR3, ranging from 30 to 90%. No expression of TLR3 was observed in vascular endothelial cells or fibroblasts in any CNV membrane. In the donor eyes, the RPE cells near the ora serrata stained stronger than those at the posterior pole, where no staining was observed in 3 out of 4 cases. Conclusion: TLR3 was found in all CNV membranes and was expressed exclusively in RPE cells. The observed difference in RPE staining for TLR3 in donor eyes and CNV membranes suggests a possible role for this receptor in human neovascular AMD.

PURPOSE: Fresh biopsied ocular tumor tissues are difficult to obtain for the purpose of performing microarray experiments on extracted nucleic acids. Present technology allows for extraction of total RNA from formalin-fixed paraffin embedded (FFPE) tissue analyzed by the cDNA mediated Annealing Sectioning and Ligation (DASL) method. We aimed to correlate gene transcript differences between two uveal melanoma (UM) clinical-histopathological parameters (metastasis, cell type). METHODS: A total of 43 FFPE UM were used. The expression of RPL13a, a ribosomal protein gene, for each sample was used to evaluate the quality of RNA extracted from FFPE tissue. Gene expression values generated from the array were analyzed using the GeneSpring GX software (Agilent). Immunohistochemistry was used in order to validate transcriptional findings at the protein level. RESULTS: A total of 106 genes were identified with (P < 0.05, Welch ANOVA test) a difference in transcript abundance for the metastasis clinical parameter. Furthermore, we identified 64 genes with a statistically significant (P < 0.05) difference in transcript abundance between the spindle and epithelioid cell types. Each individual sample for both groups (metastasis, cell type) exhibited distinct transcriptional profiles that were separated on a PCA. Positive nuclear immunostaining for LIG4-metastasis, ErbB3-cell type was found to be associated with better patient prognosis and outcome. CONCLUSIONS: To the best of our knowledge, this is the first time that a successful retrospective analysis has been done with UM FFPE RNA. This data may lead to future customized therapeutic targets, which may improve the now unchanged mortality rate of this particular malignancy.

The objective of this study was to investigate the expression of cyclooxygenase (COX)-2 in human choroidal neovascular membranes.Paraffin-embedded sections of choroidal neovascular membranes excised from 16 patients with wet age-related macular degeneration were used for this study. Sections were subjected to immunohistochemistry using a monoclonal mouse antihuman COX-2 antibody. Staining was classified as either negative or positive in retinal pigment epithelial cells, vascular endothelial cells, and fibroblasts. Serial sections were stained for vimentin expression to confirm tissue antigenicity.Eleven of 16 (69%) choroidal neovascular membranes stained positive for COX-2 in retinal pigment epithelial cells, with 6 (38%) of these also expressing COX-2 in vascular endothelial cells and 6 (38%) in fibroblasts. None of the sections that were negative for COX-2 in the retinal pigment epithelial cells showed COX-2 expression in the other cell types assessed. There was a statistically significant difference (P = 0.0097) in the mean ages between the COX-2 positive group (65.6 years) and COX-2 negative group (76.8 years) as determined by a two-tailed, unpaired Student's t-test.The expression of COX-2 in human choroidal neovascular membranes suggests a possible role for this modulator in age-related macular degeneration pathogenesis. The age-dependent expression observed is novel and warrants further investigation.

PURPOSE: To compare the effect of preserving sclera samples in either 95% ethanol or freeze-dried. METHODS: Ninety-six samples of human sclera were studied. Half of them were freeze-dried and half preserved in 95% ethanol. Preservation periods of 18, 45, 90 or 174 days were studied. Automated immunostaining was carried out in the Ventana BenchMarkR LT platform using collagen 1 and fibronectin antibodies. Histological staining was also performed with hematoxilin-eosin and Masson trichrome. Samples were classified according to the degree of collagen fiber parallelism (0-2), intensity of Masson staining (0-2), and the expression of both antibodies (0-3). Friedman and Wilcoxon tests were applied to compare preservation methods and p-values below 0.05 were considered to ensure statistical significance. RESULTS: Relevant results were found in three situations: (i) Friedman's test showed better collagen fiber integrity in the freeze-dried group rehydrated after 174-days as compared to the 90-day group; (ii) Wilcoxon's test showed better collagen fiber integrity in the freeze-dried group after 18 and 174 days as compared to the ethanol group; (iii) the freeze-dried group disclosed higher immunohistochemical expression for COL-1 antibody in the sclera samples rehydrated after 45, 90 and 174 days as compared to the ones rehydrated after 18 days. CONCLUSION: Histological and immunohistochemical analysis showed freeze-drying to be a superior method for sclera preservation as compared to 95% ethanol. This technique provides an easy method to manipulate tissue, with longer shelf life, and storage at room temperature.OBJETIVO: Comparar dois métodos de preservação de esclera humana, liofilização e álcool 95%, em diferentes períodos de tempo. MÉTODOS: Foram avaliados 96 fragmentos de seis escleras humanas. Metade das amostras foi submetida ao processo de liofilização e metade conservada em álcool 95%. Dois fragmentos de cada grupo foram avaliados pelas colorações de hematoxilina-eosina e tricrômio de Masson e submetidos a técnica de imuno-histoquímica para os anticorpos fibronectina e colágeno 1, após 18, 45, 90 e 174 dias de preservação. Os espécimens foram classificados de acordo com o paralelismo (PF:0-2) e integridade (IF:0-1) das fibras de colágeno e expressão imuno-histoquímica para os anticorpos fibronectina (FIB:0-3) e colágeno 1 (COL-1:0-3). A análise estatística foi realizada por meio dos testes de Friedman e Wilcoxon e o valor de p menor que 0,05 foi considerado estatisticamente significante. RESULTADOS: Verificaram-se diferenças significantes em três situações: (i) maior integridade das fibras de colágeno das escleras liofilizadas após 174 dias quando comparado aos 90 dias; (ii) maior expressão imuno-histoquímica para o anticorpo COL-1 nas amostras de escleras liofilizadas após os 18 dias iniciais de preservação; (iii) maior integridade das fibras de colágeno das escleras liofilizadas após 18 e 174 dias quando comparado às escleras preservadas em álcool. CONCLUSÕES: A preservação de tecido escleral por liofilização mostrou-se técnica tão eficaz quanto a preservação em álcool, apresentado vantagem quando considerada a integridade das fibras de colágeno. A liofilização mostra-se benéfica por permitir a estocagem do tecido em temperatura ambiente e com prazo de validade estendido.

