P. de Vos

Considerations for successful transplantation of encapsulated pancreatic islets

Encapsulation of pancreatic islets allows for transplantion in the absence of immunosuppression. The technology is based on the principle that transplanted tissue is protected for the host immune system by an artificial membrane. Encapsulation offers a solution to the shortage of donors in clinical islet transplantation because it allows animal islets or insulin-producing cells engineered from stem cells to be used. During the past two decades three major approaches to encapsulation have been studied. These include intravascular macrocapsules, which are anastomosed to the vascular system as AV shunt; extravascular macrocapsules, which are mostly diffusion chambers transplanted at different sites; and extravascular microcapsules transplanted in the peritoneal cavity. The advantages and pitfalls of these three approaches are discussed and compared in the light of their applicability to clinical islet transplantation. All systems have been shown to be successful in preclinical studies but not all approaches meet the technical or physiological requirements for application in human beings. The extravascular approach has advantages over the intravascular because since it is associated with less complications such as thrombosis and infection. Microcapsules, due to their spatial characteristics, have a better diffusion capacity than macrocapsules. Recent progress in biocompatibility of microcapsules has brought this technology close to clinical application. Critical issues such as limitations in the functional performance and survival are being discussed. The latest results show that both issues can be solved by the transplantation of microencapsulated islets close to blood vessels in prevascularized solid supports. [Diabetologia (2002) 45: 159–173]

Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets

Summary Graft failure of alginate-polylysine microencapsulated islets is often interpreted as the consequence of a non-specific foreign body reaction against the microcapsules, initiated by impurities present in crude alginate. The aim of the present study was to investigate if purification of the alginate improves the biocompatibility of alginate-polylysine microcapsules. Alginate was purified by filtration, extraction and precipitation. Microcapsules prepared from crude or purified alginate were implanted in the peritoneal cavity of normoglycaemic AO-rats and retrieved at 1, 2, 3, 6, 9, and 12 months after implantation. With crude alginate, all capsules were overgrown within 1 month after implantation. With purified alginate, however, the portion of capsules overgrown was usually less than 10 %, even at 12 months after implantation. All recipients of islet allografts in capsules prepared of purified alginate became normoglycaemic within 5 days after implantation, but hyperglycaemia reoccurred after 6 to 20 weeks. With intravenous and oral glucose tolerance test, all recipients had impaired glucose tolerance and insulin responses were virtually absent. After graft failure, capsules were retrieved (80–100 %) by peritoneal lavage. Histologically, the percentage of overgrown capsules was usually less than 10 % and maximally 31 %. This small portion cannot explain the occurrence of graft failure. The immunoprotective properties of the capsules were confirmed by similar if not identical survival times of encapsulated islet allo- and isografts. Our results show that purification of the alginate improves the biocompatibility of alginate-polylysine microcapsules. Nevertheless, graft survival was still limited, most probably as a consequence of a lack of blood supply to the encapsulated islets. [Diabetologia (1997) 40: 262–270]

Factors influencing the properties and performance of microcapsules for immunoprotection of pancreatic islets

