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K Dono

Publications and source records attributed to K Dono.

72 records · Page 4Linked to original sources
PubMed1991

Improvement in islet yield from a cold-preserved pancreas by pancreatic ductal collagenase distention at the time of harvesting.

This study tried to improve the number of viable islets isolated from a pancreas because a sufficient number cannot be obtained when the organ is preserved in the manner used for pancreas transplantation. The mechanism involved in the decrease in islet yield during preservation was studied to try to develop a better method for islet preparation. First, the integrity of the ductal system was compared between fresh and 6-hr simply preserved (in Hanks' balanced salt solution) rat pancreases. The ductal pressure after ductal injection of HBSS reached a plateau earlier and was significantly lower for the preserved pancreases (0.073 +/- 0.026 min, 410 +/- 17 mmHg, n = 5) than for the fresh ones (0.176 +/- 0.086 min, 561 +/- 103 mmHg, n = 7, P less than 0.05). Second, the extent of pancreatic distention was examined following ductal injection of barium gelatin solution. Solution leakage occurred earlier and distention was less in the preserved pancreas. In addition, the gelatin was found in the capillaries within some islets of the preserved pancreas. These results indicated that the preservation led to a rapid loss of integrity of the ductal system before collagenase injection. We therefore tested the efficacy of ductal collagenase injection at the time of harvesting: 15 ml of 1.0 mg/ml collagenase HBSS was intraductally injected and the pancreas was preserved at 4 degrees C for 2, 4, 6, and 24 hr. The isolation procedure was similar to that used for the fresh pancreas. The yield was significantly better than that of the simply preserved pancreas at 4 hr (241 +/- 22, n = 3, vs. 140 +/- 58, n = 3, P less than 0.05) and at 6 hr (171 +/- 58, n = 14, vs. 32 +/- 33, n = 6, P less than 0.01). These isolated islets were spherical-oval and their viability was confirmed by the ability to reverse STZ-induced diabetes in mice. These results indicated that the integrity of the ductal system, which is necessary for distention of the whole pancreas, was lost during preservation. To solve this problem, ductal collagenase injection should be done at the time of pancreas harvesting and then followed by simple preservation. This method is recommended to obtain viable islets from a preserved pancreas.

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PubMed1990

Successful islet isolation from preserved rat pancreas following pancreatic ductal collagenase at the time of harvesting.

In an attempt to develop an improved method for preserving the pancreas prior to islet isolation, the effects of warm and cold ischemia were examined on rat pancreases, from which reproducible high yields of islets can be obtained when fresh. Both warm and cold preservation rapidly decreased islet yield. Use of Hanks' or modified Sacks' solution also led to marked decrease in islet yield. After 6 hrs of preservation, the islet yield was 1/5-1/10 of those of fresh pancreases (374 +/- 74, n = 14), and no islets were obtained after 24 hrs of preservation regardless of the preservation solution. Monitoring of ductal pressure during forced injection of Hanks' solution in 6 hrs-preserved pancreas with HBSS showed a significantly earlier and lower peak of pressure than those of fresh pancreases. On the other hand, simple hypothermic preservation after pancreatic ductal distention with collagenase Hanks' solution at the time of harvesting resulted in a significantly higher islet yield up to 6 hours (171 +/- 58, n = 14, P less than 0.01), as compared with conventional methods. The viability of the islets isolated by this method was confirmed by the ability to restore normoglycemia of STZ-induced diabetic B6 mice on transplantation of 400 islets in the renal subcapsular space. These findings indicated that loss of the integrity of the ductal system against forced collagenase injection during cold preservation led to poor distention and digestion of the pancreas, ductal collagenase injection at the time of pancreas harvesting followed by simple preservation is recommended to obtain viable islets from the preserved pancreas.

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PubMed1990

Gamma-irradiation as a tool to reduce immunogenicity of islet allo- and xenografts.

Sensitivity of pancreatic endocrine cells to gamma-irradiation and alteration of islet immunogenicity by irradiation were examined. Syngeneic, allogeneic (DBA/2) and xenogeneic (rat) islets were irradiated and transplanted at varying doses (8, 24, 40, 80 and 160 Gy) into streptozotocin-induced diabetic B6AF1 mice. In an isograft model, late loss of graft function was observed in some animals given 200 24-Gy-irradiated islets. However, larger numbers (400-500) of islets given 40 Gy irradiation did not show reversal of normoglycemia. With higher doses (80 or 160 Gy) significant early as well as late graft loss was observed. In an allograft model, the irradiated islet allografts survived beyond controls. Marked prolongation was achieved with a broad range of irradiation doses between 8 Gy and 120 Gy with 30-90% of recipients maintaining normoglycemia over 50 days. Late loss of graft function was observed between 78 and 180 days with a dose over 24 Gy. Increasing dose resulted in a better allograft survival rate in the early postoperative period, but tended to curtail longterm survival. In an xenograft model, irradiation of islets with 8 to 24 Gy led to prolongation of graft survival. Maximum graft prolongation was achieved with 24 Gy. Higher doses were much less effective and, in some recipients, caused shortening of graft survival.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMedDate not supplied

Grafting of mitomycin C-treated islet xenograft under the kidney capsule produces a clear bleb.

