How Does Blood-Retinal Barrier Breakdown Relate to Death and Disability in Pediatric Cerebral Malaria?

BACKGROUND: In cerebral malaria, the retina can be used to understand disease pathogenesis. The mechanisms linking sequestration, brain swelling, and death remain poorly understood. We hypothesized that retinal vascular leakage would be associated with brain swelling. METHODS: We used retinal angiography to study blood-retinal barrier integrity. We analyzed retinal leakage, histopathology, brain magnatic resonance imaging (MRI), and associations with death and neurological disability in prospective cohorts of Malawian children with cerebral malaria. RESULTS: Three types of retinal leakage were seen: large focal leak (LFL), punctate leak (PL), and vessel leak. The LFL and PL were associated with death (odds ratio [OR] = 13.20, 95% confidence interval [CI] = 5.21-33.78 and OR = 8.58, 95% CI = 2.56-29.08, respectively) and brain swelling (P < .05). Vessel leak and macular nonperfusion were associated with neurological disability (OR = 3.71, 95% CI = 1.26-11.02 and OR = 9.06, 95% CI = 1.79-45.90). Large focal leak was observed as an evolving retinal hemorrhage. A core of fibrinogen and monocytes was found in 39 (93%) white-centered hemorrhages. CONCLUSIONS: Blood-retina barrier breakdown occurs in 3 patterns in cerebral malaria. Associations between LFL, brain swelling, and death suggest that the rapid accumulation of cerebral hemorrhages, with accompanying fluid egress, may cause fatal brain swelling. Vessel leak, from barrier dysfunction, and nonperfusion were not associated with severe brain swelling but with neurological deficits, suggesting hypoxic injury in survivors.

Secretory proteostasis of the retinal pigmented epithelium: Impairment links to age-related macular degeneration.

Secretory proteostasis integrates protein synthesis, processing, folding and trafficking pathways that are essential for efficient cellular secretion. For the retinal pigment epithelium (RPE), secretory proteostasis is of vital importance for the maintenance of the structural and functional integrity of apical (photoreceptors) and basal (Bruch's membrane/choroidal blood supply) sides of the environment it resides in. This integrity is achieved through functions governed by RPE secreted proteins, which include extracellular matrix modelling/remodelling, angiogenesis and immune response modulation. Impaired RPE secretory proteostasis affects not only the extracellular environment, but leads to intracellular protein aggregation and ER-stress with subsequent cell death. Ample recent evidence implicates dysregulated proteostasis as a key factor in the development of age-related macular degeneration (AMD), the leading cause of blindness in the developed world, and research aiming to characterise the roles of various proteins implicated in AMD-associated dysregulated proteostasis unveiled unexpected facets of the mechanisms involved in degenerative pathogenesis. This review analyses cellular processes unveiled by the study of the top 200 transcripts most abundantly expressed by the RPE/choroid in the light of the specialised secretory nature of the RPE. Functional roles of these proteins and the mechanisms of their impaired secretion, due to age and genetic-related causes, are analysed in relation to AMD development. Understanding the importance of RPE secretory proteostasis in relation to maintaining retinal health and how it becomes impaired in disease is of paramount importance for the development and assessment of future therapeutic advancements involving gene and cell therapies.

Histopathological Changes and Clinical Outcomes following Intervention for Sub-Internal Limiting Membrane Haemorrhage.

INTRODUCTION: Haemorrhage confined to the sub-internal limiting membrane (ILM) space can be associated with good visual recovery. There is controversy as to the best management of purely sub-ILM haemorrhage, which ranges from observation to immediate surgical intervention. METHODS: We studied a retrospective case series of patients with sub-ILM haemorrhage who underwent vitrectomy with subsequent histological analysis of the removed ILM. RESULTS: Sixteen patients underwent vitrectomy for sub-ILM haemorrhage. Five patients had underlying Terson syndrome, 6 had ruptured macro-aneurysms, and 5 had Valsalva retinopathy. Seven patients demonstrated cellular proliferation on the retinal surface of the ILM with staining for glial fibrillary acidic protein and cytokeratin 7, as well as CD68pg and Prussian blue. All but 1 of these cases were isolated from patients undergoing surgery >4 weeks following initial symptoms, the other presented at >2 weeks. Serial optical coherence tomography (OCT) was available in 8 patients; serial OCT in patients with delayed intervention demonstrated persistent inner retinal layer hyper-reflectance. Fourteen of 15 patients demonstrated symptomatic recovery and showed visual improvement with acuity ranging from -0.1 to 1.8 (mean 0.43) within 3 months of intervention (1 was lost to follow-up). The post-operative vision was 0.11 logMAR (mean; range -0.1 to 0.4) at 3 months in the group with intervention within 2 weeks of symptoms, and 0.9 logMAR (mean; range 0.0 to HM) in the group with delayed surgery. CONCLUSIONS: Early surgical intervention for sub-ILM haemorrhage resulted in good visual outcomes; delayed surgery may lead to proliferative vitreoretinopathy-like changes on the inner retinal surface of the ILM, and untreated cases may demonstrate persistent inner retinal changes potentially limiting visual prognosis despite subsequent surgical intervention.

