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Biomedical subjects

R F James

Publications and source records attributed to R F James.

At least 91 records · Page 5Linked to original sources
PubMed1993

Human islet purification: a prospective comparison of Euro-Ficoll and bovine serum albumin density gradients.

Euro-Ficoll (EF) and bovine serum albumin (BSA) are the two most commonly used media for the density gradient purification of human pancreatic islets. The aim of this study was to compare these two media with respect to the efficiency of human islet isolation. Ten human pancreata were collagenase-digested, and samples of digest were separated on either a continuous linear density gradient of BSA or a discontinuous gradient of EF (1.108/1.096/1.037/Euro-Collins). Efficiency of islet purification was assessed by insulin and amylase assay of aliquots aspirated from the BSA gradients, and from the interfaces of the EF gradients. Islets were obtained from two interfaces in the EF gradients. Islet yield from the upper interface was generally poor (median 28% of total insulin; range 2-71%), but purity was better than for an equivalent yield using BSA [1% (0-3%) amylase contamination for EF versus 6% (0-37%) for BSA; P = 0.013]. Pooling both EF interfaces increased yield to 66% (17-81%) but markedly reduced purity [46% (0-50%) amylase for EF versus 31% (0-52%) for BSA]. In conclusion, the efficiency of human islet purification is similar, though disappointingly low, with BSA and with EF. Considerable scope exists, therefore, for improvement in the density gradient purification of human islets.

Adolescent↗
PubMed1993

The optimization of large-scale density gradient isolation of human islets.

The use of the COBE 2991 cell processor (COBE Laboratories, Colorado) for large-scale islet purification using discontinuous density gradients has been widely adopted. It minimizes many of the problems such as wall effects, normally encountered during centrifugation, and avoids the vortexing at interfaces that occurs during acceleration and deceleration by allowing the gradient to be formed and the islet-containing interface to be collected while continuing to spin. We have produced cross-sectional profiles of the 2991 bag during spinning which allow the area of interfaces in such step gradients to be calculated. This allows the volumes of the gradient media layers loaded on the machine to be adjusted in order to maximize the area of the gradient interfaces. However, even using the maximal areas possible (144.5 cm2), clogging of tissue at such interfaces limits the volume of digest which can be separated on one gradient to 15 ml. We have shown that a linear continuous density gradient can be produced within the 2991 bag, that allows as much as 40 ml of digest to be successfully purified. Such a system combines the intrinsic advantages of the 2991 with those of continuous density gradients and provides the optimal method for density-dependent islet purification.

Cell Separation↗
PubMed1993

A method to transluminally seed angioplasty sites with endothelial cells using a double balloon catheter.

Acute arterial reocclusion and late restenosis following angioplasty may be partially caused by loss of the endothelial cell monolayer during balloon dilatation. Rapid restoration of the endothelial cell monolayer by endothelial cell transplantation has the potential to increase the antithrombotic nature of the angioplasty site and also to prevent myointimal hyperplasia which is the cause of late restenosis. We have investigated a method to transluminally deliver endothelial cells to angioplasty sites using a double balloon catheter with a central instillation port. Inflation of the balloons allows a segment of artery to be isolated from the circulation which may then be incubated with infused endothelial cells. The external iliac arteries of New Zealand white rabbits were dilated for 30 s at 8 atm pressure using a 3 mm balloon angioplasty catheter. Indium-111 labelled allogeneic rabbit endothelial cells were seeded onto the angioplasty site and retained in contact with the vascular surface for 30 min by continued inflation of the isolation balloons. After 30 min, the balloons were deflated and flow restored. Percentage cell retention was calculated by determining the gamma activity of the seeded angioplasty site, and referencing this to the contralateral sham seeded site. Results are expressed as a median with 95% confidence intervals and suggest that endothelial cells may be successfully delivered and retained on damaged vascular surfaces [table: see text].

Angioplasty, Balloon↗
PubMed1993

Storage of porcine pancreatic digest prior to islet purification. The benefits of UW solution and the roles of its individual components.

