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.

Transgenic pig expressing the red fluorescent protein kusabira-orange as a novel tool for preclinical studies on hepatocyte transplantation.

INTRODUCTION: Research on hepatocyte transplantation as an alternative or supplementary treatment for liver transplantation is progressing. However, to advance to clinical trials, confidence in the technique must be established and its safety must be validated by conducting experiments using animals of comparable sizes to humans, such as pigs. We used transgenic pigs expressing red fluorescence protein for investigating the distribution and survival of transplanted cells. MATERIALS AND METHODS: Donor hepatocytes were isolated from transgenic Kusabira-Orange (KO)-expressing pigs (age, 41 days; weight, 10 kg) created by in vitro fertilization using sperm from a transgenic-cloned KO pig by Matsunari et al. and ova from a domestic pig. The hepatocyte transplant recipients were the nontransgenic, KO-negative littermates. In these recipient pigs, double lumen cannulae were inserted into the supramesenteric veins to access the hepatic portal region. KO-positive donor hepatocytes from the transgenic male pig were isolated using collagenase perfusion. Hepatocytes (1 × 10(9) cells) were transplanted through the cannula. For estimating allogeneic immunogenicity, full-thickness skin (3 × 3 cm) from the same donor was grafted orthotopically on the neck region of the recipients. Immunosuppressive treatment was not implemented. The recipient pigs were humanely killed at 7 and 39 days after transplantation, and the organs were harvested, including the lungs, heart, liver, pancreas, and kidneys. RESULTS: Strong red fluorescence was detected in both the parenchymal and nonparenchymal hepatocytes of the transgenic male donor pig by fluorescent microscopy. Transplanted cells were detected in the liver and lung of the recipient pigs at 7 days after perfusion. Hepatocytes remained in the liver and lung of recipients on day 39, with lower numbers than that on day 7. CONCLUSION: Transgenic pigs expressing the fluorescent protein KO serve as a useful model of cell transplantation in preclinical studies.

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.

Prevalence of Japanese dialysis patients with an A-to-G mutation at nucleotide 3243 of the mitochondrial tRNA(Leu(UUR)) gene.

BACKGROUND: A high prevalence of an A-to-G mutation at nucleotide 3243 of the mitochondrial genome in patients with diabetes mellitus (DM) and/or deafness has been reported previously. We investigated the prevalence of this mutation in Japanese dialysis patients with associated DM and/or deafness. METHODS: We studied 106 dialysis patients with DM, 26 with DM and deafness, and 26 with deafness alone, using peripheral leucocytes to detect an A-to-G transition at nucleotide 3243 of the mitochondrial gene. RESULTS: We identified this transition in 1 of 26 patients with DM and deafness. None of the 106 DM or 26 dialysis patients with deafness but no DM was positive for this mutation. A 42-year-old male patient on continuous ambulatory peritoneal dialysis (CAPD) who carried this mutation had a 20-year history of sensory hearing loss as well as hypertrophic cardiomyopathy. CONCLUSION: We found that a mitochondrial gene mutation at nucleotide 3243 was present in one dialysis patient with NIDDM and deafness. The prevalence of this mutation was found to be below 1% in diabetic end-stage renal disease patients in Japan.

Hepatocyte injury in tyrosinemia type 1 is induced by fumarylacetoacetate and is inhibited by caspase inhibitors.

Tyrosinemia type 1, caused by mutations in the fumarylacetoacetate hydrolase gene (Fah), is characterized by severe liver injury. We earlier developed a tyrosinemic mouse model with two genetic defects, Fah and 4-hydroxyphenylpyruvate dioxygenase (Hpd) deficiencies. Apoptosis of hepatocytes was induced and an acute onset of liver failure occurred after administration of homogentisic acid (HGA), the intermediate metabolite between the enzymes HPD and FAH. Cytochrome c was released from mitochondria prior to liver failure in the Fah-/- Hpd-/- double-mutant mice after the administration of HGA. In a cell-free system, the addition of fumarylacetoacetate induced the release of cytochrome c from the mitochondria. We also found that caspase inhibitors were highly effective in preventing the liver failure induced by HGA in the double-mutant mice. Therefore, fumarylacetoacetate apparently induces the release of cytochrome c, which in turn triggers activation of the caspase cascade in hepatocytes of subjects with hereditary tyrosinemia type 1.