OBJETIVO: Comparar dois metodos de preservacao de esclera humana, liofilizacao e alcool 95%, em diferentes periodos de tempo. METODOS: Foram avaliados 96 fragmentos de seis escleras humanas. Metade das amostras foi submetida ao processo de liofilizacao e metade conservada em alcool 95%. Dois fragmentos de cada grupo foram avaliados pelas coloracoes de hematoxilina-eosina e tricromio de Masson e submetidos a tecnica de imuno-histoquimica para os anticorpos fibronectina e colageno 1, apos 18, 45, 90 e 174 dias de preservacao. Os especimens foram classificados de acordo com o paralelismo (PF:0-2) e integridade (IF:0-1) das fibras de colageno e expressao imuno-histoquimica para os anticorpos fibronectina (FIB:0-3) e colageno 1 (COL-1:0-3). A analise estatistica foi realizada por meio dos testes de Friedman e Wilcoxon e o valor de p menor que 0,05 foi considerado estatisticamente significante. RESULTADOS: Verificaram-se diferencas significantes em tres situacoes: (i) maior integridade das fibras de colageno das escleras liofilizadas apos 174 dias quando comparado aos 90 dias; (ii) maior expressao imuno-histoquimica para o anticorpo COL-1 nas amostras de escleras liofilizadas apos os 18 dias iniciais de preservacao; (iii) maior integridade das fibras de colageno das escleras liofilizadas apos 18 e 174 dias quando comparado as escleras preservadas em alcool. CONCLUSOES: A preservacao de tecido escleral por liofilizacao mostrou-se tecnica tao eficaz quanto a preservacao em alcool, apresentado vantagem quando considerada a integridade das fibras de colageno. A liofilizacao mostra-se benefica por permitir a estocagem do tecido em temperatura ambiente e com prazo de validade estendido.

Uveal melanoma (UM) is the most common malignant intraocular tumor in adults. Despite the high accuracy of clinical diagnosis and advances in local treatment, more than 50% of UM patients develop metastasis within 10 years of initial diagnosis. NM23 is one of the human metastasis suppressor genes. Reduced nm23-H1 expression is correlated with high metastatic potential in many different cancers. The purpose of this study is to investigate the expression of nm23-H1 in UM and its potential value as a prognostic marker. Immunostaining of nm23-H1 was verified in five human UM cell lines with different metastatic potentials. The expression level of nm23-H1 mRNA was evaluated with one-step quantitative real-time PCR. The invasion ability of the cell lines was assessed before and after silencing nm23-H1 with small interference RNA. Thirty-two cases of paraffin-embedded specimens of human UM were immunostained with nm23-H1 monoclonal antibody. The immunostaining was evaluated in a semiquantitative fashion based on extent and intensity. The real-time PCR results of five human UM cell lines showed that expression of nm23-H1 was higher in cell lines with low metastatic potential compared with those with high metastatic potential (P

OBJETIVO: Avaliar a possibilidade de identificação de células malignas circulantes nas amostras de sangue periférico de pacientes brasileiros com melanoma maligno de coróide enviadas para análise no exterior. MÉTODOS: Os marcadores melan-A e tirosinase foram usados para detectar a presença de células malignas circulantes, pela transcrição reversa seguida de reação em cadeia da polimerase e análise seqüencial de DNA (RT-nested-PCR), em seis pacientes com melanoma maligno de coróide, diagnosticados no Brasil. RESULTADOS: Cinco pacientes deste grupo (83,33%) foram considerados positivos. A reação de RT-nested-PCR foi positiva para melan-A em quatro (66,7%) e positiva para tirosinase em quatro (66,7%) dos seis pacientes testados. Três (50%) pacientes foram positivos para os dois marcadores. Um (16,7%) paciente foi negativo para ambos marcadores. CONCLUSÃO: A pesquisa de células malignas circulantes usando RT-nested-PCR, foi positiva na maioria dos pacientes estudados. A qualidade das amostras de sangue periférico dos pacientes brasileiros foi mantida no material preservado mesmo após ter sido enviado ao exterior.PURPOSE: The purpose of our study was to detect circulating malignant cells (CMCs) in oversea-shipped blood samples of patients with uveal melanoma diagnosed in Brazil. METHODOS: Melan-A and tyrosinase were the two markers used for the detection of CMCs, using reverse transcriptase nested polymerase chain reaction (RT-nested-PCR) in 6 patients with uveal melanoma. The expression of beta-actin and GAPDH were used to assess the quality of the material. RESULTS: Five patients (83.33%) tested positive for the presence of CMCs. The RT-nested-PCR was positive for melan-A in 4 patients (66.7%) and positive for tyrosinase in 4 (66.7%) of the 6 patients. Three (50%) patients were positive for both markers. One (16.7%) patient was negative for both markers. All negative controls were negative. CONCLUSION: The quality of the blood samples shipped overseas, from patients with uveal melanoma, was preserved. The detection of CMCs using RT-nested-PCR was positive in the majority of the patients.

