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Biomedical subjects

M J Jager

Publications and source records attributed to M J Jager.

At least 19 recordsLinked to original sources
PubMed2006

A new model for limbal transplantation using E-GFP for follow-up of transplant survival.

Limbal transplants in humans show a high rate of rejection even under local and systemic immunotherapy. In order to test immunomodulatory treatments a new limbal transplant model in the rat was developed using enhanced green fluorescent protein (E-GFP) as marker for follow-up. Sixty E-GFP-positive limbal transplants from Sprague-Dawley TgN(act-EGFP)Osb4 rats were transplanted onto 18 wild-type inbred Sprague-Dawley (isografts) rats, six wild-type litter mate Sprague-Dawley (sibling) rats, 18 Fischer 344 (allografts) rats, and 18 Fischer 344 rats depleted from monocytes and macrophages by subconjunctival treatment with clodronate liposomes. All rats were monitored three times a week with fluorescence microscopy, until fluorescence had disappeared. At postoperative days 6, 9, 12, and 15, three rats of all groups were killed for immunohistochemical analysis of infiltrating cells. Using a modified digital fluorescence microscope, we were able to monitor transplant behavior over time without disturbance of the ocular surface. The average days of rejection were 14 days in the isograft group, the sibling group, and the untreated allograft group. However, the average day of rejection in the allogeneic macrophage-depleted group was 27 days. Marked infiltration of macrophages and lymphocytes was seen in the untreated isografts and allografts. In the clodronate liposome-treated allografts infiltration was minor. A successful new limbal transplant model is described. The transplant can be accurately followed up in vivo by E-GFP labeling of the donor tissue without disturbing the corneal surface. Although E-GFP itself proved to be immunogenic, local clodronate liposome injections significantly increased graft survival. So the model seems to be useful for testing immunosuppressive or modulatory agents in limbal transplantation studies.

Animals↗
PubMed2006

Immunophenotypic markers to differentiate between benign and malignant melanocytic lesions.

BACKGROUND/AIMS: The authors investigated the expression of S100A1, S100A6, S100B, MelanA, and CEA in conjunctival naevi, primary acquired melanosis (PAM), conjunctival melanoma, and uveal melanoma in order to assess their potential usefulness in the pathological differential diagnosis of these entities. METHODS: Paraffin embedded sections of 18 conjunctival naevi, 14 PAM, 16 conjunctival melanomas, and 20 uveal melanomas were immunostained for S100A1, S100A6, S100B, MelanA, and CEA, and expression was scored semiquantitatively. RESULTS: Expression of S100A1 differed significantly between conjunctival naevi and conjunctival melanoma, with percentages of positive cells of 30.6% and 71.4%, respectively. Conjunctival melanomas had high average scores for S100A1 and S100B (71.4%, 62.9%, respectively), while uveal melanomas also had high S100A1 but low S100B scores (88.5%, 18.5%, respectively). MelanA was highly variable; naevi and uveal melanoma had higher average scores than conjunctival melanoma. CEA was hardly detectable in all four groups. CONCLUSION: S100A1 seems to be a possible candidate to differentiate conjunctival naevi from conjunctival melanoma. S100B seems to differentiate between uveal melanoma and conjunctival melanoma. However, the study size was small and therefore the data have to be confirmed by others.

Antigens, Neoplasm↗
PubMed2005

Normal HLA class I, II, and MICA gene distribution in uveal melanoma.

