An Effective New Cryopreservation Procedure for Pancreatic Islets Using Hollow Fiber Vitrification.

The present study aimed at establishing a new cryopreservation method for mouse pancreatic islets by vitrification using hollow fibers as a container. A unique feature of the hollow fiber vitrification (HFV) method is that this method achieves stable vitrification using a minimum volume of cryoprotectant (CPA) solution, thereby ensuring high viability of the islets. The cytotoxicity, optimum composition, and concentration of the CPAs for vitrifying islets were examined. The viability, functional-integrity of vitrified islets were evaluated in comparison with those vitrified by conventional methods. Insulin secretion was measured in vitro by a static incubation assay and the metabolic functions was tested after transplantation into Streptozotocin-induced diabetic mice. The combination of 15% dimethyl sulfoxide+15% ethylene glycol resulted in the best CPA solution for the HFV of islets. HFV showed the highest viability in comparison to 2 vitrification methods, open pulled straws and vitrification with EDT324 solution. The vitrified islets stably expressed ß-cells markers NeuroD, Pancreatic and duodenal homeobox-1, and MafA. Transplantation of the vitrified islets achieved euglycemia of the host diabetic mice and response to an intraperitoneal glucose tolerance test to a similar extent as non-vitrified transplanted islets. The HFV method allows for efficient long-term cryopreservation of islets.

Advancing pig cloning technologies towards application in regenerative medicine.

Regenerative medicine is expected to make a significant contribution by development of novel therapeutic treatments for intractable diseases and for improving the quality of life of patients. Many advances in regenerative medicine, including basic and translational research, have been developed and tested in experimental animals; pigs have played an important role in various aspects of this work. The value of pigs as a model species is being enhanced by the generation of specially designed animals through cloning and genetic modifications, enabling more sophisticated research to be performed and thus accelerating the clinical application of regenerative medicine. This article reviews the significant aspects of the creation and application of cloned and genetically modified pigs in regenerative medicine research and considers the possible future directions of the technology. We also discuss the importance of reproductive biology as an interface between basic science and clinical medicine.

Cancer-associated alterations of blood group antigen expression in the human pancreas.

Certain alterations of blood group substance (BGS) expression have been observed in gastrointestinal cancer tissues. However, in the pancreas little is known about BGS expression by normal or malignant tissue. The present immunohistochemical study analyzed simultaneously the expression of A, B, H, Lewisa (Lea), and Lewisb (Leb) antigens in specimens of normal pancreas, chronic pancreatitis, and pancreatic carcinoma (primary and metastatic). In normal pancreas all five antigens were expressed in ducts, ductules, and acini, but not in islets. Acinar cells expressed A, B, H, and Leb in supranuclear cytoplasm, whereas Lea was found mainly on centroacinar cells. Only BGSs that were appropriate for the host's blood type were expressed, except for one case of Lea deletion. BGS expression by chronic pancreatitis tissue closely resembled that by normal tissue. In primary pancreatic cancer two cancer-associated alterations were noted that were not found in either normal pancreas or chronic pancreatitis. Deletion of an expected A, B, H, or Leb antigen occurred in approximately 25% of cases, particularly in more poorly differentiated cancers. Incompatible expression of an unexpected A or B antigen occurred in 33% of cases, regardless of degree of differentiation. Metastatic pancreatic cancers also exhibited BGS deletion and incompatibility. In both primary and metastatic cancers the incidence of incompatible A or B expression was higher in cancers from the United States than in cancers from Japan, but the incidence of BGS deletion was similar between the two countries. It was concluded that deletion of A, B, H, or Leb antigens and incompatible expression of A or B antigens are cancer-associated events in the pancreas.

Lex and Ley antigen expression in human pancreatic cancer.