C-kit is a transmembrane tyrosine kinase protein thought to play an important role in tumourigenesis. With the development of the compound imatinib mesylate, which specifically inhibits tyrosine kinase receptors, C-kit has emerged as a potential therapeutic target. This study aims to determine the immunoexpression of C-kit in retinoblastoma and correlate this expression with histopathological prognostic features.Eighty-four paraffin-embedded retinoblastomas were collected from the Henry C Witelson Ocular Pathology Registry. C-kit immunostaining was used according to the protocol provided by Ventana Medical System Inc., Arizona. Immunoreactivity was correlated with the presence or absence of invasion into the choroid and optic nerve and the degree of tumour differentiation. Odds ratios were calculated to quantify differences in C-kit expression between tumours with different patterns of invasion and differentiation.Twenty-one slides (25%) were excluded from analysis because of the presence of extensive tissue necrosis or the absence of sufficient optic nerve tissue for analysis. Overall, C-kit expression was identified in 33/63 specimens analysed (52.38%). Two of the 13 tumours without choroidal or optic nerve invasion (15.4%) were positive for C-kit. C-kit expression was seen in 31 of the 50 tumours with extraretinal invasion (62%, p0.01), 26 of 44 specimens with choroidal involvement (59.9%, p0.2), and 20 of the 29 with optic nerve involvement (68.96%, p0.02). Fourteen of 25 moderate or well-differentiated specimens (56%) and 19 of 38 undifferentiated specimens (50%) displayed positivity for C-kit (p0.5).More than half the retinoblastomas in this study expressed C-kit. The expression of C-kit strongly correlated with histopathological features of a worse prognosis including optic nerve and choroidal invasion.

Most uveal melanoma patients (UMP) do not show evidence of metastases upon diagnosis. However, despite local tumour control, approximately 50% of them will develop metastases. These findings suggest that malignant cells may have already disseminated by the time of initial diagnosis. The purpose of the study was to detect circulating malignant cells (CMCs) in UMP and to correlate them with prognostic factors and therapy.Nested reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect CMCs. In each UMP, blood was collected every 3 months. In each visit, 20 RT-PCR tests were performed. The date of diagnosis, largest tumour dimension, type, and date of treatment were obtained.A total of 30 UMP were enrolled. Five patients were enrolled at the time of diagnosis and 25 patients between 1 and 17 years following diagnosis. No UMP showed clinical evidence of metastasis. A total of 136 visits were registered, 1360 samples collected, and 2720 RT-PCRs performed. CMCs were identified in 29 patients in 119 visits (87.5%). However, in each visit, a low number of positive tests were recorded. CMCs were found in newly diagnosed, irradiated, enucleated, and observed patients regardless of tumour size and time period following treatment.Uveal melanoma (UM) is not a localized ocular disease. CMCs were recorded at initial diagnosis confirming the early metastatic nature of UM. CMCs were present following treatment, including enucleation, demonstrating that CMCs are capable of disseminating and surviving, possibly as micrometastasis, which would contribute to the pool of CMCs at a later stage. Systemic therapy should be evaluated.

Objective: The aim of this study was to establish a proliferation profile of uveal melanoma cell lines, using different methods, and to compare it with their previously determined metastatic potential (MP). Methods: Four human uveal melanoma and one transformed human uveal melanocytic cell line were ranked according to proliferation profiles. The proliferation profiles of the cell lines were compared to their MPs, which were previously determined from an immunosuppressed rabbit model. Results: Ranking of the cell lines using pulse labeling with tritiated thymidine was similar to the MP of the cell lines. Conclusion: The correlation between the proliferative rate of the uveal melanoma cell lines and their previously determined MP resulted in the proposal of a new classification scheme: high proliferation/high MP, low proliferation/low MP, and high proliferation/no MP. High proliferative capacity of a cell line did not necessarily confer MP; therefore, further cellular functions/adaptations must be required for tumor cell dissemination, survival, and growth at a metastatic site.

Chronic ocular inflammation can be due to a disease process or to iatrogenic factors that attract inflammatory cells to the anterior chamber. This study was conducted to investigate the effect of commonly used ophthalmic preparations on leukocyte migration.A modified multi-well Boyden chamber was used to study the chemotactic and chemokinetic effects of 33 commercial ophthalmic preparations. To determine whether the chemotactic effect was a property of the commercial ophthalmic preparation or other chemicals present in the products, experiments were also done with some common preservatives and excipients.Of the drugs, 14 (42.4%) showed chemokinetic and/or chemotactic activity and 19 (57.6%) had either no effect or decreased neutrophil migration. Of the preservatives and excipients, 5 (62.5%) were found to be chemotactic. Eleven of 14 chemotactic drugs (78.6%) and 8 of 19 non-chemotactic drugs (42.1%) were positive for at least one chemotactic excipient. The correlation between chemotactic ophthalmic preparations and the presence of a chemotactic excipient in their composition was significant (p0.05).Chemotactic activity was commonly found in commercial ophthalmic preparation. Furthermore, the presence of certain chemotactic preservatives and/or excipients was a contributing factor enhancing this property. Avoiding known chemotactic compounds or adjusting the intervals of the treatment may help to eliminate this iatrogenic component of the inflammatory process especially in patients with chronic ocular inflammation.

Familial amyloidosis of the Finnish type (FAF) is an autosomal dominant form of systemic amyloidosis showing marked geographic clustering in Finland. The disease is caused by a point mutation, 654G-A, in the gelsolin gene. The Danish-subtype of FAF has been previously described in three families, the patients present clinical findings similar to FAF, and the mutation 654G-T in the gelsolin gene. Three members from two generations of the same family, with familial amyloidosis, were screened for mutations in the GSN gene. Genomic DNA was extracted from peripheral blood lymphocytes and the polymerase chain reaction (PCR) was carried out under standard conditions, using appropriate primers. Sequence analysis showed the presence of a G to T transition at nucleotide 654 of the gelsolin gene. This is the first report of gelsolin-related familial amyloidosis in a Brazilian family, and the result is particularly significant as this pedigree presents an unusual mutation, described previously in three families, with no known Finnish ancestors (Danish type).

