Hiromu Kaji

A newly developed three-layer agarose microcapsule for a promising biohybrid artificial pancreas : rat to mouse xenotransplantation

We examined the effectiveness of an improved version of a three-layer agarose microcapsule in islet xenotransplantation. The microcapsule is composed of a mixture of 5% agarose and 5% polystyrene sulfonic acid. The other two outer layers are polybrene and carboxymethyl cellulose. The agarose/polystyrene sulfonic acid membrane is for the purpose of immunoisolation, suppression of complement activity and reinforcement of the microcapsule. The polybrene layer suppresses the polystyrene sulfonic acid leakage by forming a polyionic complex at the surface of the agarose/polystyrene sulfonic acid membrane. The outermost layer, a carboxymethyl cellulose coating, improves the biocompatibility of the microcapsule. In vitro static incubation study showed that the insulin secretion from rat islets in microcapsules in response to 16.7 mM glucose stimulation was more than four times higher than that on 3.3 mM glucose stimulation (n = 8). In an in vivo study, 500 rat islets in microcapsules were xenogenically implanted in the abdominal cavity of mice with streptozotocin-induced diabetes. The graft survival times ranged from 2 to 5 mo, the average being 75 days (n = 5). Our results demonstrate that the improved version of the three-layer agarose microcapsule can effectively prolong the xenograft survival time without employing immunosuppressants, suggesting that this microcapsule could provide a promising biohybrid artificial pancreas for future clinical applications.

Effects of gastric inhibitory polypeptide and glucagon on portal venous and hepatic arterial flow in conscious dogs

Gastric inhibitory polypeptide (GIP) has considerable structural homology with glucagon, which is known to increase liver blood flow. We compared the effects of GIP on portal venous and hepatic arterial flow with those of glucagon in conscious dogs. Injection of GIP significantly increased portal venous flow in a dose-related manner (by 7%, 15%, and 46% at doses of 1, 100, and 500 pmol/kg, respectively). The increase in portal venous flow induced by GIP and glucagon was comparable; however, the increase in portal venous flow after GIP injection reached its peak significantly earlier than that after glucagon injection. Hepatic arterial flow decreased after GIP injection (by 17%, 21%, and 35% at doses of 1, 100, and 500 pmol/kg, respectively), whereas it was not altered by glucagon. Thus, GIP causes significant changes in both portal venous and hepatic arterial flow in conscious dogs. Although structurally related, GIP and glucagon may influence liver blood flow through different mechanisms.

Application of a novel B cell line MIN6 to a mesh-reinforced polyvinyl alcohol hydrogel tube and three-layer agarose microcapsules: An in vitro study

This study examines the function of a novel B cell line (MIN6) enclosed in hybrid bioartificial pancreas with mesh-reinforced polyvinyl alcohol hydrogel tube (MRPT) or with improved, three-layer agarose microcapsules. MIN6 was established from insulinomas obtained by targeted expression of the simian virus 40 T-antigen gene in transgenic mice. MIN6 retains the ability to secrete insulin in response to physiological glucose concentrations. The MRPT and the three-layer agarose microcapsules, which were developed to act as an artificial pancreas, were readily permeated by insulin, glucose, and other nutrients. Both can immunoisolate enclosed MIN6 cells from the recipients humoral and cellular immunosystems, which causes a xenogeneic rejection response. MIN6 cells (5.0 x 10(6) or 1.5 x 10(6)) were enclosed in MRPT or in a hundred three-layer microcapsules and subjected to an in vitro perifusion study or a static incubation study to observe the insulin release from each bioartificial pancreas in response to glucose stimulation. In vitro study revealed that the insulin secretion in response to 16.7 mM glucose stimulation was twice that with 3.3 mM glucose stimulation with both MRPT and the three-layer agarose microcapsules. The present study demonstrates that MIN6 effectively functions as a bioreactor for the hybrid bioartificial pancreas. The application of MIN6 cells to the hybrid bioartificial pancreas may offer a solution to the current serious dearth of organs.

Effect of human epidermal growth factor (hEGF) on splanchnic circulation in dogs

The effect of intravenous administration of human epidermal growth factor on the splanchnic blood flows was examined in anesthetized dogs, using an ultrasonic transit-time volume flow meter. Human epidermal growth factor (0.1, 0.5 and 1 microgram/kg) significantly increased blood flows in the portal vein (36.9 +/- 7.4% at 1 microgram/kg) and the superior mesenteric artery (49.0 +/- 16.8% at 1 microgram/kg). Systemic blood pressure monitored simultaneously was significantly decreased (8.4 +/- 1.2% at 1 microgram/kg). This study is the first to demonstrate that intravenous administration of epidermal growth factor increases the portal venous blood flow.

