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GM-CSF production by RPE cells, which form part of the blood–retina barrier, is upregulated by IL-1β and this increase can be reversed by IFN-γ. IL-1β up-regulation is not dependent on PKC but the PKC activator PMA induces low levels of GM-CSF production and acts synergistically with IL-1β to further increase GM-CSF. Although A23187 and ionomycin stimulated low levels of GM-CSF production, the IL-1β pathway was cyclosporin A insensitive and did not interact with the calcium pathway. IL-1β-stimulated GM-CSF mRNA expression and production was strongly dependent on NF-κB. IFN-γ inhibition of the GM-CSF response to IL-1β acted via NF-κB, reducing the translocation of NF-κB to the nuclei of RPE cells treated with IL-1β and IFN-γ. The results show that IFN-γ down-regulation acts either directly on NF-κB or its activation or by blockade of a pathway upstream of NF-κB. However, any such blockade does not involve PKC or intracellular calcium.

Experimental studies of ocular inflammation induced by endotoxin have been performed almost exclusively in rabbits. Although the rat has been considered refractory to both the systemic and local effects of endotoxin, the present study has shown that intravitreal injection of endotoxin evoked a characteristic acute inflammatory response which was reproducible and dose-dependent. In addition, a consensual ocular response in the control eye occurred which was less severe but also dose-dependent. Preliminary investigations support the view that consensual responses of this nature may be mediated by a nervous reflex arc mechanism. Sequential histological analysis revealed a marked polymorphonuclear cell infiltration of most ocular tissues in the early stages, while mononuclear cells predominated after 3 days. Widespread intravascular microthrombi were also observed, particularly in the ciliary vessels. Recovery, however, was usually complete by 7 days.

Reorientation and migration of cultured bovine corneal epithelial cells (CECs) in an electric field were studied. Electric field application was designed to model the laterally directed, steady direct current electric fields which arise in an injured corneal epithelium. Single cells cultured in media containing 10% foetal bovine serum showed significant galvanotropism, reorienting to lie perpendicular to electric field vector with a threshold field strength of less than 100 mV/mm. Cells cultured in serum-free medium showed no reorientation until 250 mV/mm. Addition of EGF, bFGF or TGF-beta 1 singly or in combination to serum free medium significantly restored the reorientation response at low field strengths. Both the mean translocation rate and directedness of cell migration were serum dependent. Cultured in medium with serum or serum plus added EGF, single cells showed obvious cathodal migration at 100 mV/mm. Increasing electric field strength enhanced the cathodal directedness of single cell migration. Supplementing serum free medium with growth factors restored the cathodal directed migration of single cells and highest directedness was found for the combination of EGF and TGF-beta 1. Corneal epithelial sheets also migrated towards the cathode in electric fields. Serum or individual growth factors stimulated CEC motility (randomly directed). Applied fields did not further augment migration rates but added a vector to stimulated migration. Electric fields which are present in wounded cornea interact with other environmental factors and may impinge on CECs migration during wound healing. Therapies which combine the application of growth factors and electric fields may be useful clinically.

The significantly different signals from the various structures within the orbit suggest that NMR tomography will prove to be a valuable method for the diagnosis of both ocular and orbital disease.The resolution of the technique is initially illustrated by the demonstration of the signal patterns from the normal globe and orbit. The characteristic features of some pathological conditions, including vitreous haemorrhage and retinal detachment, are then shown. The value and limitations of the technique in differentiating conditions such as choroidal haemorrhage from malignant melanoma, and intra-orbital lymphoma from pseudo-tumour, are illustrated and discussed. The absenc of artefact from bone, synthetic lens implants and non-ferromagnetic metallic foreign bodies is stressed.

Sixty patients presenting with retinal detachment have been examined using a Nuclear Enterprises Diasonograph 4102; 25 with no identifiable cause (primary), the remainder due to causes such as vitreous haemorrhage, trauma, inflammatory eye disease, aphakia and neoplasm.In the diagnosis and management of retinal detachment echography has a definite but limited application, particularly in patients who have cloudy or opaque media and in whom detachment secondary to a malignant melanoma is suspected. In patients with trauma or vitreous haemorrhage important therapeutic considerations depend upon a precise knowledge of the structure of the globe and complicating features such as vitreous fibrosis or retinal detachment. In such patients the value of echo amplitude measurements in combination with B-scan echography is described. In primary retinal detachment because of vitreous haziness, the extent, site and degree of displacement of the retinal detachment maybe more precisely demonstrated echographically than by conventional ophthalmoscopy, facilitating surgical management.

