Protein Plug Formation Research Articles

Alterations in the pH of pancreatic juice are associated with chymotrypsin C inactivation and lithostathine precipitation in chronic pancreatitis patients: a proteomic approach

BackgroundThe progression of chronic pancreatitis (CP), an inflammatory disease of the pancreas, causes pancreatic stones to form within the pancreatic ductal lumen/parenchyma, which occurs via protein plug formation. Pain is the most common symptom that necessitates clinical attention, and pain relief is the therapeutic goal for these patients. Endoscopic therapy and surgery are complimentary forms of therapy for pain relief. This study was envisaged to clarify the mechanism by which protein plug/soft stones form in pancreatic ducts prior to undergoing calcification.MethodsProtein plugs were obtained from twenty CP patients undergoing therapeutic ERCP for stone removal. Pancreatic juice was obtained from five CP patients without stones. Proteins were isolated by TCA/acetone precipitation, SDS PAGE and 2-D gel electrophoresis to determine the protein profile. Protein spots from the 2-D gel were excised and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) for identification. The effect of altered pH and elevated concentrations of trypsin on pancreatic juice protein was assessed by SDS‒PAGE to determine the protein profile. Differentially expressed protein bands were excised and subjected to MALDI-TOF. In silico analysis was performed by docking lithostathine with the calcite molecule using AutoDock Vina and PyMOL to clarify their interaction during stone formation.ResultsTwenty-three and twenty-nine spots from 2D gels of protein plugs and pancreatic juice, respectively, revealed that lithostathine (Reg1A) was the only protein in the protein plugs, whereas digestive enzymes and lithostathine were identified in pancreatic juice. Altered pH levels and increased trypsin concentrations in the pancreatic juice caused a protein to degrade via an unknown mechanism, and this protein was identified as chymotrypsin C (CTRC) by MALDI-TOF. Docking studies showed that the binding affinity of calcite was higher with the cleaved lithostathine, explaining the deposition of calcium that was observed around the protein plugs after calcified stones were formed through precipitation.ConclusionOur results suggest that chymotrypsin C (CTRC) is degraded in an acidic environment, leading to the precipitation of lithostathine in the ductal lumen.Graphical

Two Cases of Plug or Stone in Remnant Intrapancreatic Choledochal Cysts Treated with Endoscopic Retrograde Cholangiopancreatography.

Incomplete resection of choledochal cysts (CCs) that extend deep into the pancreas can lead to protein plug or stone formation, pancreatitis, and cholangiocarcinoma. We encountered two cases of choledocholithiasis in remnant intrapancreatic CCs, in which the patients exhibited symptoms after 3 and 21 years of cyst excision. A 21-year-old woman who had undergone excision of a CC, as a neonate, presented with epigastric pain. Abdominal computed tomography (CT) revealed stones inside the remnant pancreatic cyst, which were removed by endoscopic retrograde cholangiopancreatography (ERCP), and her symptoms improved. A 33-year-old woman, who underwent cyst excision 3 years ago, presented with pancreatitis. Abdominal CT showed a radiolucent plug inside the remnant pancreatic cyst. The soft, whitish plug was removed by ERCP, and the pancreatitis improved. These cases indicate that plugs and stones in CCs have the same pathogenetic mechanism, and their form depends on the time since the incomplete excision surgery.

Bicarbonate-rich fluid secretion predicted by a computational model of guinea-pig pancreatic duct epithelium.

The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3- concentrations as high as 140mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts. The model was readily able to secrete 140mm HCO3- . Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO3- concentrations is to minimize the secretion of Cl- ions. These findings help to clarify the mechanism responsible for pancreatic HCO3- secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3- ions are secreted at concentrations in excess of 140mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+ -HCO3- cotransporter (NBC1) and apical Cl- /HCO3- exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl- and HCO3- permeabilities (CFTR), and reducing the activity of the basolateral Cl- /HCO3- exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140mm HCO3- at a rate of ∼3nlmin-1 mm-2 , which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl- /HCO3- exchange via SLC26A6 at the apical membrane were able to support a HCO3- -rich secretion. Raising the HCO3- /Cl- permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3- concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl- concentration following cAMP stimulation and thereby maximizing the secreted HCO3- concentration. The addition of a basolateral Na+ -K+ -2Cl- cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl- and resulted in a lower secreted HCO3- concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl- secretion is the main requirement for secreting 140mm HCO3- .

