Jun Ida

Effect of a new immunosuppressive agent, FTY720, on survival of islet allografts.

A newly developed immunosuppressant, FTY720, has a unique mechanism that is quite different from those of conventional immunosuppressants, and is presumed to be mediated through decreases in the number of peripheral lymphocytes, especially helper T cells. This study was performed to ascertain whether this innovative drug could prolong islet allograft survival. The donors were inbred Lewis rats and the recipients were ACI rats rendered hyperglycemic with intravenous streptozotocin. In the study group, FTY720 dissolved in distilled water was orally administered at a dose of 5 mg/kg to the recipient ACI rats 1 day before and on the day of grafting. In the control group, only distilled water was orally administered to the recipient ACI rats on the day before and the day of grafting. Two thousand islets were transplanted into the portal vein of the recipient rats in the study and control groups immediately after isolation. The graft survival time in the study group was significantly longer than that in the control group, indicating that FTY720 retains a potent effect on the prolongation of islet allograft survival. FTY720 could become a useful immunosuppressant for future clinical islet allotransplantation.

Expression of pancreatic duodenal hoemobox-1 in pancreatic islet neogenesis after surgical wrapping in rats

BACKGROUND Surgical wrapping (SW) of the pancreas causes islet neogenesis in rodents. Pancreatic duodenal hoemobox-1 (PDX-1) is one of the transcriptional factors needed by pancreatic stem cells to develop a mature pancreas. The purpose of this study was to determine whether islet neogenesis arises from ductal cells and whether PDX-1 is involved in this process. METHODS SW consisted of nonocclusive wrapping of the pancreas in rats. The wrapped pancreas was then harvested, insulin content was measured, and immunohistochemical analysis for insulin, cytokeratin, and PDX-1 was performed. RESULTS The endocrine area of the wrapped pancreas significantly increased after SW. Double immunostaining identified cells positive for both insulin and cytokeratin in or along the epithelial cell lining of the ductal structures and in the centroacinar cells. PDX-1-positive cells were detected in both control islets and islets examined after SW, but these cells were observed in the exocrine area only after SW. Double staining also showed that cells positive for PDX-1 but negative for insulin were present in the exocrine area 1 day after SW and that cells positive for both PDX-1 and insulin had developed 3 days after SW. CONCLUSIONS In the process of adult islet neogenesis after SW, cells in the acini and ductal structures developed into PDX-1-expressing cells, supposedly progenitor cells, which in turn became insulin-producing cells and thus might be the origin of small islets.

Improved Large-Scale Isolation of Breeder Porcine Islets: Possibility of Harvesting From Nonheart-Beating Donor

To establish a large-scale isolation procedure for adult porcine islets usable as a donor source for xenotransplantation and as a model of human islet isolation, we improved several characteristics of the conventional isolation procedure. At a slaughterhouse we first selected a breeder pig over 1.5 years old (and over 200 kg in weight) with warm ischemic time (WIT) of 15 +/- 2 minutes as nonheart-beating donors. Then, we made a special enzymic mixture that consisted of collagenase S-1 (260 U/mg, NittaZelatin, Japan), collagenase P (1.86 U/ml Lyo Boehringer-Mannheim, USA), DNase (Sigma, St. Louis, Mo), Disparse (NittaZelatin, Japan), and protease inhibitor (Sigma). Third, this mixture was injected very gently into the pancreatic duct at the time of pancreatic harvesting. To prevent overdigestion of the pancreas, the mixture was first cooled to less than 10 degrees C. Fourth, during the warm digestion of pancreas, the pancreas with the enzymic mixture was quietly put in a water bath at 37 degrees C without mechanical shaking. Fifth, we purified the islets with a COBE 2991 cell processor by the Dextran 70 gradient method, because Dextran 70 is very cheap and has the same purification effect as the Ficoll gradient. The results of 10 consecutive breeder porcine islet isolations are reported. The total yield of isolations of islets over 50 microm in the longest diameter after staining with Dithizone (DTZ) was 85,900 +/- 19,954 islets, 291,667 +/- 240,452 IEQ (2,900 +/- 2,324 IEQ/g). The purity of the isolated islets was very high: 90.2 +/- 3.8%. Glucose stimulation during in vitro incubation induced significant insulin release from isolated breeder porcine islets. In two of the diabetic rats receiving encapsulated islets grafts using a mesh-reinforced polyvinyl alcohol hydrogel bag (MRPB), a prominent reduction in serum glucose levels (less than 200 mg/dL) persisted for 13 and 19 days, respectively, after intraperitoneal xenotransplantation islets without immunosuppression. In conclusion, we succeeded in a more efficient and less-expensive isolation of a large amount of adult porcine islets from a nonheart-beating donor.

