Sirolimus: a potent new immunosuppressant for liver transplantation.

BACKGROUND: Sirolimus (rapamycin) is a new immunosuppressant that appears to be synergistic with cyclosporine in kidney transplantation, but with a different side-effect profile. This pilot study evaluated sirolimus in liver transplantation. METHODS: Patients undergoing orthotopic liver transplantation for primary tumors (8), and later for nonmalignant disease (7), received one of three sirolimus-based immunosuppressive regimens. Protocol A comprised sirolimus, microemulsion cyclosporine (target whole blood concentration: 100 ng/ml), and prednisolone; protocol B omitted prednisolone; and protocol C was sirolimus alone. By 3 months after transplantation, all patients were receiving sirolimus as monotherapy. RESULTS: Fifteen patients were treated with a follow-up of 117-806 days. Rejection was more common on monotherapy than double therapy, and absent on triple therapy. The drug was generally well tolerated, with only three patients discontinuing sirolimus: one for hyperlipidemia, one for pneumocystis pneumonia, and one for inability to tolerate the taste of the drug. Two patients discontinued cyclosporine early, both as a result of neurological complications; they continued on sirolimus monotherapy. Five patients died; one suffered a cardiac arrest, and four died from sepsis in association with graft-versus-host disease, recurrent tumor, a paralyzed right hemidiaphragm, and primary nonfunction. CONCLUSIONS: Sirolimus combined with cyclosporine provided potent immunosuppression of liver allografts, and sirolimus monotherapy was adequate and well tolerated as maintenance therapy. Side effects of sirolimus over the short period of follow-up were uncommon and reversible with dose reduction or cessation of therapy.

Drug-induced pancreatitis.

Few data exist about the incidence of drug-induced pancreatitis in the general population. 20 cases of drug-related pancreatitis were reported in Switzerland over a period of 12 years. The proportion of cases of pancreatitis caused by drugs is estimated to be around 2% in the general population, with much higher proportions in specific subpopulations, such as children and patients who are HIV positive. The literature about drug-induced pancreatitis consists mainly of anecdotal case reports. Clear evidence of a definite association with pancreatitis, by means of rechallenge tests, or consistent case reports, supported by animal experiments or data on the incidence of acute pancreatitis in drug trials exists for didanosine, valproic acid (sodium valproate), aminosalicylates, estrogen, calcium, anticholinesterases and sodium stibogluconate. An association with drug-induced pancreatitis is likely but not definitely proven for thiazide diuretics, pentamidine, ACE inhibitors, asparaginase, vinca alkaloids, some nonsteroidal anti-inflammatory drugs and clozapine. Pancreatitis is possibly caused by azathioprine, furosemide (frusemide), tetracycline, metronidazole, isoniazid, rifampicin (rifampin), sulphonamides, cyclosporin and some antineoplastic drugs. Many drugs have been reported to be associated with acute pancreatitis. However, lack of rechallenge evidence, consistent statistical data, or evidence from experimental studies on a possible mechanism prohibit definitive conclusions about most of them. The high incidence of concurrent illnesses known to induce acute pancreatitis, makes a trigger role or co-factor role for the drug seem most likely.

High-level secretion of native rat pancreatic lithostathine in a baculovirus expression system.

We have constructed a recombinant baculovirus expression vector containing rat pancreatic lithostatin cDNA. Baculovirus infection of Spodoptera frugiperda (sf9) insect cells resulted in the de novo synthesis and secretion of a recombinant protein demonstrating an apparent molecular weight of about 16.5 kDa. Under optimal conditions [multiplicity of infection of 5 plaque-forming units (pfu)/cell and culture times of 48-56 h postinfection] recombinant protein was secreted into the culture medium at 5-10 mg/L. The secretory form of the recombinant protein was judged to be rat pancreatic lithostatin by the following criteria: (a) Trypsin cleavage resulted in limited proteolysis of the secreted product giving rise to a trypsin-resistant 15.5-kDa peptide, consistent with the size of the "pancreatic stone/thread protein"; (b) polyclonal antibodies raised against the recombinant protein identified 16.5-kDa secretory proteins in both rat pancreatic juice and sf9 culture medium; and (c) immunohistochemistry indicated that the native antigen resides within zymogen granules in pancreatic acinar cells.