Intracellular distribution of the Wilson's disease gene product (ATPase7B) after in vitro and in vivo exogenous expression in hepatocytes from the LEC rat, an animal model of Wilson's disease.

In patients with Wilson's disease, both copper incorporation into ceruloplasmin and excretion of this metal into bile are impaired. These conditions are caused by a genetic defect in the Wilson's disease gene (ATP7B). To investigate the Wilson's disease gene protein (ATPase7B) in hepatocytes, we constructed an expression plasmid carrying full-length complementary DNA for human Wilson's disease gene and attempted to express the gene in hepatocytes of LEC rats, an animal model of Wilson's disease. Transfection of hepatocytes, either in vitro or in vivo, was done using a newly developed cationic liposome containing 1,4-bis(3-(N-hexadecyl) aminopropyl) piperazine. Immunological analyses of human ATPase7B with specific monoclonal antibodies showed human ATPase7B to be a membrane protein with a molecular mass of 155 kd. Analysis of human ATPase7B expressed in hepatocytes from LEC rats suggested that this protein is present in the trans-Golgi network and at the plasma membrane, a distribution pattern similar to that of Menkes' disease protein (ATPase7A). These findings suggest that these two putative copper-transporting P-type ATPases function similarly at the cellular level. Cotransfection and coexpression of the human Wilson's disease gene and ceruloplasmin gene in cultured hepatocytes indicate that the distribution of ceruloplasmin is always accompanied by ATPase7B at the perinuclear region, but that part of ATPase7B localizes irrespective of the distribution of ceruloplasmin. Based on these investigations, we propose that ATPase7B exists in the trans-Golgi network and transports copper into this compartment. This seems to ensure an appropriate delivery of copper to the apoceruloplasmin. On the other hand, part of ATPase7B that is not accompanied by ceruloplasmin in the perinuclear region and at the plasma membrane seems to contribute to efflux of this metal from the hepatocytes. Thus the distribution patterns of ATPase7B in hepatocytes may explain the dual roles of this P-type ATPase in hepatocytes.

Usnic acid and diffractaic acid as analgesic and antipyretic components of Usnea diffracta.

Diffractaic acid and usnic acid were identified as the analgesic and antipyretic components of a lichen, Usnea diffracta. Both compounds showed an analgesic effect by the acetic acid-induced writhing and tail-pressure methods in mice. Regarding the effect on normal body temperature and LPS-induced hyperthermia in mice, diffractaic acid showed a significant effect only on the former and usnic acid only on the latter.

Pharmacologically active components from a Peruvian medicinal plant, huira-huira (Culcitium canescens H. & B.).

The methanol extract of Huira-Huira (Culcitium canescens) showed analgesic effects in acetic acid-induced writhing and tail pressure tests, and it also produced potent prolongation of hypnosis induced by pentobarbital. The latter activity was used as an isolation-guide to determine the active components which were identified as dehydrocacalohastine, cacalohastine and cacalonol.

Ascaridole as a pharmacologically active principle of "Paico," a medicinal Peruvian plant.

"Paico," Chenopodium ambrosioides L., is a traditional Peruvian medicine which is considered to be nervine, antirheumatic, anthelmintic, etc. An attempt was made to isolate the component having sedative and/or analgesic properties from "Paico" and "Aritasou" (the Japanese name for C. ambrosioides). Ascaridole was identified as the active principle in both materials.

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.

Establishment of an enzyme immunoassay using monoclonal antibody (HG1-219) and its application for the diagnosis of hepatocellular carcinoma.

A monoclonal antibody (MoAb HG1-219) against a human gastric cancer cell line (HuG-1) and its shedding antigen (HG1-219 Ag) was generated and a solid-phase sandwich enzyme immunoassay (EIA-219) was developed. The mean serum HG1-219 Ag concentration in normal individuals was 30.5 +/- 14.5 U/ml measured by EIA-219. When the mean +3 SD of the antigen concentration in normal individuals was used as a cut-off level, 4.3% (2/47) of patients with chronic hepatitis, 9.1% (4/44) of cirrhotic patients and 37.5% (18/48) of patients with hepatocellular carcinoma (HCC) had HG1-219 Ag above the cut-off value. The positive rates of a-fetoprotein (AFP) (> 400 ng/ml) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) for HCC were 26.7% (12/45) and 33.3% (12/36), respectively. There was no significant correlation between HG1-219 Ag and AFP or PIVKA-II in patients with HCC. The combination assay of EIA-219, AFP and PIVKA-II for HCC gave the positive rate of 75% (27/36). The effect of periodic acid on the HG1-219 Ag and the inhibition of EIA-219 by CA 19-9 suggest that the epitope of HG1-219 Ag is a suger chain similar to CA 19-9.

