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T W Frick

Publications and source records attributed to T W Frick.

At least 19 recordsLinked to original sources
PubMed2001

A family of 16-kDa pancreatic secretory stress proteins form highly organized fibrillar structures upon tryptic activation.

A group of 16-kDa proteins, synthesized and secreted by rat pancreatic acinar cells and composed of pancreatic stone protein (PSP/reg) and isoforms of pancreatitis-associated protein (PAP), show structural homologies, including conserved amino acid sequences, cysteine residues, and highly sensitive N-terminal trypsin cleavage sites, as well as conserved functional responses in conditions of pancreatic stress. Trypsin activation of recombinant stress proteins or counterparts contained in rat pancreatic juice (PSP/reg, PAP I and PAP III) resulted in conversion of 16-kDa soluble proteins into 14-kDa soluble isoforms (pancreatic thread protein and pancreatitis-associated thread protein, respectively) that rapidly polymerize into insoluble sedimenting structures. Activated thread proteins show long lived resistance to a wide spectrum of proteases contained in pancreatic juice, including serine proteases and metalloproteinases. In contrast, PAP II, following activation with trypsin or pancreatic juice, does not form insoluble structures and is rapidly digested by pancreatic proteases. Scanning and transmission electron microscopy indicate that activated thread proteins polymerize into highly organized fibrillar structures with helical configurations. Through bundling, branching, and extension processes, these fibrillar structures form dense matrices that span large topological surfaces. These findings suggest that PSP/reg and PAP I and III isoforms consist of a family of highly regulated soluble secretory stress proteins, which, upon trypsin activation, convert into a family of insoluble helical thread proteins. Dense extracellular matrices, composed of helical thread proteins organized into higher ordered matrix structures, may serve physiological functions within luminal compartments in the exocrine pancreas.

Amino Acid Sequence↗
PubMed2001

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.

Acute-Phase Proteins↗
PubMed2000

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.

Adaptation, Physiological↗
PubMed1999

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.

Adult↗
PubMed1999

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.

Adaptation, Physiological↗
PubMed1997

TNF receptor p55 controls early acute graft-versus-host disease.

Allogeneic bone marrow transplantation is frequently associated with graft-vs-host disease (GVHD). To understand the effector mechanisms of GVHD, we investigated the role of the TNF receptor p55 (TNFRp55), which is known to be important in inflammation and cytotoxicity. After the transplantation of allogeneic bone marrow and spleen cells to lethally irradiated mice, all wild-type recipients developed early lethal GVHD within 1 wk, whereas TNFRp55-deficient recipients had much reduced GVHD and survived for at least 3 wk. No defect in alloantigen presentation was found, since T cell proliferation and cytotoxicity were similar to allogeneic wild-type and TNFRp55-deficient stimulator and target cells. Also, TNF alpha release did not differ significantly between the two types of recipients. Therefore, early acute GVHD in wild-type mice was primarily due to TNFRp55-mediated tissue damage. Interestingly, lethal GVHD was not entirely dependent upon the TNFRp55. In experimental conditions using sublethal irradiation and high donor spleen cell numbers, TNFRp55-deficient recipient mice developed lethal GVHD with similar kinetics and frequency as the control mice. These data suggest that the effector mechanisms leading to organ damage in murine acute GVHD can be dissected in a cytokine pathway through the TNFRp55, as demonstrated here, and in a cellular pathway through direct interaction of cytotoxic lymphocytes with target tissues involving perforin and Fas/Fas ligand, as reported previously.

Animals↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

Elevated calcium and activation of trypsinogen in rat pancreatic acini.

