[Three cases of enteroenteric intussusception examined by three-dimensional computed tomography enteroclysis].

Three-dimensional computed tomography (3D CT) enteroclysis or virtual enteroscopy is a novel technique to explore the entire small bowel using a modified protocol of virtual colonoscopy by inflating the small bowel with air. In our hospital, the procedure is performed routinely for cases with suspected gross lesions. We performed 3D CT enteroclysis for three cases with enteroenteric intussusception bowel. The lesions associated with intussusception were identified, single-incision laparoscopic surgery was performed, and diagnoses of lipoma and Peutz-Jeghers polyp were made in two cases. 3D CT enteroclysis did not reveal any associated lesion in the third case. This was followed by an intraoperative exploration during gastrectomy for stomach cancer, but no intestinal lesion was found. A diagnosis of idiopathic intussusception and its spontaneous release was made, and no recurrence was observed during the follow-up period. 3D CT enteroclysis seems to be an appropriate modality for the evaluation of enteroenteric intussusception.

[Meckel's diverticulum detected by virtual enteroscopy in a patient with nonsteroidal anti-inflammatory drug-induced enteropathy].

A 68-year-old female presented to our hospital with abdominal discomfort and obscure gastrointestinal bleeding. She had been prescribed aspirin for retinal venous occlusion. Video capsule endoscopy (VCE) revealed multiple erosions, annular ulcers, and bleeding, confirming a diagnosis of nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy. Virtual enteroscopy (VE) was performed to evaluate stenosis of the small intestine, during which a 5-cm long diverticulum was incidentally detected at a site 99cm from the ileocecal valve. On the basis of the location, size, and shape, a diagnosis of Meckel's diverticulum was made. Second look of the VCE images could not detect the Meckel's diverticulum. After the cessation of taking aspirin, the patient had no more abdominal symptoms, and we concluded NSAID-induced enteropathy was the cause of the symptoms. Meckel's diverticula are sometimes difficult to diagnose, but VE was able to depict the lesion clearly. Meckel's diverticulum is one of the best indications for VE.

[Virtual enteroscopy for the evaluation of stenosis in a case of chronic multiple ulcers of the small intestine].

A 39-year-old female presented to our hospital with diarrhea, vomiting, anemia, and hypoalbuminemia. Virtual enteroscopy was performed to evaluate the small bowel; we found annular stenoses at 89, 100, 116, 147, and 154 cm from the ligament of Treitz. Small bowel resection was performed, and annular ulcers were confirmed at 58, 71, 90, 130, 138, 218, and 225 cm from the ligament of Treitz. Clinical records and pathological examination failed to determine the cause of these ulcers, and we diagnosed chronic multiple ulcers of the small intestine. Thus, we believe that virtual enteroscopy can be beneficial in preoperatively diagnosing multiple ulcers and stenoses in the small bowel.

Corticosteroids correct aberrant CFTR localization in the duct and regenerate acinar cells in autoimmune pancreatitis.

BACKGROUND & AIMS: Corticosteroids are now widely accepted as a treatment for autoimmune pancreatitis (AIP). However, the molecular mechanism by which steroid treatment improves AIP remains largely unknown. The aim of this study was to elucidate cellular mechanisms by which corticosteroids improve both pancreatic exocrine function and histopathology in AIP. METHODS: Pancreatic exocrine function was evaluated by the secretin-stimulated function test and pancreatic biopsy specimens were processed for histologic analysis at the time of diagnosis and 3 months after initiation of steroid treatment. Expression and localization of proteins was assayed by immunohistochemistry. Analysis of immunoglobulin (Ig)G4-positive plasma cells was used to verify inflammation in AIP. RESULTS: The number of IgG4-positive plasma cells in pancreatic sections was decreased by steroid treatment, indicating reduced inflammation. Fluid, bicarbonate (HCO(3)(-)), and digestive enzyme secretions all were impaired in most patients with AIP. Corticosteroids improved both HCO(3)(-) and digestive enzyme secretion. A large fraction of the cystic fibrosis transmembrane conductance regulator (CFTR), which plays a central role in pancreatic duct HCO(3)(-) secretion, was mislocalized to the cytoplasm of duct cells before treatment. Corticosteroids corrected the localization of CFTR to the apical membrane, accounting for the improved HCO(3)(-) secretion. Steroid treatment resulted in regeneration of acinar cells, accounting for restored digestive enzyme secretion. CONCLUSIONS: Corticosteroids reduce inflammation and restore both digestive enzyme and HCO(3)(-) secretion in patients with AIP by regenerating acinar cells and correcting CFTR localization in pancreatic duct cells. Mislocalization of CFTR may explain aberrant HCO(3)(-) secretion in other forms of pancreatitis.

