Immunology of ocular tumours.
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Eye Neoplasms↗
Biomedical subjects
M J Jager
Publications and source records attributed to M J Jager.
Frequent loss of heterozygosity on chromosome 6p in uveal melanoma.
Lack of expression of HLA class I antigens is frequently observed on primary uveal melanoma, and is correlated with improved patient survival. Several mechanisms may contribute to the observed loss of HLA class I expression, including changes at the DNA level. In this study, we used microsatellite analysis as a molecular genetic approach to examine loci on chromosome 6p for loss of heterozygosity (LOH). Three pairs of microsatellite markers were used to screen 20 formalin-fixed, paraffin-embedded uveal melanomas for LOH on the short arm of chromosome 6. In all cases, normal adjacent scleral tissue was used as a control. We identified LOH in eleven cases from microsatellite locus D6S105 to the telomere, in eight cases from microsatellite locus D6STNFa to the telomere (area includes D6S105), and in seven cases from microsatellite locus D6S291 to the end of chromosome 6p (includes D6STNFa and D6S105). In seven cases, retention of heterozygosity was found at all three loci using these primers. Our results suggest that loss of heterozygosity on chromosome 6p is a common feature in uveal melanoma. We did not find a correlation between the presence of LOH and locus-specific HLA-A and -B expression.
Choroid Neoplasms↗
Effects of transpupillary thermotherapy on immunological parameters and apoptosis in a case of primary uveal melanoma.
Transpupillary thermotherapy (TTT) is a new treatment modality for uveal melanoma. We studied whether application of TTT influences the immunogenicity of the tumour cells in vivo or the expression of molecules related to apoptosis. Immunohistochemistry using monoclonal antibodies directed against HLA molecules, HMB45, P53, Fas ligand (FasL), Fas, Bcl-2 and tumour-infiltrating cells was applied to sections of an enucleated eye containing a uveal melanoma that received TTT 1 week before enucleation. The innermost part of the tumour which had been exposed directly to the laser treatment showed no staining for HLA antigens, nor for Fas or FasL epitopes. The intermediate part of the tumour showed a wet necrosis and HLA expression similar to the expression in the peripheral tumour. A large number of macrophages were observed in the necrotic as well as the intact tumour tissue, especially bordering the wet necrotic area. FasL and Bcl-2 were only expressed in the viable, outer part of the tumour. This immunological evaluation of one case of uveal melanoma treated with TTT revealed that TTT may not only have a direct destructive effect on the primary tumour, but may also influence the immunogenicity of uveal melanoma cells, induce infiltration of macrophages into the tumour, and induce apoptosis. The presence of many macrophages suggests that they play a role in the removal of the TTT-treated tumour tissue by phagocytosis.
Adult↗
Expression of angiogenic and immunosuppressive factors by uveal melanoma cell lines.
Dissemination of uveal melanomas is almost exclusively haematogenous, making angiogenesis of the tumour a prerequisite for the formation of metastases. Uveal melanomas must employ strategies to evade the immune system in order to escape immune surveillance. We therefore determined the expression of the following angiogenic and immunosuppressive factors in seven human uveal melanoma cell lines using reverse transcriptase-polymerase chain reaction (RT-PCR): secreted interleukin-1 receptor antagonist (sIL-1ra), interleukin (IL)-6, IL-8, IL-10, transforming growth factor (TGF)-alpha, TGFbeta, vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and angiopoietin-2. In addition, the secretion of sIL-1ra, IL-6, IL-8, IL-10, TGFbeta and VEGF was assayed by enzyme linked immunosorbent assay (ELISA). The potential of uveal melanoma cell lines to convert plasminogen to angiostatin was tested in an in vitro assay. All the factors except angiopoietin-1 were determined in one or more cell lines using RT-PCR, although these results were not necessarily confirmed by ELISA. Expression of VEGF and angiopoietin-2 was found in all seven cell lines. Production of angiostatin was observed in one cell line. All seven cell lines examined expressed angiogenic factors and most cell lines expressed immunosuppressive factors. The expression of VEGF and angiopoietin-2 in combination with a lack of angiopoietin-1 expression suggest high vascular remodelling capacity and could be of great relevance for the metastatic potential of uveal melanoma.
Angiopoietin-1↗
Corneal transplantation in antibody-deficient hosts.