The aim of this study was to establish the beneficial effect of storage of pancreatic digest in University of Wisconsin solution on porcine islet purification, the mechanism of this effect, and the components of UW responsible. Ten porcine pancreata were collagenase-digested, and samples of digest were washed and stored for 1 hr in either UW or minimum essential medium at 4 degrees C, prior to separation on continuous linear density gradients of bovine serum albumin. Samples of digest from a further ten pancreata were similarly treated, comparing storage in MEM, UW, and five solutions varying in lactobionate:chloride ratio and raffinose content. The purity of the islet preparations and the densities of islets and exocrine tissue were determined from insulin and amylase assay of aliquots aspirated from these gradients. Washing and storage of digest in UW markedly improved islet purity, compared with MEM, due to an increase in the density of exocrine tissue. Exocrine tissue density following storage was dependent upon the control of acinar cell volume, rather than exocrine enzyme discharge, and was determined primarily by the chloride:lactobionate ratio of the storage solution. Raffinose was of little additional benefit, while the beneficial effect of UW was greater than that due to its lactobionate and raffinose content alone. In conclusion, inadequate purification of islets results from exocrine tissue swelling. This swelling is reduced by storage of the pancreatic digest in UW solution, due primarily to the replacement of chloride by lactobionate in UW.

Adenosine↗
PubMed1993

Concentration-dependent effects of adrenaline on the profile of insulin secretion from isolated human islets of Langerhans.

The effects of the mixed alpha/beta-agonist adrenaline on insulin secretion from isolated human islets of Langerhans were studied. In static incubation experiments, adrenaline (0.1 nmol/l to 10 mumol/l) caused a concentration-dependent inhibition of glucose-induced insulin secretion from isolated human islets. However, perifusion experiments revealed that the time-course of the secretory changes induced by adrenaline was complex. When employed at a high concentration (1 mumol/l), adrenaline caused a sustained inhibition of glucose-induced insulin secretion, which could be relieved by the addition of the alpha 2-antagonist yohimbine (10 mumol/l). By contrast, infusion of adrenaline at a lower concentration (10 nmol/l), produced a large initial potentiation of glucose-induced insulin secretion. This response was, however, short-lived and followed by sustained inhibition of secretion, which could be relieved by yohimbine (10 mumol/l). The initial stimulation of insulin secretion provoked by 10 nmol adrenaline/l was abolished when islets were incubated in the presence of the beta-antagonist, propranolol (1 mumol/l), consistent with activation of beta-adrenoceptors. In support of this, treatment of human islets with the selective beta 2-agonist clenbuterol, was also associated with marked stimulation of insulin secretion. By contrast, each of two selective beta 3-agonists tested failed to alter insulin secretion from human islets. The results indicate that human pancreatic B-cells are equipped with both alpha 2- and beta 2-adrenoceptors which can affect insulin secretion. Adrenaline interacts with both of these but the alpha 2-response is predominant and can overcome the tendency of beta 2-adrenoceptors to potentiate insulin release.

Clenbuterol↗
PubMed1992

Storage of human pancreatic digest in University of Wisconsin solution significantly improves subsequent islet purification.

Density-gradient purification of human pancreatic islets from the collagenase-digested pancreas relies on the exocrine tissue being denser than the islets. Cold storage of the pancreas before and after digestion causes cell swelling, which can decrease the density of pancreatic exocrine tissue and adversely affect subsequent purification. Using 14 human pancreata (seven perfused in situ with hyperosmolar citrate (HOC) and seven with University of Wisconsin solution (UW)), it is shown that storage of the pancreatic digest in UW significantly increases the density of pancreatic exocrine tissue compared with storage in minimal essential medium (MEM) (P = 0.009). This results in an improvement in islet purity (P = 0.036) for HOC- but not UW-perfused pancreata. Storage in UW for 1 h not only prevented the deterioration that occurred in MEM, but resulted in an improvement in islet purity for five of the seven HOC-perfused pancreata. Most pancreata in the UK are perfused with HOC, but storage of the digest in UW results in significantly better islet purity and, when islets cannot be purified immediately, a period of storage will often improve separation and allow islets to be purified.

Adenosine↗
PubMed1992

Prostacyclin production from seeded prosthetic vascular grafts.

