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Biomedical subjects

R F James

Publications and source records attributed to R F James.

At least 73 records · Page 4Linked to original sources
PubMed1994

Storage of pancreatic digest before islet purification. The influence of colloids and the sodium to potassium ratio in University of Wisconsin-based preservation solutions.

The density-dependent purification of islets from several species of mammalian pancreata is improved by prior storage of the dispersed, collagenase-digested pancreas in suitable storage solutions, such as University of Wisconsin (UW) solution. The optimal composition of such solutions, however, is not fully established, although previous investigations have suggested separately that cellular impermeants and colloids are important components. To investigate this issue further, dispersed tissues from 7 porcine and 7 human pancreata were stored in UW or in solutions containing the impermeants lactobionate and raffinose, with either no added colloid or in the presence of the colloids hydroxyethyl starch, dextran 40, dextran 250, or Ficoll 400; hydroxyethyl starch-containing solutions in which the principal cation was sodium, rather than potassium, were also studied. Subsequent purification of islets on continuous linear density gradients of BSA was then assessed by insulin/amylase assay of gradient fractions. Islet purity was slightly reduced using solutions containing impermeants but lacking a colloid, compared with using UW. In the combined presence of impermeants and a colloid, however, islet purity was similar to that obtained with UW, and for porcine pancreata, solutions containing Ficoll 400 or dextran 40 were slightly superior to UW. Purity was not, however, influenced by the sodium to potassium ratio of storage media. In conclusion, impermeants and colloids are both essential components of solutions used to preserve pancreatic tissue before islet purification, findings which may be relevant when designing media for use during other phases of islet isolation, e.g., during collagenase digestion/density gradient purification.

Adenosine↗
PubMed1994

Effect of pulsatile shear stress on endothelial attachment to native vascular surfaces.

An in vitro model of vascular damage was used to investigate the ability of seeded endothelial cells to resist shear stresses generated in a perfusion circuit. At perfusion rates of 100 ml/min the maximum shear stress reached 16.5 dyn/cm2. At this level the rate of cell detachment from the damaged vascular surface was 88 per cent per h for the first 20 min of flow but gradually decreased to 5 per cent per h after 90 min. These findings suggest that endothelial cells may be retained on damaged vascular surfaces in conditions that approximate to arterial flow.

Blood Flow Velocity↗
PubMed1994

ATP-induced intracellular Ca2+ signals in isolated human insulin-secreting cells.

Using isolated beta-cells from human islets of Langerhans we have demonstrated that purinergic receptor agonists are functionally coupled to rises in the intracellular calcium ion concentration ([Ca2+]i). The effects of ATP, ADP and AMP have been examined over a range of concentrations, 0.5 to 500 microM. The actions of ATP were more potent than those of either ADP or AMP suggesting that a P2-type of purinergic receptor operates in these cells. Responses to ATP were concentration-related, but exhibited marked desensitisation at high concentrations (> 100 microM). Purinergic receptor agonists elevate [Ca2+]i by mechanisms that involve both Ca2+ influx and Ca2+ mobilisation from intracellular stores. The physiological significance of our data has been discussed, and related to previous studies carried out upon rodent and clonal insulin-secreting cells.

Adenosine Diphosphate↗
PubMed1994

Platelet deposition after angioplasty is abolished by restoration of the endothelial cell monolayer.

PURPOSE: Percutaneous transluminal angioplasty of an atheromatous plaque causes endothelial desquamation and intimal dissection with the consequent formation of a thrombogenic flow surface. In this study we investigated the hypothesis that platelet deposition after balloon angioplasty may be decreased by rapid restoration of the endothelial cell monolayer, achieved by transluminally seeding angioplasty sites with endothelial cells. METHODS: Bilateral external iliac angioplasty was performed in eight New Zealand white rabbits. One angioplasty site was isolated from the circulation and incubated with a supraconfluent endothelial cell suspension with a double balloon catheter; the contralateral angioplasty site was sham seeded with culture medium. The deposition of autologous indium 111-labeled platelets on the angioplasty sites was quantified 30 minutes after restoration of flow and was referenced to an undamaged segment of aorta that acted as a negative control. RESULTS: Platelet deposition on the nonseeded angioplasty site (13.1 x 10(4) platelets/mm2) was significantly higher than on nondilated segments (3.4 x 10(4) platelets/mm2; p = 0.014). Restorationof endothelial cell coverage by endothelial seeding significantly reduced platelet deposition on dilated arterial segments to levels not significantly higher than in controls (3.6 x 10(4) platelets/mm2; p = 0.014). CONCLUSIONS: These results illustrate that rapid reendothelialization of angioplasty sites decreases subsequent platelet deposition and may reduce the rate of acute arterial reocclusion complicating endovascular techniques.

