Elevated plasma CD105 and vitreous VEGF levels in diabetic retinopathy.

Diabetic retinopathy is the leading cause of blindness in the industrialized world. Hyperglycaemia induces retinal hypoxia that upregulates a range of vasoactive factors which may lead to macular oedema and/or angiogenesis and hence potentially sight threatening retinopathy. In this study, we have focused on the association of CD105 and vascular endothelial growth factor (VEGF) with the development and progression of diabetic retinopathy by means of quantifying their expression in the plasma and vitreous of diabetic patients. CD105 levels were quantified in the plasma of 38 type I diabetic patients at various stages of retinopathy and 15 non-diabetic controls. In an additional cohort of 11 patients with advanced proliferative retinopathy and 23 control subjects, CD105 and VEGF were measured in the vitreous. The values were expressed as median (range) and statistical analysis was carried out using the non-parametric Mann-Whitney U test. Plasma CD105 levels were significantly increased in diabetic patients [1.8 (1.1-2.4) ng/ml] compared with non-diabetic controls [0.7 (0.3-1.8) ng/ml] (p<0.01). Plasma CD105 levels were elevated in diabetic patients with all stages of retinopathy, the highest level was observed in background retinopathy [2.3 (2.1-2.5) ng/ml] followed by proliferative retinopathy [2.1 (0.9-2.8) ng/ml] and advanced proliferative retinopathy [1.4 (0.6-1.8) ng/ml]. Vitreous contents of CD105 did not differ between controls and patients with advanced proliferative retinopathy, but vitreous levels of VEGF were elevated by approximately 3-fold in patients with advanced proliferative retinopathy [7.2 (1.90-15.60) ng/ml] compared with the control subjects [1.80 (1.10-2.210)] (p<0.01). These observations indicate that plasma levels of CD105 and vitreous levels of VEGF are associated with diabetic retinopathy, suggesting that CD105 and the angiogenic factor VEGF may play a critical role in the development and progression of diabetic retinopathy. Further studies are required to determine whether circulating CD105 levels could serve as a surrogate marker for early stage retinopathy and for monitoring disease progression.

How should we train physicians for remote and rural practice? What the present incumbents say.

OBJECTIVE: To obtain the views of the current remote and rural consultant physicians with regards to their opinion on components of an ideal training programme for an aspirant remote and rural physician. DESIGN: A questionnaire was designed to elicit information in three main areas: experience and training prior to appointment, current pattern of service provision and opinions on components of an ideal training programme for remote and rural physicians. SETTING: Five Scottish rural hospitals in Shetland, Wick, Stornoway, Fort William and Oban. SUBJECTS: Thirteen consultant physicians based in the five rural hospitals chosen. RESULTS: The response rate to the questionnaire was 85%. All had previous experience in acute general medicine, and most in one of a variety of subspecialties. Each physician had developed interests and skills in other branches of medicine following appointment in order to meet local service needs. Most felt that there was a need for expansion of consultant numbers in the future, 45% citing the European Working Time Directive as the major reason. There was an encouraging degree of commonality between the current consultants as to what they felt should be included in a training programme for remote and rural physicians. CONCLUSION: There are challenges in meeting training needs for consultant physicians intending to work in a remote setting. Development of broader-based training than offered by most current dual training programmes is essential. Only imaginative approaches to training will produce physicians who are fit for purpose.

A comparative evaluation of digital imaging, retinal photography and optometrist examination in screening for diabetic retinopathy.

AIMS: To compare the respective performances of digital retinal imaging, fundus photography and slit-lamp biomicroscopy performed by trained optometrists, in screening for diabetic retinopathy. To assess the potential contribution of automated digital image analysis to a screening programme. METHODS: A group of 586 patients recruited from a diabetic clinic underwent three or four mydriatic screening methods for retinal examination. The respective performances of digital imaging (n=586; graded manually), colour slides (n=586; graded manually), and slit-lamp examination by specially trained optometrists (n=485), were evaluated against a reference standard of slit-lamp biomicroscopy by ophthalmologists with a special interest in medical retina. The performance of automated grading of the digital images by computer was also assessed. RESULTS: Slit-lamp examination by optometrists for referable diabetic retinopathy achieved a sensitivity of 73% (52-88) and a specificity of 90% (87-93). Using two-field imaging, manual grading of red-free digital images achieved a sensitivity of 93% (82-98) and a specificity of 87% (84-90), and for colour slides, a sensitivity of 96% (87-100) and a specificity of 89% (86-91). Almost identical results were achieved for both methods with single macular field imaging. Digital imaging had a lower technical failure rate (4.4% of patients) than colour slide photography (11.9%). Applying an automated grading protocol to the digital images detected any retinopathy, with a sensitivity of 83% (77-89) and a specificity of 71% (66-75) and diabetic macular oedema with a sensitivity of 76% (53-92) and a specificity of 85% (82-88). CONCLUSIONS: Both manual grading methods produced similar results whether using a one- or two-field protocol. Technical failures rates, and hence need for recall, were lower with digital imaging. One-field grading of fundus photographs appeared to be as effective as two-field. The optometrists achieved the lowest sensitivities but reported no technical failures. Automated grading of retinal images can improve efficiency of resource utilization in diabetic retinopathy screening.

