M. Monden

Protection of islet allografts transplanted together with Fas ligand expressing testicular allografts

Summary Fas ligand (FasL) is highly expressed in testicular tissues and thought to be responsible for protection from allograft rejection by inducing apoptosis of anti-graft activated T cells. FasL-expressing islets have been shown to induce a granulocyte-mediated inflammatory reaction. We investigated whether a graft can be protected from alloimmune responses by manipulating the Fas/FasL-system. We transplanted allogeneic islets under the kidney capsule of streptozotocin-induced diabetic mice together with testicular tissue. Significant prolongation of survival of C3H islet allograft was observed in C57BL/6 (B6) recipients transplanted with C3H testicular tissue, but not in those transplanted with C3H-gld testicular tissue expressing non-functional FasL. No significant prolongation was observed in B6-lpr recipients expressing non-functional Fas. Immunohistochemical staining of C3H testicular tissue in the composite graft showed a high expression of FasL, but not that of the C3H-gld testicular tissue. In situ terminal deoxynucleotidyl transferase-mediated dUDP-biotin catalysed DNA nick-end labelling (TUNEL) staining of a composite graft of C3H islet and testicular tissue in B6 recipients demonstrated extensive apoptosis of infiltrating mononuclear cells around the graft. The protective effect of C3H testicular tissue was abrogated when anti-FasL monoclonal antibody was administered i. p. postoperatively. Our results suggest that FasL-positive testicular allografts protect composite islet allografts and indicate that manipulation of Fas/FasL mediated apoptosis is a suitable strategy for controlling rejection of islet allografts. [Diabetologia (1998) 41: 315–321]

Low temperature collagenase digestion for islet isolation from 48-hour cold-preserved rat pancreas

We examined the efficacy of relatively low temperature collagenase digestion at 20 degrees C on the yield and viability of islets after long-term cold preservation. Wistar rat pancreases were distended with University of Wisconsin solution via a pancreatic duct at the time of harvesting to which collagenase and 2.5 mM calcium chloride were added. The pancreases were cold-preserved at 4 degrees C for 24 or 48 hr. After storage, they were incubated for collagenase digestion at 37 degrees C or 20 degrees C for various incubation periods to obtain the peak yield. At 20 degrees C, in vitro collagenase activity measured by the FALGPA method was one fourth of that at 37 degrees C, and pancreases were well digested with a prolonged digestion period (60-90 min vs. 15-20 min for the 37 degrees C group). In vitro insulin secretion of islets isolated from freshly removed pancreases was maintained at 20 degrees C for 120 min in University of Wisconsin solution as compared with 30 min at 37 degrees C. Therefore, the preserved pancreases used in this study were incubated either at 37 degrees C or 20 degrees C at various times in order to obtain peak islet yields. The islet yields from 24-hr cold-preserved pancreases at 37 degrees C and 20 degrees C digestion were 573 +/- 59/rat (n = 6) and 497 +/- 84/rat (n = 11), respectively, and those from 48-hr cold-preserved pancreases were 395 +/- 113/rat (n = 6) and 414 +/- 75/rat (n = 6), respectively. The yields from 24- and 48-hr cold-preserved pancreases were significantly low compared with 635 +/- 52/rat for fresh pancreases (n = 15), but there was no significant difference between the 2 methods. The viability of the isolated islets, which was examined by transplantation to streptozotocin-induced diabetic C57BL/6 mice, showed a significant difference in the capacity to ameliorate diabetes. The functional success rate of islet transplantation after 24-hr cold preservation was equally good (8/8 for 37 degrees C group vs. 9/10 for 20 degrees C group), but the rate for those from 48-hr cold-preserved pancreases was significantly better with digestion at 20 degrees C than at 37 degrees C (1/8 for 37 degrees C group vs. 7/8 for 20 degrees C group, P < 0.05). We concluded that viable islets can be isolated from 48-hr cold-preserved pancreases with the low temperature collagenase digestion method, which shows promise as a modality for successful clinical islet transplantation.