We have previously shown that mitomycin C (MMC) treatment of donor tissue resulted in significant prolongation of graft survival in allo- and xenotransplantation models. However, the mechanisms involved in this prolongation are not clearly understood. This study aims to shed light on the immune responses to MMC-treated islet xenografting under the kidney capsule. Collagenase-digested WS (RT1k) rat islets incubated for 30 min with MMC and subsequently cultured for 20 h were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (B6;H-2b) mice. The grafts were harvested on postgrafting day 7 and sections were prepared and stained by hematoxylin and eosin (H&E). Histological study of the grafts in a group not treated with MMC showed marked cellular infiltration and destruction of islet clusters, whereas that of MMC-treated grafts demonstrated a bleb formation under the kidney capsule, in which islet cell clusters were reorganized, creating a layer of cells fixed to the interior of the bleb. Minimal invasion by inflammatory cells was observed only at the edge of the bleb, and most islet cells were protected from these infiltrating cells. In conclusion, MMC treatment induces remodeling of islet structure and forms a bleb under the kidney capsule, where no inflammatory cell infiltration occurs, suggesting that this site is a kind of immunologically privileged environment for xenografted islets.

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PubMedDate not supplied

Role of the liver in alloimmune response following inoculation of donor spleen cells.

The liver is thought to be an immunologically privileged organ in the response to inoculated antigens. We previously demonstrated that it is possible to localize inoculated antigens to the liver alone or only to extrahepatic tissue using orthotopic syngeneic liver transplantation (OSLT). In this study, we analyzed more detailed mechanisms of the anti-alloimmune response in the liver. DA rat spleen cells were systemically injected into WS rats (donor spleen cell inoculation, DSI). In the sensitized liver-grafted (SLG) group, after DSI, liver grafts were retrieved from sensitized WS rats, then transplanted into naive WS rats. In the sensitized liver-removed (SLR) group, after DSI, WS rats were totally hepatectomized and given livers transplanted from naive WS rats. All the rats were challenged with heterotopic heart grafts 10 days after DSI. Mean heart graft survival in the control, DSI, SLG, and SLR groups were 11.6+/-1.6, 10.7+/-2.4, 4.4+/-1.0, and 24.6+/-6.3 days, respectively. Accelerated rejection in the SLG group as well as graft prolongation in the SLR group disappeared when OSLT was performed 2 days after DSI or later. Irradiation of DA splenocytes before inoculation did not alter graft survival in SLG. However, pretreatment with gadolinium chloride prior to DSI attenuated the antidonor response in the SLG group. In conclusion, a vigorous antidonor response occurred in the liver after systemic inoculation of spleen cells. It peaked I day after DSI and disappeared rapidly. Kupffer cells seemed to play an important role in this phenomenon.

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PubMedDate not supplied

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.

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PubMedDate not supplied

Pretreatment of crude pancreatic islets with mitomycin C (MMC) prolongs islet graft survival in a xenogeneic rat-to-mouse model.

In this study, we examined the effect of mitomycin C (MMC) treatment on graft survival and evaluated its efficacy in immunomodulation of islet graft for transplantation. Male WS rats were used as islet donors and streptozotocin-induced diabetic C57BL/6 mice as recipients. The isolated islets were treated with MMC at concentrations of 0, 0.1, 1, 3.2, 10, 32, 100, 320, and 1000 microg/mL for 30 min, and were cultured for 20 h. Then, 300-400 islets were transplanted into the renal subcapsular space of diabetic mice. Significant prolongation of graft survival was obtained when the islets were treated with MMC at a concentration of 10, 32, or 100 microg/mL (MST 23 +/- 7.4, 17.5 +/- 5.4, 29.6 +/- 9.7 days: p < 0.003, p < 0.012, p < 0.001, respectively, vs. 12.3 +/- 2.7 days for culturing alone). Islets treated with MMC at a concentration of 320 microg/mL or more failed to restore normoglycemia in the diabetic recipient mice after transplantation. Viability of islets incubated with doses up to 100 microg/mL, assessed under the confocal microscope after propidium iodide and Hoechst 33342 staining, was maintained well comparable to that of freshly isolated islets, while those treated at 320 microg/mL was significantly decreased. Thus, a therapeutic window for MMC efficacy was found at concentrations from 10 microg/mL to 100 microg/mL. This modality is simple and effective and underlying molecular mechanisms need to be determined in the future.

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