Biomedical subjects

Peter F Sharp

Publications and source records attributed to Peter F Sharp.

12 recordsLinked to original sources

PubMed2006

Automatic detection of retinal anatomy to assist diabetic retinopathy screening.

Screening programmes for diabetic retinopathy are being introduced in the United Kingdom and elsewhere. These require large numbers of retinal images to be manually graded for the presence of disease. Automation of image grading would have a number of benefits. However, an important prerequisite for automation is the accurate location of the main anatomical features in the image, notably the optic disc and the fovea. The locations of these features are necessary so that lesion significance, image field of view and image clarity can be assessed. This paper describes methods for the robust location of the optic disc and fovea. The elliptical form of the major retinal blood vessels is used to obtain approximate locations, which are refined based on the circular edge of the optic disc and the local darkening at the fovea. The methods have been tested on 1056 sequential images from a retinal screening programme. Positional accuracy was better than 0.5 of a disc diameter in 98.4% of cases for optic disc location, and in 96.5% of cases for fovea location. The methods are sufficiently accurate to form an important and effective component of an automated image grading system for diabetic retinopathy screening.

Automation↗

PubMed2006

Automated microaneurysm detection using local contrast normalization and local vessel detection.

Screening programs using retinal photography for the detection of diabetic eye disease are being introduced in the UK and elsewhere. Automatic grading of the images is being considered by health boards so that the human grading task is reduced. Microaneurysms (MAs) are the earliest sign of this disease and so are very important for classifying whether images show signs of retinopathy. This paper describes automatic methods for MA detection and shows how image contrast normalization can improve the ability to distinguish between MAs and other dots that occur on the retina. Various methods for contrast normalization are compared. Best results were obtained with a method that uses the watershed transform to derive a region that contains no vessels or other lesions. Dots within vessels are handled successfully using a local vessel detection technique. Results are presented for detection of individual MAs and for detection of images containing MAs. Images containing MAs are detected with sensitivity 85.4% and specificity 83.1%.

Algorithms↗

PubMed2006

Automated assessment of diabetic retinal image quality based on clarity and field definition.

PURPOSE: To evaluate the performance of an automated retinal image quality assessment system for use in automated diabetic retinopathy grading. METHODS: Algorithmic methods have been developed for assessing the quality of 45 degrees single field retinal images for use in diabetic retinopathy screening. For this purpose, image quality was defined by two aspects: image clarity and field definition. An image with adequate clarity was defined as one that shows sufficient detail for automated retinopathy grading. The visibility of the macular vessels was used as an indicator of image clarity, since these vessels are known to be narrow and become less visible with any image degradation. An image with adequate field definition was defined as one that shows the desired field of view for retinopathy grading, including the full 45 degrees field of view, the optic disc, and at least two optic disc diameters of visible retina around the fovea. From 489 patients attending a diabetic retinopathy screening program, 1039 retinal images were obtained. The images were graded by a clinician for image clarity and field definition, with a comprehensive image-quality grading scheme. RESULTS: The sensitivity and specificity were, respectively, 100% and 90.9% for inadequate clarity detection, 95.3% and 96.4% for inadequate field definition detection, and 99.1% and 89.4% for inadequate overall quality detection. CONCLUSIONS: The automated system performs with sufficient accuracy to form part of an automated diabetic retinopathy grading system.

Adolescent↗

PubMed2005

Involvement of CCR5 in the passage of Th1-type cells across the blood-retina barrier in experimental autoimmune uveitis.

Although the recruitment of T helper cell type 1 (Th1)/Th2 cells into peripheral tissues is essential for inflammation and the host response to infection, the traffic signals that enable the distinct positioning of Th1/Th2 cells are unclear. We have determined the role of CC chemokine receptor 5 (CCR5) in this using experimental autoimmune uveitis (EAU) as a model system. In EAU, Th1-like cells are preferentially recruited into the retina across the blood-retina barrier, partly as a result of expression of the adhesion molecules P-selectin glycoprotein ligand 1 and lymphocyte function-associated antigen-1 on these cells. CD3+ T cells, infiltrating the retina, also expressed the chemokine receptor CCR5, and CCR5 ligands, macrophage-inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated on activation, normal T expressed and secreted (RANTES), were strongly expressed in the retina at peak EAU. Th1-like cells, polarized in vitro, expressed high levels of CCR5. The trafficking of these CCR5+ cells was examined by tracking them after adoptive transfer in real time in vivo at an early disease stage using scanning laser ophthalmoscopy. Treatment of the cells with antibody against CCR5 prior to transfer resulted in a reduction in their infiltration into the retina. However, rolling velocity, rolling efficiency, and adherence of the cells to retinal endothelium were not reduced. CCR5 is clearly important for Th1 cell recruitment, and this study demonstrates for the first time in vivo that CCR5 may act at the level of transendothelial migration rather than at the earlier stage of rolling on the endothelium.