Usefulness of virtual enteroscopy for the detection of small polypoid lesion in the small bowel, a case report.

INTRODUCTION: Virtual enteroscopy (VE) has been developed to explore the entire small bowel. We have previously reported that VE can reveal elevated lesions measuring >10 mm in diameter. However, data on the existence of smaller polypoid lesions is scarce. This study aimed to report a case of pyogenic granuloma in the ileum detected by VE. PRESENTATION OF CASE: A 55-year-old woman presented to our hospital with iron deficiency anemia. Esophagogastroduodenoscopy, colonoscopy, and abdominal contrast-enhanced computed tomography did not indicate any bleeding sources. Video capsule endoscopy revealed a small polypoid lesion in the small bowel. VE was subsequently performed and a polypoid lesion was detected at 119 cm from the ileocecal valve. Its size was estimated to be 6 mm. Based on VE findings, laparoscopic-assisted surgery for the small bowel tumor was performed. During surgery, the polypoid lesion, at 120 cm from the end of the ileum, was barely palpable. The resected specimen showed a 5.5 × 5.0 mm polypoid lesion. Microscopically, the polypoid lesion was diagnosed as pyogenic granuloma. DISCUSSION: We detected a 5.5 × 5.0 mm polypoid lesion in the small bowel, and this is the minimum size of the lesion visualized on VE. This imaging technique provides surgeons with data on the location, number, and size of polypoid lesions. CONCLUSION: VE is a new useful tool for the preoperative collection of data on small polypoid lesions in the small bowel.

An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells.

We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.

Transcriptome analysis of mouse stem cells and early embryos.

Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization.

Mammalian preimplantation embryos provide an excellent opportunity to study temporal and spatial gene expression in whole mount in situ hybridization (WISH). However, large-scale studies are made difficult by the size of the embryos ( approximately 60mum diameter) and their fragility. We have developed a chamber system that allows parallel processing of embryos without the aid of a microscope. We first selected 91 candidate genes that were transcription factors highly expressed in blastocysts, and more highly expressed in embryonic (ES) than in trophoblast (TS) stem cells. We then used the WISH to identify 48 genes expressed predominantly in the inner cell mass (ICM) and to follow several of these genes in all seven preimplantation stages. The ICM-predominant expressions of these genes suggest their involvement in the pluripotency of embryonic cells. This system provides a useful tool to a systematic genome-scale analysis of preimplantation embryos.

Fecal pancreatic elastase: a reproducible marker for severe exocrine pancreatic insufficiency.

BACKGROUND: In order to apply fecal pancreatic elastase for follow-up of exocrine pancreatic function in chronic pancreatitis and cystic fibrosis, we examined the sensitivity, specificity, and long-term variability of a new polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA). METHODS: Patients with definite chronic pancreatitis (n = 23), probable or possible chronic pancreatitis (n = 14), autoimmune pancreatitis (n = 7), or acute pancreatitis (n = 11), and 51 healthy subjects and 11 healthy infants participated in this study. Pancreatic function was graded as normal (n = 3), mild (n = 18), moderate (n = 9), or severe (n = 18) exocrine insufficiency on the basis of secretin tests. Fecal pancreatic elastase was measured by a new ELISA. RESULTS: Fecal pancreatic elastase concentration in control subjects varied widely, with a median of 478 microg/g. The specificity of this test was 90.2% with a cutoff value of >200 microg/g. The sensitivities were 60.9% for detecting definite chronic pancreatitis, 76.5% for calcifying pancreatitis, 71.4% for autoimmune pancreatitis, and 7.1% for probable or possible chronic pancreatitis. The sensitivities were 16.7% for mild, 12.5% for moderate, and 72.2% for severe exocrine pancreatic insufficiency. Forty patients were reexamined after a median interval of 347 days. The fecal pancreatic elastase levels between the first and second tests were not significantly different. Two infants, 4.5 and 5 months old, had abnormally low values, but after a median of 304 days all infants showed normal levels (median, 444 microg/g). CONCLUSIONS: Fecal pancreatic elastase is a reproducible marker for severe exocrine pancreatic insufficiency. This test is valuable for longitudinal follow-up of exocrine pancreatic function.