Neurovascular sequestration in paediatric P. falciparum malaria is visible clinically in the retina.

Retinal vessel changes and retinal whitening, distinctive features of malarial retinopathy, can be directly observed during routine eye examination in children with P. falciparum cerebral malaria. We investigated their clinical significance and underlying mechanisms through linked clinical, clinicopathological and image analysis studies. Orange vessels and severe foveal whitening (clinical examination, n = 817, OR, 95% CI: 2.90, 1.96-4.30; 3.4, 1.8-6.3, both p<0.001), and arteriolar involvement by intravascular filling defects (angiographic image analysis, n = 260, 2.81, 1.17-6.72, p<0.02) were strongly associated with death. Orange vessels had dense sequestration of late stage parasitised red cells (histopathology, n = 29; sensitivity 0.97, specificity 0.89) involving 360° of the lumen circumference, with altered protein expression in blood-retinal barrier cells and marked loss/disruption of pericytes. Retinal whitening was topographically associated with tissue response to hypoxia. Severe neurovascular sequestration is visible at the bedside, and is a marker of severe disease useful for diagnosis and management.

Human Conjunctival Stem Cells are Predominantly Located in the Medial Canthal and Inferior Forniceal Areas.

PURPOSE: The conjunctiva plays a key role in ocular surface defence and maintenance of the tear film. Ex vivo expansion of conjunctival epithelial cells offers potential to reconstruct the ocular surface in cases of severe cicatrising disease, but requires initial biopsies rich in stem cells to ensure long-term success. The distribution of human conjunctival stem cells, however, has not been clearly elucidated. METHODS: Whole human cadaveric conjunctiva was retrieved and divided into specific areas for comparison. From each donor, all areas from one specimen were cultured for colony-forming efficiency assays and immunocytochemical studies; all areas from the other specimen were fixed and paraffin embedded for immunohistochemical studies. Expression of CK19, p63, and stem cell markers ABCG2, ΔNp63, and Hsp70 were analyzed. Results were correlated to donor age and postmortem retrieval time. RESULTS: Conjunctiva was retrieved from 13 donors (26 specimens). Colony-forming efficiency and expression of stem cell markers ABCG2, ΔNp63, and Hsp70 in cultures and ABCG2 in fixed tissue were all consistently demonstrated throughout the tissue but with highest levels in the medial canthal and inferior forniceal areas (P < 0.01 for each). Both increasing donor age and longer postmortem retrieval times were associated with significantly lower colony-forming efficiency, stem cell marker expression in cell cultures and ABCG2 expression in fixed tissue. CONCLUSIONS: Biopsies from the medial canthus and inferior forniceal areas, from younger donors, and with short postmortem retrieval times offer the greatest potential to developing conjunctival stem cell-rich epithelial constructs for transplantation.

Severity of retinopathy parallels the degree of parasite sequestration in the eyes and brains of malawian children with fatal cerebral malaria.