Carbohydrate antigens are useful markers for the serological detection of pancreatic cancer. However, data concerning the expression of structurally well-defined carbohydrate antigens in normal and malignant pancreatic tissue is quite limited. The Lex and Leg antigens are closely related carbohydrate antigens synthesized on type 2 blood group oligosaccharide side chains of glycolipids and glycoproteins. Monoclonal antibodies anti-SSEA-1 and AH6 recognize "simple" Lex and Ley epitopes, respectively, regardless of the length of the carrier carbohydrate. Other monoclonal antibodies recognize Lex (FH4), sialyl Lex (FH6, IB9) or Ley (KH1, CC-1, CC-2) carried only by elongated type 2 side chains with or without internal alpha 1,3 fucosyl substitution. The present comparative immunohistochemical study used tissues of normal pancreas, chronic pancreatitis, and pancreatic cancer to determine the normal expression of Lex and Ley antigens in the pancreas and to elucidate any cancer-associated alterations. Lex-related antigens were not expressed in normal pancreas, expressed in only 10-20% of chronic pancreatitis tissues, but expressed in 50-70% of pancreatic cancer tissues. The frequency of Lex-related antigen expression in pancreatic cancer tissues was lowest in poorly differentiated cancers. Within a given specimen, at least three or all four of the Lex recognizing monoclonal antibodies were simultaneously expressed. Unlike Lex antigens, Ley-related antigens were expressed in 32-77% of specimens of normal pancreas, with similar frequencies in specimens of chronic pancreatitis and pancreatic cancer. In normal pancreas, simple Ley was expressed by both ductal and acinar cells, but extended Ley antigens were expressed only by acinar cells. In pancreatic cancer, extended Ley antigen expression was found in less than 10% of poorly differentiated tumors. Coexpression among the Ley-related antigens was less common than with the Lex-related antigens. Also in cancer specimens, simple Lex and simple Lex antigens were often concordantly expressed, whereas extended Lex and extended Ley antigen expression was often discordant. Hyperplastic ducts and ductules associated with pancreatic cancer expressed Lex-related antigens more frequently than morphologically similar lesions associated with chronic pancreatitis. These results demonstrate that Lex-related antigens are cancer-associated determinants in the human pancreas. The discrepant expression between Lex and Ley antigens in these tissues implies altered regulation of fucosyltransferase activity associated with the malignant state.

New radioimmunoassay for pancreatic cancer-associated antigen span-1 with reference to differential-diagnosis and monitoring in pancreatic-cancer.

Sera from patients with various malignant benign disorders, as well as from a large number of healthy blood donors (2197). were analyzed with a newly developed SPan-1 coated bead radioimmunoassay. Only 0.9% of the healthy patients had SPan-1 levels above the cut-off value. SPan-1 antigen levels were elevated in 89.2% of sera from pancreatic cancer patients and in 75% of T1 and T2 cancers. High diagnostic accuracy was also recognized in differentiating benign pancreatic diseases from pancreatic cancers. SPan-I levels decreased after pancreatic resection and rose again at recurrence of disease. SPan-1 could be distinguished from other tumor markers such as DU-PAN-2 and OC-125 by competition radioimmunoassays and was elevated in individuals with malignant gastrointestinal diseases who had normal levels of either or both CEA and CA 19-9 antigens. This rapid. reproducible and sensitive assay for SPan-I appears to be a useful procedure for the detection and follow-up of pancreatic cancer.

Radioimmunoreactive serum elastase levels and histologic changes during experimental pancreatitis in rats.

A radioimmunoassay system showed elastase levels of 128 to 232 ng/mL in normal rat serum. The average normal value was approximately 64-fold greater than the minimal detectable amount. No cross-immunoreactivity was found with human elastase 1, porcine elastase, and trypsin. The elastase and amylase levels in rat serum were measured during pancreatitis induced by trypsin (group 1) and normal saline solution (group 2). Within 15 minutes the serum elastase and amylase levels increased significantly and remained elevated. The serum elastase levels in group 1 were significantly higher than in group 2. Hemorrhagic pancreatitis was found in group 1 and edematous pancreatitis was found in group 2 five hours after induction of pancreatitis. Elastase levels in these tissues were significantly lower than those in normal tissue. The levels in ascitic fluid were higher than those found in serum.

Effects of bethanechol on the pancreas in antrectomized and normal rats.