PURPOSE: The molecules of the HLA class I and II molecules as well as the MHC class I chain-related gene A (MICA), a polymorphic and stress-induced cell surface molecule, are involved in T-cell and natural killer-cell (NK-cell) mediated immune responses. In this study we looked for any genetic susceptibility contributed by HLA class I, class II, or MICA genes with regard to the development of uveal melanoma. METHODS: Between 1998 and 2001, 159 uveal melanoma patients were typed for HLA class I and II, and 168 uveal melanoma patients were evaluated for MICA by microsatellite typing. The HLA antigen and MICA allele frequencies were compared with control groups of, respectively, 2,440 and 247 healthy Dutch individuals. RESULTS: HLA class I, HLA class II, and MICA gene frequencies in uveal melanoma patients and healthy Dutch controls showed no significant deviations after correction for the number of comparisons. CONCLUSIONS: We conclude that there is no genetic susceptibility or increased risk attributed to any HLA class I, class II, and MICA polymorphism with regard to the development of uveal melanoma.

Adolescent↗
PubMed2005

Ciliary tissue transplantation in the rabbit.

Irreversible damage of the ciliary body can be responsible for prolonged ocular hypotony and phthisis bulbi, which, currently, cannot be treated. The aim of this study was to achieve survival of morphologically normal ciliary tissue (CT) transplants in the anterior chamber of a rabbit's eye. Outbred female New Zealand albino rabbits received CT allografts, which were placed on to the surface of the host iris. We evaluated the influence of ciclosporin (CsA), VEGF and donor perfusion on graft survival. Operated eyes were assessed clinically and histologically, and revascularization of the grafts was determined by fluorescein angiography. All grafts became dark and ischemic during the first five to seven days after transplantation. Reperfusion of the grafted tissue was complete at approximately ten days after transplantation. In untreated animals, transplants became infiltrated by inflammatory cells, which led to destruction of the tissue. This was prevented by systemic use of CsA. Transplants treated with VEGF prior to transplantation had fewer ischemic areas but epithelial cell survival was not improved. Whole body donor perfusion prior to preparation of the grafts resulted in less inflammation and, histologically, in a better quantity and quality of the epithelial cells in the CT transplants. Ciliary tissue can be successfully transplanted but the ciliary epithelium suffers from ischemia and in untreated animals the whole transplant is rejected in the classical fashion. If the donor is perfused and the host immunosuppressed, histologically normal ciliary epithelium can be preserved together with rapid revascularization, minimal inflammation and good survival of the transplant, although fibrosis continued to occur during the two months after transplantation.

Animals↗
PubMed2005

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.

Blotting, Western↗
PubMed2004

The efficacy and safety of topical polymyxin B, neomycin and gramicidin for treatment of presumed bacterial corneal ulceration.

AIM: To evaluate the clinical efficacy and safety of topical polymyxin B, neomycin, and gramicidin for the treatment of suspected bacterial corneal ulceration at the Leiden University Medical Center. METHODS: Patients with a diagnosis of a suspected bacterial corneal ulcer between April 1995 and February 2002 were retrospectively identified and reviewed; clinical and microbiological features and response to therapy were analysed. All patients were treated with Polyspectran eye drops. RESULTS: In total, 91 patients were included in this analysis. Bacteriological cultures of 46 patients (51%) were positive and revealed 51 microorganisms. Staphylococcus aureus (29.4%) and Pseudomonas aeruginosa (23.5%) were the most frequently encountered bacteria. Eighteen patients switched therapy before complete healing of the corneal ulceration, four patients were lost to follow up. Of the 69 patients who completed Polyspectran treatment, re-epithelialisation occurred in 68 patients (99%) and on average took 12.6 (median 8) days. Among 91 patients, there were four perforations and one evisceration. Seven toxic or allergic reactions were reported. CONCLUSION: This study shows that the combination of polymyxin B, neomycin, and gramicidin is an effective and safe treatment of suspected corneal ulceration.

Adult↗
PubMed2003

Gene expression profiling identifies tumour markers potentially playing a role in uveal melanoma development.

