The short-term effects of prescribed burning on biomass removal and the release of nitrogen and phosphorus in a treatment wetland.

Nutrient removal by constructed wetlands can decline over time due to the accumulation of organic matter. A prescribed burn is one of many management strategies used to remove detritus in macrophyte-dominated systems. We quantified the short-term effects on effluent water quality and the amount of aboveground detritus removed from a prescribed burn event. Surface water outflow concentrations were approximately three times higher for P and 1.5 times higher for total Kjeldhal nitrogen (TKN) following the burn event when compared to the control. The length of time over which the fire effect was significant (P < 0.05), 3 d for TKN and up to 23 d for P fractions. Over time, the concentration of soluble reactive phosphorus (SRP) in the effluent decreased, but was compensated with increases in dissolved organic phosphorus (DOP) and particulate phosphorus (PP), such that net total P remained the same. Total aboveground biomass decreased by 68.5% as a result of the burn, however, much of the live vegetation was converted to standing dead material. These results demonstrate that a prescribed burn can significantly decrease the amount of senescent organic matter in a constructed wetland. However, short-term nutrient releases following the burn could increase effluent nutrient concentrations. Therefore, management strategies should include hydraulically isolating the burned area immediately following the burn event to prevent nutrient export.

Increased allergen concentration enhances IFN-gamma production by allergic donor T cells expressing a peripheral tissue trafficking phenotype.

BACKGROUND: Clinically effective allergen-specific immunotherapy correlates with decreased circulating allergen-specific IL-4+ T cells but increased IFN-gamma+ cells at sites of allergen challenge. Whether immunotherapy promotes trafficking of IFN-gamma+ T cells to peripheral tissues is unknown. As aeroallergen is administered at higher concentrations during immunotherapy than those encountered naturally, the effect of allergen concentration on adhesion molecule (CD62L and CD49d) and chemokine receptor (CCR3 and CCR5) expression by peripheral-blood T cells was analysed in parallel with cytokine production. METHODS: House dust mite-allergic donor peripheral blood mononuclear cells were cultured for 14 days with different allergen concentrations. Cytokine profiles of were analysed by flow cytometry. RESULTS: Cultures stimulated with 100 microg/ml house dust mite extract compared with 1 microg/ml had increased proportions and numbers of CD62Llo, CD49dhi or CCR5+ T cells expressing IFN-gamma. CCR3-positive CD4+ and CD8+ T cell numbers were very low and did not differ between cultures. In contrast the proportions of 'peripheral tissue trafficking' CD4+ T cells expressing IL-4 were decreased in cultures stimulated with high in comparison with low allergen concentration. CONCLUSION: These results indicate the importance of achieving high allergen doses during immunotherapy to promote IFN-gamma production and expression of a 'peripheral tissue trafficking' phenotype by allergen-specific CD4+ and CD8+ T cells. The net change in cytokine milieu at sites of allergen encounter would then down-regulate clinical manifestations of allergic disease.

Induction of T 'regulatory' cells by standardized house dust mite immunotherapy: an increase in CD4+ CD25+ interleukin-10+ T cells expressing peripheral tissue trafficking markers.

