Biomedical subjects
M Boulton
Publications and source records attributed to M Boulton.
Late diagnosis of HIV infection in children causes distress.
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The effects of growth factors and conditioned media on the proliferation of human corneal epithelial cells and keratocytes.
BACKGROUND: As growth factors play an important role in epithelial wound repair, we evaluated the effect of exogenous growth factors in the presence and absence of corneal epithelial and keratocyte conditioned medium on human corneal epithelial cell and keratocyte proliferation. METHODS: Preconfluent cultures of human corneal epithelial cells or stromal keratocytes were exposed to varying concentrations of EGF, TGF-beta or bFGF in the presence or absence of human corneal epithelial or stromal keratocyte conditioned medium. Cell numbers were determined after 48 h incubation. RIA and ELISA were used to quantify the levels of EGF, TGF-beta and bFGF in conditioned media. RESULTS: EGF and bFGF increased, while TGF-beta decreased, the proliferation of both cell types in a dose-dependent manner. Epithelial cell conditioned medium inhibited, and keratocyte conditioned medium stimulated, the proliferation of both cell types. The proliferative effects of EGF, TGF-beta and bFGF in the presence of keratocyte conditioned medium were additive for both cell types. By contrast, the addition of exogenous growth factors was unable to overcome the inhibitory potential of epithelial conditioned medium. Both conditioned media contained significant levels of bFGF, but TGF-beta levels in epithelial conditioned medium were up to 5 times greater than that in keratocyte conditioned medium. CONCLUSIONS: The results indicate that corneal cells maintain tissue homeostasis and modulate the wound healing response via paracrine/autocrine pathways.
Blue light-induced singlet oxygen generation by retinal lipofuscin in non-polar media.
Accumulation of lipofuscin (LF) is a prominent feature of aging in the human retinal pigment epithelium (RPE) cells. This age pigment exhibits substantial photoreactivity, which may increase the risk of retinal photodamage and contribute to age-related maculopathy. In a previous study, we detected singlet oxygen generation by lipofuscin granules excited with blue light. In this paper we investigated the ability of hydrophobic components of lipofuscin to photogenerate singlet oxygen in non-polar environments. Singlet oxygen was detected directly by monitoring its characteristic phosphorescence at ca 1270 nm. The action spectrum of singlet oxygen formation indicated that this process was strongly wavelength-dependent and its efficiency decreased with increasing wavelength by a factor of ten, comparing 420 nm and 520 nm. The quantum yield of singlet oxygen increased with increasing concentration of oxygen. Using laser flash photolysis we studied the possible mechanism of singlet oxygen formation. The observed transient, with a broad absorption spectrum peaking at around 440 nm, was identified as a triplet with lifetime ca 11 microseconds. It was quenched by both molecular oxygen and beta-carotene with concomitant formation of a beta-carotene triplet state. These results indicate the potential role of hydrophobic components of lipofuscin in blue light-induced damage to the RPE.
Prediction of functional regions of the maize streak virus replication-associated proteins by protein-protein interaction analysis.
The replication of the geminiviruses depends on the viral encoded early (complementary-sense) proteins and on host genome encoded factors. Additionally, some of the early proteins (the AL2 protein of subgroup III, and the RepA (formerly known as C1) or Rep (C1:C2) proteins of subgroup I geminiviruses) can function as transcriptional activators of virion- (V-)sense gene expression. The yeast two-hybrid system has allowed us to predict some of the functionally important regions of the maize streak virus (MSV) early proteins RepA and Rep. Defined domains of these proteins were shown to act as transactivators in yeast cells. We detected the association of the RepA and Rep proteins, and their subfragments, with the maize retinoblastoma protein (ZmRb1) which is likely to be one of the interacting host proteins. We showed the self-association capability of the MSV proteins and suggest that homo- or hetero-oligomerization may play an important role in virus replication. These results provide new insights into the role of different regions of the MSV proteins in relation to transcriptional activation and regulation of viral DNA replication.
Adhesion of IOLs to the posterior capsule.