Aims To determine the expression of breast metastasis suppressor 1 (BRMS1) in human uveal melanoma (UM) tissues and cell lines. In addition, we intend to establish a possible association between BRMS1 expression and the presence of metastatic disease. Methods Thirty-one formalin-fixed paraffin-embedded tissues from enucleated eyes of patients with UM were immunostained. Clinical–pathological data were obtained, including age, tumour location, largest dimension, cell type, and occurrence of metastasis. The expression of BRMS1 mRNA in four human UM cell lines was determined by real-time reverse transcriptase polymerase chain reaction, and protein expression was assessed by immunocytochemistry and western blot. The association between BRMS1 immunostaining and location, largest tumour dimension, and tumour cell type was determined using the correlation coefficient test. The association between BRMS1 immunostaining and the incidence of metastasis was assessed using Kaplan–Meier analysis. Results Of the 31 cases of UM, 24 (77.42%) stained positive and seven (22.58%) negative for BRMS1. From the positively stained tumours, 21 (87.50%) showed cytoplasmatic staining. Macrophages were usually positive when present in the tumour and staining intensity was generally higher than in UM cells. BRMS1 mRNA was present in all four human UM cell lines, as well as cytoplasmatic immunoexpression of BRMS1. Immunoblotting showed variable BRMS1 protein levels between the different cell lines. No statistically significant correlation was found between BRMS1 protein expression and survival (P = 0.69), tumour cell type (P = 0.68), largest tumour dimension (P = 0.75), and tumour location (P = 0.11). Conclusions BRMS1 is expressed in UM both at the mRNA and protein level; however, neither was associated with any of the prognosticor outcome parameters that we tested.

PROBLEM: There is considerable controversy concerning the root cause and mechanisms of early embryo loss. It has been suggested that most pregnancy losses occur due to morphogenetic anomalies of the embryo. It has also been suggested that the maternal specific immune system rejects the embryo. METHODS: Existing data on the cell and molecular biology of early embryo loss in murine experimental models is reviewed. RESULTS: Using the CBA? x DBA/2? model of early embryo loss, it has been established that maternal inflammatory cells infiltrate the decidua basalis of all implantation sites within 48 hr after implantation. For most embryos, the relatively low numbers of macrophages (MΦ) and natural killer-like (NK-like) cells of maternal origin remain relatively constant after day 8, whereas 20-30% of the embryos show a significant increase in inflammatory cells in the maternal decidua, corresponding to the incidence of early embryo resorption visible at day 12. Evidence will be reviewed to suggest that decidual NK-like cells are not cytolytic but may be producing the MΦ-activating cytokine interferon gamma (IFN-γ), which activates decidual MΦ and other cells. Furthermore, embryo loss is ameliorated by in vivo treatment with anti-IFNγ or anti-NK antisera, indicating that NK-like cells and/or IFNγ are required for embryo loss, but not for embryo survival. In resorbing embryos, the inflammatory MΦ show evidence of having been primed during early pregnancy, in that in vitro incubation with lipopolysaccharide induced the production of tumor necrosis factor alpha and nitric oxide. CONCLUSION: These findings support the concept that early embryo loss is a nonspecific event mediated by the triggering of cytotoxin production by primed decidual macrophages.

The anterior chamber of the eye is known to be a site of immune privilege. Particularly, the aqueous humor (AqH) appears to possess unique immunoregulatory properties. The authors have previously shown that human AqH (HAqH) may increase or decrease the proliferation of different cell types. Although no single factor has been established as solely responsible for these effects, much attention has been given to the 24-30 kD fraction of AqH, where transforming growth factor-beta (TGF-β) is found. The purpose of this study was to determine the changes occurring in the rabbit AqH (RAqH) in relation to intraocular inflammation. Heterologous lens or human serum albumin (HSA) immunization-induced uveitis models were used in two groups of New Zealand albino rabbits to study the relationship between uveitis and TGF-β. AqH and serum samples were obtained serially before, during and after the induction of ocular inflammation. Systemic humoral immunity to HSA or lens antigens was monitored using enzyme-linked immunosorbent assay (ELISA). A mink lung epithelial cell (CCL-64) bioassay for TGF-β was used to quantify the amount of this cytokine in RAqH. TGF-β levels in RAqH increased fourfold after the first immunization. A sharp decrease in RAqH TGF-β levels was found in association with the development of acute intraocular inflammation. The implications of this finding to the etiology of uveitis are discussed.

Background/Aims: Nuclear receptor corepressor 1 (NCoR1) is a protein complex with diverse functions in development and tumorigenesis. We investigated the pattern of expression and histopathological correlation of NCoR1 in 41 retinoblastoma tumor samples, 1 retinoblastoma cell line (WERI-Rb-1) and human retinal progenitor cells (hRPCs). Methods: Tissue sections from 41 retinoblastoma cases, the retinoblastoma cell line WERI-Rb-1 and hRPCs were stained with rabbit polyclonal anti-NCoR1 H76 antibody. Percentage and intensity of staining were classified by an ocular pathologist from 0 to 3. The paired t test was used to test for differences. Results: In the nonneoplastic retina, NCoR1 was expressed mainly in cell nuclei. The retinoblastoma tumor cells similarly had nuclear NCoR1 but also had a higher level of cytoplasmic NCoR1 expression compared to all 3 normal retinal cell layers (p < 0.002). In contrast to the normal retina, NCoR1 was mainly expressed in the cytoplasm of the proliferating WERI-Rb-1 cells. This cytoplasmic expression pattern was also seen in the undifferentiated hRPCs. Conclusions: The aberrant cytoplasmic expression of NCoR1 in retinoblastoma appears to be associated with the proliferative and/or dedifferentiated properties of retinoblastoma. Further investigation into the role of NCoR1 in retinoblastoma may provide insight into how therapeutically inhibiting its nucleocytoplasmic shuttling may affect retinoblastoma tumor biology.