A demonstration of the B-scan echographic appearances of some commonly encountered lens abnormalities is presented. Examples of intumescent, traumatic and calcified cataracts, congenital and traumatic displacements and other abnormalities are shown. The significance of these findings is discussed and the importance of pre-operative ultrasonic examination of the patient for cataract extraction to exclude vitreous or retinal pathology is stressed. The value of echographic examination in traumatic cases, where vitreous haemorrhage has occurred, in the demonstration of intraocular abnormalities, such as retinal detachment or a non-radio-opaque foreign body is also discussed.

The locomotory behavior of human blood neutrophil leukocytes was studied at a boundary between two surfaces with different chemokinetic properties. This was achieved by time-lapse cinematography of neutrophils moving on coverslips coated with BSA, then part-coated with immune complexes by adding anti-BSA IgG with a straight-line boundary between the BSA and the immune complexes. Cell locomotion was filmed in microscopic fields bisected by the boundary, and kinetic behavior was assessed by comparing speed (orthokinesis), turning behavior (klinokinesis), and the rate of diffusion of the cells on each side of the boundary, using a recently described mathematical analysis of kinesis. In the absence of serum or complement, the proportion of motile cells and their speed and rate of diffusion were greater on BSA than on antiBSA, but there was no consistent difference in turning behavior between cells on the two surfaces. The immune complexes were therefore negatively chemokinetic in comparison with BSA, and this resulted from a negative orthokinesis with little or no contribution from klinokinesis. As would be predicted theoretically, this resulted in gradual accumulation of cells on the immune complexes even in the absence of a chemotactic factor. In further studies, a parallel plate flow chamber was used to show that, under conditions of flow, neutrophils accumulated much more rapidly on a surface coated with BSA-anti-BSA than on BSA alone. Moreover, neutrophils on immune complex-coated surfaces lost their ability to form rosettes with IgG-coated erythrocytes. This suggests that neutrophils on immune complex-coated surfaces redistribute their Fc receptors (RFc gamma) to the under surface, and that the lowered speed of locomotion is due to tethering of neutrophils by substratum-bound IgG-Fc.

By passing a suspension of polymorphonuclear leucocytes through a parallel-plate chamber their adhesiveness can be assessed by scoring the number of cells trapped on the lower plate, and the fluid shear stress can be defined for a given flow rate. Since the adhesiveness of the cell at the instant of collision must exceed the distractive shear if the cell is to stop, the kinetics of cell accumulation provide a measure of the adhesiveness of the leucocytes and the adhesive interaction can be quantified. This measure of adhesion does not suffer from the complication that the force required to remove the cells from the surface will be greater if the cells have the opportunity to spread before the distractive force is applied. The assay is described in detail and the results of modifying the surface of the flow chamber and altering the composition of the suspension medium are used to illustrate the method. Plasma proteins generally seemed to reduce the adhesiveness of neutrophil leucocytes, whether they were present as a coat of adsorbed protein or in the suspension medium during perfusion. Neutrophil leucocytes, unless suspended in relatively high concentrations of plasma, were considerably more adhesive than other cells that have been tested in this assay system.

Ninety-two patients with various ocular and retro-orbital abnormalities have had orbital B-scanning carried out using the Nuclear Enterprises Diasonograph 4102. Using the 5 MHz transducer and a simple direct water bath, satisfactory results have been obtained in a wide variety of pathological conditions in the lens, vitreous and retroorbital structures. Several illustrations are shown which demonstrate that standard ultrasonic equipment can be used successfully for ophthalmic investigation.

The effect of glycosaminoglycans on the invasion of choroid fibroblasts into type I collagen gels was studied. Both hyaluronate and chondroitin sulphate, when incorporated into the gel, facilitated invasion of the collagen matrix, although hyaluronate was considerably more effective. Hyaluronate-induced fibroblast invasion was markedly concentration-dependent, being reduced at both high and low concentrations. Increased cell invasion appeared to correlate with denser packing of collagen fibrils within the gel, since the same effect could be achieved by increasing the collagen concentration of native, i.e. glycosaminoglycan-free gels. Scanning electron microscopy of the interior of the collagen gels suggested that changes in packing arrangement of fibrils in gels that had polymerized in the presence of glycosaminoglycans might account in part for different rates of cell invasion.

