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Biomedical subjects

M G Evans

Publications and source records attributed to M G Evans.

At least 37 records · Page 2Linked to original sources
PubMed1991

Transplantation of purified frozen/thawed canine pancreatic islet allografts with cyclosporine.

We studied the survival of defined volumes of highly purified frozen/thawed islets transplanted into the spleen of 4 groups of apancreatic mongrel dogs (total, 21), with or without cyclosporine from day -4 to day 30: group 1 (controls), without CsA, autograft of mean (+/- SE) islet volume 5 +/- 1 microliters/kg body weight; group 2, single-donor allograft, and group 3, multiple-donor allograft (both, 6 +/- 1 microliters/kg), both with CsA; and group 4, large-volume multiple-donor allograft (24 +/- 1 microliters/kg) with CsA. Grafts were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. The CsA dose was adjusted to maintain whole-blood trough values of 600-800 micrograms/L, and allograft recipients manifesting early hyperglycemia were given insulin to maintain plasma glucose concentration below 150 mg/dl. In group 1, two dogs died early (graft failure in 1, intussusception in 1), but the remaining five were normoglycemic at 30 days. In groups 2 and 3, hyperglycemia ensued from about day 2.5, but 4 of 5 grafts in group 2 and all grafts in group 3 secreted insulin into the splenic vein at 30 days. In group 4, normoglycemia ensued within 24 hr of transplantation and was maintained in all 6 dogs for 30 days; the grafts failed 15 +/- 2 days after CsA was stopped. The data demonstrate prolonged function of purified frozen/thawed islets after transplantation into these outbred dogs but indicate a need for a greater volume of allogeneic islets from multiple donors than of autologous islets to achieve normoglycemia.

Animals↗
PubMed1991

The actions of calcium on the mechano-electrical transducer current of turtle hair cells.

1. Mechano-electrical transducer currents evoked by deflections of the hair bundle were recorded in turtle isolated hair cells under whole-cell voltage clamp. The outcome of perfusing with solutions of reduced Ca2+ concentration was investigated. 2. The transducer current was roughly doubled by lowering the concentration of divalent cations from normal (2.2 mM-Mg2+, 2.8 mM-Ca2+) to 0 Mg2+, 0.5 mM-Ca2+. No significant effects on the current's kinetics or reversal potential, or on the current-displacement relationship, were noted. 3. If the Ca2+ concentration was lowered to 50 microM (with no Mg2+), there was about a threefold increase in the maximum current but other changes, including loss of adaptation and a decreased slope and negative shift in the current-displacement relationship, were also observed. As a result, more than half the peak transducer current became activated at the resting position of the hair bundle compared to about a tenth in the control solution. 4. The extra changes manifest during perfusion with 50 microM-Ca2+ had also been seen when the cell was held at positive potentials near the Ca2+ equilibrium potential. This supports the view that some consequences of reduced external Ca2+ stem from a decline in its intracellular concentration. 5. With 20 microM-Ca2+, a standing inward current developed and the cell became unresponsive to mechanical stimuli, which may be explained by the transducer channels being fully activated at the resting position of the bundle. 6. The results are interpreted in terms of a dual action of Ca2+: an external block of the transducer channel which reduces the maximum current, and an intracellular effect on the position and slope of the current-displacement relationship; the latter effect can be modelled by internal Ca2+ stabilizing one of the closed states of the channel. 7. During perfusion with 1 microM-Ca2+, the holding current transiently increased but then returned to near its control level. There was a concomitant irreversible loss of sensitivity to hair bundle displacements which we suggest is due to rupture of the mechanical linkages to the transducer channel. 8. Following treatment with 1 microM-Ca2+, single-channel currents with an amplitude of -9 pA at -85 mV were sometimes visible in the whole-cell recording. The probability of such channels being open could be modulated by small deflections of the hair bundle which indicates that they may be the mechano-electrical transducer channels or conductance about 100 pS. 9. Open- and closed-time distributions for the channel were fitted by single exponentials, the mean open time at rest being approximately 1 ms. The mean open time was increased and the mean closed time decreased for movements of the hair bundle towards the kinocilium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Evolution of the MHC class I genes of a New World primate from ancestral homologues of human non-classical genes.

