H. J. Hahn

Biocompatibility of mannuronic acid-rich alginates

Highly purified algin preparations free of adverse contaminants with endotoxins and other mitogens recently became available by a new purification process (Klöck et al., Appl. Microbiol. Biotechnol., 1994, 40, 638-643). An advantage of this purification protocol is that it can be applied to alginates with various ratios of mannuronic acid to guluronic acid. High mannuronic acid alginate capsules are of particular practical interest for cell transplantation and for biohybrid organs, because mannuronate-rich alginates are usually less viscous, allowing one to make gels with a higher alginate content. This will increase their stability and reduce the diffusion permeability and could therefore protect immobilized cells more efficiently against the host immune system. Here we report the biocompatibility of purified, mannuronic acid-rich alginate (68% mannuronate residues) in a series of in vitro, as well as in vivo, assays. In contrast to raw alginate extracts, the purified product showed no mitogenic activity towards murine lymphocytes in vitro. Its endotoxin content was reduced to the level of the solvent. Animal studies with these new, purified algin formulations revealed the absence of a mitogen-induced foreign body reaction, even when the purified material (after cross-linking with Ba2+ ions) is implanted into animal models with elevated macrophage activity (diabetes-prone BB/OK rat). Thus, alginate capsules with high mannuronic acid content become available for applications such as implantation. In addition to the utilization as implantable cell reactors in therapy and biotechnology, these purified algins have broad application potential as ocular fillings, tissue replacements, microencapsulated growth factors and/or interleukins or slow-release dosage forms of antibodies, surface coatings of sensors and other invasive medical devices, and in encapsulation of genetically engineered cells for gene therapy.

Anti-CD4 monoclonal antibody-induced allograft tolerance in rats despite persistence of donor-reactive T cells.

Although CD4-targeted therapy abrogates acute rejection and may induce permanent graft acceptance in rodents, little is known about the mechanisms of long-term graft survival in these models. Recently, we have shown that treatment with a nondepleting anti-CD4 monoclonal antibody (mAb) (RIB-5/2) induces long-term survival of renal, heart, and skin allografts in strong major histocompatibility complex I/II incompatible rat strains. Here, we demonstrate that the development of major histocompatibility complex-specific and tissue-nonspecific tolerance rather than graft adaptation is responsible for long-term anti-CD4 mAb-induced transplant survival. Donor-specific but not third-party heart and pancreatic islet grafts were accepted permanently without adjunctive therapy in long-term kidney allograft recipients, and infusion of naive or alloimmune splenocytes failed to break the tolerant state. Interestingly, alloreactive T cells were not depleted in these long-term survivors, as ex vivo donor-specific mixed lymphocyte reaction was largely unaffected. The reverse transcriptase-polymerase chain reaction analyses of long-term renal allografts before and after donor-specific antigen challenge revealed no changes in CD3 mRNA level, but showed up-regulation of CD25, interleukin (IL) 2, interferon (IFN) gamma, IL-4, and IL-10 mRNA in the early phase, suggesting the presence of alloreactive T cells in tolerant rats. At later time points, the expression of IFN-gamma declined rapidly, whereas IL-4 persisted, resulting in a reversal of IFN-gamma/IL-4 ratio. Our data demonstrate the stability of anti-CD4 mAb-induced tolerance despite persistence of alloreactive T cells, suggesting the role of active tolerance-maintaining mechanisms. The T helper (Th) 1/Th2 shift may be involved in this regulatory process, as anti-CD4 mAb prevents acute graft-deteriorating rejection by effectively blocking Th1 responses, and well-functioning grafts may tolerize themselves by inducing regulatory cells.

Stress Response of Pancreatic Islets from Diabetes Prone BB Rats of Different Age

Protective and/or repair mechanisms are thought to be activated in pancreatic beta cells in response to injury during insulitis. Manifestation of type-1 diabetes may depend on an imbalance between beta cell damage and repair. To prove this hypothesis, the ability of collagenase-isolated islets to respond to heat stress depending on the age of BB rats was investigated. The islets were exposed either to 44°C (HS) or 37°C (control) for 30 min and then kept at 37°C for 5 h. Immediately and 5 h after heat shock, insulin secretion in response to 20 mmol/l glucose and total protein synthesis of heat-exposed islets were significantly diminished as compared with controls. The islet proteins were separated by SDS-PAGE followed by immunoblotting. Islets from BB rats at an age of 6-90 days responded to heat shock with the expression of major heat shock protein 70 (HSP 70). Islets from 3-day old rats, however, did not respond with induction of HSP 70. In contrast we could detect inducible HSP 70 in islets from 3-day old diabetes-resistant LEW rats. In islets from 90-day old BB rats we observed a decreased amount of HSP 70 compared with islets from 9-, 12-, 30- and 60-day old animals. There was also a higher extent of HSP 70 to observe in islets from 90-day old LEW rats as compared with 90-day old BB rats. Differences in HSP 70 expression between islets of 3-day old BB and LEW rats and other age groups of BB rats might represent distinct stages of maturation of islets whereas diminished expression of HSP 70 in islets of 90-day old BB rats at the age of high probability of developing diabetes might result from reduced ability to induce protective mechanisms.