BACKGROUND: Clinically effective subcutaneous allergen-specific immunotherapy (SIT) is associated with altered circulating T cell cytokine production and altered local cytokine responses with increased IL-10 following allergen challenge in target organs. OBJECTIVE: This study aimed to elucidate mechanisms for these T cell changes, by examining surface expression of markers for peripheral tissue trafficking on circulating cytokine-positive T cells following standardized house dust mite- (HDM-) SIT. METHODS: A randomized conventional HDM immunotherapy study was performed on a panel of 12 HDM-allergic subjects. Nine subjects received treatment with conventional HDM immunotherapy using a standardized extract and three subjects were treated by standard pharmacotherapy alone. Symptom and medication scores and allergen-induced cutaneous late-phase responses were assessed before and 9 months after institution of therapy. Before and at 3 and 9 months of SIT, peripheral blood mononuclear cells were cultured for 14 days with HDM extract and CD4+ and CD8+ T cell expression of CD62L, CD49d and CCR5 and production of IL-10, IFN-gamma and IL-4 were analysed by flow cytometry. Allergen-specific T cell proliferation was assessed by 3H-thymidine incorporation. RESULTS: At 9 months, all SIT-treated patients showed reduced symptom scores and late-phase cutaneous responses to HDM compared with baseline levels. The proportions of CD4+ T cells which were IL-10+ were increased (P < 0.01), and the proportions of CD4+ and CD8+ T cells which were IL-4+ decreased (P < 0.05) compared with baseline. CD4+ and CD8+ T cell IFN-gamma production, expression of surface markers for peripheral tissue trafficking and allergen-specific proliferation remained unchanged during SIT treatment. However, increased proportions of CD4+CD62L(-), CD4+CD49d(hi), CD4+CCR5+ T cells expressing IL-10 were detected at 9 months of SIT compared with baseline (P < 0.05). IL-10 staining co-localized with CD4+CD25+ T cells. CONCLUSION: Clinically effective subcutaneous immunotherapy with a standardized HDM Dermatophagoides pteronyssinus preparation results in decreased numbers of IL-4+ T cells and expansion of CD4+IL-10+ T cells expressing a peripheral tissue trafficking phenotype. The co-localization of IL-10+ staining to CD4+CD25+ T cells is consistent with the induction of a T regulatory cell population by SIT.

The hevein domain of the major latex-glove allergen Hev b 6.01 contains dominant T cell reactive sites.

BACKGROUND: Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupational asthma and rhinitis affecting frequent latex-glove users. Hev b 6.01, a known major latex allergen, is cleaved naturally into hevein (4.7 kDa) and a C-terminal fragment (14 kDa). Hevein is an abundant protein in latex-glove extracts. As the immune response to allergens is initiated by activation of allergen-specific CD4(+) T cells, identification of dominant T cell epitopes is crucial for the development of specific immunotherapy. OBJECTIVE: To identify dominant T cell epitopes of Hev b 6.01 in latex-allergic glove users. METHODS: Ten latex-allergic frequent glove users and six non-latex-allergic atopic control subjects were selected, based on clinical symptoms and positive latex-specific serum IgE. Serum IgE reactivity to glove extract and recombinant Hev b 6.01 (rHev b 6.01) were analysed by ELISA. Latex-specific short-term oligoclonal T cell lines were generated from peripheral blood of latex-allergic subjects. These lines were tested for proliferative responses to overlapping 20-mer peptides of the Hev b 6.01 molecule. CD4(+) T cell intracellular cytokines, IL-4 and IFN-gamma were assessed following stimulation with immobilized anti-CD3 in the presence of IL-2. RESULTS: All ten of the latex-allergic patients showed serum IgE binding to glove extract while eight of these also showed IgE binding to rHev b 6.01 by ELISA. Western blotting confirmed reactivity with rHev b 6.01 at around 20 kDa. T cell proliferation assays showed that latex-specific T cell lines from all subjects responded to one or more peptides, with greatest frequency of reactivity to peptides Hev b 6.01 p(10-29) and Hev b 6.01 p(19-38) in the hevein domain. An allergic-type cytokine profile with considerable IL-4 in addition to IFN-gamma was evident from intracellular cytokine staining. CONCLUSION: Hevein is an important T cell as well as B cell immunogen and contains dominant T cell reactive sites.

The tumour suppressor gene maspin is differentially regulated in cytotrophoblasts during human placental development.