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VEGF localisation in diabetic retinopathy.
AIM: To determine the staining pattern of vascular endothelial growth factor (VEGF) at different stages of diabetic retinopathy (including post-laser photocoagulation) and to compare staining in excised fibrovascular and fibrocellular (non-diabetic) preretinal membranes. METHODS: Immunohistochemical localisation of VEGF, using antibodies raised against VEGF165 and VEGF121,165,189, was carried out on specimens of normal human retina (n = 15), diabetic retinas ((a) with no overt retinopathy (n = 19), (b) with intraretinal vascular abnormalities but no proliferative retinopathy (n = 6), (c) with active proliferative retinopathy (n = 6), (d) with no residual proliferative retinopathy after photocoagulation therapy (n = 15)), excised diabetic fibrovascular membranes (n = 19), and non-diabetic fibrocellular membranes (n = 7). The degree and pattern of immunostaining was recorded. RESULTS: In general, VEGF was absent from the majority of normal retinas. VEGF staining was apparent in most diabetic tissues but the staining pattern was dependent on both the specificity of the antibody used and the category of tissue. Staining with the VEGF165 antibody was generally confined to endothelial cells adn perivascular regions while the VEGF121,165,189 antibody was also associated with extravascular components of the inner retina. Intensity of immunostaining of diabetic eyes was dependent on the severity of retinopathy being least in diabetics with no overt retinopathy and greatest in retinas with proliferative retinopathy. Interestingly, the intensity of immunostaining in diabetic retinas which had undergone laser surgery for proliferative retinopathy was reduced to basal levels. Moderate to intense immunostaining was observed in all fibrovascular and fibrocellular membranes examined. CONCLUSIONS: This study supports a circumstantial role for VEGF in the pathogenesis of both the preclinical and proliferative stages of diabetic retinopathy.
Lens epithelial cell proliferation, migration, and metaplasia following capsulorhexis.
AIM: To study the behaviour of residual lens epithelial cells after capsulorhexis and expression of material from the isolated lens. METHODS: Human and bovine lens capsules were isolated, sterile non-toxic silicone rings inserted, and the preparations placed in organ culture for up to 12 weeks. Cell coverage of the posterior lens capsule was recorded and the capsules were examined, both pre- and post-coverage, for (a) proliferative activity and (b) cytoskeletal components. RESULTS: After a lag period outgrowth was observed across the posterior capsule. The rate of cell coverage was dependent upon both species and the presence or absence of serum. The proliferative activity of the cells was greatest at or near the leading edge and decreased once covered. Wrinkles became visible in the posterior capsule during the late stages of precoverage and increased in both number and complexity. All cells on both the human and bovine posterior capsules contained F-actin and vimentin and the majority were immunolabelled for alpha-smooth muscle actin (alpha-SMA). CONCLUSIONS: This model exhibits many of the in vivo characteristics of the lens capsule after extracapsular surgery and may prove useful in further elucidating the cellular mechanisms of posterior capsule opacification.
Determination of volumetric cerebrospinal fluid absorption into extracranial lymphatics in sheep.
We estimated the volumetric clearance of cerebrospinal fluid (CSF) through arachnoid villi and extracranial lymphatics in conscious sheep. Catheters were inserted into both lateral ventricles, the cisterna magna, multiple cervical lymphatics, thoracic duct, and jugular vein. Uncannulated cervical vessels were ligated. 125I-labeled human serum albumin (HSA) was administered into both lateral ventricles. 131I-HSA was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. From mass balance equations, total volumetric absorption of CSF averaged 3.37 +/- 0.38 ml/h, with 2.03 +/- 0.29 ml/h (approximately 60%) removed by arachnoid villi and 1.35 +/- 0.46 ml/h (approximately 40%) cleared by lymphatics. With projected estimates for noncannulated ducts, total CSF absorption increased to 3.89 +/- 0.33 ml/h, with 1.86 +/- 0.49 ml/h (48%) absorbed by lymphatics. Additionally, we calculated total CSF drainage to be 3.48 +/- 0.52 ml/h, with 54 and 46% removed by arachnoid villi and lymphatics, respectively, using previously published mass transport data from our group. We employed estimates of CSF tracer concentrations that were extrapolated from relationships observed in the study reported here. We conclude that 40-48% of the total volume of CSF absorbed from the cranial compartment is removed by extracranial lymphatic vessels.