Microarray is a powerful tool to compare the gene expression of different tumour specimens and cell lines simultaneously and quantitatively. To get a better insight into genes that are involved in uveal melanoma tumorigenesis, we compared the gene expression profiles of 12 different uveal melanoma cell lines with three melanocyte cell cultures obtained from healthy donor eyes. Gene expression profiles were obtained by nylon filter arrays, containing 1176 gene spots related to cancer development. The expression levels of selected genes were validated on cell lines and primary uveal melanomas by real time RT-PCR, and were subsequently included in cluster analysis. Four candidate tumour markers, Laminin Receptor 1, Endothelin 2, Von Hippel Lindau Binding protein 1 and Cullin 2, have been selected from genes that were differentially expressed in the uveal melanoma cell lines compared to the normal uveal melanocytes. In primary uveal melanomas, these four markers could discriminate between two classes of uveal melanoma, which may be indicative of a differential disease process.

Biomarkers, Tumor↗
PubMed2002

[HLA class I antigen expression correlates with histological cell type in uveal melanoma].

PURPOSE: The prognosis of uveal melanoma is correlated with its histologic cell type. The epithelioid cell type is associated with a higher metastatic rate than the spindle cell type. The Human Leucocyte Antigen Class I (HLA-I) expression of the melanoma also correlates with the prognosis. In this study, we analyzed HLA-I antigen expression of uveal melanomas to determine whether a relationship exist between antigenic expression and melanoma cell type. METHODS: Formalin-fixed, paraffin-embedded spindle cell type (n = 11) and epithelioid cell type (n = 11) uveal melanomas were immunostained with the HC10 antibody (1:80) for HLA-I antigen expression with appropriate positive and negative controls. Sections were assessed semiquantitatively according to the percentage of stained cells. RESULTS: Among the spindle cell type melanomas, 2 out of 11 (18%) stained with HC10 antibodies. The staining intensity was less than 25% of the cells in these two melanomas. Among the epithelioid cell type melanomas, 9 out of 11 (82%) stained with HC10. The staining intensity was more than 25% of the cells in 5 of these 9 melanomas. CONCLUSIONS: It is unknown why spindle and epithelioid cell type uveal melanomas have different prognoses. Human uveal melanoma cell lines with low HLA-I expression are susceptible to NK cell-mediated lysis in vitro and in murine studies. The prognostically more favorable spindle cell type melanoma expresses less HLA-I than the epithelioid cell type melanoma. These results stress the role of NK cells in the rejection of uveal melanoma.

Histocompatibility Antigens Class I↗
PubMed2001

Rejection of intraocular tumors by CD4(+) T cells without induction of phthisis.

Immune privilege of the eye protects against sight-threatening inflammatory events, but can also permit outgrowth of otherwise nonlethal immunogenic tumors. Nonetheless, ocular tumor growth can be controlled by cellular immune responses. However, this will normally result in phthisis of the eye, in case tumor rejection is mediated by a delayed-type hypersensitivity response orchestrated by CD4(+) T cells. We now show that intraocular tumors can be eradicated by CD4(+) Th cells without inducing collateral damage of neighboring ocular tissue. Injection of tumor cells transformed by the early region 1 of human adenovirus type 5 in the anterior chamber of the eye leads to intraocular tumor formation. Tumor growth is transient in immunocompetent mice, but lethal in immunodeficient nude mice, indicating that T cell-dependent immunity is responsible for tumor clearance. Tumor rejection has all the characteristics of a CD8(+) T cell-mediated immune response, as the tumor did not express MHC class II and only tumor tissue was the subject of destruction. However, analysis of the molecular and cellular mechanisms involved in tumor clearance revealed that perforin, TNF-alpha, Fas ligand, MHC class I, and CD8(+) T cells did not play a crucial role in tumor eradication. Instead, effective tumor rejection was entirely dependent on CD4(+) Th cells, as CD4-depleted as well as MHC class II-deficient mice were unable to reject their intraocular tumor. Taken together, these observations demonstrate that CD4(+) T cells are able to eradicate MHC class II-negative tumors in an immune-privileged site without affecting surrounding tissues or the induction of phthisis.