Identification of factors that play a role in regulating the highly invasive ability of human placental cells throughout gestation will contribute to a better understanding of this unique developmental process. The aims of this study were to determine whether the tumour suppressor gene maspin is present in the human placenta and plays a putative role in the regulation of cytotrophoblast invasion during placental development. The data showed that the expression of maspin mRNA was maximum in term placentae compared to the first and second trimester tissues, and absent in the HTR-SVneo (immortalized extravillous cytotrophoblast), JEG-3 and JAR (choriocarcinoma) cell lines. Maspin protein, detected by Western blot analysis, was twofold higher in the second trimester and 4.4-fold higher in the third trimester compared to the first trimester. Maspin immunohistochemical staining was localized in cytotrophoblasts with increased and more diffuse staining in the second and third trimesters. Corresponding to the period of maximum maspin expression, cytotrophoblasts isolated from term placentae had significantly lower invasive ability as compared to first and second trimester cytotrophoblasts (P< 0.03). Further, addition of recombinant maspin significantly decreased cytotrophoblast invasion in vitro by 40-50 per cent in all three trimesters of gestation. This study provides the first evidence of the temporal expression of maspin during human gestation and suggests a putative role for maspin in regulating the invasive activity of cytotrophoblasts at term. The down-regulation of maspin expression may be critical at the time of implantation and early placental development, whereas upregulation of maspin may serve as a signal for the end of cytotrophoblast invasion and gestation.

Regulation of human cytotrophoblast morphogenesis by hepatocyte growth factor/scatter factor.

In vitro morphogenesis of epithelial cells to form tube-like structures is regulated by hepatocyte growth factor-scatter factor (HGF/SF). The placenta is a rich source of HGF/SF, and its absence in mice has been shown to lead to impaired placental growth and embryonic death. There is no information in the literature regarding in vitro morphogenesis of human cytotrophoblasts or the effect of HGF/SF on this process. In this study, cytotrophoblasts were isolated from human placentae obtained from all three trimesters of gestation and cultured on the recombinant basement membrane matrix (Matrigel). Under these conditions, cytotrophoblasts participated in morphogenetic events including formation of spheroid-like structures, radial linear processes with branching, and invaded Matrigel and formed large, tube-like structures. The presence of a developing lumen was documented in the linear projections arising from spheroids and in the tube-like structures by both confocal and transmission electron microscopy. Immunohistochemistry was used to characterize the phenotype of the cells, and staining with anti-cytokeratin and anti-E-cadherin antibodies confirmed the presence of cytotrophoblasts in both the spheroids and tube-like structures. Recombinant HGF (rHGF) significantly increased the invasive activity of cytotrophoblasts isolated from the first and second (P < 0.001) and third trimesters (P < 0.01). In addition, rHGF significantly increased the percentage of spheroids with branching processes in the first and second trimesters (P < 0.05). Anti-HGF antibody inhibited both these effects in a dose-dependent manner, indicating the specificity of the above findings. This study provides new evidence indicating that HGF/SF regulates invasion and branching morphogenesis of cytotrophoblasts throughout gestation, with maximum effects in the first and second trimester. These findings may help to elucidate the importance of the reduced expression of HGF/SF identified in placentae from women with preeclampsia or intrauterine growth restriction and suggest that HGF/SF may serve as an important candidate in therapeutic intervention strategies.

Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.

Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.

Macular pigment and lutein supplementation in retinitis pigmentosa and Usher syndrome.

PURPOSE: To determine macular pigment (MP) in patients with inherited retinal degeneration and the response of MP and vision to supplementation of lutein. METHODS: Patients with retinitis pigmentosa (RP) or Usher syndrome and normal subjects had MP optical density profiles measured with heterochromatic flicker photometry. Serum carotenoids, visual acuity, foveal sensitivity, and retinal thickness (by optical coherence tomography [OCT]) were quantified. The effects on MP and central vision of 6 months of lutein supplementation at 20 mg/d were determined. RESULTS: MP density in the patients as a group did not differ from normal. Among patients with lower MP, there was a higher percentage of females, smokers, and light-colored irides. Disease expression tended to be more severe in patients with lower MP. Inner retinal thickness by OCT correlated positively with MP density in the patients. After supplementation, all participants showed an increase in serum lutein. Only approximately half the patients showed a statistically significant increase in MP. Retinal nonresponders had slightly greater disease severity but were otherwise not distinguishable from responders. Central vision was unchanged after supplementation. CONCLUSIONS: Factors previously associated with lower or higher MP density in normal subjects showed similar associations in RP and Usher syndrome. In addition, MP in patients may be affected by stage of retinal disease, especially that leading to abnormal foveal architecture. MP could be augmented by supplemental lutein in many but not all patients. There was no change in central vision after 6 months of lutein supplementation, but long-term influences on the natural history of these retinal degenerations require further study.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry.

