Barbara J. Gilligan

A telemetry-instrumentation system for monitoring multiple subcutaneously implanted glucose sensors

An implantable potentiostat-radiotelemetry system for in vivo sensing of glucose is described. An enzyme electrode sensor measures the oxidation current of hydrogen peroxide formed by the stoichiometric conversion of glucose substrate and oxygen cofactor in an immobilized glucose oxidase layer. The sensor current is converted to a frequency and transmitted at programmable intervals (4, 32, 256 s) to a remote receiver. Low power CMOS circuitry is employed and device operation for up to 1.5 years is predicted using two series connected 250 mAh lithium cells. Crystal controlled RF frequencies uniquely identify each sensor allowing over 10 sensors within the same 10 m radius. A custom interface card allows a PC to program the receiver and handle the transmitted sensor data using software written in Microsoft C and QuickBasic. Software control allows on-the-fly sensor addition or subtraction to the sensor group being monitored. Over 10 sensors can be tracked long-term using the longest transmit interval, or four sensors can be tracked during short-term infusion studies when the transmit interval is reduced to 4 s. The design, construction, operation, and performance of the system hardware and software are described and evaluated.<<ETX>>

Evaluation of a Subcutaneous Glucose Sensor out to 3 Months in a Dog Model

OBJECTIVE To advance the feasibility of an impiantatile long-term glucose sensor with bioprotective sensor membranes and test protocols using a somatostatin analog (octreotide). RESEARCH DESIGN AND METHODS Implantable sensors were constructed with one of eight bioprotective membranes and screened in vitro for stable response to glucose. Sensors were implanted subcutaneously into nondiabetic mongrel dogs and monitored at 4-min intervals via radiotelemetry. When implanted sensor responses showed evidence of tracking blood glucose after glucagon challenge (8–21 days postimplant), a glucose infusion protocolwas used to assess performance. Sensor data were collected every 4 s after octreotide inhibition of endogenous insulin release. Reference plasma glucose samples were taken every 4–10 min. RESULTS Preimplant in vitro testing of sensors verified linearity to 33.3 mM glucose and response times to 90% of equilibrium in 2–7 min. Ten implanted sensors tracked glucose for 20–114 days, during which 25 separate glucose infusion studies were conducted. The resulting regression data yielded a mean slope of 0.99 ± 0.06, an intercept of 0.24 ± 0.53 mM glucose, and a correlation coefficient 0.98 ± 0.01. Long-term sensor stability was not judged adequate for clinical application, although two sensors tracked within ±15% for 33 and 42 days. In vivo oxygen delivery was shown toaffect sensor performance. On expiant, two of eight tested bioprotective membranes were found to be biostable and to fully protect the sensors enzyme membrane. The foreign body capsule was adequately vascularized adjacent to the sensor up to 91 days postimplant. Sensor units eventually failed because of electronic problems (package leakage) or because of biodegradation or biofouling of test bioprotective membranes. CONCLUSION Further development of this type of sensor may provide diabetic patients with a better means of monitoring blood glucose.

Feasibility of Continuous Long-Term Glucose Monitoring from a Subcutaneous Glucose Sensor in Humans

The feasibility of continuous long-term glucose monitoring in humans has not yet been demonstrated. Enzyme-based electrochemical glucose sensors with telemetric output were subcutaneously implanted and evaluated in five human subjects with type I diabetes. Subject-worn radio-receiver data-loggers stored sensor outputs. Every 1-4 weeks the subjects glucose levels were manipulated through the full clinical range of interest using standard protocols. Reference blood glucose samples were obtained every 5-10 min and analyzed in our hospital clinical laboratory and/or on glucose meters. The sensor data were evaluated versus the reference data by linear least squares regression and by the Clarke Error Grid method. After surgical explantation and device inspection, the tissue-sensor interface was evaluated histologically. The remaining sensor-membranes were also recalibrated for comparison with preimplant performance. Four of the five glucose sensors tracked glucose in vivo. One sensor responded to manipulated glucose changes for 6.2 months with clinically useful performance (>/=90% of sensor glucose values within the A and B regions of the Clarke Error Grid). For this sensor, recalibration was required every 1-4 weeks. The other three transiently responding sensors had electronic problems associated with packaging failure. The remaining sensor never tracked glucose because of failure to form any sustained connection to adjacent subcutaneous tissue. Thus, stable, clinically useful sensor performance was demonstrated in one of five subjects with diabetes for a sustained interval of greater than 6 months. While this glucose sensor implant technology shows promise in humans, it needs to be made more reliable and robust with respect to device packaging and sensor-tissue connection.