BACKGROUND: Malarial retinopathy (MR) has diagnostic and prognostic value in children with Plasmodium falciparum cerebral malaria (CM). A clinicopathological correlation between observed retinal changes during life and the degree of sequestration of parasitized red blood cells was investigated in ocular and cerebral vessels at autopsy. METHODS: In 18 Malawian children who died from clinically defined CM, we studied the intensity of sequestration and the maturity of sequestered parasites in the retina, in nonretinal ocular tissues, and in the brain. RESULTS: Five children with clinically defined CM during life had other causes of death identified at autopsy, no MR, and scanty intracerebral sequestration. Thirteen children had MR and died from CM. MR severity correlated with percentage of microvessels parasitized in the retina, brain, and nonretinal tissues with some neuroectodermal components (all P < .01). In moderate/severe MR cases (n = 8), vascular congestion was more intense (ρ = 0.841; P < .001), sequestered parasites were more mature, and the quantity of extraerythrocytic hemozoin was higher, compared with mild MR cases (n = 5). CONCLUSIONS: These data provide a histopathological basis for the known correlation between degrees of retinopathy and cerebral dysfunction in CM. In addition to being a valuable tool for clinical diagnosis, retinal observations give important information about neurovascular pathophysiology in pediatric CM.

Cerebral malaria in children: using the retina to study the brain.

Cerebral malaria is a dangerous complication of Plasmodium falciparum infection, which takes a devastating toll on children in sub-Saharan Africa. Although autopsy studies have improved understanding of cerebral malaria pathology in fatal cases, information about in vivo neurovascular pathogenesis is scarce because brain tissue is inaccessible in life. Surrogate markers may provide insight into pathogenesis and thereby facilitate clinical studies with the ultimate aim of improving the treatment and prognosis of cerebral malaria. The retina is an attractive source of potential surrogate markers for paediatric cerebral malaria because, in this condition, the retina seems to sustain microvascular damage similar to that of the brain. In paediatric cerebral malaria a combination of retinal signs correlates, in fatal cases, with the severity of brain pathology, and has diagnostic and prognostic significance. Unlike the brain, the retina is accessible to high-resolution, non-invasive imaging. We aimed to determine the extent to which paediatric malarial retinopathy reflects cerebrovascular damage by reviewing the literature to compare retinal and cerebral manifestations of retinopathy-positive paediatric cerebral malaria. We then compared retina and brain in terms of anatomical and physiological features that could help to account for similarities and differences in vascular pathology. These comparisons address the question of whether it is biologically plausible to draw conclusions about unseen cerebral vascular pathogenesis from the visible retinal vasculature in retinopathy-positive paediatric cerebral malaria. Our work addresses an important cause of death and neurodisability in sub-Saharan Africa. We critically appraise evidence for associations between retina and brain neurovasculature in health and disease, and in the process we develop new hypotheses about why these vascular beds are susceptible to sequestration of parasitized erythrocytes.

Age-related changes of cystatin C expression and polarized secretion by retinal pigment epithelium: potential age-related macular degeneration links.

PURPOSE: Cystatin C, a potent cysteine proteinase inhibitor, is abundantly secreted by the RPE and may contribute to regulating protein turnover in the Bruch's membrane (BrM). A cystatin C variant associated with increased risk of developing AMD and Alzheimer's disease (AD) presents reduced secretion levels from RPE. The purpose of this study was to analyze the effects of age and the accumulation of advanced glycation end-products (AGEs) on the expression and secretion of cystatin C by the RPE. METHODS: Confluent monolayers of human fetal RPE (hfRPE) cells were cultured using an in vitro model mimicking extracellular AGE accumulation. Cystatin C expression, secretion, and its polarity were analyzed following culture on AGE-containing BrM mimics (AGEd versus non-AGEd). Monolayer barrier properties were assessed by transepithelial resistance measurements. The relative level of cystatin C protein expression in human RPE in situ was assessed immunohistochemically in relation to age. RESULTS: Advanced glycation end product-exposed RPE monolayers presented significantly decreased cystatin C expression and secretion. Basolateral secretion was fully established by week 8 in non-AGEd conditions. In AGEd cultures, polarity of secretion was impaired despite maintenance of physiological barrier properties of the monolayer. In the macula region of RPE/choroid segments from human eyes, the level of cystatin C protein was reduced with increasing donor age. CONCLUSIONS: Exposure to AGEs reduces expression of cystatin C and affects its normal secretion in cultured RPE. Age-related changes of cystatin C in the RPE from the posterior pole may compromise its extracellular functions, potentially contributing to AMD pathogenesis.

Fibrous membranes in diabetic retinopathy and bevacizumab.

