Inflammation-dependent expression of SPARC during development of chronic pancreatitis in WBN/Kob rats and a microarray gene expression analysis.

The pathophysiology of human chronic pancreatitis is not well understood and difficult to follow on a molecular basis. Therefore, we used a rat model [Wistar-Bonn/Kobori (WBN/Kob)] that exhibits spontaneous chronic inflammation and fibrosis in the pancreas. Using microarrays we compared gene expression patterns in the pancreas during development of inflammation and fibrosis of WBN/Kob rats with age-matched healthy Wistar rats. The extracellular matrix protein SPARC (secreted protein, acidic, and rich in cysteines) and other transcripts of inflammatory genes were quantified by real-time PCR, and some were localized by immunohistochemistry. When pancreatic inflammation becomes obvious at the age of 16 wk, several hundred genes are increased between 3- and 50-fold in WBN/Kob rats compared with healthy Wistar rats. Proteins produced by acinar cells and characteristic for inflammation, e.g., pancreatitis-associated protein, are highly upregulated. Other proteins, derived from infiltrating inflammatory cells and from activated stellate cells (fibrosis) such as collagens and fibronectins are also significantly upregulated. SPARC was localized to acinar cells where it increased in the vicinity of inflammatory foci. However, acinar expression of SPARC was lost during destruction of acinar cells. In human pancreatic specimens with chronic pancreatitis, SPARC exhibited a similar expression profile. During chronic inflammation and fibrosis in the WBN/Kob rat, inflammatory genes, growth factors, and structural genes exhibit a high increase of expression. A temporal profile including pre- and postinflammatory phases indicates a concurrent activation of inflammatory and fibrotic changes. Inflammation dependent expression of SPARC appears to be lost during acinar-to-duct metaplasia both in rat and human pancreas.

A selective COX-2 inhibitor suppresses chronic pancreatitis in an animal model (WBN/Kob rats): significant reduction of macrophage infiltration and fibrosis.

INTRODUCTION: Therapeutic strategies to treat chronic pancreatitis (CP) are very limited. Other chronic inflammatory diseases can be successfully suppressed by selective cyclooxygenase 2 (COX-2) inhibitors. As COX-2 is elevated in CP, we attempted to inhibit COX-2 activity in an animal model of CP (WBN/Kob rat). We then analysed the effect of COX-2 inhibition on macrophages, important mediators of chronic inflammation. METHODS: Male WBN/Kob rats were continuously fed the COX-2 inhibitor rofecoxib, starting at the age of seven weeks. Animals were sacrificed 2, 5, 9, 17, 29, 41, and 47 weeks later. In some animals, treatment was discontinued after 17 weeks, and animals were observed for another 24 weeks. RESULTS: Compared with the spontaneous development of inflammatory injury and fibrosis in WBN/Kob control rats, animals treated with rofecoxib exhibited a significant reduction and delay (p<0.0001) in inflammation. Collagen and transforming growth factor beta synthesis were significantly reduced. Similarly, prostaglandin E(2) levels were markedly lower, indicating strong inhibition of COX-2 activity (p<0.003). If treatment was discontinued at 24 weeks of age, all parameters of inflammation strongly increased comparable with that in untreated rats. The correlation of initial infiltration with subsequent fibrosis led us to determine the effect of rofecoxib on macrophage migration. In chemotaxis experiments, macrophages became insensitive to the chemoattractant fMLP in the presence of rofecoxib. CONCLUSION: In the WBN/Kob rat, chronic inflammatory changes and subsequent fibrosis can be inhibited by rofecoxib. Initial events include infiltration of macrophages. Cell culture experiments indicate that migration of macrophages is COX-2 dependent.

The bifunctional rat pancreatic secretory trypsin inhibitor/monitor peptide provides protection against premature activation of pancreatic juice.