The products of the classical human major histocompatibility complex (MHC) class I genes (HLA-A, -B, -C) are highly polymorphic molecules that bind peptides and present them to T lymphocytes. The non-polymorphic, non-classical MHC class I gene products (HLA-E, -F, -G) are not restricting elements for the majority of T lymphocytes. The evolutionary relationship of the non-classical and classical MHC class I genes is unclear. Here we present the cloning and sequencing of the MHC class I genes of a New World primate, the cotton-top tamarin (Saguinus oedipus). The expressed MHC class I genes of this species are more closely related to the human non-classical HLA-G gene than they are to genes of the human classical HLA-A, -B, and -C loci. These observations imply that classical and non-classical genes do not necessarily constitute mutually exclusive groups over evolutionary time.

Amino Acid Sequence↗
PubMed1990

Prolonged function of canine pancreatic fragments autotransplanted to the spleen by venous reflux.

The long-term viability and function of pancreatic islets transplanted as nonvascularized dispersed grafts has not been well established. We report maintenance of nondiabetic carbohydrate metabolism for up to 36 months (17.8 +/- 1.4 months) in 77% of 40 consecutive canine recipients of nonpurified islet grafts transplanted to the spleen by venous reflux. Spontaneous loss of graft function occurred in 9 dogs during the follow-up period. Failure usually occurred within 1 year of implantation and was predicted by low K value and low insulin output on intravenous glucose tolerance tests 1 and 3 months postimplant. High K values and insulin output correlated strongly with maintenance of function beyond 24 months. A sufficient mass of implanted islets can provide satisfactory metabolic control for prolonged periods.

Animals↗
PubMed1990

Reversal of diabetes in dogs by transplantation of pure cryopreserved islets.

We have autotransplanted highly purified cryopreserved canine islets into pancreatectomized dogs. Islets were frozen by slow cooling (0.25 degrees C/min) to -40 degrees C, stored at -196 degrees C and thawed rapidly (200 degrees C/min). A total of 7868 +/- 665 (means +/- SE) cryopreserved islets/kg body weight were implanted to the spleen (n = 7) by venous reflux, and seven other control dogs received 6811 +/- 653 fresh islets/kg (P versus cryo., NS). Fasting plasma glucose [( PG] mg/dl, +/- SEM) and intravenous glucose tolerance were determined before and at 1 and 3 months postimplantation. Six dogs that received cryopreserved islets and all seven control animals promptly became normoglycemic, with PGs of 133 +/- 23 and 110 +/- 4, respectively, at 1 week postimplantation (NS). During follow-up one normoglycemic animal from each group died due to small bowel volvulus and one recipient of fresh islets became diabetic at 14 days. The remaining dogs remained normoglycemic with PGs of 89 +/- 3 and 92 +/- 3 in dogs receiving cryopreserved and fresh islets, respectively, at 3 months postimplantation (NS). Mean K value (decline of glucose, %/min +/- SEM) at ivGTT for all dogs was 2.5 +/- 0.3 preoperatively. At 1 and 3 months postimplantation, the K values of dogs receiving cryopreserved islets were 1.3 +/- 0.1 and 1.4 +/- 0.2, respectively, compared with 1.3 +/- 0.2 and 2.0 +/- 0.2 for control dogs (NS). These data demonstrate prompt and sustained function of a defined quantity of frozen-thawed purified islets in a large mammal. Cryopreservation is an effective method for the long-term storage of purified islet tissue.

Animals↗
PubMed1990

Potassium currents in hair cells isolated from the cochlea of the chick.

