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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-μm sized-islets) were sequentially perifused at 37°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°C culture in CMRL 1066 medium with or without the addition of either 200 or 400 μU/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perufusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) μU/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

Mass vaccination with reduced dose 1/50 Rev 1 strain live vaccine (1-2 10(9) colony forming units), administered subcutaneously, over a four and a half year period reduced the prevalence of Brucella melitensis in Kuwait's small ruminant population from 5.8% in 1993 to 2.02% in 1997. Serological test results using the Rose Bengal Plate Test, Rivanol Agglutination Test and Complement Fixation showed no evidence of persistence of positive serology in animals nine or more months after vaccination. Questionnaires and post-vaccination flock inspections found that the effects on gestation (abortions) were minimal--and not proven to be due to the vaccine. The conclusion from these findings is that mass vaccination with reduced dose Rev 1 administered by the subcutaneous route is a practical field strategy for control of Brucella melitensis.

INCE 1985,27 transplants have occurred at Washington University School of Medicine (WUMS) in St. Louis and 4 transplants in conjunction with the University of Western Ontario, London, Ontario. The majority of suc- cessful transplants have occurred since 1989 when we entered into the era of better immunosuppression, early intensive insulin therapy, purer preparations, immunoalter- ation by 24°C culture, and cryopreservation resulting in larger transplanted islet preparations. The two groups of patients most extensively followed at WUMS were patients with established kidney grafts followed by islet transplant (EKI, n = 8) and those receiving simultaneously trans- planted kidney and islet transplant (SKI, n = 9). Several controversies concerning islet transplantation still exist. For example, should islet transplantation occur simultaneous with or following renal transplant? Does cryopreservation and/or islet culture make a difference in islet function? What is the role of human leukocyte antigen (HLA) matching? Does islet mass improve or hamper long-term islet function? Presented here are the results of a retrospec- tive study addressing these questions in regard to our EKI and SKI patients.

A QUESTION that has plagued islet transplantation since its inception is whether islet failure is the result of exhaustion or rejection. Attempts have been made to immunomodulate islet preparations prior to transplantation’ as well as improve immunosuppression’,’ to lessen the risk of rejection. However, clinical islet transplantation has not achieved the desired success rate and the cause of failure still remains unanswered. We have retrospectively analyzed seven of our islet recipients for anti-donor HLA sensitization. Our data suggest that there is a strong correlation between islet allograft failure, documented by a decrease in Sustacal Stimulation Indices (SSI), and a rise in anti-donor HLA sensitization as detected by panel reactive antibody (PRA) testing. Furthermore, the data imply that donor-specific T cells are present in these patients and may be responsible for loss of islet function.

1. BSA-containing solutions improve islet yields using standard collagenase digestion techniques. 2. The BSA effect on islet isolation is independent of source and lot of collagenase. 3. The BSA effect on islet isolation is not due solely to its colloid action, as HES failed to achieve the same level of improvement seen with albumin. 4. BSA can protect islets from warm ischemic injury, and the protective action appears to be unique to albumin, as HES was not as effective.

Streptozocin-induced diabetic nude mice (blood glucose 493 +/- 14 mg/dl) received aliquots of 2000 human islet equivalents (IE) under the kidney capsule and were then followed for up to 30 days with measurement of blood glucose concentration and body weight. Characterization of islet aliquots before the implantation included the assessment of the endocrine beta cell and nonbeta cell mass, estimated by point counting morphometry of immunostained sections. Islet transplantation was followed by a rapid decrease in blood glucose levels and by a progressive increase in body weight; 15 days after transplantation mean glycemic levels were 102 +/- 11 mg/dl and further decreased to 70 +/- 3 mg/dl at 30 days. Despite the progressive improvement in the glucose levels, the beta cell mass of the islet grafts significantly decreased over time from 2.63 +/- 0.2 mg, at the time of transplantation, to 1.16 +/- 0.1 and 0.86 +/- 0.1 mg 15 and 30 days, respectively, after transplantation. In contrast, the endocrine nonbeta cell mass remained stable from before the implantation to 30 days after. Therefore, the endocrine nonbeta cell/beta cell ratio increased from 14% at the time of transplantation, to 35% and 37%, 15 and 30 days, respectively, after transplantation. The rate of replication of the transplanted beta cells was similar in the grafts harvested at 15 and 30 days, with the percentage of beta cells positive for bromo-2' deoxyuridine (BrdU) incorporation being in the range of approximately 0.1% 6 hr after the BrdU injection. These data demonstrate that an important decrease in beta cell mass takes place immediately after islet transplantation--the most dramatic decrease occurring in the first 15 days and persisting even after revascularization has occurred. However, endocrine nonbeta cell mass remained stable indicating that engrafted nonbeta cells are less likely to die than beta cells. The finding that the nonbeta/beta cell ratio of a human islet graft can increase over time, raises questions about whether such a change in islet structure could have an influence upon function.

