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Biomedical subjects

P E Lacy

Publications and source records attributed to P E Lacy.

At least 19 recordsLinked to original sources
PubMed1996

Prolonged survival of discordant porcine islet xenografts.

Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degree C were rejected after : 9+/-0.1 (mean+/-SE) days in control mice: 14+/-3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43+/-6 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36+/-4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47+/-3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P<0.01) and similarly longer than in the control and MLS plus PLS groups. Then, we transplanted islets that had been either cultured at 24 degrees C for 7 days or cryopreserved into 7-day aCD4-treated mice, to evaluate whether low temperature culture or the freezing-thawing procedure could affect survival. Neither 7-day, low temperature culture (mean survival time: 37+/-2 days) nor cryopreservation (mean survival time: 39+/-2 days) prolonged islets function further. Thus, the present study demonstrates that prolonged survival can be achieved with discordant porcine islet xenografts, and shows the greater efficacy of aCD4 treatment, which was not improved by additional immunosuppressive therapies we tested, nor by culture or cryopreservation of the islets.

Animals↗
PubMed1996

Successful transfer of immune unresponsiveness to concordant rat islet xenografts.

Indefinite survival of concordant xenogeneic Wistar Furth (WF) rat islet survival was obtained by intrahepatic transplants of cultured WF islets and a single injection of antilymphocyte sera in C57BL/6 mice. Adoptive transfer of splenocytes from mice with established WF islet xenografts produced a marked prolongation of survival of WF islets transplanted under the kidney capsule of diabetic irradiated (600 rads), naive C57BL/6 recipients (mean survival time = 48.9 +/- 17.1 days), and three of the recipients were still normoglycemic at 100 days after transplantation. Adoptive transfer of an equal mixture (3 x 10(7) cells each) of these splenocytes with normal splenocytes also prolonged survival of the kidney capsule islet xenografts (mean survival time = 26.5 +/- 7.8 days vs. 15.2 +/- 5.3 days for controls). In vitro studies on lymphocyte proliferation demonstrated a low rate of proliferation of splenocytes from established islet xenografts in the presence of irradiated WF splenocytes (stimulation index = 1.6 vs. 16.2 for naive C57Bl/6 mice), and mixing the cells with control splenocytes also decreased the proliferation of splenocytes as compared with controls (stimulation index = 5.4 vs. 16.2 in controls). The inhibitory effect was not species specific, since splenocytes from mice with established islet xenografts also produced a 42% inhibition of proliferation in the presence of irradiated Lewis splenocytes. These findings demonstrate that concordant, islet xenograft, immune unresponsiveness can be adoptively transferred by splencotyes from mice with established islet xenografts.

Animals↗
PubMed1996

Induction of allogeneic islet survival by intrahepatic islet preimmunization and transient immunosuppression.

Induction of tolerance to fully major histocompatibility complex (MHC)-mismatched rat islet allografts implanted at two different islet transplant sites (liver and kidney capsule [KC]) was examined. Streptozotocin-induced diabetic Lewis (RT1(1)) rats remained hyperglycemic (> 200 mg/dl) after intrahepatic preimmunization by injection of 200 low-temperature cultured (24 degrees C for 7 days) Wistar-Furth (WF, RT1u) rat islets into the portal vein with one injection (1 ml) of rat antilymphocyte serum intraperitoneally. Three weeks later, 1,200 WF islets that had been cultured to remove passenger lymphoid cells were transplanted into the liver via the portal vein or under the KC. The intrahepatic transplants survived 60.2 +/- 11.9 days, and all six of the KC transplants maintained normoglycemia for > 100 days after the preimmunization regimen. In contrast, survival of fresh islet transplants was not significantly improved by this preimmunization protocol at either transplantation site. This study demonstrates that indefinite islet allograft survival can be achieved across a full MHC mismatch by intrahepatic preimmunization with a small number of cultured donor islets and a brief period of immunosuppression followed by transplantation of low-temperature cultured donor islets.

Animals↗
PubMed1996

Continued insulin dependence despite normal range insulin sensitivity and insulin connecting peptide levels in a kidney/islet transplant patient.

OBJECTIVE: The majority of islet transplant recipients remain insulin-requiring, although many have near-normal connecting peptide (CP) levels. Insulin resistance may be one possible cause of the continuing need for exogenous insulin in islet transplant recipients. To assess this, we have studied the insulin sensitivity index (S1) in one patient with near-normal CP levels after islet transplant who remained insulin-requiring. RESEARCH DESIGN AND METHODS: The islet transplant recipient is a 36-year-old woman with no residual CP who received a kidney transplant, followed 7 days later by an islet transplant. The islets were infused into the liver via the umbilical vein. Induction immunosuppression consisted of OKT3, prednisone, cyclosporin A, and azathioprine, with maintenance on the latter three. RESULTS: Maximum CP levels after a standardized Sustacal meal were 2.09, 1.18, 0.85, and 0.81 nmol/l at 1,6,18, and 24 months posttransplant, respectively. Insulin requirements at the same times were 0.27, 0.45, 0.49, and 0.62 U.kg(-1).d(-1), while S1 was 36.3, 53.3, and 13.2 min (-1).nmol(-1).ml at 6,18, and 24 months, respectively. This compares with S1 values of 43.3+/- 10.0 min (-1).nmol(-1).ml for normal subjects. CONCLUSIONS: This patient had near-normal S1 and CP levels, but she was unable to discontinue insulin therapy, suggesting that other factors are critical. Despite this, she maintained normal or near-normal glycated hemoglobins, indicating metabolic benefit from the islet transplant.