Conformational changes of pancreatitis-associated protein (PAP) activated by trypsin lead to insoluble protein aggregates.

Pancreatitis-associated protein (PAP), a secretory acute-phase protein of the pancreatic acinar cell, is highly up-regulated early in acute pancreatitis. PAP expression returns to undetectable levels when the pancreas recovers. In the rat, three isoforms of PAP are known, all of which are upregulated during acute pancreatitis. Their functions remain obscure. Pancreatic stone protein (PSP/reg), which shows strong sequence homology to PAP, is secreted into pancreatic juice under physiologic and pathologic conditions. PSP/reg is highly susceptible to trypsin cleavage at its ARG11-ILE12 bond. Cleavage results in an N-terminal undecapeptide and a C-terminal peptide called pancreatic thread protein (PTP). PTP forms oligomeric fibrillar structures, which spontaneously sediment in vitro. PTP can be found in protein plugs or stones from patients with chronic pancreatitis. Rat PAP contains a trypsin cleavage site at the same position as PSP/reg. We hypothesize that PAP is susceptible to tryptic cleavage, and that the C-terminal cleavage product of PAP spontaneously precipitates at neutral pH. To test our hypothesis, we generated and purified recombinant PAP. Here we report the production of rat PAP I, II, and III in a yeast expression system using Pichia pastoris. We demonstrate in vitro the tryptic cleavage of rat PAP and the formation of a spontaneously precipitating peptide, which we call pancreatitis-associated thread protein (PATP). PATP displays pH-dependent solubility characteristics very similar to those of PTP.

Regulation of PSP/reg in rat pancreas: immediate and steady-state adaptation to different diets.

Pancreatic stone protein/reg protein (PSP/reg) is a secretory pancreatic protein of hitherto unknown function. It is precursor to a spontaneously precipitating peptide called pancreatic thread protein, which is found in protein plugs within the pancreatic ductal system. Increasing PSP/reg concentrations in pancreatic juice might augment the risk of intraductal plug formation and therefore be a condition predisposing to chronic pancreatitis. Malnutrition is associated with a high incidence of chronic pancreatitis in tropical countries. In a diet study with rats, we tested the hypothesis that protein malnutrition leads to increased PSP/reg concentrations in pancreatic juice. A highly sensitive and reliable enzyme-linked immunosorbent assay (ELISA) for rat PSP/reg was newly established. Male Sprague-Dawley rats were allocated to three nearly isocaloric experimental diets, which contained 0, 45, or 82% casein, respectively, or to a control diet (22% casein). We evaluated PSP/reg expression under these four dietary conditions on the RNA and on the protein level, performing a time-course study over a period of 28 days. Our results demonstrate that PSP/reg expression is not increased because of a protein-deficient diet if investigated under steady-state conditions. After a temporary increase in PSP/reg levels due to a carbohydrate-deficient high-protein diet, we could not find signs of a diet-dependent regulation of this protein. The regulation of PSP/reg thus differs from that of most other pancreatic secretory proteins. Our findings contradict earlier reports that had drawn conclusions based solely on messenger RNA levels.

Ultrastructure of the guinea pig pancreas in acute hypercalcemia.

The effect of acute hypercalcemia on pancreatic ultrastructure and the ultrastructural localization of calcium during hypercalcemia were studied in the guinea pig pancreas. After 3 h of i.v. calcium infusion (0.6 mmol/kg/h), hypertrophy and distention of the Golgi apparatus and an increased number of condensing vacuoles were seen. At 6 h, vacuolar fusion and displacement of zymogen granules occurred. At 9 h, irregular distribution of zymogen granules, indentation of the nucleus with chromatin clumping, and inclusion of intact cell organelles were present. Disruption of the plasma membrane and release of cell organelles into the interstitial space were seen. Control animals receiving saline solution (0.9% NaCl) revealed normal pancreatic ultrastructure. The serum ionized calcium values were 0.65 +/- 0.36 mM in controls and 0.71 +/- 0.14, 0.79 +/- 0.21, and 1.22 +/- 0.50 mM at 3, 6, and 9 h of calcium infusion, respectively. The ultrastructural localization of calcium was performed with the pyroantimonate staining technique after 3 h of calcium and saline infusion. Large calcium deposits were found in calcium-treated animals along the plasma membrane and in the Golgi region. The findings indicate that calcium exerts a strong stimulatory effect that eventually leads to the degeneration of the pancreatic acinar cell.