Severe combined immunodeficient (SCID) mice have become a promising tool for the development of models of human immunologic process. We report the development of a reproducible technique for engrafting SCID mice with human PBL (hu-PBL-SCID). Our results show that a booster injection of anti-CD3 antibody stimulated human lymphocytes given 2 days after the initial injection of lymphocytes will improve the efficiency of chimera establishment to 86.7% (13 out of 15). There was also good correlation among detection of human Ig, human CD3+ cells, and human DNA by polymerase chain reaction amplification in the circulation of hu-PBL-SCID mice. Questions remain concerning the immune function of the human lymphoid cells in the SCID mouse. In this study, we analyzed the ability of human T cells in SCID mice to reject human islet allografts transplanted under the kidney capsule. Human islet allograft function assessed by human C-peptide levels demonstrated failure of islet allografts within 21 days after transplantation in hu-PBL-SCID. In contrast, human islets grafted in unreconstituted SCID mice continued to function for greater than 60 days. Recovered human T cells from rejected islets of hu-PBL-SCID mice displayed specific cytolytic activity against HLA class I-matched islets, while the recovered cells from spleen of hu-PBL-SCID mice showed minimal specific cytotoxicity against islets. These results suggest that graft-infiltrating lymphocytes were activated by the engrafted islets within the hu-PBL-SCID, causing the eventual rejection of the human islet allograft. Thus, engraftment of the anti-CD3 antibody-primed human PBL results in a mouse-human chimera with a functionally competent human immune system that is capable of rejecting a human islet allograft.

We evaluated the effect of immunosuppressive concentrations of cyclosporin A (CsA), given intramuscularly, on the levels of glucose, insulin, C-peptide, glucagon, pancreatic polypeptide (PP), lactate, alanine and beta-hydroxy-butyrate in a group of pancreatectomized mongrel dogs with intrasplenic islet autografts, given mixed meal and oral glucose, while on or off CsA therapy. In whole blood, HPLC-measured drug levels ranged from 412 to 803 ng/ml. Basal glucose and insulin concentrations did not differ significantly between non-pancreatectomized, control dogs and transplanted animals, whether on or off CsA. After the meal challenge, glucose levels were significantly higher in transplanted animals than in normal dogs, and no additional deleterious effect of CsA was observed. Similar insulin and C-peptide responses were found in animals either on or off CsA treatment. Fasting and post-meal concentrations of glucagon, PP and intermediate metabolites were not affected by the drug. These results suggest that intramuscular CsA, given at doses known to sustain islet allograft function, has no detrimental effect on the hormonal and metabolic responses to mixed meal and oral glucose in dogs with intrasplenic islet autografts.

Few data have been published so far on the long-term metabolic and endocrine consequences of intrasplenic islet autografts in dogs and the available information mainly deals with glucose response and, more rarely, insulin secretion during intravenous glucose tolerance tests. The effect of islet transplantation on glucagon levels has never been reported. In this study we measured glucose, insulin, total glucagon, and pancreatic glucagon in dogs before and after (up to 1 year) intrasplenic islet autografts. Pancreata were retrieved from 21 adult dogs and the islets were isolated by collagenase digestion. The endocrine tissue was transplanted into the spleen by direct pulp injection. Autografts resulted in normoglycaemia in 19 out of 21 dogs (90%). Among the successfully transplanted dogs, the percentage of functioning grafts was 71% at 1 year. The metabolic and hormonal results of the follow-up showed that fasting glucose and insulin concentrations did not differ significantly before and after transplantation. However, glucose tolerance and insulin secretion on intravenous glucose tolerance testing were significantly reduced in transplanted animals. Fasting total and pancreatic glucagon, and their integrated releases during the test did not change significantly after islet transplantation. These results demonstrate that: 1) long-term function of intrasplenic canine islet autografts can be achieved in high percentages; 2) autotransplanted animals have normal or near normal fasting glucose and insulin levels, but reduced glucose disappearance and insulin secretion on intravenous glucose tolerance tests; 3) normal glucagon secretion is a feature of successful islet grafts.