BACKGROUND: In the rat, two forms of the pancreatic secretory trypsin inhibitor, PSTI-I and PSTI-II, are secreted into pancreatic juice. It is assumed that their role is to protect the pancreas from premature activation of the protease-rich pancreatic juice. In the small intestine, PSTI-I, also called 'monitor peptide', is thought to have a different role: PSTI-I competes with protein for activated trypsin. In the presence of a protein-rich meal, free PSTI induces a release of cholecystokinine from the intestine. METHODS: To investigate whether its role as monitor peptide is compatible with the inhibitory, protective function in the pancreas, PSTI-I was chemically synthesized and then renatured. RESULTS: The peptide was almost completely trypsin resistant and exhibited a dose-dependent inhibitory activity to bovine and partially purified rat trypsin. Furthermore, experiments with trypsin- and endopeptidase-activated pancreatic juice demonstrated that its inhibitory capacity was sufficient to prevent premature activation. Binding studies of (125)I-labeled PSTI-I with the putative intestinal receptor using isolated membranes indicated the presence of high-affinity binding sites (k(d) = 5 x 10(-8)M). Binding of PSTI-I could be competed with excess PSTI-I or trypsin. In a biological assay system, injections of PSTI-I displayed monitor peptide activity by inducing a dose-dependent trypsinogen release from the pancreas. CONCLUSION: Our experiments support a dual function of PSTI-I: monitoring protein in the gut due to its 'moderate' affinity for trypsin and a protective role in the pancreas.

Increased secretion of the pancreatic secretory trypsin inhibitor (PSTI-I, monitor peptide) during development of chronic pancreatitis in the WBN/Kob rat.

BACKGROUND: Recent genetic investigations into cationic trypsinogen and pancreatic secretory trypsin inhibitor (PSTI) led to the conclusion that mutations in either gene can contribute to the development of (hereditary) chronic pancreatitis. Since genetic animal models are not available yet, we have studied the Wistar-Bonn/Kobori (WBN/Kob) rat, a model for chronic pancreatitis (CP). To explore the possibility that PSTI may be secreted at lower levels or contain a mutation in the WBN/Kob rat, we investigated the masses of PSTI-I and -II and asked whether the ratio of PSTI/trypsinogen is decreased in animals with CP. METHODS: We collected pancreatic juice from WBN/Kob and Wistar rats aged 6-36 weeks and measured PSTI-I (ELISA) and trypsin. RESULTS: PSTI-I and -II were identified in Wistar and WBN/Kob rats by mass spectrometry and N-terminal sequencing. Using a newly developed PSTI-I ELISA, we can show that the PSTI-I/trypsinogen ratio is not decreased but rather increased in WBN/Kob rats compared to healthy Wistar rats. No evidence for a PSTI mutation was found. CONCLUSION: Our data does not support the hypothesis that a dysbalance of PSTI/trypsinogen ratio is a causative factor for CP.

Coordinate regulation of secretory stress proteins (PSP/reg, PAP I, PAP II, and PAP III) in the rat exocrine pancreas during experimental acute pancreatitis.

BACKGROUND: Pancreatic stone protein (PSP/reg) is a constitutively secreted protein in pancreatic juice. Pancreatitis-associated protein (PAP) belongs to the same family of proteins. PAP is highly increased during acute pancreatitis, while no exact data exist regarding PSP/reg protein synthesis and secretion. Recently, an attempt to determine PSP/reg and PAP levels in sera of rats with acute pancreatitis showed a significant increase in PAP but failed to demonstrate changes in PSP/reg. Others reported that surgical manipulation of the pancreas, including sham controls, affected mRNA levels of PSP/reg. Neither report determined protein levels of PSP/reg. METHODS: Rats were treated intraperitoneally with a supramaximal dose of caerulein to induce pancreatitis, a physiological dose of caerulein, or a saline injection. Pancreata were analyzed for PAP and PSP/reg using ELISAs. RNA was extracted for Northern blot analysis of PAP I, II, and III and PSP/reg mRNA. RESULTS: Experimental induction of acute pancreatitis caused a coordinate increase in both PSP/reg and PAP. PAP showed an acute response and returned to low levels within 48 h while PSP/reg exhibited a more sustained response. Intraperitoneal application of a physiological dose of caerulein and even a saline injection caused an increase in PSP/reg. CONCLUSION: PSP/reg and PAP levels are increased through similar mechanisms by physiological and supramaximal doses of caerulein. However, PSP/reg regulation appears to sustain high levels while PAP levels are more transient. Since the regulation of this protein family is affected even under mild stress, we define them as secretory stress proteins.

Late oesophageal perforation after intraoperative transoesophageal echocardiography.