There are several approaches of immunoprotection of pancreatic islets for the purpose of successful allo- or xenotransplantation in the absence of immunosuppressive medication. Extravasculair approaches are either macroencapsulation (large numbers of islets together in one device) or microencapsulation. The latter approach is to envelop each individual islet in a semipermeable immunoprotective capsule. Quite promising results have been achieved with polylysine-alginate microencapsulated islet grafts in rodents, but clinical application is still restricted to a very small number of cases. Relevant considerations regard the following aspects. The biocompatibility of the microcapsules is influenced by the chemical composition of the materials applied and by mechanical factors related to the production process. With purified instead of crude alginates, the percentage of capsules with fibrotic overgrowth is reduced to approximately ten percent, and the remaining overgrowth is mainly explained by mechanical factors, i.e. inadequate encapsulation of individual islets. Even with purified alginates, however, the duration of encapsulated graft function is limited to a period of six to twenty weeks. Obviously, other factors than bioincompatibility play a role, which factors have to be identified. The limited duration of graft survival cannot be explained by rejection since, in rats, survival times of encapsulated isografts are similar, if not identical, to those of encapsulated allografts. An important factor is probably insufficient nutrition as a consequence of insufficient blood supply of the encapsulated and thus isolated islet. This also influences the functional performance of encapsulated islet grafts. Although normoglycemia can be readily obtained in streptozotocin diabetic rat recipients, glucose tolerance remains severely impaired, as a consequence of an insufficient increase of insulin levels in response to intravenous or oral glucose challenge. Important factors are the characteristics of the capsules applied in view of optimal diffusion kinetics, and the fact that an encapsulated islet graft can only be implanted in the peritoneal cavity because of its volume. Further studies should focus on finding a practically applicable method to reduce the barrier between encapsulated islets and the bloodstream, in order to improve both the functional performance and the survival of encapsulated islet grafts.There are several approaches of immunoprotection of pancreatic islets for the purpose of successful allo- or xenotransplantation in the absence of immunosuppressive medication. Extravasculair approaches are either macroencapsulation (large numbers of islets together in one device) or microencapsulation. The latter approach is to envelop each individual islet in a semipermeable immunoprotective capsule. Quite promising results have been achieved with polylysine-alginate microencapsulated islet grafts in rodents, but clinical application is still restricted to a very small number of cases. Relevant considerations regard the following aspects. The biocompatibility of the microcapsules is influenced by the chemical composition of the materials applied and by mechanical factors related to the production process. With purified instead of crude alginates, the percentage of capsules with fibrotic overgrowth is reduced to approximately ten percent, and the remaining overgrowth is mainly explained by mechanical factors, i.e. inadequate encapsulation of individual islets. Even with purified alginates, however, the duration of encapsulated graft function is limited to a period of six to twenty weeks. Obviously, other factors than bioincompatibility play a role, which factors have to be identified. The limited duration of graft survival cannot be explained by rejection since, in rats, survival times of encapsulated isografts are similar, if not identical, to those of encapsulated allografts. An important factor is probably insufficient nutrition as a consequence of insufficient blood supply of the encapsulated and thus isolated islet. This also influences the functional performance of encapsulated islet grafts. Although normoglycemia can be readily obtained in streptozotocin diabetic rat recipients, glucose tolerance remains severely impaired, as a consequence of an insufficient increase of insulin levels in response to intravenous or oral glucose challenge. Important factors are the characteristics of the capsules applied in view of optimal diffusion kinetics, and the fact that an encapsulated islet graft can only be implanted in the peritoneal cavity because of its volume. Further studies should focus on finding a practically applicable method to reduce the barrier between encapsulated islets and the bloodstream, in order to improve both the functional performance and the survival of encapsulated islet grafts.

Induction of organ dysfunction and up-regulation of inflammatory markers in the liver and kidneys of hypotensive brain dead rats: a model to study marginal organ donors.

BACKGROUND Marginal donors exposed to the full array of effects induced by brain death are characterized by low success rates after transplantation. This study examined whether organs from marginal brain dead animals show any change in organ function or tissue activation making them eventually more susceptible for additional damage during preservation and transplantation. METHODS To study this hypothesis we first focused on effects of brain death on donor organ quality by using a brain death model in the rat. After induction of brain death, Wistar rats were ventilated for 1 and 6 hr and then killed. Sham-operated rats served as controls. Organ function was studied using standard serum parameters. Tissue activation of liver and kidney was assessed by staining of immediate early gene products (IEG: FOS, JUN), and inflammatory markers; cell adhesion molecules (Intercellular adhesion molecule-1, vascular cell adhesion molecule-1), leukocyte infiltrates (CD45, T cell receptor, CD8, CD4), and MHC class II. RESULTS During brain death progressive organ dysfunction was observed that coincided with a significant increase in activation of immediate early genes, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD45, and MHC class II versus nonbrain dead controls. In liver tissue also the markers for T cell receptor and CD8 significantly increased. CONCLUSIONS These findings suggest that an immune activation with increased endothelial cell activation and immediate early gene expression occurs in marginal donors after brain death induction. We suggest that brain death should not longer be regarded as a given nondeleterious condition but as a dynamic process with potential detrimental effects on donor organs that could predispose grafts for increased alloreactivity after transplantation.

