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Biomedical subjects

J C Edwards

Publications and source records attributed to J C Edwards.

At least 19 recordsLinked to original sources
PubMed1998

Functional expression of p64, an intracellular chloride channel protein.

p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity.

Cell Membrane Permeability↗
PubMed1998

Differential distribution of Fc gamma RIIIa in normal human tissues and co-localization with DAF and fibrillin-1: implications for immunological microenvironments.

Fc gamma RIIIa is a cytokine-inducible IgG Fc receptor implicated in the activation of macrophages by immune complexes. Differential expression of Fc gamma RIIIa by macrophages in different tissues may therefore modulate local immune responsiveness. Fc gamma RIIIa expression in normal human tissues was assessed semiquantitatively using microdensitometry. Synovial intimal, serosal, alveolar, salivary gland and placental macrophages, Kupffer cells, and macrophages in mechanically stressed dermis expressed high levels of Fc gamma RIIIa. Less consistent expression was seen in skeletal muscle and lymphoid organs. No significant expression was observed in brain, thyroid, spine, intestine, myocardium, prostate, uterus, flexor forearm dermis, uterus, or kidney. Staining for Fc gamma RIII was also observed on extracellular matrix, and co-localized with both complement decay-accelerating factor and fibrillin-1. It is proposed that differential levels of both cellular and extracellular Fc gamma RIIIa, by modulating the response to immune complexes, may contribute to relative tissue susceptibility to infection and autoimmune disease.

CD55 Antigens↗
PubMed1998

Foot pain: specific indications for scintigraphy.

Bone scintigraphy is requested as part of the investigation of foot pain, but its contribution to clinical management has not been comprehensively documented. Previously published data are limited; the most comprehensive series identified scintigraphic abnormalities in patients with primarily orthopaedic problems and a control group was not included (Maurice HD et al. J Bone Joint Surg 1987;69B:448 52). The aim of this study was to evaluate whether bone scintigraphy may be useful in different clinical circumstances indicated by referral request details. Regions of scintigraphic abnormality were scored and compared with clinical details drawn from case notes of 60 patients with foot pain. The commonest clinical indications for scans were: confirmation of the clinical suspicion of plantar fasciitis, documentation of the extent of inflammatory arthritis and location of focal pathology. A group of 30 asymptomatic, age- and sex-matched controls were also studied. In 14 out of 19 symptomatic feet in the plantar fasciitis group, focal uptake at the medial calcaneal tubercle was present, confirming the diagnosis. In patients with non-specific, diffuse foot pain, the bone scan identified focal abnormalities in 11 out of 14 cases, thus directing the clinician to the site of pathology. Scintigraphy also proved useful in mapping local inflammatory disease. Technetium-99m methylene diphosphonate image abnormalities occurred in the control group most commonly in the midfoot (16 regions in 13 subjects) and first metatarsophalangeal joint (19 regions in 14 subjects).

Adolescent↗
PubMed1998

An essential role for the interaction between hyaluronan and hyaluronan binding proteins during joint development.

We studied the expression of hyaluronan binding proteins (HABPs) during the development of embryonic chick joints, using immunocytochemistry and biotinylated HA. The expression of actin capping proteins and of actin itself was also studied because the cytoskeleton is important in controlling HA-HABP interactions. Three cell surface HABPs were localized in the epiphyseal cartilage, articular fibrocartilage, and interzone that comprise the developing joint. Of these three HABPs, CD44 was associated with the articular fibrocartilages and interzone, whereas RHAMM and the IVd4 epitope were associated with all three tissues. Biotinylated HA was localized to interzone and articular fibrocartilages before cavity formation and within epiphyseal chondrocytes post cavitation. Actin filament bundles were observed at the developing joint line, as was the expression of the actin capping protein moesin. Manipulation of joint cavity development, using oligosaccharides of HA, disrupted joint formation and was associated with decreases in CD44 and actin filament expression as well as decreased hyaluronan synthetic capability. These results suggest that HA is actively bound by CD44 at the developing joint line and that HA-HABP interactions play a major role in the initial separation events occurring during joint formation.

Actins↗
PubMed1997

Characterization of the osteoclast ruffled border chloride channel and its role in bone resorption.