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which "mimics" embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Molecular regulation of tumor cell vasculogenic mimicry by tyrosine phosphorylation: role of epithelial cell kinase (Eck/EphA2).

During embryogenesis, blood vessels are formed initially by the process of vasculogenesis, the in situ differentiation of mesenchymal cells into endothelial cells, which form a primitive, patterned vasculogenic network. This is followed by angiogenesis, the sprouting of new vessels from preexisting vasculature, to yield a more refined microcirculation. However, we and our collaborators have recently described a process termed "vasculogenic mimicry," which consists of the formation of patterned, tubular networks by aggressive melanoma tumor cells (in three-dimensional cultures in vitro), that mimics endothelial-formed vasculogenic networks and correlates with poor clinical prognosis in patients. Previous microarray analysis from our laboratory comparing the highly aggressive versus the poorly aggressive melanoma cells revealed a significant increased expression of tyrosine kinases associated with the aggressive melanoma phenotype. Because of the important role of protein tyrosine kinases in phosphorylating various signal transduction proteins that are critical for many cellular processes (e.g., cell adhesion, migration, and invasion), we examined whether protein tyrosine kinases are involved in melanoma vasculogenic mimicry. Immunofluorescence analysis of aggressive melanoma cells forming tubular networks in vitro showed that tyrosine phosphorylation activity colocalized specifically within areas of tubular network formation. A phosphotyrosine profile of the aggressive melanoma cells capable of forming tubular networks indicated differences in tyrosine phosphorylated proteins compared with the poorly aggressive melanoma cells (incapable of forming tubular networks). Most notably, we identified epithelial cell kinase (EphA2) as being one receptor tyrosine kinase expressed and phosphorylated exclusively in the aggressive metastatic melanoma cells. Furthermore, general inhibitors of protein tyrosine kinases hindered tube formation, and transient knockout of EphA2 abrogated the ability of tumor cells to form tubular structures. These results suggest that protein tyrosine kinases, particularly EphA2, are involved in the formation of tubular networks by aggressive melanoma tumor cells in vitro, which may represent a novel therapeutic target for further clinical investigation.

Molecular determinants of ovarian cancer plasticity.

During development, the formation and remodeling of primary vascular networks occurs by vasculogenesis and angiogenesis. Recently, the term "vasculogenic mimicry" has been used by our laboratory and collaborators to reflect the embryonic-like ability of aggressive, but not nonaggressive, melanoma tumor cells to form a pattern of matrix-rich networks (containing channels) surrounding spheroids of tumor cells in three-dimensional culture, concomitant with their expression of vascular cell markers. Ovarian cancer is usually diagnosed as advanced stage disease in most patients when widespread metastases have already been established within the peritoneal cavity. In this study, we explored whether invasive ovarian carcinoma cells could engage in molecular vasculogenic mimicry reflected by their plasticity, compared with their normal cell counterparts. The data revealed that the invasive ovarian cancer cells, but not normal ovarian surface epithelial cells, formed patterned networks containing solid and hollow matrix channels when grown in three-dimensional cultures containing Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. Immunohistochemical analysis showed that matrix metalloproteinases (MMP)-1, -2, and -9, and MT1-MMP were discretely localized to these networks, and the formation of the networks was inhibited by treatment with MMP inhibitors. Furthermore, the RNase protection assay revealed the expression of multiple vascular cell-associated markers by the invasive ovarian cancer cells. In patient tumor sections from high-stage, high-grade ovarian cancers, 7 to 10% of channels containing red blood cells were lined by tumor cells. By comparison, all vascular areas in benign tumors and low-stage cancers were endothelial lined. These results may offer new insights and molecular markers for consideration in ovarian cancer diagnosis and treatment strategies based on molecular vascular mimicry by aggressive tumor cells.