Adult↗
PubMed1995

Islet cell transplantation for insulin-dependent diabetes.

About 20 years after investigators began work on the isolation of animal islets, we are now poised to use such cells to control blood sugar levels in humans. Studies in various laboratories have led to development of transplantation techniques that obviate the need for chronic immunosuppression; equally important, the techniques also appear to avoid the problem of graft rejection.

Animals↗
PubMed1995

Insulin inhibits its own secretion from isolated, perifused human pancreatic islets.

It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification. Aliquots of 200 islet equivalents (IE, 150-microns sized-islets) were sequentially perifused at 37 degrees C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37 degrees C culture in CMRL 1066 medium with or without the addition of either 200 or 400 microU/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perifusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) microU/ml added insulin, basal Cp release was 0.12 +/- 0.03, 0.14 +/- 0.02 and 0.14 +/- 0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47 +/- 0.13 ng/ml, P < 0.02) and Ins400 (0.68 +/- 0.15 ng/ml, P < 0.05) than C (0.83 +/- 0.15 ng/ml). The second-phase secretion peak was also significantly (P < 0.05) reduced with added insulin (Ins200: 0.47 +/- 0.08 ng/ml; Ins400: 0.45 +/- 0.07 ng/ml) than in its absence (C: 0.65 +/- 0.09 ng/ml). Accordingly, total Cp secretion was lower with Ins200 (10.6 +/- 2.3 ng/ml, P = 0.03) and Ins400 (11.8 +/- 2.3 ng/ml) than with C (16.0 +/- 2.2 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

C-Peptide↗
PubMed1994

Cryogenic storage of isolated, purified porcine pancreatic islets.

Highly purified islets of Langerhans were prepared in the present study from adult pigs by collagenase digestion and density gradient purification. After overnight culture, the tissue was equilibrated with DMSO at 25 degrees C, supercooled to -7.5 degrees C, nucleated, slowly cooled at 0.25 degrees C/min to -40 degrees C, and stored at -130 degrees C. Then, after variable periods of storage, the islets were rapidly thawed at 37 degrees C. Postthaw actual islet and islet equivalent (150-microns sized islets) recovery were 75 +/- 7% and 66 +/- 4%, respectively. The frozen-thawed porcine islets maintained good morphology on histological staining by hematoxylin-eosin and aldehyde-fuchsin. Upon perifusion, basal insulin secretion was 43 +/- 10 and 67 +/- 18 pmol/L from noncryopreserved, control islets, and cryopreserved islets, respectively (P = 0.2). Peak insulin release at 16.7 mmol/L glucose was 85 +/- 28 pmol/L from noncryopreserved islets and 157 +/- 48 pmol/L from the frozen-thawed islets (P = 0.1). When 10 mmol/L theophylline was added to 16.7 mmol/L glucose, the secretion of the hormone peaked to 221 +/- 83 (control islets) and 479 +/- 140 pmol/L (cryopreserved islets, P = 0.1). Total insulin secretion differed significantly for the noncryopreserved and the cryopreserved islets at both 16.7 mmol/L (1412 +/- 306 vs. 3756 +/- 764 pmol/L, respectively, P = 0.007) and 16.7 mmol/L glucose plus 10 mmol/L theophylline (2161 +/- 371 vs. 7505 +/- 2075 pmol/L, respectively, P = 0.011). Normoglycemia was restored within 7 days from implantation in temporarily immunosuppressed (aL3T4 antibody) mice with streptozotocin-induced diabetes by transplanting 1500-2000 cryopreserved porcine islets under the kidney capsule. Mean survival time of frozen-thawed islet xenografts (39 +/- 3 days) was similar to that of noncryopreserved islet xenografts (43 +/- 6 days). This study demonstrates that cryogenic storage is feasible of isolated porcine islets, with the frozen-thawed pancreatic endocrine tissue maintaining morphological integrity and both in vitro and in vivo viability. Further studies are needed to define the effect of cryopreservation on the immunogenic properties of porcine islets.

Animals↗
PubMed1994

Induction of immune unresponsiveness to concordant islet xenografts by intrahepatic preimmunization and transient immunosuppression.