Enzymatic glucose sensors. Improved long-term performance in vitro and in vivo.

We studied the long-term in vitro and in vivo performance of enzyme electrode glucose sensors. Single commercially produced enzyme-active membranes remained functional for estimating glucose in vitro for 14-36 months. These membranes were implanted subcutaneously in rats for 1 year and, upon explanation, remained functional for measuring glucose in vitro. Sensors with these membranes plus an additional outer membrane with lower glucose permeability allowed glucose monitoring in the low oxygen tension of subcutaneous tissue. These sensors were surgically implanted in three nondiabetic dogs. Each sensor implant was coupled to a radio transmitter to allow continuous long-term glucose monitoring in these awake unrestrained dogs. In vivo sensor performance was evaluated by intravenous glucose infusion, with reference blood glucose determinations made in the clinical laboratory. These subcutaneously implanted sensors tracked changes in plasma glucose for up to 12 weeks. The in vivo initial response for three sensor implants was approximately 35 sec (n=8). Sensor peak response to glucose after bolus infusion ranged from 3 to 14 min. Stability of sensor sensitivity within ± 15% for more than 1 month was demonstrated in two of the dogs. Sensor lifetime was limited not by loss of enzyme activity, but by biodegradation of the outermost polyurethane membrane. The findings suggest that long-term continuous monitoring of blood glucose using a subcutaneously implanted enzyme electrode sensor may be possible.

Prolonged Hypothermia Causes Primary Nonfunction in Preserved Canine Renal Allografts due to Humoral Rejection

Canine kidney preservation models have historically used autotransplants to avoid the complications of rejection, although clinically all transplants are allografts. This study investigated the effects of preservation time and method on early kidney function in a canine allograft vs. autograft model. Kidneys were harvested from beagles, preserved by cold storage (CS) in UW solution for 0, 24 or 72 h, or by machine perfusion (MP) with Belzer MPS for 72 h. In some experiments 45 min of warm ischemia (WI) was performed in situ before harvest. Allograft recipients received steroid immunosuppression. Kidney function was assessed by serum creatinine and survival for 7 days. Allografts preserved for 0 and 24 h performed as well as autografts. Allografts preserved for 72 h by either CS or MP had a higher incidence of primary nonfunction (PNF) compared with autografts, as determined by survival (50% vs. 100%, p < 0.003). Primary nonfunction kidneys had thrombotic microangiopathy, vascular and peritubular capillary binding of IgM and complement C4d, and evidence of circulating donor‐specific antibodies; all consistent with humoral rejection. These responses were dependant on hypothermia time and were not attributable to ischemia, immunosuppression, preservation solution, or cellular rejection. In conclusion, prolonged hypothermia can cause PNF in allografts owing to acute humoral rejection.

The effects of donor brain death on renal function and arachidonic acid metabolism in a large animal model of hypothermic preservation injury

Background. Donor brain death (BD) has been implicated as a risk factor for the poor performance of kidneys after transplantation in small but not large animal models. This study determined the effects of donor BD on renal function and lipid mediator metabolism in a large animal model of renal hypothermic preservation injury. Methods. Adult beagle donors were subjected to explosive BD for 16 hr. After BD, the kidneys were removed, cold stored for 24 hr in cold University of Wisconsin solution, and allotransplanted into recipient dogs for either 4 hr (group 1) or 7 days (group 2). Controls for both groups consisted of kidneys obtained from living donors. Renal allograft function and tissue arachidonic acid (AA) metabolism were determined after reperfusion. Results. Short-term renal function after transplantation was generally unaffected by BD. Renal blood flow decreased after reperfusion but was not altered during the 16-hr BD period. Neutrophil infiltration significantly increased in kidneys from brain-dead donors before storage and after 4 hr of reperfusion. Renal cortex and medulla AA metabolism were not significantly affected by BD after short-term reperfusion except when thiol-ether leukotrienes (LTC4/D4/E4) were increased with BD. Serum creatinine was elevated during 7 days, but, surprisingly, BD significantly attenuated this injury. Conclusion. BD in large mammals does not significantly affect renal allograft function or AA metabolism after transplantation. The role of BD in human renal preservation injury and inflammation should be reevaluated.

Primitive endothelial cell lines from the porcine embryonic yolk sac.