Sirtuin 1 (SIRT1) is a deacetylase that can regulate various biological processes via repression of transcription. Its activity has been linked to the differentiation of neural progenitor cells, although little is known about its function during retinal development. The study described herein was undertaken to evaluate the expression of SIRT1 and its innate inhibitor, DBC1, in retinal tissues and progenitor cells. We found both SIRT1 and DBC1 to be widely expressed in mouse and human retinas, with subtle differences in subcellular distribution of each protein. We further demonstrate that nuclear-localized SIRT1 is only seen in human-derived retinal progenitor cells and not in adult retinas, suggesting that this nuclear localization may be important in retinal development. Moreover, we observed cytoplasmic DBC1 in a subset of progenitor cells as well as in mature ganglion cells, indicating that the progenitor cell subset, which was comprised predominantly of small cells, may represent a population of ganglion cell precursors. Collectively, the data presented in this study provide support for SIRT1 and DBC1 as regulators of retinal development and normal retinal physiology.

BACKGROUND The class III histone deacetylase SIRT1 is overexpressed in many malignancies and has been implicated in inactivating proteins that are involved in tumor suppression and DNA damage repair. In the current study, we examined the expression of SIRT1 in normal epithelium (NE) compared with ocular surface squamous neoplasia (OSSN) to elucidate a possible role for SIRT1 in the development or progression of this malignancy. METHODS We examined SIRT1 expression by immunohistochemistry in 47 cases of OSSN and 10 specimens of NE. Our sample included 11 benign lesions (papillomas), 25 cases of conjunctival intraepithelial neoplasia, and 11 malignant lesions of squamous cell carcinoma. The extent of staining and intensity was scored and the combined raw data were then converted to the German Immunoreactive Score. RESULTS Nuclear and cytoplasmic expression of SIRT1 was observed in all cases of OSSN. For the NE specimens, 50% showed negative expression and 30% weak expression, and 20% were considered significantly immunoreactive. The differential expression of SIRT1 between NE and OSSN was statistically significant (P < 0.0001). Additionally, when the staining pattern in cases of conjunctival intraepithelial neoplasia was evaluated, the staining of the more differentiated surface cells was remarkably weaker compared with the cells located closer to the basal membrane. CONCLUSIONS SIRT1 may play an important role in the development and progression of epithelial tumors of the conjunctiva. Further research into the potential of SIRT1 as a novel therapeutic target is warranted.

The use of biological assays in research often creates complex Problems of data analysis. Due to the nature of bioassays which respond to the intricate interplay between multiple factors that may either increase or decrease the readout, simple arithmetic or standard linear regression analysis often fails to provide accurate values when extrapolation from a standard curve is employed. Non-linear polynomial analysis could provide a better way for expressing such complex relationships. Using a bioassay for transforming growth factor-beta (TGF-β) employing the mink lung epithelium cell line CCL-64, the authors present a new way to analyze experimental data based on non-linear polynomial statistics to provide accurate quantitative results when other simpler techniques have failed. This technique for data analysis provided accurate consistent results with acceptably small experimental variance. Using this approach, the bioactive concentration of TGF-β in normal rabbit aqueous humor was found to be 2.72 ± 0.61 ng/ml, that is, corresponding to 2. 72 ng of PURE TGF-β2, when referring to a standard curve.

The liver is a major site of metastasis for human malignancies, yet the factors that regulate tumor cell survival and growth in this organ remain elusive. Previously, we reported that M-27(IGF-IR) murine lung carcinoma cells with ectopic insulin-like growth factor-1 (IGF-I) receptor overexpression acquired a site-specific, liver-metastasizing potential. Gene expression profiling and subsequent RNA and protein analyses revealed that this was associated with major changes to the expression of extracellular matrix (ECM) protein-encoding genes including type III, IV and XVIII collagen genes, and these changes were also observed in the respective tumors in vivo. Because type IV collagen was the most prominently altered ECM protein in this model, we further analyzed its functional relevance to liver metastasis. M-27 cells stably overexpressing type IV collagen α1 and α2 chains were generated and their growth and metastatic properties investigated. We found that these cells acquired a site-selective growth advantage in the liver and this was associated with cell rescue from anoikis in a collagen IV/α2 integrin/FAK-dependent manner and increased responsiveness to IGF-I. Conversely, collagen IV or focal adhesion kinase (FAK) silencing by small-interfering RNA in highly metastatic tumor cells enhanced anoikis and decreased liver metastases formation. Moreover, analysis of human surgical specimens revealed uniformly high collagen IV expression in 65/65 hepatic metastases analyzed, regardless of tissue of origin, whereas it was variable and generally low in 50/50 primary colorectal carcinoma specimens examined. The results suggest that collagen IV-conveyed signals are essential cues for liver metastasis in diverse tumor types and identify mediators of collagen IV signaling as potential therapeutic targets in the management of hepatic metastases.

Lysyl oxidase is a marker of poor prognosis in several malignancies and is hypothesized to promote a migratory phenotype in hypoxic breast carcinomas. This study aims to characterize the expression of the lysyl oxidase and lysyl oxidase-like proteins in human uveal melanoma cell lines and archival choroidal melanomas using immunohistochemistry. The transcriptional control of lysyl oxidase will also be investigated under simulated hypoxic conditions using cobalt chloride. Lastly, changes in cellular proliferation and invasion will be assessed after the treatment of cell lines with beta-aminopropionitrile, a lysyl oxidase catalytic inhibitor. Retrospective analysis of lysyl oxidase expression in primary human uveal melanoma showed 82% (27 of 33) of tumors being stained positive. High lysyl oxidase expression correlated with the aggressive epithelioid cell type and was associated with shorter metastasis-free survival. Simulated hypoxia resulted in a significant increase in lysyl oxidase mRNA expression. Inhibiting lysyl oxidase's catalytic activity significantly reduced cellular invasion but had no effect on cell proliferation. Our study is the first to show lysyl oxidase expression in primary choroidal melanomas. This protein may represent a potential therapeutic target that warrants further study in this malignancy.