Automated detection of microaneurysms in digital red-free photographs: a diabetic retinopathy screening tool.

AIMS: To develop a technique to detect microaneurysms automatically in 50 degrees digital red-free fundus photographs and evaluate its performance as a tool for screening diabetic patients for retinopathy. METHODS: Candidate microaneurysms are extracted, after the image has been modified to remove variations in background intensity, by algorithms that enhance small round features. Each microaneurysm candidate is then classified according to its intensity and size by the application of a set of rules derived from a training set of 102 images. RESULTS: When 3,783 individual images were analysed and the results compared with the opinion of a clinical research fellow examining the same images, the program achieved a sensitivity of 81% and a specificity of 93% for the detection of images containing microaneurysms. Nine hundred and twenty-five sets of 4 images per patient were then analysed and the total number of microaneurysms detected compared with the overall patient retinopathy grade derived by the clinician examining the same images. In this context, intended to mimic a screening situation, the program achieved a sensitivity of 85% and a specificity of 76% for the detection of patients with (any) retinopathy (positive predictive value 0.71, negative predictive value 0.88). CONCLUSIONS: An automated technique was developed to detect retinopathy in digital red-free fundus images that can form part of a diabetic retinopathy screening programme. It is believed that it can perform a useful role in this context identifying images worthy of closer inspection or eliminating 50% or more of the screening population who have no retinopathy.

The preprocessing of retinal images for the detection of fluorescein leakage.

Images of the human retina are routinely used in clinical practice for the diagnosis and management of eye disease. Increased permeability of retinal blood vessels, which is a clinically significant feature, can be visualized with a process known as fluorescein angiography as leakage of fluorescence dye into the surrounding tissues. Analyses of such images can be quantified but significant degradation of images due to uneven illumination or occluded optical pathways is often incurred during image capture. We describe a procedure to restore fluorescein angiographic retinal images so that quantitative computation can be reliably performed. Analysis of the image acquisition system reveals that captured images are composed of two functions, one describing the true underlying image and the other the degradation incurred. These two functions are independent of one another and it is possible to estimate the degradation from an isolated captured image and restore it appropriately. Any leakage of fluorescein dye is then detected by analysing the restored angiographic sequence over time and finding areas of the image that do not have the usual decrease in fluorescence intensity.

A comparison of computer based classification methods applied to the detection of microaneurysms in ophthalmic fluorescein angiograms.

We compared the performance of three computer based classification methods when applied to the problem of detecting microaneurysms on digitised angiographic images of the retina. An automated image processing system segmented 'candidate' objects (microaneurysms or spurious objects), and produced a list of features on each candidate for use by the classifiers. We compared an empirically derived rule based system with two automated methods, linear discriminant analysis and a learning vector quantiser artificial neural network, to classify the objects as microaneurysms or otherwise. ROC analysis shows that the rule based system gave a higher performance than the other methods (p = 0.92) although a much greater development time is required.

A fully automated comparative microaneurysm digital detection system.

A fully automated digital image processing system, which provides an objective and repeatable way to quantify microaneurysms in digitised fluorescein angiograms, has been developed. The automated computer processing includes registration of same-eye retinal images for serial studies, cutting of regions-of-interest centred on the fovea, the detection of microaneurysms and the comparison of serial images for microaneurysm turnover. The microaneurysm detector was trained against a database of 68 images of patients with diabetes containing 394 true microaneurysms, as identified by an ophthalmologist. The microaneurysm detector achieved 82% sensitivity with 2.0 false-positives per image. An independent test set, comprising 20 images containing 297 true microaneurysms, was used to compare the microaneurysm detector with clinicians. The microaneurysm detector achieved a sensitivity of 82% for 5.7 false-positives per image, whereas the clinician receiver-operator-characteristic (ROC) curve gives 3.2 false-positives per image at a sensitivity of 82%. It is concluded that the computer system can reliably detect microaneurysms. The advantages of the computer system include objectivity, repeatability, speed and full automation.