Adaptive response of the rat pancreas to dietary substrates: parallel regulation of trypsinogen and pancreatic secretory trypsin inhibitor.

Chronic pancreatitis has been associated with malnutrition in alcoholic patients and malnourished juveniles. The composition of the diet, especially the protein content, regulates the synthesis of secretory proteins in the rat pancreas. Adaptive responses of the pancreas have shown that anionic proteases (e.g., trypsinogen) are upregulated during protein deprivation. We hypothesize that the (cationic) pancreatic secretory trypsin inhibitor (PSTI) is down-regulated after a protein-deficient diet. Low PSTI levels might cause a lack of protection from prematurely activated trypsin and therefore enhance the risk for pancreatic inflammation. Over a period of 1 month, rats were fed one of four isocaloric diets with a casein content varying from 0 to 82%. PSTI and trypsinogen mRNA remained fairly constant, irrespective of the diet composition. Trypsinogen and elastase secreted into pancreatic juice were upregulated after a protein-deficient diet relative to a control diet. Contrary to our hypothesis, PSTI was also upregulated. Parallel secretion of trypsinogen and PSTI appears to ensure protection against premature activation even under extreme dietary conditions.

Effect of the organophosphate iso-OMPA on amylase release by pancreatic lobules of dog, guinea pig, and cat.

Organophosphates (OPs) cause irreversible inhibition of cholinesterases (ChEs) and profound cholinergic stimulation. There are major differences in the response of the dog and cat pancreas to the in vivo administration of Diazinon (O,O-diethyl O-2-isopropyl-4-methyl-6-pyrimidyl phosphothioate), a butyrylcholinesterase (BuChE) inhibitor. Acute edematous pancreatitis is found in the dog but not in the cat. The present experiments were designed to see what effect OP had in vitro on pancreatic exocrine function of dog, cat, and guinea pig, and whether the effects were consistent with an anti-ChE activity. A water-soluble OP agent, tetraisopropyl pyrophosphoramide (iso-OMPA) at 10(-3) M, which like Diazinon inhibits BuChE, was used. Minced pieces of fresh whole pancreata 3 mm in size were taken from 3 dogs, 4 guinea pigs, and 2 cats. The tissues were placed in flasks containing Eagle's solution and gassed with 100% O2. Cumulative amylase release was measured by Phadebas method up to 3 h. At half-maximal acetylcholine (ACH) concentration (10(-5) M), the canine pancreas pretreated with iso-OMPA (10(-3) M) showed a 42-87% greater release of amylase than tissues receiving ACH alone (p less than 0.001). The same potentiated response to ACH was seen in guinea pig pancreas pretreated with iso-OMPA (p less than 0.001), but iso-OMPA pretreatment did not augment the ACH response in the cat. Atropine pretreatment effectively blocked all ACH responses, and there was no effect seen with iso-OMPA alone. In the dog, iso-OMPA in combination with half-maximal carbachol (10(-6) M), or in combination with half-maximal cholecystokinin (CCK-8) stimulation (10(-9) M), provided no potentiated amylase release.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypercalcemia causes acute pancreatitis by pancreatic secretory block, intracellular zymogen accumulation, and acinar cell injury.