Immunohistochemical analysis of apoptosis-related proteins in human embryonic and fetal pancreatic tissues

SummaryBackground. The growth of both cancer cells and fetal tissue is rapid; however, cancer cells de-differentiate and proliferate in a disorderly manner, whereas fetal tissues differentiate and proliferate in an orderly manner. Thus, there may be both common and different factors that are involved in the process of the uncontrolled cell growth of pancreatic cancers and the development of the fetal pancreas. The common part of the mechanisms should be in the regulation of the cell cycle, resulting in rapid proliferation via such mechanisms as growth stimulation and avoidance of apoptosis. Therefore, in the current study we investigated the expression of apoptosis-related proteins in fetal pancreatic tissues.Methods. Sixteen human embryonic and fetal pancreatic tissues obtained between 6 and 32 wk of gestation were used. We immunohistochemically examined the protein expression of Bcl-2, Bcl-XL, Mcl-1, and Bax. Further, the expression of insulin, glucagon, and proliferting cell nuclear antigen (PCNA), and TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining were examined.Results. In embryonic and fetal pancreatic tissues, Bcl-2 was not detected in any type of pancreatic cell (acinar, ductal, or islet). Bcl-XL was expressed in all types of pancreatic cells throughout the gestation. Mcl-1 was expressed in all types of pancreatic components, and strongly expressed in the margin of the islets. Bax, a pro-apoptotic protein, was expressed in all components. PCNA was strongly expressed in the embryonic and fetal pancreas, especially in early stages of gestation; however, TUNEL staining was negative in all samples. At least one antiapoptotic protein was expressed in all types of pancreatic cells.Conclusion. The results of the current study indicate that active proliferation and avoidance of apoptosis take place in embryonic and fetal pancreatic tissues, which may be controlled by particular combinations of apoptosis-related proteins. Among these proteins, Bcl-XL and Mcl-1 may play an important role in the proliferation and differentiation of the embryonic and fetal pancreas.

Expression of METH-1 and METH-2 in pancreatic cancer

PURPOSE METH-1/hADAMTS-1 and METH-2/hADAMTS-8 are recently identified genes that inhibit angiogenesis, and the murine homologue, ADAMTS-1, shows metalloproteinase function. Because the significance of METH-1 and METH-2 has not been determined in solid tumors, we examined the mRNA expressions of these molecules in pancreatic cancer and hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN METH-1 and METH-2 mRNA expressions were identified in six pancreatic cancer cell lines and were quantified by TaqMan reverse transcription-PCR in 18 paired samples of pancreatic cancer and surrounding noncancerous pancreas, and in 14 samples of pancreatic cancer. METH-1 mRNA expression was also examined in 16 pairs of HCC and cirrhotic liver. Vascularity was estimated by CD34 staining. The correlation between clinicopathological factors and METH-1 expression was additionally analyzed. RESULTS Four of six pancreatic cancer cell lines expressed METH-1, and 1/6 expressed METH-2. METH-1 was substantially expressed in both pancreatic cancer and noncancerous pancreas, but METH-2 was not. METH-1 expression in pancreatic cancer tissue was significantly lower than that in noncancerous pancreas (P = 0.002), and a similar result was obtained between HCC and cirrhotic liver (P = 0.003). METH-1 expression did not show a significant correlation with vascularity in pancreatic cancer or in HCC. However, pancreatic cancer with higher expression of METH-1 showed significantly severe lymph node metastasis or retroperitoneal invasion (P = 0.033 and P = 0.018, respectively) and worse prognosis (P = 0.038). CONCLUSIONS METH-1, which was initially reported to have a potent antiangiogenic effect, does not seem to be a predominant determinant of tumor vascularity in pancreatic cancer. Rather, METH-1 seems to be involved in progression of pancreatic cancer through local invasion and lymph node metastasis.

