The C1q binding activity of IgG is modified in vitro by reactive oxygen species: implications for rheumatoid arthritis

IgG can be denatured in vitro by reactive oxygen species (ROS). Native IgG activates the complement cascade through C1q. Using a modified ELISA, C1q binding activity of rheumatoid IgG has been compared to IgG denatured by neutrophil-derived ROS. The C1q binding activity of rheumatoid synovial fluid IgG is greater than the corresponding serum IgG (P < 0.01). Denaturation of IgG by activated polymorphs or the Fenton reaction decreased its C1q binding activity (P < 0.01). In vitro exposure of IgG to OH. and ROO. increased its interaction with C1q (P < 0.01). Hypochlorous acid had no effect. ROS-induced alteration to IgG-C1q binding activity may promote the inflammatory response in rheumatoid arthritis.

Computer simulation of radio-frequency methane/hydrogen plasmas and their interaction with GaAs Surfaces

The following thesis describes the computer modelling of radio frequency capacitively coupled methane/hydrogen plasmas and the consequences for the reactive ion etching of (100) GaAs surfaces. In addition a range of etching experiments was undertaken over a matrix of pressure, power and methane concentration. The resulting surfaces were investigated using X-ray photoelectron spectroscopy and the results were discussed in terms of physical and chemical models of particle/surface interactions in addition to the predictions for energies, angles and relative fluxes to the substrate of the various plasma species. The model consisted of a Monte Carlo code which followed electrons and ions through the plasma and sheath potentials whilst taking account of collisions with background neutral gas molecules. The ionisation profile output from the electron module was used as input for the ionic module. Momentum scattering interactions of ions with gas molecules were investigated via different models and compared against results given by quantum mechanical code. The interactions were treated as central potential scattering events and the resulting neutral cascades were followed. The resulting predictions for ion energies at the cathode compared well to experimental ion energy distributions and this verified the particular form of the electrical potentials used and their applicability in the particular geometry plasma cell used in the etching experiments. The final code was used to investigate the effect of external plasma parameters on the mass distribution, energy and angles of all species impingent on the electrodes. Comparisons of electron energies in the plasma also agreed favourably with measurements made using a Langmuir electric probe. The surface analysis showed the surfaces all to be depleted in arsenic due to its preferential removal and the resultant Ga:As ratio in the surface was found to be directly linked to the etch rate. The etch rate was determined by the methane flux which was predicted by the code.

Lipoidal species in ocular spoilation processes

Tear component deposition onto contact lenses is termed `spoilation' and occurs due to the interaction of synthetic polymers with their biological fluid environment. Spoilation phenomena alter the physico-chemical properties of hydrophilic contact lenses, diminishing the optical properties of the lens; causing discomfort and complications for the wearer. Eventually these alterations render the lens unwearable. The primary aim of this interdisciplinary study was to develop analytical techniques capable of analysing the minute quantities of biological deposition involved, in particular the lipid fraction. Prior to this work such techniques were unavailable for single contact lenses. It is envisaged that these investigations will further the understanding of this biological interfacial conversion. Two main analytical techniques were developed: a high performance liquid chromatography (HPLC) technique and fluorescence spectrofluorimetry. The HPLC method allows analysis of a single contact lens and provided previously unavailable valuable information about variations in the lipid profiles of deposited contact lenses and patient tear films. Fluorescence spectrophotofluorimetry is a sensitive non-destructive technique for observing changes in the fluorescence intensity of biological components on contact lenses. The progression and deposition of tear materials can be monitored and assessed for both in vivo and in vitro spoiled lenses using this technique. An improved in vitro model which is comparable to tears and chemically mimics ocular spoilation was also developed. This model allows the controlled study of extrinsic factors and hydrogel compositions. These studies show that unsaturated tear lipids, probably unsaturated fatty acids, are involved in the interfacial conversion of hydrogel lenses, rendering them incompatible with the ocular microenvironment. Lipid interaction with the lens surface then facilitates secondary deposition of other tear components. Interaction, exchange and immobilisation (by polymerisation) of the lipid layer appears to occur before the final and rapid growth of more complex, insoluble discrete deposits, sometimes called `white spots'.

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.