Adoptive Transfer↗

PubMed2005

Ultra-wide-field fluorescein angiography of the ocular fundus.

PURPOSE: To evaluate the clinical applicability of a novel scanning laser ophthalmoscopy imaging system for ultra-wide-field fluorescein angiography. DESIGN: Observational case report. METHODS: Wide-field fluorescein angiography images were captured in normal volunteers and in patients with a variety of clinical disorders with a newly developed scanning laser ophthalmoscope (Optomap; Optos PLC., Dunfermline, Scotland, United Kingdom) that produces high-resolution images of the fundus up to a 200-degree field. Fluorescein angiograms were performed using standard sequence times. RESULTS: Peripheral vascular perfusion of all four quadrants of a normal fundus was observed with good detail of the capillary architecture. Patients with diabetes and ocular inflammatory eye disease showed evidence of peripheral retinal nonperfusion. The relative degrees of retinal perfusion and nonperfusion could be compared in single images. CONCLUSIONS: Ultra-wide-field fundus fluorescein angiography offers the possibility of evaluating and quantitating peripheral retinal perfusion and vascular pathology in fundus disease.

Fluorescein Angiography↗

PubMed2004

Recruitment of IFN-gamma-producing (Th1-like) cells into the inflamed retina in vivo is preferentially regulated by P-selectin glycoprotein ligand 1:P/E-selectin interactions.

Although there is evidence that altering the Th1/Th2 balance toward Th2 cells may be important in the resolution of Th1-type autoimmune disease, adoptive transfer of Th2 cells is not effective in protecting against Th1-type disease and may cause disease. Therefore, we examined the recruitment of Th1- and Th2-like cells into the retina in the murine autoimmune disease experimental autoimmune uveoretinitis. CD4 T cells were polarized in vitro to IFN-gamma-producing Th1-like cells and non-IFN-gamma-producing Th2-like cells, labeled, and adoptively transferred. Trafficking to the retina in vivo was evaluated by scanning laser ophthalmoscopy and infiltration by confocal microscopy. There were more rolling and adherent Th1-like cells and they rolled more slowly than did Th2-like cells. Th1-like cells were preferentially recruited into the retinal parenchyma at both initiation and resolution. Surface P-selectin glycoprotein ligand 1 (PSGL-1) and LFA-1 were up-regulated on both populations but were expressed at higher levels on Th1-like cells. Up-regulation of CD44 expression was higher on Th2-like cells. P-selectin, E-selectin, and ICAM-1 are up-regulated on postcapillary venules in the retina. Pretreatment of Th1-like cells with anti-PSGL-1 inhibited rolling and infiltration of Th1-like cells but not Th2-like cells, providing direct in vivo evidence for the inability of Th2 to respond to P/E-selectin despite increased expression of PSGL-1. Anti-LFA-1 pretreatment inhibited infiltration of both Th1- and Th2-like cells, but more so Th-1. We suggest that random trafficking of activated T cells (both Th1 and Th2) across the blood-retina barrier is mediated by CD44:CD44R and LFA-1:ICAM-1, whereas preferential recruitment of Th1 cells is mediated by PSGL-1:P/E-selectin.

Animals↗

PubMed2003

Reduction in shear stress, activation of the endothelium, and leukocyte priming are all required for leukocyte passage across the blood--retina barrier.