The effect of chronic administration of bethanechol and pentagastrin on the pancreas was examined. Rats were either antrectomized or subjected to a sham operation. Three weeks after surgery, rats received a daily intraperitoneal injection of either bethanechol (12 mg/kg) or pentagastrin (250 micrograms/kg) for 14 days. The fasting serum gastrin after bethanechol treatment increased to 1.89 times that of controls treated with saline. Although antrectomy decreased fasting serum gastrin to approximately 40% of controls, serum gastrin increased by 2.17 times that of the antrectomized rats and 1.37 times that of controls in the bethanechol group. After 14 days of bethanechol treatment, weight and amylase in the pancreas increased significantly compared with control; DNA and protein also increased 1.3 and 1.5 times that of control. The increase in DNA and pancreatic weight indicated that hyperplasia was the predominant mechanism. The pancreas showed atrophy in antrectomized rats but this was reversed by both bethanechol and pentagastrin. The results indicate that either endogenous gastric or extragastric gastrin release by cholinergic stimuli may have an important role in the regulation of pancreatic growth.

Effects of cerulein on the normal pancreas and on experimental pancreatic carcinoma in the Syrian golden hamster.

The effects of cerulein on normal pancreas and on N-nitrobis (2-hydroxypropyl) amine (BHP)-induced experimental pancreatic carcinoma in Syrian golden hamsters were studied. Twenty hamsters received a subcutaneous injection of cerulein (20 micrograms/kg). The results showed that when cerulein was injected subcutaneously for 10 days, pancreatic weight and amylase increased. DNA and the pancreatic weight/DNA ratio were also increased significantly in treated hamsters compared with controls (p less than 0.02 versus p less than 0.01). These results indicated that chronic cerulein injection had hypertrophic and hyperplastic effects. DNA synthesis, as measured by histoautoradiography of tritiated thymidine-labeled tissue, increased in pancreatic acinar cells (p less than 0.01) and increased slightly in islet cells and in ductal cells. Tritiated thymidine uptake in the pancreas of the treated group indicated a rather selective exocrine gland incorporation by acinar rather than ductal cells. Sixty hamsters received a subcutaneous injection of BHP (500 mg/kg) once a week, while 63 hamsters received BHP (500 mg/kg) plus cerulein (20 micrograms/kg). Twenty-seven hamsters received cerulein (20 micrograms/kg) alone. All animals were killed from 8 to 27 weeks later, and no cancer-bearing hamsters were observed during the eighth and ninth week following administration. From the 10th to 14th weeks after administration of BHP and cerulein, 87.9% (13 of 15) had tumors compared with 18.7% (3 of 16) after BHP alone (p less than 0.01). One of three and two of 13 tumors were adenoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a new synthetic protease inhibitor on beta-endorphin release during acute pancreatitis in dogs.

The effect on endogenous beta-endorphins of a new synthetic protease inhibitor was studied in acute pancreatitis. Pancreatitis was induced by the injection of autologous bile mixed with trypsin into the main pancreatic duct after ligation of the accessory duct. Plasma beta-endorphin concentrations and cardiovascular function were measured. Ten dogs (control group) were given 10 ml/kg/h of lactate Ringer's solution intravenously beginning 1 h before the induction of pancreatitis and continuing throughout the experiments. Six dogs received an intravenous infusion of 3 mg/kg/h of a new synthetic protease inhibitor, E-3123 (4-(2-succinimidoethylthio)4-geranidinobenzoate methanesuLfonate), in lactate Ringer's solution soon after the induction of pancreatitis. Plasma beta-endorphin concentrations in the control group increased significantly. However, plasma beta-endorphin levels in the protease inhibitor group did not increase as in the control group. The protease inhibitor infusion improved hypotension, myocardial depression, and plasma lactate, suggesting that the inhibitory effect of the protease inhibitor on beta-endorphin release contributed to the improvement.

Malignant T-cell lymphoma of the pancreas.

A case of malignant T-cell lymphoma of the pancreas is presented. Previously reported histological data confirming this disease are reviewed to elucidate features that may suggest this disease and how to differentiate between T- and B-cell lymphoma of the pancreas.

Various tumor markers for small pancreatic cancer with special reference to the present status of pancreatic cancer in Japan and our experience over the past 2 years.

