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.

Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease.

Hirschsprung disease (HSCR) is sometimes associated with a set of characteristics including mental retardation, microcephaly, and distinct facial features, but the gene mutated in this condition has not yet been identified. Here we report that mutations in SIP1, encoding Smad interacting protein-1, cause disease in a series of cases. SIP1 is located in the deleted segment at 2q22 from a patient with a de novo t(2;13)(q22;q22) translocation. SIP1 seems to have crucial roles in normal embryonic neural and neural crest development.

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.

Single pancreatic beta cells co-express multiple islet hormone genes in mice.

AIMS/HYPOTHESIS: It is widely accepted that production of insulin, glucagon, somatostatin and pancreatic polypeptide in islet cells is specific to beta, alpha, delta and pancreatic polypeptide cells, respectively. We examined whether beta cells express other genes encoding islet hormones. METHODS: Nested RT-PCR was performed on single beta cells of transgenic mice with green fluorescent protein (GFP) driven by mouse insulin I promoter (MIP-GFP). RESULTS: Only 55% of adult beta cells expressed the insulin gene alone, while others expressed two or more islet hormone genes; 4% expressed all four hormone genes. In embryonic and neonatal cells, 60% to 80% of GFP(+) cells co-expressed pancreatic polypeptide and insulin genes in contrast to 29% in adult. To clarify cell fate, we conducted lineage tracing using rat insulin II promoter-cre mice crossed with reporter mice Gt(ROSA)26Sor-loxP-flanked STOP-cassette-GFP. All GFP(+) cells expressed insulin I and II genes, and showed similar heterogeneity of co-expression to that seen in MIP-GFP mice. Although we report expression of other hormone genes in a significant proportion of beta cells, our lineage tracing results demonstrate that after inducing InsII (also known as Ins2) expression, beta cell progenitors do not redifferentiate to non-beta cells. CONCLUSIONS/INTERPRETATION: This study shows co-expression of multiple hormone genes in beta cells of adult mice as well as in embryos and neonates. This finding could: (1) represent residual expression from beta cell precursors; (2) result from alternative developmental pathways for beta cells; or (3) denote the differentiation potential of these cells. It may be linked to functional heterogeneity. This heterogeneity in gene expression may provide a means to characterise the functional, cellular and developmental heterogeneity seen in beta cells.

Determination of three enolase isozymes and S-100 protein in various tumors in children.

Three enolase isozymes (alpha alpha, alpha gamma, and gamma gamma) and S-100 protein in the extract of neuroendocrine tumors (neuroblastoma, ganglioneuroblastoma, ganglioneuroma, and pheochromocytoma) and nonneuroendocrine tumors (Wilms' tumor, rhabdomyosarcoma, and hepatoblastoma) were determined by means of enzyme immunoassay systems. All of the tumors examined showed a high level of alpha alpha-enolase (1.71 to 19.0 micrograms/mg protein). Levels of nervous system-specific enolases (NSE; alpha gamma and gamma gamma) in the neuroendocrine tumors were also rather high (alpha gamma, 1.64 to 7.45 micrograms/mg protein; gamma gamma, 0.052 to 5.56 micrograms/mg protein). However, the NSE concentration in the extract of nonneuroendocrine tumors was low (alpha gamma, less than 0.88 micrograms/mg protein; gamma gamma, 0 microgram/mg protein). The level of S-100 protein was relatively high in ganglioneuroma (greater than 500 ng/mg protein) and ganglioneuroblastoma (greater than 100 ng/mg protein), but low in neuroblastoma (less differentiated neuroendocrine tumor) and nonneuroendocrine tumors. Serum levels of enolase isozymes were also determined in neuroblastoma patients before and after resection of primary tumor or effective chemotherapy. The elevated level of serum NSE (alpha gamma and gamma gamma) was markedly decreased with little change in the alpha alpha level by the treatment.

Changes in Muscle Strength and Six-Minute Walk Distance Before and After Living Donor Liver Transplantation.