Serious haemodynamic instability occurred during emergency surgery for a perforated duodenal ulcer in a 72-year-old man with acute myocardial infarction. Intraoperative transoesophageal echocardiography was crucial for diagnosis of the location of myocardial infarction in the right ventricle and the subsequent haemodynamic management. Postoperatively, a thrombus in the right coronary artery was removed by coronary angiography. The patient's trachea was extubated on the fourth postoperative day. Another 4 days later a leak in the lower oesophagus was suspected because of pleural empyema, and verified. The patient's trachea had to be re-intubated and an oesophageal stent was inserted. The patient was discharged, fully recovered, 2 months after the operation.

Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine.

The serum lipoprotein high-density lipoprotein (HDL), which is a ligand of scavenger receptors such as scavenger receptor class B type I (SR-BI) and cluster determinant 36 (CD36), can act as a donor particle for intestinal lipid uptake into the brush border membrane (BBM). Both cholesterol and phospholipids are taken up by the plasma membrane of BBM vesicles (BBMV) and Caco-2 cells in a facilitated (protein-mediated) process. The protein-mediated transfer of cholesterol from reconstituted HDL to BBMV depends on the lipid composition of the HDL. In the presence of sphingomyelin, the transfer of cholesterol is slowed by a factor of about 3 probably due to complex formation between cholesterol and the sphingolipid. It is shown that the mechanism of lipid transfer from reconstituted HDL to either BBMV or Caco-2 cells as the acceptor is consistent with selective lipid uptake: the lipid donor docks at the membrane-resident scavenger receptors which mediate the transfer of lipids between donor and acceptor. Selective lipid uptake implies that lipid, but no apoprotein is transferred from the donor to the BBM, thus excluding endocytotic processes. The two BBM models used here clearly indicate that fusion of donor particles with the BBM can be ruled out as a major mechanism contributing to intestinal lipid uptake. Here we demonstrate that CD36, another member of the family of scavenger receptors, is present in rabbit and human BBM vesicles. This receptor mediates the uptake of free cholesterol, but not of esterified cholesterol, the uptake of which is mediated exclusively by SR-BI. More than one scavenger receptor appears to be involved in the uptake of free cholesterol with SR-BI contributing about 25% and CD36 about 35%. There is another yet unidentified protein accounting for the remaining 30 to 40%.

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.

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.

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.

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.

Surgical treatment of the ulnar nerve entrapment neuropathy: submuscular anterior transposition or simple decompression of the ulnar nerve? Long-term results in 79 cases.

The surgical treatment of the ulnar nerve entrapment neuropathy at the elbow is controversial. None of the presently advocated procedures (simple decompression of the ulnar nerve, medial epicondylectomy, subcutaneous or submuscular anterior transposition of the ulnar nerve) has proven optimal regarding long-term results. We studied the outcome in 79 patients whose ulnar nerve had been operated on for the first time, either by simple decompression (31 cases) or by submuscular anterior transposition (48 cases). The mean follow-up was 76 months. Patients were classified according to McGowan pre- and postoperatively; we also applied a more detailed scoring system of our own. Preoperatively, the patients were distributed almost equally between the three McGowan classes. Postoperatively, about one out of three patients in both treatment groups experienced a distinct improvement, i.e. was upgraded to a better McGowan class. Using our own scoring system, the overall rate of objective improvement was 73% after transposition and 55% after simple decompression. Irrespective of the surgical method, roughly 90% of the patients considered their postoperative condition to be improved. However, one specific group of patients (people with habitual ulnar luxation or subluxation of the ulnar nerve) experienced a distinctly better result when treated by anterior transposition than by simple decompression. Our results show that simple decompression of the ulnar nerve can be recommended in all patients without cubital (sub)luxation of the nerve, whereas people with a tendency of cubital (sub)luxation of the ulnar nerve should be treated by submuscular anterior transposition.

[Thoracoscopic resection of a schwannoma]. / Thorakoskopische Resektion eines Schwannoms.

Schwannomas are most often benign tumors originating from the sheath of peripheral nerves. Most are asymptomatic and detected incidentally on a routine chest X-ray. Resection is generally recommended to confirm the diagnosis. In a 35-year-old patient we resected and removed an intrathoracic schwannoma of the 6th intercostal nerve thoracoscopically. The technique of resection and protected removal is described.

[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.