1. Potassium currents were characterized in tall hair cells of the chick's cochlea. Outward potassium currents were found to flow through two distinct classes of channels. 2. Individual hair cells were isolated from 200 microns long segments of the apical half of the chick's cochlea. Whole-cell voltage-clamp and current-clamp recordings were made from these cells. 3. Voltage responses to injected current ranged from high-frequency (100-250 Hz) oscillations in some cells, to slowly repetitive Ca2+ action potentials or slow oscillations (5-20 Hz) in others. 4. Ionic currents recorded in voltage clamp also varied in different hair cells. Cells with high-frequency voltage oscillations had rapidly activating Ca2(+)-dependent outward K+ current, IK(Ca). Cells that generated action potentials had slow delayed rectifier outward K+ current, IK, and inward rectifier current, IIR. All hair cells had inward Ca2+ current. 5. IK(Ca) activated positive to -45 mV. Tail currents reversed at the K+ equilibrium potential. This current was eliminated in Ca2(+)-free solutions, or when exposed to 10 mM-TEA. This outward current was fully activated within 1-3 ms at 0 mV. The whole-cell current was noisy and ensemble variance analysis suggested a single-channel conductance of 63 pS near 0 mV. 6. IK activated positive to -50 mV. Tail currents reversed at the K+ equilibrium potential. This current was not eliminated in Ca2(+)-free solutions, and was relatively resistant to external TEA. IK activated slowly, reaching peak values in 10-20 ms at 0 mV. This current showed little variance and the average single-channel conductance based on macroscopic noise near 0 mV was 8 pS. 7. External tetraethylammonium (TEA) or Ca2(+)-free saline eliminated the high-frequency voltage oscillations seen in many basal cells. In contrast TEA had little effect on slow action potentials (or low-frequency oscillations) seen in cells with IK. 8. IK(Ca) was prominent in hair cells originating 1.0-2.0 mm from the cochlear apex. IK and IIR dominated the membrane conductance of tall hair cells originating within 0.5 mm of the cochlear apex. 9. The frequency of voltage oscillation in apical cells was temperature-dependent, nearly doubling for each 10 degrees C rise in temperature. 10. IIR activated at membrane potentials negative to -75 mV. The average time constant of activation at -100 mV was 2 ms. Tail currents reversed at the K+ equilibrium potential and did not depend on the external Na+ concentration. IIR was blocked by 5 mM-Cs+ or 100 microM-Ba2+ in the external saline.

Action Potentials↗
PubMed1990

Calcium currents in hair cells isolated from the cochlea of the chick.

1. Calcium currents were characterized in tall hair cells isolated from the chick's cochlea to determine what types of Ca2+ channels existed and if these varied in cells with differing voltage responses to current injection. 2. Whole-cell, tight-seal recordings showed that the current-voltage relation of cochlear hair cells of the chick was dominated by K+ current. However, when outward K+ current was blocked it was found that all hair cells had a smaller, maintained inward current. 3. This inward current was a Ca2+ current since it required Ca2+ in the external medium, could also be carried by Ba2+, and was blocked reversibly by 5 mM-Co2+ and by Ni2+ and Cd2+ at micromolar concentrations. The Ca2+ channels were opened at membrane potentials positive to -50 mV, and the current was maximal near 0 mV. 4. The dihydropyridine BayK8644 (0.5 microM) produced a voltage-dependent increase of inward current. Ten micromolar nifedipine partially blocked the inward current. The outward Ca2(+)-activated K+ current was also reduced in the presence of 10 microM-nifedipine. These effects of dihydropyridines were completely reversible. 5. The Ca2+ current had rapid activation kinetics, reaching steady-state levels within 1 ms. If all outward currents were completely blocked the Ca2+ current showed no inactivation during depolarization lasting 200 ms. 6. No differences in voltage activation range, pharmacology, or kinetics of the Ca2+ current were found in tall hair cells from apical and basal regions of the cochlea. This is in contrast to the marked differences in K+ currents amongst cells from these two widely separated regions of the cochlea.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Isolation of purified large mammal and human islets of Langerhans.