BACKGROUND: Impaired exercise capacity and muscle weakness are important characteristics of liver transplantation recipients. Perioperative rehabilitation has been introduced to promote early mobilization of patients and to prevent postoperative pulmonary complications. However, it is unknown how physical status recovers during the hospital stay after a liver transplant. The purpose of this study was to evaluate the changes in clinical indicators that represent the functional exercise capacity and muscle strength before and after living donor liver transplantation (LDLT). METHODS: We retrospectively reviewed 21 consecutive patients who underwent LDLT with perioperative rehabilitation from April 2014 to December 2015. Twelve patients who were tested for 6-minute walk distance, hand-grip strength, and isometric knee extensor muscle strength before and 4 weeks after LDLT were enrolled. RESULTS: At the preoperative baseline, the 6-minute walk distance significantly correlated with the Model for End-stage Liver Disease score and pulmonary functions (vital capacity, forced vital capacity, and forced expiratory volume in 1 second of predictive values). Comparisons between the preoperative and postoperative values revealed significant decreases in weight, Barthel Index, hand-grip strength, and isometric knee extensor muscle strength. Changes in hand-grip strength and isometric knee extensor muscle strength after LDLT correlated with the preoperative Model for End-stage Liver Disease score. CONCLUSIONS: Physical functional status had not been fully recovered 4 weeks after LDLT. Further investigation regarding developing a strategy for prevention of muscle atrophy before LDLT and recovery of physical fitness after LDLT would be helpful.

Cloning of a sensory-kinase-encoding gene that belongs to the two-component regulatory family from the cyanobacterium Synechococcus sp. PCC7942.

A screening method employing Escherichia coli was adopted to clone a sensory-kinase (SK)-encoding gene directly from a phylogenetically distant species, the phototrophic cyanobacterium Synechococcus sp. PCC7942. From the Synechococcus chromosomal DNA, we searched for DNA clones which are able to complement phenotypically not only an E. coli envZ mutant for the expression of ompC, but also an E. coli phoR/creC mutant for the expression of alkaline phosphatase. These E. coli genes are known to encode SK. A 0.75-kb DNA fragment was thus cloned under the control of the E. coli lac promoter carried on an E. coli plasmid vector. A larger DNA fragment encompassing an entire open reading frame was then cloned and its complete nucleotide (nt) sequence determined. The nt sequence corresponds to a gene that encodes a 43,280-Da protein of 387 amino acids with a high degree of homology to the bacterial SK. Thus, we succeeded in cloning a SK-encoding gene, which most likely functions in signal transduction in Synechococcus sp. PCC7942. Hence, the gene was designated sasA (Synechococcus adaptive-response SK A). The purified SasA protein was demonstrated in vitro to undergo autophosphorylation.

Late infantile Hirschsprung disease-mental retardation syndrome with a 3-bp deletion in ZFHX1B.

A 48-year-old woman with late infantile onset mental retardation developed megacolon. Although the patient had no typical clinical features of Hirschsprung disease-mental retardation syndrome, a new 3-base pair deletion, eliminating an Asn, was identified in the responsible gene ZFHX1B. This suggests that screening for ZFHX1B mutations is warranted even in the absence of typical clinical features of the syndrome.

Specific quantitation of secretory immunoglobulin A with enzyme immunoassay using activated thiol--Sepharose for separation method.

A specific and sensitive enzyme immunoassay system for human secretory IgA was developed using anti-alpha-chain antibodies coupled to activated thiol-Sepharose, and anti-secretory component antibodies labeled with beta-D-galactosidase from Escherichia coli. The dose response of the enzyme activity in eluate was observed between 3 and 1000 ng of secretory IgA with little cross-reactions with IgA, IgG, IgM and secretory component. The assay method could be employed for the measurement of secretory IgA in saliva, urine, feces, intestine and serum without interferences by the abundant IgA in the same samples.

Nervous system-specific enolase in serum as a marker for neuroblastoma.

Serum levels of nervous system-specific enolase (alpha gamma form plus gamma gamma form) were determined in 18 patients with neuroblastoma and in 40 control infants by means of a sandwich enzyme immunoassay method specific to the gamma subunit (or 14-3-2 protein) of enolase isozymes. Levels in patients with neuroblastoma were elevated (mean, 70.3; range, 6.2 to 330.0 ng/mL) when compared with those of control subjects (4.3 +/- 1.7 ng/mL). Most of the patients (6/7), whose serum nervous system-specific enolase level increased more than 100 ng/mL, died within 1 month. Serial measurements in patients with neuroblastoma receiving various therapies have revealed that there was a good correlation between serum nervous system-specific enolase levels and the course of the disease. These results indicate that the nervous system-specific enolase in serum may be a valuable marker for therapeutic monitoring of patients with neuroblastoma, as reported recently in patients with small-cell carcinoma of the lung.

Ontogeny of immunoreactive and bioactive microsomal steroidogenic enzymes during adrenocortical development in rats.