Early cancer of the gastric remnant--with special emphasis on the importance of follow-up of gastrectomized patients.

Seven patients with early cancer of the gastric remnant (group 1) and nine with gastric remnant cancer which developed after surgery for early gastric cancer (group 2), were studied clinicopathologically. The findings can be summarized as follows: In group 1, the majority of cases were classified as type I by gross type. Most lesions were in the posterior wall of the remnant stomach. All lesions were differentiated carcinomas. In group 2, of the nine early gastric tumors resected at the initial surgery, six were present at site A and three at site M. The gross type of the tumor was varied. Six patients underwent a Billroth II, and two a Billroth I, resection. The findings at the second operation (for gastric remnant cancer) showed that five lesions were located in the gastric stump or anastomosed region and four in the posterior or anterior wall of the remnant stomach. Histologically, the lesions included four undifferentiated and five differentiated carcinomas. Of the nine lesions involving the gastric remnant, only two were at an early stage, the remaining seven being at an advanced stage. The outcome was extremely poor in this group. Many of the patients died of carcinomatous peritonitis. In summary, a large percentage of the patients of group 1 had a type I lesion in the remnant stomach. In group 2, more than half of the lesions were located in the gastric stump or at the site of the anastomosis, and the majority of the lesions were at an advanced stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Surgical curability and prognosis for standard versus extended resection for T1 carcinoma of the pancreas.

The prognosis of 185 patients who underwent surgical resection of small carcinomas of less than 2.0 centimeters (T1) of the pancreas in whom either standard or extended resection was used was analyzed. Pancreatic resection was performed upon 98.3 percent of the patients who were more than 49 years old. The location of the tumor was the head of the pancreas in 83.4 percent of the patients, the body of the pancreas in 13.2 percent and the tail of the pancreas in 3.3 percent. Tumor staging revealed 42.9 percent in stage I and the remainder in advanced stage, that is 33.1 percent stage II, 18.8 percent stage III and 5.1 percent stage IV. When standard resection was performed, surgical cure rates were more than 80 percent in stage I, II and III, but in stage IV, it was only 16.6 percent. After extended resection, the surgical cure rates increased even in stage IV (100 percent). There were no significant differences in the overall postoperative cumulative survival rates between standard and extended operation. In stage II, however, a significant better survival rate was observed after extended resection than after standard resection.

[Etiology of idiopathic portal hypertension (IPH)--the role of immunological mechanism in IPH].

The pathogenesis of IPH is not yet clear, but immunological mechanism is suspected to play an etiologic role in IPH. In this study, we investigated immunological abnormalities in human IPH and attempted to produce experimental models of IPH in rabbits sensitized with extract of IPH or normal rabbit spleen. We examined the role of the spleen in this disease. In the peripheral blood of human IPH, we found a reduction in suppressor T lymphocyte activity, increase in the Leu3a/Leu2a or OKT4/OKT8 ratio and some autoantibodies, e.g., antinuclear antibody or antilymphocyte antibody. After splenectomy, these immunological abnormalities tended to normalize. Furthermore, we have experienced some IPH cases with autoimmune diseases. In animal experiment, we succeeded in producing models in rabbits resembling human IPH by repeat injection of IPH or normal rabbit spleen-extract and found elevation factors of the portal vein pressure in the mitochondrial and microsomal fractions of the fixed cells of the spleen. From these results, IPH may be etiologically associated with an immunological mechanism and the spleen may play an important role in this disease.

[Clinicopathological and histochemical studies of linitis plastica type gastric cancer with special reference to early gastric cancer in the region of the fundic gland].

Early gastric cancer in the region of the fundic gland was found to occur predominantly in women. Many lesions of this type were identified in the posterior wall of the middle portion of the stomach. Histologically, the lesions were classified as poorly differentiated carcinomas in all cases of early cancer. In 9 patients with cancer of linitis plastica type having IIc lesions located in the region of the fundic gland, the tumor focus was also in posterior wall of the middle portion of the stomach. Giant folds were present diffusely in all other portions of the gastric mucosa in these latter cases. These cases also had diffuse infiltration of cancer cells into the submucosa, muscle layer and extraserosal regions. Histologically, the lesions were classified in all cases of linitis plastica type as poorly differentiated carcinomas including signet ring cells. Acid mucopolysaccharides (AMPS) and sialic acid were histochemically detected in the interstitial tissues of early gastric carcinomas and linitis plastica type gastric cancers. The AMPS digestion rates were higher for early cancer tissue. The present results support the hypothesis that early gastric cancer in the region of the fundic gland may occur in conditions favoring tumor growth and may develop into cancer of linitis plastica type.