The passage of leukocytes across the blood-retina barrier at the early stages of an inflammatory reaction is influenced by a complex series of interactions about which little is known. In particular, the relationship between hydrodynamic factors, such as shear stress and leukocyte velocity, to the adherence and subsequent extravasation of leukocytes into the retina is unclear. We have used a physiological method, scanning laser ophthalmoscopy, to track labeled leukocytes circulating in the retina, followed by confocal microscopy of retinal flatmounts to detect infiltrating cells at the early stage of experimental autoimmune uveitis. This has shown that retinal vessels are subjected to high shear stress under normal circumstances. During the inflammatory reaction, shear stress in retinal veins is reduced 24 h before leukocyte infiltration. This reduction is negatively correlated with leukocyte rolling and sticking in veins and postcapillary venules, the sites of leukocyte extravasation. Activation of vascular endothelial cells is also a prerequisite for leukocyte rolling and infiltration. In addition, antigen priming of leukocytes is influential at the early stage of inflammation, and this is seen clearly in the reduction in rolling velocity and adherence of the primed leukocytes in activated retinal venules, 9 days postimmunization.

Animals↗

PubMed2003

Effect of anti-macrophage inflammatory protein-1alpha on leukocyte trafficking and disease progression in experimental autoimmune uveoretinitis.

This study has enabled us to identify the influence of the chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha), on leukocyte behavior at the blood-retina barrier in vivo and its link with the inflammatory process and disease pathogenesis. MIP-1alpha has not previously been thought to be effective under conditions of physiological shear flow. However, short-term anti-MIP-1alpha treatment inhibited leukocyte slowing and accumulation and subsequent extravasation of leukocytes at the blood-retina barrier in animals with experimental autoimmune uveoretinitis. This was effective predominantly in the post-capillary venules which have been shown to be the main site of passage of leukocytes across the blood-retina barrier. Long-term anti-MIP-1alpha treatment also prevented decreased leukocyte velocity and reduced disease severity as measured clinically, histologically and in terms of blood-retina barrier breakdown.

Animals↗

PubMed2003

Requirements for passage of T lymphocytes across non-inflamed retinal microvessels.

Although activated T lymphocytes can migrate through unstimulated neural endothelium to perform immune surveillance or initiate inflammation, the precise mechanism by which this occurs is not clear. In this study, we have used intravital scanning laser ophthalmoscopy to show that circulating, activated T cells induce early changes in the retinal venules that enable T cell diapedesis in the absence of cell rolling, and without any reduction in shear stress within the venules. Concanavalin A (Con A)-activated T cells, but not naive T cells, were able to penetrate the normal blood-retinal barrier (BRB) 8-16 h after adoptive transfer. A minimum number (> or =1 x 10(5) cells/mouse) of Con A-activated T cells needed to be transferred before lymphocytes crossed the normal BRB. Cell rolling and reduction of shear stress did not occur in normal retinal venules and post-capillary venules. In contrast, in mice with experimental autoimmune uveoretinitis (EAU), in which the BRB has broken down, 45% of blast cells were rolling in retinal venules. Cell rolling correlated with significantly reduced shear stress. Both naive and Con A-activated T cells could cross the disabled barrier, with Con A-activated T cells migrating faster and in greater numbers than naive cells. Adoptive transfer of Con A-activated cells into normal recipient mice induced limited and transient breakdown of the BRB and up-regulation of ICAM-1 but not P-selectin. Pretreatment of Con A-activated cells with anti-LFA-1 significantly suppressed T cell infiltration in normal recipient mice. Our data indicate that critical to immune surveillance in the central nervous system (CNS) is the ability of activated T cells to interact with the endothelium, up-regulating ICAM-1 and inducing transient breakdown of the barrier.

Adoptive Transfer↗

PubMed2003

Automated measurement of microaneurysm turnover.

PURPOSE: An automated system for the measurement of microaneurysm (MA) turnover was developed and compared with manual measurement. The system analyses serial fluorescein angiogram (FA) or red-free (RF) fundus images; fluorescein angiography was used in this study because it is the more sensitive test for MAs. Previous studies have shown that the absolute number of MAs observed does not reflect the dynamic temporal nature of the MA population. In this study, almost half of the MAs present at baseline had regressed after a year and been replaced by new lesions elsewhere. METHODS: Two clinical datasets were used to evaluate the performance of the automated turnover measurement system. The first consisted of 10 patients who had two fluorescein angiograms acquired a year apart. These data were analyzed, both manually and using the automated system, to investigate the inter- and intraobserver variations associated with manual measurement and to assess the performance of the automated system. The second dataset contained FAs from a further 25 patients. This dataset was analyzed only with the automated system to investigate some properties of microaneurysm turnover, in particular the differing detection sensitivities of new, static and regressed microaneurysms. RESULTS: Manual measurements exhibited large inter- and intraobserver variation. The sensitivity and specificity of the automated system were similar to those of the human observers. However, the automated measurements were more consistent-an important condition for accurate turnover quantification. Regressed MAs were more difficult to detect reliably than new MAs, which were themselves more difficult to detect reliably than static MAs. CONCLUSIONS: The automated system was shown to be fast, reliable, and repeatable, making it suitable for processing large numbers of images. Performance was similar to that of trained manual observers.