Secreted protein acidic and rich in cysteine (SPARC) has been shown to play an integral role in the progression of numerous malignancies. The aim of this study was to investigate the expression of SPARC in uveal melanoma (UM).SPARC expression was assessed in UM cell lines using RT-PCR and immunocytochemistry. Small interfering RNA directed against SPARC was used to transfect each of the cell lines, which were subsequently run in proliferation assays. SPARC expression was further investigated in 19 cases of human UM and 11 primary and 8 metastatic tumors from a rabbit xenograft model.The cell lines transfected with SPARC siRNA showed a significant decrease in proliferation compared to controls. All cases of human uveal melanoma demonstrated positive staining for SPARC as did all primary and metastatic tumors from the xenograft model.SPARC may represent a novel target to inhibit growth of UM.

Purpose: To examine the immunohistochemical profile of heat shock protein 90 (Hsp90) in uveal melanoma and the cytotoxicity of an Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in uveal melanoma cell lines. Experimental Design: Hsp90 expression was determined by immunohistochemistry in 44 paraffin-embedded sections of primary human uveal melanoma and in five uveal melanoma cell lines (92.1, OCM-1, MKT-BR, SP6.5, and UW-1). Sulforhodamine B–based proliferation assay was used to compare uveal melanoma cell growth with a range of concentrations of 17-AAG. Changes in cell migration, invasion, cell cycle fractions, and apoptotic activity were also evaluated. Expression of intracellular proteins was determined by Western blot analysis after 17-AAG exposure. Results: Immunohistochemical expression of Hsp90 was identified in 68% of the paraffin-embedded sections and significantly associated with largest tumor dimension (P = 0.03). 17-AAG significantly reduced the proliferation rates of uveal melanoma cell lines, with concentrations of 100 to 0.1 μmol/L. 17-AAG also significantly reduced the migratory and invasive capabilities of uveal melanoma cell lines. Cell cycle analysis showed that 17-AAG induced accumulations of cells in G1. Caspase-3 protease activity analysis, a marker for apoptosis, showed a significant increase after drug exposure. The cytotoxic effect of 17-AAG was associated with decreased levels of phosphorylated Akt and cyclin-dependent kinase 4. Conclusions: The immunohistochemical expression of Hsp90 in uveal melanoma indicates worse prognosis. To the best of our knowledge, this is the first report showing the inhibitory effect on uveal melanoma cells using 17-AAG to target Hsp90. Therefore, Hsp90 may be used as a potential target for treatment of patients with uveal melanoma.

Purpose: To examine the immunohistochemical profile of Hsp90 in uveal melanoma and the effects of Hsp90 inhibitor 17-AAG, in uveal melanoma cell lines. Methods: Hsp90 expression was studied in 44 sections of human uveal melanoma and in five uveal melanoma cell lines (92.1, OCM-1, MKTBR, SP6.5 and UW-1). Sulfurhodamine-B based proliferation assay was assessed with a range of concentrations of 17-AAG. Changes in cell migration and invasion were evaluated in vitro in the presence or absence of 17-AAG. Cell cycle fractions were determined by flow cytometry and the caspase 3 protease activity was detected using the BD ApoAlertTM Caspase Colorimetric Assay. Expression of intracellular proteins was determined by Western blot analysis. Results: Immunohistochemical expression of Hsp90 was identified in 68 % of the paraffin embedded sections and was significantly associated with largest tumor dimension. A statistically significant reduction on proliferation rate of uveal melanoma cell lines was observed with drug concentrations of 100 μM to 1 μM and also a reduction of their migratory and invasive capabilities. Exposure to 17-AAG induced accumulations of cells in G1 and loss of cells in S phase and also a significant increase in Caspase 3 protease activity. The cytotoxic effect of 17-AAG was associated with decreased levels of phospho-AKT after 24 h exposure to the drug. Conclusions: The immunohistochemical expression of Hsp90 in uveal melanoma indicates worse prognosis. Inhibition of HSP90 function by 17-AAG had an effect on cell motility and invasive potential, inducing cell cycle arrest and promoting apoptosis. Further trials in uveal melanoma models should be undertaken to study the effect of 17 AAG to target Hsp90 in vivo.

Purpose: We previouly demonstrated the feasibility of a human uveal melanoma GFP model in nude mice. We were able to monitor the preferential colonization of the liver by malignant cells during a 20-day period. In this study we extended the observation period in order to observe disease progression and metastatic growth. Methods: Five hundred thousand, GFP transfected, human primary uveal melanoma cells (92.1) were injected into the tail vein of 30 nude mice at time zero. Starting at day one, five mouse per week was imaged, using an abdominal incision to expose the liver, for up to 60 minutes before sacrifice. The liver was imaged in vivo and post mortem, while the lungs, spleen, and kidney were all examined post-mortem for GFP expression. Histopathological examination of all organs was performed to ensure that the GFP signal was arising from malignant cells. Results: In vivo imaging of the liver revealed positive GFP signal in all mice throughout the experiment. Malignant cells seeded the liver after injection, but there was no apparent tumor growth upon completion of the experiment. The micrometastatic foci remained viable without changes in size or intensity of the signal. No tumors were seen in any other organ at the end of the experiment. Histopathology confirmed that the cells emitting GFP were malignant cells. Conclusions: The results of this uveal melanoma model show great promise for understanding the previously unobservable interactions between single human uveal melanoma cells and native liver tissue. The fact that our cells successfully colonize the liver yet remain dormant for six weeks should actually mirror the disease in humans; metastases in patients takes roughly five to ten years to develop.