Adhesion of BHK cells to a variety of polymer surfaces carrying measured densities of hydroxyl and carboxyl groups was studied. The effects on cell adhesion of blocking hydroxyl groups by acetylation and carboxyl groups with diazomethane were measured. Hydroxyl groups were required for cell adhesion, though the very high surface densities of these groups diminished cell adhesion. The optimal surface density of OH groups for BHK adhesion was 2000 per 1× 10−11 cm2. Carboxyl groups slightly inhibit cell adhesion, since blocking of these groups by methylation increased adhesion. The role of oxidizing systems of cellular origin in conditioning of the substrate, in serum-free conditions, was demonstrated for leucocytes and BHK cells, in particular by the result that oxidizable substrates such as phytane and poly(l,2-butadiene) could be made suitable for cell adhesion by contact with cells.

Binding curves for the adsorption of plasma fibronectin, alpha-l-antitrypsin, alpha-2-macroglobulin, ceruloplasmin, transferrin and bovine serum albumin to plain and to hydroxylated polystyrene surfaces were measured. These curves were correlated with the adhesion of BHK cells and leucocytes to these adsorbed protein surfaces in protein-free culture media. Hydroxylated polystyrene adsorbed less of alpha-l-antitrypsin, alpha-2-macroglobulin and albumin than the plain polystyrene. On the other hand the hydroxylated surfaces bound more fibronectin than the plain polystyrene surfaces. Hydroxylated polystyrene surfaces were also more adhesive for both BHK cells and leucocytes than plain polystyrene: a result confirming earlier work. The competition of fibronectin for adsorption to plain polystyrene with alpha-l-antitrypsin, alpha-2-macroglobulin and ceruloplasmin was measured and correlated with effects on cell adhesion. The results suggest that the low adhesiveness of BHK cells and leucocytes on plain polystyrene in sera-containing media is due both to the low binding of fibronectin and to the binding of serum albumin, alpha-l-antitrypsin and alpha-2-macroglobulin. The relative unimportance of fibronectin in adhesion to these surfaces is shown by the finding that cell attachment will not occur to polystyrene surfaces that have bound high levels of the antiadhesive proteins in the presence of fibronectin, even though attachment will occur in the absence of fibronectin provided that the antiadhesive proteins are lacking.

Neutrophil leucocytes circulate in plasma, which contains broad-spectrum antiproteases such as alpha-2-macroglobulin and alpha-1-antitrypsin, and move into environments in which antiproteases are present. Plasma antiproteases significantly reduce the adhesiveness of leucocytes in both static and dynamic adhesion assays, although their action does not seem to depend upon their anti-protease capacity. Contrary to expectation, leucocyte movement over surfaces and through both rigid and deformable three-dimensional matrices (micropore filters and collagen gels) is unaffected by antiproteases. This renders the idea that forward movement depends upon the proteolytic cleavage of redundant adhesion sites less probable. Various non-physiological antiproteases (chloromethyl ketones, soya bean trypsin inhibitor, and ε-aminocaproic acid are also ineffective in reducing locomotion, although they do cause minor changes in adhesiveness.

The effect of hyaluronate on neutrophil motility in vitro was studied by the micropore filter technique and by direct visual analysis of the locomotion of neutrophils on glass. Both directed and random locomotion of neutrophils were inhibited by physiological concentrations (0·5–6·0 mg ml−1) of hyaluronate in adose- and molecular weight-dependent manner. Inhibition of cell movement was more pronounced for high molecular weight chemoattractants such as casein than for small chemotactic peptides such as f-Met-Leu-Phe. Chemotactic factor gradient formation in filter chambers was profoundly retarded by hyaluronate, which may partly explain the inhibitory effects of hyaluronate on directed neutrophil locomotion. In addition, hyaluronate inhibited the binding of chemotactic factor to the neutrophil surface. This effect, together with a reduction in cell-to-substratum adhesion, may provide an additional explanation for hyaluronate-induced inhibition of random neutrophil locomotion. Inhibition of locomotion by hyaluronate was easily reversed by washing the cells free of hyaluronate; thus competition by hyaluronate for cell-surface binding sites is unlikely, and physical effects such as steric exclusion or molecular sieving by the large hyaluronate polymer provide the most probable explanations of its inhibitory effect on cell locomotion. Since hyaluronate is a major constituent of tissue matrices, these results draw attention to the importance of the extracellular environment in regulating inflammatory cell movement in vivo.

The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin.