Adjustment of digestion enzyme composition improves islet isolation outcome from marginal grade human donor pancreata

Despite improvements and recent attempts to standardize techniques to isolate islets from human donor pancreata, there still exists the problem of consistently recovering sufficient quantities of high quality islets. Moreover, achieving consistent recoveries of high numbers of good quality islets becomes even more challenging from marginal grade human donor pancreata with prolonged cold ischemic times. In this study, we investigate whether addition of Pefabloc SC, a serine protease inhibitor, in combination with Pulmozyme, a recombinant human DNase I, to Liberase HI improves islet isolation outcome from marginal grade human donor pancreata (cold ischemic time > 12 h). Twenty-three marginal grade human donor pancreata were randomly digested using four different enzyme preparations: (1) Liberase alone (n = 6), (2) +Pefabloc (n = 7), (3) +Pefabloc/Pulmozyme (n = 5), and (4) +Pulmozyme (n = 5). Overall, there were no significant differences in donor age, body mass index (BMI), pancreas weight, and cold ischemic time. After purification, significantly higher islet yields (3,281 ± 590 IE/g) were obtained with the Pefabloc/Pulmozyme group as compared to the Liberase alone (1,615 ± 305 IE/g) or the Pefabloc group (1,255 ± 261 IE/g) (P < 0.05). Significant improvements in islet viability were also noted from the Pefabloc/Pulmozyme group (87.3 ± 4.4%) as opposed to islets isolated from the Pefabloc group (75.2 ± 3.9%) (P < 0.05). No significant differences in insulin secretory response to glucose stimulation among the four groups were observed, which indicates that the addition of Pefabloc and/or Pulmozyme does not have a detrimental effect on the functionality of islets. It is concluded that the addition of Pefabloc in combination with Pulmozyme to the Liberse HI significantly improves islet isolation outcome and potentially impacts the viability and morphology of the islets obtained from marginal grade human donor pancreata with prolonged cold ischemic times. This study was presented in a poster session at the American Transplant Congress 2005 in Seattle, Washington, USA (May 2005).     

University of Wisconsin Solution with Trypsin Inhibitor Pefabloc Improves Survival of Viable Human and Primate Impure Islets During Storage

Background. Recent studies suggest that impure islets (islets which have not been isolated from exocrine tissue and other parts of the pancreas) have great potential for successful transplantation. The evidence that supports this view includes findings that embedded islets (islets surrounded by exocrine tissue) undergo less apoptosis, peripancreatic lymph nodes prevent recurrence of IDDM (insulin dependent diabetes mellitus), and that islet yields and insulin content decrease during the purification process. Improved protocols have also been developed to prevent allorejection of impure islets. Despite these promising results, the storage of impure islets remains difficult, and was a method sought to decrease storage losses. Methods. Storage methods of impure human and non-human primate islets were compared, using either culture media or University of Wisconsin solution (UW). The effects of trypsin inhibition using Pefabloc (Roche Molecular Biochemicals, Indianapolis, IN) during storage period were also examined. Results. Low temperature and inhibition of trypsin activity during storage of impure islets improved both islet yield and viability. It was found that using UW solution and trypsin inhibition allowed perfect preservation of viable impure islets up to 48h. A functional assay by glucose stimulation test showed these impure islet responded to glucose stimulation after 24h. Conclusion. The benefits of storing impure islets using UW solution and Pefabloc at low temperature have been established. This improved method of preserving impure islets makes this model of transplantation even more viable.     

Method for isolation of pancreatic blood vessels,their culture and coculture with islets of langerhans

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 μm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.     

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients ¹, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome ². At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient ³. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) ^4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al⁸. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification ³. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods ⁹. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37°C overnight culture before islets were transplanted or used for research.Open in a separate windowClick here to view.^{(52M, flv)}     

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients ¹, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome ². At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient ³. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) ^4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al ⁸. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37°C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4°C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.     