Partial cytochrome b sequences for six Hymenoptera of the eastern United States.

Mitochondrial DNA (mtDNA) haplotypes have been commonly used to determine honeybee subspecies relationships. To see if these markers would also be useful for comparisons of other Hymenoptera, we collected workers of six local species: Vespa crabro, the European hornet; Bombus impatiens, a bumblebee; Vespula germanica, the German yellow jacket; Polistes fuscatus, a paper wasp; Halictus ligatus, an alkali bee; and an unspecified Megachile, a leafcutting bee. MtDNA was isolated and digested with six endonucleases (AvaI, BglII, EcoRI, HindIII, HinfI, XbaI). The digested DNA was electrophoresed and visualized on agarose gels with comparison to a standard fragment marker and similarly treated honeybee mtDNA. The fragments obtained were also purified and sequenced. Phylogenetic relationships between six wasp and bee species, Apis mellifera, and several other similar aculeate Hymenoptera were determined. Newly defined DNA sequences were posted to GenBank (AF281169-AF281174).

Down-regulation of HP1Hsalpha expression is associated with the metastatic phenotype in breast cancer.

We previously identified a down-regulation in heterochromatin-associated protein 1 (HP1)Hsalpha expression in MDA-MB-231 breast carcinoma cells (highly invasive/metastatic) compared with MCF-7 cells (poorly invasive/nonmetastatic). In this study, we demonstrate that HP1Hsalpha, but not HP1Hsbeta or HP1Hsgamma, is down-regulated at the mRNA and protein levels in highly invasive/metastatic breast cancer cell lines. In agreement, little to no nuclear HP1Hsalpha staining was observed in these cell lines. In contrast, poorly invasive/nonmetastatic cell lines showed HP1Hsalpha localization to the nucleus and nuclear membrane. Transfection of MDA-MB-231 cells with a green fluorescent protein-HP1Hsalpha expression vector decreased their ability to invade a collagen IV/laminin/gelatin matrix compared with green fluorescent protein-transfected controls. Consistent with the cell culture studies, immunohistochemical analysis of HP1Hsalpha protein localization in distant metastatic tissues from breast cancer patients revealed a decrease in the staining intensity and percentage of cells expressing HP1Hsalpha in seven of nine distant metastatic lesions compared with normal mammary and primary tumors. These results demonstrate a role for HP1Hsalpha in breast cancer invasion and metastasis. Given the role of HP1 in transcriptional silencing in Drosophila, we propose a model in which HP1Hsalpha normally silences genes involved in breast cancer invasion and metastasis.

Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry.

Tissue sections from aggressive human intraocular (uveal) and metastatic cutaneous melanomas generally lack evidence of significant necrosis and contain patterned networks of interconnected loops of extracellular matrix. The matrix that forms these loops or networks may be solid or hollow. Red blood cells have been detected within the hollow channel components of this patterned matrix histologically, and these vascular channel networks have been detected in human tumors angiographically. Endothelial cells were not identified within these matrix-embedded channels by light microscopy, by transmission electron microscopy, or by using an immunohistochemical panel of endothelial cell markers (Factor VIII-related antigen, Ulex, CD31, CD34, and KDR[Flk-1]). Highly invasive primary and metastatic human melanoma cells formed patterned solid and hollow matrix channels (seen in tissue sections of aggressive primary and metastatic human melanomas) in three-dimensional cultures containing Matrigel or dilute Type I collagen, without endothelial cells or fibroblasts. These tumor cell-generated patterned channels conducted dye, highlighting looping patterns visualized angiographically in human tumors. Neither normal melanocytes nor poorly invasive melanoma cells generated these patterned channels in vitro under identical culture conditions, even after the addition of conditioned medium from metastatic pattern-forming melanoma cells, soluble growth factors, or regimes of hypoxia. Highly invasive and metastatic human melanoma cells, but not poorly invasive melanoma cells, contracted and remodeled floating hydrated gels, providing a biomechanical explanation for the generation of microvessels in vitro. cDNA microarray analysis of highly invasive versus poorly invasive melanoma tumor cells confirmed a genetic reversion to a pluripotent embryonic-like genotype in the highly aggressive melanoma cells. These observations strongly suggest that aggressive melanoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis.