Various tumor markers for detection of small pancreatic cancer less than 4.0 cm of diameter were evaluated and our recent 2 year experience is presented. Even in T1 cancer, 62.5% of the patients (N = 24) had elevated serum CA 19-9 and 56.5% of the patients also had elevated serum SPan-1. However, that of other markers was less than 30% except for CA 50 (60%). In T2 cancer, the highest sensitivity was observed for SPan-1 (79.6%, N = 54) and that of Ca 19-9 (N = 54) was also high (77.7%). That of other markers was less than 50%. The combination assay of CA 19-9 and SPan-1 in T1 cancer increased sensitivity from a single assay of 62.5% and 56.5%, respectively, to 70%. In T2 cancer, the sensitivity of the combination was 97.1%. All of our cases showed positive results using serum SPan-1 and/or CA 19-9 before confirming the diagnosis with imaging procedures. By applying measurements accurately, the test is a very useful adjunct to the diagnosis of small pancreatic cancer and therefore improves its prognosis.

Pancreatic microcirculation of dogs measured by hydrogen gas generated by electrolysis.

Electrochemically generated hydrogen gas was used to measure pancreatic regional blood flow in dogs by Koshu's method. This method is not necessary to inhale H2 gas and it is possible to generate H2 gas from H3O- in tissue in precise areas and to measure pancreatic regional blood flow easily, immediately, and steadily. Reliability and reproducibility were as good as for the H2 gas clearance method. Although the H2 gas clearance method has disadvantages in measuring pancreatic regional blood flow, especially in hypocirculation and in small animals, this new method can be used effectively. It will be useful in the investigation and examination of pathologic conditions in small animals and in humans.

Serial histologic study of the development, progression, and healing of acute pancreatitis in the rat.

This study was undertaken to investigate serially the development, progression, and healing of acute pancreatitis which was induced in rats by the closed duodenal loop technique. Edematous changes appeared within 2 to 4 h. Pancreatic blood flow increased at 1 h and tended to decrease after 3 h. After injection of fesin into the loop, fesin appeared in the periacinar space and acinar cells after 4 h. Electron microscopy showed that partial destruction of the plasma membrane occurred and serum amylase increased considerably at the same time, suggesting that the initial inflammatory changes caused the release of pancreatic enzymes at approximately 4 h. After elimination of the causal factors, edematous pancreatitis healed completely but hemorrhagic acute pancreatitis was not completely healed 6 months later.

Human monoclonal antibody prepared from lymphocytes of pancreatic cancer patients.

We have succeeded in establishing a human monoclonal antibody (MoAb) OC2D against pancreatic cancer cells using lymphocytes from pancreatic cancer patients and a human lymphoblastoid cell line HO323. The reactivity of OC2D using various cell lines and immunoperoxidase staining showed a rather specific effect for malignant cells such as those of pancreatic, gastric, and colon cancers. The antigen recognized by OC2D appeared to be nonsecreting and was mainly found on the cell surface, and various enzyme treatments showed that the epitope was an asialosylated carbohydrate. The hybridoma OC2D has continued to produce human MoAb for 10 months after fusion and grew stably in serum-free medium as well as in serum-containing medium. These results suggest that this human MoAb OC2D is useful for both imaging and targeting therapy.

Electron microscopic studies of human pancreatic adenocarcinoma.

Tissue samples from 12 patients with pancreatic carcinoma were studied by light and electron microscopy. Ten were diagnosed as adenocarcinoma by light microscopy. However, three different electron microscopic findings were observed among these ten adenocarcinomas. One showed that the tumour cells had large nuclei with poorly developed intracellular organelles. Many mucinous granules, well developed cellular projections and intracellular microcysts were observed. In the second findings were different in that these cells had no granules. The intracellular organelles were developed poorly and abundant microvilli and cellular projections were observed. These observations suggest that the tumour cells may arise from pancreatic ductular cells. The third specimen showed a completely different appearance. There were pleomorphic nuclei with enlarged nucleoli and the cytoplasm contained swollen mitochondria, a well developed rough endoplasmic reticulum and varying numbers of zymogen-like granules. Occasionally, the zymogen-like granules were absent. These characteristics resemble the de-differentiation of acinar cells which has been repeated in experimental pancreatic carcinoma. These results suggest that careful examination of human pancreatic carcinoma may show more cells of acinar origin.