Recent advances in pancreatology.

Recent advances in pancreatology.

Molecular Mechanisms of Pancreatic Stone Formation in Chronic Pancreatitis

Chronic pancreatitis (CP) is a progressive inflammatory disease in which the pancreatic secretory parenchyma is destroyed and replaced by fibrosis. The presence of intraductal pancreatic stone(s) is important for the diagnosis of CP; however, the precise molecular mechanisms of pancreatic stone formation in CP were left largely unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel expressed in the apical plasma membrane of pancreatic duct cells and plays a central role in secretion. In previous studies, we have found that CFTR is largely mislocalized to the cytoplasm of pancreatic duct cells in all forms of CP and corticosteroids normalizes the localization of CFTR to the proper apical membrane at least in autoimmune pancreatitis. From these observations, we could conclude that the mislocalization of CFTR is a cause of protein plug formation in CP, subsequently resulting in pancreatic stone formation. Considering our observation that the mislocalization of CFTR also occurs in alcoholic or idiopathic CP, it is very likely that these pathological conditions can also be treated by corticosteroids, thereby preventing pancreatic stone formation in these patients. Further studies are definitely required to clarify these fundamental issues.

Lactoferrin in Gastrointestinal Disease

Lactoferrin, a major whey protein, is a red iron-binding protein present mainly in external secretions such as breast milk and in polymorphonuclear neutrophils. The presence of lactoferrin in body fluids is proportional to the flux of neutrophils and its assessment can provide a reliable biomarker for inflammation. In gastrointestinal diseases increased fecal lactoferrin is a sensitive and specific surrogate marker for inflammatory bowel diseases in patients with chronic diarrhea and pain, and ascites lactoferrin can also provide a promising and reliable biomarker for bacterial peritonitis. Lactoferrin in pancreatic juice and stone could provide pathophysiological information of protein plug and stone formation in the pancreatic duct. Serum anti-lactoferrin autoantibody might contribute to the clarification of the pathogenetic mechanisms of autoimmune pancreatitis and liver diseases, although its diagnostic and prognostic value appears to be limited. Further studies will be necessary to elucidate the exact details.

Recent Developments in the Treatment of Alcoholic Chronic Pancreatitis

Chronic pancreatitis is a progressive inflammatory condition characterized by repeated attacks of abdominal pain, and the destruction and fibrosis of the pancreatic parenchyma which causes to reduced exocrine and endocrine functions. Alcohol is the most common cause of chronic pancreatitis. Although abstinence is usually considered a prerequisite for successful treatment of alcoholic chronic pancreatitis, we often encounter patients who have repeated attacks from the compensated stage through the transitional stage. In alcoholic chronic pancreatitis, continued alcohol consumption causes changes in the digestive hormones and vagal nerve function that induce the pancreatic acinar cells to oversecrete protein, increasing the protein concentration and viscosity of the pancreatic juice. This induces protein sedimentation from the pancreatic juice and formation of protein plugs within the pancreatic duct, triggering repeated attacks of acute pancreatitis. The treatment of alcoholic chronic pancreatitis includes alleviation of symptoms, particularly abdominal pain, elimination of trigger factors, prevention of recurrence and disease progression, adjuvant therapies for pancreatic exocrine and endocrine failure. Recently, the main constituent proteins in these protein plugs have been identified, enabling trials of several therapies, such as the administration of secretin formulations and endoscopic removal. Bromhexine hydrochloride, a bronchial mucolytic, has an affinity for the pancreatic acinar cells, inducing them to secrete pancreatic juice of low viscosity. In this review, we summarize the most recent thoughts about alcoholic chronic pancreatitis, and the new treatments, and in particular, we present our findings concerning the efficacy of bromhexine hydrochloride in the treatment of this disease.