Possible modulatory effect of endogenous islet catecholamines on insulin secretion

Background

The possible participation of endogenous islet catecholamines (CAs) in the control of insulin secretion was tested.

Methods

Glucose-induced insulin secretion was measured in the presence of 3-Iodo-L-Tyrosine (MIT), a specific inhibitor of tyrosine-hydroxylase activity, in fresh and precultured islets isolated from normal rats. Incubated islets were also used to measure CAs release in the presence of low and high glucose, and the effect of α2-(yohimbine [Y] and idazoxan [I]) and α1-adrenergic antagonists (prazosin [P] and terazosin [T]) upon insulin secretion elicited by high glucose.

Results

Fresh islets incubated with 16.7 mM glucose released significantly more insulin in the presence of 1 μM MIT (6.66 ± 0.39 vs 5.01 ± 0.43 ng/islet/h, p < 0.02), but did not affect significantly the insulin response to low glucose. A similar enhancing effect of MIT upon insulin secretion was obtained using precultured islets devoid of neural cells, but absolute values were lower than those from fresh islets, suggesting that MIT inhibits islet rather than neural tyrosine hydroxylase. CAs concentration in the incubation media of fresh isolated islets was significantly higher in the presence of 16.7 than 3.3 mM glucose: dopamine 1.67 ± 0.13 vs 0.69 ± 0.13 pg/islet/h, p < 0.001, and noradrenaline 1.25 ± 0.17 vs 0.49 ± 0.04 pg/islet/h, p < 0.02. Y and I enhanced the release of insulin elicited by 16.7 mM glucose while P and T decreased such secretion.

Conclusion

Our results suggest that islet-originated CAs directly modulate insulin release in a paracrine manner.     

Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III

The purpose of the present study was to evaluate whether AFPs protect the heart from freezing and improve survival and viability in subzero cryopreservation. Hearts were subject to 5 preservation protocols; University of Wisconsin solution (UW) at 4 degrees C, UW at -1.3 degrees C without nucleation, UW at -1.3 degrees C with nucleation, UW AFP I (15 mg/cm(3)) at -1.3 degrees C with nucleation, and in UW AFP III (15 mg/cm(3)) at -1.3 degrees C with nucleation. Hearts were preserved for 24, 28, and 32 h, rewarmed and connected to the working isolated perfusion system. Data [heart rate (HR), coronary flow (CF), and developed pressure (dP)] was collected 30 and 60 min after reperfusion. Hearts preserved at -1.3 degrees C without AFPs froze, while hearts preserved with AFP did not freeze when nucleation was initiated and survived. Survival and dP of hearts preserved for 24h at -1.3 degrees C using AFP III was better than those preserved at 4 degrees C, (dP; 1.4 vs. 0.8, p<0.05). Four of six hearts and six of six hearts died when preserved at 4 degrees C for 28 and 32 h, respectively, all of the hearts that were preserved at -1.3 degrees C with or without AFPs survived after 28 h (n=18) and 32 h (n=18). CF was higher in UW -1.3 degrees C group without attempted nucleation than in AFP I and AFP III groups after 28 and 32 h (3.4 vs. 1.7, p<0.05, and 3.4 vs. 1.7, p<0.05, respectively). In conclusion, AFPs were found to protect the heart from freezing and improve survival and dP (AFP III) in prolonged subzero preservation.     