Association between macrophage activation and function of micro-encapsulated rat islets

Aims/hypothesisSurvival of microencapsulated islet grafts is limited, even when inflammatory reactions against the capsules are restricted to a small portion of less than 10%.MethodsThis study investigates both in vivo in rat recipients and in vitro whether cellular overgrowth on this minority of the capsules contributes to limitations in the functional survival of the 90% of the encapsulated islets which remain free of any cellular overgrowth.ResultsIn successful rat recipients of an allogenic microencapsulated islet graft we found that the vast majority of cells in the capsular overgrowth were activated ED-1 and ED-2 positive macrophages which were found in numbers of approximately 1500 per capsule. Co-culture of encapsulated islets with 1500 (nr8383) rat-macrophages per capsule showed that the activation of macrophages was caused by islet-derived bioactive factors since TNF-α and IL-1β secretion by macrophages was induced by islet-containing capsules and not by empty capsules. This activation of macrophages was associated with a decrease in function of the encapsulated islets as evidenced by a quantitatively reduced (35%) insulin response in static incubation and a slower response in perifusion.Conclusion/interpretationPresent research aims to design strategies for the temporary inhibition of macrophage activation since macrophages are predominantly present in the first two months after implantation. These strategies will serve as a pertinent basis for future clinical application of microencapsulated islets.

Factors influencing isolation of functional pancreatic rat islets.

Yields and function of isolated islets vary considerably in spite of the introduction of new or improved methods for isolation. In most studies, these variations have been attributed to inadequacies of the applied collagenase preparations. However, when we retrospectively analyzed our rat islet isolations, we found large variations in yield and function in spite of application of identical collagenase sources. Therefore, in the present study, we determined the effect of rat donor strain, the source of inhibition of proteolytic activity (by bovine serum albumin), and the culture conditions on yield and function. AO rats showed a twofold higher islet yield than Wistar and Lewis rats. However, a higher yield was not associated with a higher response on glucose load since this response was more pronounced with Lewis islets than with Wistar and AO islets. Rats with a higher weight donate more islets but have a lower insulin secretory capacity. Islet yield and function also vary with application of different sources of bovine serum albumin during digestion. Moreover, the culture conditions influence the functional survival of isolated rat islets. CMRL1066 preserves the insulin secretory capacity of rat islets better than RPMI1640. Finally, the number of islets surviving the culture is higher when 4 instead of 12 and 24 islets were applied per square centimeter. Our observations indicate that strain and weight of the rat donor, the source of bovine serum albumin, and the culture conditions of islets are pertinent factors in efficacious isolation of islets.

Susceptibility of human pancreatic β cells for cytomegalovirus infection and the effects on cellular immunogenicity.

Objectives Human cytomegalovirus (HCMV) infection has been suggested to be a causal factor in the development of type 1 diabetes, posttransplantation diabetes, and the failure of islet allografts. This effect of CMV has been interpreted as an indirect effect on the immune system rather than direct infection–induced cell death. In the present study, we investigated (i) the susceptibility of &bgr; cells to HCMV infection, (ii) regulation of immune cell–activating ligands, (iii) release of proinflammatory cytokines, and (iv) the effects on peripheral blood mononuclear cell (PBMC) activation. Methods CM insulinoma cells and primary &bgr; cells were HCMV-infected in vitro using a laboratory and a clinical HCMV strain. The susceptibility to infection was measured by the expression of viral genes and proteins. Furthermore, expression levels of Major Histocompatibility Complex I, Intracellular Adhesion Molecule-1, and Lymphocyte Function Associated Antigen-3 and the release of proinflammatory cytokines were determined. In addition, PBMC activation to HCMV-infected &bgr; cells was determined. Results &bgr; Cells were susceptible to HCMV infection. Moreover, the infection increased the cellular immunogenicity, as demonstrated by an increased MHC I and ICAM-1 expression and an increased proinflammatory cytokine release. Human cytomegalovirus–infected CM cells potently activated PBMCs. The infection-induced effects were dependent on both viral “sensing” and viral replication. Conclusions In vivo &bgr;-cell HCMV infection and infection-enhanced cellular immunogenicity may have important consequences for native or transplanted &bgr;-cell survival.