PURPOSE: To examine the role of donor-specific antibodies, with or without complement, in rejection of orthotopic corneal transplants by using mice as recipients in which the genes for the heavy chain of immunoglobulin or the third complement component have been eliminated by homologous recombination. METHODS: BALB/c corneas were transplanted into eyes of B-cell-deficient (n=17) or wild-type control C57BL/6 (n=30) mice and into eyes of complement (C3)-deficient (n=15) or wild-type control 129-C57BL/6 (n=13) mice. After surgery all grafts were evaluated over 8 weeks in a masked manner by biomicroscopy for signs of rejection. RESULTS: The rates of corneal transplant rejection were similar among B-cell-deficient and C3-deficient mice compared with rejection rates in their respective wild-type control subjects. This similarity applied to the time course of rejection and to cumulative survival rates. CONCLUSIONS: Neither donor-specific antibody nor the third component of complement play essential roles in acute rejection of orthotopic corneal allografts in mice.
Animals↗
Distribution of tumors in the retina in hereditary retinoblastoma patients.
We have compared the location of the ocular tumors in hereditary retinoblastoma in relation to the age of the patients at time of diagnosis. Eighty fundus drawings were analyzed from 59 hereditary patients containing I 59 tumors. At the time of diagnosis, indirect ophthalmoscopy was performed under general anaesthesia in all patients and standard drawings of the retina were made depicting the number and relative location of all tumors. The distance between the center of the tumor and the center of the macula was measured and plotted against the age of the patients at time of diagnosis. The results show that the distance between the center of the tumor and the center of the macula at time of diagnosis increases with age during the first seven months after birth.
Child, Preschool↗
Expression of MAGE, gp100 and tyrosinase genes in uveal melanoma cell lines.
In order to determine the possible use of uveal melanoma cell lines as stimulators in immunotherapy, we evaluated the expression of the human genes for MAGE-1, -2 and -3, gp100 and tyrosinase in uveal melanoma cell lines. mRNA expression of the MAGE-1, -2 and -3, gp100 and tyrosinase genes and the HLA class I specificity were determined in five primary and three metastatic uveal melanoma cell lines. Expression of the examined genes was heterogeneous in the primary and metastatic cell lines. The cell lines OCM-1 and OMM-1 expressed MAGE-1, -2 and -3, whereas EOM-3, MEL202, 92-1 and OMM-3 were negative for these antigens. gp100 was expressed in all cell lines, and tyrosinase in all but three (EOM-29, OMM-2 and OMM-3). Except for EOM-3, the HLA-A type of all the cell lines could be determined by complement-dependent microlymphocytotoxicity assay. Since at least two melanoma-associated antigens can be found in uveal melanoma cell lines, as well as the HLA class I molecules, these cell lines may be applicable as immunogens for specific immunotherapy against metastatic uveal melanoma.
Antigens, Neoplasm↗
Occurrence of cutaneous and uveal melanoma in patients with uveal melanoma and their first degree relatives.
Uveal and cutaneous melanomas are rare tumours, but have been described to occur together in one patient or in members of the same family. A group of 109 consecutive uveal melanoma patients from one specialized ocular tumour clinic were investigated dermatologically. The patient's own history and medical data and the family history of skin or eye problems were recorded. A total of three cutaneous melanomas were found as a result of this study--two in ocular melanoma patients and one in a first-degree relative. Four patients had first-degree relatives with a skin melanoma (in three of these families dysplastic naevus syndrome was also found), and one patient had a first-degree relative with an uveal melanoma. To find cutaneous and uveal melanoma coexisting in two cases and cutaneous melanoma in first-degree relatives in four cases out of a total of 109 uveal melanoma patients seems more than a coincidence. A linking factor in three cases was the familial atypical multiple mole melanoma syndrome, suggesting a common genetic predisposition to both malignancies in these families. In our only family with familial uveal melanoma, cutaneous melanoma and atypical naevi did not occur. A different genetic mechanism for these cases is probable.
Aged↗
A small molecular weight factor in aqueous humor acts on C1q to prevent antibody-dependent complement activation.