Increased superoxide anion production is associated with early atherosclerosis and cardiovascular dysfunctions in a rabbit model

Objective Hypercholesterolemia (HC) has been associated with impairment of vascular and myocardial functions. As HC could generate an alteration in the oxidative status, we studied the effects of a 1-month cholesterol diet on cardiovascular oxidative stress. Methods and Results New Zealand rabbits received cholesterol (1%) or normal chow for 1 month. At 30 days, superoxide anion levels, assessed by ESR spectroscopy, NAD(P)H oxidase (NOX) activity, and dihydroethidium (DHE) staining of aortas were higher in the cholesterol-fed (CF) group compared with control (respectively, 4.0 ± 0.6 Arbitrary Units/mg (AU/mg) vs. 2.6 ± 0.3, p < 0.05; 4231 ± 433 vs. 2931 ± 373 AU/mg, p < 0.05; 21.4 ± 1.2 vs. 12.9 ± 1.7% fluorescence/mm², p < 0.001). NOX gp91phox and p67phox expression in the aortas were higher in the CF group vs. control (1.5 ± 0.2 vs. 0.5 ± 0.2, p < 0.001; 0.9 ± 0.2 vs. 0.3 ± 0.2, p < 0.05). The endothelium-dependent relaxation evaluated on the iliac arteries was higher in control than in the CF group (64.8 ± 10.1 vs. 13.1 ± 3.70%, p < 0.001). The cardiac diastolic pressure estimated on isolated hearts was higher in the CF group than in control (21.1 ± 4.1 vs. 10.3 ± 1.4 mmHg, p < 0.05) after 60 min of ischemia. Conclusions Hypercholesterolemia induced increased levels of superoxide in the aortas and a higher expression of NOX subunits, associated with altered vasorelaxation. The increased diastolic pressure observed in hearts, consistent with a post-ischemic contractile dysfunction might be mediated by the production of superoxide.     

Pancreatic islets isolation using different protocols with in situ flushing and intraductal collagenase injection

Human islet transplantation seems to be a very promising clinical procedure for patients with type I diabetes mellitus. The aim of our study was to investigate the influence of in situ intravascular flushing with University of Wisconsin (UW) solution and intraductal collagenase injection at the time of pancreas procurement on the isolated islets and exocrine tissue injury. Our experiments indicated that in situ perfusion with the UW solution has a beneficial effect on pancreatic islets and intraductal distention results in an increase in the concentration of pancreatic enzymes released into the cold preservation solution during ischemic conditions. Cold ischemia reduced islet yield, but pancreas perfusion with the UW solution showed better ischemic tolerance of isolated islets during glucose static incubation. We conclude that intravascular pancreas flushing has a crucial effect on recovery and yield of pancreatic islets and protects against exocrine tissue injury.     

Cardiorespiratory fitness influences the blood pressure response to experimental weight gain

Objective: We tested the hypothesis that with similar weight gain the increase in blood pressure (BP) would be smaller in men with higher cardiorespiratory fitness (HCRF) than in men with lower cardiorespiratory fitness (LCRF). Research Methods and Procedures: Thirteen men (age = 23 ± 1, BMI = 24 ± 1) were overfed by ～1000 kcal/d over ～8 weeks to achieve a 5‐kg weight gain. Resting BP and 24‐hour ambulatory BP, body composition, and fat distribution were measured. Results: Cardiorespiratory fitness (CRF) was higher in the HCRF group compared with the LCRF group (49.9 ± 1.2 vs. 38.1 ± 1.4 mL/kg per minute, p < 0.001). At baseline, body weight was similar in the HCRF and LCRF groups, whereas the HCRF group displayed lower levels of total body fat (13.0 ± 1.7 vs. 16.9 ± 1.3 kg, p = 0.049) and abdominal visceral fat (49 ± 6 vs. 80 ± 14 cm², p = 0.032). Resting BP and 24‐hour ambulatory BP were similar in the two groups at baseline. After weight gain, body weight increased ～5 kg (p < 0.05) in both groups; the changes in body composition and regional fat distribution were similar. As hypothesized, the increases in resting systolic (1 ± 2 vs. 7 ± 2 mm Hg; p = 0.008) and diastolic (?1 ± 4 vs. 5 ± 1 mm Hg; p = 0.005) BP were smaller in the HCRF group. CRF was correlated with the increases in resting systolic (r = ?0.64; p = 0.009) and diastolic BP (r = ?0.80; p < 0.001). Furthermore, the relationship between CRF and BP remained significant after adjusting for the changes in the proportion of total abdominal fat gained as visceral fat. Discussion: These findings suggest that higher levels of CRF are associated with a smaller increase in BP with weight gain, independently of changes in abdominal visceral fat.     