BACKGROUND: Acute pancreatitis associated with hypercalcaemia has been described in humans and experimental animals. It has been demonstrated that calcium dose dependently accelerates trypsinogen activation, and it is generally believed that ectopic activation of digestive enzymes is an early event in the pathophysiology of acute pancreatitis. AIMS AND METHODS: Trypsinogen activation peptide (TAP) was measured in isolated rat pancreatic acini exposed to elevated extracellular calcium in order to investigate the association between calcium and trypsinogen activation in living cells. TAP was determined in the culture medium either before (extracellular compartment) or after (intracellular compartment) cell homogenisation. RESULTS: Neither secretory stimulation nor elevated calcium alone caused an increase in TAP levels. Maximal cerulein or carbachol stimulation superimposed on high medium calcium, however, significantly increased intracellular trypsinogen activation twofold. This increase was inhibited by either NG-monomethyl-L-arginine (L-NMMA) or verapamil. Acinar cell morphology and function remained intact as demonstrated by electron microscopy and secretagogue dose-response studies. CONCLUSIONS: These results support the hypothesis that increased intracellular trypsinogen activation is an early step in the pathogenesis of hypercalcaemia induced pancreatitis. The model may have a bearing on other types of pancreatitis as elevated cytosolic calcium is thought to be an early event in the pathogenesis of acute pancreatitis in general.

Acute Disease↗
PubMed1996

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.

Animals↗
PubMed1995

[Immunological aspects of kidney transplantation in Switzerland 1981-1992. Swiss Transplant Work Group Kidney Transplantation].

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.

Cyclosporine↗
PubMed1995

Acute hypercalcemia causes acute pancreatitis and ectopic trypsinogen activation in the rat.

BACKGROUND & AIMS: Clinical and experimental observations have associated acute and chronic hypercalcemia with pancreatitis. The aim of this study was to determine whether acute hypercalcemia can induce acute pancreatitis and, if so, whether the pathogenesis involves premature protease activation. METHODS: Rats given bolus infusions of CaCl2 (200 mg/kg; n = 76) were compared with saline-treated controls (n = 40). Serum [Ca2+], serum amylase activity, trypsinogen activation peptide (TAP) concentration in serum and pancreatic tissue, pancreatic wet/dry weight ratio, and histology were assessed for 24 hours. For dose-response analysis, CaCl2 was injected at a dose of 50-200 mg/kg, and the aforementioned indices were assayed for 1 hour (n = 5 each). RESULTS: There were no significant changes in the controls. Calcium infusion increased serum [Ca2+] 3-fold after 5 minutes (P < 0.001). Within 1 hour, serum amylase (2.5-fold) and tissue TAP (3-fold) levels increased along with macroscopic and microscopic edema formation and leukocytic infiltration. The extent of the changes at 1 hour correlated with the calcium dose. Amylase and tissue TAP concentrations remained elevated until 24 hours when serum TAP concentration had increased (P < 0.001) and focal acinar necrosis became evident. CONCLUSIONS: Acute experimental hypercalcemia induces dose-dependent morphological alterations characteristic of acute pancreatitis, acute hyperamylasemia, and early ectopic trypsinogen activation. This supports the pathophysiological relevance of excess calcium and offers a possible pathogenetic mechanism for its association with clinical pancreatitis.

Acute Disease↗
PubMed1995

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.

Acute Disease↗
PubMed1995

Calcium administration augments pancreatic injury and ectopic trypsinogen activation after temporary systemic hypotension in rats.