Effect of fat emulsion in patients with liver disorders.

The effect of fat emulsion in preoperative patients (n = 17) with liver disorders was studied by liver-function tests, intraoperative liver biopsy specimens, intravenous fat tolerance tests, and serum fatty acid analyses. There was no difference between liver-function tests before and after the administration of fat emulsion. Before the administration of fat emulsion, the levels of serum lipids were relatively low, and serum fatty acid patterns were abnormal. However, after the administration of fat emulsion, serum lipid levels increased, and serum fatty acid patterns became nearly normal. There was a significant correlation between the total fat intake and serum linoleic acid change before and after the administration of fat emulsion. From this equation, the total fat intake required for a normal range of serum linoleic acid was calculated to be 7 g/kg fat. These results suggest that the administration of fat emulsion improves serum fatty acid patterns without a deterioration of liver function in preoperative patients with liver disorders, but total intake of 10% fat emulsion greater than 70 ml/kg might induce higher serum fatty acids than in healthy control subjects and cause overload in these patients.

The possibility of diagnosing small pancreatic cancer (less than 4.0 cm) by measuring various serum tumor markers. A retrospective study.

Comparative studies measuring various tumor markers such as SPan-1, CA 19-9, carcinoembryonic antigen (CEA), DUPAN-2, and elastase I were done in 74 patients with small pancreatic cancer including 23 cases of T1 pancreatic cancer (tumor size less than 2.0 cm) and 51 cases of T2 pancreatic cancer (tumor size between 2.1 to 4.0 cm), retrospectively. Although the mean value of these tumor markers in T1 and T2 pancreatic cancer were higher than those of the control cutoff levels, their sensitivities were different. In T1 pancreatic cancer, 13 of 23 cases (56.5%) of SPan-1 and 14 of 23 cases of (60.7%) of CA 19-9 had levels above normal. Although the numbers of patients were small, sensitivities of CEA, DUPAN-2, and elastase I were 30.8%, 22.2%, and 28.6%, respectively. In T2 pancreatic cancer, 41 of 51 cases (80.4%) of SPan-1 and 40 of 51 cases (78.4%) of CA 19-9 showed higher levels than normal, but only 46.9% of CEA, 40.0% of DUPAN-2, and 52.6% of elastase I were positive. The overall sensitivities in small pancreatic cancer (less than 4.0 cm) were 73% for SPan-1 and CA 19-9 but were less for CEA, DUPAN-2, and elastase I. These results indicate that even small pancreatic cancers release detectable pancreatic cancer-associated antigens in serum in more than 70% of cases, especially SPan-1 and CA 19-9. The measurement of these two tumor markers makes it possible to detect small pancreatic cancers after using imaging diagnostic procedures.

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.

Clinicopathological and histochemical studies of linitis plastica type gastric cancer with special reference to early gastric cancer in the region of the fundic gland.

In this study, early gastric cancer in the region of the fundic gland was found to occur predominantly in women. Many lesions of this type were identified in the posterior wall of the middle portion of the stomach. Histologically, the lesions were classified as poorly differentiated carcinomas in all cases of early cancer. On the other hand, in 9 patients with cancer of linitis plastica type having IIc lesions (less than 2.0 cm in diameter) located in the region of the fundic gland, the tumor focus was also in the posterior wall of the middle portion of the stomach. Giant folds were present diffusely in all other portions of the gastric mucosa in these latter cases. These cases also had diffuse infiltration of cancer cells into the submucosa, muscle layer and extraserosal regions. Histologically, the lesions were classified, in all cases of linitis plastica type, as poorly differentiated carcinomas including signet ring cells. Acid mucopolysaccharides (AMPS) and sialic acid were histochemically detected in the interstitial tissues of early gastric carcinomas and linitis plastica type gastric cancers. The AMPS digestion rates were higher for early cancer tissue. The present results support the hypothesis that early gastric cancer in the region of the fundic gland may occur in conditions favorable to tumor growth and may develop into cancer of linitis plastica type.