Obese reproductive-age women exhibit a proatherogenic inflammatory response during hyperglycemia

Objective: The objective was to determine if physiological hyperglycemia induces a proatherogenic inflammatory response in mononuclear cells (MNCs) in obese reproductive‐age women. Research Methods and Procedures: Seven obese and 6 age‐matched lean women (20 to 39 years of age) underwent a 2‐hour 75‐g oral glucose tolerance test. The release of interleukin‐6 (IL‐6) and interleukin‐1β (IL‐1β) from MNCs cultured in the presence of lipopolysaccharide (LPS) was measured after isolation from blood samples drawn fasting and 2 hours after glucose ingestion. Reactive oxygen species (ROS) generation and intra‐nuclear nuclear factor κB (NFκB) from MNCs were quantified from the same blood samples. Insulin resistance was estimated by homeostasis model assessment of insulin resistance (HOMA‐IR). Total body fat and truncal fat were determined by DXA. Results: Obese women had a higher (p < 0.03) total body fat (42.2 ± 1.1 vs. 27.7 ± 2.0%), truncal fat (42.1 ± 1.2 vs. 22.3 ± 2.4%), and HOMA‐IR (3.3 ± 0.5 vs. 1.8 ± 0.2). LPS‐stimulated IL‐6 release from MNCs was suppressed during hyperglycemia in lean subjects (1884 ± 495 vs. 638 ± 435 pg/mL, p < 0.05) but not in obese women (1184 ± 387 vs. 1403 ± 498 pg/mL). There was a difference (p < 0.05) between groups in the hyperglycemia‐induced MNC‐mediated release of IL‐6 (?1196 ± 475 vs. 219 ± 175 pg/mL) and IL‐1β (?79 ± 43 vs. 17 ± 12 pg/mL). In addition, the obese group exhibited increased (p < 0.05) MNC‐derived ROS generation (39.3 ± 9.9 vs. ?1.0 ± 12.8%) and intra‐nuclear NFκB (9.4 ± 7.3 vs. ?23.5 ± 13.5%). Truncal fat was positively correlated with the MNC‐derived IL‐6 response (ρ = 0.58, p < 0.05) and intra‐nuclear NFκB (ρ = 0.64, p < 0.05). Discussion: These data suggest that obese reproductive‐age women are unable to suppress proatherogenic inflammation during physiological hyperglycemia. Increased adiposity may be a significant contributor to this pro‐inflammatory susceptibility.     

Co-transplantation of islets with mesenchymal stem cells in microcapsules demonstrates graft outcome can be improved in an isolated-graft model of islet transplantation in mice

Background aimsCo-transplantation of islets with mesenchymal stem cells (MSCs) has been shown to improve graft outcome in mice, which has been partially attributed to the effects of MSCs on revascularization and preservation of islet morphology. Microencapsulation of islets provides an isolated-graft model of islet transplantation that is non-vascularized and prevents islet aggregation to preserve islet morphology. The aim of this study was to investigate whether MSCs could improve graft outcome in a microencapsulated/isolated-graft model of islet transplantation.MethodsMouse islets and kidney MSCs were co-encapsulated in alginate, and their function was assessed in vitro. A minimal mass of 350 syngeneic islets encapsulated alone or co-encapsulated with MSCs (islet+MSC) were transplanted intraperitoneally into diabetic mice, and blood glucose concentrations were monitored. Capsules were recovered 6 weeks after transplantation, and islet function was assessed.ResultsIslets co-encapsulated with MSCs in vitro had increased glucose-stimulated insulin secretion and content. The average blood glucose concentration of transplanted mice was significantly lower by 3 weeks in the islet+MSC group. By week 6, 71% of the co-encapsulated group were cured compared with 16% of the islet-alone group. Capsules recovered at 6 weeks had greater glucose-stimulated insulin secretion and insulin content in the islet+MSC group.ConclusionsMSCs improved the efficacy of microencapsulated islet transplantation. Using an isolated-graft model, we were able to eliminate the impact of MSC-mediated enhancement of revascularization and preservation of islet morphology and demonstrate that the improvement in insulin secretion and content is sustained in vivo and can significantly improve graft outcome.     