The potential of anticomplement synthetic sulfonic polymers for xenotransplantation

IN IMMEDIATELY vascularized xenografts, the presence of species-specific preformed natural antibodies associated with the activation of the host complement system has been reported to induce hyperacute humoral rejection in discordant combinations. Furthermore, complement activation on the endothelial cells of xenografts has been suggested to play a major role in hyperacute rejection. On the other hand, in neovascularized xenografts, such as pancreatic islets of Langerhans, the absence of initial vascularization has been suggested to avert hyperacute humoral rejection and that the rejection might be cell mediated. Nevertheless, islet xenografting in higher mammals has been reported to result in an exceedingly brief period of graft functioning, which suggests that humoral immunity might play a significant role in islet demise. Thus, we hypothesized that, in islet xenotransplantation, islets might be hyperacutely rejected by antibody-dependent and/or antibody-independent activation of complement, not by cell-mediated mechanisms only. We have regarded humoral immunity, such as complement activation, as an important trigger of vascularized or neovascularized xenograft rejection. Based on this viewpoint, we have conducted various experiments on xenotransplantation of immediately vascularized organ or neovascularizing tissue, such as islets employing sulfonic polymer poly(styrene sulfonic acid) (PSSa) carrying a strong anticomplementary effect in which we have originally focused on its chemical characteristics. PSSa (Toyo Soda Manufacturing Co, Tokyo, Japan) is a synthetic polymer that has a molecular weight of about 50,000 d (Fig 1), and is used clinically as an ion-exchange resin, sodium polystyrene sulfonate (Kayexalate, Torii Pharmaceutical Co, Tokyo), which is administered anally into patients’ lower intestinal cavity.

Effects of Ethanol and Wine on Hepatic Arterial and Portal Venous Flows in Conscious Dogs

The effects of ethanol and wine on hepatic arterial and portal venous flows were examined in conscious dogs. Ethanol was given intravenously or intragastrically, and red wine (ethanol: 14%) was given intragastrically over 30 min. Intravenous ethanol (0.8 g/kg) and intragastric ethanol (14% vol/vol) increased hepatic arterial flow, which remained elevated for 60 min after the cessation of ethanol administration. Ethanol also increased portal venous flow. Portal venous flow returned gradually toward basal levels after the cessation of intravenous ethanol infusion, whereas it remained elevated even after the cessation of intragastric ethanol. Intragastric wine increased hepatic arterial and portal venous flows. In contrast to intragastric ethanol, hepatic arterial flow continued to rise after the cessation of intragastric wine infusion, while portal venous flow returned toward basal levels. We conclude that, though both ethanol and wine increase hepatic blood flow, the responses of hepatic arterial and portal venous flows differ substantially among intravenous ethanol, intragastric ethanol and intragastric wine.

Effect of synthetic porcine neuropeptide Y (NPY) on splanchnic blood flows and exocrine pancreatic secretion in dogs.

This study examined the effect of synthetic porcine neuropeptide Y on the splanchnic blood flows and the exocrine pancreatic secretion in dogs. Graded doses of neuropeptide Y (0.1–5 μg/kg, intravenous) caused dose-dependent reduction of the secretin-stimulated exocrine pancreatic secretion and of the blood flows in the superior mesenteric artery, the portal vein, and the pancreatic tissue. Neuropeptide Y at 5 μg/kg reduced the blood flows to 45.9±13.3% (superior mesenteric artery), 63.0±10.5% (portal vein), and 77.9±4.8% (pancreatic tissue), respectively. This dose also reduced secretin-stimulated pancreatic juice volume and CCK-8 plus secretin-stimulated protein output to 65.2±9.3 and 63.3±14.0%, respectively. This study shows a potent vasoconstrictor effect of neuropeptide Y on splanchnic vessels. Neuropeptide Y also inhibited exocrine pancreatic secretion in a significant correlation with the reduction in pancreatic tissue blood flow, which suggests that reduction in the blood flow may be one of the possible mechanisms of the inhibitory action of neuropeptide Y on exocrine secretion.

Qualitative and quantitative changes in islet cells of autotransplanted pancreas in dogs in relation to glucose metabolism.

This study was conducted to clarify the quantitative and qualitative changes in the cells of the islets of Langerhans in the autotransplanted pancreas in dogs and to correlate these changes with alterations of glucose tolerance. The left lobe of a canine pancreas was transplanted into the left iliac fossa. The splenic vessels were anastomosed to the left iliac vessels. The pancreatic duct was left open to the peritoneal cavity. Open biopsies of the autotransplanted pancreas were performed 3, 7, 11, and 14 weeks after transplantation. The islets in the transplanted pancreas were examined ultrastruc-turally; B-cells, A-cells, and D-cells were identified immunohistochemically and their percentages were determined. Intravenous glucose tolerance tests were performed 3, 5, 7, 11, and 14 weeks after operation, and several indexes (K values and integrated response and increased values for baseline serum and immunoreactive insulin) were calculated. Fibrosis of the transplanted pancreas progressed after transplantation. The percentage of B-cells fell significantly (p <0.01), whereas that of A-and D-cells did not have significant changes. The ultrastructural study revealed an increase in collagen bundles, degranulation of B-cells, and marked preservation of A-cell granules 3 weeks after transplantation. B-Cells with dark cytoplasm were found 7 weeks after operation. In contrast, clusters of immature B-cells were seen in some sections of the pancreas 14 weeks after autotransplantation. The integrated response in baseline serum was increased significantly (p <0.01), and the K value was decreased significantly (p <0.01) 11 weeks after autotransplantation when compared with the initial posttransplant period. On the other hand, 14 weeks after autotransplantation, neither the integrated response in baseline serum nor the K value showed significant difference from the initial posttransplant period. It may be that the trend toward slight recovery of glucose metabolism 14 weeks after autotransplantation was associated with the clusters of immature B-cells observed ultrastructurally, although the number of B-cells was relatively decreased. Further studies must be done to clarify this phenomenon.