The functional development of the neonatal rat adrenal cortex is characterized by a triphasic response to adrenocorticotropic hormone (ACTH), with a nadir in responsiveness around neonatal day 10 (d10). In this study, the hypothesis was tested that hyporesponsiveness to ACTH partly results from deficiencies in steroidogenic enzyme content. Immunoreactive (ir) levels of mitochondrial cytochrome P450 enzymes (side chain cleavage (P450scc) and 11 beta-hydroxylase (P450c11)) did not change during neonatal development. Immunoreactive levels of microsomal 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), however, were significantly and comparably lower in both day 1 (d1) and d10 neonates compared to adult rats. Activity of 3 beta-HSD did not parallel changes in ir 3 beta-HSD content. Enzyme activity was low on d1 (approximately 39% of adult activity), but by d10 was statistically equivalent to that of microsomes from adult adrenal glands. Immunoreactive levels of microsomal cytochrome P450 21 alpha-hydroxylase (P450c21) were significantly lower in d1 glands than in adult glands (by approximately 50%), but by d10 were statistically indistinguishable from adults. On the other hand, P450c21 activity was equivalent on d1 and d10 and both were significantly lower compared to adults (approximately 62% of adult activity). ACTH injections from d3-d10 facilitated the adrenocortical steroidogenic response to ACTH on d10. This treatment increased levels of ir 3 beta-HSD, but not ir P450c21. The results suggest that rat adrenocortical 3 beta-HSD and P450c21 are developmentally and differentially regulated, and that ir levels of the proteins are not correlated with enzyme activity during the neonatal period. One possible explanation for these observations is that multiple isoforms of the two enzymes, with different antigenic and enzymatic properties, may be expressed during development at different times. In addition, the combined decreased activities of these two enzymes can almost entirely account for the decreased steroidogenic output of rat adrenocortical cells on d1, but not during the later neonatal period.

Enhancement of S-100 beta protein in blood of patients with Down's syndrome.

The human gene encoding the beta subunit of S-100 protein (S-100 beta) was mapped on chromosome 21. In order to confirm the expression of gene-dosage effect of S-100 beta in patients with Down's syndrome (DS), concentrations of immunoreactive S-100 alpha and S-100 beta proteins were determined in the blood plasma and lymphocytes fraction of the patients and control subjects. Cu/Zn-superoxide dismutase (SOD), a protein that is known to show the gene-dosage effect on the trisomy of chromosome 21, also was immunoassayed in the same blood samples as control proteins. In blood plasma, S-100 beta protein as well as Cu/Zn SOD was enhanced (P less than 0.001) in the patients (160 +/- 70 pg S-100 beta/ml and 87 +/- 83 ng SOD/ml, N = 44) as compared with control individuals (76 +/- 25 pg/ml, and 18 +/- 11 ng/ml, respectively, N = 28). However, concentrations of S-100 alpha in blood plasma of DS patients were similar to those of normal subjects. Concentrations of S-100 beta in lymphocyte fractions of DS patients (24.7 +/- 10.9 ng/mg protein) were also higher (P less than 0.001) than those of control subjects (10.1 +/- 5.8 ng/mg protein). These results indicate that gene-dosage effect of S-100 beta levels are expressed in DS patients.

Partial splenic embolization decreases the serum bilirubin level in patients with hypersplenism following the Kasai procedure for biliary atresia.

BACKGROUND: Partial splenic embolization (PSE) has been used in the palliative treatment of adults and children with hypersplenism. However, there has been no previous report of its use in the treatment of patients with jaundice. The purpose of this study was to investigate the utility of PSE in the treatment of jaundice in patients with biliary atresia and hypersplenism. STUDY DESIGN: Partial splenic embolization was performed in eight patients with biliary atresia and hypersplenism that developed following the Kasai procedure. Seven of them had experienced complete resolution of jaundice postoperatively, but became icteric thereafter. Jaundice remained unchanged in the initial postoperative period in the last patient but subsequently worsened. White blood cell, platelet, and red blood cell counts, hematocrit, and hemoglobin concentrations, and serum concentrations of glutamic-oxaloacetic transaminase, glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenase, albumin, and total bilirubin were evaluated one month before PSE and one, two, three, six, nine, 12, 18, and 24 months after PSE. RESULTS: The total bilirubin concentration decreased in all patients after PSE from 8.6 +/-3.6 mg/dL to 3.0 +/-1.0 mg/dL. This change was noted within three months of PSE, and correlated with an increase in the red blood cell count. CONCLUSIONS: Partial splenic embolization is a useful method for reducing serum bilirubin concentrations in patients with hypersplenism following the Kasai procedure for biliary atresia.

Spontaneous perforation of choledochal cyst.