SummaryEndothelial cell lines have been established from cells that were isolated from porcine yolk sacs from day 18 and day 22 embryos and propagated in vitro under various growth conditions. After expansion in vitro, the general properties of the cells proved similar for the different media used. The endothelial cells expressed cell surface receptors for acetylated low-density lipoprotein and also expressed cell surface-associated angiotensin-converting enzyme. The cells showed a characteristically high level of binding for Bandeiraea simplicifolia lectin I and Dolichos biflorus agglutinin but did not bind significant amounts of Ulex europaeus lectin I. The cells expressed low but serologically detectable levels of Class I major histocompatibility complex (MHC) antigens but failed to bind antibodies directed against Class II MHC antigens. α5β1 integrins were weakly expressed, whereas vascular cell adhesion molecule-1 (CD106) and αvβ3 integrins were not detected. Three-dimensional tube formation was readily observed in cultures grown on Matrigel and occurrel even in uncoated plastic dishes in the absence of Matrigel. In contrast to most of the adult porcine endothelial cells, yolk sacderived endothelial cells did not posses serologically detectable receptors for porcine growth hormone (GH), an observation consistent with the finding that GH did not increase the proliferative rate of these cells. Electron microscopic examination demonstrated the presence of Weibel-Palade bodies, tight endothelial cell junctions, and typical rough endoplasmic reticulum. Exposure of the cells to either concanavalin-A-stimulated porcine splenocyte culture supernatants or to human tumor necrosis factor α did not cause upregulation of VCAM-1 or Class II MHC antigens. Addition of prrcine interferon-γ led to an increase in the level of expression of Class I MHC. Yolk sac endothelial cells from day 22 embryos showed a low but detectable level of expression of Class II MHC antigens, whereas the endothelial cells from day 18 embryos showed no expression of Class II antigens after interferon-γ stimulation. The cells maintained competence to develop vascular structures in vitro and could do so after coinjection with murine tumor cells into adult, immunocompromised mice.

Role of endothelium in the control of mouse yolk sac stem cell differentiation

Studies in our laboratory have shown that as early as day 8.5 of development, mouse yolk sac cells can generate T cells when placed in a thymic microenvironment. At this stage, yolk sac cells can also differentiate into myeloid cells in vitro. B cell differentiation in vitro was achieved with day 9 yolk sac by providing a bone marrow stromal feeder layer. We have now established endothelial cell lines and clones from yolk sacs of day 8-12 mouse embryos. These vary in their ability to support stem cell maintenance and differentiation. Our principal work has been carried out with day 12 cloned endothelial cell lines. One clone supported the > 100 fold expansion of yolk sac hematopoietic stem cells that subsequently could generate B cells, T cells and myeloid cells both in vitro and in vivo. Preliminary experiments with endothelial cells from younger embryos are also described.

Role of peroxynitrite anion in renal hypothermic preservation injury

Background. Peroxynitrite anions may play a role in normothermic renal ischemia and reperfusion. The purpose of this study was to determine if endogenous peroxynitrite anion is involved in renal preservation injury. Methods. Experiments were conducted in isolated canine renal tubules and in a canine autotransplant model of hypothermic preservation injury. Results. Isolated renal tubules demonstrated progressive loss of membrane transport function after reperfusion with increasing cold storage times in UW solution as assessed by tetraethylammonium cation transport (TEA). This transport defect was not altered by reperfusion in the presence of WW85, a peroxynitrite decomposition catalyst. Likewise, tubule LDH release was not altered by WW85. Renal tubules did not demonstrate any evidence of peroxynitrite formation after cold storage (0–120h) or after subsequent reperfusion in vitro as measured by nitrotyrosine adduct formation. Addition of exogenous peroxynitrite (1 mM) directly to freshly isolated renal tubules produced strong nitrotyrosine signals but failed to alter membrane function (TEA uptake). Conversely, SIN-1, a peroxynitrite generator molecule, failed to produce a nitrotyrosine signal in extracted renal tubule proteins but significantly impaired transport function. Finally, function of cold stored canine autografts was not affected by the scavenging of peroxynitrite anions (WW85) before kidney harvest and immediately at reperfusion. Tissue biopsies from cold stored kidney autografts also failed to show evidence of peroxynitrite synthesis either after cold storage (72 h) or after kidney transplantation (60 min reperfusion). Conclusions. This study concludes that peroxynitrite anions are not formed and are not involved in renal preservation injury.