Adult↗

PubMed2002

Improved leukocyte tracking in mouse retinal and choroidal circulation.

The purpose of this study is to develop a new method with which to visualize leukocyte dynamics in murine choroidal and retinal circulation. Both pigmented (B10.RIII) and non-pigmented (BALB/c) mice were used in this study. One hundred microl of 0.05% sodium fluorescein was injected via the mice tail vein to outline the vessel, followed by 150 microl (10(7) cells) C-AM labelled leukocytes. Fundus images were obtained with a confocal scanning laser ophthalmoscope. The dynamic image sequences were recorded simultaneously on videotape (S-VHS) and digitally at 25 frames per sec. The digital images were later analysed with a custom-made personal computer-based image analysis system. Both the choroidal and retinal circulation can be visualized in non-pigmented mice in the first few seconds of fluorescein angiography. However, the view of the choroidal and the retinal capillary circulation is soon blurred due to the rapid fluorescein leakage in the choroid. In contrast, in pigmented mice, retinal circulation is clear against the dark background of the choroid, while choroidal circulation is masked behind the pigment epithelial layer and cannot be seen at all. C-AM labelled leukocytes were clearly seen in the retinal circulation of all experimental mice and in the choroidal circulation of non-pigmented mice for as long as 30 min. The number of labelled circulating cells decreased as time clasped. Cells moved rapidly in the retinal arteries, slowing down or even stopping for a few seconds in the capillary system, and then moved slightly faster again through the postcapillary venules and veins. In non-pigmented mice, significant number of cells were seen to have arrested in the choroidal circulation. There was no difference between B10.RIII mice and BALB/c mice in vessel diameters, leukocyte velocities and shear stresses. This method allows the visualization of leukocytes and provides data on their behavior as they move through the choroidal and retinal circulation of non-pigmented mice, and in the retinal circulation of pigmented mice. It provides a valuable new tool for the investigation of real time leukocyte dynamics in murine retinal and choroidal microcirculations both under physiological conditions and during the development of ocular disease.

Animals↗

PubMed2002

Involvement of CD44 in leukocyte trafficking at the blood-retinal barrier.

In the present study, we investigated the involvement of CD44 in leukocyte trafficking in vivo at the blood-retinal barrier using experimental autoimmune uveoretinitis (EAU) as a model system. Leukocyte trafficking was evaluated using adoptive transfer of calcein-AM (C-AM)-labeled spleen cells harvested from syngeneic mice at prepeak severity of EAU to mice at a similar stage of disease. CD44 and its ligand hyaluronan were up-regulated in the eye during EAU. CD44-positive leukocytes were found sticking in the retinal venules and postcapillary venules but not in the retinal arterioles nor in mesenteric vessels. Preincubation of in vitro C-AM-labeled leukocytes with anti-CD44 monoclonal antibodies (mAb; IM7) or high molecular weight hyaluronic acid (HA) before transfer significantly suppressed leukocyte rolling but not sticking in retinal venules and also reduced cell infiltration in the retinal parenchyma. Administration of the HA-specific enzyme hyaluronidase to mice before cell transfer also reduced leukocyte infiltration, suggesting that CD44-HA interactions are involved in leukocyte recruitment in EAU. This was further supported by the observation that disease severity was reduced by administration of anti-CD44 mAb (IM7) at the early leukocyte-infiltration stage. Further studies also indicated that CD44 activation was associated with increased levels of apoptosis, and this may also be in part responsible for the reduction in disease severity. These findings demonstrate that CD44 is directly involved in leukocyte-endothelial interaction in vivo and influence the trafficking of primed leukocytes to the retina and their overall survival.

Animals↗