Biomedical subjects
Publications and source records attributed to J Miyazaki.
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The properties of human fetal alkaline phosphatase (ALP) have been compared with those of purified human adult hepatic and intestinal ALPs. The ALPs from different human fetal organs were divided into two groups. One terminated universal type was presented in fetal liver, spleen, adrenal, kidney, lung, heart, pancreas, thymus and bone, and was similar to adult hepatic-type ALP. The other terminated intestinal type was presented in fetal intestine and meconium and was similar to adult intestinal ALP. The activity of human fetal intestinal ALP increased markedly with the advance of intra-uterine life during fetal development, and that of human fetal thymus ALP increased slightly.
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BACKGROUND: High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the 'hydrodynamics-based procedure'. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research. METHODS: We tested this technique for the delivery of a therapeutic protein in normal rats, using a rat erythropoietin (Epo) expression plasmid vector, pCAGGS-Epo. RESULTS: We obtained maximal Epo expression when the DNA solution was injected in a volume of 25 ml (approximately 100 ml/kg body weight) within 15 s. We observed a dose-response relationship between serum Epo levels and the amount of injected DNA up to 800 microg. Using quantitative real-time PCR, the vector-derived Epo mRNA expression was mainly detected in the liver. When a lacZ expression plasmid was injected similarly, beta-galactosidase was exclusively detected in the liver, mainly in hepatocytes. Toxicity attributable to the technique was mild and transient, as assessed by histochemical analysis. Epo gene expression and erythropoiesis occurred with Epo gene transfer in a dose-dependent manner, and persisted for at least 12 weeks, the last time point examined. Repeated administration of the plasmid DNA also effectively led to erythropoiesis. CONCLUSIONS: These results demonstrate that gene transfer into the liver via rapid tail vein injection can easily be achieved in the rat, which is more than 10 times larger than the mouse, and has significant value for gene function analysis in rats.
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 recipient's 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.
This experiment was designed to investigate whether MIN6-cells, which are derived from transgenic mouse insulinoma cells, could be a useful tool for transplantation. An implantable diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP) was constructed by placing a pancreatic B-cell line (MIN6) in a mixed matrix in the center of a ring holder sandwiched between two nuclepore membranes (pore size 0.2 microns), which were held in place by a silicon seal. Nine streptozotocin (STZ)-induced diabetic rats were each implanted with the diffusion chamber containing mouse insulinoma cells (xenograft-implantation) as a Bio-AEP, but without any immunosuppressant. In three of the STZ-diabetic rats with a Bio-AEP, a return to normoglycemia was observed up to 20 week after implantation (good control). Four of the nine STZ-diabetic rats, which had received diffusion chambers with MIN6, also showed a return to normoglycemia for up to 10 wk (fair control). In two of the rats, blood glucose levels showed poor control. The results indicate that MIN6 cells should be useful for the implantation of xenographic cells in diabetic animals.
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Embedded-culture of pancreatic beta-cells derived from a transgenic mouse insulinoma (MIN6 cells) was studied in vitro and in vivo. The MIN6 cells were enmeshed in an agarose-PVMA-collagen matrix for long-term maintenance. The cells formed islet-like cell clusters (ICCs) in the mixed matrix. When 10 mmol/L nicotinamide was added to these cultures the cells secreted insulin in response to various concentrations of glucose, whereas the untreated control cells were unresponsive. Both control and nicotinamide-treated MIN6 cells exhibited normal beta-cell function for up to 35 days in the mixed matrix, and the cells were much better preserved with nicotinamide than without it. MIN6 cells were suspended in the mixed matrix with nicotinamide and transferred into diffusion chambers to create a bio-artificial endocrine pancreas (Bio-AEP). In streptozotocine-induced diabetic rats with implanted Bio-AEP but without any immunosuppressants, a return to normoglycaemia was observed for up to 12 wk or more after transplantation. Our results indicate that nicotinamide-treated MIN6 cells embedded in a mixed matrix should be useful for the study of xenotransplantation and the development of a bioartificial endocrine pancreas.
