Chromosomal aberrations in iris melanomas.

BACKGROUND: Uveal melanomas can develop in the choroid, ciliary body and iris. In choroidal and ciliary body melanomas, specific chromosomal changes correlate with metastatic disease. Iris melanomas have a better prognosis than choroidal melanomas, and it would be interesting to know if they share chromosomal changes. In addition, iris melanomas might harbour UV-induced mutations of tumour suppressor genes, such as PTEN and CDKN2A. METHODS: Twenty iris melanomas were analysed for chromosome 1p, 3, 6, 8, 9p and 10q abnormalities using fluorescence in situ hybridisation. These results were correlated to clinical follow-up data using statistical analyses. RESULTS: (Partial) loss of chromosome 3 was observed in nine iris melanomas, and gain of 8q was present in seven tumours. Loss of chromosome 9p was demonstrated in seven tumours, but no deletions of the PTEN region on chromosome 10 were found. Three patients died of metastatic disease, and one patient developed liver metastases, but is still alive. Univariate analysis indicated a lower disease-free survival for patients with diffuse growing melanomas (p=0.01), melanomas that lost a copy of chromosome 3 (p=0.03), or invading the ciliary body (p=0.01). In a multivariate analysis, none of the correlations were significant. CONCLUSION: Loss of chromosome 3 as well as loss of chromosomal region 9p21 (that entails tumour suppressor gene CDKN2A) plays a role in iris melanoma. A firm correlation with disease-free survival could not be established, possibly due to the small sample size.

Spinocerebellar ataxia type 7 (SCA7): widespread brain damage in an adult-onset patient with progressive visual impairments in comparison with an adult-onset patient without visual impairments.

Spinocerebellar ataxia type 7 (SCA7) represents a rare and severe autosomal dominantly inherited ataxic disorder and is among the known CAG-repeat, or polyglutamine, diseases. In contrast to other currently known autosomal dominantly inherited ataxic disorders, SCA7 may manifest itself with different clinical courses. Because the degenerative changes evolving during these different clinical courses are not well known, many neurological disease symptoms still are unexplained. We performed an initial pathoanatomical study on unconventional thick tissue sections of the brain of a clinically diagnosed and genetically confirmed adult-onset SCA7 patient with progressive visual impairments. In this patient we observed loss of myelinated fibres in distinct central nervous fibre tracts, and widespread degeneration of the cerebellum, telencephalon, diencephalon and lower brainstem. These degenerative changes offer appropriate explanations for a variety of less-understood neurological symptoms in adult-onset SCA7 patients with visual impairments: gait, stance and limb ataxia, falls, dysarthria, dysphagia, pyramidal signs, Parkinsonian features, writing problems, impairments of saccades and smooth pursuits, altered pupillary functions, somatosensory deficits, auditory deficits and mental impairments.

In situ adenocarcinoma ex pleomorphic adenoma of the lacrimal gland.

A case of an in situ adenocarcinoma of the lacrimal gland that developed in a recurring pleomorphic adenoma is presented. Six years after incomplete resection of a pleomorphic adenoma, the patient complained of a progressive swelling of her right upper eyelid without pain or diplopia. An orbital computerized tomography scan showed an inhomogeneous mass in the right lacrimal gland region without bone involvement. After local excision of the in situ adenocarcinoma ex pleomorphic adenoma, the tumour has been in control for over 6 years.

Unintentional inversion of corneal buttons during penetrating keratoplasty: clinico-pathological report of two cases.

AIM: This report describes the clinico-pathological features of unintentionally inverted corneal buttons in two patients. METHODS: A clinico-pathological report. RESULTS: Two patients who underwent repeat keratoplasty for failed grafts were found to have inverted corneal buttons on histopathological examination. A detailed description of the pathological features of the inverse keratoplasty and the clinical outcome after repeat keratoplasty is presented. CONCLUSION: Inadvertent inverse keratoplasty should be considered as a rare cause of corneal graft failure. The serious complication of anterior chamber epithelialization seems to be unlikely and the prognosis following repeat penetrating keratoplasty appears to be very good.

Autologous peripheral retinal pigment epithelium translocation in patients with subfoveal neovascular membranes.