PURPOSE: The purpose of this study was to determine the histopathologic characteristics of bevacizumab-treated human proliferative diabetic retinopathy (PDR) membranes with particular regard to membrane vasculature as a step toward addressing the effects of the drug on PDR membranes. Intravitreous injection of bevacizumab, an antivascular endothelial growth factor monoclonal antibody, has recently been advocated as an adjunct in surgery for PDR. In this context, a clinically observed decrease in PDR epiretinal membrane vascularity (vascular regression) occurs from 24 hours to 48 hours after injection, but the exact mechanisms of drug action are unknown. METHODS: A consecutive series of seven PDR membrane specimens that had been removed sequentially from seven bevacizumab-treated patients were studied retrospectively. The membrane specimens were examined using light microscopic methods, including immunohistochemistry. RESULTS: Five of the seven membranes were clinically avascular (one contained "ghost" vessels) and did not hemorrhage during excision. Of these 5 specimens, which included 1 removed 7 days after a total of 6 intravitreous injections of 1.25 mg bevacizumab, 4 contained histologically detectable capillaries (1 did not). These blood vessels were lined by endothelial cells as determined by immunohistochemistry for the endothelial markers CD31 and CD34. The two remaining membranes were clinically and histologically still vascularized despite bevacizumab treatment. All the specimens also contained smooth muscle actin-containing fibroblastic cells within the collagenous stroma. CONCLUSION: The findings do not support the concept that the clinical phenomenon of vascular regression in PDR membranes after bevacizumab injection in the vitreous is resulting from obliteration of the membrane blood vessels. Another mechanism appears to be involved in at least some patients, possibly a vasoconstrictive response. Such a mechanism might explain reversal of the effects of bevacizumab that has been reported after this treatment.

Human retinal pigment epithelial SPARC expression and age: an immunohistochemical study.

The aim of this study was to investigate the temporal and spatial expression of the matricellular protein SPARC (Secreted Protein, Acidic and Rich in Cysteine; also known as osteonectin) in human retinal pigment epithelial (RPE) cells, and compare the results with Bruch's membrane thickness, employing immuno-histochemistry. Eyes from 36 human donors, 16 being 65 years old, were included in the study. Intensity of SPARC immunoreactivity was evaluated using Aequitas Image Analysis software with two-way analysis of variance (ANOVA). Bruch's membrane thickness was assessed by profile analysis and the association between RPE SPARC immunoreactivity and Bruch's membrane thickness was investigated by fitting a linear mixed effects model to the data set. Intensity of SPARC immunostaining in RPE cells was significantly lower in older donors (p<0.05 and p<0.001 for posterior and peripheral RPE cells, respectively). The anatomical localisation of the RPE cells also affected the intensity of SPARC staining, which was lower in posterior compared to peripheral cells (p<0.01). No correlation was observed between SPARC immunoreactivity in RPE cells and the thickness of the underlying Bruch's membrane, in either posterior or peripheral regions. Our results suggest that RPE cell SPARC levels decline with age, a change that may play a role in the pathogenesis of age-related diseases such as age-related macular degeneration.

The use of silicone oil-RMN3 (Oxane HD) as heavier-than-water internal tamponade in complicated inferior retinal detachment surgery.

BACKGROUND: Conventional silicone oil provides suboptimal support of the inferior retina. In this study we evaluated the efficacy of Oxane HD in the management of complex retinal detachments involving lower quadrants of the retina. METHODS: A prospective, interventional, comparative study. Eighteen patients were recruited. Treatment outcomes were compared with a historical control group of 14 patients. Patients with grade C3 PVR or greater and inferior retinal breaks, recurrent inferior retinal detachments (with or without PVR) and giant retinal tears were included. In those patients who re-detached under heavy silicone oil (n = 4), retro-oil epiretinal membranes (ERMs) were obtained at the time of subsequent surgery to analyse the immunopathological response to oxane HD. Immunohistochemistry was used to detect glia, retinal pigment epithelium cells (RPE), macrophages, T lymphocytes, or neural elements in the tissue using well-characterised monoclonal antibodies. RESULTS: Retinal attachment of the posterior pole following removal of silicone oil was achieved in 66.6% of the treatment group (n = 12) and 64.3% of controls (n = 9) (p = 1.0). Post-operative PVR developed in five patients in the treatment group (27.8%) and five control patients (35.8%). Following removal of silicone oil, residual oil was observed in 27.8% of the treatment group and 7.1% of controls. Median visual acuity, 3 months following removal of silicone oil, was 2.0 (IQR 0.9-2.0) in the treatment group and 1.0 (IQR 0.6-1.8) in the control group. Complications in the treatment group included, hypotony (n = 3), uveitis (n = 2), glaucoma (n = 1). All ERMs analysed demonstrated microscopic appearances typical of PVR. The membranes were fibrocellular in nature, contained RPE and glial cells, and variable amounts of intracellular and extracellular pigment. In addition, all had a dense infiltrate of vacuolated (presumed oil-filled) macrophages. CONCLUSION: We failed to observe an advantage following the use of Oxane HD in the treatment of inferior retinal detachments. Moreover, Oxane HD was difficult to remove and was associated with a higher incidence of complications.