Streptozotocin-induced, diabetic mice (C57BL/6) were preimmunized by injecting 25 low temperature, cultured Wistar-Furth (WF) rat islets into the portal vein, and the recipients received one injection of mouse and rat antilymphocyte sera. 3 wk later, fresh WF islets were transplanted under the kidney capsule of the preimmunized recipients, and normoglycemia was maintained in all 13 recipients for 60 d. Removal of the grafts at 60 d returned the mice to a diabetic state. Transplants of fresh WF islets under the kidney capsule without pretreatment of the recipients had a mean survival time of 16.5 +/- 2.5 d. These findings demonstrate that immune unresponsiveness can be achieved across a concordant, islet xenograft barrier within 3 wk after intrahepatic preimmunization with a small number of donor rat islets and transient immunosuppression with antilymphocyte sera.

Animals↗
PubMed1994

Induction of tolerance to islet xenografts in a concordant rat-to-mouse model.

Induction of tolerance to concordant rat islet xenografts (150 Wistar-Furth [WF] islets) in streptozocin-induced (STZ) diabetic mice (C57BL/6) was determined at three different sites for islet implantation (thymus, kidney capsule, and liver). Islets transplanted into the thymus or kidney capsule were either fresh or cultured at 24 degrees C for 7 days, and the mice received a single injection of either anti-mouse lymphocyte serum (MALS) alone or anti-rat lymphocyte serum (RALS) and MALS. Islets transplanted into the liver via the portal vein were cultured at 24 degrees C for 7 days, and the mice received a single injection of MALS and RALS. To document the induction of tolerance, recipients with islet xenografts surviving > 100 days were made diabetic again by STZ (thymus and liver) or nephrectomy (kidney capsule) and received a second transplant of 150 fresh WF islets in the kidney capsule. Kidney capsule placement of fresh or cultured islets with MALS alone or MALS and RALS did not induce tolerance in a significant number of recipients. The intrathymic transplantation of fresh or cultured islets with MALS alone resulted in prolonged WF islet xenograft survival (mean survival time of 39.7 +/- 7.9 days) but did not result in tolerance, whereas the administration of MALS and RALS with the intrathymic placement of fresh or cultured islets induced tolerance in approximately 50% of the mice. Intrahepatic transplantation of cultured islets with MALS and RALS resulted in tolerance to donor islets in 90% of the recipients. Donor specificity was evaluated by a third major histocompatibility complex-disparate fresh Lewis islet xenograft.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Pulsatile insulin secretion from isolated human pancreatic islets.

Insulin secretion from the pancreas is pulsatile. The precise site and function of the pacemaker that regulates insulin periodicity in humans have not been determined. We isolated human pancreatic islets from five cadaver organ donors by collagenase digestion and density gradient purification. After 24 h of culture in CMRL-1066 medium at 37 degrees C, aliquots of 200 islets were perifused (1 ml/min for 120 min) with glucose and other secretagogues in oxygenated Krebs-Ringer bicarbonate solution at 37 degrees C. Samples for insulin measurement were taken every minute, and insulin secretion was analyzed by the Clifton and Steiner cycle detection technique. With 3.3 mM glucose (n = 17), insulin oscillations were demonstrated with a periodicity of 9.8 +/- 0.1 min (means +/- SE), mean amplitude was 16.8 +/- 1.8 pM, and overall mean insulin release was 43.8 +/- 4.2 pM. With 16.7 mM glucose (n = 14), no change of insulin periodicity was observed (10.2 +/- 0.9 min), mean amplitude was 41.4 +/- 10.2 pM (P < 0.01 vs. 3.3 mM glucose), and mean insulin release was 118.2 +/- 19.2 pM (P < 0.01 vs. 3.3 mM glucose). Both at 3.3 and 16.7 mM glucose, the addition of 1.4 mM glucagon (n = 4), 15 mM arginine (n = 4), or 100 micrograms/ml tolbutamide (n = 4) caused no change of insulin periodicity but enhanced mean amplitude and mean insulin release compared with glucose alone. These results show that a pacemaker is located within the islets that regulates pulsatile insulin secretion in humans; the pacemaker is remarkably stable, because its periodicity is not affected by factors altering insulin secretion.

Adult↗
PubMed1994

Protection of encapsulated human islets implanted without immunosuppression in patients with type I or type II diabetes and in nondiabetic control subjects.

Human islets were macroencapsulated in permselective hollow fiber membrane devices and successfully allotransplanted subcutaneously with > 90% viability after 2 weeks in situ. Recipients were patients with type I or type II diabetes and normal control subjects; none was immunosuppressed. Between 150 and 200 islet equivalents were implanted in each of the nine patients. No adverse patient complications were observed. Biocompatibility of devices was excellent. Insulin-positive beta-cells were confirmed in encapsulated islets recovered from the implanted devices in all patient populations including the type I diabetic patients. Glucose-stimulated insulin release could be demonstrated in vitro from recovered islets. These data demonstrate that macroencapsulated human islets can survive at the subcutaneous site and that permselective membranes can be designed to protect against both allogeneic immune responses as well as the autoimmune component of type I diabetes.

Biocompatible Materials↗