New technique for the cytologic identification of presumed Acanthamoeba from corneal epithelial scrapings.

PURPOSE: To describe a cytologic technique for the rapid identification of presumed Acanthamoeba organisms from corneal epithelial scrapings. METHODS: After administering topical anesthesia, we removed the affected corneal epithelium with a scalpel blade. The tip of the blade, containing the scrapings, was washed off into a cuvette with a solution of an alcohol-based fixative for cytology specimens. The blade was immersed in the cuvette and agitated to ensure that the sample was collected. The specimen was fixed for at least 10 minutes and processed by cytospin centrifugation. RESULT: Seventy-five patient samples have been studied with this technique, with excellent preservation of the organism. CONCLUSIONS: The organism preservation with this technique is superior to that of conventional smears and permits confirmatory organism identification by immunohistochemistry.

Distribution of prognostically important vascular patterns across multiple levels in ciliary body and choroidal melanomas.

PURPOSE: To investigate the validity of assigning patients whose eyes have been removed for ciliary body or choroidal melanoma to risk groups for metastasis based on the identification of microcirculatory patterns in one cross-section taken from the center of the tumor. METHODS: Multiple levels were cut through the blocks of 15 ciliary body or choroidal melanomas until the tumor was exhausted. Each level was examined for the presence of microvascular networks and parallel vessels with cross-linking histologic features strongly associated with death from metastatic melanoma. RESULTS: The central histologic section did not contain either microvascular networks or parallel vessels with cross-linking in eight tumors, nor were these patterns encountered in any of the more peripheral levels of the tumor. Seven tumors contained at least one focus of either microvascular networks or parallel vessels with cross-linking in the central histologic section. In two tumors, at least one of these patterns appeared in all histologic levels; in five tumors, at least one of these patterns appeared through multiple levels until just before the tumor was exhausted from the block (0.24 to 0.85 mm from the edge of the tumor). CONCLUSIONS: This study suggests that the prognostic classification of uveal melanoma based on the histologic profile of the microcirculation may be consistent throughout the tumor depth.

Regulation of uveal melanoma interconverted phenotype by hepatocyte growth factor/scatter factor (HGF/SF).

Human uveal melanoma disseminates initially and preferentially to the liver. This study describes the relationship between the expression of the c-met proto-oncogene (receptor for hepatocyte growth factor/scatter factor (HGF/SF)) in interconverted uveal melanoma cells (co-expressing vimentin and keratin intermediate filaments) and the regulation of their motogenic response to HGF/SF, a key step in local invasion and targeted dissemination to the liver. Expression of c-met in uveal melanoma cell lines correlates with both the appearance of an interconverted phenotype and invasive ability (measured in vitro). Using chemotactic checkerboard analysis, the greatest motogenic response to HGF/SF was achieved by invasive, interconverted, c-met-positive uveal melanoma cells. C-met was observed histologically in a uveal melanoma containing interconverted cells but was absent in a tumor composed of non-interconverted cells (vimentin positive/keratin negative). The c-met ligand, HGF/SF, although not expressed by uveal melanoma cell lines, was localized in tissue sections of primary uveal melanomas and metastatic melanoma to the liver. In the primary tumor, staining for HGF/SF was most intense at the level of the choriocapillaris, a finding that is significant because 1) highly remodeled neovascular loops and networks, which appear in tumors likely to disseminate, can be traced to the choriocapillaris and the draining vortex veins and 2) HGF/SF plays a role in tumor angiogenesis. Foci of metastatic melanoma to the liver stain diffusely for HGF/SF. Regulation of the uveal melanoma interconverted phenotype by HGF/SF may play an important role in the dissemination of this tumor.

