Ragnar Källén

Two doses of daclizumab in conjunction with low-dose cyclosporine, mycophenolate mofetil and steroids resulted in a low incidence of acute rejection after renal transplantation.

Five doses of daclizumab, given initially after kidney transplantation, reduce the rate of acute rejection (AR). Without cyclosporin A (CsA), a protocol, including daclizumab, mycophenolate mofetil (MMF) and corticosteroids (CSs), has recently shown efficacy in terms of graft function and survival. The rate of AR was relatively high, however. In this single‐centre study, a CsA low‐dose regimen was combined with two doses of daclizumab (1 mg/kg day 0 and 14), plus MMF (2 g) and CS. Forty‐three cadaver donor renal recipients were included. Following the onset of graft function, target trough levels of CsA were 150–200 ng/ml for 90 days, then 100–150 ng/ml. One year AR rate was 23% (n = 10) and events occurred at a median of 2.9 months (range from 9 days to 9.6 months). Delayed graft function (DGF) (absent spontaneous reduction of serum creatinine day 1) was 51%. Graft survival was 95% and patient survival 98% after 1 year. With respect to our previous experience, we used CsA, azathioprine and CSs (n = 223) from 1988 to 1995, and the rate of AR was 57%. From 1996 to 1998, standard CsA doses, MMF and CS (n = 67) resulted in 31% AR. Median time to AR was 0.8 and 1.0 month, and the rate of DGF was 20 and 22%, respectively. This CsA low‐dose protocol, including two doses of daclizumab, MMF and CS, resulted in a reduction and delay of AR episodes and excellent graft function, graft survival and patient survival, despite an increase in DGF.

Pancreatic enzymes in serum and urine as indicators of pancreatic allograft rejection in the pig.

We have studied the reliability of serum and urinary immunoreactive anionic trypsin (irAT), immunoreactive cationic trypsin (irCT), and amylase activity as rejection indicators in a porcine whole-organ pancreaticoduodenal transplantation model with exocrine drainage to the urinary bladder. No immunosuppressive therapy was administered. Exocrine tissue integrity and function were studied by measuring these enzymes in serum and urine. Urine analyses were performed before and after an intravenous secretin-cholecystokinin stimulation. Of 16 transplanted pigs, 10 became diabetic during a 2-week observation period while six remained normoglycemic. Serum irAT was found to predict rejection while serum amylase and serum irCT did not. An increase in irAT was seen in rejecting pigs preceding the onset of hyperglycemia by a median of 2 days (range 1-9). Secretion of irAT into the urine remained high during the observation period in nondiabetic pigs while the output declined in diabetic pigs. This decline was seen after the increase in serum irAT. When urine was sampled after a secretin-CCK stimulation, these findings were clearly evident, but less unequivocal results were obtained without stimulation. IrAT measurements were superior to measurements of amylase, irCT, or bicarbonate. Thus rejection of a pancreatic allograft was first indicated by a temporary rise in serum immunoreactive anionic trypsin, probably due to the onset of tissue damage. Thereafter, stimulated urinary enzyme output levels gradually declined and finally, hyperglycemia developed.

Biochemical characterization of reperfusion pancreatitis in porcine pancreatic allografts after six hours of cold storage.

Biochemical signs of pancreatitis in plasma and pancreatic exudates were determined in 22 pigs subjected to pancreatic allograft transplantation after the graft had been in cold storage for 6 hr. Two perfusion and preservation media were used. We found signs of protease activation in the pancreatic exudate during the first hour after reperfusion. The local protease protection barrier was, however, not broken and no plasma changes indicating pancreatitis were seen during this period. On the first and second postoperative days, mild biochemical signs of pancreatitis were seen in the plasma, including a decrease in kininogen and C3 concentration as well as in plasma kallikrein inhibitory activity and the appearance of trypsin-protease inhibitor complexes. No correlation was seen between these biochemical signs of pancreatitis and graft appearance or function, indicating that the reperfusion pancreatitis seen after 6 hr of cold storage is of minor significance. No significant differences were seen between the two preservation media used (Perfadex and EuroCollins solution).