Induction of organ dysfunction and activation of inflammatory markers in donor liver and kidney during hypotensive brain death

ESULTS after transplantation with kidneys received from living-related or living-unrelated donors are far superior to the outcome after cadaveric organ transplantation. 1 This fact cannot be explained only by HLA matching or shorter cold ischemia times and might be due to the state of brain death (BD) in the donor. Although BD is still considered a static and given condition, it is known to have definite effects on hemodynamic stability, hormone regulation, and inflammatory reactivity. BD may initiate a cascade of events that predisposes for additional injury during preservation, reperfusion, and after transplantation. The latter may be enhanced in marginal cadaveric donors which are subjected to BD and to proven detrimental effects of hypotension. We have developed a rat BD model, mimicking the marginal donor situation in humans. In this study, we used this model to investigate the combined effects of BD and hypotension on endothelial cell expression of cell adhesion molecules (ICAM-1, VCAM-1). Also, we studied the presence of leukocyte infiltrates (CD45, TcR, CD8, CD44) and the expression of MHC class II in kidney and liver after BD induction.

LPS Promotes Pre-osteoclast Activity by Up-regulating CXCR4 via TLR-4

Lipopolysaccharide (LPS) has been shown to be a prominent pathogenic factor in inflammatory bone loss. However, knowledge of the mechanisms involved is limited. The role of the SDF-1/CXCR4 (Stromal-derived factor-1 and its unique chemokine receptor) axis in LPS-induced bone loss has not been studied. The aim of this study was to investigate the role of the SDF-1/CXCR4 axis in LPS-stimulated inflammatory bone loss. The results show that LPS does not influence the expression of SDF-1/CXCR4 in osteoblasts, but up-regulates the expression of CXCR4 in pre-osteoclasts via Toll-like receptor 4, which subsequently enhances pre-osteoclast migration. Moreover, LPS promoted RANKL-induced osteoclast differentiation partially through CXCR4 up-regulation. In conclusion, the present study demonstrated, for the first time, that the up-regulated expression of CXCR4 in pre-osteoclasts by LPS stimulation is involved in LPS-induced bone resorption.

Effects of acute cytomegalovirus infection on rat islet allograft survival.

Transplantation of pancreatic islets is a promising therapy for the treatment of type 1 diabetes mellitus. However, long-term islet graft survival rates are still unsatisfactory low. In this study we investigated the role of cytomegalovirus (CMV) in islet allograft failure. STZ-diabetic rats received an allogenic islet graft in combination with either an acute CMV infection or control infection. A third group received ganciclovir treatment in addition to the CMV infection. Graft function was assessed by measuring basal blood glucose levels. After sacrifice, the islet grafts were retrieved for analysis of infection and leukocyte infiltration. CMV-infected recipients demonstrated accelerated islet graft failure compared to noninfected controls. CMV infection of the graft was only observed prior to complete graft failure. Quantification of the leukocyte infiltration demonstrated increased CD8+ T-cell and NK cell infiltration in the CMV-infected grafts compared to the controls. This suggests that CMV infection accelerates immune-mediated graft destruction. Antiviral ganciclovir treatment did not prevent accelerated graft failure, despite effectively decreasing the grade of infection. Our data confirm the recently published CITR data, which state that CMV is an independent risk factor for failure of islet grafts. Also, our data demonstrate that new approaches for preventing virus-induced islet allograft failure may be required.