An immunohistological study of gastric cancer--with special reference to the expression of carbohydrate antigens and nuclear DNA ploidy patterns.

The expression of carbohydrate antigens specific to Span-1, CA 19.9, and SLEX in cancer tissues and nuclear DNA ploidy patterns were studied from tissue specimens of lesions excised from 137 patients with gastric cancer. The frequency of detection of each antigen was augmented with advanced invasion depth and progress in regional lymph node metastasis. In the cases which were positive for all three kinds of antigens, lymph node metastasis and lymphatic or venous invasion were detected with significantly higher frequencies than in the negative cases. The DNA histograms showed a DNA ploidy pattern of Type Ia or Ib in the cases negative for these antigens and a non-diploid or aneuploid pattern of Type II or III in many of the positive cases. These findings suggest that an immunohistological study of gastric cancer using monoclonal antibodies, combined with a nuclear DNA ploidy analysis, might be useful for understanding malignancy of the tumour.

Gastric carcinoma in young adults in Japan.

Forty-nine cases of young adults with carcinoma of the stomach were reviewed. The occurrence of this tumor had a percentage rate of 2.7 of the total of 1767 Japanese patients with stomach carcinoma, with a male to female ratio of 1.0:0.88. The most common complaint observed was epigastric pain (45.7%). In the majority of patients, the period from the onset of symptoms to the time of diagnosis was over two years (49%). Thirty-nine out of 49 patients had resection of the stomach and a consequent operation was curative in 55.1% of them. The overall 5-year survival rate was 26.8%. However, the 5-year survival rate for the curative operation was 42.6% particularly in early gastric carcinoma without any serosal invasion and no patients died from carcinoma of the stomach. These results indicate that early diagnosis is very important to improve the curing rate of this disease in young adults.

Serum elastase I levels in pancreatic disease.

The radioimmunoassay for human elastase I used in this study is accurate, sensitive, and specific, which we have confirmed. The assay can be done within 4 hours, which is important for clinical purposes. A total o 103 subjects were examined, and levels of 99 to 370 ng/dl (mean 200) in normal human sera were determined. The serum elastase levels in acute, acute relapsing, and chronic relapsing pancreatitis were significantly higher than normal. Although serial determinations returned to normal within 5 days after the onset of the attack, they decreased gradually and remained high on the 7th, 10th, and 11th days in patients who still had residual signs of pancreatitis. The values in patients with chronic pancreatitis and various other diseases were normal. The values in patients with acute pancreatitis were significantly higher than in those with hyperamylasemia of nonpancreatic origin. Twelve of 19 patients with pancreatic cancer had abnormal serum elastase levels; this was especially true in patients with cancer of the pancreatic head. We believe that the measurement of serum elastase levels by radioimmunoassay will become a useful diagnostic method for pancreatic disease in the future.

Pancreatic cystadenoma of the spleen.

A cystadenoma of the pancreas within the spleen from pancreatic heterotopia is described. The tumor was located in the hilum of the spleen and completely separated from the normal pancreatic tail. This fact strongly supports the possibility that this tumor arose from pancreatic heterotopia.

Carcinoembryonic antigen-like activity in gastric juice and plasma in patients with gastric disorders.

Carcinoembryonic antigen (CEA)-like activity in gastric juice and plasma was examined in patients with gastric disease. There was no statistical difference in plasma CEA in normal subjects and in patients with benign gastric cancer. However, four patients with advanced gastric cancer, one with stage III and three with stage IV disease had a very high plasma CEA level. There was significant differences in CEA-like activity in gastric juice in patients with benign gastric disease and early gastric cancer (p less than 0.01) and patients wih benign and advanced gastric cancer (p less than 0.001). These results suggest that measurement of CEA-like activity in gastric juice is a useful adjunct to the diagnosis of early malignant changes in the stomach.