PURPOSE: Aqueous humor inhibits activation of the classic complement pathway; however, the mechanism of this inhibition is unknown. We have examined at the molecular level potential factors responsible for the inhibition, and we have tried to determine where in the complement pathway the inhibition takes place. METHODS: Fresh rabbit aqueous humor was size fractionated by centrifuge concentrators and by size exclusion column chromatography, and each fraction was assayed for inhibition of the classic complement pathway in a standard CH50 hemolytic assay. Fractions with inhibitory activity were assayed for protein and the presence of ascorbic acid and were subjected to heat treatment. To identify where in the pathway the inhibitor(s) function, the expression of activated complement components bound to the surface of antibody-coated erythrocytes was analyzed by flow cytometry using fluorescein isothiocyanate-labeled antibodies to specific complement components. In addition, hemolytic assays were performed for the function of individual complement components. RESULTS: The most potent inhibition of the classic pathway was in a fraction of aqueous humor of less than 1.3 kDa. The inhibitory activity in the fraction was unassociated with detectable protein or ascorbic acid, and it remained present after heat treatment. The functional analysis through flow cytometry and hemolytic assays for individual complement components showed that the inhibitor in the less than 1.3-kDa fraction caused a blockade in the complement pathway at the level of C1q. CONCLUSIONS: The aqueous humor contains a unique potent anticomplementary factor that has a molecular weight less than 1.3 kDa. This heat-stable inhibitory factor inhibits the classic pathway at the level of C1q. These results imply that within the eye the complement pathway is inhibited at the earliest steps of its initiation. Such inhibition would prevent production of complement products that mediate inflammation and chemotaxis of inflammatory cells. Therefore, as part of the adaptation of immune privilege, the ocular microenvironment is protected from inflammation induced by antigen-antibody complexes.
Animals↗
Role of mucosal IgA in the resistance to Acanthamoeba keratitis.
PURPOSE: To determine whether oral immunization with Acanthamoeba castellanii antigens elicits mucosal antibodies of the IgA isotype and whether mucosal antibodies affect parasite adhesion to the corneal epithelium. METHODS: Chinese hamsters were immunized with 100 microg aqueous Acanthamoeba antigen mixed with cholera toxin (Ac-CT) and subsequently challenged with parasite-laden contact lenses that were applied to abraded corneal surfaces. Tears and stool samples were examined for the presence of Acanthamoeba-specific IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The effect of mucosal antibody on trophozoite binding to corneal epithelium and viability of trophozoites was examined in vitro. RESULTS: Hamsters immunized orally with Ac-CT showed significantly lower infection rates than did control groups (21.4% versus 72.6%). ELISA analysis of mucosal specimens showed the presence of parasite-specific IgA in stool samples and tears from hamsters orally immunized with Ac-CT, but not in control animals. In vitro assays showed that anti-Acanthamoeba IgA did not affect parasite viability. However, mucosal anti-Acanthamoeba IgA profoundly inhibited (>75%) the binding of parasites to corneal epithelial cells in vitro. CONCLUSIONS: Oral immunization with Ac-CT induces the production of parasite-specific IgA in mucosal secretions and prevents corneal infection. Mucosal antibody does not affect the viability of Acanthamoeba trophozoites but seems to prevent infection by inhibiting parasite binding to the corneal epithelium.
Acanthamoeba↗
Decreased expression of HLA class I on ocular melanoma cells following in vitro photodynamic therapy.
Our objective was to investigate whether photodynamic therapy (PDT) influences the expression of HLA Class I and beta 2-microglobulin molecules on cultured uveal melanoma cells. Uveal melanoma cells were incubated with hematoporphyrin esters (HPE) and illuminated using red light. HLA expression on cells was determined by flowcytometry. PDT treatment induced an immediate reduction in expression of HLA Class I and beta 2-microglobulin, followed by a transient increase in expression after 2 h. Normalization occurred after 6 h. Treatment of ocular melanoma cells with PDT temporally alters the expression of HLA Class I and beta 2-microglobulin, which may affect anti-tumor-immune responses.
Antigens, Neoplasm↗
Inverse correlation between expression of HLA-B and c-myc in uveal melanoma.
HLA class I is expressed in 75-85 per cent of uveal melanoma and cytoplasmic c-myc expression has been reported in 78 per cent of uveal melanoma. In skin melanoma, an inverse relationship has been observed between HLA class I expression and c-myc. The purpose of this study was to determine whether a similar correlation occurred between high expression of c-myc and low expression of HLA class I in uveal melanoma. The expression of c-myc, HLA-A, and HLA-B was determined by immunohistochemistry on formalin-fixed and paraffin-embedded sections of 30 uveal melanomas. Cell cultures from four primary uveal melanomas (lines 92-1, MEL 202, OCM-1, and EOM-3) and one uveal melanoma metastasis (line OMM-1) were tested for mRNA levels of c-myc and HLA-A and HLA-B in Northern blot assays. The high level of expression of cytoplasmic c-myc was significantly correlated with low expression of HLA-B (P = 0.03) and vice versa. High expression of HLA-B was significantly correlated with the presence of epithelioid cells (P = 0.004). The inverse correlation observed between c-myc and HLA-B expression is similar to previous observations in cutaneous melanoma. By downregulating HLA-B expression, c-myc may influence the immune response in uveal melanoma. Tumours containing epithelioid cells showed a significantly higher expression of HLA-B than tumours of the spindle cell type.