The expression of cyclooxygenase-2 (COX-2) has been reported as an indicator of poor prognosis in a wide variety of human tumors, including colon, breast and uveal melanoma (UM). COX-2 inhibitors have shown promise in controlling the malignancy of several types of tumors. Previous studies have demonstrated the efficacy of a COX-2 inhibitor on the proliferation rates of human UM cells. The goal of this experiment was to investigate the efficiency of Nepafenac, a topically administered COX-2 inhibitor, in a rabbit model of UM. The animals were divided into two groups of 14 animals for the duration of the 12-week experiment. One animal per group was killed each week to evaluate disease progression and for histopathological studies. The experimental group received drops containing 0.3% Nepafenac solution. Intraocular tumor growth was evaluated weekly by fundoscopic examination and each animal was weighed prior to examination. Blood samples were taken weekly from all rabbits to detect circulating malignant cells (CMCs) throughout the experiment. After the second week of inoculation, the experimental group weighed significantly more than the control group. The control group developed more intraocular tumors and presented with metastases and higher detectable levels of CMCs before the treated group. These results indicate that the topical administration of a COX-2 inhibitor delayed the progression of this malignancy in our animal model. A clinical trail using an anti-COX-2 inhibitor for patients with UM should be considered.

Background Melan-A and tyrosinase are new immunohistochemical markers that can be used in the diagnosis of melanocytic lesions. The aim of this study was to investigate the correlation between radiotherapy or clinicohistopathological parameters and the expression of melan-A and tyrosinase in uveal melanoma. Methods Thirty-six enucleated cases of uveal melanoma were studied. The formalin-fixed, paraffin-embedded specimens were immunostained with monoclonal antibodies against melan-A and tyrosinase. The samples were classified as either positive or negative. The chi-square or the Student-t tests were used to test for the correlation of the expression rates of melan-A and tyrosinase with clinico-pathological parameters. Results Melan-A and tyrosinase were positive in 33 (91.7%) and 35 (97.2%) of the specimens, respectively. There was no significant association between the expression of melan-A or tyrosinase and radiotherapy or any clinico-pathological parameter. All specimens were positive for at least one of the immunohistochemical markers. Conclusion To the best of our knowledge this is the first study concluding that the expression of melanocytic markers such as melan-A and tyrosinase is not influenced by radiotherapy or any clinico-pathological parameter. Moreover, when tyrosinase and melan-A are used together, 100% of the formalin-fixed, paraffin-embedded uveal melanoma samples tested positive for one of those markers.

The purpose of our study was to detect circulating malignant cells (CMCs) in oversea-shipped blood samples of patients with uveal melanoma diagnosed in Brazil. METHODOS: Melan-A and tyrosinase were the two markers used for the detection of CMCs, using reverse transcriptase nested polymerase chain reaction (RT-nested-PCR) in 6 patients with uveal melanoma. The expression of beta-actin and GAPDH were used to assess the quality of the material.Five patients (83.33%) tested positive for the presence of CMCs. The RT-nested-PCR was positive for melan-A in 4 patients (66.7%) and positive for tyrosinase in 4 (66.7%) of the 6 patients. Three (50%) patients were positive for both markers. One (16.7%) patient was negative for both markers. All negative controls were negative.The quality of the blood samples shipped overseas, from patients with uveal melanoma, was preserved. The detection of CMCs using RT-nested-PCR was positive in the majority of the patients.

To compare the chemotactic activity induced by polymethyl methacrylate (PMMA), acrylic, and silicone intraocular lens (IOL) materials, regardless of the surgical and host factors.The chemotactic effect of five different IOLs was studied using a modified multi-well Boyden chamber. Two different non-coated PMMA IOLs, a heparin-coated PMMA IOL, an acrylic IOL, and a silicone IOL were assessed. Each mean migration distance of the IOLs was compared with the others using Student's t test.All IOLs induced some amount of leukocyte chemotaxis. There was no statistically significant difference between the chemotactic activity of PMMA IOLs. However, chemotactic activity was significantly higher in acrylic (p0.05) and silicone (p0.05) IOLs compared to one of the PMMA lenses.This limited study has proven that chemotaxis assay is a useful tool to assess the biocompatibility of IOLs. The IOL material itself might attract inflammatory cells to its surface in the absence of surgical and host factors. As PMMA IOLs do not cause an increase in inflammatory reaction, they may be considered safe to use. Further improvement in chemical composition and surface characteristics of IOLs should reduce the inflammatory reaction and increase the biocompatibility and safety of IOLs.

Purpose To characterize, in detail, tumor development, malignant cell dissemination, and metastasis in a 10-week animal model of uveal melanoma. Methods One million 92.1 human primary uveal melanoma cells were injected into the suprachoroidal space of the right eye of 27 immunosuppressed albino rabbits. Intraocular tumor growth was monitored weekly by fundoscopy and by ultrasonography at the end of the experiment. To document the progression of the disease, one animal per week was killed. The enucleated eyes, lungs, and livers were macroscopically examined and histopathologically studied by hematoxylin and eosin, periodic acid-Schiff, and immunohistochemistry. Mononuclear layers isolated from the rabbits' blood samples were cultured. Results Histopathology showed intraocular tumors in 89% of the animals. Tumor growth was found 1 week after cell inoculation, and by the end of the experiment large tumor masses were observed. Microscopic pulmonary metastatic foci were first observed 4 weeks after cell injection. By the end of the experiment, all the animals had metastasis to the lungs. Interestingly, 18% of the animals also had micrometastasis to the liver. Viable adherent uveal melanoma cells were successfully isolated from peripheral blood and grown in vitro. Conclusions In this study, most rabbits developed intraocular tumors followed by lung metastasis, and some of these rabbits later developed liver micrometastases. This novel source of research material warrants a follow-up longer than 10 weeks to further explore the pathophysiologic bases of liver involvement commonly encountered in humans. The success in the isolation and culture of circulating malignant cells in this animal model suggests that it might be worthwhile to explore the application of this technique to the management of patients with primary uveal melanoma.