Role of micro-chimerism in inducing immunological tolerance by intraportal injection of donor spleen cells in rats

Recently, we reported that intraportal (IP) injection of donor spleen cells (SPCs) prevented liver allograft rejection. Moreover, we developed a new method using polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis, and demonstrated micro-chimerism (MC) at the DNA level in the spleen 14 days after IP injection. In the present study, the long-term presence of injected allogeneic SPCs was investigated at the cellular level by immunofluorescence staining as well as the DNA level using RFLP analysis. Male ACI (RTla) rats were used as the donors and Lewis (RT11) rats as the recipients. After DNA preparation from the lymphoid organs, RT1Bβ domain 1 region was amplified by PCR, and RFLP analysis was performed with PVMII restriction enzyme. In the immunofluorescence staining, the monoclonal antibody, MN4-91-6, was used to detect the injected donor ACI SPCs in a frozen specimen. We did not detect MC in Lewis rats intravenously injected with 5 × 107 ACI SPCs on day 14. On the other hand, stable chimerism in the spleen was observed in intraportally injected rats up to 28 days after injection at not only the DNA level but also the cellular level. No chimerism was detected in other organs (including the thymus, lymph nodes, and liver). In conclusion, the long-term presence of injected allogeneic SPCs in the spleen was demonstrated after IP injection but not after IV injection, and this phenomenon may be one of the mechanisms involved in portalvenous immunosuppression.

Microchimerism and hyporesponsiveness induced by intraportal injection of donor spleen cells in rats

It is controversial whether or not microchimerism (MC) is responsible for the induction and maintenance of donor-specific tolerance. We have shown that intraportal injection (i.p.) of donor splenocytes induces a long-term graft survival of liver and heart in rats. In this study, we examined by polymerase chain reaction (PCR) the status of MC in the liver, spleen, and blood of rat cardiac recipients following i.p. or intravenous injection (i.v.) of donor splenocytes. Male DA (RT1a) and Wistar (RT1k) rats were used as donors and recipients, respectively. Heterotopic heart transplantation was performed 10 days after i.p. or i.v. injection of 5 x 10(7) DA spleen cells. DA cardiac allografts were rejected with a mean survival time (MST) of 11.9 +/- 1.6 (n = 10) days in nontreated recipients. Injections (i.v.) led to no significant prolongation of graft survival (MST: 11.2 +/- 1.9 days, n = 6), while i.p. or i.v. injection alone resulted in significant MC in these organs throughout the observation time over 60 days. MC was detected in the spleen, liver, and blood of cardiac recipients 7 days after transplantation and also even after cessation of cardiac heartbeat 21 days after transplantation. This was the case with either i.p. or i.v. group, which showed MC on day 7 after transplantation and persistent MC after cessation of the heartbeat. These data suggests that the presence of MC in the liver, spleen and blood of transplant recipients may not be responsible for immunological unresponsiveness to donor antigens.

