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Biomedical subjects

R G Bretzel

Publications and source records attributed to R G Bretzel.

At least 271 records · Page 15Linked to original sources
PubMed1978

[Autotransplantion of Langerhans islets after total duodeno-pancreatectomy in a patient with chronic pancreatitis (author's transl)].

Total duodenopancreatectomy was performed in a 36-year-old man because of global chronic pancreatitis. Islets cells were isolated by collagenase digestion from the resected pancreas and re-implanted into the liver via the portal vein. Until the sixth postoperative day the amount of insulin required fell from 54 IU initially to 10 IU per day. Serial insulin determination demonstrated secretion by the implanted cells.

Adult↗
PubMedDate not supplied

Percutaneous transhepatic catheterization of the portal vein: A combined CT- and fluoroscopy-guided technique.

Combined CT- and fluoroscopy-guided transhepatic portal vein catheterization was performed in 44 patients selected for pancreatic islet cell transplantation. The method allowed catheterization with a single puncture attempt in 39 patients. In four patients two attempts and in one patient four attempts were necessary. One minor hematoma of the liver capsule occurred that required no further treatment. Compared with other methods the average number of puncture attempts was reduced.

Adult↗
PubMedDate not supplied

Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas.

BACKGROUND: Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. METHODS: After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. RESULTS: Purified islet yield decreased from 3670 +/- 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 +/- 180 and 2080 +/- 290 IEQ/g in TLM-preloaded or postloaded organs, respectively (P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 +/- 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 +/- 0.15) and TLM-postloaded pancreata (1.81 +/- 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. CONCLUSIONS: Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.

Adenosine↗
PubMedDate not supplied

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation.

BACKGROUND: Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10). METHODS: Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed (n = 6) as previously described or stored by TLM or OLM. RESULTS: Compared to unstored (429200 +/- 86700 IEQ) and OLM-stored pancreases (338600 +/- 42100 IEQ), (P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 +/- 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 +/- 0.4) was significantly decreased after TLM storage (1.8 +/- 0.2, P < .05) but not after OLM storage (2.3 +/- 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups. CONCLUSIONS: This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.

Adenosine↗
PubMedDate not supplied

Timing of hyperthermic preconditioning affects islet resistance against inflammation.

BACKGROUND: Heat exposure of isolated islets enhances resistance against inflammation but decreases islet graft function. In contrast, donor preconditioning by whole-body hyperthermia increases islet ischemic tolerance and improves viability of pancreatic isografts. This study aimed to compare yield, viability, and inflammatory resistance of rat islets subjected to heat shock prior to (pre-HS) or after isolation (post-HS). METHODS: Islets were isolated as previously described. HS (42 degrees C/45 min) was induced 12 hours before islet isolation (pre-HS, n = 31) or in freshly isolated islets prior to 12 hours of recovery at 37 degrees C (post-HS, n = 12). Islets continuously incubated at 37 degrees C served as controls (n = 33). Proinflammatory treatment included incubation with 0.05 mmol/L H(2)O(2), 1.0 mmol/L DETA-NO or cytokines (interleukin-1beta + tumor necrosis factoralpha + interferongamma). RESULTS: Purified islet yield was 1200 +/- 80 IEQ in unconditioned donors (n = 45) and 980 +/- 80 IEQ after pre-HS (ns). Islet viability was not affected by post-HS, but the glucose stimulation index (P < 0.001, P < 0.01) and formazan production (P < 0.05) were significantly lower compared to pre-HS or sham treatment. The expression of heat shock protein HSP70 in pre-HS islets was slightly higher compared to controls (ns) but lower compared to post-HS islets (P < 0.05), correlating with the resistance against H(2)O(2) and DETA-NO compared to post-HS islets (P < 0.05) or controls (ns). Cytokines did not affect mitochondrial formazan production. CONCLUSIONS: The findings indicate that hyperthermic islet treatment is less harmful if performed in the native pancreatic environment. This beneficial effect is associated with a decreased HSP70 expression resulting in a reduced resistance against inflammatory mechanisms.

Animals↗
PubMedDate not supplied

Influence of neutral protease activity on human islet isolation outcome.