Adenovirus E1 Proteins↗
PubMed2001

Promoter hypermethylation: a common cause of reduced p16(INK4a) expression in uveal melanoma.

Tumors often display unrestricted cell cycling attributable to a dysfunctional G(1)-S checkpoint. One of the mechanisms leading to such a defect is the inactivation of the cyclin-dependent kinase inhibitor p16(INK4a). Although inactivation of p16(INK4a) is observed in a wide range of tumors, including cutaneous melanoma, genetic alteration of p16(INK4a) is reportedly uncommon in uveal melanoma. Here we show that the p16(INK4a) promoter is hypermethylated in 6 of 12 uveal melanoma cell lines and in 7 of 22 primary uveal melanomas analyzed. Five of seven patients with a methylated primary tumor died of metastatic disease compared with 2 of 15 patients with a nonmethylated primary tumor. We also show that all uveal melanoma cell lines with a hypermethylated p16(INK4a) promoter have lost p16(INK4a) expression but have maintained the expression of p14(ARF). Treatment of uveal melanoma cell lines with 5-aza-2'-deoxycytidine results in demethylation of p16(INK4a) and in reexpression of p16(INK4a) mRNA, which is maintained upon withdrawal of the 5-aza-2'-deoxycytidine. In conclusion, p16(INK4a) promoter methylation appears to be a common event in uveal melanoma and is accompanied by the loss of p16(INK4a) expression.

Adult↗
PubMed2001

Characterization of melanocortin-1 receptor gene variants in uveal melanoma patients.

PURPOSE: Allelic variations of the melanocortin-1 receptor (MC1R) gene have been linked to red hair and sun-sensitive skin types and may play a role in the susceptibility to develop cutaneous malignant melanoma (CMM). To define the role of MC1R gene in uveal melanoma, a case control study was performed, in which the presence of MC1R gene variations in uveal melanoma patients was compared with that of healthy controls. METHODS: MC1R gene variants were analyzed in 162 uveal melanoma patients and 255 healthy controls. After genomic DNA was isolated from venous blood, the MC1R gene was amplified by polymerase chain reaction (PCR) and examined for the presence of variants by single-strand conformation polymorphism (SSCP) analysis. Participants were asked to complete a questionnaire regarding skin type, eye color, and hair color. RESULTS: No disparity was found between the distribution of the MC1R gene variants in both groups. Furthermore, no associations between MC1R genotype and pigment phenotype were found. In contrast to CMM, uveal melanoma patients did not show specific MC1R gene variants. Compared with controls, most uveal melanoma patients had blue eyes (65%, P = 0.060) and skin type III (56%); however, in the uveal melanoma group the presence of dark blond hair was significantly elevated (46%, P = 0.030). These findings are in contrast with studies on CMM, where most patients have skin type II and red/fair hair. CONCLUSIONS: These data suggest that MC1R variants do not play a role in the susceptibility to develop uveal melanoma. Furthermore, most uveal melanoma patients share phenotypic characteristics that differ from findings in CMM patients.

Adult↗
PubMed2001

Uveal melanoma: no expression of HLA-G.

PURPOSE: To investigate whether uveal melanoma cells express HLA-G, a nonclassical HLA class I molecule that has been shown to be a critical mediator in the inhibition of natural killer (NK) cell-mediated cytolysis. METHODS: Eleven human uveal melanoma cell lines were analyzed for the expression of HLA-G by flow cytometry, immunocytochemistry, Western blot analysis, and RT-PCR followed by Southern blot analysis. Two HLA-G-specific monoclonal antibodies were used, 87G and MEM-G/1. In addition, HLA-G expression was determined on frozen tissue sections of 17 primary uveal melanomas. RESULTS: With all HLA-G detection methods, no evidence for HLA-G expression by uveal melanoma cells was found. In contrast, the trophoblast cell line JEG-3 clearly expressed HLA-G transcripts and protein in all cases. Furthermore, interferon-gamma did not induce HLA-G expression in the uveal melanoma cell lines. Notably, all cell lines expressed HLA-E, and this expression was significantly enhanced by interferon-gamma. CONCLUSIONS: Because none of the uveal melanoma cell lines nor any of the primary uveal melanomas displayed expression of HLA-G, it is unlikely that HLA-G plays a role, direct or indirect, in the modulation of cellular immunity against uveal melanoma tumors.