Biologic determinants of uveal melanoma metastatic phenotype: role of intermediate filaments as predictive markers.

The long-range goal of our research is to develop intervention strategies based on newly discovered biologic mechanisms responsible for the invasive dissemination of metastatic uveal melanoma. To accomplish this goal, we have focused on the biologic relevance of novel marker proteins contributing to the uveal melanoma metastatic phenotype. The expression of vimentin intermediate filaments (IFs), a mesenchymal marker, is typical of melanomas, whereas carcinomas typically express keratin IFs, which are markers for epithelia. Thus, cells that coexpress both IFs are regarded as "interconverted" in that they display both mesenchymal and epithelial phenotypes. Although the biologic functions of IFs have remained enigmatic, there is substantial support to suggest that the significance of vimentin/keratin coexpression is linked with poor patient outcome in cutaneous melanoma. Our data demonstrate that human uveal melanoma cell lines (isolated from primary choroidal or ciliary body melanomas and from foci of metastatic uveal melanoma to the liver), which contain predominant populations of cells that coexpress vimentin/keratins 8 and 18 (keratins 8,18) IFs, were 6-fold more invasive through collagenous extracellular matrices in vitro, compared with uveal melanoma cells expressing vimentin only, and were 8- to 13-fold more invasive than normal uveal melanocytes. Colocalization of vimentin/keratins 8,18 in cell cultures was corroborated by immunohistochemistry in histologic sections of tumors from which the cell lines were derived. Minor populations of these cells also coexpressed keratins 13 and 17. Experimental down-regulation of the predominant keratins 8,18 in the interconverted cells, using 16-mer antisense oligonucleotides, resulted in a significant decrease in the migratory ability of the cells-similar to levels achieved by cells positive only for vimentin. These findings provide justification for additional studies of the association between coexpression of IFs vimentin/keratins 8,18 and uveal melanoma metastasis.

Imaging the microvasculature of choroidal melanomas with confocal indocyanine green scanning laser ophthalmoscopy.

OBJECTIVE: To image the microvasculature of choroidal melanoma with a new confocal scanning laser ophthalmoscope. METHODS: Eighteen consecutive patients, each with a unilateral choroidal melanoma, were examined prospectively. Indocyanine green angiography was performed with a new confocal scanning laser ophthalmoscope that enabled serial optical sectioning through the tumor. Two additional patients were studied with indocyanine green angiography and confocal scanning laser ophthalmoscopy just before enucleation for posterior choroidal melanomas. The histologic identification of microvasculature patterns was compared with the angiograms for these patients. RESULTS: In the series of 18 patients, 16 (89%) indocyanine green angiograms with optical sectioning revealed tubular structures within the melanoma that were identified as tumor vessels based on their angiographic appearance. The microvasculature patterns identified by indocyanine green angiography correlated well with the histologic appearance of these microvasculature patterns in both patients for whom histologic verification was available. CONCLUSIONS: This preliminary study suggests that indocyanine green angiography with confocal scanning laser ophthalmoscopy images the microvasculature of choroidal melanomas and may be capable of detecting microvasculature patterns that have been shown to be prognostically significant from histopathological studies.

The microcirculation of choroidal and ciliary body melanomas.

The microcirculation of ciliary body and choroidal melanomas is remodelled into patterns. The presence of microvascular networks, composed of back-to-back loops that encircle microdomains of tumour, and parallel vessels with cross-linking, are associated with death from metastatic melanoma. The formation of these complex vascular patterns may result from reciprocal interactions between the tumour cell and the extracellular matrix, and pattern formation may reflect an invasive tumour cell phenotype. Ciliary body and choroidal melanomas are among the few forms of cancer treated before a pathologist assigns a grade to indicate whether tumour is likely to follow a benign or aggressive course. There is evidence to suggest that prognostically significant microcirculatory patterns may be detectable by non-invasive imaging techniques that may provide a substitute for biopsy to guide the clinical management of patients with these sight- and life-threatening tumours.