Angioplasty, Balloon↗
PubMed1994

The effect of transluminal endothelial seeding on myointimal hyperplasia following angioplasty.

Myointimal hyperplasia develops as a generalised response to vascular injury, and may cause stenoses in 40% of all peripheral arterial reconstructions. Disruption of the endothelial monolayer is a prerequisite for the development of intimal hyperplasia, and may be the initiating event in this process. This study examined the hypothesis that restenosis following balloon angioplasty may be reduced by rapid restoration of the endothelial monolayer, achieved by endothelial seeding. Bilateral iliac angioplasties were performed in 11 rabbits. A double balloon catheter was used to seed one angioplasty site with autogenous endothelial cells; the contralateral site was sham seeded with culture medium and acted as a control. Arterial patency rates, the degree of intimal hyperplasia (IH/IEL), and the extent of endothelialisation were quantified at 1 (n = 5) and 3 (n = 6) weeks following balloon dilatation. The results suggest that transluminal endothelial seeding may be a therapeutically applicable technique as it decreases myointimal hyperplasia, and increases patency following angioplasty. This study also illustrates the protective effect of the vascular endothelium following arterial injury, and indicates that intensive efforts should be made to preserve the endothelium during vascular reconstruction.

Angioplasty, Balloon↗
PubMed1994

The effectiveness of components of University of Wisconsin solution in improving human pancreatic islet purification.

The purification of human pancreatic islets before transplantation relies on the density-dependent separation of islets from exocrine fragments after collagenase digestion of the donor pancreas. The results vary among pancreases despite increasing automation of the digestion and purification processes, reflecting variations in the overlapping densities of islets and contaminating exocrine tissue. Hypothermic storage of both the pancreas and the pancreatic digest alters cell volumes and tissue densities, thereby affecting islet purification. By biochemical analysis of the isopycnic distribution of islets and exocrine tissue fragments from 23 human pancreases on linear continuous density gradients, the effect of various solutions for cold storage of pancreatic digest was studied. The use of the University of Wisconsin cold storage solution, which resulted in a significant decrease in digest volume (P = 0.006) and increase in the densities of both exocrine tissue (P = 0.001) and islets (P = 0.005), produced a significant improvement in islet purity compared with tissue culture medium (P = 0.035), predominantly due to the inclusion of a colloid, which increased the difference in density between exocrine tissue and islets. The addition of large molecular weight cellular impermeants without alteration in the concentration of permeable anions produced no effect. The results of this study support the concept that the use of solutions that minimize cell swelling throughout the process of islet purification would result in significant improvements in density-dependent islet separation, and that such solutions should contain a colloid.

Adenosine↗
PubMed1994

The nature of the epithelium in acquired cholesteatoma: Part 3--Cytokeratin patterns in aural epithelial and cholesteatoma cells grown in cell culture.

The nature and origin of the epithelial layers in acquired cholesteatoma is still unclear. Although previous morphological studies comparing external meatal and cholesteatoma epithelium have shown no significant difference, bone resorption is generally much more severe with cholesteatoma than with chronic otitis media without cholesteatoma. It is possible that cholesteatoma epithelium has undergone transformation leading to its enhanced bone destroying role. In this study the cytokeratin patterns of aural and cholesteatoma epithelia grown in cell culture were compared using monoclonal antibodies. No significant difference in staining patterns were found suggesting that there has been no change in cell phenotype which maintains that of external auditory meatus epithelium. This study therefore supports the immigration theory of cholesteatoma genesis.

Antibodies, Monoclonal↗
PubMed1993

Porcine islet isolation: prospective comparison of automated and manual methods of pancreatic collagenase digestion.