Adolescent↗
Lack of effect of different cytokines on expression of membrane-bound regulators of complement activity on human uveal melanoma cells.
Tumor cells are protected from antibody-dependent complement-mediated lysis by membrane-bound regulators of complement activation (m-RCA). m-RCA are expressed on uveal melanoma cells. We determined whether cytokine treatment affects expression of m-RCA on these cells in vitro. m-RCA expression on uveal melanoma cell lines was studied by flow cytometry, using monoclonal antibodies directed against CD46, CD55, and CD59. Cytokines studied were interferon-alpha (IFN-alpha), IFN-gamma, interleukin-1B (IL-1B), IL-12, and tumor necrosis factor-alpha (TNF-alpha). All three m-RCA were expressed on the uveal melanoma cell lines (CD59>>CD46>CD55), although in variable amounts. With a few exceptions, the cytokines had no effect on m-RCA expression. CD55 expression was not influenced by any of the cytokines. IFN-gamma downregulated expression of CD46 on one cell line (p < 0.01). TNF-alpha upregulated CD59 expression on two of the five cell lines (p < 0.012 and p < 0.001, respectively), which effect was dose dependent. IFN-alpha, IFN-gamma, IL1-beta, IL12, and TNF-alpha had limited effects on m-RCA expression on uveal melanoma cells in vitro.
Antineoplastic Agents↗
Effect of hyperthermia on expression of histocompatibility antigens and heat-shock protein molecules on three human ocular melanoma cell lines.
Hyperthermia is used as a new treatment modality for ocular melanoma. We wondered whether this treatment would affect the antigenicity of melanoma cells and studied the effect of hyperthermia on the expression of histocompatibility antigens (HLA), beta 2-microglobulin, as well as heat-shock proteins (HSP-60 and HSP-70) on choroidal melanoma cells. Uveal melanoma cell lines were exposed to different temperatures (39-45 degrees C) in a waterbath. Antigen expression was determined with fluorescence-activated cell sorting analysis, using monoclonal antibodies against HLA and HSP. In a 51Cr-release cytotoxicity assay we studied the effect of heat on natural killer (NK) cell susceptibility. Exposure to 45 degrees C for 30 min reduced expression of HLA class I antigens and beta 2-microglobulin. A greater reduction was observed after longer exposure times. Expression of HSP-70 was increased after exposure to 45 degrees C at all time intervals, while expression of HSP-60 was not induced by heat treatment. We did not find a significant difference in the NK cell susceptibility between heated and unheated cells. Hyperthermia has a time- and temperature-dependent effect on expression of HLA class I and HSP-70 molecules on the cell surface of uveal melanoma cells. Hyperthermia did not alter the susceptibility to NK cell lysis.
Cells, Cultured↗
HLA expression in a primary uveal melanoma, its cell line, and four of its metastases.
BACKGROUND: The level of HLA expression on a tumour may influence the immunological response against this tumour, and vice versa. HLA expression was determined in a primary uveal melanoma, its metastases, and on a cell line derived from this melanoma, and the presence and type of infiltrate in tissue sections were also studied. METHODS: Immunohistochemistry with monoclonal antibodies (MAbs) against HLA class I and II, T cells, NK cells, and macrophages. RESULTS: Primary and metastatic lesions, as well as the cell line showed high levels of expression of the monomorphic determinants of HLA class I. Expression of the polymorphic HLA-A2 and HLA-A3 antigens was decreased on metastases to the skin and liver. HLA-Bw4 expression was low on all lesions, as well as expression of HLA class II. Tumour infiltrating cells consisted mainly of CD3, CD4, and CD8 positive cells. Expression on the cell line corresponded to expression on the primary tumour. CONCLUSION: The primary uveal melanoma as well as the cell line showed a high expression of monomorphic and polymorphic HLA-A antigens, while metastases showed a high expression of monomorphic and a lower expression of polymorphic antigens. This variation in expression may support tumour cell escape from NK cells as well as CTL mediated lysis.
Aged↗
Systemic immune response to Acanthamoeba keratitis in the Chinese hamster.
