Akira Shiroi

Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.

Background and Aims. Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently identified the emergence of cellular clusters within differentiated ES cell cultures by staining with dithizone (DTZ). DTZ is a zinc‐chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. The aim of the present study was to investigate the characteristics of DTZ‐stained cellular clusters originating from ES cells.

Potential Use of Embryonic Stem Cells for the Treatment of Mouse Parkinsonian Models: Improved Behavior by Transplantation of In Vitro Differentiated Dopaminergic Neurons from Embryonic Stem Cells

Background and Aims. The purpose of the present study was to examine the efficacy of transplantation of mouse embryonic‐stem‐(ES)‐cell‐derived tyrosine hydroxylase‐positive (TH+) cells into Parkinsonian mice using behavioral tests and immunohistochemical evaluation.

Development of hepatocytes from ES cells after transfection with the HNF-3beta gene.

We have attempted to generate embryonic stem (ES) cell‐derived hepatocytes expressing liver‐specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)‐3β‐transfected ES cells are cultured in α‐MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)‐2 in the three‐dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF‐3β‐transfected ES cells also had hepatocyte‐like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.

Cronkhite-Canada syndrome with colon cancer, portal thrombosis, high titer of antinuclear antibodies, and membranous glomerulonephritis.

A 64-year-old man, who came to us with diarrhea, presented with ectodermal changes such as hyperpigmentation, alopecia, and onychatrophy, and was affected by polyposis in the colorectum and stomach. The polyps were histologically consistent with those in Cronkhite-Canada syndrome (CCS). Interestingly, the patient also had colon cancer, as well as portal thrombosis and a high concentration of antinuclear antibody. Treatment with prednisolone ameliorated the symptoms and the gastrointestinal polyposis, while the cancer was successfully treated with a hemicolectomy. Six months after the surgery, the patient developed nephropathy, with nephrotic-range proteinuria, without recurrence of the cancer. The biopsied renal specimen showed membranous glomerulonephritis. This is a rare case of CCS associated with various complications such as colon cancer, portal vein thrombosis, a high titer of antinuclear antibodies, and membranous glomerulonephritis. Although the pathogenesis of CCS is essentially unknown, these complications might have been indicative of an underlying immunological abnormality.

In vitro differentiation of hepatocyte-like cells from embryonic stem cells promoted by gene transfer of hepatocyte nuclear factor 3 β

We investigated the ability of a genetically altered embryonic stem (ES) cell line to promote endodermal differentiation toward hepatocytes in vitro by transfecting the hepatocyte nuclear factor-3beta (HNF-3beta) gene. Parental and HNF-3beta-transfected ES cells were initiated toward differentiation in embryoid bodies (EBs) for 5 days and the resulting EBs were transferred to an attached culture system. Albumin production was observed using an immuno-cytochemical method 7 days after induction of differentiation in almost all differentiating HNF-3beta-transfected ES cells, whereas scant immuno-reactivity against albumin was found on the same day in the cultures of differentiating parental ES cells. An analysis using a reverse transcriptase polymerase chain reaction revealed the HNF-4alpha expression in the HNF-3beta-transfected ES cells and also demonstrated that the expression of endodermal and hepatocyte-related markers, such as transthyretin, alpha-fetoprotein, albumin, alpha-1 antitrypsin, tryptophan-2,3-dioxygenase and phosphoenol-pyruvate carboxykinase, could be observed at an early stage in the outgrowths of HNF-3beta-transfected ES cells compared to the parental ES cells. These results suggest that HNF-3beta-transfected ES cells may be useful for the efficient induction of hepatocytes in vivo.

Spontaneous elimination of serum HCV-RNA after total gastrectomy for early gastric cancer in a patient with chronic hepatitis C

