Search PubMed⌕ Search

Biomedical subjects

Ian Grierson

Publications and source records attributed to Ian Grierson.

At least 19 recordsLinked to original sources
PubMed2007

Polydimethylsiloxane as a substrate for retinal pigment epithelial cell growth.

Retinal pigment epithelial (RPE) cell transplantation represents potential treatment for age-related macular degeneration (AMD). Because delivery of isolated cells can cause serious complications, it is necessary to develop a suitable transplant membrane that could support an intact functioning RPE monolayer. Polydimethylsiloxane (PDMS) possesses the physical properties required for a transplanting device and is widely used clinically. We have investigated the use of PDMS as a potential surface for the growth of healthy RPE monolayers. PDMS discs were surface modified by air and ammonia gas plasma treatments. Dynamic contact angles were measured to determine the changes in wettability. Human ARPE-19 cells were seeded onto untreated and treated samples. Cell number, morphology and monolayer formation, cytotoxicity, and phagocytosis of photoreceptor outer segments (POS) were assessed at set time-points. Air plasma treatment increased the wettability of PDMS. This significantly enhanced cell growth, reaching confluence by day 7. Immunofluorescence revealed well-defined actin staining, monolayer formation, and high cell viability on air plasma treated and untreated surfaces, and to a lesser extent, on ammonia plasma treated. Furthermore, RPE monolayers were able to demonstrate phagocytosis of POS in a time-dependent manner similar to control. PDMS can support an intact functional monolayer of healthy differentiated RPE cells.

Cell Line↗
PubMed2006

Expression of p53-induced apoptosis effector PERP in primary uveal melanomas: downregulation is associated with aggressive type.

Expression of PERP (p53 apoptosis effector related to PMP-22) was investigated in primary uveal melanomas and its variation was analyzed in relation to clinico-pathological and cytogenetical characteristics of these tumors. The transcriptional level of PERP gene was measured by quantitative real-time RT-PCR in 26 uveal melanomas with known chromosomes 3 and 8 status. PERP protein levels were assessed by Western blot analysis of 22 fresh-frozen tumors and by immunohistochemical analysis of 16 paraffin-embedded tumor specimens. Differential expression of PERP was identified in primary choroidal melanoma specimens, both at transcriptional and protein level. Reduced PERP mRNA level was significantly associated with monosomy 3 (two-way ANOVA and t-test, p=0.004) but not with gains in chromosome 8. Transcriptional downregulation of PERP did not present a statistically significant association with ciliary body involvement, size, PAS-positive loops or cell type. Immunoblotting and immunohistochemistry further demonstrated significantly reduced PERP protein level in monosomy 3 melanomas, as compared with disomy 3 tumors. The altered expression of PERP highlighted this apoptosis-specific target of p53 as a possible contributor to apoptosis in uveal melanoma with PERP downregulation being particularly relevant to the aggressive (monosomy 3) type of uveal melanoma. As PERP is a novel type of p53 effector that is likely to stimulate apoptosis through a mechanism distinct from that of Bcl-2-related mitochondrial effectors, further elucidation of its role in uveal melanoma pathogenesis will assist in the design of novel therapeutic approaches aimed at increasing the rate of apoptosis in this tumor.

Adult↗
PubMed2006

A dual Golgi- and mitochondria-localised Ala25Ser precursor cystatin C: an additional tool for characterising intracellular mis-localisation leading to increased AMD susceptibility.

An artificial mutant Ala25Ser precursor cystatin C was created to help elucidate the cause of intracellular mis-localisation of the biochemically related variant B (Ala25Thr) precursor cystatin C to the mitochondria. Homozygotes of variant B precursor cystatin C were reported to carry an increased susceptibility to developing the exudative form of AMD. Ala25Ser precursor cystatin C shows a dual distribution to the Golgi apparatus and to the mitochondria. This localisation is thus intermediary between that of wild-type cystatin C (targeted to ER/Golgi compartment) and that of variant B precursor cystatin C. Furthermore, the level of secretion of Ala25Ser cystatin C by RPE cells is intermediary between wild type and variant B cystatin C. Ala25Ser precursor cystatin C thus represents a biochemical intermediate between the wild type and the AMD-associated cystatin C and as such, is a novel tool for the investigation of the mechanism of intracellular mis-localisation of variant B cystatin C. Our findings further support the hypothesis that substitution of the alanine residue in the penultimate position of precursor cystatin C signal sequence with a less hydrophobic amino acid residue, such as threonine (as in variant B cystatin C) or serine is sufficient to impair the intracellular trafficking and processing of the protein.

Cystatin C↗
PubMed2006

Modification of the surface properties of a lens material to influence posterior capsular opacification.