Hyperbranched Polyglycerol as a Colloid in Cold Organ Preservation Solutions

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.     

Probiotic containing Lactobacillus reuteri DSM 17648 as an adjunct treatment for Helicobacter pylori infection: A randomized,double-blind,placebo-controlled trial

Background

Despite multiple therapy regimens, the decline in the Helicobacter pylori eradication rate poses a significant challenge to the medical community. Adding Lactobacillus reuteri probiotic as an adjunct treatment has shown some promising results. This study aims to investigate the efficacy of Lactobacillus reuteri DSM 17648 in H. pylori eradication and its effect in ameliorating gastrointestinal symptoms and adverse treatment effects.

Materials and Methods

This randomized, double-blinded, placebo-controlled trial involved treatment-naïve H. pylori-positive patients. Ninety patients received standard triple therapy for 2 weeks before receiving either a probiotic or placebo for 4 weeks. The posttreatment eradication rate was assessed via a ¹⁴C urea breath test in Week 8. The Gastrointestinal Symptom Rating Scale (GSRS) questionnaire and an interview on treatment adverse effects were conducted during this study.

Results

The eradication rate was higher in the probiotic group than in the placebo group, with a 22.2% difference in the intention-to-treat analysis (91.1% vs. 68.9%; p = 0.007) and 24.3% difference in the per-protocol analysis (93.2% vs. 68.9%; p = 0.007). The probiotic group showed significant pre- to post-treatment reductions in indigestion, constipation, abdominal pain, and total GSRS scores. The probiotic group showed significantly greater reductions in GSRS scores than the placebo group: indigestion (4.34 ± 5.00 vs. 1.78 ± 5.64; p = 0.026), abdominal pain (2.64 ± 2.88 vs. 0.89 ± 3.11; p = 0.007), constipation (2.34 ± 3.91 vs. 0.64 ± 2.92; p = 0.023), and total score (12.41 ± 12.19 vs. 4.24 ± 13.72; p = 0.004). The probiotic group reported significantly fewer adverse headache (0% vs. 15.6%; p = 0.012) and abdominal pain (0% vs. 13.3%; p = 0.026) effects.

Conclusions

There was a significant increase in H. pylori eradication rate and attenuation of symptoms and adverse treatment effects when L. reuteri was given as an adjunct treatment.     

Comparing the effects of Dextran 70 and Hydroxyethyl starch in an intestinal storage solution

Introduction

Our lab has developed a novel strategy for intestinal preservation involving the intraluminal delivery of a nutrient-rich preservation solution. The aim of this study was to compare the effectiveness of two impermeant agents for use in our solution: Dextran 70 (D70; Mw = 70 kDa) and Hydroxyethyl starch (HES; Mw = 2200 kDa).

Methods

Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a ‘backtable’ intraluminal flush with: UW solution (group 1, UW), or an amino acid-based nutrient-rich preservation solution (AA solution) containing either 5% D70 (group 2, AA-D70) or HES (group 3, AA-HES). Tissue samples (n = 6) were taken at 2, 4, 8, and 12 h cold storage; histology, energetic, end-product, and oxidative parameters were assessed. In separate groups (n = 4), D70 and HES were fluorescently labeled with fluorescein isothiocyanate (FITC) in order to directly observe mucosal penetration of the starch and dextran.