Azathioprine-prednisone immunosuppression has proven ineffective in rodent, canine, and human islet transplantation. ALS has demonstrated beneficial effects in rodent islet transplantation, but high dose CsA monotherapy remains the most effective immunosuppressive modality in canine islet allotransplantation. Nephrotoxicity prevents effective utilization of such an approach in human islet transplantation. We have been unable to demonstrate additional benefit from the addition of azathioprine and prednisone to CsA immunosuppression in large animal islet allotransplantation. CsA, azathioprine and prednisone all appear to have the potential for adverse effects variously on islet engraftment, insulin secretion, and/or peripheral insulin activity. The effects of CsA appear to be dose related and may be reversible. Our approach in clinical islet transplantation has been to achieve potent induction immunosuppression with Minnesota antilymphocyte globulin, delayed CsA administration, and ongoing maintenance with low dose triple immunotherapy. Initial results are encouraging.

Inseltransplantation als eine zuverlassige alternative Therapie fur Typ I-Diabetes Mellitus bleibt eine vielversprechende Moglichkeit. Es gibt aber keine gut dokumentierten Falle von erfolgreichen klinischen Versuchen. Trotzdem wurden entscheidende Fortschritte gemacht, um die Isolation der menschlichen Langerhansschen Inseln zu verbessern. Inselautotransplantation nach vollstandiger oder nahezu vollstandiger Pankreatektomie macht eine Langzeitfunktion transplantierter Inseln wahrscheinlich. Anfangliche klinische Versuche mit allogenen Inseltransplantaten haben eine kurzfristige endokrine Funktion aufgezeigt.

The primary objective of islet transplantation is to transplant sufficient quantities of normal, functional islet tissue in to Type 1, IDDM patients to achieve normal metabolic function without insulin therapy sufficiently early in their disease to prevent their complications while avoiding the use of tong-term immunosuppress ion and autoimmune recurrence. A total of 14 patients with kidney transplants have reached insulin independence after islet transplantation using immunosuppression with the longest at 30 months. Many others have had partial islet function in clinical trials. Immunoalteration studies in which islets are altered in vitro prior to transplantation to eliminate donor immune cells that cause rejection have succeeded in rodent studies. Immunoisolation studies in which islets are encapsulated with specific biocompatible membranes and transplanted without immunosuppression are also showing promise in animals and beginning in human studies. These kinds of approaches will be necessary to make this potential therapy a practical approach for the patient with diabetes.

Recent biochemical studies have shown that the cytoplasmic islet cell–antibody autoantigen has properties of a monosialoganglioside (GM). To characterize islet glycolipids and ascertain whether islets express unique gangliosides, we determined the pattern of ganglioside expression in whole human pancreas and isolated human islets using high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC). The major gangliosides detected in glycolipid extracts of whole human pancreas were GM3, GD3 (disialoganglioside), and in a lesser amount, a GD1a-comigrating ganglioside. In contrast to whole human pancreas, isolated human islets were found to predominantly express GM3, an acidic glycolipid comigrating with GM2, and a ganglioside with mobility between GM2 and GM1 by both HPLC and HPTLC. Quantitation of the major ganglioside UV peaks seen on HPLC gave the following results. In whole pancreas, GM3 represented 66.7% of total gangliosides detected; an asialoglycolipid comigrating with GM2, 2.0%; a ganglioside migrating between GM2 and GM1, 2.6%; GD3, 22.6%; and a GD1a-comigrating ganglioside, 6.1%. In isolated islets, these components were found at the following levels: GM3, 14.9%; GM2-comigrating glycolipid, 74.2%; a ganglioside migrating between GM2 and GM1, 9.8%; GD3, 1.1%; and the GD1a-comigrating ganglioside, not detectable.

Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-Km, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. We isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus.

Islets of Langerhans from unrelated donor rhesus monkeys were transplanted into the portal vein of five rhesus monkeys which had been made diabetic by partial pancreatectomy and Streptozotocin. There was definite improvement of the diabetic state with an increase in the mean KT1/2 from 0.71 to 1.52 percent per minute. The islets were isolated by a more efficient digestion-filtration process than previously had been described. This process has allowed, for the first time, successful isolation of large masses of islets permitting islet transplants in subhuman primates.