Recrudescence is a common and troubling feature of Acanthamoeba keratitis and suggests that corneal infection with this organism fails to stimulate the systemic immune apparatus. The present study examined the cell-mediated and humoral immune responses to Acanthamoeba keratitis in the Chinese hamster. Corneal infection with A. castellanii failed to induce either delayed-type hypersensitivity (DTH) or serum IgG antibody against parasite antigens. The failure to induce cell-mediated and humoral immunity did not result in anergy or tolerance since subsequent intramuscular (i.m.) immunization with parasite antigens elicited robust DTH and IgG antibody responses. The inability of corneal infections to induce primary cell-mediated immune responses was due to the absence of resident antigen-presenting cells in the central cornea because induction of Langerhans cell (LC) migration into the central cornea prior to infection with Acanthamoeba promoted the development of parasite-specific DTH. Although the presence of resident LC did not promote the development of a primary humoral immune response, subsequent i.m. immunization elicited heightened parasite-specific IgG antibody production which was indicative of an anamnestic response. Collectively, the results indicate that in the absence of resident antigen-presenting cells, corneal infection with Acanthamoeba fails to stimulate primary cell-mediated or humoral immunity. Induction of peripheral LC into the central corneal epithelium promotes the development of parasite-specific DTH, but does not exacerbate corneal disease.
Acanthamoeba↗
Human leukocyte antigen class I expression. Marker of poor prognosis in uveal melanoma.
PURPOSE: Because the expression of human leukocyte antigen (HLA) antigens is important for immunologic recognition of tumor cells, we determined expression of locus-specific HLA class I antigens in uveal melanoma and tested whether the level of HLA expression was related to prognosis or associated with known prognostic parameters. METHODS: Expression of HLA-A and -B antigens was determined on 30 formalin-fixed and paraffin-embedded sections of uveal melanoma by immunohistochemistry with locus-specific monoclonal antibodies and scored semiquantitatively. RESULTS: The level of expression of HLA-A and -B varied between uveal melanomas. Expression levels of HLA-A and -B were significantly correlated (P = 0.02). High HLA-B expression was significantly correlated with the presence of epithelioid cells (P = 0.04) in the tumor. Expression levels of HLA-A as well as of HLA-B, cell type, mitotic rate, Mib-1 score, and largest tumor diameter were significant predictive factors for survival. High expression of HLA-A and -B was associated with a decreased survival. Multiple Cox regression analysis with stepwise selection of covariates showed that the contribution of HLA-A expression to survival (P = 0.0003) exceeded that of tumor diameter (P = 0.02) and Mib-1 score (P = 0.04). CONCLUSIONS: Lack of expression of HLA-A as well as of HLA-B antigens on uveal melanoma is correlated with a better patient survival. Our data suggest that shedding of uveal melanoma micrometastases with a low expression of HLA class I into the systemic circulation may facilitate their removal and prevent the development of metastases. These findings support a protective role for natural killer cells in the development of metastatic disease in uveal melanoma.
Adolescent↗
Multidrug resistance-related proteins in primary choroidal melanomas and in vitro cell lines.
PURPOSE: Metastatic uveal melanoma is strongly resistant to chemotherapy, and multidrug resistance (MDR) may be involved. To investigate the role of MDR, the presence of the MDR-associated proteins P-glycoprotein (Pgp), MRP, and lung resistance protein (LRP) was determined on primary choroidal melanomas and cell lines. METHODS: A panel of primary choroidal melanomas was examined for the presence of MDR-associated proteins by immunohistochemical analysis. In cell lines established from four primary choroidal melanomas and one metastatic choroidal melanoma, the expression of MDR-associated proteins was determined with monoclonal antibodies in cytospin preparations and flow cytometry. In addition, the functional capacities of transporter proteins Pgp and MRP as adenosine triphosphate-driven efflux pumps were determined by measuring the cellular accumulation and efflux of the fluorescent dyes rhodamine 123 and calcein-AM, with and without the presence of specific pump inhibitors PSC833 and probenecid. RESULTS: Low levels of Pgp and MRP were detected in most primary tumors and in some cell lines. Measurable transporter function of Pgp could be determined in cell line OCM-1. Lung-resistance protein was present in all primary tumors and cell lines and showed high expression levels. CONCLUSIONS: This study revealed the involvement of LRP and at least a minor role of Pgp and MRP in chemoresistance of choroidal melanoma. Compared with cutaneous melanomas, uveal melanomas appear to express slightly higher levels of Pgp. These findings provide insights into the drug-resistant phenotype of this disease and can aid in the design of therapeutic protocols.
ATP Binding Cassette Transporter, Subfamily B, Mem↗