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D R Chadwick

Publications and source records attributed to D R Chadwick.

29 records · Page 2Linked to original sources
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↗
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

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↗
PubMed1986

The localisation of immunoglobulin in chronic periaortitis.

An immunohistochemical study was undertaken in an attempt to localise immunoglobulin in sections of human advanced atherosclerosis with thinning of the media (sub-clinical periaortitis) and without thinning of the media as well as sections of artery from patients with clinical periaortitis. The findings were that in routinely processed sections of advanced atherosclerosis showing medial attenuation and in sections from cases of clinical periaortitis IgG, and to a lesser extent IgM, was localised to insoluble lipid, ceroid, within the atheroma itself. It is suggested that these observations support the hypothesis that chronic periaortitis has an auto-allergic cause and that the allergen may be a component of ceroid, which is elaborated within the atheroma.

Adult↗
PubMedDate not supplied

Cerebral melioidosis in Singapore: a review of five cases.

A variety of neurological manifestations of infection with Burkholderia pseudomallei have been described including cerebral abscesses, which are a well-recognized form of neurological melioidosis. The optimal antibiotic therapy for this condition has not been defined; however, combinations of intravenous antibiotics are frequently used in the early stages. Five cases of melioidosis involving brain abscesses are described which presented in Singapore over the past 3 years (1997-2000), 4 of which cases had evidence of disseminated infection. Despite profound neurological deficits and low Glasgow Coma Scale scores at presentation in 3 of these cases, all survived after surgical drainage and prolonged courses of intravenous ceftazidime or imipenem, and only 2 of whom had residual neurological impairment. One incidental finding on computed tomography (CT) or magnetic resonance imaging (MRI) scans not described before as an association with cerebral melioidosis was sinusitis in 4 out of the 5 cases.

Adult↗
PubMedDate not supplied

Pancreatic islet purification using bovine serum albumin: the importance of density gradient temperature and osmolality.

Euro-Ficoll and bovine serum albumin (BSA) are two of the most commonly used density gradient media for the purification of pancreatic islets. Euro-Ficoll is based upon Euro-Collins, a cold storage medium, and must, therefore, be used at 4 degrees C. The ionic composition of BSA, however, is likely to contribute to hypothermic cellular swelling, and this may influence the efficiency of islet purification using this medium at 4 degrees C. Experience in this laboratory also suggested that batch-to-batch variation in islet purity using BSA was related to differences in BSA osmolality. The aim of this study was to assess the effect of gradient medium temperature and osmolality on the purification of human and porcine islets using BSA. Pancreata were collagenase-digested, and islets were purified on continuous linear density gradients of BSA. The distribution of insulin and amylase in each gradient was assayed, and used to calculate the median density of islets and exocrine tissue, and the efficiency of islet purification (% amylase contamination at a fixed insulin yield), using: 1) gradient osmolalities of 300, 400, and 500 mOsm/kg H2O (seven porcine pancreata), and 2) gradients at 4 degrees C and at 22 degrees C (eight human and seven porcine pancreata). Increase in density gradient osmolality produced increases in porcine exocrine tissue density which exceeded changes in islet density, resulting in improved islet purity, maximal at a BSA osmolality of 400 mOsm/kg H2O. For human pancreata there was no significant change in pancreatic tissue densities nor islet purity with temperature.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMedDate not supplied

The effect of UW solution and its components on the collagenase digestion of human and porcine pancreas.

University of Wisconsin (UW) solution is used extensively as a cold storage solution during the procurement and transport of the pancreas prior to islet isolation. However, it has been observed that UW inhibits the collagenase digestion phase of human but not porcine islet isolation, resulting in poor islet yields and islets of poor viability. The aim of this study was, therefore, to confirm this species difference and to determine which components of UW are responsible for the inhibition in the human. In the initial experiment, blocks of human and porcine pancreas (n = 7) were incubated in test tubes containing collagenase at a concentration of 4 mg/mL at 37 degrees C dissolved in 4 mL of either Hanks' solution or UW. Every 5 min the tubes were manually shaken and the degree of tissue dissociation scored on a scale of + and 3+. Our results confirm the inhibition of collagenase digestion in the human but not the pig. Using the same methodology, we then investigated the components of UW that were causing the observed inhibition in the human pancreas (n = 7). This time the collagenase was dissolved in individual or combinations of UW components. Using Hank's as a control, the results were then expressed as a median ratio. The components found to be most inhibitory were magnesium, the Na+/K+ ratio, hydroxyethyl starch (HES), and adenosine. Allopurinol in combination with either lactobionate or glutathione was markedly inhibitory (i.e., median ratio 1.8 and 1.9, respectively). The most inhibitory solution tested was combination of the three components raffinose, glutathione, and lactobionate (median ratio 2.1). This combination was almost as inhibitory as UW itself (median ratio 2.7). These findings are essential for the development of effective cold-storage solutions for the human pancreas that do not inhibit the subsequent collagenase digestion phase of islet isolation.

Adenosine↗