BACKGROUND: Because hypercalcemia is a known etiologic factor for human acute pancreatitis, studies of the pancreatic pathophysiology and pathomorphology of experimental hypercalcemia have potential clinical significance. MATERIALS AND METHODS: Rats received central venous infusion of either 0.6 mmol/kg per hour CaCl2 or 0.9% NaCl infusion for 12 hours. Pancreatic tissue samples were obtained and prepared for electron microscopy. Tissue homogenates were examined for DNA, lactate dehydrogenase (LDH), protein, amylase, and calcium contents. Basal or stimulated (cerulein 0.25 microL/kg per hour) pancreatic secretions were analyzed for volume, protein, and amylase output, as well as protein composition on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The tissue calcium content and the ratio of LDH to DNA was unchanged after calcium infusion, but the ratios of total protein to DNA and of amylase to DNA were significantly larger. Basal output of pancreatic juice volume, protein, and amylase were significantly lower. SDS-PAGE of pancreatic juice revealed weakening of a 70,000-d band and appearance of lower molecular weight bands in two samples. Ultrastructural examination demonstrated accumulation of zymogen granules in the acinar cell, large autophagic vacuoles containing remnants of condensing vacuoles. CONCLUSIONS: These findings suggest that hypercalcemia induces pancreatic injury via a secretory block, accumulation of secretory proteins, and possibly activation of proteases.

Hypercalcemia associated with pancreatitis and hyperamylasemia in renal transplant recipients. Data from the Minnesota randomized trial of cyclosporine versus antilymphoblast azathioprine.

The incidence and possible etiologic factors of acute pancreatitis and hyperamylasemia were statistically evaluated in renal transplant recipients. Two hundred twenty-four patients were randomized in a prospective trial of cyclosporine and antilymphoblast azathioprine immunosuppressive regimens. They had a median follow-up of 20 months. Pancreatitis developed in 8 patients and hyperamyl asemia developed in 20 patients. There were no statistical relationships between the incidences of pancreatitis and hyperamylasemia and the immunosuppressive drugs or viral infections. However, pancreatitis developed in 11 percent of the transplant patients with repeatedly elevated serum calcium levels (37 patients, p less than 0.01) and hyperamylasemia developed in 19 percent (p less than 0.025). Other etiologic factors, such as gallstones, alcoholism, and corticosteroids, played a minor role in this patient population. These results suggest that hypercalcemia is a major etiologic factor for pancreatitis in renal transplant recipients.

Effects of insecticide, diazinon, on pancreas of dog, cat and guinea pig.

The organophosphate insecticide Diazinon has been reported to cause acute pancreatitis in dogs. Based on histochemical examination of the acinar tissue, it was suggested that pancreatic tissue-fixed butyrylcholinesterase (BuChE) is the target enzyme of organophosphate toxicity. To further evaluate this theory, we exposed dogs, cats, and guinea pigs to a single sublethal dose of the organophosphate insecticide Diazinon (75 mg/kg). In cats, which lack pancreatic BuChE, no pathological changes occurred after two, three, and six hours, whereas in the guinea pigs as in dogs, both having abundant pancreatic BuChE, vacuolization of the acinar cells, interstitial edema and vasculitis indicate acute edematous pancreatitis as early as two hours. Atropine pretreatment (0.2 mg/kg) gave complete protection against pancreatitis. It was concluded that inhibition of pancreatic BuChe leads to cholinergic hyperstimulation of the acinar cell, which results in acute pancreatitis, and that pancreatic BuChE is essential for dogs and guinea pigs to downregulate cholinergic excitation. The insecticide pancreatitis model is considered a simple, non-invasive, reproducible, and cheap and useful method to evaluate early changes and methods of treatment in acute pancreatitis. Pancreatitis in humans has also been reported after accidental insecticide exposure.

Electrohydraulic extracorporeal non-water bath shock-wave lithotripsy of gallstones: two years' experience.

A prospective study was performed to evaluate the effectiveness of extracorporeal shockwave lithotripsy (ESWL) using a non-water bath lithotripter in combination with oral chemolitholysis on gallstone clearance. Patients were treated without general anesthesia or parenteral analgesia. We treated 74 patients selected according to the widely accepted criteria. Only 2 patients could not be sufficiently treated because of pain. After a 2 year period, 24 (32%) patients showed complete stone clearance, 35 (47%) patients had residual fragments, 5 (7%) patients underwent cholecystectomy, 2 (3%) patients were lost to follow up, and 8 (11%) patients discontinued the treatment before fragment clearance. According to the life-table estimate, 77% of our patients with successful ESWL and uncomplicated oral chemolitholysis are stonefree after 1 year. We consider the major advantage of this nonsurgical treatment of gallstone disease is that general anesthesia or parenteral analgesia has become unnecessary.

Obstructive ileus and acute pancreatitis.