PURPOSE: To study the effect of surface properties of materials on cellular behaviour and the formation of posterior capsular opacification (PCO). METHODS: Polymethylmethacrylate, silicone and a hydrophobic acrylic were plasma treated and used in tissue culture. The changes in surface properties were quantified by dynamic contact angle measurements. Bovine lens epithelial cells (BLECs) were seeded onto these materials and cultured for 1 month. Serial photographs were taken. The cells were then fixed and stained to facilitate counting. RESULTS: Plasma treatment significantly increased the hydrophilicity of surfaces. BLECs grew on all surfaces but significantly more cells adhered to the treated than the untreated surfaces. On the untreated surfaces the BLECs had a fibroblastic morphology whereas on the treated surfaces the cells maintained their epithelial morphology. CONCLUSIONS: Posterior capsular opacification is a form of wound healing and the behaviour of lens epithelial cells is central to its progression. Emphasis has been on the elimination of residual lens epithelial cells to combat PCO. This study demonstrated that the phenotype of BLECs was influenced by the surface properties of the intraocular lens materials. Gas plasma treatment of the materials increased their hydrophilicity and allowed the adhered BLECs to maintain their normal epithelial morphology. We believe that controlled growth of lens epithelial cells may reduce the incidence of PCO.

Acrylic Resins↗
PubMed2005

Melanin in the trabecular meshwork is associated with age, POAG but not Latanoprost treatment. A masked morphometric study.

We wished to conduct a light and electron microscopic investigation of pigmentation within the trabecular meshwork of normals and primary open angle glaucoma (POAG) patients. In particular we wished to get a precise determination of whether there was a relationship between pigmentation and age. In addition we wanted to know if there was a difference between normals and POAGs and whether trabecular meshwork hyperpigmentation was associated with topical latanoprost medication. A total of 25 sham trabeculectomies conducted on post mortem donor eyes provided the age-matched normals and there were 62 trabeculectomy specimens from POAG patients. These were masked and the meshwork subjected to qualitative and quantitative morphological investigation. Light and electron microscopy confirmed that most of the trabecular meshwork melanin was phagocytosed and within meshwork cells. The granules were measured and found to be of the large iris epithelial type. Light microscopic morphometric analysis showed that the number of meshwork cell profiles that contained melanin increased both in normals and POAGs with age. However there was nearly three times more pigmented meshwork cells in the POAGs than the normals. The POAGs were divided into three groups of (1) minimal or no medication prior to surgery, (2) maximal medical therapy and (3) maximum medical therapy including latanoprost (12 specimens). All groups were significantly greater that the normals but of the three it was the maximal medical therapy group (without latanoprost) that had the highest pigmentation. We concluded that pigmentation of the meshwork is age-related and it is elevated in POAG by mechanisms unknown. The melanin accumulation seems to be partly due to the disease process, partly as a consequence of chronic antiglaucoma medication but interestingly not due to latanoprost even in patients where there is iris darkening (four specimens).

Administration, Topical↗
PubMed2005

Suppression of thrombospondin 1 and 2 production by herpes simplex virus 1 infection in cultured keratocytes.

PURPOSE: Stromal vascularization is a frequent occurrence in herpes simplex keratitis (HSK) and carries a poor prognosis for penetrating keratoplasty. The pathogenesis may involve disruption of the normal equilibrium between angiogenic and anti-angiogenic factors in and around the cornea. Thrombospondin (TSP) 1 and 2 are multifunctional matricellular glycoproteins with potent anti-angiogenic properties and are expressed by human keratocytes in a stromal wound repair model. We hypothesize that the synthesis of these anti-angiogenic proteins by keratocytes is inhibited by HSV1 and that such a mechanism may contribute to stromal vascularization in HSK. METHODS: Nonconfluent monolayers of human keratocytes were infected with HSV1 at a multiplicity of infection of 5 virus particles/keratocyte. Expression of TSP1 and TSP2 was determined by immunohistochemistry and SDS-polyacrylamide gel electrophoresis at 0, 2, 4, 6, 8, 24, 48, and 72 h after infection (ai). Expression of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) served as a control. Expression of immediate early and late viral proteins was also determined. Protein expression was quantified by densitometric analysis of the immunoblot bands. RESULTS: Human keratocytes supported the growth of HSV1 at all times ai. TSP1 and TSP2 were downregulated as early as 4 h ai to a 50% reduction by 8 h (p<0.002), and were absent from 24 h ai (p<0.001). There was no change in the level of expression of GAPDH throughout the duration of the experiment. Immediate early viral proteins (HSV1:ICP27) could be detected from 6 h ai reaching maximum intensity 24 h ai and late proteins (HSV:1gD) were expressed from 24 h. CONCLUSIONS: The synthesis of TSP1 and TSP2 is selectively downregulated by HSV1 infection in human keratocytes. Addition of these proteins or their angio-active peptides in early stage HSK therapy may be an important adjuvant in controlling HSV1 induced corneal vascularization.

