Reinout van Schilfgaarde

Effect of the alginate composition on the biocompatibility of alginate-polylysine microcapsules

Alginate-polylysine (PLL) capsules are commonly applied for immunoprotection of endocrine tissues. Alginate is composed of mannuronic acid (M) and guluronic acid (G). Different types of alginate have different ratios of G to M, but little is known of the influence of these differences on biocompatibility. Therefore, we have investigated in vivo the effect of the G-content of the alginate on the biocompatibility of the capsules. Capsules prepared of commercially available alginates with either a high or an intermediate G-content were implanted in the peritoneal cavity of rats and retrieved one month later for histological evaluation. The fibrotic reaction was more severe against high-G alginate capsules than to intermediate-G alginate capsules. The majority of the high-G capsules proved to be overgrown and adherent to the abdominal organs whereas with intermediate-G alginate most capsules were found freely floating in the peritoneal cavity and free of any adhesion of cells. This was not caused by the alginate as such but rather by inadequate binding of high-G alginate to PLL since in the absence of PLL, i.e. with beads instead of capsules, no fibrotic reaction was observed. As high-G alginates have beneficial effects for islet encapsulation, efforts should be made to apply polycations which more effectively interact with high-G alginate than PLL.

Effects of Brain Death and Hemodynamic Status on Function and Immunologic Activation of the Potential Donor Liver in the Rat

ObjectiveTo assess the effect on the function and immunologic status of potential donor livers of the duration of brain death combined with the presence and absence of hemodynamic instability in the donor. Summary Background DataBrain death, regarded as a given condition in organ transplantation, could have significant effects on the donor organ quality. MethodsBrain death was induced in Wistar rats. Short or long periods of brain death in the presence or absence of hemodynamic instability were applied. Sham-operated rats served as controls. Organ function was studied by monitoring standard serum parameters. The inflammatory status of the liver was assessed by determining the immediate early gene products, the expression of cell adhesion molecules, and the influx of leukocytes in the liver. ResultsProgressive organ dysfunction was most pronounced in hemodynamically unstable brain-dead donors. Irrespective of hemodynamic status, a progressive inflammatory activation could be observed in brain-dead rats compared with controls. ConclusionsBrain death causes progressive liver dysfunction, which is made worse by the coexistence of hemodynamic instability. Further, brain death activates the inflammatory status of the potential donor liver, irrespective of the presence of hypotension. The changes observed may predispose the graft to additional damage from ischemia and reperfusion in the transplant procedure.

The capsular overgrowth on microencapsulated pancreatic islet grafts in streptozotocin and autoimmune diabetic rats

Abstracts This study investigates whether capsular overgrowth on alginate‐polylysine microencapsulated islets is influenced by (1) the presence of islet tissue, (2) MHC incompatibility between donor and recipient, or (3) the presence of autoimmune diabetes. Encapsulated Albino Oxford (AO, n= 6, isografts) and Lewis (n= 6, allografts) rat islets, and encapsulated human islets (n= 5, xenografts) were implanted intraperitoneally into streptozotocin‐dia‐betic AO rats. Also, encapsulated AO islets were implanted into autoimmune diabetic Bio Breeding/Organon (BB/O) rats (n= 5, allografts). Five isografts, five allografts, and three xenografts in AO recipients and five allografts in BB/O recipients resulted in normoglycemia. Two weeks after implantation, islets containing capsules were retrieved by peritoneal lavage, after which all animals that had become normoglycemic after transplantation returned to a state of hyperglycemia. Recovery rates of the capsules of these successful grafts, expressed as percentages of the initially implanted graft volume, varied from 72%± 7% to 80%± 9%. The associated pericapsular infiltrates (PCI) were similar in all groups and varied from 3.2%± 1.4% to 8.3%+ 2.6% Similar recovery rates and PCI were also found with empty capsules. However, the recovery rates of recipients with graft failures were lower and showed more PCI. Immunohistological staining of PCI showed no differences in the types of cells in the PCI on capsules with or without islets. We conclude that this early PCI is a capsule‐induced foreign body reaction that is not influenced by MHC incompatibility or by the presence of autoimmune diabetes, and it should be avoided by improving the biocompatibility of the capsules.

Brain death induces apoptosis in donor liver of the rat

Background. A difference in short- and long-term function between living-related and cadaveric donor organs is consistently shown in kidney- and liver-transplant studies. We hypothesize that this is caused by induction of apoptosis and inflammation of the potential graft because of the phase of brain death (BD) in the cadaveric donor that predisposes for additional transplant injury. Previously, we have shown inflammation in the liver of brain-dead donors by increased expression of cell adhesion molecules and influx of leukocytes. The key inflammatory mediator in inflammation is tumor necrosis factor (TNF)-&agr;. In addition to being involved in inflammation, TNF-&agr; also activates the potential detrimental process of apoptosis and, on the other hand, activates an antiapoptotic survival pathway by way of NF-kB. The aim of the present study was to investigate whether the inflammatory response in the liver of brain-dead donors is accompanied by changes in apoptosis and in expression of apoptosis-related proteins, in particular those regulated by NF-kB. Methods. BD was induced by inflation of an intracranially placed balloon. Apoptosis was assessed by caspase-3 enzyme activity and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay. Changes in expression of proteins involved in pathways leading to apoptosis were studied by determination of mRNA levels using semiquantitative reverse-transcriptase polymerase chain reaction followed by image analysis. TNF-receptor (TNFR), Fas, and Fas-ligand (FasL) were used as indicators for activation of the death receptor mediated pathway. Bcl-2, Bax, Bak, Bid, and A1 were used as indicators for activation of the mitochondrial pathway. Results. After 6 hours of normotensive BD, the number of apoptotic cells and caspase-3 activity were significantly increased compared with non–brain-dead control rats. TUNEL staining revealed that the apoptotic cells were primarily hepatocytes. mRNA levels of all NF-&kgr;B induced activators (Fas, bid) and inhibitors (A1, BCl-xl, cIAP2) of both apoptotic pathways were significantly increased in liver tissue of BD donors versus non-BD controls. Conclusions. The phase of BD in the donor induces increased apoptosis of hepatocytes despite an enhanced expression of NF-kB-dependent antiapoptotic genes.