UNLABELLED: Observations in rat pancreata have revealed that enzymatic islet release is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. Since no information is available about the effect of NP activity on islet release from the human pancreas, the present study evaluated the effect of various NP concentrations on the outcome of human islet isolation. METHODS: Following intraductal collagenase distension, pancreata obtained from adult multiorgan donors were digested using 2000 PZ-U of purified Serva collagenase NB 1 supplemented with 2.6 (n = 10) or 4.5% (DMC-U/PZ-U) (n = 10) of NP. RESULTS: Increasing NP from 2.6% to 4.5% reduced the amount of undigested tissue from 22 +/- 2 to 17 +/- 2 g (P < .05) while simultaneously increasing the volume of digested tissue (26 +/- 2 vs 40 +/- 3 mL, P < .01). Increased NP concentrations increased the islet yield prepurification (459,800 +/- 22,900 vs 587,600 +/- 69,000 IEQ, P < .05), but simultaneously affected islet purification, resulting in equal islet yields (345,700 +/- 31,200 vs 391,500 +/- 35,400 IEQ, NS) and less purity (70 +/- 6 vs 49% +/- 5%, P < .01). A NP concentration of 4.5% reduced the stimulation index (4.7 +/- 1.2 vs 2.0 +/- 0.5, P < .01) and viability (100 +/- 1 vs 95% +/- 3%, P < .05). CONCLUSIONS: Although increased NP activity seems to improve islet release from adult human pancreata, it significantly affects islet viability and function. The reduction in purity reflected damage to acinar tissue by increased NP activity presumably affecting islet integrity.

Adult↗
PubMedDate not supplied

The ratio between class II and class I collagenase determines the amount of neutral protease activity required for efficient islet release from the rat pancreas.

UNLABELLED: Previous investigations clearly showed that the successful release of islets from the pancreas is mediated by both neutral protease (NP) and collagenase, consisting of subclasses I and II showing different capacities to cleave islets from the pancreas. Since no informations about the optimal ratio between class II and class I collagenase (II/I-ratio) are available yet, the present study sought to evaluate the efficient range for the II/I-ratio. METHODS: Following intraductal pancreas collagenase distension, rat islets were isolated utilizing 20 PZ-U Serva collagenase NB 1 and 1.0 or 0.4 DMC-U NP. After purification we determined the islet yield (IEQ), viability (trypan-blue exclusion) and function in diabetic nude mice. RESULTS: At 1.0 DMC-U NP, a II/I-ratio of 2.6, 1.5 or 0.7 yielded 2200 +/- 280, 2185 +/- 420, and 2205 +/-90 IEQ, respectively (ns). Viability varied between 70% and 80% (ns). Digestion time was significantly lowest (P < .05) using a II/I-ratio of 0.7. Utilization of 0.4 DMC-U NP resulted in a viability of >98% among all experimental groups (P < .001 vs 1.0 DMC-U). Islet yield decreased at a II/I-ratio of 2.6 (1520 +/- 120 IEQ, P < .05) and 1.5 (1780 +/- 130 IEQ, ns), but not at 0.7 (2310 +/- 160 IEQ, ns). Again, digestion time was lowest (P < .001) using a II/I- ratio of 0.7. Transplantation into diabetic nude mice demonstrated islet function in all experimental groups. CONCLUSIONS: NP significantly affects islet viability. This study indicates that the minimal amount of NP required for efficient islet cleavage depends on the II/I-ratio.

Animals↗
PubMedDate not supplied

Prevention and slowing down the progression of the diabetic nephropathy through antihypertensive therapy.

Diabetic nephropathy is the major cause of illness and premature death in people with diabetes, largely through accompanying cardiovascular disease and end-stage renal failure. Diabetic patients are several times as prone to kidney disease as nondiabetic people and the accumulative risk of diabetic nephropathy in insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) is about 30%-50% after 25 years of disease. Diabetic nephropathy is a progressive disease that takes several years to develop, ending in chronic renal insufficiency. Proteinuria heralds the onset of diabetic nephropathy, and the worsening of proteinuria parallels the progression of renal disease. The main risk factors for the frequency, severity, and progression of diabetic nephropathy are the degree of hyperglycemia and associated metabolic disturbances, hypertension, protein overload, cigarette smoking, as well as the duration of diabetes. Interventional strategies for primary, secondary, and tertiary prevention of diabetic nephropathy therefore include meticulous glycemic control, appropriate treatment of associated lipid abnormalities, rigorous control of the blood pressure, reduction in dietary protein intake, in particular animal protein, and of fat intake, and stopping cigarette smoking. Randomized clinical trials indicate that antihypertensive therapy is beneficial in preventing and slowing down the progression of diabetic nephropathy. There is now increasing evidence that angiotensin-converting enzyme inhibitors and certain calcium antagonists produce a more beneficial effect on diabetic nephropathy in terms of reducing proteinuria and slowing the progression to diabetic renal failure. These drugs are attributed nephroprotective capacity beyond their blood pressure lowering capacity and initial clinical trials with combinations have revealed even additive protective effects on end organs.