Blotting, Western↗
PubMed2005

Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue.

Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.

Actin Cytoskeleton↗
PubMed2005

Differential expression of angioregulatory matricellular proteins in posterior uveal melanoma.

Metastases from uveal melanoma, the most common primary malignant eye tumour in adults, develop solely via their vascular bed due to the absence of intraocular lymphatics. The present study investigated the expression in this tumour of three matricellular proteins--Secreted Protein Acidic and Rich in Cysteine (SPARC), thrombospondin 1 (TSP1) and thrombospondin 2 (TSP2)--with putative contrasting roles in the regulation of angiogenesis. Immunohistochemical analysis of the three proteins was carried out in paraffin-embedded specimens from 27 posterior uveal melanomas and was corroborated with Western blot analysis of fresh-frozen samples from seven of the tumours. SPARC immunoreactivity was detected in all specimens and defined two categories of tumour: SPARC-rich (21 of 27 specimens) and SPARC-patchy (six of 27 specimens) uveal melanomas. SPARC-rich tumours had a significantly higher proportion of specimen area occupied by blood vessels (P=0.04) and showed a positive association with the presence of epithelioid-type tumoral cells (P=0.101). TSP1 was not detected by either of the methods in any of the tumours analysed. Some immunopositivity for TSP2 was detected in tumour cells in approximately 40% of specimens, but was not associated with survival, tumour vascularity or any other histopathological indices of survival. The pattern of expression of these matricellular proteins in uveal melanoma is consistent with a cooperative mechanism for establishing an enhanced environment favourable to angiogenesis. Interventions inducing TSP1 expression and/or inhibiting SPARC expression may be candidates for therapies directed towards the inhibition of angiogenesis in posterior uveal melanoma.

Adult↗
PubMed2004

Unexpected intracellular localization of the AMD-associated cystatin C variant.

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.

Aging↗
PubMed2003

Effects of the matricellular protein SPARC on human retinal pigment epithelial cell behavior.

PURPOSE: To determine the effects of the matricellular protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) on human retinal pigment epithelial (HRPE) cell behavior in vitro. METHODS: Proliferation and migration assays were performed on HRPE cells exposed to various concentrations of SPARC. Additionally, HRPE cells were seeded on top of collagen matrices (a 2D model of the retinal scarring disorder known as proliferative vitreoretinopathy or PVR) and were exposed to SPARC over a 7 day period. Changes in matrix contraction were recorded. RESULTS: HRPE cell proliferation was significantly inhibited at 1 and 10 microg/ml SPARC (p<0.01). SPARC protein did not stimulate HRPE cell migration at any of the concentrations used. SPARC did not significantly affect fibronectin-induced HRPE cell migration at SPARC concentrations up to 10 microg/ml. HRPE cell-seeded collagen matrices demonstrated a significant inhibition of matrix contraction by 1 and 10 microg/ml SPARC (t-test; p<0.02 and 0.001, respectively) compared to controls. CONCLUSIONS: SPARC protein has anti-proliferative effects on HRPE cells in vitro. In addition, SPARC appears to have an inhibitory effect on HRPE-mediated contraction of 2D collagen matrices. These results are consistent with an important role for SPARC in modulating cell behavior in vitro and may indicate a role for SPARC in modifying HRPE cell activities during the development of PVR and other proliferative retinal diseases.

Adult↗
PubMed2003

Fine structural evaluation of the iris after unilateral treatment with latanoprost in patients undergoing bilateral trabeculectomy (the Mainz II study).

OBJECTIVE: To investigate by masked electron microscopy whether 6 months of topical latanoprost caused pathological changes in the peripheral iris of patients with glaucoma. METHODS: Seventeen patients with bilateral primary open-angle glaucoma requiring trabeculectomy were recruited for this study. The iridectomy taken during surgery on the first eye served as a control. The second test eye was treated topically with latanoprost for 6 months before its trabeculectomy. Fourteen patients completed the treatment arm of the study, and 1 of these underwent marked color change. As a result, 31 iridectomy specimens were fixed, coded, and evaluated. RESULTS: The specimens were evaluated for evidence of stromal inflammation, vascular alterations, and stromal and posterior epithelial degeneration. None of these features was evident in any of the 31 iridectomy specimens. There was evidence of the incidence of free melanin granules in the stroma, melanin turnover, abundance of stromal clump cells, atypical cellular features in melanocytes, and prominence of the anterior border. After code breaking, it was evident that none of these features distinguished the test from the fellow irises in our group. The patient with color change in the test iris did not stand out from the others in this analysis. Qualitative reexamination of further sections after unmasking gave the impression of increased melanin granule numbers in the melanocytes of the anterior border region. CONCLUSIONS: The ultrastructure of the iridectomies from the latanoprost-treated eyes and the fellow eyes conformed to published standards for normal iris. There was no evidence of early ultrastructural changes, which might have been the harbingers of latanoprost-induced iris abnormality.