BACKGROUND: Spontaneous perforation of the common bile duct in children is very rare and its etiology is unknown. We describe herein five patients treated for the spontaneous perforation of choledochal cyst and suggest the important factors leading to perforation. STUDY DESIGN: All patients were initially treated with T-tube drainage through the perforated site. Cholangiography through the T tube was performed intraoperatively and the important factors leading to perforation were examined. Furthermore, histological examination of the perforated wall of the common bile duct was performed. RESULTS: Cholangiography through a T tube revealed the presence of a pancreaticobiliary junction malformation and filling defects (protein plugs) in the common channel in all patients. Postoperatively, the T tube was gently irrigated with a physiological salt solution until the free flow of bile into the duodenum was established. Histological examination showed that the wall near the perforation was covered with a granulation tissue that was present only at the limited area. CONCLUSIONS: Perforation of the common bile duct was related to the abrupt increase in intraluminal pressure due to obstruction by protein plugs at the common channel.

Complete excision of the intrapancreatic portion of choledochal cysts.

BACKGROUND: Cyst excision is the treatment for patients with choledochal cysts. In general, many authors recommend intramural dissection between the outer and inner layers of the cyst or partial excision leaving part of the cyst in the pancreas to avoid pancreatic injury. However, because there are few large series with long-term follow-up periods, it remains unclear how much of the intrapancreatic portion of the cyst should be resected and what resection technique should be used. STUDY DESIGN: During an 18-year period, 104 patients underwent excision of choledochal cysts at our hospitals. Twelve patients had partial excision of the cyst above the pancreas, and 17 had intramural dissection of the intrapancreatic portion. Seventy-five patients underwent complete excision of the intrapancreatic portion of the cyst by our new technique, in which the outer plane of the epicholedochal plexus is dissected, exposing the narrow distal segment connecting the cyst to the pancreatic duct. Our new technique was compared retrospectively with the other two techniques. RESULTS: With our technique, the intrapancreatic cyst could be excised completely in 75 patients without any complications. Blood loss was significantly decreased when our technique was used compared to intramural excision. A pancreatic fistula occurred after intramural excision in one patient, and pancreatic stones formed several years after partial excision and intramural excision in three patients who proved to have residual cystic material in the pancreas. CONCLUSIONS: Our operative technique is safe and effective for the complete excision of the intrapancreatic portion of a choledochal cyst.

High levels of immunoreactive nervous system-specific enolase in sera of patients with neuroblastoma.

Serum levels of nervous system-specific enolase (NSE, gamma gamma form plus alpha gamma form) in patients with neuroblastoma and in control subjects were determined with a sensitive solid-phase sandwich enzyme immunoassay system. Serum levels of NSE in healthy adults ranged from 1.4-5.7 ng/ml (2.87 +/- 1.18 ng/ml, n = 20), and in control children (1-7 years old) from 2.6 to 10.8 ng/ml (5.76 +/- 2.42 ng/ml, n = 20). Serum samples (n = 13) from patients with neuroblastoma contained high levels of NSE, range 13.6 to 330 ng/ml (mean 96 ng/ml); however those (n = 7) from ganglioneuroblastoma patients were within a normal range (3.0-25.0 ng/ml; mean 8.3 ng/ml). These results suggested that the NSE in serum might be a valuable marker substance for screening and therapeutic monitoring of neuroblastoma.

Effect of respiratory movement on cerebrospinal fluid dynamics in hydrocephalic infants with shunts.

The authors report a study of the effect of respiratory movement on intracranial, auricular, and intraperitoneal cerebrospinal fluid (CSF) pressure in hydrocephalic infants with shunts. Postoperative intraventricular pressures were also recorded for comparison. The intraventricular, right auricular, and intraperitoneal pressures rose during expiration and dropped during inspiration; the pressure changes were most marked while the infants were crying or straining. All pressures dropped simultaneously at the time of inspiration, but the auricular pressure was most significantly affected. It dropped to -100 to -200 mm H2O when the patients cried, while intraventricular and intraperitoneal pressures remained above O mm H2O. The postoperative intracranial pressures were in accord with these results; the pressures after ventriculoatrial shunt were significantly lower than those after ventriculoperitoneal shunt when the same pressure valves were used.

Late versus early surgical correction for congenital diaphragmatic hernia in newborn infants.