Cystatin C in macular and neuronal degenerations: implications for mechanism(s) of age-related macular degeneration.

Cystatin C is a strong inhibitor of cysteine proteinases expressed by diverse cells. Variant B cystatin C, which was associated with increased risk of developing age-related macular degeneration, differs from the wild type protein by a single amino acid (A25T) in the signal sequence responsible for its targeting to the secretory pathway. The same variant conveys susceptibility to Alzheimer disease. Our investigations of the trafficking and processing of variant B cystatin C in living RPE cells highlight impaired secretion of extracellular modulators and inappropriate protein retention in RPE cells as potential molecular mechanisms underpinning macular, and possibly neuronal, degeneration.

Does the presence of an epiretinal membrane alter the cleavage plane during internal limiting membrane peeling?

PURPOSE: To determine whether the presence of a clinically and/or microscopically detectable epiretinal membrane (ERM) alters the cleavage plane during internal limiting membrane (ILM) peeling. DESIGN: Retrospective, observational, immunohistochemical study of ILM specimens using archival formalin-fixed, paraffin-embedded tissue. PARTICIPANTS: Fifty-one patients who had had ILM excision. METHODS: Fifty-one ILM specimens peeled during vitrectomy for various etiologies were examined by light microscopy. The removal of ILM was assisted using Trypan blue (n = 30), indocyanine green (n = 7), or brilliant blue G (n = 14). Monoclonal antibodies to glial fibrillary acidic protein and to neurofilament protein were used to detect glial or neuronal cells respectively on the vitreous or retinal surfaces of the ILM. Specimens were divided into 2 groups: ILM peeled for full-thickness macular hole (MH; n = 31) and ILM peeled after removal of clinically detectable ERM (n = 20). MAIN OUTCOME MEASURES: Primary outcome measure was the localization of immunohistochemical markers to neuronal or glial cells on the vitreous or retinal surfaces of ILM. The secondary outcome measure was the correlation of the results of the primary measure with the dyes used to facilitate ILM peeling. RESULTS: Glial and/or neuronal cells were detected on the retinal surface of the ILM in 10 of 31 (32%) of the MH ILM specimens and in 13 of 20 (65%) of the ILM peeled after ERM excision; the difference was significant (P = 0.02). There was no association between the presence of neuronal and glial cells with the type of dye used (P = 0.2). Of the 23 ILM specimens with cells attached to the retinal surface, 21 (91%) were associated with clinical and/or histologic evidence of ERM and 2 (9%) were not. The correlation between the presence of cells on the vitreous and the retinal surfaces of ILM was high (P<0.0001). CONCLUSIONS: The findings suggest that ERM may be associated with sub-ILM changes that alter the plane of separation during ILM peeling. This study does not confirm any influence of dyes on the cleavage plane during surgery.

Expression of hypoxia-inducible factor-1alpha and -2alpha in human choroidal neovascular membranes.