Blotting, Western↗
PubMed2000

Adoptive T cell immunotherapy of human uveal melanoma targeting gp100.

HLA-A*0201-restricted CTL against human gp100 were isolated from HLA-A*0201/K(b) (A2/K(b))-transgenic mice immunized with recombinant canarypox virus (ALVAC-gp100). These CTL strongly responded to the gp100(154-162) epitope, in the context of both the chimeric A2/K(b) and the wild-type HLA-A*0201- molecule, and efficiently lysed human HLA-A*0201(+), gp100(+) melanoma cells in vitro. The capacity of the CTL to eradicate these tumors in vivo was analyzed in A2/K(b)-transgenic transgenic mice that had received a tumorigenic dose of human uveal melanoma cells in the anterior chamber of the eye. This immune-privileged site offered the unique opportunity to graft xenogeneic tumors into immunocompetent A2/K(b)-transgenic mice, a host in which they otherwise would not grow. Importantly, systemic (i.v.) administration of the A2/K(b)-transgenic gp100(154-162)-specific CTL resulted in rapid elimination of the intraocular uveal melanomas, indicating that anti-tumor CTL are capable of homing to the eye and exerting their tumoricidal effector function. Flow cytometry analysis of ocular cell suspensions with HLA-A*0201-gp100(154-162) tetrameric complexes confirmed the homing of adoptively transferred CTL. Therefore, the immune-privileged state of the eye permitted the outgrowth of xenogeneic uveal melanoma cells, but did not protect these tumors against adoptive immunotherapy with highly potent anti-tumor CTL. These data constitute the first direct indication that immunotherapy of human uveal melanoma may be feasible.

Animals↗
PubMed2000

High frequency of allele-specific down-regulation of HLA class I expression in uveal melanoma cell lines.

Uveal melanoma is the most common primary intra-ocular tumor in adults and has a high mortality rate due to liver metastases, for which no effective treatment is available. To investigate whether immunotherapy might be feasible in uveal melanoma, the HLA class I surface expression of 6 uveal melanoma cell lines was analyzed by flow cytometry using a broad panel of allele-specific monoclonal antibodies. To up-regulate HLA expression, cells were also cultured with IFN-alpha or -gamma. In general, expression of HLA-A alleles was high (except for cell line EOM-3) and could be further up-regulated by both IFN-alpha and -gamma. In cell line EOM-3, IFN-gamma treatment resulted in significant HLA-A expression while IFN-alpha treatment did not. Expression of HLA-B alleles was low or even negative. Variable effects were observed after IFN treatment. In 3 cell lines, expression of some HLA-B alleles could not be induced by IFN-alpha or -gamma: HLA-B44 in cell line 92-1, HLA-B15 in cell line OCM-1 and HLA-B5 in cell line MEL-202. The other B alleles of these cell lines showed enhanced expression levels upon IFN stimulation. In OMM-1 cells, IFN-alpha and -gamma increased the expression of HLA-A but did not induce expression of the 2 B alleles, indicating an HLA-B locus-specific loss. We thus found a high frequency of allele-specific and locus-specific down-regulation of HLA expression in uveal melanoma cell lines. Some of these defects were not restored by IFN-alpha or -gamma treatment. The lack of HLA expression may explain why uveal melanoma cells escape immune surveillance by cytotoxic T cells and complicate the development of immunotherapy in uveal melanoma.

Adult↗
PubMed2000

Expression of epidermal growth factor receptor: risk factor in uveal melanoma.