Activated Pancreatic Enzyme and Pancreatic Stone Protein (PSP/reg) in Bile of Patients with Pancreaticobiliary Maljunction/Choledochal Cysts

Symptoms of pancreaticobiliary maljunction/choledochal cysts are caused by the obstruction of bile and pancreatic ducts due to protein plugs compacted in the common channel. However, the mechanism of protein plug formation remains unknown. Pancreatic stone protein (PSP) is reported to be a key protein to form protein plugs in chronic pancreatitis. Bile from 13 patients with pancreaticobiliary maljunction and bile from two normal controls were analyzed. Activity of pancreatic enzymes and the concentration of PSP were measured. The mean concentrations of PSP were 76.9+/-30.9 ng/mL in the bile-duct bile, and 76.9+/-29.8 ng/mL in the gallbladder bile. PSP was not detected in the controls (P < 0.05). In the bile-duct bile of the patients, trypsin(ogen) was detectable in nine patients, of which seven patients had activated trypsin. In the gallbladder bile, trypsin(ogen) was detectable in 12 patients, of which 9 patients had activated trypsin. Neither activated trypsin nor trypsinogen was detected in the controls. Bile in pancreaticobiliary maljunction patients contained both activated trypsin and PSP. Activated trypsin cleaves soluble PSP and creates insoluble PSP. Protein plugs in pancreaticobiliary maljunction may be formed by assembled insoluble PSP.

Mediastinal Pancreatic Pseudocyst Caused by Obstruction of the Pancreatic Duct Was Eliminated by Bromhexine Hydrochloride

A 49-year-old man, who had a 30-year history of drinking the equivalent of 80 g of ethanol per day, underwent a detailed medical examination for cough and dyspnea. Chest-abdominal computed tomography and endoscopic retrograde pancreatography led to the diagnosis of a mediastinal pancreatic pseudocyst resulting from obstruction of the pancreatic duct by a protein plug. The pseudocyst rapidly improved with conservative treatment with camostat mesilate, H2-receptor antagonist and digestive enzymes. Although the patient abstained from alcohol for approximately 6 months, he resumed drinking, leading to recurrent attacks of pancreatitis. Bromhexine hydrochloride was then administered for 6 months, with the expectation that it would have a mucolytic effect on the pancreatic juice, resulting in improvement in the clinical symptoms, pancreatic enzymes and pancreatic exocrine function, as well as elimination of the protein plug. Bromhexine hydrochloride may be a new therapy for pathological states, such as alcoholic chronic pancreatitis, in which there is increased viscosity of the pancreatic juice because of elevated protein concentration, leading to protein plug formation and temporary blockage of the pancreatic duct.

Pancreatic complications in choledochal cyst and their surgical outcomes.

Follow-up results were analyzed to evaluate the surgical managements of pancreatic complications such as pancreatitis and protein plug formation in patients with choledochal cysts. Sixty-two patients with choledochal cysts treated between 1976 and 1999 were reviewed. Twenty-four were children and 38 were adults. Fifty-four patients showed primary cases. Cyst excision and hepaticoenterostomy were finally performed in 56 patients. Surgical sphincteroplasty or endoscopic sphincterotomy was performed to prevent recurrent protein plugs in six patients. The follow-up period was 8.1 +/- 6.1 years. Acute pancreatitis and protein plug formation was observed in 18 (33.3%) and 11 (20.4%) of 54 patients showing primary cases, respectively. Both acute pancreatitis and protein plug formation were observed more frequently in children from 1 to 15 years of age (70.6% and 41.2%, respectively) than in adults (18.6% and 12.5%, respectively). Acute pancreatitis and/or protein plug formation developed in four (57.1%) of seven patients who underwent cystenterostomy. Protein plug formation in the residual cyst after cyst excision was observed in two patients, one of whom had undergone sphincteroplasty. Diabetes mellitus due to chronic pancreatitis developed in one patient who was diagnosed late. No other pancreatitis or protein plug recurred postoperatively in this series. Our results suggested that cystenterostomy did not resolve pancreatic complications of choledochal cysts, and that surgical sphincteroplasty was ineffective in preventing the recurrent protein plug formation in the residual duct. In conclusion, complete cyst excision and an early diagnosis are necessary to prevent the development of chronic or recurrent pancreatitis after surgery.