Raised intracranial pressure increases CSF drainage through arachnoid villi and extracranial lymphatics.
We demonstrated previously that about one-half of cerebrospinal fluid (CSF) removed from the cranial vault was cleared by extracranial lymphatic vessels. In this report we test the hypothesis that lymphatic drainage of CSF increases as intracranial pressure (ICP) is elevated in anesthetized sheep. Catheters were inserted into both lateral ventricles, cisterna magna, cervical lymphatics, and jugular vein. A ventriculocisternal perfusion system was employed to regulate CSF pressures and to deliver a protein tracer (125I-labeled human serum albumin) into the CSF compartment. 131I-labeled human serum albumin was injected intravenously to permit calculation of plasma tracer loss and tracer recirculation into lymphatics. ICP was controlled by adjusting the height of the inflow reservoir and the cisterna magna outflow catheter appropriately. The experimental design consisted of a 3-h period of lower pressure followed by a 3-h period of higher pressure in the same animal (10-20 or 20-30 cmH2O). We determined that incremental changes in ICP were associated with higher CSF transport through lymphatic and arachnoid villi routes in all eight animals tested (P = 0.004).
Increased expression of placenta growth factor in proliferative diabetic retinopathy.
Proliferative diabetic retinopathy is thought to be mediated by the hypoxic regulation of angiogenic growth factors, in particular the vascular endothelial growth factor (VEGF) family. The aim of this study was to determine if placental growth factor (PIGF), a recently identified member of the VEGF family, was expressed in diabetic eyes undergoing preretinal neovascularization. Rabbit anti-PIGF antiserum was raised using a 20-amino acid N-terminal sequence to PIGF and did not cross react with VEGF165. Immunohistochemistry was performed on specimens of normal retina (n = 8), diabetic retina in the absence (n = 7) and presence (n = 4) of proliferative retinopathy, scatter laser-treated diabetic retina (n = 7), excised fibrovascular preretinal membranes (n = 12), and nondiabetic fibrocellular epiretinal (n = 7) membranes. PIGF levels were also determined in vitrectomy specimens from patients with either proliferative diabetic retinopathy or macular hole. PIGF immunoreactivity was intensely localized to the endothelial and perivascular regions of newly formed blood vessels of excised fibrovascular preretinal membranes. Intense localization of PIGF protein was also observed in superficial retinal vessels in diabetic retinae adjacent to neovascular preretinal membranes. Localization of PIGF was weak or absent in diabetic retinae that showed no evidence of neovascular proliferation. PIGF protein was also absent in normal retinae, in diabetic retinae that had received extensive treatment with scatter laser photocoagulation, and in nonvascularized epiretinal membranes. PIGF was present in all diabetic vitreous samples (mean 103 pg/ml) but nondetectable in control samples. These results strongly implicate a role for PIGF in the pathogenesis of proliferative diabetic retinopathy.
Fluorescence properties of autofluorescent granules generated by cultured human RPE cells.
PURPOSE: To compare the fluorescence properties of autofluorescent granules generated by retinal pigment epithelial (RPE) cells in vitro with those of the lipofuscin of RPE in vivo. METHODS: Cultured human RPE cells were maintained in basal medium for as long as 1 year, fed rod outer segments (ROS) daily for as long as 56 days, fed ROS in the presence and absence of leupeptin, or fed liposomes consisting of the major phospholipids in ROS. At different time points, cells were examined for overall fluorescence, and their fluorescence spectra were determined. In addition, chloroform-methanol extracts were examined by thin-layer chromatography and compared with those generated from RPE lipofuscin. RESULTS: Autofluorescent granules accumulated in cultured RPE cells, regardless of the presence of an exogenous substrate or the nature of the substrate. The rate of accumulation of autofluorescent granules was greatest in cells fed ROS. The autofluorescent material generated in cultured RPE cells had some spectral similarities with RPE lipofuscin but differed in solubility and chromatographic mobility of their constituent fluorophores. CONCLUSIONS. The autofluorescent granules generated by cultured RPE, even with different specific substrates, differ from lipofuscin granules in vivo, suggesting that additional properties of RPE cells or of the materials they phagocytose are required to produce autofluorescent materials with the characteristics of lipofuscin.