Role of oxygen-derived free radicals in protease activation after pancreas transplantation in the pig.

The role of oxygen-derived free radicals in pancreatitis after pancreas transplantation was examined in a porcine pancreatic transplantation model. Trypsin activation, protease inhibitor consumption, kininogen consumption, and postoperative graft function were investigated in 24 pigs subjected to whole organ pancreaticoduodenal transplantation. The animals were divided into one control group and two groups treated with free radical scavengers. One group was given allopurinol, and one group was treated with superoxide dismutase in combination with catalase. In the early phase (within 1 hr) after reperfusion, no differences were seen between the groups as to protease activation. Neither trypsin-protease inhibitor imbalance nor any signs of kininogen consumption were seen. In a later phase (1-3 days after the transplantation), the trypsin activation, measured as high molecular weight immunoreactive cationic trypsin in plasma, was significantly less pronounced in allopurinol-treated animals. This finding indicates a less severe form of reperfusion pancreatitis in this group compared with the other groups. A tendency toward better function in the allopurinol-treated group was also seen. We conclude that oxygen-derived free radicals seem to be of importance in the development of reperfusion pancreatitis after pancreas transplantation in the pig. We also conclude that allopurinol, but not superoxide dismutase/catalase, possibly due to the administration regimens used in this series, is able to attenuate the trypsin activation and the development of pancreatitis in the later phase in this model.

Characterization of immunoreactive trypsin as a means of differentiating graft pancreatitis and allograft rejection after porcine pancreatic transplantation.

Graft pancreatitis and allograft rejection were both accompanied by increased serum levels of immunoreactive anionic trypsin (ir AT) in a porcine pancreatic allograft transplantation model. Characterization of this immunoreactivity by gel filtration revealed different elution profiles in these conditions that can be helpful in the differentiation between them. During graft pancreatitis, a major part of the immunoreactivity was found within the high-molecular-weight fraction corresponding to the formation of complexes between trypsin and protease inhibitors. During allograft rejection, virtually all serum ir AT increase could be attributed to the release of anionic trypsinogen without any evidence of activation. Since this transplantation model includes urinary diversion of the exocrine secretions, ir AT and immunoreactive cationic trypsin (irCT) can also be measured in the urine. Characterization of this immunoreactivity showed that most of both irAT and irCT was found as active trypsin but a minor part was probably complexed with some protease inhibitor (possibly pancreatic secretory trypsin inhibitor [PSTI]).

Duodenal mucosal pH as a reperfusion indicator in pancreatic-duodenal transplantation in the pig

We have studied differences in reperfusion between the pancreas and the duodenum after 6 hr of cold storage in a porcine whole-organ pancreaticoduodenal allograft transplantation model. Two different flush-out and storage solutions, Perfadex and EuroCollins, were compared. Graft duodenal mucosal pH (pHi) was measured as an indicator of duodenal mucosal reperfusion. Pancreatic reperfusion was estimated indirectly using a ratio between the release of immunoreactive cationic trypsin (irCT) to serum 10 and 60 min following reperfusion. Twelve pigs (Perfadex n = 6, EuroCollins n = 6) were transplanted and all showed preserved endocrine and exocrine function postoperatively. Our data support the concept that reperfusion of a pancreaticoduodenal graft can be estimated using duodenal pHi and that the ratio of irCT at 10 and 60 min gives an indirect estimate of pancreatic reperfusion. The results also show that grafts stored in EuroCollins reperfuse more slowly than grafts stored in Perfadex.