Pathogenesis of Alcohol-Induced Chronic Pancreatitis: Facts, Perceptions, and Misperceptions

The pathogenesis of chronic pancreatitis secondary to chronic alcoholism is not fully understood. A major hurdle in the understanding of the pathogenesis is the inability to study early lesions of the pancreas and the sequential changes. Facts are few; observations are many. Each new hypothesis argues against all previous hypotheses; however, clinical chronic pancreatitis is initiated by one or more of the mechanisms. Good experimental models for alcoholic pancreatitis are not available, limiting the ability to study the pathogenesis. Additional studies on genetic markers and immunologic mechanisms might explain acinar cell injury, which seems to be the earliest lesion in most, if not all, types of chronic pancreatitis. Opie's common channel and obstruction regurgitation theories seem unrelated to chronic pancreatitis. Although biochemical changes of the pancreatic secretion in alcoholic patients promote protein-plug formation, evidence is too weak to consider protein plug as the earliest change. The theory of necrosis of the acinar cell by some unknown mechanism, subsequently leading to fibrosis, is gaining support; however, it is clear that the pathogenesis of alcoholic pancreatitis is not yet fully understood.

Pathologic changes in the common bile duct of an experimental model with pancreaticobiliary maljunction without biliary dilatation.

Pancreaticobiliary maljunction (PBM), a congenital anomaly, causes regurgitation of pancreatic juice into the biliary tract, where it exerts a hazardous influence. However, changes in the common bile duct (CBD) remain obscure due to a lack of suitable experimental models. Using cats, we have developed an experimental model of PBM without bile-duct dilatation that allows the pure effects of PBM to be studied. Histologic and cellular kinetic changes in the CBD were analyzed in 6 controls and 9 experimental animals that survived for more than 6 months. CBD sections were stained with hematoxylin and eosin and a monoclonal antibody to the proliferating cell nuclear antigen (PCNA). Invaginations of the bile-duct epithelium or parietal sacculi increased, and peribiliary glands were well-developed. PCNA-positive cells significantly increased in the CBD, especially in the parietal sacculi and glands. It is concluded that PBM increases the cell cycle in CBD epithelium and subsequently developed peribiliary glands. These developed glands may be associated with the formation of protein plugs, often seen in patients with PBM.

Chronic ethanol administration decreases rat pancreatic GP2 content

Postulated mechanisms of alcoholic pancreatitis include (i) zymogen granule fragility facilitating intracellular activation of digestive enzymes and (ii) ductular obstruction by protein plugs. GP2, a pancreatic glycoprotein, stabilizes zymogen granule membranes and is an important constituent of pancreatic protein plugs. Therefore, this study examined the pancreatic content and messenger RNA levels of GP2 after chronic ethanol administration. Rats were fed liquid diets with or without ethanol, for four weeks. GP2 levels in pancreatic homogenates, crude zymogen granules and zymogen granule membrane fractions were assessed by immunoblotting. Messenger RNA levels for GP2 were measured by Northern and dot blotting of pancreatic RNA. Pancreatic GP2 levels were lower in ethanol-fed rats than in controls (GP2 levels expressed as % of control: 38.75±5.8, p<0.001 in homogenate; 31.28±3.5, p<0.0005 in crude zymogen granules and 22.89±5.4, p<0.0005 in zymogen granule membranes). Messenger RNA levels for GP2 were unchanged after ethanol feeding. Chronic ethanol consumption decreases GP2 content of pancreatic homogenate and zymogen granules. This decrease could (i) result from an increased release into pancreatic juice thereby favouring protein plug formation and (ii) impair zymogen granule stability. Both these mechanisms could potentiate pancreatic damage.

Massive pancreatic pleural effusion: Pathogenesis of pancreatic duct disruption

Massive pancreatic pleural effusion is believed to be caused by pancreatic duct disruption. To elucidate the cause of the duct disruption, pancreas specimens resected from three cases of massive pancreatic pleural effusion were investigated histopathologically. There was no evidence of either chronic or acute pancreatitis except focal fibrosis along a single dilated duct in the resected pancreas. From our study, the pathogenesis of pancreatic duct disruption is suggested as follows: (a) alcohol ingestion can induce focal acute inflammation on a single branch of the duct system and elicit protein plug formation; and (b) focal stenotic change occurs on this branch at a point near the main pancreatic duct, and transient obstruction can occur by means of the protein plugs. The “upstream” extent of this branch will then dilate until it ruptures. However, on the frequency of this atypical pancreatitis, we have no clues to mention.