Administration, Topical↗
PubMed2003

Latanoprost-induced iris darkening: a morphometric study of human peripheral iridectomies.

PURPOSE: This microscopic study was undertaken to compare the melanocytes of peripheral iridectomy specimens from two eyes that had latanoprost-induced iris darkening (LIID) with iridectomies taken from the fellow untreated eyes. METHODS: The two patients in this study were the ones who underwent LIID in the latanoprost treated eye from a series of 17 patients requiring bilateral trabeculectomy. The first trabeculectomy procedure provided a control peripheral iridectomy for each patient, whereas the second eye was treated with once daily 50 microg ml(-1) latanoprost drops for 6 months. The four peripheral iridectomy specimens from the two LIID patients were subjected to quantitative morphometric analysis by light microscopy of iris cellularity, and electron microscopy of iris melanocyte immature melanosomes and mature melanin granules. RESULTS: There was no significant difference in stromal cellularity between the LIIDs and their respective controls nor were there significant differences in the numbers of immature melanosomes or melanin granules in the melanocytes. However, there was a significant increase in the diameter of melanin granules that was more pronounced in the anterior border layer than the deeper stroma. With the anterior border melanocytes, the increase in melanin granule size was associated with significant increases in granule area and the percentage of cell cytoplasm occupied by melanin (granularity). CONCLUSIONS: The only morphological change identified in two peripheral iridectomies that had LIID when compared to untreated fellow eye specimens was a modest increase in the size of stromal melanocyte melanin granules that was more pronounced in the cells of the anterior border region.

Antihypertensive Agents↗
PubMed2003

Fate of cystatin C lacking the leader sequence in RPE cells.

The study made use of transiently transfected RPE cells expressing mature cystatin C (lacking the 26 amino acids leader sequence of the precursor cystatin C) fused to enhanced green fluorescent protein (EGFP) and, in control transfections, EGFP on its own. The findings demonstrate that 'leaderless' cystatin C is not processed through the secretory pathway of RPE cells. Since a polymorphism in the leader sequence has recently been associated with increased risk for development of exudative age-related macular degeneration (AMD), the present findings lend support to the hypothesis that impairment of function of the leader sequence may contribute to the aetiology of exudative AMD.

5' Untranslated Regions↗
PubMed2003

Monosomy 3 in uveal melanoma: correlation with clinical and histologic predictors of survival.

PURPOSE: To correlate monosomy 3 in uveal melanoma with clinical and histologic prognostic variables and death caused by metastatic disease. METHODS: Loss of heterozygosity (LOH) on chromosome 3 was investigated by PCR-based microsatellite analysis in 105 tumors and related to large basal tumor diameter (LBD), ciliary body (CB) involvement, tumor cell type, periodic acid-Schiff (PAS)-positive loops, and death related to metastatic disease. A model relating monosomy 3 to these was created with forward-stepwise logistic regression and used to derive a prognostic index. RESULTS: Monosomy 3 occurred in 54 (51%) tumors and regional chromosome 3 LOH in another six (6%) tumors. Monosomy 3 was associated with epithelioid cells (chi(2) test, P < 0.001), PAS-positive loops (chi(2), P = 0.001), LBD (Mann-Whitney test, P = 0.002), CB involvement (chi(2) test, P = 0.008), and metastasis-related death (log rank analysis, P = 0.0003). The regression coefficients indicated that epithelioid histology was 15 times as influential with each millimeter of increase in LBD. A prognostic score was derived: one point for each LBD category (<7.4, 7.5-12.4, 12.5-17.4, and >17.4 mm) and three points for epithelioid histology. The prevalence of monosomy 3 increased with score, from 0% in 18 tumors scoring less than 4 to 95% in 21 tumors scoring 7. CONCLUSIONS: Monosomy 3 correlates with survival but can be predicted only in patients with large epithelioid tumors. The absence of monosomy 3 is predictable only in patients who have small, spindle-cell tumors. In most patients, prediction of monosomy 3 according to tumor size and histology is unreliable.

Adult↗