Involvement of calcium in retinal pigment epithelial cell proliferation and pigmentation.
PURPOSE: The aim of this study is to explore the role of intracellular calcium in the mechanism of co-regulation of retinal pigment epithelial cells (RPE) by vitreous fluid and platelet mitogens, in order to evaluate the use of calcium modulating drugs in preventing RPE cell proliferation and contraction of fibrocellular membranes. METHODS: Monolayers of human RPE cells were loaded with Fura-2-AM and examined in a fluorimeter for changes in intracellular free calcium in response to platelet mitogens (PDGFAB or TGFbeta1) and vitreous fluid (containing vitreous substrate proteins), both alone or in combination. The effect of the calcium antagonists TMB8 and verapamil and the calmodulin antagonists J8 and tamoxifen were then examined on RPE cell proliferation and pigmentation, both in the presence and absence of vitreous substrate and platelet mitogens. RESULTS: We report that co-exposure of RPE cells to platelet mitogens and vitreous fluid produces an increase in intracellular free calcium of greater duration than that with either PDG-FAB, TGFbeta1 or vitreous fluid alone. Calcium and calmodulin antagonists significantly reduce RPE cell proliferation in both the presence and absence of vitreous substrate and platelet mitogens. Calcium antagonists also stimulate the accumulation of autofluorescent granules within RPE cells. CONCLUSIONS: Calcium signalling plays a role in the co-regulation of RPE cells by vitreous substrate and platelet mitogens. Drugs that lower intracellular calcium or inhibit calmodulin may offer an additional approach to preventing the hyperproliferation of RPE cells in PVR.
A role for vitamin A in the formation of ocular lipofuscin.
BACKGROUND: Lipofuscin granules in the retinal pigment epithelium are lipid protein aggregates which are thought to represent the lifelong accumulation of the non-degradable end products from the phagocytosis of photoreceptor outer segments. Given the increasing evidence for a key role for vitamin A in the formation of ocular lipofuscin, the fluorophores generated by reacting vitamin A with lipid were assessed. METHODS: Reaction mixtures consisting of vitamin A (retinol) or its aldehyde (retinal) and (a) isolated rod outer segments, (b) the lipid extract of rod outer segments, (c) protein, or (d) liposomes were incubated at either pH 4.5 or 7.0 for up to 42 days. The fluorescence characteristics and mobility of the chloroform soluble fluorophores generated were compared with those extracted from purified human lipofuscin. Finally, the effect of lysosomal degradation on fluorophores generated in the above mixtures was assessed. RESULTS: Major spectral changes were observed when ROS or liposomes were incubated with retinal. These changes were pH dependent and did not occur if retinal was replaced with retinol. A number of the fluorophores generated exhibited similar fluorescence characteristics and chromatographic mobility to those of lipofuscin. Neither the presence of protein nor exposure to lysosomal enzymes had any effect on the spectral profile or fluorophore mobility of the fluorophores generated. CONCLUSIONS: These results suggest that some of the chloroform soluble fluorophores of lipofuscin are formed as a direct reaction product of retinal and lipid.
Intravitreal growth factors in proliferative diabetic retinopathy: correlation with neovascular activity and glycaemic management.