Structural and Functional Effects of Long-Term Alcohol Administration on the Dog Exocrine Pancreas Submitted to Two Different Diets

Our purpose was to study the influence of two different levels of dietary protein and fat on the action of chronic alcohol feeding on the exocrine pancreatic secretion and the pancreatic morphology of conscious dogs. Ten animals were provided with gastric and duodenal cannulas. Five of them (group H) received a high-protein (39% of calories), high-fat (34%) diet, and the five others (group L) a moderately low-protein (15%), low-fat (20%) diet. Animals were housed in closed kennels lightened with artificial light and did not have free access to sunlight. Five series of experiments were performed just before and 5 and 12 months after daily alcohol administration through the gastric cannula (2 g/kg/day). Volume, bicarbonate, and protein were measured under basal conditions after intragastric ethanol infusion (1.5 g/kg), under hormonal stimulation with 1 clinical unit (CU)/kg/h secretin or 1 CU/kg/h secretin plus 3 Crick Harper Rate (CHR) U/kg/h cholecystokinin (CCK), before and after intravenous ethanol 1.3 g/kg for 20 min, and after intragastric ethanol (1 g/kg) given with a meal. Group H was the most sensitive to the action of chronic alcohol feeding. At the end of 1 year of alcohol administration, volume and bicarbonate were not affected, but protein secretion was significantly increased in basal conditions and under secretin infusion, but not under CCK infusion or in response to a meal. The secretory pattern of these dogs was different from the response of dogs studied in previous experiments having the same diet but housed in an open kennel and having free access to outside and sunlight. In group L, protein was less affected, but volume and bicarbonate were significantly decreased 1 year under secretin stimulation. Histological damages were seen in the two groups characterized by a slight periacinar fibrosis and alterations of ductal cells. Acinar and ductal luminae were dilated and filled with protein plugs also present in pancreatic juice and able to stop the flow of juice. At the difference from human beings, these plugs were built up of all secretory protein but not of an insoluble fibrillar molecular form of pancreatic stone protein. This study confirms the role of chronic alcoholism on the formation of protein plugs and shows the influence of nutritional and environmental conditions.

Alcoholic pancreatitis in rats fed ethanol in a nutritionally adequate liquid diet

In an effort to develop a model of chronic alcoholic pancreatitis in Sprague-Dawley rats fed a nutritionally adequate diet, 3 groups of 15 animals each were fed Wayne Rodent-Blox ad libitum, Lieber-DeCarli diet with 40% of carbohydrate calories replaced by ethanol ad libitum and isocaloric amounts of Lieber-DeCarli diet respectively for a period of 18 months. Rats were anesthetized and basal and secretin-stimulated pancreatic juice was obtained. Pancreatic glands were isolated and divided into portions for histology, biochemical analyses, and cell fractionation. The homogenate, zymogen granule fraction, mitochondrial-lysosomal fraction, microsomal fraction and postmicrosomal supernatant as well as aliquots of pancreatic juice were analyzed for cathepsin B, acid phosphatase, beta-D-glucoronidase, arylsulphatase and leucine naphthylamidase. All of the ethanol-fed animals developed morphological changes akin to human chronic pancreatitis. There were focal areas of parenchymal degeneration with fibrosis, protein plug formation and tubular complexes. In the pancreatic tissue of animals fed ethanol, total protein, trypsinogen (and free trypsin) were increased and amylase was decreased. While acid phosphatase was increased in all of the particulate fractions, cathepsin B was increased in the zymogen granule and mitochondrial-lysosomal fractions. Basal and post-secretin pancreatic juice did not show a significant increase in digestive or lysosomal enzymes. It is suggested that focal degenerative changes may be due to trypsin generated by intracellular activation of digestive enzymes by lysosomal enzyme cathepsin B.