Lack of disease recurrence in diabetic BB/PFD rats after syngeneic islet transplantation

Restimulation of autoreactivity in two different BB rat sublines of the same origin (Ottawa, Canada) was investigated by syngeneic islet transplantation into diabetic animals. Despite identical methods and conditions recurrence of hyperglycaemia was observed in BB/OK rats (Karlsburg, Germany) but not in BB/Pfd rats (Leuven, Belgium). Pancreatic morphology at the time of transplantation revealed significant differences in islet volume density and the degree of insulitis. Additionally, marked differences in the phenotypical composition of cells infiltrating the islets were observed. A loss of autoimmune memory in BB/Pfd rats is discussed as a probable reason for the lack of disease recurrence in those animals.

Prevention of autoimmune but not allogeneic destruction of grafted islets by different therapeutic strategies

Grafting autoimmune-diabetic recipients with allogeneic islets, graft rejection and disease recurrence as major problems of reaching indefinite survival and tolerance induction have to be solved. Anti-CD25 and anti-CD4 monoclonal antibodies were successfully used after allogeneic islet transplantation in experimentally diabetic rats. A temporary anti-CD25 therapy also prevented disease recurrence in autoimmune-diabetic BB rats, while this was not yet reported for an anti-CD4 treatment. In autoimmune-diabetic NOD mice disease recurrence can be successfully treated using an anti-CD4 monoclonal antibody. We, therefore, compared the efficacy of a short-term anti-CD25 and anti-CD4 treatment regarding the prevention of allograft rejection and disease recurrence in autoimmune-diabetic BB/OK rats. Both monoclonal antibodies were combined with low doses of Cyclosporin A. Untreated BB/OK rats relapsed into hyperglycaemia within 3 weeks independent of the islet donor, LEW.1A, LEW.1BB/OK or BB/OK rats. However, after grafting MHC-identical allogeneic (LEW.1BB/OK) or syngeneic (BB/OK) islets we observed about 30% spontaneous acceptance. Both the anti-CD25 and anti-CD4 therapy significantly prolonged the survival of allogeneic grafted islets. After MHC-identical allogeneic and syngeneic islet transplantation the temporary immunotherapy increased the proportion of permanent acceptors to 63% and 75%, respectively. The efficacy of both treatment strategies in prolonging allograft survival and prevention of disease recurrence was identical. In summary, anti-CD25 as well as anti-CD4 therapy prevented autoimmune but not allogeneic islet destruction in autoimmune-diabetic BB/OK rats. In conclusion, targeting different immune cells by monoclonal antibodies with different specificities can lead to very similar results with respect to an interruption of allograft rejection and autoimmune reaction.

Endocrine pancreas histology of congenic BB-rat strains with reduced diabetes incidence after genetic manipulation on chromosomes 4, 6 and X.

Congenic BB.SHR rat strains were established by crossing of spontaneously diabetic BB/OK rats and diabetes-resistant SHR rats. Chromosomal regions on which the genes Iddm 4 (BB.6s), Iddm6 (BB.Xs) and Iddm 2 (BB.LL) are located were exchanged. As a result of genetic manipulation diabetes incidence was markedly reduced from 80% in BB/OK to 50% in BB.SHR (Chr. X), to 14% in BB.SHR (Chr. 6) and to 0% in BB.LL rats. Pancreata of these newly generated BB.SHR rats were investigated histologically. In newly diagnosed diabetic rats of congenic strains pancreatic insulin content (BB.6s: p<0.05; BB.Xs p<0.01) and relative volume of insulin-positive cells (BB.Xs: p<0.001) were significantly higher than in BB/OK rats. The degree of insulitis was not different in 90-day-old and newly diagnosed diabetic animals. Surprisingly, in 30-day-old rats we observed an increase of the degree of insulitis with decreasing diabetes incidence. We suppose that by an earlier occurrence of the immunological β-cell destruction, a part of the animals is able to develop a secondary diabetes resistance. The exchange of the BB-lymphopenia gene by that of SHR-rats prevented the development of hyperglycaemia without altering the auto-reactive immune response, which could be observed in all animals investigated.