Previous studies have shown that the yield and viability of islets obtained from human or large mammal pancreas depends upon techniques used for islet isolation and upon factors that affect the quality of the donor pancreas. In the present studies, the efficacy of collagenase digestion by ductal perfusion or automated techniques was compared in a canine model of purified islet isolation. The ductal perfusion technique was then developed for human pancreas which was excised before (n = 8) or after (n = 8) multiple organ procurement and without in situ arterial perfusion of hypothermic preservation solution or significant cold storage. Studies with canine pancreas showed that ductal perfusion yielded 2.0 +/- 0.7 microL of islets/g of pancreas and when combined with automated digestion, yields improved to 3.6 +/- 0.8 microL/g (versus perfusion alone, not significant). The yield and viability of human islets was improved when the pancreas was excised before procurement of other donor organs. Results of islet isolation from eight consecutive human pancreases procured in this manner revealed a total yield of 355.2 +/- 44 x 10(3) islets, corresponding to 5345 +/- 600 islets/g (+/- SEM, mean islet diameter 150 microns). Six of eight Ficoll purification attempts succeeded, yielding 186.6 +/- 31 x 10(3) islets of purity ranging from 45-60%. Perifusion with glucose elicited biphasic insulin secretion with a three-fold rise. Islets from two of the isolations were utilized to initiate a clinical trial of islet transplantation in insulin-dependent diabetes mellitus.

Animals↗
PubMed1990

Critical mass of islets that function after implantation in a large mammalian.

A major problem that limits clinical islet transplantation is insufficient information on the critical quantity of islets needed to reverse insulin dependence. To address this problem, we have previously identified the critical number of purified islets of known size that consistently induced normoglycemia in a large mammal model of type I diabetes. In the present studies, we found that the dose-response relationship between the volume of islets transplanted and consistent normoglycemia in dogs corresponded to greater than 4.1 microliters islet tissue per kilogram body weight. The functional outcome of similar quantities of purified islets was then examined after autoimplantation into splenic or hepatic sites and after splenic implantation by venous reflux or pulp injection. During prolonged follow-up, four of five initially normoglycemic recipients of intrahepatic islets became hyperglycemic within 1 year and the other failed at 26 months. In contrast, function of intrasplenic islets was more durable with delayed onset of hyperglycemia observed in only two of six grafts at 13 and 18 months. Sustained normoglycemia was induced by splenic venous reflux of islets in six of seven dogs, but intrapulp injection succeeded in only two of six. Islet allografts implanted to the spleen (n = 10) or to the kidney capsule (n = 6) of Cyclosporine-treated recipients induced normoglycemia in all, but sustained function ensued in only the intrasplenic group when the islet mass was augmented by 40%. These data define the critical islet volume needed to induce normoglycemia in a large mammal. Islets implanted by venous reflux to spleen provide more durable long-term function than the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Islet cryopreservation.

As methods for human islet isolation improve and clinical trials become widespread, some form of storage will be needed. Cryopreservation would offer many advantages: storage in a tissue bank to provide sufficient islets to transplant to individual recipients; modulation of tissue immunogenicity; purification; and, facilitate shipment from center to center. Since 1976, much work has been done on islet cryopreservation with cooling rates of 0.025 degrees C/min to 75 degrees C/min being reported as optimum. In rats we have found that slow cooling to -40 degrees C with rapid thawing from -196 degrees C gives the highest survival as measured in-vitro and in-vivo. This freeze-thaw protocol also provides viable pancreatic microfragments and pure dog islets which can both induce prolonged normoglycemia. This freezing procedure also gives prolonged survival of islet xenografts and purifies the pancreatic microfragments of the unwanted exocrine contaminants. This method of freezing is now being used for human islets. Low temperature storage of human islets will greatly help the clinical use of islet transplantation.

Animals↗
PubMed1990

Islet isolation from human pancreas stored in UW solution for 6 to 26 hours.