Bone resorption by osteoclasts requires massive transcellular acid transport, which is accomplished by the parallel action of a V-type proton pump and a chloride channel in the osteoclast ruffled border. We have studied the molecular basis for the appearance of acid transport as avian bone marrow mononuclear cells acquire a bone resorptive phenotype in vitro. We demonstrate a critical role for regulated expression of a ruffled border chloride channel as the cells become competent to resorb bone. Molecular characterization of the chloride channel shows that it is related to the renal microsomal chloride channel, p64. In planar bilayers, the ruffled border channel is a stilbene sulfonate-inhibitable, outwardly rectifying chloride channel. A mechanism by which outward rectification of the single channel chloride current could allow efficient regulation of acidification by the channel is discussed.

Animals↗
PubMed1997

Detection of antigen-antibody binding events with the atomic force microscope.

An atomic force microscope (AFM) has been used to directly monitor specific interactions between antibodies and antigens employed in an immunoassay system. Results were achieved using AFM probes functionalized with ferritin, and monitoring the adhesive forces between the probe and anti-ferritin antibody-coated substrates. Analysis of the force distribution data suggests a quantization of the forces, with a period of 49 +/- 10 pN. This periodic force may be attributed to single unbinding events between individual antigen and antibody molecules. These results demonstrate that the AFM could be employed as an analytical tool to study the interactions between the molecules involved in biosensor systems. The potential of the technique to provide information relating to the manner in which the antibody molecule binds to its specific antigen is also discussed.

Animals↗
PubMed1997

Restricted expression of Fc gammaRIII (CD16) in synovium and dermis: implications for tissue targeting in rheumatoid arthritis (RA).

Interactions between immune complexes and immunoglobulin Fc receptors may contribute to inflammation in RA. Previous studies suggested that Fc gammaRIII (CD16) may be preferentially expressed in diseased synovial intima. The distribution of immunoreactive Fc gammaRIII was examined in normal fetal limb tissues, and both normal and selected abnormal samples of adult synovium and skin. In fetal limbs at 10-14 weeks gestation Fc gammaRIII was restricted to synovial intima. In normal adult synovium Fc gammaRIII was restricted to intimal cells. In inflamed synovia differential expression of Fc gammaRIII in the intima was less consistent. In both fetal and adult synovium Fc gammaRIII was largely restricted to cells expressing CD45 (leucocyte common antigen). Staining for Fc gammaRIII was, however, occasionally associated with CD45 intimal cells in fetal synovium. In both fetal and adult tissues cell membrane Fc gammaRIII was frequently closely associated with complement decay-accelerating factor (DAF), which is present on intimal fibroblasts and extracellular matrix. Fc gammaRIII expression was minimal in normal forearm dermis, but widespread on CD45+ cells in skin exposed to mechanical stress. In skin containing rheumatoid nodules, Fc-gammaRIII was preferentially expressed on palisading macrophages. These observations indicate that expression of Fc gammaRIII on macrophages may be involved in the susceptibility of connective tissues to immune complex-induced damage in RA. Colocalization of Fc gammaRIII and DAF in synovium may indicate an unrecognized functional interrelationship.

Adult↗
PubMed1997

Expression of molecules involved in B lymphocyte survival and differentiation by synovial fibroblasts.

The synovitis of rheumatoid arthritis (RA) is one of few pathological lesions in which B lymphocyte accumulation progresses to the extent of germinal centre formation. The present study was designed to assess the ability of synovial fibroblasts to express molecules implicated in B lymphocyte survival and differentiation, both in vivo, and in response to cytokines in vitro. Normal and diseased synovia were examined by indirect immunofluorescence. In all tissues synovial intimal fibroblasts showed co-expression of vascular cell adhesion molecule-1 (VCAM-1) and complement decay-accelerating factor (DAF) comparable to that of follicular dendritic cells (FDC), but not complement receptor 2 (CR2). In rheumatoid synovia, subintimal cells showed variable expression of VCAM-1 and DAF, with bright co-expression of VCAM-1, DAF and CR2 in lymphoid follicle centres. B lymphocytes, some of which were proliferating cell nuclear antigen-positive, were present in contact with subintimal cells expressing VCAM-1 with or without DAF or CR2. B lymphocytes were rarely present in the intimal layer, and, where present, showed fragmentation. In vitro, synovial fibroblasts exposed to tumour necrosis factor-alpha (TNF-alpha) in combination with interferon-gamma (IFN-gamma) showed enhanced expression of VCAM-1, in comparison with fibroblasts from skin and lung and, unlike skin and lung fibroblasts, also expressed DAF and CR2. These findings support the hypothesis that synovial targeting in RA involves an enhanced ability of synovial fibroblasts to support B lymphocyte survival. This appears to be dependent, not on the constitutive expression of VCAM-1 and DAF on intimal cells, but on the increased ability of subintimal cells to respond to proinflammatory cytokines, perhaps critically in the expression of VCAM-1.