AIM: To evaluate the possibility of translocating autologous peripheral retinal pigment epithelial (RPE) cells and enhance their adhesion to improve functional outcome after choroidal neovascular membrane extraction in patients with subfoveal neovascular membranes. METHODS: A prospective, non-controlled surgical study in eight consecutive patients operated between February and July 2001 with final data monitoring in July 2002. All patients had mixed subfoveal membranes of 2-4 disc diameters. Functional tests included Snellen vision and central fixation testing. During vitrectomy, after the extraction of the neovascular complex, 8 x 10(4)-16 x 10(4) RPE cells were removed from the periphery and translocated under the macula following the submacular injection of 2 microg of poly-L-lysine to promote adhesion of the cells. RESULTS: With a follow up ranging from 3 months to 16 months, a pigmented area was seen in the extraction bed of the neovascular membrane in only one patient. Fixation was at the edge of the extraction bed in three patients. Vision remained the same in five patients and deteriorated in three (all with retinal detachment). Retinal detachment due to proliferative vitreoretinopathy occurred in three patients. CONCLUSIONS: The translocation of autologous peripheral RPE cells after membrane extraction was technically possible in a sterile manner, but was associated with a high proliferative vitreoretinopathy rate and in the present series had no measurable positive effect on functional outcome.

Role of Fas-ligand in age-related maculopathy not established.

PURPOSE: Fas-ligand expression on retinal pigment epithelium is hypothesized to have an inhibitory effect on human ocular neovascularization. METHODS: We studied Fas-ligand expression in the aging retinal pigment epithelium and in early and late stages of age-related maculopathy. Immunohistochemistry with antibodies against Fas-ligand was performed on paraffin-embedded sections of 23 human eye bank eyes (aged 45 to 96 years) and 12 eyes with exudative age-related maculopathy. RESULTS: Fas-ligand expression in retinal pigment epithelium was not related to age or to the presence of early age-related maculopathy. Furthermore, Fas-ligand expression in retinal pigment epithelium was similar in subretinal and subretinal pigment epithelium choroidal neovascular membranes. CONCLUSION: It appears to be unlikely that Fas-ligand expressed on retinal pigment epithelium controls the extension of choroidal neovascular membranes from subretinal pigment epithelium to subretinal.

Radiotherapy of exudative age-related macular degeneration; a clinical and pathologic study.

BACKGROUND: Radiotherapy has recently been employed to treat patients with exudative macular degeneration in order to prevent severe visual loss. Radiotherapy affects the evolution of exudative macular degeneration directly by endothelial toxicity, leading to capillary closure, and/or indirectly through its attenuating effects on the inflammatory response, mediated by macrophages and other inflammatory cells. METHODS: In this study we describe the histopathologic findings in a patient with exudative age-related macular degeneration (AMD) in both eyes whose right eye was treated with radiotherapy (5 times 2 Gy) 3 years before he died. The eyes were enucleated post mortem and investigated by light microscopy. Additionally, immunohistochemical investigation with antibodies against CD34 and CD68 was performed to identify patent endothelial cells and macrophages. RESULTS: Both eyes showed neovascular AMD consisting of mixed fibrocellular and fibrovascular membranes. Capillaries in both the choriocapillaris and the neovascular membrane were patent in both eyes. Macrophages were present in the choroidal neovascularization of both eyes. Neither preexistent choroidal, intraretinal, nor neovascular vessels showed increased wall thickness as sign of radiation damage. CONCLUSION: No radiation-related histopathologic effect could be demonstrated 3 years after radiation therapy in this patient with AMD.

The APO(*)E3-Leiden mouse as an animal model for basal laminar deposit.

AIM: To investigate the APO(*)E3-Leiden mouse as an animal model for age related maculopathy (ARM) related extracellular deposits. METHODS: Eyes were obtained from APO(*)E3-Leiden transgenic mice on a high fat/cholesterol (HFC) diet (n=12) or on a normal mouse chow (n=6), for 9 months. As controls, eyes were collected from APO-E knockout mice on the same diets. From each mouse one eye was processed for microscopic evaluation and immunohistochemistry with a polyclonal antibody directed against human apo-E. Electron microscopy was also performed. RESULTS: All 12 eyes of the APO(*)E3-Leiden mice on an HFC diet contained basal laminar deposit (BLD; class 1 to class 3), whereas two of six APO(*)E3-Leiden mice on normal chow showed BLD class 1. The ultrastructural aspects of this BLD were comparable with those seen in early BLD in humans, and BLD showed immunoreaction with anti-human-apo-E antibodies. No BLD was found in any of the control mice. Drusen were not detected in any of the mice. CONCLUSION: These results indicate that APO(*)E3-Leiden mice can be used as animal model for the pathogenesis of BLD, and that a HFC diet enhances the accumulation of this deposit. Furthermore, this study supports the previously suggested involvement of dysfunctional apo-E in the accumulation of extracellular deposits in ARM.

Herpes simplex virus-specific T cells infiltrate the cornea of patients with herpetic stromal keratitis: no evidence for autoreactive T cells.