Results

Over the 12 h storage time-course, direct visualization of the fluorescently labeled D70 showed penetration of the mucosal layer as early as 2 h and progressively continued to do so throughout the 12 h period. In contrast, HES did not cross the mucosal barrier and remained captive within the lumen. As time of storage progressed, grade of injury increased in all groups, however, at 4 and 12 h the AA-HES treated tissues exhibited significantly less injury compared to UW and AA-D70, P < 0.05. AA-HES group showed on moderate villus clefting (median grade 2; P < 0.05) while the AA-D70 group exhibited complete villus denudation (grade 4) and the UW group had extensive injury into the regenerative cryptal regions (grade 6). Metabolic parameters revealed a preferential maintenance of ATP and Energy Charge; increases in lactate, alanine and ammonium supported the involvement of aerobic and anaerobic pathways for energy production.

Conclusion

The results of this study challenge the idea that oncotic support is not a fundamental requirement of static organ storage. Furthermore, our data suggests that HES is an effective oncotic agent for use in our intraluminal nutrient-rich preservation solution, while Dextran 70 is not.     

A comparison of cross sectional surface area densities between adult and juvenile porcine islets of Langerhans.

The object of this study was to evaluate differences in islet diameters, their distribution and both cross sectional surface areas and densities of insulin containing islets between adult and juvenile porcine pancreata using a computerised image analysis system (Improvision). Five adult (A) (2-3 yrs) and 5 juvenile (J) (< 12 mths) Large White pancreata were assessed. Biopsies were taken from 5 different regions (posterior lobe, duodenal lobe, along with the head, body and tail regions of the splenic lobe) of the pancreas and tissue sections stained for insulin. In both A and J pancreata islet numbers increased with decreasing islet diameter, showing a skewed distribution. There was no statistical significance between the cross sectional surface area within A (mean 5.04 x 10(3) microm2) or J (mean 5.99 x 10(3) microm2) pancreata. Assuming islets are spherical, extrapolation from pir2 showed that the mean diameter for A was 80 microm and 87 microm in J. These compared with A 77 microm and J 86 microm diameters using conventional microscopic techniques. The percentage islet volume density relative to exocrine tissue, derived from the principle of Delesse (Area density = volume density), did not significantly differ between each of the 5 areas studied, either in A or J. The percentage islet volume densities did show a significant difference between A (mean 1.83%) (P = 0.001) but not between J pancreata (mean 2.13 %). In conclusion poor islet yields can be attributed to differences in islet volume density of islets within porcine pancreata. These results also suggest that the posterior and duodenal lobes should be used along with the splenic lobe in order to improve porcine islet yields. Furthermore, the current practise of reporting porcine islet yields and the isolation index relative to 150 microm (IEQs) needs to be redefined, based on the assumption that the average size of an adult porcine islet is 80 microm.     

Anti‐lipolytic Effects of Insulin in African American and White Prepubertal Boys

Objective: Relative to whites, African Americans have lower circulating triglycerides (TG) and greater highdensity lipoprotein cholesterol. The metabolic basis for this difference is not known. This study was conducted to test the hypothesis that insulin‐induced suppression of free fatty acids (FFA) results in lower serum TG in African American versus white prepubertal children. Research Methods and Procedures: Insulin, FFA, and TG were determined at baseline and during a frequently sampled, intravenous glucose tolerance test in eight African American and eight white prepubertal males pair‐matched for whole‐body insulin sensitivity. Results: Baseline TG was lower in African Americans (0.43 ± 0.10 vs. 0.79 ± 0.37 mM/L; mean ± SD; p < 0.01). African Americans had higher peak insulin (218 ± 102 vs. 100 ± 30 pM/L; mean ± SD; p < 0.01) and a greater acute insulin response (9282 ± 4272 vs. 4230 ± 1326 pM/L × 10 minutes; mean ± SD; p < 0.05). FFA and TG values determined at the FFA nadir were lower in African Americans (0.26 ± 0.02 vs. 0.30 ± 0.03 mEq/L; mean ± SD; p < 0.01 for FFA nadir and 0.49 ± 0.07 vs. 0.77 ± 0.33 mM/L; mean ± SD; p < 0.05 for TG). Among all subjects, FFA nadir was correlated with peak insulin (r = ?0.54; p < 0.05). After adjusting for FFA nadir, neither baseline nor postchallenge TG differed with ethnicity (p = 0.073 and 0.192, respectively). The ethnic difference in FFA nadir disappeared after adjusting for peak insulin (p = 0.073). Discussion: These data suggest that hyperinsulinemiainduced suppression of FFA among African Americans is a determinant of lower TG in this group.     