PURPOSE: To investigate the prognostic significance of the expression of epidermal growth factor receptor (EGFR) in uveal melanoma. EGFR is a transmembrane glycoprotein, and its expression has been correlated with the development of metastases in various malignancies. METHODS: Frozen sections from 22 primary uveal melanomas were examined for EGFR expression by a three-step immunoperoxidase staining, using a mouse anti-human EGFR IgG2b monoclonal antibody. The results were compared with patient survival and clinical and histopathologic parameters. RESULTS: EGFR expression could not be determined on one tumor due to excessive pigmentation. Two patients died of causes unrelated to melanoma, and two patients were lost to follow-up. Out of 21 tumors, six tumors showed immunoreactivity for EGFR. Five of these six patients (83%) died due to metastases, compared with 2 (17%) of 12 patients with no EGFR expression (Kaplan-Meier analysis P = 0.0004). EGFR-positive tumors tended to have a greater tumor prominence and a higher mitotic rate. CONCLUSIONS: The expression of EGFR was significantly correlated with death due to metastatic disease and therefore can be regarded as an important prognostic factor in human uveal melanoma.

Adult↗
PubMed1999

Successful immunotherapy of an intraocular tumor in mice.

Immune privilege in the eye is considered essential in the protection against local sight-threatening inflammatory responses. However, the deviant immune responses in the eye may also provide an ideal opportunity to uncontrolled growth of viruses or tumors by inhibiting intraocular immunological attack. To establish to what extent immune privilege interferes with T cell-mediated antitumor immunotherapy, we established a new ocular tumor model in the mouse and tested whether well-defined tumor-specific CTLs can eradicate an immunogenic intraocularly growing tumor. Tumor cells, transformed by human adenovirus type 5 early region 1 (Ad5E1), injected s.c. in a dose of 10(7) cells, did not induce s.c. tumor growth in C57BL/6 mice. However, an injection of 0.3 x 10(6) of these cells into the anterior chamber of the eye led to intraocular tumor growth in 95% of mice (n = 20). Tumor growth in the eye did not induce systemic tumor-specific tolerance, because 70% of the mice were able to eradicate the tumor spontaneously after 5 weeks. Mice vaccinated s.c. with irradiated tumor cells were protected against intraocular tumor challenge, indicating that preactivated memory T cells are able to protect against intraocular tumor growth. Moreover, an i.v. injection of an Ad5E1-specific CTL clone was able to eradicate established intraocular Ad5E1-transformed tumors, whereas the anatomy of the eye remained intact. These results demonstrate that tumor-specific, CTL-mediated immunity can be used successfully for the prevention and eradication of tumors growing in the immune-privileged anterior chamber of the eye, without detectable destruction of the eye.

Adenoviruses, Human↗
PubMed1999

Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system.

After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion (TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as early as 2 days after corneal infection, reaches peak titers by 3-5 days after infection, and is undetectable by 7-10 days. During the period of HSV-1 replication, macrophages and gammadelta TCR+ T lymphocytes infiltrate the TG, and TNF-alpha, IFN-gamma, the inducible nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-alpha, IFN-gamma, and the iNOS product nitric oxide (NO) all inhibit HSV-1 replication in vitro. Macrophage and gammadelta TCR+ T cell depletion studies demonstrated that macrophages are the main source of TNF-alpha and iNOS, whereas gammadelta TCR+ T cells produce IFN-gamma. Macrophage depletion, aminoguanidine inhibition of iNOS, and neutralization of TNF-alpha or IFN-gamma all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal infection. Moreover, individually depleting macrophages or neutralizing TNF-alpha or IFN-gamma markedly reduced the accumulation of both macrophages and gammadelta TCR+ T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virus replication in the sensory ganglia is controlled by macrophages and gammadelta TCR+ T lymphocytes through their production of antiviral molecules TNF-alpha, NO, and IFN-gamma. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG.

Animals↗