A protocol with FK 506 for inducing unresponsiveness to murine islet allografts

BACKGROUNDnThe most favorable protocol for transplantation is inducing unresponsiveness before operation by means of nondangerous modalities. This would permit discontinuance of long-term use of immunosuppressants. In this study we developed a potential protocol for inducing unresponsiveness to islet allografts by preoperative donor spleen cell inoculation (DSI) and a single injection of FK 506.nnnMETHODSnBALB/c (H-2d) and C57BL/6 (H-2b) mice were used as islet donors and recipients, respectively. The streptozocin-induced diabetic mice that had been given DSI at a dose of 1 x 10(7) or 1 x 10(4) and a single injection of FK 506 (10 mg/kg intramuscularly) at different schedules (on day 1, 3, 5, or 7 relative to DSI on day 0) were subjected to islet allografting on day 10.nnnRESULTSnAll islet recipients returned to normoglycemia within a few days with no toxic effect of FK 506 treatment. DSI at a dose of 1 x 10(7) alone led to shortening of the mean survival time to 10.1 +/- 4.1 days, as compared with 13.5 +/- 6.3 days for the untreated animals. In contrast, marked prolongation of graft survival was induced when FK 506 was given on day 3 (> 84 +/- 27.3 days, p < 0.0001) or on day 5 after DSI (> 50.9 +/- 46.0 days, p < 0.05). Five of seven allografts given FK 506 on day 3 and three of seven allografts given FK 506 on day 5 survived indefinitely. Other time schedules of DSI and FK 506 treatment (on day 1 or 7 after DSI) or FK 506 treatment alone had no significant effect on mean survival time. With the same protocol, third-party islet allografts (C3H) were immediately rejected (10.6 +/- 2.6 days).nnnCONCLUSIONSnProlongation of islet allograft survival was induced by certain doses of DSI and preoperative FK 506 treatment. This modality prevents an adverse effect of FK 506 on grafted islets and can induce unresponsiveness to islet allografts, offering a protocol for successful clinical islet transplantation.

Induction of Allograft Tolerance

SummaryOrgan transplantation can be one of the therapeutic modalities for various terminal diseases. However, the morbidity and mortality associated with this procedure are still major problems. Discontinuation of immunosuppression or induction of allograft tolerance is the ideal goal in organ transplantation. In fact, there are reports of patients who have been able to stop all conventional immunosuppression and remain clinically stable and drug free over long periods. The mechanism underlying this phenomenon has not yet been clarified.Recent advances in molecular biology shed light on the molecular mechanisms involved in allograft rejection. Depletion of passenger leucocytes and the reduction of immunogenicity of the allografted tissue lead to significant prolongation of graft survival. Some of the new immunosuppressive drugs have the potential for inducing unresponsiveness when combined with appropriate antigen presentation. Adhesion molecules participating in the interactions of lymphocytes and endothelial cells, and of antigen-presenting cells and T cells, have been identified, and monoclonal antibodies against each molecule have been shown to block full T cell activation, leading to antigen-specific unresponsiveness in certain situations. Some of these modalities are applicable to a large animal model, which could lead to an end to immunosuppressive treatment in clinical organ transplantation.

Prolongation of cardiac allograft survival by intraportal injection of donor antigens—Differential mechanisms according to the timing of injection

Recently, we reported that intraportal (IP) injection of donor spleen cells (SPCs) before transplantation as well as at the time of transplantation significantly prolongs cardiac allograft survival in rats (7). Long-term establishment of chimerism induced by intraportal administration of SPCs could be a part of this prolongation. In this study, we examined the effect of irradiation of SPCs as a source of donor antigens on cardiac allograft survival. Experiments were carried out using DA (RT1a) as the donor and BUF (RT1b) as the recipient strain. Fifty million irradiated or nonirradiated SPCs were injected either intravenously (i.v.) or intraportally (i.p.) on day -10 or day 0, the day of cardiac allografting. Untreated animals rejected allografts within a mean survival time (MST) of 7.2 +/- 0.8 days (n = 5). Injection (i.p.) of SPCs on both day -10 (n = 4) and day 0 (n = 6) induced significant prolongation of MST over the control (19.0 +/- 4.7, 14.2 +/- 2.1 days; p < 0.001 vs. control), while i.v. injection of SPCs on either day -10 (n = 4) or day 0 (n = 4) failed to do so (9.2 +/- 1.0, 8.5 +/- 0.6 days). Significant prolongation was still observed when irradiated SPCs were injected on day -10 (n = 4; MST: 19.0 +/- 5.4 days, p < 0.002 vs. control), but not when injected on day 0 (n = 5; MST: 9.4 +/- 2.1 days). These data suggest that the immunosuppressive effect of i.p. injection of donor SPCs could be induced by differential mechanisms according to the timing of inoculation of donor antigens.