A prospective study was undertaken to compare an automated method of porcine pancreatic digestion with a simpler manual procedure. These techniques have not previously been compared directly. After intraductal distension with collagenase, seven porcine pancreata were divided longitudinally; half of each was digested by the automated method and half by the manual technique. Islet yield and purity were measured. Compared with the manual technique, the automated method isolated a significantly greater total volume of islet tissue (median (range) 3.56 (1.39-5.30) versus 1.07 (0.46-1.92) mm3/g, P = 0.022), increased the median (range) number of 105-microns islet equivalents isolated (5875 (2294-8746) versus 1766 (759-3168) per g, P = 0.022) and improved the islet cleavage index (median (range) 92 (89-99) versus 82 (78-92) per cent, P = 0.035). It is concluded that, although the automated method is more complicated to set up, it greatly improves the yield of intact islets from the porcine pancreas.

Animals↗
PubMed1993

Effect of seeding time and density on endothelial cell attachment to damaged vascular surfaces.

An in vitro model to facilitate the study of endothelial cell seeding of damaged vascular surfaces has been developed. This may have applications in the study of endothelial seeding of angioplasty and endarterectomy sites. Using this model, the optimum endothelial seeding time for attachment to damaged vascular surfaces should not exceed 30 min and, to achieve confluent cell attachment, a seeding density > 5 x 10(5) cells/cm2 should be used.

Angioplasty, Balloon↗
PubMed1993

Cryopreservation of rat islets of Langerhans: a comparison of two techniques.

Although a number of different protocols have been described for the cryopreservation of pancreatic islets of Langerhans, most involve equilibration with 2 M Me2SO followed by slow cooling and fast warming. This paper compares two methods with identical equilibration protocols: one with cooling at 0.25 degree C/min to -40 degrees C prior to transferring to liquid nitrogen and subsequent warming at 200 degrees C/min and sucrose dilution of Me2SO (Method A) and the other with cooling at 0.3 degree C/min to -70 degrees C prior to transferring to liquid nitrogen and warming at 50 degrees C/min followed by stepwise dilution of Me2SO (Method B). Islet viability was assessed by syngeneic transplantation to the renal subcapsular space of streptozotocin-induced diabetic rats. Seven hundred fifty fresh islets in the size range 100-200 microns consistently reversed diabetes (blood glucose < 10 mmol/liter) whereas 1000 Method A cryopreserved islets and 2000 Method B cryopreserved islets were required. Cryopreservation by either method resulted in impaired islet function compared to that of fresh islets, although this functional loss could be partially compensated by the transplantation of greater numbers of cryopreserved islets.

Animals↗
PubMed1993

Islet microencapsulation: a review.

The original report on the microencapsulation of islets of Langerhans used sodium alginate and poly-L-lysine (PLL) to form the capsules. Although several alternative materials have subsequently been used with vary-mg degrees of success, it is those studies using islets encapsulated in alginate-PLL-alginate which are reviewed in detail in this article. Since the first report of islet microencapsulation, many studies have demonstrated excellent in vitro viability of encapsulated islets. However, transplantation experiments into chemically induced diabetic recipients have yielded varied results, with some studies showing good long-term graft function whilst in others grafts failed due to pericapsular fibrosis. The use of naturally occurring animal models of type 1 (insulin-dependent) diabetes has demonstrated a decline in graft function, suggesting that this presents a more complex problem to be solved than that in chemically induced diabetic recipients. Fibrosis of capsules has been the major problem causing graft failure, and this has been demonstrated to be more severe in spontaneously diabetic models. However, recent advances in alginate purification and attempts to reduce the size of the encapsulated islets are major steps towards encapsulated islet transplants becoming a viable proposition for the treatment of type 1 diabetic patients.

Alginates↗
PubMed1993

The use of continuous density gradients for the assessment of islet and exocrine tissue densities and islet purification.

The purification of large numbers of human pancreatic islets remains one of the limiting factors in islet transplantation. This paper describes and validates a method for accurately and reproducibly determining the density of islets and exocrine tissue in pancreatic digest on the basis of their isopycnic distribution on linear continuous density gradients. The use of this data to analyse and compare the purity of a standard 60% islet yield is described. The results obtained using such gradients will enable factors responsible for the variation in yield between pancreases to be determined and optimized, improving the results and reliability of islet purification.

Cell Separation↗