B-Lymphocytes↗
PubMed1997

Subcellular distribution and targeting of the intracellular chloride channel p64.

p64 is an intracellular chloride channel originally identified in bovine kidney microsomes. Using a combination of immunofluorescent and electron microscopic technique, we demonstrate that p64 resides in the limiting membranes of perinuclear dense core vesicles which appear to be regulated secretory vesicles. Heterologous expression of p64 in PancI cells, a cell type which does not normally express p64, results in targeting to a similar compartment. Mutagenesis experiments demonstrate that both the N- and C-terminal domains of the protein independently contribute to subcellular distribution of the protein. The C-terminal domain functions to prevent expression of p64 on the plasma membrane and the N-terminal domain is necessary to deliver p64 to the appropriate membrane compartment.

Animals↗
PubMed1997

Measurement of synovial lining volume by magnetic resonance imaging of the knee in chronic synovitis.

OBJECTIVES: Current methods of monitoring chronic synovitis in a single joint rely on clinical examination derived indices, such as the detection of synovial thickening. This study aimed to develop a reproducible method for quantifying the volume of synovial lining in chronic synovitis using contrast enhanced magnetic resonance (MR) imaging. METHODS: The knees of 18 patients with chronic synovitis were examined (34 studies). A 2D T1 weighted FLASH sequence was used to evaluate the temporal enhancement of synovial structures after intravenous contrast. Synovial lining volume was calculated from subtraction of pre and post-enhancement 3D T1 weighted MP RAGE images with thresholding and pixel counting. Eleven patients were examined before and after intra-articular glucocorticoid (mean interval 14 weeks) and MR data compared with changes in clinical examination derived indices of disease activity. RESULTS: Synovial lining volume varied from 52-267 ml. The coefficient of variation in volume calculation was 3.5% for a single observer and was 3.8% for two observers. Synovial lining volume was quantified in all patients where synovial lining thickening could not be detected clinically. A decrease in synovial lining volume of > 40% was associated with an improvement in synovial lining thickening, detected clinically. Decreases in synovial lining volume were quantified by MR in two of three patients where changes in clinical examination derived indices were inconsistent. CONCLUSIONS: A reproducible method of estimating the volume of synovial lining in patients with chronic synovitis has been developed. MR measurement of synovial lining volume may quantify changes in chronic synovitis that remain unidentified by clinical measures.

Adult↗
PubMed1996

In situ observation of streptavidin-biotin binding on an immunoassay well surface using an atomic force microscope.

Polystyrene microtitre wells are commonly used as supports for the enzyme-linked immunosorbent assay (ELISA) method of biomolecular detection, which is employed in the routine diagnosis of a variety of medical conditions. We have used an atomic force microscope (AFM) to directly monitor specific molecular interactions between individual streptavidin and biotin molecules on such wells. This was achieved by functionalising an AFM probe with biotin and monitoring the adhesive forces between the probe and a streptavidin coated immunoassay well. The results demonstrate that the AFM may be employed as an analytical tool to study the interactions between biomolecules involved in immunoassay systems.

Bacterial Proteins↗
PubMed1996

The discrimination of IgM and IgG type antibodies and Fab' and F(ab)2 antibody fragments on an industrial substrate using scanning force microscopy.