Soluble leucocyte adhesion molecules in diabetic retinopathy stimulate retinal capillary endothelial cell migration.

Diabetic retinal neovascularisation is considered to be a consequence of retinal ischaemia caused by capillary occlusion. Capillary occlusion is the result of microvascular thrombi in which erythrocytes, platelets and leucocytes each may play a role. We investigated the role of leucocytes in this process and the subsequent angiogenic response. We studied the serum levels of the soluble leucocyte adhesion molecules soluble E-Selectin, soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) in the serum of 93 patients with insulin-dependent diabetes (IDDM) and varying degrees of retinopathy and 47 healthy age and sex matched control subjects. We also measured the ability of serum to stimulate retinal capillary endothelial cell migration using an assay of angiogenesis in vitro. Soluble E-Selectin and sVCAM-1 levels were raised in all patients with IDDM (p < 0.001; p < 0.001) particularly those with retinopathy (p < 0.001; p < 0.001). Soluble E-Selectin levels were highest in the patients with severe non-proliferative diabetic retinopathy (p < 0.001) and sVCAM-1 levels were highest in patients with proliferative diabetic retinopathy (p < 0.01). In contrast soluble ICAM-1 levels were the same in patients and control subjects (p > 0.05). Soluble E-Selectin levels in diabetic patients were correlated with the level of glycated haemoglobin (p < 0.05). Retinal endothelial cell migration-inducing (ECMI) activity was increased in patients with IDDM (p < 0.01) in particular in those with retinopathy (p < 0.01). Furthermore, in vitro ECMI activity could be blocked by antibodies to sVCAM-1 and sE-Selectin. These data point to a functional role for leucocyte adhesion in the microvasculopathy of diabetic retinopathy and may have implications for the induction of retinal angiogenesis.

An image-processing strategy for the segmentation and quantification of microaneurysms in fluorescein angiograms of the ocular fundus.

Digital image-processing techniques can provide an objective and highly repeatable way of quantifying retinal pathology. This study describes an image-processing strategy which detects and quantifies microaneurysms present in digitized fluorescein angiograms. After preprocessing stages, a bilinear top-hat transformation and matched filtering are employed to provide an initial segmentation of the images. Thresholding this processed image results in a binary image containing candidate microaneurysms. A novel region-growing algorithm fully delineates each marked object and subsequent analysis of the size, shape, and energy characteristics of each candidate results in the final segmentation of microaneurysms. The technique is assessed by comparing the computer's results with microaneurysm counts carried out by five clinicians, using Receiver Operating Characteristic (ROC) curves. The performance of the automated technique matched that of the clinicians' analyses. This strategy is valuable in providing a way of accurately monitoring the progression of diabetic retinopathy.

Urinary free cortisol in the assessment of hydrocortisone replacement therapy.

Glucocorticoid replacement therapy should be tailored to individual patients' requirements to avoid the risks of over and under treatment. Serum and urinary free cortisol profiles over 24 hours were compared as a means of assessing replacement therapy in nine patients on long-term twice daily hydrocortisone. Both indices varied in relationship to the timing and dose of hydrocortisone and there was a close correlation between individual measurements of serum and urinary free cortisol (r = 0.885, p < 0.0001). Collection of urine samples offers certain advantages over repeated serum sampling and urinary free cortisol measurement may have a role in the assessment of hydrocortisone replacement therapy.

The early effects of radioiodine therapy for hyperthyroidism on biochemical indices of bone turnover.

OBJECTIVE: We investigated the early changes following radioiodine therapy for hyperthyroidism, in biochemical indices of bone synthesis and degradation, and their relationship to circulating thyroid hormone concentrations. DESIGN: Prospective follow-up over the first 12 weeks after radioiodine therapy. PATIENTS: Six women with clinical and biochemical evidence of hyperthyroidism. MEASUREMENTS: Serum concentrations of T4, free T3 and osteocalcin, and urinary excretion of the pyridinium cross-links, pyridinaline and deoxypyridinaline, measured before and weekly for 12 weeks after administration of radioiodine therapy. RESULTS: Biochemical indices of bone metabolism were elevated prior to treatment. There was a brisk reduction in circulating thyroid hormones levels paralleled by a similar fall in pyridinium cross-link excretion, which had returned to normal in five patients by the end of the study. There was a positive correlation between pyridinium cross-link excretion and thyroid hormone concentrations. There was no significant change in serum osteocalcin. CONCLUSIONS: Treatment of hyperthyroidism results in prompt correction of the associated increased rate of bone collagen degradation suggesting that effective early correction of hyperthyroidism is desirable to limit its detrimental effect on skeletal mass.