Computed tomographic enteroclysis with air and virtual enteroscopy: protocol and feasibility for small bowel evaluation.

BACKGROUND AND AIMS: We describe our optimized protocol for computed tomographic enteroclysis using air as the contrast material and report an early assessment of its clinical performance. METHODS: Thirty-one examinations of computed tomographic enteroclysis with air were performed in 30 patients in our hospital from September 2008 to September 2010. The volume of injected air and intra-intestinal pressure were monitored in 16 cases. The data were reviewed for ratios of successful whole small bowel depictions out of the total number of examinations for patients without stenosis. Efforts were made to confirm depicted abnormal findings when possible by other imaging techniques, intra-operative findings, histopathological findings, and subsequent history. RESULTS: The injected air volume and final intra-intestinal pressure were 2925 ± 686 ml and 24.5 ± 7.1cm H2O in cases without stenosis. In 19 examinations with anterograde air injection for patients without stenosis, whole small bowel depiction was achieved in 16 (84.2%). Computed tomographic enteroclysis with air was useful for detecting strictures (in Crohn's disease, malignant lymphoma, metastatic carcinoma), Meckel's diverticulum, and for excluding other obstructive conditions in ileus. CONCLUSIONS: Computed tomographic enteroclysis with air has a potential to enable the exploration of the whole small bowel, thereby providing information of small bowel lesions that complements other techniques.

Molecular mechanisms of pancreatic stone formation in chronic pancreatitis.

Chronic pancreatitis (CP) is a progressive inflammatory disease in which the pancreatic secretory parenchyma is destroyed and replaced by fibrosis. The presence of intraductal pancreatic stone(s) is important for the diagnosis of CP; however, the precise molecular mechanisms of pancreatic stone formation in CP were left largely unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel expressed in the apical plasma membrane of pancreatic duct cells and plays a central role in [Formula: see text] secretion. In previous studies, we have found that CFTR is largely mislocalized to the cytoplasm of pancreatic duct cells in all forms of CP and corticosteroids normalizes the localization of CFTR to the proper apical membrane at least in autoimmune pancreatitis. From these observations, we could conclude that the mislocalization of CFTR is a cause of protein plug formation in CP, subsequently resulting in pancreatic stone formation. Considering our observation that the mislocalization of CFTR also occurs in alcoholic or idiopathic CP, it is very likely that these pathological conditions can also be treated by corticosteroids, thereby preventing pancreatic stone formation in these patients. Further studies are definitely required to clarify these fundamental issues.

Aquaporin 1 water channel is overexpressed in the plasma membranes of pancreatic ducts in patients with autoimmune pancreatitis.

Chronic pancreatitis with all kinds of etiologies is characterized by pancreatic exocrine dysfunction especially impaired fluid secretion from pancreatic ducts. However, the molecular mechanism of this impaired fluid secretion in chronic pancreatitis is largely unknown. Aquaporin water channels are intrinsic membrane proteins expressed most of the cell types which have high osmotic water permeability. Among them aquaporin 1 (AQP1) is a predominant water channel expressed in the plasma membranes of human pancreatic ducts. Exocrine function test revealed that fluid secretion was severely impaired in AIP. immunohistochemical analysis revealed that AQP1 is localized mainly in the apical and lateral membranes of small pancreatic ducts in control subjects. AQP1 expression was significantly increased in plasma membranes of pancreatic ducts in AIP. Upregulation of AQP1 expression seen in pancreatic ducts of patient with AIP may be caused by the reduced fluid secretion from the pancreas as compensation. Further study would be required to elucidate the precise molecular mechanism for the role of AQP1 in pancreatic fluid secretion from the pancreas in diseases characterized by the impaired ductal fluid secretion such as cystic fibrosis.

Esg1, expressed exclusively in preimplantation embryos, germline, and embryonic stem cells, is a putative RNA-binding protein with broad RNA targets.