Albuminuria↗
PubMedDate not supplied

Pretransplant induction of HSP-70 in isolated adult pig islets decreases early islet xenograft survival.

The heat-induced HSP-70 expression protects rat islet single cells against lysis mediated by nitric oxide (NO), reactive oxygen, and streptozotocin. The present study was performed to investigate the potential antiinflammatory effect of pretransplant heat shock in adult pig islets for subsequent early islet xenograft survival. Maximum HSP-70 expression in freshly isolated pig islets was induced by hyperthermia at 43 degrees C for 90 min prior to islet regeneration at 37 degrees C for 4-6 h. Heat-stressed and sham-treated islets were incubated in 0.6 mM H2O2 or 1.5 mM Na-nitroprusside at 37 degrees C for 20 h. Early graft survival was evaluated in normoglycemic Lewis rats after simultaneous, contralateral transplantation of heat-shocked islets and sham-treated islets into the renal subcapsular space of the same recipient. Prior hyperthermia significantly reduced specific lysis of islets exposed to NO or H2O2, although protection was only marginal. No differences were observed between viability of heat-shocked and sham-treated islets after NO exposure. In contrast, prior heat shock increased islet viability after H2O2 treatment. The finding that hyperthermia reduced recovery of initially grafted pig insulin 48 h after transplantation by 30% compared to controls contrasted significantly with an increased insulin recovery in heat-exposed islets at the end of simultaneous 37 degrees C culture. The observation, that the heat-induced HSP-70 expression decreases early islet xenograft survival as reflected by recovery of grafted insulin, implies an enhancement of islet immunogenicity and the induction of apoptosis. Future experiments aiming at augmentation of intrinsic defense mechanisms should consider detrimental effects associated with induction of heat shock proteins.

Animals↗
PubMedDate not supplied

Clinical islet transplantation--registry report, accomplishments in the past and future research needs.

This review provides the results of a recent analysis of the Islet Transplant Registry on clinical adult islet transplants performed worldwide through June 30, 1992. Between December 12, 1893 and June 30, 1992, 167 adult islet transplants were performed at 25 institutions worldwide, including 104 at 9 institutions in North America, 62 at 15 institutions in Europe, and 1 elsewhere. The total number of diabetic patients reported to be insulin independent after adult islet allotransplantation through June 30, 1992, was 19. In an analysis by era, the percentage of patients that showed positive basal C-peptide levels (i.e., > or = 1 ng/mL at > or = 1 mo) posttransplant, and that became insulin independent (> 1 wk) in the 1985-1989 era (n = 35 cases) were 20% and 6%, and in the 1990-1992 era (n = 69 cases) were 64% and 20%, respectively, and thus have improved significantly (p < 0.001 and p < 0.05). For the 1990-1992 period, the percentage of patients who showed positive basal C-peptide levels post-transplant, and who became insulin independent in the single donor pancreas group (n = 31 cases) were 52% and 13%, and in the multiple donor pancreata group (n = 36 cases) were 75% and 28%, respectively. Islet graft function rates were nearly identical for grafts prepared from pancreata stored < or = 6 h (n = 27) and > 6 < or = 12 h (n = 29), so that 67% and 72% showed positive basal C-peptide levels, and 30% and 21% of the recipients became insulin independent, respectively. No single patient showed islet graft function sufficient to allow withdrawal from insulin, if the pancreata have been stored for more than 12 h. In regard to recipient category for the six groups, namely IAK (islet after kidney), SIK (simultaneous islet kidney transplantation), SIL (simultaneous islet liver transplantation), SIL(C) (simultaneous islet liver transplantation after cluster operation), SIKL (simultaneous islet kidney liver transplantation), and SIH-L (simultaneous islet heart-lung transplantation), the number of patients who showed positive basal C-peptide levels post-transplant was 11 (58%), 17 (57%), 5 (83%), 8 (80%), 1 (50%), and 0 (0%), and the number of insulin independent patients was 4 (21%), 4 (13%), 0 (0%), 6 (60%), 0 (0%), and 0 (0%), respectively. Comparing the two largest recipient categories, namely IAK and SIK, no difference in the outcome of these transplants was apparent.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMedDate not supplied

Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas.

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the libration of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.

Animals↗
PubMedDate not supplied

Assessment of intracellular insulin content during all steps of human islet isolation procedure.

This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p < 0.001). This increase of insulin during pancreas digestion was attributed to the asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.

Cell Separation↗