Altered platelet stearic to oleic acid ratio in malignancy.

Alteration of lipid metabolism associated with malignant disease is well-documented and some studies have suggested a reduced stearic to oleic acid ratio occurs in erythrocytes in cancer patients. In this study, the fatty acid composition was measured in platelets, which are capable of lipid synthesis and have a much shorter lifespan. While demonstrating any malignancy related change in the platelet stearic to oleic acid ratio the study aimed to assess whether it could be of value as a tumour marker. Patients with active malignancy (n = 46) had a lower ratio of stearic to oleic acid than those with malignant disease in clinical remission [mean (S.D.) 1.08 (0.22) vs. 1.26 (0.30), P less than 0.01], and 22 healthy controls [1.29 (0.24), P less than 0.001]. However in a group of 17 patients with chronic, non-malignant diseases the ratio was also lower than in normal controls and similar to that seen in the active malignancy group [0.97 (0.29)]. Thus while a reduction in platelet stearic to oleic acid ratio was found in active malignancy, it is not specific to neoplastic disease.

The effect of insulin suppression on postprandial nutrient metabolism: studies with infusion of somatostatin and insulin.

Utilization of fat, carbohydrate and protein before and after feeding was studied in six healthy subjects using simultaneous respiratory gas exchange measurement and [1-13C] leucine infusion. The role of insulin was investigated by repeating a control study with the addition of an infusion of somatostatin, a hormone which can suppress insulin release. Where near-complete insulin suppression was effected, subjects were studied on a third occasion with the further addition of exogenous insulin infusion. The normal switch on feeding from fat to carbohydrate as principal energy source was reproduced at insulin levels of only 17%-33% of control values, which were inadequate to prevent hyperglycaemia. At fed levels below 10%, a fat-predominant pattern persisted unless insulin was infused. Protein degradation was reduced and synthesis unaffected by feeding, regardless of insulin concentration. Leucine oxidation was dependent on its plasma concentration in the presence of circulating insulin. Thus insulin appears to be necessary for the normal switch to carbohydrate oxidation on feeding but not for postprandial changes in protein metabolism.

A simple approach to screening for microalbuminuria in a type 1 (insulin-dependent) diabetic population.

The reproducibility of albumin concentration in first-morning samples of urine was assessed in 334 insulin-dependent diabetic patients aged 18-60 years. The albumin excretion rate was determined immunoturbidimetrically in three sterile, Albustix-negative, first-morning urine samples submitted over a week. An abnormally high mean value, greater than or equal to 2.5 mg albumin per mmol creatinine (Ua/Uc), was found in 33 patients (9.9%). These patients were older (mean 42 vs 34 years, P less than 0.01), had longer disease duration (18 vs 14 years, P less than 0.01) and higher HbA1c values (6.8 vs 6.3%, P less than 0.05) than those without microalbuminuria. Although triplicate samples were collected within 7 days, Ua/Uc showed considerable intraindividual variation, with a mean coefficient of variation of 49%. Despite this it was found that Ua/Uc values greater than 1 mg/mmol on the first specimen had a sensitivity of 97% and a specificity of 82% for detecting those with a three-sample mean value greater than 2.5 mg/mmol. Thus virtually all those with microalbuminuria (32/33) had a single first-morning result greater than 1 mg/mmol, and in those with a lower ratio microalbuminuria was excluded with more than 99% certainty.

Measurement of bone collagen degradation in hyperthyroidism and during thyroxine replacement therapy using pyridinium cross-links as specific urinary markers.

UNLABELLED: Urinary excretion of the bone collagen derived pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) was measured in 19 patients (4 M:15 F) with untreated thyrotoxicosis, and 20 pre-, and 20 postmenopausal women taking T4 100-200 micrograms daily for autoimmune hypothyroidism. Both PYD and DPD excretion (nanomoles per mmol creatinine) was elevated in the thyrotoxic patients compared to 287 controls; median 131 vs. 26 and 37.5 vs. 7.2, respectively, P less than 0.0001. In premenopausal women mean urinary pyridinium cross-link excretion and serum osteocalcin levels were similar in both T4-treated and matched control groups, despite suppression of serum TSH concentrations to below 0.1 mU/L in 14 of the 20 taking T4. In postmenopausal women mean (+/- 1 SE) urinary PYD excretion (nanomoles per mmol creatinine) was raised in those taking T4, relative to euthyroid controls; 40.0 +/- 2.7 vs. 32.1 +/- 2.3, P less than 0.05. DPD excretion and serum osteocalcin levels were also higher, but not significantly. When only the T4-treated women with a subnormal serum TSH were considered the difference in PYD excretion was more marked, and mean DPD excretion was also significantly elevated; 13.7 +/- 1.3 vs. 10.1 +/- 0.8, P less than 0.05. CONCLUSION: bone collagen breakdown is increased in thyrotoxicosis, and in postmenopausal women taking sufficient T4 to suppress serum TSH. Similarly treated premenopausal women appear to be at lower risk.