A 48-year-old patient presented with a 24 hour history of diffuse abdominal pain and diarrhea. Based on elevated serum amylase and lipase levels, a CT-scan, and a history of chronic alcohol intake, acute alcoholic pancreatitis was diagnosed. The patient clinically improved under conservative therapy, but after restarting enteral nutrition on the fourth day, he developed full blown mechanical ileus. Intraoperatively, an adhesive band and acute edematous pancreatitis and fat necrosis was found. Retrospectively, the initial clinical symptoms and plain abdominal x-ray findings suggest coincidence of obstructive ileus and acute pancreatitis. We hypothesize that obstructive ileus had triggered pancreatitis.

Drug-induced acute pancreatitis: further criticism.

A comprehensive literature search was performed to collect all available data on drug-induced pancreatitis. Strong evidence for an association with acute pancreatitis has been described for anticholinesterases, calcium 2',3'-dideoxyinosine, estrogen, L-asparaginase, salicylates, thiazide-diuretics, valproic acid, and vinca alkaloids. Weak evidence has been found for antituberculous agents, azathioprine, biguanides, cisplatinum, cyclosporine A, H2-blocking agents, loop diuretics, 6-mercaptopurine, metronidazole, pentamidine, steroids, sulfonamides, sulindac and tetracycline. Many cases were associated with underlying conditions known to induce acute pancreatitis themselves. It is concluded that for none of the drugs studied the available data are consistent enough to support a definite association with acute pancreatitis. Nevertheless, the data suggest that drugs may be a trigger or a cofactor in inducing pancreatitis.

[Results of surgical therapy of ulnar sulcus syndrome. Submuscular anterior transposition versus simple decompression of the ulnar nerve]. / Ergebnisse der operativen Therapie des Sulcus-ulnaris-Syndroms. Submuskuläre Vorverlagerung versus einfache Dekompression des N. ulnaris.

The operative treatment of the ulnar neuropathy at the elbow is controversial. We studied the course of 79 patients who had been operated for the first time, either by simple decompression (31 cases) or by submuscular anterior transposition (48 cases) of the ulnar nerve. Our results show that the simple decompression can be recommended in all patients without cubital (sub)luxation of the ulnar nerve. The submuscular anterior transposition should be preferred if a tendency of cubital (sub)luxation of the ulnar nerve has been found.

[Immunological aspects of kidney transplantation in Switzerland 1981-1992. Swiss Transplant Work Group Kidney Transplantation]. / Immunologische Aspekte der Nierentransplantation in der Schweiz 1981-1992.Swisstransplant Arbeitsgruppe Nierentransplantation.

Every year some 200-260 kidney transplants are performed in Switzerland, improving the quality of life of patients with end stage renal disease. The current organ shortage is delaying transplantation of the 400 patients on the waiting list, a situation which calls for optimal utilization of the available donor kidneys. It is well established that AB0-compatibility, negative cytotoxic crossmatch, and optimal immunosuppressive therapy including cyclosporin A are important for a favorable clinical outcome. To identify further factors influencing transplant outcome, we undertook a retrospective study of all 1656 transplants to which the above criteria applied. We defined transplants matched for at least 1A, 1B, and 1DR HLA antigen as the better matched, and the remainder as the less well matched grafts. In patients who were not or only weakly immunized to alloantigens, the 5-year graft survival probability was 0.78 versus 0.69 for the better versus the less well matched transplants (p < 0.005). The strongly immunized patients did not, however, show a significant association between the degree of HLA matching and graft survival, presumably because there were more immunized patients in the HLA matched group. As expected, the patients previously immunized to alloantigens showed significantly reduced graft survival early after transplantation. Positive CMV serology, sex mismatch, and cold ischemia time did not correlate with graft survival. Compared to results obtained in the USA or Germany, the survival time of donor kidneys transplanted in Switzerland was significantly increased. Factors contributing to the good results in Switzerland are discussed. Future goals are reduction of alloimmunization and optimized HLA compatibility.

Hypercalcaemia and pancreatic ultrastructure in cats.