We have previously employed scanning force microscopy (SFM) to study antibody-antigen molecular interactions on microtiter wells used for enzyme linked immunosorbant assays (ELISA). Here we demonstrate the ability of SFM to image and discriminate different types of antibody and antibody fragments bound to an ELISA well surface. The samples studied include a type IgG antibody with a proportion of bound IgM and two-dimensional films of whole IgG antibody, and Fab' and F(ab)2 antibody fragments. Molecular resolution is achieved in each case despite the size of substrate features exceeding most of the molecular dimensions observed. Analysis of the data shows that the SFM overestimates molecular dimensions by an approximately constant amount, which is proposed to principally result from the effects of a finite probe size and not from deformation of the molecular species due to the imaging forces employed.

Animals↗
PubMed1996

Cellular distribution and regulation of NHE-1 isoform of the NA-H exchanger in the avian osteoclast.

The sodium-hydrogen exchanger (NHE) has been implicated in bone resorption by osteoclasts. We have studied expression of NHE-1, an isoform of the NHE, in chicken bone marrow mononuclear phagocyte precursors during differentiation into the osteoclast phenotype in culture. A monoclonal anti-body raised against the carboxy-terminus of NHE-1 detected the presence of a 100 kDa protein (similar to the mammalian form of NHE-1) in the osteoclasts. Laser scanning confocal microscopy revealed association with the alpha(v)beta(3) integrin and focal adhesion kinase (pp(125)FAK) at the basolateral membrane (BLM) of the osteoclast in addition to a more generalized cellular distribution. A fragment of avian NHE-1 cDNA was obtained by polymerase chain reaction cloning, and it was used to characterize expression of NHE-1 transcripts in cultured chicken osteoclast precursors. The avian NHE-1 message was a 3.9 kB band on Northern analysis, which differed from the mammalian message. Retinoic acid (RA) elicited an increase in the steady-state intracellular pH (pH(1)) from 6.87 to 7.10 in the absence of bicarbonate and was inhibited by ethylisopropylamiloride, an inhibitor of Na-H exchange. Using ribonuclease protection assays, we found that NHE-1 transcripts are induced as cells differentiate in vitro and in response to 13-cis-RA. Western blot analysis indicated that protein levels also increased in response to 13-cis-RA. Our results demonstrate expression of NHE-1 in avian osteoclasts with a complex cellular distribution in culture, and NHE-1 expression is induced as cells differentiate into mature osteoclasts in response to 13-cis-RA.

Amino Acid Sequence↗
PubMed1996

Patterns of cellular activation after tendon injury.

Mechanisms which lead to disabling adhesions following flexor tendon surgery of the hand were investigated in a rabbit model which was used to assess the relative response of the cells of the synovial sheath, epitenon and the endotenon to injury. A transverse laceration, cutting through 50% of the tendon, was made just outside the synovial sheath on the flexor aspect of the flexor digitorum profundus tendon. The synovial sheath was preserved intact. Using monoclonal antibodies for localizing specific inflammatory markers, we were able to follow the response and activity of the synovial sheath, epitenon and endotenon with respect to these markers at various times after surgery. Our findings suggest that the synovial sheath and the epitenon are relatively more reactive in the early period after injury, as judged by a range of inflammatory indices with the notable exception of the expression of the potent neovascularizing agent, basic fibroblast growth factor (bFGF).

Animals↗
PubMed1996

Clinical outcome after one year following samarium-153 particulate hydroxyapatite radiation synovectomy.

The clinical outcome and tolerability following treatment with samarium-153 particulate hydroxyapatite was evaluated in patients with persistent rheumatoid knee synovitis. The clinical review of 18 patients treated with intra-articular samarium-153 particulate hydroxyapatite combined with triamcinolone hexacetonide who had failed to obtain more than 4 weeks symptom relief from a prior intra-articular glucocorticoid injection was undertaken. No unwanted effects from the treatment were observed. Symptom relief was maintained in 56% patients at 6 months and in 44% of patients at 12 months following treatment. Median duration of symptom relief was 9 months. There was a significantly higher mean baseline Ritchie Articular Index in patients relapsing within 3 months and a trend towards earlier relapse in patients with higher indices of disease activity at the time of treatment. There was a trend towards earlier relapse in patients with a poor range of knee flexion at baseline and with worse indices of intra-articular radiopharmaceutical distribution. Samarium-153 particulate hydroxyapatite knee synovectomy is well tolerated and may be an effective treatment for carefully selected patients with persistent rheumatoid knee synovitis.

Adult↗