Elevated levels of corticotrophin-releasing factor binding protein in the blood of patients suffering from arthritis and septicaemia and the presence of novel ligands in synovial fluid.

In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis that from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF-41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease.

Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites

Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H2O2) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint. (c) 2006 Elsevier Inc. All rights reserved.

Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Hydrogen sulfide (H(2)S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H(2)S in response to pro-inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H(2)S. HACs and MPCs were exposed to IL-1β, IL-6, TNF-α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H(2)S synthesizing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) were determined by Western blot and zinc-trap spectrophotometry, respectively. Cellular oxidative stress was induced by H(2)O(2), the peroxynitrite donor SIN-1 and 4-hydroxynonenal (4-HNE). Cell death was assessed by 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H(2) S synthesis was inhibited by siRNA-mediated knockdown of CSE and CBS and pharmacological inhibitors D,L-propargylglycine and aminoxyacetate, respectively. Exogenous H(2)S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H(2)S in a CBS-dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL-1β, TNF-α, IL-6 or LPS. Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment. These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs. H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

General relationships between pressure, weight, and mass of a hydrostatic fluid

In curved geometries the hydrostatic pressure in a fluid does not equal the weight per unit area of the fluid above it. General weight–pressure and mass–pressure relationships for hydrostatic fluids in any geometry are derived. As an example of the mass–pressure relationship, we find a geometric reduction in surface pressure as large as 5 mbar on Earth and 39 mbar on Titan. We also present a thermodynamic interpretation of the geometric correction which, as a corollary, provides an independent proof of the hydrostatic relationship for general geometries.

Diagnostic analysis of atmospheric moisture and clear-sky radiative feedback in the Hadley Centre and Geophysical Fluid Dynamics Laboratory (GFDL) climate models

The interannual variability of the hydrological cycle is diagnosed from the Hadley Centre and Geophysical Fluid Dynamics Laboratory (GFDL) climate models, both of which are forced by observed sea surface temperatures. The models produce a similar sensitivity of clear-sky outgoing longwave radiation to surface temperature of ∼2 W m−2 K−1, indicating a consistent and positive clear-sky radiative feedback. However, differences between changes in the temperature lapse-rate and the height dependence of moisture fluctuations suggest that contrasting mechanisms bring about this result. The GFDL model appears to give a weaker water vapor feedback (i.e., changes in specific humidity). This is counteracted by a smaller upper tropospheric temperature response to surface warming, which implies a compensating positive lapse-rate feedback.

The acoustic signature of fluid flow in complex porous media

Effective medium approximations for the frequency-dependent and complex-valued effective stiffness tensors of cracked/ porous rocks with multiple solid constituents are developed on the basis of the T-matrix approach (based on integral equation methods for quasi-static composites), the elastic - viscoelastic correspondence principle, and a unified treatment of the local and global flow mechanisms, which is consistent with the principle of fluid mass conservation. The main advantage of using the T-matrix approach, rather than the first-order approach of Eshelby or the second-order approach of Hudson, is that it produces physically plausible results even when the volume concentrations of inclusions or cavities are no longer small. The new formulae, which operates with an arbitrary homogeneous (anisotropic) reference medium and contains terms of all order in the volume concentrations of solid particles and communicating cavities, take explicitly account of inclusion shape and spatial distribution independently. We show analytically that an expansion of the T-matrix formulae to first order in the volume concentration of cavities (in agreement with the dilute estimate of Eshelby) has the correct dependence on the properties of the saturating fluid, in the sense that it is consistent with the Brown-Korringa relation, when the frequency is sufficiently low. We present numerical results for the (anisotropic) effective viscoelastic properties of a cracked permeable medium with finite storage porosity, indicating that the complete T-matrix formulae (including the higher-order terms) are generally consistent with the Brown-Korringa relation, at least if we assume the spatial distribution of cavities to be the same for all cavity pairs. We have found an efficient way to treat statistical correlations in the shapes and orientations of the communicating cavities, and also obtained a reasonable match between theoretical predictions (based on a dual porosity model for quartz-clay mixtures, involving relatively flat clay-related pores and more rounded quartz-related pores) and laboratory results for the ultrasonic velocity and attenuation spectra of a suite of typical reservoir rocks. (C) 2003 Elsevier B.V. All rights reserved.

Variation in dynamic elastic shear modulus of sandstone upon fluid saturation and substitution

Experimental acoustic measurements on sandstone rocks at both sonic and ultrasonic frequencies show that fluid saturation can cause a noticeable change in both the dynamic bulk and shear elastic moduli of sandstones. We observed that the change in dynamic shear modulus upon fluid saturation is highly dependent on the type of saturant, its viscosity, rock microstructure, and applied pressures. Frequency dispersion has some influence on dynamic elastic moduli too, but its effect is limited to the ultrasonic frequency ranges and above. We propose that viscous coupling, reduction in free surface energy, and, to a limited extent, frequency dispersion due to both local and global flow are the main mechanisms responsible for the change in dynamic shear elastic modulus upon fluid saturation and substitution, and we quantify influences.

Weathering microenvironments on feldspar surfaces: implications for understanding fluid-mineral reactions in soils

The mechanisms by which coatings develop on weathered grain surfaces, and their potential impact on rates of fluid-mineral interaction, have been investigated by examining feldspars from a 1.1 ky old soil in the Glen Feshie chronosequence, Scottish highlands. Using the focused ion beam technique, electron-transparent, foils for characterization by transmission electron microscopy were cut from selected parts of grain surfaces. Some parts were bare whereas others had accumulations, a few micrometres thick, of Weathering products, often mixed with mineral and microbial debris. Feldspar exposed at bare grain surfaces is crystalline throughout and so there is no evidence for the presence of the amorphous 'leached layers' that typically form in acid-dissolution experiments and have been described from some natural Weathering contexts. The weathering products comprise sub-mu m thick crystallites of an Fe-K aluminosilicate, probably smectite, that have grown within an amorphous and probably organic-rich matrix. There is also evidence for crystallization of clays having been mediated by fungal hyphae. Coatings formed within Glen Feshie soils after similar to 1.1 ky are insufficiently continuous or impermeable to slow rates Of fluid-feldspar reactions, but provide valuable insights into the complex Weathering microenvironments oil debris and microbe-covered mineral surfaces.

The unsteady flow of a weakly compressible fluid in a thin porous layer. I: Two-dimensional theory

We consider the problem of determining the pressure and velocity fields for a weakly compressible fluid flowing in a two-dimensional reservoir in an inhomogeneous, anisotropic porous medium, with vertical side walls and variable upper and lower boundaries, in the presence of vertical wells injecting or extracting fluid. Numerical solution of this problem may be expensive, particularly in the case that the depth scale of the layer h is small compared to the horizontal length scale l. This is a situation which occurs frequently in the application to oil reservoir recovery. Under the assumption that epsilon=h/l<<1, we show that the pressure field varies only in the horizontal direction away from the wells (the outer region). We construct two-term asymptotic expansions in epsilon in both the inner (near the wells) and outer regions and use the asymptotic matching principle to derive analytical expressions for all significant process quantities. This approach, via the method of matched asymptotic expansions, takes advantage of the small aspect ratio of the reservoir, epsilon, at precisely the stage where full numerical computations become stiff, and also reveals the detailed structure of the dynamics of the flow, both in the neighborhood of wells and away from wells.