OBJECTIVE: To study the effects of local and systemic infusions of calcium on the ultrastructure of the pancreas in cats. DESIGN: Controlled study. INTERVENTIONS: Three groups of four cats each had local infusions (into the splenic artery) of calcium gluconate 0.6 mmol/kg.hour or potassium chloride 1.1 mmol/kg.hour, or sodium chloride 0.9%, for three hours. Two groups of eight cats each had systemic infusions (into the jugular vein) of either calcium gluconate 0.6 mmol/kg.hour or sodium chloride 0.9%, for twelve hours. In the group that was given calcium, the infusion rate was reduced after three hours to 0.3 mmol/kg.hour to maintain the hypercalcaemic state for a further nine hours. RESULTS: Local infusion of calcium caused destruction of acinar cells with hydropic degeneration of nuclei, discharge of cell organelles into the interstitial spaces, and extravasation of red blood cells but no apparent damage to the capillaries. There were no ultrastructural changes of any importance in the groups that received potassium or sodium chloride. Systemic infusion of calcium resulted in a 1.8 fold increase in the ionised calcium concentration in the serum, progressive signs of overstimulation of the Golgi apparatus with hypertrophy, fusion of condensing vacuoles, and disruption of the acinar cell polarization. This was followed by clumping of nuclear chromatin and destruction of acinar cells. CONCLUSION: Acute pancreatitis in cats can result from stimulation and destruction of acinar cells by hypercalcaemia.

Pancreatic stone protein (lithostathine), a physiologically relevant pancreatic calcium carbonate crystal inhibitor?

Apart from digestive enzymes, pancreatic juice contains several proteins that are not directly involved in digestion. One of these, lithostathine, has been reported to exhibit calcite crystal inhibitor activity in vitro. As pancreatic juice is supersaturated with respect to calcium carbonate, it was hypothesized that lithostathine stabilizes pancreatic juice. Lithostathine is cleaved by trace amounts of trypsin, resulting in a C-terminal polypeptide and an N-terminal undecapeptide, which has been identified as the active site of lithostathine regarding crystal inhibition. We produced rat lithostathine in a baculovirus expression system. In order to test its functional activity, the protein was purified using a nondenaturing multi-step procedure. In the low micromolar range, recombinant rat lithostathine in vitro exhibited calcite crystal inhibitor activity, confirming earlier reports. Limited tryptic proteolysis of recombinant lithostathine was performed, and the two cleavage products were separated; the C-terminal polypeptide was precipitated by centrifugation, and the N-terminal undecapeptide was purified by high performance liquid chromatography. Only the C-terminal peptide displayed measurable calcite crystal inhibitory activity. Furthermore, synthetic undecapeptides with identical sequence to the N-terminal undecapeptides of rat or human lithostathine were inactive. However, when tested in the same in vitro assays, other pancreatic or extra-pancreatic proteins show inhibitory activity in the same concentration range as lithostathine, and inorganic phosphate is active as well. Based on these findings it seems unlikely that lithostathine is a physiologically relevant calcite crystal inhibitor. The name "lithostathine" is therefore inappropriate, and the protein's key function remains to be elucidated.

Acute hypercalcemia induces acinar cell necrosis and intraductal protein precipitates in the pancreas of cats and guinea pigs.

The effect of local and systemic calcium administration was tested on the pancreas of cat and guinea pig. After 3 h of local calcium infusion (0.6 mmol/kg x h) via the splenic artery of the cat hemorrhagic pancreatitis could be shown. Control animals treated with potassium (1.1 mmol/kg x h) or 0.9% NaCl alone showed no morphological change in the pancreas. Intravenous administration of calcium (0.6 mmol/kg x h) led to a 1.8-fold increase in serum ionized calcium levels in the cat and a 1.6-fold increase in levels in the guinea pig. The cat showed necrosis of acinar and ductal cells throughout the gland at 12 h. In the guinea pig, acinar cell vacuolisation and cell necrosis started at 3 h, and at 9 h degeneration of entire acini, hydropic swelling and degeneration of ductal cells, and perivascular leukocytic infiltration was present. In both species, a significant increase in the number of intraductal precipitates and a significant increase in urinary amylase output was present in calcium treated animals. The findings suggest that hypercalcemia has a deleterious effect on the pancreas that causes acinar and ductal cell necrosis and eventually pancreatitis.