Nutrient oxidation patterns and protein metabolism in lean and obese subjects.

The immediate metabolic response to eating has been compared in a group of grossly obese subjects (W/H2 = 45) with that in lean controls (W/H2 = 22). Dietary intake of energy for obese subjects was based on their estimated basal energy expenditure for ideal body weight (given at an hourly rate of 3 X BMR over a 4-h period). Lean subjects were measured twice: control 1 with the same intake of energy as the obese in terms of ideal body weight and control 2 with the same energy intake in relation to each subject's measured resting energy expenditure (2.2 X REE). The changes in energy expenditure and nutrient disposal with the onset of eating have been assessed by a method of combined respiratory gas analysis and intravenous infusion of 13C-labelled leucine. Leucine kinetics were used to quantitate rapid changes in protein oxidation and to assess protein synthesis and degradation. 1) Total energy expenditure was 20-30 per cent greater in obese subjects than lean subjects in fasting and feeding. Energy expenditure expressed per kg fat-free mass, from D2O dilution, was similar in obese and lean subjects in both fasting (5.8 v. 5.5 kJ/kg FFM/h) and feeding [6.7 v. 6.3 (Control 2) kJ/kg FFM/h]. 2) The onset of eating was associated with increased carbohydrate and protein oxidation with decreased fat oxidation in both lean and obese individuals. In obese subjects, however, both the decrease in fat oxidation and the increase in protein oxidation were significantly smaller (P less than 0.05) than the corresponding increments in lean subjects (Control 2). 3) The rate of protein synthesis was significantly (P less than 0.05) higher in obese subjects both in the fasting state (99 v. 84 mumols leucine/kg FFM/h) and in the fed state [94 v. 67 (Control 2) mumols leucine/kg FFM/h]. The rate of protein degradation was also higher in obese individuals in fasting (117 +/- 6 v. 106 +/- 4 mumol leucine/kg FFM/h) and feeding [65 +/- 4 v. 54 +/- 6 (Control 2) mumol leucine/kg FFM/h] though these differences are not statistically significant (P greater than 0.05). 4) The observed differences between obese and lean individuals in protein and energy metabolism in the fasted state and in the immediate response to eating do not support a hypothesis of greater metabolic efficiency in obesity.

Residual abnormalities of insulin secretion and sensitivity after weight loss by obese women.

Changes in plasma insulin and glucose concentrations before and after feeding were measured in six female subjects (post-obese) who had regained a normal body mass after a history of severe obesity (mean weight loss, 37.1 +/- 2.6 kg). The responses of the post-obese group were compared with a group of weight- and age-matched subjects who had not been obese (lean). After an overnight fast subjects were fed a meal at 09.00 h and 13.10 h. Fasting and post-prandial insulin concentrations were lower in post-obese than in lean subjects. Immediately after beginning to eat at 13.10 h all subjects showed a rise in insulin concentration with no change in glucose concentration. In this pre-absorptive period there was no significant difference in insulin concentration between post-obese and lean subjects, although the increment in insulin concentration over baseline values was greater in post-obese subjects (P less than 0.05). It is concluded that abnormalities of insulin secretion and action remain after weight loss by obese subjects. These abnormalities may predispose to hyperphagia and accumulation of excess adiposity.

Diagnostic problems caused by spurious elevation of thyrotrophin measured by sensitive assay.

Thyrotrophin measured by the Amerwell immunoradiometric assay was found to be falsely elevated, causing potential diagnostic confusion in hypothyroid, euthyroid and hyperthyroid individuals. Non-specific antibodies in patients' sera may be responsible, by cross-linking the mouse monoclonal antibodies of the assay. Interference was overcome in the sera from these patients by addition of mouse serum (20 microliters per ml test serum) over and above that already incorporated in the kit. Problems with spuriously elevated thyrotrophin levels are not confined to one manufacturer's kit and can cause diagnostic confusion in several situations.