Microparticles are small subcellular vesicles, which function in inter-cellular communication by transferring RNA, bioactive lipids, proteins and receptors to target cells. Neutrophil microparticles are abundant in rheumatoid arthritis synovial fluids. Current dogma dictates that cartilage is an impenetrable avascular matrix through which metabolites from the synovial fluid must diffuse; we present the first evidence that microparticles can access chondrocytes through the cartilage. Addition of neutrophil microparticles to chondrocytes in vitro and in vivo afforded protection from arthritogenesis, evidenced by reduced extracellular matrix degradation, increased expression of genes involved in cartilage matrix synthesis and reduced inflammatory mediator production. Adoptive transfer of fluorescently labelled neutrophils into mice with inflammatory arthritis migrated to the inflamed joints and released microparticles, which could be found abundantly within the cartilage. We propose a mechanism whereby microparticles deliver the pro-resolving protein Annexin A1, which engages the receptor FPR2/ALX on the chondrocyte, eliciting tissue protection. This protection could be blocked in part by TGF-β neutralising antibodies and by preventing microparticle phosphatidylserine interaction with chondrocytes by coating with Annexin V. Intriguingly, microparticle treatment directly inhibited the phosphorylation of Hsp27. Hsp27 exists as large oligomers within the resting cell, and upon phosphorylation are released as monomers which function to stabilise the mRNA of certain proinflammatory genes such as IL-8, IL-6 and COX-2; which were effects seen during microparticle/chondrocyte coculture. Thus, microparticles may directly reprogram chondrocytes to prevent the expression of pro-inflammatory cytokines and mediators, but also they exert anti-inflammatory effects via the exposure of phosphatidylserine and induce the production of protective TGF-β by chondrocytes. As cartilage has limited capacity for self-repair, there is an unmet need for therapies that actively repair or protect cartilage, especially in Rheumatoid Arthritis. We envisage these microparticles could offer therapeutic possibilities in the protection of cartilage in situ in these difficult-to-treat patients.

Microparticles (MP) are submicron structures produced by all cells upon activation or apoptosis that act as a non-soluble means of communication between cells. They ferry proteins, bioactive lipids, RNA and receptors, as well as ridding cells of redundant organelles and toxins. They have been recently investigated for their pathophysiological role and as potential biomarkers/effectors in many diseases. In severe sepsis, studies of MP so far have produced inconsistent and even conflicting results. In this project, it was demonstrated that cell derived MP subsets vary according to the cause of severe sepsis (community acquired pneumonia (CAP) or faecal peritonitis (FP)), where CAP patients had higher levels of circulating MP. Surprisingly, FP patients MP levels were comparable to healthy volunteers. Further stratification of MP subsets according to their expression of the protein alpha-2-macroglobulin (A2M) has yielded better differentiation between the two diseases. The A2M expressing MP were significantly higher in survivors of community acquired pneumonia sepsis, but there was no similar association in patient with FP. Granulocyte macrophage colony-stimulating factor (GM-CSF) and interferon γ (IFN-γ) are being studied as possible adjuvant therapies in sepsis. They seem to reverse the immune-paresis that ensues after the initial insult. MP produced from whole blood stimulated with GM-CSF and IFN-γ were studied in this project. Both GM-CSF and IFN-γ increased MP expressing A2M over control. These MP elicited a pro-inflammatory phenotype when incubated with neutrophils or endothelial cells which may contribute to the potential benefits of GM-CSF and IFN-γ in severe sepsis.

Aim: Endothelial microparticles (EMPs) are released from dysfunctional endothelial cells. We hypothesised that patients with unstable carotid plaque have higher levels of circulating microparticles compared to patients with stable plaques, and this may correlate with serum markers of plaque instability and inflammation. Method: Circulating EMPs and inflammatory markers were measured in twenty healthy controls and seventy patients undergoing carotid endarterectomy. EMPs were quantified using flow cytometry. Bioplex assays profiled systemic inflammatory and bone-related proteins. Immunohistological analysis detailed the contribution of differentially-regulated systemic markers to plaque pathology. Alizarin red staining showed calcification. Results: EMPs were significantly higher in patients with carotid stenosis (greater than or equal to 70%) compared to controls, with no differences between asymptomatic vs symptomatic patients. Asymptomatic patients with unstable plaques exhibited higher levels of EMPs compared to those with stable plaques, with a similar trend observed in symptomatic patients. CXCL9 and SCGF-β were significantly elevated in asymptomatic patients with unstable plaques, with IL-16 and macrophage inhibitory factor significantly elevated in the stable plaque group. CXCL9, CTACK and SCGF-β were detected within all plaques, suggesting a contribution to both localised and systemic inflammation. Osteopontin and osteoprotegerin were significantly elevated in the symptomatic vs asymptomatic group, while osteocalcin was higher in asymptomatic patients with stable plaque. All plaques exhibited calcification, which was significantly greater in asymptomatic patients. This may impact on plaque stability. Conclusions: Circulatory EMP, CXCL9 and SCGF-β levels are raised in asymp-tomatic patients with unstable plaques, which could be important in identifying patients at most benefit from intervention.