Agonistic anti-Fas antibody has the growth inhibitory effect on pancreatic cancer cells independent of DcR3

(BACKGROUND) Agonistic anti-Fas antibody (CH-11) has been reported to have little growth inhibiting potency to almost all pancreatic cancer cell lines (PCCs). DcR3 is antagonistic soluble receptor against Fas ligand, which belongs to a new TNF superfamily. Genomic amplification and mRNA expression of DcR3 have been reported in colon and lung cancers. However no information is available of the DcR3 status in PCCs. Here in the study we examined the effect of CH-11 in the presence of IFN,yand DcR3 genomic amplification and mRNA expression in PCCs. (METHORD) 1) PCCs (AsPC-1, BxPC-3, Capan-2, CFPAC-1, HPAC, MIA PaCa-2) were incubated with various concentrations of CH-11 after pretreatment with IFN~. Attached ceils were harvested and counted using Coulter Counter. 2) Genomic DNA was extracted from PCCs and quantitative PCR was performed using TaqMan system. The ratio of DNA copy number of DcR3 to beta actin was calculated. 3) mRNA was isolated from subconfluent PCCs and converted to cDNA. Quantitications of mRNA of DcR3 and beta actin were carried out with TaqMan RT-PCR. (RESULT) 1) CH-11 suppressed the growth of PCCs except for AsPC-1 to various extents when pretreated with IFN-/. 2) BxPC-3 and HPAC showed DcR3 genomic amplification (2.6 + 0.4, 2.9 + 0.4 times). Genomic amplification of DcR3 and the sensitivity to CH-11 were not correlated in PCCs. 3) Capan-2 and CFPAC-1 strongly expressed DcR3 mRNA. DcR3 mRNA expression and the sensitivity of CH-11 were not correlated in PCCs. Furthermore genomic amplification and mRNA expression of DcR3 were not correlated each other in PCCs. (CONCLUSION) Agonistic anti-Fas antibody inhibited the growth of pancreatic cancer cell lines in the presence of IFN~. Several pancreatic cancer cell lines strongly expressed DcR3 mRNA, while DcR3 genomic amplification and mRNA expression are not related to the sensitivity to CH-11 in pancreatic cancer cells. The expression of DcR3 is not involved in the inhibitory effect of CH-11 on pancreatic cancer cell growth.

Desulfation vs. deprotection: direct solid-phase synthesis of Tyr(SO3 H)-containing peptides using the Sn1-type deprotection procedure

Recent advances in the Fmoc-chemistry made the direct synthesis of the sulfated peptide possible using the solid-phase method, however, even when the Fmocstrategy is adopted, conditions for the final cleavage/deprotection with acidic reagents becomes very crucial. During the course of our efforts to find a general synthetic method for the Tyr(SO3 H)-containing peptides, we noticed that TFA is not a destructive acid towards Tyr(SO3 H) as long as the treatment is conducted at low temperature [1]. Based on this finding, we used 90% aqueous TFA at low temperature as the deprotection system for Tyr(SO3 H)-containing peptides. In this study, we also performed kinetic studies on the desulfation reaction and the deprotection reactions to understand this deprotection system.