PURPOSE: Up-regulation of pro-angiogenic cytokine expression occurring secondary to hypoxia in physiologic and pathophysiologic conditions is mediated by the family of transcription regulators know as hypoxia inducible factors (HIF). The present study was undertaken to investigate the expression of HIF occurring in human choroidal neovascularization (CNV) and the posterior segment of young and old eyes. METHODS: Surgically excised CNV from patients with either age-related macular degeneration (AMD; n = 9), punctuate inner choroidopathy (PIC; n = 3) and young normal eyes were immunohistochemically probed with monoclonal antibodies against HIF-1alpha and -2alpha and compared to that for cell markers specific for vascular endothelial cells (CD34), macrophages (CD68), retinal pigment epithelial cells (RPE; panel cytokeratins/CK18) and VEGF. Following secondary antibody amplification, reactions were visualized with fast red chromogen. RESULTS: Cellular immunoreactivity of membranes for HIF-2alpha was strong in eight out of nine AMD specimens but it was only weakly positive for HIF-1alpha in five specimens. In contrast, two out of three PIC specimens were weakly positive for HIF-1alpha but demonstrated no staining for HIF-2alpha. Immunohistochemical analysis revealed areas within the CNV membranes that were predominantly immunopositive for CD68 and cytokeratin indicating the presence of RPE and/or macrophages and that these cells strongly co-localized with the presence of HIF and VEGF. No immunochemical co-localization was observed with HIF and the endothelial cell marker CD34 in any membranes studied. Normal globes also demonstrated HIF-2 positivity to be predominantly localized to the central RPE rather than peripheral RPE irrespective of age of donor. CONCLUSIONS: The localization of HIF expression supports the concept that hypoxia is a major stimulus for the development of submacular wound healing and within this context CNV is but one component of this process.

Labeling of stem cells with monocrystalline iron oxide for tracking and localization by magnetic resonance imaging.

Precise localization of exogenously delivered stem cells is critical to our understanding of their reparative response. Our current inability to determine the exact location of small numbers of cells may hinder optimal development of these cells for clinical use. We describe a method using magnetic resonance imaging to track and localize small numbers of stem cells following transplantation. Endothelial progenitor cells (EPC) were labeled with monocrystalline iron oxide nanoparticles (MIONs) which neither adversely altered their viability nor their ability to migrate in vitro and allowed successful detection of limited numbers of these cells in muscle. MION-labeled stem cells were also injected into the vitreous cavity of mice undergoing the model of choroidal neovascularization, laser rupture of Bruch's membrane. Migration of the MION-labeled cells from the injection site towards the laser burns was visualized by MRI. In conclusion, MION labeling of EPC provides a non-invasive means to define the location of small numbers of these cells. Localization of these cells following injection is critical to their optimization for therapy.

Clinicopathological case series of four patients with inherited macular disease.

PURPOSE: To correlate the phenotype of four patients with inherited macular disease with the immunohistopathology of retinal tissue collected at the time of retinal pigment epithelium (RPE)-choroidal transplantation. METHODS: A clinicopathologic case series describing the phenotype of four patients, including confocal immunohistochemistry and electron microscopy (EM), and the results of genetic testing. RESULTS: In Case 1, electrophysiology showed only macular dysfunction. Confocal microscopy revealed minor abnormalities. EM showed abnormal cone inner segments with swollen mitochondria. In case 2 (R172W mutation in RDS), electrophysiology demonstrated generalized cone system dysfunction with severe macular involvement. Peripherin labeling of outer segments was nonuniform, and EM showed discs arranged in whorllike structures. Case 3 showed severe central macular dysfunction on multifocal electroretinogram (ERG). Peripherin staining was irregular and disorganized. EM revealed abnormal inner segment morphology, particularly in rods, and disorganized irregular outer segments. Case 4 had localized central macular dysfunction on multifocal ERG. Confocal microscopy was grossly normal, with evidence of early redistribution of cone opsin to the inner segment. EM showed variable rod morphology and normal cones. CONCLUSIONS: RPE transplantation provides a unique opportunity to gain insight into retinal disorders by enabling phenotypic correlation with the immunohistopathology of retinal tissue collected during surgery.

Cathepsin S and its inhibitor cystatin C: imbalance in uveal melanoma.

The present study aimed to investigate, as a follow-up of microarray profiling, the expression of the lysosomal cysteine protease cathepsin S and that of its endogenous inhibitor cystatin C in the most common primary intraocular tumor in adults, uveal melanoma. The expression pattern unveiled was characterized by a relative increase in the active form of the elastolytic and collagenolytic cathepsin S that was not counterbalanced by the expression of its strongest endogenous inhibitor cystatin C in the aggressive, highly metastatic uveal melanomas. The study provides evidence for a novel correlation between a specific cysteine protease activity and the strongest predictive factor for metastatic behavior in primary uveal melanoma and documents the first investigation of both a specific protease activity and its endogenous inhibitor in uveal melanoma. The results indicate that the shift in the balance between cathepsin S and cystatin C may be part of deregulated proteolytic pathways contributing to the invasive phenotype of uveal melanoma.