AIM: Many growth factors are implicated in proliferative diabetic retinopathy (PDR). It was decided to test the hypothesis that no one factor is predominant but that a regular profile of levels of different growth factors might be operating, and that the profile might differ according to whether or not insulin therapy was part of the patient's glycaemic management. The levels of several growth factors in vitrectomy samples were therefore determined from diabetic patients with tractional, non-haemorrhagic sequelae of PDR and these levels were correlated with (a) each other (growth factor profile), (b) neovascular activity, and (c) the method of glycaemic management (insulin treated (IT) or non-insulin treated (NIT)). METHODS: 72 samples of vitreous were obtained from either diabetic patients with PDR (n = 51) or non-diabetic (control) patients (n = 21). Levels of bFGF, IGF-I, EGF, and insulin were determined by radioimmunoassay; levels of TGF-beta 2 by ELISA; and levels of IGF-I binding protein by western ligand blotting. The data were analysed using appropriate statistics. RESULTS: There was no regular growth factor profile. bFGF levels were significantly greater in vitreous from NIT patients compared with IT patients and controls. The highest levels of bFGF were found in NIT patients with actively vascularised membranes. TGF-beta 2 levels were significantly greater in vitreous from IT patients compared with NIT patients and controls The highest levels of TGF-beta 2 were found in IT patients with actively vascularised membranes. IGF-I levels were significantly greater in diabetics (irrespective of insulin treatment) than non-diabetics and the highest levels of IGF-I were found in IT patients with actively vascularised membranes. A 34 kDa IGFBP was the predominant IGFBP identified in vitreous and was found to be elevated in diabetics patients. CONCLUSION: In PDR there is a correlation between intravitreal growth factor levels and both disease state (whether active or fibrotic) and method of glycaemic management.
Lymphatic drainage of the CNS: effects of lymphatic diversion/ligation on CSF protein transport to plasma.
The plasma recovery of an intraventricularly administered protein was compared before and after lymph diversion/ligation in the same conscious sheep to determine the relative roles of arachnoid villi and lymphatics in the clearance of a cerebral spinal fluid tracer. 125I-human serum albumin was injected into both lateral ventricles, and venous blood was sampled. One day later, multiple cervical vessels and the thoracic duct were cannulated for lymph collection. Uncannulated vessels were ligated. The experiment was repeated with 131I-human serum albumin as the tracer. Before lymph diversion/ligation, the time-averaged tracer transport into the plasma was 6.4 +/- 1.0%/h, with an average 6-h plasma recovery of 38.2 +/- 5.7% (percentage of injected dose). After lymph diversion/ligation, the values dropped to 2.9 +/- 0.5%/h and 17.7 +/- 2.7%, respectively. The collected lymph contained 8.7 +/- 2.6% of the tracer. No significant differences were observed in sham-operated animals. In conclusion, extracranial lymphatic vessels in sheep transport approximately one-half of the protein tracer from the cerebral spinal fluid compartment into plasma.
Cell attachment to, and contraction of, the retina in vitro.
PURPOSE: To examine the behavior of fibroblasts and retinal pigment epithelial cells after attachment to the retinal surface in vitro to elucidate the pathobiology of the early stages of epiretinal membrane formation. METHODS: Human retinal pigment epithelial (HRPE) cells and bovine Tenon's capsule fibroblasts (BTFs) were seeded onto the surface of bovine retinal explants maintained in organ culture. The changes induced in the underlying retina, including contraction, were assessed during a period of up to 10 days. Immunohistochemistry was used to assess proliferation of the seeded cells and to determine deposition of extracellular matrix. RESULTS: Explants of bovine neuroretina were maintained in organ culture, with good morphologic preservation of the inner limiting lamina and inner retinal layers, for 7 to 10 days. The HRPE cells and the BTFs attached to the retinal surface and exerted tractional forces, producing partial- and full-thickness retinal folding. Contraction commenced within 24 hours of attachment of the cells and continued for several days, with most of the contraction occurring within the next 48 to 72 hours. The HRPE cells and BTFs were found to be equally contractile. Deposition of cellular fibronectin (but not collagen type I) was demonstrated. CONCLUSIONS: The contractile cellular membranes generated in this organ culture system exhibit many of the morphologic and functional features of epiretinal membranes found in the early stages of proliferative vitreoretinopathy.
Qualitative research.
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