Fluorophotometry was used to evaluate the integrity of the blood-ocular barriers to fluorescein in experimental diabetes mellitus in rats. This technique allowed quantitation of ocular fluorescein concentrations following intravenous injection. Streptozotacin-induced diabetes resulted in an increased fluorescein accumulation in the anterior chamber (1.52 +/- 0.17 microgram/ml, mean +/- S.E.M.) and vitreous (0.82 +/- 0.11) over baseline nondiabetic levels (0.68 +/- 0.80 and 0.40 +/- 0.03, respectively). Fluorophotometry was repeated at 5, 13, and 20 days following portal vein pancreatic islet transplantation. At 5 days anterior chamber (1.40 +/- 0.17) and vitreous (0.61 +/- 0.08) fluorescein concentrations remained elevated. However, at 13 and 20 days following islet transplantation, ocular fluorescein concentrations were identical to levels observed prior to the induction of diabetes. Intravenous glucose (0.5 gm/kg) tolerance testing was performed 5 and 13 days following transplantation. The glucose responses to the tolerance test were normal and similar at both times. However, at 5 days the insulin response was abnormal with a decreased initial peak and an absent second peak. At 13 days there was a normal biphasic insulin response. In experimental diabetes mellitus ocular vascular permeability was more closely correlated with insulin than blood glucose abnormalities.

Islets were isolated from human pancreases by the distention method and purified by centrifugation on Ficoll or Percoll gradients. The purity of the final preparations was 60% to 90% islets, making it possible to culture the islet preparations in vitro. Perifusion of the islet preparations after 1, 3, or 7 days of culture at 24 degrees C indicated that the islets were viable and would respond to glucose stimulation. If the islets were returned to 37 degrees C culture for 1 day, then the insulin secretory response at 7 days was comparable with the response during the first day of culture at 37 degrees C. Extending the culture period at 24 degrees C to 14 days damaged the islets as indicated by the lack of response to glucose stimulation. The Ficoll technique was a much more effective method for purifying the islets, since it provided approximately twice the yield of islets and insulin compared with the Percoll procedure. The mean yield of purified islets by the Ficoll technique was 2180 +/- 325 islets/gm of pancreas with a calculated islet mass of 3.7 +/- 0.8 mm3/gm. The findings that massive numbers of purified human islets can be obtained by centrifugation on Ficoll gradients and that the purified islet preparations remain morphologically and functionally intact during 7 days' culture at 24 degrees C make it possible to assess the function of donor islets before human islet transplantation, transplant the islets via the portal vein, and use low-temperature culture as a possible approach for altering the immunogenicity of donor human islets.

Twenty-four patients who had undergone radical pancreatic resection were evaluated by CT one week to 11 years after surgery. Eighteen patients had had the Whipple procedure; six had had total pancreatectomy. The region between the aorta and superior mesenteric artery, previously occupied by the uncinate process of the pancreas, is an important area to evaluate for tumor recurrence because periampullary tumors tend to metastasize to the lymph nodes in this region. Tumor recurrence here is readily detectable by CT since radical pancreatectomy leaves this area free of soft tissue attenuation material. Administration of glucagon prior to CT scanning helps fill the afferent jejunal loop with oral contrast material, and thus helps define right upper quadrant anatomy. CT demonstrated postoperative complications or tumor recurrence in 16 of the 24 patients and was 100% accurate in patients who had follow-up.

The extrahepatic diversion of essential splanchnic hepatotrophic factors may cause the liver atrophy and insufficiency that follows portacaval shunting. To investigate this, control dogs with end-to-side portacaval shunts (control-PCS, n = 6) were compared with dogs shunted 1 month after intraportal pancreatic islet autotransplantation (islet-Tx-PCS, n = 5). From the distal pancreas of each experimental dog, 1.95 +/- 0.49 X 10(5) islets were isolated by collagenase digestion and retransplanted within 3 hours. Assays of hepatocellular function (caffeine clearance) and hepatic blood flow (indocyanine green), conventional biochemical liver function tests, and glucose, insulin, and glucagon responses to intravenous glucose challenge were measured monthly and when dogs were killed. Four of six control-PCS dogs were killed 32 +/- 9 days after shunting because of more than 20% body weight loss; one control-PCS dog lost only 7% of body weight by day 56 and stabilized. No significant loss of body weight occurred in islet-Tx-PCS dogs (n = 5). Liver function test abnormalities seen in control-PCS dogs were absent in islet-Tx-PCS dogs. Both control-PCS and islet-Tx-PCS indocyanine green half-life measurements were significantly (p less than 0.05) prolonged at all times, indicating equally reduced hepatic blood flow after shunting for both groups. In contrast, islet-Tx-PCS caffeine half-life periods were significantly (p less than 0.05) shorter than in control-PCS dogs and were similar to those in normal dogs, indicating a protective effect of the transplanted islets on hepatocellular function. We conclude that intraportal pancreatic islet autotransplants prevent, by the local release of hepatotrophic factors within the liver, the metabolic abnormalities and loss of hepatic function after PCS.