BACKGROUND: Congenital diaphragmatic hernia, although rare (1 per 2-4,000 births), is associated with high mortality and cost. Opinion regarding the timing of surgical repair has gradually shifted from emergent repair to a policy of stabilization using a variety of ventilatory strategies prior to operation. Whether delayed surgery is beneficial remains controversial. OBJECTIVES: To summarize the available data regarding whether surgical repair in the first 24 hours after birth rather than later than 24 hours of age improves survival to hospital discharge in infants with congenital diaphragmatic hernia who are symptomatic at or immediately after birth. SEARCH STRATEGY: Search of Medline (1966-1999), Embase (1978-1999) and the Cochrane databases using the terms "congenital diaphragmatic hernia" and "surg*"; citations search, and contact with experts in the field to locate other published and unpublished studies. SELECTION CRITERIA: Studies were eligible for inclusion if they were randomized or quasi-randomized trials that addressed infants with CDH who were symptomatic at or shortly after birth, comparing early (<24 hours) vs late (>24 hours) surgical intervention, and evaluated mortality as the primary outcome. DATA COLLECTION AND ANALYSIS: Data were collected regarding study methods and outcomes including mortality, need for ECMO and duration of ventilation, both from the study reports and from personal communication with investigators. Analysis was performed in accordance with the standards of the Cochrane Neonatal Review Group. MAIN RESULTS: Two trials met the pre-specified inclusion criteria for this review. Both were small trials (total n<90) and neither showed any significant difference between groups in mortality. Meta-analysis was not performed because of significant clinical heterogeneity between the trials. REVIEWER'S CONCLUSIONS: There is no clear support for either immediate (within 24 hours of birth) or delayed (until stabilized) repair of congenital diaphragmatic hernia, but a substantial advantage to either one cannot be ruled out. A large, multicenter randomized trial would be needed to answer this question.

Late versus early surgical correction for congenital diaphragmatic hernia in newborn infants.

BACKGROUND: Congenital diaphragmatic hernia, although rare (1 per 2-4,000 births), is associated with high mortality and cost. Opinion regarding the timing of surgical repair has gradually shifted from emergent repair to a policy of stabilization using a variety of ventilatory strategies prior to operation. Whether delayed surgery is beneficial remains controversial. OBJECTIVES: To summarize the available data regarding whether surgical repair in the first 24 hours after birth rather than later than 24 hours of age improves survival to hospital discharge in infants with congenital diaphragmatic hernia who are symptomatic at or immediately after birth. SEARCH STRATEGY: Search of MEDLINE (1966-2002), EMBASE (1978-2002) and the Cochrane databases using the terms "congenital diaphragmatic hernia" and "surg*"; citations search, and contact with experts in the field to locate other published and unpublished studies. SELECTION CRITERIA: Studies were eligible for inclusion if they were randomized or quasi-randomized trials that addressed infants with CDH who were symptomatic at or shortly after birth, comparing early (<24 hours) vs late (>24 hours) surgical intervention, and evaluated mortality as the primary outcome. DATA COLLECTION AND ANALYSIS: Data were collected regarding study methods and outcomes including mortality, need for ECMO and duration of ventilation, both from the study reports and from personal communication with investigators. Analysis was performed in accordance with the standards of the Cochrane Neonatal Review Group. MAIN RESULTS: Two trials met the pre-specified inclusion criteria for this review. Both were small trials (total n<90) and neither showed any significant difference between groups in mortality. Meta-analysis was not performed because of significant clinical heterogeneity between the trials. REVIEWER'S CONCLUSIONS: There is no clear evidence which favors delayed (when stabilized) as compared with immediate (within 24 hours of birth) timing of surgical repair of congenital diaphragmatic hernia, but a substantial advantage to either one cannot be ruled out. A large, multicenter randomized trial would be needed to answer this question.

Gastroesophageal reflux occurring after repair of congenital diaphragmatic hernia.

Over the past 2 decades, 110 patients with congenital diaphragmatic hernia (CDH) were treated in the authors' hospital. Eighty-six survived; of these, 10 patients (11.6%) had gastroesophageal reflux (GER) after repair of CDH. Seven occurred in the past 5 years, during which time advanced intensive care including extracorporeal membrane oxygenation (ECMO) was used. Vomiting started within 4 weeks after repair of CDH in eight cases, and hiatal hernia was demonstrated in six cases. Three patients responded to conservative therapy; the other seven required antireflux surgery. Several factors are believed to be possible causes of the development of GER in CDH cases. Among them, slow pulmonary expansion of the affected side was thought to be the most important. Namely, in a case of CDH associated with severe hypoplastic lung, the esophagus may be deviated to the affected side before the lung is expanded. After expansion, the abdominal esophagus shortens, and GER or a hiatal hernia can occur in severe cases. There were seven such patients in our series of 10. With the increase in the survival rate of CDH cases associated with severe hypoplastic lung, the number of such patients also may increase. Therefore, some additional procedure to prevent the lower esophagus from sliding will be necessary in the repair of diaphragmatic hernia.