We have previously shown that both priming and triggering signals were needed for nitric oxide production by decidual macrophages and that nitric oxide was responsible for embryo wastage. In this study, we investigated the role of IFN-gamma as the primary signal for macrophage activation in early embryo loss. IFN-gamma-deficient (GKO) and heterozygous F1 control mice were injected with lipopolysaccharide (LPS) at day 7 of gestation. The results showed that the GKO mice were more resistant to LPS-induced embryo loss than the wild type. This suggested that IFN-gamma was needed for LPS-induced embryo resorption and that decidual macrophages from pregnant GKO mice were not primed and could not be activated when given LPS. Further, the results showed that IFN-gamma mRNA was simultaneously expressed in the same embryos that also expressed mRNA markers for macrophage activation (TNF-alpha and iNOS), indicating that macrophage activation could be a consequence of IFN-gamma production. Similarly, we investigated the role of IL-12 as a switch cytokine capable of eliciting TH1-associated cytokine production including IFN-gamma. The results showed that IL-12 mRNA expression was correlated with IFN-gamma expression and macrophage activation. In this in vivo study, we showed for the first time that spontaneously increased decidual IFN-gamma expression is detrimental to embryo survival.

AIMS--To predict 5 year survival in patients with uveal malignant melanomas DNA indices were studied. METHODS--Using 45 paraffin embedded uveal malignant melanomas, the DNA index and S phase fraction of each tumour were the predictor variables recorded. RESULTS--Using the Cox proportional hazards model, aneuploid tumours and tumours which had an S phase fraction greater than 4% were significant predictors of early death. In order to demonstrate a biological gradient between a larger DNA index and shorter survival time, linear regression and transformed linear regression models were used. However, no such gradient could be demonstrated. CONCLUSION--Although this study shows promise for the use of DNA studies in the prognosis of uveal malignant melanoma, the exact role of these techniques remains to be determined.

Purpose To provide insight into the relationship between cyclooxygenase-2 (COX-2) expression and histopathologic features of retinoblastoma specimens treated either by primary or secondary enucleation. Design Laboratory investigation. Methods Twenty-five retinoblastoma specimens received between 1994 and 2003 were retrieved for this study from the Ocular Pathology Registry, Federal University of Sao Paulo, Brazil and the Henry C. Witelson Ocular Pathology Laboratory and Registry, McGill University, Montreal, Canada. The specimens retrieved were divided into two groups: Group I, enucleation was performed as a form of primary treatment (n = 15) and Group II, enucleation after failure of conservative treatment (n = 10). Patient information and formalin-fixed, paraffin-embedded specimens were obtained. New sections of these blocks were used for hematoxylin and eosin stain and for immunoassay using a monoclonal mouse anti–COX-2 antibody. Two independent ophthalmic pathologists reviewed all of the microslides. Results Twenty-three specimens (92%) presented a high expression of COX-2 (15 in Group I; eight in Group II) and there was no statistical difference in COX-2 expression between the two groups ( P = .07). However, all specimens expressed COX-2 to a different degree. The areas of tumor invasion were positive for COX-2 in 87.5% of the two groups. Conclusion Most retinoblastoma specimens revealed a high COX-2 expression. Future studies will be necessary to correlate the high expression of COX-2 in retinoblastoma and a possible applicability of anti–COX-2 medications in the treatment of these tumors.

Choroidal neovascular membrane (CNVM) is one of the leading causes of severe visual loss and is often associated with age-related macular degeneration (AMD). Various modalities of treatment, including photocoagulation and surgery, are being considered as options, but with limited success. Prostate-specific membrane antigen (PSMA) is a type II membrane glycoprotein expressed in benign and malignant prostatic tissues, in some non-prostatic tissues, and in the endothelium of tumor-associated neovasculature of non-prostatic neoplasm. Some studies have suggested that the expression of PSMA is restricted to endothelium from tumor-associated neovasculature and might be stimulated by some tumor-secreted angiogenic factors. However, no previous study demonstrating PSMA expression in non-related tumor neovasculature, such as CNVM, has been performed to date. Furthermore, demonstration of PSMA expression in CNVM in AMD patients could reveal a novel target for antineovascular therapy. The purpose of this study was to evaluate the immunohistochemical expression of PSMA in CNVM from AMD.

Background To examine the immunohistochemical expression of heat shock protein 90 (Hsp90) and Ki-67 protein in human pterygium. Materials and methods Tissues obtained during pterygium surgery of 15 patients who underwent the bare-sclera procedure and 10 normal conjunctivae were studied. All of these pterygia were primary ones. Recurrent pterygia were excluded. Normal bulbar conjunctivas (2 x 2 mm) were obtained from the nasal region close to the limbus from patients during their cataract and retina surgeries. Immunohistochemical detection of Hsp90 and Ki67 was done using the streptavidin–biotin method in paraffin embedded tissue sections. Results The percentage of cells stained for Hsp90 was greater for pterygium epithelium (76 ± 10.8) than for normal conjunctiva (1.4 ± 0.8). In each pterigyum sample more than 60% of cells were positive. The differences in positive cells between normal and pterigyum epithelium were highly significant for Hsp90 (P < 0,001). Pterygium epithelium also showed a higher percentage of cells that stained for Ki67 (10.1 ± 9.5) than for normal conjunctiva (2.1 ± 1.9). The differences in positive cells were also statistically significant for Ki67 (P < 0.01). Although there were significant differences in the majority of samples observed. It was noted that in some samples there was no difference between normal and pterygium epithelium for Ki67. Conclusion Our results indicate an abnormal expression of Hsp90 and ki-67 in pterygium samples when compared to normal conjunctiva.The finding of abnormal expression of levels of Hsp90 in pterygium samples can stimulate new research into pterygium and its recurrence. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1128478792898812