newly developed drug synergistically combining antisecretory and anti–H. pylori properties of ranitidine and bismuth (5). Modern investigators demonstrated that RBC had synergistic activity with clarithromycin and nitromidazoles (6, 7). Unfortunately, there is no information on the efficiency of combined treatment regimens including RBC in pediatric practice at present. We studied 39 consecutive pediatric patients infected with H. pylori who were presenting with persistent abdominal complaints (chronic epigastric pain, nausea, vomiting). All of the children underwent endoscopy with gastric biopsies at our hospital. These biopsies were studied for H. pylori colonization (the modified Giemsa technique, urease test, and polymerase chain reaction) and for the presence of gastric inflammation (8). None of our patients responded to the preceding antiacid therapy including a 4-wk course of ranitidine alone (5–10 mg/kg). Duodenal ulcers were detected at endoscopy in eight children (20.5%). Mean age at diagnosis was 13.7 yr (range 12–15 yr); the male/female ratio was 0.85. Outpatients were treated with RBC (400 mg b.i.d.) for 4 wk pus clarithromycin (7.5 mg/kg/day) and tinidazole (20 mg/kg/day) for the first 2 wk. For ethical reasons no placebo group was included. Bismuth blood levels were monitored to avoid bismuth toxicity. The treatment regimen was well tolerated. The potentially drug-related adverse events (blackening of the tongue, 11; dry metallic taste, two; anorexia, one) were registered in 12 (30.7%) patients, and dark stools were observed in all. All of the above symptoms were self-limiting. No child had toxic values of blood bismuth levels. Thus, there were no serious adverse reactions, and therefore there were no withdrawals due to any side effects during the course of the study. Follow-up endoscopic examination with biopsies was performed in all the patients 4 wk after the completion of therapy. The control endoscopy demonstrated healing of duodenal ulcers in all cases. In 35/39 patients (89.7%) antral biopsy specimens revealed disappearance of H. pylori (Giemsa stain1 urease test). Per protocol analysis, 35/39 patients (89.7%) (95% CI 5 80–99%) were cured, but intention-to-treat analysis results were the same. Cure ofH. pylori infection was accompanied by considerable improvement in endoscopic appearance, histology, and resolution of symptoms. No relapses of ulcer disease were found in theH. pylori–negative children during the repeated endoscopy 6 months after successful treatment. We therefore suggest that our treatment regimen combining RBC with clarithromycin and tinadzole eradicates H. pylori from the antrum in children with duodenal ulcers and gastritis in a significant number of cases. Moreover, the present data indicate that RBC-based treatment of H. pylori infection in childhood is a potentially useful and safe eradication regimen. Obviously, the RBC triple therapy can be suitable for curing H. pylori in adults as well as in children.

Development of hepatocytes from embryonic stem cells after transfection with the HNF-3b gene

We have attempted to generate embryonic stem (ES) cell‐derived hepatocytes expressing liver‐specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)‐3β‐transfected ES cells are cultured in α‐MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)‐2 in the three‐dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF‐3β‐transfected ES cells also had hepatocyte‐like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.

Hepatic adenomas treated with percutaneous ethanol injection in a patient with glycogen storage disease type Ia

Abstract: We report a 20-year-old man with glycogen storage disease type Ia (GSD Ia) who presented multiple hepatocellular adenomas (HCAs) in 1993 and in whom percutaneous ethanol injection (PEI) was conducted as treatment for some enlarging tumors beneath the liver surface. In a 6-year follow-up period, we observed gradual enlargement of some of the HCAs, and the rapid growth of a newly developed tumor. In August 1996, one slow-growth HCA was 52 mm in diameter and was located beneath the surface of the liver. We conducted PEI therapy to prevent its spontaneous rupture. During the following year, another tumor developed beneath the liver surface, but showed extremely rapid growth, reaching 51 mm in diameter, from being undetectable, within 12 months. PEI therapy was again conducted for this newly developed tumor. Although additional PEI therapy was required for each tumor, because of suspected recurrence, no findings of discrete recurrence have been detected by computed tomography and magnetic resonance imaging for more than 2 years, up to the time of this study. We consider PEI to be a useful and effective therapeutic modality for individual HCAs in patients with GSD Ia.

Primary biliary cirrhosis exacerbated by a course of acute hepatitis C and subsequent interferon therapy

Primary biliary cirrhosis exacerbated by a course of acute hepatitis C and subsequent interferon therapy

CASE OF HEMORRHAGE FROM A DUODENAL DIVERTICULUM TREATED SUCCESSFULLY BY ENDOSCOPIC INJECTIONS OF EPINEPHRINE

Although duodenal diverticula are found relatively frequently in adult gastrointestinal tracts, the majority are asymptomatic. We report a case of duodenal diverticulum complicated with hemorrhage. A 74‐year‐old woman developed hematemesis and tarry stools. An emergent upper gastrointestinal endoscopy revealed a diverticulum, about 3 cm in diameter, in the posteromedial aspect of the second duodenal segment, right oral to the papilla. The diverticulum was filled with blood clots. After removing them by gentle suction, a linear ulcer became visible and an actively oozing site was seen at one edge of the ulcer. Three injections of epinephrine in a 2.5% sodium chloride solution (epinephrine concentration 0.05 mg/mL), each 1.0 mL for a total volume of 3.0 mL, were made at the oozing site. The exuding ceased immediately after the third injection and bleeding did not reappear. In our patient, successful and complete hemostasis was obtained by this endoscopic injection of epinephrine, although most cases of duodenal diverticulum complicated with hemorrhage had been treated surgically. We think that endoscopic, instead of surgical treatment is considerably becoming another choice for treating patients with a bleeding duodenal diverticulum.