The Effect of Human Amniotic Fluid on DNA Synthesis of Rat Pancreatic Islets in Tissue Culture

In this study we investigated the effect of human amniotic fluid (HAF) on the incorporation of 3H-thymidine into cultured rat islets. The addition of 1% HAF (1% HAF, 99% TCM) did not alter the incorporation of labeled thymidine, in comparison to TCM 199 alone (100%). The culture in the presence of 50% HAF (1:1 diluted with TCM, v/v) resulted in a significantly increased DNA synthesis. To exclude any effect of ionic composition or substrate dilution by the HAF, the TCM 199 was also either diluted (1:1) with saline or Hanks balanced salt solution (HBSS). Islets cultured under these conditions did not show any stimulated thymidine incorporation into islet DNA. From these results we conclude that HAF contains an activity which stimulates islet replication. In order to concentrate this stimulatory activity, HAF was tested after it had been lyophilized or evaporated. Independent of the method used, in no case a stimulatory effect of concentrated HAF was found. In contrast, the concentrated form of HAF inhibited the incorporation of labeled thymidine into islet DNA.

In Vitro Stimulation by Islet Antigen Facilitates the Detectability of Beta-cell Reactive Cells in Diabetes-prone BB/OK Rats

It has been supposed that beta-cell destruction in man and animals is due to autoreactive T-cells. We used the [51Cr]-release assay to identify the presence of beta-cell reactive cells in the spleen of diabetes-prone BB/OK rats before and after diabetes manifestation as well as in long-term normoglycaemic rats with a reduced diabetes risk of 3%. Splenic mononuclear cells (MNCs) obtained from diabetes-resistant LEW.1W and the majority of long-term normoglycaemic BB/OK rats (86.4%) showed no reactivity to pancreatic islets in vitro. In contrast, beta-cell reactive cells were identified in dependence on age in 30.4-65.0% of 75-120 days old normoglycaemic rats and in relation to diabetes duration (1 and 20 days) in 75.0% and 16.0% of diabetic BB/OK rats. Islet antigen-specific stimulation of splenic MNCs, that showed no spontaneous islet-directed reactivity, resulted in a concentration-dependent activation of cytolytically reactive cells in BB/OK but not in LEW.1W rats. Splenic MNCs derived from all diabetic, from 82.4% of young normoglycaemic and from 46.2% of long-term normoglycaemic BB/OK rats developed an islet-directed reactivity in vitro. Phenotyping of MNCs showed a significant increase of activated IL2R+ T-lymphocytes in diabetic BB/OK rats, but without any correlation to their cytolytic potential in the [51Cr]-release assay. Despite this fact, IL2R+ cells enriched from the pool of MNCs mediated an enhanced [51Cr]-release from islets, indicating their relevance in the beta-cell destruction. These data suggest, that functional reactivity rather than phenotypic characterization of MNCs is useful to identify the existence of beta-cell reactive cells. Furthermore, for screening diabetes risk in young normoglycaemic BB/OK rats besides the detection of beta-cell reactive cells the occurrence of regulatory cells seems to be decisive.

Überleben von Inselallotransplantaten nach temporärer Empfängerbehandlung mit Anti-IL-2-Rezeptor Antikörper

Bisherige immunsuppressive Therapieregime, die insbesondere bei Allotransplantationen zur Anwendung kommen, basieren auf einer unspezifischen Unterdruk kung der Immunantwort. So sind Nebenwirkungen und Komplikationen nach Gabe konventioneller Immunsuppressiva nicht zu vermeiden. Kurzlich wurde bei Ratten [1, 2, 3] und Mausen [4] ein verlangertes Allotransplantatuberleben be obachtet, wenn die Empfanger mit einem monoklonalen Antikorper gegen IL-2-Rezeptor (Anti-IL-2R mAK) behandelt wurden. Die zusatzliche Gabe von Cyclosporin A (CsA) in subtherapeutischen Dosen verbessert die Resultate weiter [1, 3]. Der Anti-IL-2R mAK reagiert mit aktivierten T-Lymphozyten, die in der Phase der Aktivierung den IL-2R auf ihrer Oberflache exprimieren. Damit wird spezifisch die klonale Expansion der aktivierten T-Lymphozyten gehemmt, die im Prozes der Transplantatabstosung eine zentrale Rolle spielen. Ein weiterer Vorteil dieser Behandlungsform ist durch die temporare Anwendung gegeben.