Endothelial cell seeding has been proposed as a method of improving patency rates in small-calibre prosthetic vascular grafts. In vivo, endothelial cells normally produce prostacyclin (PGI2), a potent antiplatelet agent. The aim of this study was to determine whether seeded grafts show significant PGI2 production after in vivo implantation. Grafts were seeded with either autologous canine venous endothelial cells or autologous microvascular endothelial cells. After 12 weeks, PGI2 production was assessed under basal and stimulated conditions. Seeded grafts were compared with non-seeded controls and the corresponding aorta. The overall patency rate in seeded grafts was 80 per cent compared with 10 per cent in non-seeded grafts (P < 0.01). Grafts seeded with cells from either source produced significantly more PGI2 than unseeded grafts in both basal and stimulated states (P < 0.05). The aorta produced significantly more PGI2 than seeded grafts under both conditions (P < 0.01). Endothelial cell seeding produces a functional graft and leads to an improved patency rate.

Animals↗
PubMed1992

Endothelial cell seeding of damaged native vascular surfaces: prostacyclin production.

Endothelial cell seeding has been successful in reducing the thrombogenicity of prosthetic vascular grafts in animal and clinical studies. The reduction in thrombogenicity may be attributed to the intrinsic properties of endothelial cells themselves, and their ability to produce anti-thrombogenic mediators such as prostacyclin, and endothelium-derived relaxing factor. Endothelial seeding of damaged vascular surfaces produced during percutaneous transluminal angioplasty and endarterectomy is an attractive possibility due to the excellent attachment characteristics of the sub-endothelial tissue exposed during these procedures. The ability of endothelial seeded damaged vascular surfaces to produce prostacyclin was measured in an in vitro model of vascular injury. Endothelial-seeded damaged surfaces produced significantly higher prostacyclin release than did vessels damaged by balloon dilatation (265.5 pg cm-2 min-1 and 87.5 pg cm-2 min-1 respectively). This study provides evidence that endothelial seeding of damaged native vascular surfaces is technically feasible and that seeding may reduce the thrombogenicity of vascular surfaces following balloon dilatation.

Angioplasty, Balloon↗
PubMed1992

Monoclonal antibodies raised against semi-purified preparations of human islets define subpopulations of pancreatic cells.

Monoclonal antibodies were produced from fusions between splenocytes from BALB/c mice immunised with purified human islets and the mouse myeloma cell line NS-0/Uncl. Supernatants from uncloned hybrids were screened by immunohistology on frozen sections of human pancreas. The range of specificities appeared to reflect the relative purity of the human islet preparations used. Eight monoclonal antibodies were investigated further, four of these bound to islet cells, two to acinar cells, one to ductal cells and one to occasional cells. The antigens recognised by these antibodies were characterised by immunohistology using a number of different tissues, as well as haemagglutination, immunoblotting and radioimmunoassay for insulin. Seven of the eight antibodies studied were IgM. One acinar cell antibody (IgG2a) precipitated proteins of 200Kd and 11OKd molecular weight. None of the antibodies bound directly to insulin. Seven of the antibodies appear to have defined previously unreported epitopes in the pancreas and will prove useful in further studies of human pancreatic cells.

Animals↗
PubMed1991

Autoantigen recognition by thyroid-infiltrating T cells in Graves disease.

Graves disease is a common form of human autoimmune thyroiditis. It shares many pathological features and HLA associations with other, less easily studied, organ-specific autoimmune conditions such as insulin-dependent diabetes mellitus, and hence it is also a useful model for understanding these other diseases. We have previously shown that thyroid-infiltrating T cells in Graves disease that have been recently activated in vivo specifically recognize autologous thyroid epithelial cells. However, the autoantigens involved were not defined. In this study, we have made use of antigen-independent T-cell cloning techniques to show that at least three different thyroid antigens, three different epitopes on a single antigen, and two HLA class II elements are involved in this recognition process in a single individual. This demonstrates that T cells that are present and activated at the site of a human autoimmune disease may show considerable heterogeneity in their recognition of autoantigen on the target tissue. This contrasts with the limited heterogeneity recently reported in some animal models and has potentially important implications for both our understanding of the autoimmune process in humans and the design of immunotherapies to reverse it.

Adult↗