Protein Content of Precipitates Present in Pancreatic Juice of Alcoholic Subjects and Patients With Chronic Calcifying Pancreatitis

Chronic calcifying pancreatitis is characterized by the formation of intraductal protein plugs or precipitates and calcified stones in ducts. Similar precipitates may be collected by endoscopic retrograde catheterization of the main pancreatic duct. They are present in the pancreatic juice of alcoholic subjects and patients with chronic calcifying pancreatitis. Protein analysis of these precipitates was performed to try to elucidate the mechanism of stone formation. Two protein fraction were separated by extraction of precipitates. One fraction was easily soluble in saline and contained a small amount of most of the proteins of pancreatic juice. The other fraction was soluble in citrate or ethylenediaminetetraacetate and contained a few proteins with close isoelectric points and identical molecular weight (13,500). These proteins showed immunological identity with the "stone protein" isolated from human pancreatic calculi. Our data demonstrate that the major citrate-soluble protein of precipitates in pancreatic juice is identical with "stone protein". They are strongly support the concept that this protein is the organic matrix of pancreatic stones. Different mechanisms are proposed to explain the phenomenon of protein precipitation that frequently occurs in alcoholic subjects and patients with chronic calcifying pancreatitis.

Pancreatic citrate and protein secretion of alcoholic dogs in response to graded doses of caerulein.

Previous studies have shown dissolution of human pancreatic stones in vitro by citrate solution which bind considerable amounts of ionized calcium. Pancreatic citrate secretion has been demonstrated in canine and human pancreatic juices. This study compares pancreatic citrate secretion of chronic alcohol-fed dogs with controls in response to graded doses of caerulein, in order to evaluate possible differences in factors favouring pancreatic lithogenicity. The dose-response-relation of citrate outputs after graded doses of caerulein revealed significantly reduced maximal secretory capacity of citrate in alcoholic dogs. Protein concentrations in juices from alcoholic dogs were reduced for all doses of caerulein but protein outputs were not different. Bicarbonate concentrations and outputs, as well as volumes, were significantly greater in alcoholic dogs. Linear relation were found between citrate and protein secretion. Chronic alcohol consumption in the dog leads to reduced citrate secretion, which is consistent with recent results in humans suffering from chronic calcifying pancreatitis, who secrete significantly less citrate than healthy subjects. The decrease of calcium-chelating citrate could be an additional factor causing increased calcium levels in the pancreatic juice of chronic alcoholics, a circumstance that might favour protein-plug formation and subsequently pancreatic stone formation.

Scanning electron-microscopic studies on protein plugs obtained from patients with chronic pancreatitis.

The previous histochemical study on the endoscopically obtained protein plugs revealed the process of protein plug formation. The present scanning electron-microscopic study was performed to clarify the fine structure of the protein plug and was quite in accordance with the results of the histochemical study: (1) protein plugs were constructed of interlacing networks and amorphous material stuffed in the networks; (2) protein plug formation started in the form of random and loose interlacing of cottony network fibrils and poor stuffing with degenerating epithelial cells and amorphous material, and in this stage protein plugs enlarged by adherence of smaller aggregates; (3) with maturation, protein plugs came to be covered by stratiform fine net works which were more compactly built up with uniformly thick, dense and smooth fibers and were stuffed more densely with amorphous material; (4) mature protein plugs were considered to enlarge by repeated process of stratiform deposit of reticular substance and amorphous material and subsequent maturation. However, it remains to be clarified which factors induce the changes of networks from random interlacing to stratiform arrangement.

Histochemical studies on protein plugs obtained by endoscopic retrograde catheterization of the papilla.

In order to elucidate mechanisms of protein plug formation, histochemical studies were performed on aggregates and protein plugs present in pancreatic juice. Pancreatic juice was obtained from three control subjects and five patients with chronic pancreatitis through endoscopic retrograde catheterization of the papilla. Specimens for staining were prepared in two ways: (1) fixed with 10 per cent formaldehyde, embedded in paraffin and sectioned, and (2) placed on slide glass and fixed with isopropylalcohol. Staining included hematoxylin-eosin, periodic-acid Schiff, von Kossa, alcian blue, toluidine blue and double staining with PAS and AB. The process of protein plug formation can be as follows: (1) a prerequisite for aggregate formation, consisting of clusters of desquamated epithelial cells, highly concentrated sulfated acidic mucopolysaccharide and neutral mucopolysaccharide, (2) formation of aggregates in which epithelial cells and amorphous substance are interlaced with developing fine reticular substance, (3) enlargement of aggregates by fusion with adjacent aggregates through bridging action of the reticular substance sprouting, like prickles, from their surface, and (4) "maturity" of aggregates, taking a three-dimensional form which result in a spherical, spheroidal or cylindrical protein plug.