PURPOSE: Herpetic stromal keratitis (HSK) is a T-cell-mediated inflammatory disease initiated by a herpes simplex virus (HSV) infection of the cornea. Recently, studies in the HSK mouse model have shown that the immunopathogenic T cells are directed against the HSV protein UL6 cross-reacting with an unknown corneal autoantigen. Whether this type of autoimmunity plays a role in human HSK was analyzed. METHODS: T-cell lines (TCLs) were generated from corneal buttons of 12 patients with different clinical stages of HSV-induced necrotizing stromal keratitis (n = 9) or immune stromal keratitis (n = 3). The initiating virus was identified by polymerase chain reaction and immunohistology performed on the corneal buttons. Peripheral blood mononuclear cells (PBMCs) were isolated, and B cell lines (BLCLs) were generated by transformation with Epstein-Barr virus. Proliferative responses of these intracorneal TCLs were determined by culturing T cells with autologous BLCLs infected with HSV-1, HSV-2, wild-type vaccinia virus (VV-WT), or VV expressing HSV-1 UL6 (rVV-UL6). Alternatively, T cells were incubated with PBMCs pulsed with human cornea protein extract. RESULTS: Irrespective of clinical diagnosis or treatment, T cells were recovered from the corneal buttons of all the 12 HSK patients. The intracorneal TCLs of 9 of the 12 HSK patients showed HSV-specific T-cell reactivity. In none of the TCLs, T-cell reactivity against HSV-1 UL6 or human corneal antigens was detected. CONCLUSIONS: These data suggest that the potentially immunopathogenic intracorneal T-cell response in HSK patients is directed to the initiating virus and not to a human corneal autoantigen or HSV-1 UL6.

Apoptosis is present in the primate macula at all ages.

BACKGROUND: It has become increasingly clear that apoptosis is a main event in photoreceptor cell death in a variety of retinal degenerations. We investigated the role of apoptosis in the physiologically aging primate macula. METHODS: Twenty maculae of rhesus monkeys, aged 6-34 years, were investigated. Apoptosis was determined in formalin-fixed, paraffin-embedded eyes using the TUNEL (TdT-mediated dUTP-biotin nick end labeling) method and quantitatively analyzed. Morphology of TUNEL-positive cells was studied by confocal laser microscopy and transmission electron microscopy. The thickness of the outer nuclear layer (ONL) was determined by image analysis. Furthermore, expression of apoptosis-regulating proteins Bcl-x, Fas and Fas Ligand was studied by immunohistochemistry. RESULTS: TUNEL-positive nuclei showed apoptotic features on confocal laser microscopy. They were scattered and sparsely found in the macula, most frequently in the ONL. The thickness of the ONL decreased with increasing age. Apoptosis was found equally distributed at all ages, although in the two oldest maculae up to 13 times more apoptosis was found. Expression of Bcl-x, Fas and Fas Ligand was equal at all ages. CONCLUSION: Our findings indicate that apoptosis in the primate macula occurs at all ages at similar rates, possibly increasing in the oldest age group, and may account for the decreasing thickness of the primate macula with age.

Somatostatin receptor 2A expression in choroidal neovascularization secondary to age-related macular degeneration.

PURPOSE: The growth of ocular neovascularization is regulated by a balance between stimulating and inhibiting growth factors. Somatostatin affects angiogenesis by inhibiting the growth hormone-insulin-like growth factor axis and also has a direct antiproliferative effect on human retinal endothelial cells. The purpose of our study is to investigate the expression of somatostatin receptor (sst) subtypes and particularly sst subtype 2A (sst2A) in normal human macula, and to study sst2A in different stages of age-related maculopathy (ARM), because of the potential anti-angiogenic effect of somatostatin analogues. METHODS: Sixteen eyes (10 enucleated eyes, 4 donor eyes, and 2 surgically removed choroidal neovascular [CNV] membranes) of 15 patients with eyes at different stages of ARM were used for immunohistochemistry. Formaldehyde-fixed paraffin-embedded slides were incubated with a polyclonal anti-human sst2A antibody. mRNA expression of five ssts and somatostatin was determined in the posterior pole of three normal human eyes by reverse transcriptase-polymerase chain reaction. RESULTS: The immunohistochemical expression of sstA in newly formed endothelial cells and fibroblast-like cells was strong in fibrovascular CNV membranes. mRNA of sst subtypes 1, 2A, and 3, as well as somatostatin, was present in the normal posterior pole; sst subtypes 4 and 5 were not detectable. CONCLUSIONS: Most early-formed CNV in ARM express sst2A. The presence of mRNA of sst subtype 2A was observed in normal human macula, and subtypes 1 and 3 and somatostatin are also present. sst2A receptors bind potential anti-angiogenic somatostatin analogues such as octreotide. Therefore, somatostatin analogues may be an effective therapy in early stages of CNV in ARM.

