Histochemical analysis of the role of class I and class II Clostridium histolyticum collagenase in the degradation of rat pancreatic extracellular matrix for islet isolation

To understand why class II Clostridium histolyticum collagenase is much more effective than class I in the isolation of rat pancreatic islets, we analyzed the role of these collagenases in pancreatic tissue dissociation. Crude collagenase was purified and then fractionated into class I and II with different enzyme activities and protein compositions, Pancreatic tissue was incubated,vith either class I, class II, or class I + II, with or without added protease, under conditions that eliminated endogenous proteolytic activity, The degradation of pancreatic extracellular matrix was monitored by selective histochemical staining of tissue samples, Class I and LI showed similar capacities to degrade glycoproteins and degraded about one-third of the glycoproteins during 120 min of incubation, The degradation of collagens by class I and II was relatively more effective, 80 to 95% of the collagens being removed in 120 min, and also class dependent. Bath in the presence and absence of protease, class II was more effective at degrading collagens than class I, but this difference in efficacy was less apparent than with islet isolation, Class I + II degraded collagens faster and more complete than did the individual classes, indicating a synergistic effect of class I and LI. Evaluation of collagen degradation at various pancreatic locations did not show a selective degradation of collagens by any of the collagenase classes, The present data offer a partial explanation for the major role of class II in islet isolation. (C) 1997 Elsevier Science Inc

Relationship between duration of brain death and hemodynamic (in)stability on progressive dysfunction and increased immunologic activation of donor kidneys

Relationship between duration of brain death and hemodynamic (in)stability on progressive dysfunction and increased immunologic activation of donor kidneys.BackgroundConsistent difference in graft survival after renal transplantation has been shown when cadaveric transplants are compared to the living related donor situation, in favor of the latter. Recently, evidence has been put forward that brain death has significant effects on the donor organ quality. In this study, we aimed to assess the relation between brain death–induced hemodynamic instability in combination with the duration of brain death on the function and immunogenicity status of potential donor kidneys.MethodsIn Wistar rats, short-term (1hour) or long-term (6hours) brain death in the presence or absence of hemodynamic stability was applied. Sham-operated rats served as controls (1hour and 6hours). Organ function was studied by monitoring serum creatinine, lactate dehydrogenase (LDH), lactate, and total protein content. Expression of cell adhesion molecules [intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)] and the influx of leukocytes in the kidney assessed the immunologic status of the kidney.ResultsProgressive organ dysfunction was most pronounced in hemodynamically unstable brain-dead donors reflected by increased serum creatinine levels. Regardless of hemodynamic status, a progressive inflammatory activation by cell adhesion molecule expression and an influx of leukocytes could be observed in kidneys of brain-dead rats compared with nonbrain-dead controls.ConclusionBrain death causes progressive kidney dysfunction. Also, inflammatory responses reflecting tissue injury are caused by brain death. When hemodynamic instability in the brain-dead donor is not corrected, kidney dysfunction is enhanced and immune activation occurs faster and is more profound. The observed changes may predispose the graft for additional ischemia/reperfusion injury during the transplant process and hence accelerate rejection of the graft after transplantation

Causes of limited survival of microencapsulated pancreatic islet grafts

Successful transplantation of pancreatic tissue has been demonstrated to be an efficacious method of restoring glycemic control in type 1 diabetic patients. To establish graft acceptance patients require lifelong immunosuppression, which in turn is associated with severe deleterious side effects. Microencapsulation is a technique that enables the transplantation of pancreatic islets in the absence of immunosuppression by protecting the islet tissue through a mechanical barrier. This protection may even allow for the transplantation of animal tissue, which opens the perspective of using animal donors as a means to solve the problem of organ shortage. Microencapsulation is not yet applied in clinical practice, mainly because encapsulated islet graft survival is limited. In the present review we discuss the principal causes of microencapsulated islet graft failure, which are related to a lack of biocompatibility, limited immunoprotective properties, and hypoxia. Next to the causes of encapsulated islet graft failure we discuss possible improvements in the encapsulation technique and additional methods that could prolong encapsulated islet graft survival. Strategies that may well support encapsulated islet grafts include co-encapsulation of islets with Sertoli cells, the genetic modification of islet cells, the creation of an artificial implantation site, and the use of alternative donor sources. We conclude that encapsulation in combination with one or more of these additional strategies may well lead to a simple and safe transplantation therapy as a cure for diabetes

A versatile alginate droplet generator applicable for microencapsulation of pancreatic islets

Alginate beads for immunoisolation of pancreatic islets by microencapsulation should be small, smooth, and spherical in order to ensure that around the islets a strong alginate-polylysine-alginate capsule will be formed with optimal biocompatibility and diffusion of nutrients and hormones. However, the preparation of small capsules around islets is difficult. Our newly designed air jet droplet generator allows for variations in the length and diameter of the alginate nozzle and the air jacket and is in this way adaptable to a required bead size. Alginate droplets are converted into rigid beads in a 100 mM CaCl2 solution. Their size depends upon the diameter of the jacket, the air flow rate, and the outer diameter of the nozzle, whereas the production rate depends upon the pressure on the alginate, and on the diameter and the length of the nozzle. When the air flow or the alginate flow surpasses a certain rate, the droplets are fragmented. This study describes the mutual relationship of these variables and defines their optimal range for reproducible production of smooth and spherical beads for microencapsulation of islets at an acceptable production rate

CHANGING PATTERNS IN GRADUATE SURGICAL EDUCATION IN THE NETHERLANDS

An overview is presented of the organization, outline, and contents of graduate surgical training programs in The Netherlands. Adaptations of these programs to changes in demands in surgical practice and health care delivery systems are discussed. Special emphasis is given to the relation of surgery and the surgical specialties, additional training, theoretic training, and manpower planning. The principal questions regarding the optimal way to respond to changing concepts in the field of surgery are presented and discussed in the context of the situation in The Netherlands, but they seem to be similar in the surgical communities of many countries

Factors influencing the adequacy of microencapsulation of rat pancreatic islets

The observation that only a portion of all alginate-polylysine microcapsules are overgrown after implantation suggests that physical imperfections of individual capsules, rather than the chemical composition of the material applied, are responsible for inducing insufficient biocompatibility and thereby fibrotic overgrowth of those capsules. We recently developed a lectin binding assay that allows for quantifying the portion of inadequately encapsulated islets, and demonstrated that inadequately encapsulated islets induce a fibrotic response associated with graft failure. The present study investigates factors influencing the adequacy of encapsulation of pancreatic islets, We applied our lectin binding assay and found that the number of inadequate, and particularly incomplete, capsules is influenced by the following factors. (1) A capsule diameter of 800 mu m is associated with a lower percentage of inadequate capsules than smaller (500 mu m and 600 mu m) or larger (1800 mu m) capsules. (2) A high rather than low guluronic acid content of the alginate is associated with a lower percentage of inadequate capsules, This can be explained, at least in part, by smaller ranges of swelling and subsequent shrinkage during the encapsulation procedure. (3) An increase in viscosity caused by applying a higher alginate concentration compensates for a low guluronic acid content, This effect of increased viscosity cannot be explained by a reduced range of swelling and shrinkage during the encapsulation procedure. We conclude that alginates with a high guluronic acid content and a viscosity near the filtration limit are preferable in order to minimize the number of inadequate capsules