BACKGROUND: Calcium infusion and hypotension have been described as the most important risk factors for pancreatic injury after cardiopulmonary bypass. METHODS: Rats were randomly allocated to three experimental groups undergoing either sham operation and saline infusion (Control, n = 30), hemorrhagic reduction of mean arterial pressure to 30 mmHg for 30 min alone (hypotension, n = 51), or hypovolemic hypotension followed by bolus infusion of CaCl2 (200 mg.kg-1; hypercalcemia, n = 85). Serum ionized calcium, amylase activity, trypsinogen activation peptide in pancreatic tissue homogenates, pancreatic wet/dry weight ratio, histologic changes, and mortality were assessed for 24 h. RESULTS: Control rats showed no significant changes of any parameter throughout the experiments. In contrast, hypotension significantly increased serum amylase (P < 0.001), tissue trypsinogen activation peptide (P < 0.01), wet/dry weight ratio (P < 0.001), and histologic scores for edema (P < 0.001) and pancreatic necrosis (P < 0.05). Subsequent CaCl2 administration transiently increased [Ca2+] (P < 0.001) with the concentration rapidly returning to baseline within 3 h. That infusion of CaCl2 further increased amylase (P < 0.05), tissue trypsinogen activation peptide (P < 0.05), wet/dry weight ratio (P < 0.001), and histologic evidence of pancreatic edema (P < 0.05) and acinar necrosis (P < 0.05) when compared with hypotension alone. Whereas all Control animals survived the experiments, 22% (P < 0.05) and 47% (P < 0.05 vs. hypotension) of animals died in the hypotension and hypercalcemia groups, respectively. CONCLUSIONS: Temporary hypotension alone causes ectopic trypsinogen activation and lethal acute pancreatitis. Super-imposed hypercalcemia significantly aggravates hypotension-induced pancreatic injury and mortality in rats.

Animals↗
PubMed1995

Increased intrapancreatic trypsinogen activation in ischemia-induced experimental pancreatitis.

OBJECTIVE: The potential of pancreatic ischemia to cause acute pancreatitis as indicated by morphologic changes and ectopic trypsinogen activation was investigated. BACKGROUND: Experimental evidence has shown that pancreatic ischemia is important in the evolution of severe pancreatitis, but whether ischemia can initiate pancreatitis has been disputed. METHODS: Pancreatic ischemia was induced in rats by hemorrhagic hypotension (30 mm Hg for 30 min; n = 64). Changes of pancreatic microcirculatory perfusion were studied using diffuse reflectance spectroscopy. Serum amylase, trypsinogen activation peptide (TAP) in serum and pancreatic tissue, wet/dry weight ratio, and histology were determined over 24 hours and compared with sham-operated control subjects (n = 35). RESULTS: In control animals, serum amylase (47.9 +/- 2.1 units/L), serum (7.9 +/- 0.7 nmol/L) and tissue TAP (63.0 +/- 5.4 nmol/L x g), wet/dry weight ratio (2.8 +/- 0.1), and histology remained unchanged. Temporary hypotension markedly decreased pancreatic perfusion with incomplete recovery after reperfusion. Pancreatic isoamylase activity increased within 1 hour (110 +/- 5 units/L, p < 0.05) and further to 151 +/- 18 units/L at 24 hours. Tissue TAP was elevated at 1 hour (134 +/- 16 nmol/L x g, p < 0.05) and increased to 341 +/- 43 nmol/L x g (p < 0.001) after 24 hours, whereas serum TAP remained unchanged (8.3 +/- 0.5 nmol/L). Morphologic alterations included elevated wet/dry weight ratio (4.1 +/- 0.3, p < 0.01) and increased histologic scores for edema (p < 0.05) and acinar necrosis (p < 0.05) at 24 hours. Trypsinogen activation preceded the development of pancreatic necrosis. CONCLUSIONS: In addition to its potentiating role, severe pancreatic ischemia can play a pathogenetic role in the initiation of acute pancreatitis.

Amylases↗
PubMed1995

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.

Animals↗
PubMed1995

Twenty-seven years of liver transplantation at Addenbrooke's Hospital, Cambridge.

Twenty-seven years ago, liver transplantation at Addenbrooke's Hospital was a very experimental procedure. It is now recognized as an accepted mode of treatment for end-stage liver failure. In the coming year, over 600 liver transplants will take place in the United Kingdom. All western European countries now have an active liver transplantation program and more than 17,000 liver transplants have already been performed. As results improve and the procedure becomes more readily and widely accepted, the donor shortage is likely to get worse, and will be only partially met by the introduction of split livers and living-related donors.

Adolescent↗