Protease Activation in the Porcine Pancreatic Allograft During Preservation

A porcine pancreatic transplantation model was used to investigate possible protease activation in the pancreatic graft during preservation. After perfusion with Perfadex and cold ischemia for 24 h, but prior to reperfusion, activated carboxypeptidase B was demonstrated in tissue samples from the graft parenchyma with a Western blot technique, indicating that graft pancreatitis may already be initiated during the preservation phase. A higher degree of carboxypeptidase B activation was observed in grafts perfused at a pressure of 130 cm H2O than after perfusion at 70 cm H2O. During reperfusion, the fraction of activated carboxypeptidase B gradually declined but was still detectable after 2 h. One group of pigs received aprotinin intravenously during reperfusion, but the protease inhibitor did not influence the degree of carboxypeptidase B activation in the biopsy specimen. Immunoblotting against cationic trypsinogen/trypsin was also performed. When activated trypsin was detectable, it never represented more than a few percent of the total amount of uncomplexed immunoreactive trypsinogen/trypsin.

Exocrine pancreatic proteins in serum during pancreatic allograft rejection.

The serum concentrations of immunoreactive pancreatic secretory trypsin inhibitor, cationic elastase, anionic trypsin, cationic trypsin, and total amylase activity, were studied after pancreatic allograft transplantation. Nine patients received whole organ pancreatico-duodenal allografts with exocrine drainage to the bladder. Eight of the patients received simultaneously a renal allograft. The serum concentration of immunoreactive cationic elastase increased gradually during the first postoperative days to a peak on the fifth day after surgery; all other proteins decreased in concentration after the first day. Eighteen episodes of pancreatic and/or kidney rejection, diagnosed by means of kidney biopsy, urinary cytology, kidney function, and urinary amylase levels, were analyzed. The pancreatic proteins displayed different patterns in serum concentration during the days immediately before diagnosis of rejection. The pancreatic secretory trypsin inhibitor showed the most pronounced peak in serum concentration during rejection and even reacted during some episodes without a decline in urinary amylase output. Cationic elastase on the other hand showed no reaction at all. From this homogeneous material it is possible to conclude that changes in serum concentrations during rejection are not the same for all pancreatic exocrine proteins.

Protease activation following reperfusion of porcine pancreatic allografts

To study the degree of protease activation at reperfusion of a pancreatic allograft after cold storage for 24 hr, 18 porcine whole-organ pancreaticoduodenal allograft transplantations were performed. Twelve grafts were flushed with and stored in Perfadex. In six of these, a hyperosmotic salt solution was injected into the graft aorta at reperfusion. Six grafts were flushed and stored in UW solution. Eleven of twelve grafts in the Perfadex groups were functioning on the first postoperative day, compared with one of six in the UW solution group. There was a significantly more pronounced protease activation among grafts stored in UW solution than in the other groups, with a subsequent breakthrough of the local protease protection barrier made up of protease inhibitors. In surviving pigs (n = 14), biochemical signs of protease activation evolved in plasma, including formation of trypsin-protease inhibitor complexes, a decline in C3 and kininogen levels, and a decline in functionally active alpha 2-macroglobulin, functionally active antithrombin III, and plasma kallikrein inhibitory activity. These biochemical signs of pancreatitis correlated with a deteriorated graft function on the second postoperative day, indicating that graft tissue damage occurred due to protease activation.

Early Indicators of Allograft Rejection in a Porcine Pancreatic Transplantation Model

Urine cytology and blood lymphocyte blastogenesis were evaluated as indicators of allograft rejection in a porcine pancreatic transplantation model. The percentage of activated lymphocytes and/or blasts was significantly increased during the rejection phase. Positive cytology was present in all rejection episodes. An increased thymidine uptake of blood lymphocytes and a decreased uridine/thymidine uptake quotient were seen prior to the onset of rejection. The reported dissociation of anionic and cationic trypsin levels in serum and urine after transplantation was not seen after simple urinary diversion of the pancreatic juice. This supports the hypothesis that a decreased synthesis of cationic trypsinogen compared with anionic trypsinogen occurs after porcine pancreatic transplantation.