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Microparticles (KEMs) made from the sorghum prolamin protein, kafirin, have internal vacuoles. Hence, they have potential as delivery vehicles for nutraceuticals. However, their physico-chemical properties need to be improved for this application. The influence of kafirin extracted from white tan-plant and red non-tannin sorghum types of 81% and 84% protein content respectively and the rate of water addition on the formation of KEMs from kafirin in acetic acid solution by coacervation on their morphology was investigated. A water flow rate of 1.4 and 0.7 ml/min during coacervation using 81% kafirin resulted in spherical-shaped KEMs between 1 and 10 μm in diameter and vacuoles up to 2 μm. KEMs made with 84% kafirin at a flow rate of 0.7 ml/min were large and oval-shaped with an average length and width of 43 and 21 μm respectively and numerous vacuoles up to 3 μm. At a flow rate of 1.4 ml/min, the KEMs were oval-shaped with larger vacuole sizes (5 μm), a length and width of 91 and 30 μm respectively. However, SDS-PAGE indicated that neither the source of kafirin, nor the conditions of microparticle preparation had an effect on KEMs protein molecular size.As the presence of phenolic compounds in the kafirins might have been responsible for the differences in KEMs morphology, the effect of sorghum-derived polyphenols (extracted from condensed-tannin and non-tannin black sorghum brans) on the physico-chemical properties of KEMs was then investigated using 81% kafirin. Aqueous condensed tannin (10.1 mg CE (catechin equivalent)/100 mg extract) and black non-tannin (4.6 mg CE/100 mg extract) extracts in varying concentrations, were substituted for the water used for coacervation. KEMs made with condensed tannin extracts were oval-shaped and much larger, than control KEMs ranging from 20 to 400 μm, with rough surfaces and enlarged vacuoles. The enlarged vacuoles were probably due to more air being trapped within the particles during formation. However, KEMs made from non-tannin phenolic extracts were smaller and spherical with average diameters up to 18 μm. Tannins are known to bind strongly to kafirin through hydrogen and hydrophobic bonds, which probably resulted in the larger microparticles. The KEMs made from condensed tannins also had high antioxidant capacities compared to KEMs made from non-tannin phenolic extracts, attributed to tannins being more potent antioxidants. Thus, condensed tannin extracts are the most beneficial as they contributed towards the antioxidant activity of the KEMs, resulting in the development of innovative KEMs with added antioxidant benefits and enlarged size. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Food Science / unrestricted

In an effort to understand the resuspension phenomena, interactions of spherical micro-meter particles (glass beads (GB) and Stainless steel (SS)) were investigated experimentally on different surfaces (glass, ceramic, hardwood, metal and chemical agent resistant coated metal (CARC)). Particles were deposited on the lower surface of a 10 cm square wind tunnel by gravitational settling. Air flows were imposed from an open entrance at average velocities up to 16 m/s. Individual particle trajectories obtained by high-speed imaging reveal three different types of motion: rolling/bouncing, immediate liftoff and complex motion. Surface roughness significantly affects the particle initial motion prior to liftoff. The majority of particle trajectories from the glass substrate were parallel to the surface with complex motion, covering 25% of the total distance traveled in rolling/bouncing motion before liftoff. Hardwood substrates took the longest time for initial particle movement (t >1 s) causing a more rapid liftoff. The ceramic substrate showed the most rolling/bouncing motion, for 80% of the particles. Additionally, single layer detachment showed that the detachment percentage initially follow an exponentially increasing trend for a period of ~ 1 s, followed by a plateau phase for a period of 5 s. Changing velocity, substrate and particle size significantly affects GB particle detachment. Furthermore, detachment from the metal substrate was consistently higher than the CARC substrates. However, particle density is not a significant difference in the bigger particle size studied. Initial 3-D particle tracking showed that particles seem to travel in a constant angle to the left rather than going straight in the flow direction. A detachment mode model showed that the detachment by direct liftoff required a much higher speed than rolling motion with a minimum of 14 m/s for both GB70 and SS70 on glass and metal surface, and the velocity increased to 21 m/s for the smaller particle. Incorporating the different types of particle motion prior to liftoff into resuspension models, and how their relative contributions change with different particle and substrate materials, can potentially yield improved predictive capabilities.

Ultrafine grained (UFG), and nanocrystalline (nc) materials are of interest because of the high strength, compared with coarse grained counterparts. Several current methods to fabricate UFG and nc materials result in samples too small for practical use. In addition, the fabrication of nc materials, in particular, is difficult, and defects in the material causes significant reduction in strength and ductility of these materials. The present study uses Equal Channel Angular Extrusion (ECAE) to produce relatively large consolidates of UFG and nc materials. ECAE has been used to consolidate micro and nanocrystalline powders. The behavior of consolidated pure Cu and aluminum alloys in the nano and micron size were explored. The effects of different routes, extrusion temperature, and post-ECAE processing on microstructure and mechanical behavior were studied. Processing parameters were explored to determine the influence of each parameter on the consolidation performance. The goals of experimenting with different processing parameters were to increase the ductility of the material, while maintaining relatively strong specimens. Comparisons were made with a recently developed powder compaction constitutive model and corresponding simulations. ECAE of microcrystalline powders produced relatively ductile materials, with high strength. Swaging of these consolidated powders produced samples which were softer and less ductile in tension, than the non-swaged samples. ECAE produced effective consolidation of Cu nanoparticles with average sizes of 100 nm, with an ultimate tensile strength of 680 MPa, with a fracture strain as much as 10%, which is higher than previously reported 7% [Haouaoui, 2005].

Ph.D.