Upscaling the production of microencapsulated pancreatic islets.

Abstract Presently used single-needle air-driven droplet generators are incapable of producing sufficient numbers of islet-containing droplets in a sufficiently short time-period to allow for successfully grafting alginate-poly- l -lysine encapsulated islets in large animals or humans. We have designed an air-driven multineedle droplet generator, which increases the production rate by simultaneously producing multiple droplets. Although we have tested a four-needle device, the construction is such that the number of needles, and thereby the production rate, can be readily extended. The production rate can be further extended by increasing the number of islets per millilitre alginate in the reservoir. When tested with 500 and 800 μm capsules, an increase in the number of islets per millilitre alginate was found to be associated with an increase in the number of inadequately encapsulated islets in a diameter-dependent fashion. When small instead of large capsules are produced from a given volume of alginate, larger numbers of capsules are obtained, but also a larger portion of inadequate capsules. With 10 000 islets per millimetre alginate, these combined effects can be calculated to result in a two-fold increase in the production rate of adequate capsules when 500 μm instead of 800 μm capsules are produced. Hence, substantial upscaling of the production can be achieved by combining an increase in the number of needles with a decrease in the capsule diameter.

The Efficacy of Intraperitoneal Pancreatic Islet Isografts in the Reversal of Diabetes in Rats

The peritoneal cavity is of renewed interest for pancreatic islet transplantation, since it is the preferable site for transplantation of immunoisolated islets. In this study we investigated the minimum islet graft volume needed to restore normoglycemia after free intraperitoneal isogenic transplantation in streptozotocin diabetic rats. Furthermore, graft function was tested by measuring glucose and insulin response to an intravenous glucose load and spontaneously ingested carbohydrate-rich meal. Three graft volumes were used: 8.0–10.0 (group A); 4.0–5.0 (group B); and 2.0–2.3 μl (group C); 1 μl contained about 300 islets. All 10 rats in group A and 7 out of 9 rats in group B became normoglycemic for at least 6 months posttransplant, with blood glucose levels not significantly different from normal control animals. Only 3 out of 9 animals in group C became

Rat Islet Isograft Function. Effect of Graft Volume and Transplantation Site

Islet isograft function was analyzed after transplantation of 4 well-defined endocrine volumes (12.5%, 25%, 50%, and 100% of the endocrine volume in the normal adult rat pancreas) to 3 different sites (kidney, liver, and spleen). Graft function was tested in unanesthetized, unstressed rats by the responses to glucose infusion and to a meal. All animals with grafts > or = 25% of the endocrine volume of the rat pancreas returned to normoglycemia after transplantation. The minimal graft volume for restoring normoglycemia is probably between 12.5% and 25%, since also a small number of grafts of 12.5% were successful. At 1 month, basal glucose and insulin levels were similar to those of controls in rats with grafts to the spleen, but higher in rats with grafts to the kidney or liver. Irrespective of the transplantation site, recipients had higher glucose and lower insulin levels than controls in response to glucose infusion. In response to a meal, however, only the first-phase insulin response was reduced, but the total insulin output during the entire test was similar to that of controls. Graft performance was found to be graft-size dependent. Results of tests performed at 2 months showed a tendency of increasing responsiveness compared with the results of tests at 1 month.

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 with 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 II 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. Both 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 II. 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.

Peri-insular presence of collagenase during islet isolation procedures

Reportedly, higher islet yields are obtained by ductal collagenase administration and subsequent digestion of the pancreas than by the chopped tissue collagenase digestion technique. However, the exact mechanism of islet isolation is not known. This study aims to understand the underlying mechanism of a favorable effect of ductal collagenase administration. To this end, we investigated if the higher yields can be explained by a different distribution of the collagenase enzymes in the pancreatic tissue after ductal application as compared to during chopped tissue digestion. India ink was used to mimic and visualize the distribution of collagenase in histological sections of pancreases of several species. Ink particles were seen around and even within the islets both after ductal application and during chopped tissue collagenase digestion. Thus, collagenase enzymes are not restricted to the exocrine tissue compartment with either technique. In view of this observation, we compared the efficacy of both techniques in islet isolation procedures in paired experiments in rats. Both techniques gave similar islet yields to those reportedly obtained with the ductal collagenase method. However, with either technique, the islet yield was only approximately 50% of the endocrine volume of the pancreas, indicating that a substantial loss of islet tissue had occurred. We conclude that, irrespective of the route of collagenase administration, collagenase enzymes are present in the peri-insular space during islet isolation procedures. This is pertinent in view of the finding that both methods have similar islet yields in rats and that collagenase digestion, as such, is associated with loss of islet tissue.