Immunohistochemical identification of pancreatic hormones,neuropeptides and cytoskeletal proteins in pancreas of the camel (Camelus dromedarius)

The patterns of distribution of insulin (INS), glucagon (GLU), atrial natriuretic peptide (ANP), neuropeptide-Y (NPY), cholecystokinin-octapeptide (CCK-8), neurofilament-200 protein (NF), S-100 protein (S-100), and vimentin (VIM) in the pancreas of the one-humped camel (Camelus dromedarius) were investigated using immunohistochemical techniques. INS-immunoreactive cells were observed in the central and peripheral parts of the islets of Langerhans, but some solitary INS-positive cells were found outside the islets. INS-positive cells constituted 44.26–90.91% [mean ± standard deviation (std): 67.34 ± 14.20] of the total number of islet cells. GLU-immunopositive cells were located mainly in the peripheral region of the islets, and they constituted 11.43–44.44% [mean ± std: 23.54 ± 8.27] of the total number of islet cells. ANP and CCK-8 immunoreactivity was observed in neurons and perivascular nerves fibers. NPY was identified in pancreatic neurons and in some peripheral and central cells of the islets of Langerhans. VIM immunoreactivity was observed in the endothelial cells of blood vessels and the nerves located in the perivascular, interlobular and periacinar regions. VIM was also detected immunohistochemically in the periductal nerves of the pancreas. NF occurred only in nerves. S-100 was discerned mainly in the nerves of the interlobular connective tissue and in nerves lying close to blood vessels and acinar tissue. It is concluded that INS, GLU, ANP, NPY, CCK-8, NF, S-100, and VIM are well distributed in the pancreas of the camel. J. Morphol. 231:185–193, 1997. © 1997 Wiley-Liss, Inc.     

Liver-Fat Accumulation and Insulin Resistance in Obese Women with Previous Gestational Diabetes

Objective: We determined whether fat accumulation in the liver is associated with features of insulin resistance independent of obesity. Research Methods and Procedures: We recruited 27 obese nondiabetic women in whom liver fat (LFAT) content was determined by proton spectroscopy, intra-abdominal and subcutaneous fat by magnetic resonance imaging, and insulin sensitivity by the euglycemic insulin clamp technique. The women were divided based on their median LFAT content (5%) to groups with low (3.2 ± 0.3%) and high (9.8 ± 1.5%) liver fat. The groups were almost identical with respect to age (36 ± 1 vs. 38 ± 1 years in low vs. high-LFAT), body mass index (32.2 ± 0.6 vs. 32.8 ± 0.5 kg/m²), waist-to-hip ratio, intra-abdominal, subcutaneous, and total fat content. Results: Women with high LFAT had features of insulin resistance including higher fasting serum triglyceride (1.93 ± 0.21 vs. 1.11 ± 0.09 mM, p < 0.01) and insulin (14 ± 3 vs. 10 ± 1 mU/L, p < 0.05) concentrations than women with low LFAT. The group with high LFAT also had higher 24-hour blood pressures, and lower whole-body insulin sensitivity compared with the low-LFAT group. Discussion: In obese women with previous gestational diabetes, LFAT, rather than any measure of body composition, is associated with features of insulin resistance.