The biohybrid artificial pancreas is designed to enclose pancreatic tissues with a selectively permeable membrane that immunoisolates the graft from the host immune system, allowing those endocrine tissues to survive and control glucose metabolism for an extended period of time. The pancreatic B cell line MIN6 is established from a pancreas B cell tumor occurring in transgenic mice harbouring the human insulin promoter gene connected to the SV40 T-antigen hybrid gene. It has been proven that glucose-stimulated insulin secretion in MIN6 cells retains a concentration-dependent response similar to that of normal islets. In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation to evaluate this device as bioartificial pancreas. MIN6 cells were microencapsulated in three-layer microbeads formulated with agarose, polystyrene sulfonic acid, polybrene, and carboxymethyl cellulose. Microbeads were xenogenically implanted in the subcutaneous tissue of the back of Lewis rats with streptozotocin-induced diabetes. One week after implantation, microbeads were retrieved and cultured for 24 h before the static incubation. There was no evidence of adhesion to the graft and the fibrosis in the transplantation site as determined by gross visual inspection. Microscopic examination demonstrated that retrieved microbeads maintained normal shape, containing intact MIN6 cells. Histological study showed that these MIN6 cells in the microbeads appeared to be viable without cellular infiltration within or around the microbeads. Immunohistochemical analysis of the microbeads clearly revealed the intense staining of insulin in the cytoplasm of encapsulated MIN6 cells. Insulin productivity of MIN6 cells in the microbeads is strongly suggested to be preserved. In response to 16.7 mM glucose stimulation, static incubation of microbeads 1 wk after implantation caused the 2.3 times increase in insulin secretion seen after 3.3 mM glucose stimulation (84.3 +/- 10.0 vs. 37.4 +/- 10.7 microU/3 x 10(6) cells/hr, n = 5 each, p < 0.01). This study demonstrates that three-layer microbeads encapsulating MIN6 cells retain excellent biocompatibility and maintain good insulin secretion even after subcutaneous xenotransplantation, suggesting the possible future clinical application of this unique bioartificial pancreas to subcutaneous xenotransplantation.
In Japan the incidence of atypical mycobacteriosis has steadily increased, with Mycobacterium avium-intracellulare complex (MAC) the most common infecting organism. A standard chemotherapy regimen for MAC infection has not been established because of significant resistance to anti-mycobacterial drugs. Sparfloxacin has good antimicrobial activity against several acid-fast bacteria and is expected to be an effective drug for treating mycobacteriosis. We examined the effects of adding sparfloxacin to anti-tuberculotic combination therapy in six patients with MAC pulmonary disease. Drug susceptibility was also assessed using the agar dilution method. The minimum inhibitory concentrations (MICs) for sparfloxacin, levofloxacin, isoniazid, rifampicin, streptomycin, ethambutol and clarithromycin was measured in clinical isolates from all patients; sparfloxacin showed the lowest MIC. Bacteriological and clinical improvements were observed in the four patients who completed the study. Dosing was discontinued in two patients because of pruritic skin eruptions. Sparfloxacin shows promise as an anti-mycobacterial agent for treating MAC pulmonary disease.
The efficacy of sparfloxacin (SPFX) for the control of bronchial asthma was evaluated in 26 patients with suspected Chlamydia pneumoniae infection. Patients were randomly allocated to receive SPFX 200 mg/day (n = 14) or control treatment (n = 12) for 21 days. Significant improvements in serum C-reactive protein levels, and significant decreases in peripheral eosinophil counts, serum eosinophil cationic protein (ECP) and sputum ECP were observed in the SPFX-treated group at day 21. SPFX-treated patients also had a significantly reduced frequency of asthma symptoms, reduced inhalant beta2-stimulant use, and significant increases in morning peak expiratory flow. At the end of the study, C. pneumoniae was undetectable in two SPFX-treated patients who underwent polymerase chain reaction testing, but one control patient who was tested still had detectable levels of C. pneumoniae. These results suggest that SPFX could be used to control bronchial asthma in patients with suspected persistent C. pneumoniae infection.