1. Viable and functional islets can be recovered from human pancreata stored for prolonged periods (up to 18 hours) with either the UW solution or EC. 2. Function of islets from pancreas stored in UW solution appeared superior. However, the difference in these preliminary studies does not appear nearly so dramatic as in transplantation of vascularized pancreas. 3. Function of islets from human pancreas stored for longer than 18 hours after UW in situ flush was uniformly poor. 4. Considerable room for improvement in the storage of human pancreas before isolation exists. Progress in this area will be important for the development of any large-scale program of clinical islet transplantation.

Adenosine↗
PubMed1990

Comparison of automated and manual methods for islet isolation.

The authors used the principal features of a collagenase perfusion technique and an automated dissociation technique to determine if islets could be isolated from the large mammal pancreas and to compare the effects of the two methods on isolated islets. The pancreases of 16 dogs were cannulated and perfused with collagenase at 4 degrees C, then warmed to 37 degrees C. Group 1 (eight) pancreases were perfused at 37 degrees C until digested, then dissociated manually by teasing and trituration. Group 2 (eight) pancreases were transferred to a closed chamber for continued collagenase digestion and dissociation at 37 degrees C. Islets were purified using identical Ficoll gradients. Aliquots were stained with dithizone and evaluated for number, size and purity. Total islet volume was calculated. Group 2 pancreases were thoroughly digested leaving only a few residual ducts, but undigested fragments persisted in group 1 pancreases. Islet size was similar in both groups. There was a greater islet volume before and after Ficoll purification in group 2, but the difference was not significant. Purity was greater than 90% in both groups. Perifusion with 28 mM glucose elicited a biphasic insulin release from islets in both groups. The data show that the combined protocol enables mass isolation of purified islets from the canine pancreas. Compared with the manual technique, the automated protocol for pancreas dissociation tends to improve the yield of islets without compromising islet size and viability. It provides the advantages of a closed system with increased control over the extent of collagenase digestion.

Animals↗
PubMed1989

Activation and adaptation of transducer currents in turtle hair cells.

1. Transducer currents were recorded in turtle cochlear hair cells during mechanical stimulation of the hair bundle. The currents were measured under whole-cell voltage clamp in isolated cells that were firmly stuck to the floor of the recording chamber. 2. Stimuli were calibrated by projecting the image of the hair bundle onto a rapidly scanned 128 photodiode array. This technique showed that, while the cell body was immobilized, the tip of the bundle would follow faithfully the motion of an attached glass probe up to frequencies of more than 1 kHz. 3. The relationship between inward transducer current and bundle displacement was sigmoidal. Maximum currents of 200-400 pA were observed for deflections of the tip of the bundle of 0.5 microns, equivalent to rotating the bundle by about 5 deg. 4. In response to a step deflection of the bundle, the current developed with a time constant (about 0.4 ms for small stimuli) that decreased with the size of displacement. This suggests that the onset of the current was limited by the gating kinetics of the transduction channel. The onset time course was slowed about fourfold for a 20 degrees C drop in temperature. 5. For small maintained displacements, the current relaxed to about a quarter of the peak level with a time constant of 3-5 ms. This adaptation was associated with a shift of the current-displacement relationship in the direction of the stimulus. The rate and extent of adaptation were decreased by lowering external Ca2+. 6. Adaptation was strongly voltage sensitive, and was abolished at holding potentials positive to the reversal potential of the transducer current of about 0 mV. It was also diminished by loading cells with 10 mM of the Ca2+ chelator BAPTA. These observations suggest that adaptation may be partly controlled by influx of Ca2+ through the transducer channels. 7. Removal of adaptation produced asymmetric responses, with fast onsets but slow decays following return of the bundle to its resting position; the offset time course depended on both the magnitude and duration of the prior displacement. 8. In some experiments, hair bundles were deflected with a flexible glass fibre whose motion was monitored using a dual photodiode arrangement. Positive holding potentials abolished adaptation of the transducer currents, but had no influence on the time course of motion of the fibre. We have no evidence therefore that adaptation is caused by a mechanical reorganization within the bundle.

Adaptation, Physiological↗