Perinatally ruptured dermoid cyst presenting as congenital oculomotor palsy.

An eight-month-old girl presented with congenital exotropia and latent nystagmus. Further evaluation revealed congenital ptosis of the left eye and restriction of the elevation, depression and adduction of the left eye. A diagnosis of congenital oculomotor palsy was made. At the age of three months she had been examined by the neurologist because of retarded psychomotor development. All laboratory investigations were normal. At the age of eight months, a CT scan of the brain and orbit was found to be normal. The patient was treated for amblyopia. At the age of five, strabismus surgery was performed, and a large fibrous tumor encapsulating the superior and lateral rectus muscle was found. A biopsy was taken and pathology showed fibrous tissue containing a hair. Based on the clinical history, the diagnosis of a perinatally ruptured orbital dermoid cyst was made. Review of the previous CT and an additional CT showed enlargement of the left lateral orbital wall with a notch in the lateral wall, indicative of a dermoid cyst.

CHARGE association-related ocular pathology in a newborn with partial trisomy 19q and partial monosomy 21q, from a maternal translocation (19;21) (q13.1;q22.3).

We report a novel case of partial trisomy 19q and concomitant partial monosomy 21q, segregated from a maternal translocation (19;21) (q13.1;q22.3), identified by spectral karyotyping. Clinical examination revealed dysmorphic features of the face and limbs, cleft palate, bilateral colobomas with associated bilateral colobomatous optic nerve cysts, hearing loss, and a cardiac anomaly. At autopsy, the dysmorphic features and cleft palate were confirmed. The ocular histopathology is described in detail and the cardiac anomaly was further specified. The combination of phenotype features is diagnostic of the CHARGE (coloboma, heart malformation, atresia choanae, retarded growth and development, and/or CNS anomalies, genital hypoplasia, ear anomalies and/or deafness) association. This case also has some phenotypic features in common with previous cases of partial trisomy 19q. The importance of a complete autopsy in cases with multiple congenital anomalies and/or genetic abnormalities is emphasized. This will allow optimal genetic counseling and contribute to our understanding of developmental biology.

New biodegradable networks of poly(N-vinylpyrrolidinone) designed for controlled nonburst degradation in the vitreous body.

Polymers of N-vinylpyrrolidinone (NVP) are known to have excellent biocompatibility when implanted in the vitreous body or used as a vitreous substitute. Although poly(NVP) is capable of absorbing relatively large amounts of water, it is not prone to hydrolysis. Yet intraocular degradation of several crosslinked poly(NVP) hydrogels has been reported recently, but some ambiguity remains about the exact mechanism of degradation of these materials. To date there is no biomaterial that combines the excellent intraocular biocompatibility on the one hand and controlled kinetics of degradation on the other hand. We attempted to design and prepare such materials through the chemical synthesis of a novel dimethacrylate crosslinker molecule. The essential feature of this molecule is that its core contains two carbonate groups, which are evidently susceptible to hydrolytic scission. We studied a series of 3-dimensional networks of poly(NVP), which were crosslinked by this molecule. This approach offers several advantages: the hydrolysis of the carbonate groups in the crosslinks leads to liberation of poly(NVP) and/or oligo(NVP) chains that can probably be cleared from the eye via phagocytosis; hydrolysis generates two alcohols and CO(2) (i.e., there is no catalytic burst effect); when these materials are implanted in dry form, swelling and degradation will progress from the exterior of the material toward its interior. Therefore, these materials can be designed such that surface degradation rather than bulk degradation occurs; the hydrolysis rate can be controlled via the crosslink density or through synthesis of other crosslink molecules with either more (>2) or less (1) carbonate groups or alternatively with one or more other labile groups. We report on the chemical synthesis of the crosslinker molecule, as well as the preparation and degradation of a series of poly(NVP)-based hydrogels in vitro and in vivo (rabbit eyes). We found that these materials indeed displayed excellent biocompatibility in the rabbit eye. Further, the experiments confirmed that degradation occurs without the burst effect. The results are in line with the idea that the rate of intraocular swelling and degradation depends on the crosslink density, but this is only a preliminary conclusion that must be strengthened by much more experimental work. Nonetheless, we foresee several applications of these or related materials in ophthalmology, for example, as biodegradable matrix materials for controlled drug delivery of ganciclovir in the vitreous body.