In our earlier attempt to identify genes involved in the maintenance of cellular pluripotency, we found that KH-domain protein Embryonal stem cell-specific gene 1 (Esg1) showed similar expression patterns to those of Oct3/4 (Pou5f1), whereas the forced repression of Oct3/4 in mouse embryonic stem cells immediately downregulated the expression of Esg1. Here we further confirm this overlap by in situ hybridization and immunohistochemical analyses. Both Esg1 transcript and protein exist in the egg and preimplantation embryos. At embryonic day 3.5, blastocyst stage, however, ESG1 protein was more abundant in the inner cell mass (ICM) than in trophectoderm (TE), whereas Esg1 transcript was detected in both the ICM and the TE, particularly in the polar trophectoderm. The presence of an RNA-binding KH-domain in ESG1 led us to search for and identify 902 target transcripts by microarray analysis of immunoprecipitated ESG1 complex. Interaction of 20 target mRNA with ESG1, including Cdc25a, Cdc42, Ezh2, Nfyc and Nr5a2, was further validated by reverse transcriptase-polymerase chain reaction of the immunoprecipitation material, supporting the notion that ESG1 is an RNA-binding protein which associates with specific target transcripts.

Dual effects of n-alcohols on fluid secretion from guinea pig pancreatic ducts.

Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular Ca(2+) concentration ([Ca(2+)](i)) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [Ca(2+)](i) was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3-10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [Ca(2+)](i) in secretin- or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [Ca(2+)](i) in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3-10 mM concentrations significantly reduced fluid secretion and decreased [Ca(2+)](i) in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [Ca(2+)](i) response to ACh as well as the [Ca(2+)](i) elevation induced by thapsigargin, suggesting that they inhibit Ca(2+) influx. Removal of extracellular Ca(2+) reduced [Ca(2+)](i) in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [Ca(2+)](i) in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane Ca(2+) channel.

Distribution of aquaporin 1 in the rat pancreatic duct system examined with light- and electron-microscopic immunohistochemistry.

The pancreatic duct is the major site for the secretion of pancreatic fluid, but the pathway of water transport in this system is not known. Recently, intense signal for mRNA of aquaporin 1 (AQP1) water channels was detected in isolated rat interlobular ducts. Therefore, we performed light- and electron-microscopic (EM) immunohistochemistry for AQP1 in the rat pancreatic ducts. AQP1 immunoproducts were not observed in the acinar cells, centroacinar cells or intercalated ducts. In the smaller intralobular ducts less than 10 microm in diameter (the lumen plus duct cells), most cells were immunonegative. AQP1-positive cells appeared in intralobular ducts 10-15 microm in diameter. In small and medium-sized interlobular ducts 15-70 microm in diameter surrounded by periductal connective tissue 2-40 microm thick, most cells were AQP1 positive with various degrees of immunoreactivity. In the larger interlobular ducts, the expression of AQP1 was variable, ranging from immunopositive to negative. In the main pancreatic duct, most cells were negative for AQP1. EM immunohistochemistry of the intralobular and small interlobular ductal epithelial cells showed that the AQP1 immunoproducts were more abundant in the basolateral membrane than in the apical membrane, though they were present in both membranes. In the medium-sized interlobular ducts, AQP1 immunoproducts were distributed densely along the apical, lateral interdigitation and basal membrane of the epithelial cells. In the various sizes of interlobular ducts, immunoproducts were associated not only with the plasma membrane, but also with the caveolae and vesicle-like structures. Secretin did not induce any significant difference in AQP1 expression and cellular and subcellular localization. These results indicate that the expression and subcellular localization of AQP1 vary considerably depending on the duct size, which may reflect water transport characteristics in the different divisions of the pancreatic duct system.

Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells.

Transplantation of nuclei (NT) from engineered mouse ES cells is a potentially powerful and rapid route to create knockout mice, obviating the need for matings to obtain germ-line chimeras. However, such an application is currently impossible, because NT often results in abnormalities in embryo and placenta. Although the epigenetic instability of several imprinted genes in ES cells and ES-derived NT mice has been demonstrated, it is not clear yet what causes the abnormalities. To gain perspective on the extent and types of changes, we have done gene expression profiling for mouse placentas produced by NT of ES cells and compared them with the expression profiles of placentas produced by NT of one-cell embryos. Based on microarray studies with the NIA 15K mouse cDNA collection, we report five principal aberrant events: (1) inappropriate expression of imprinted genes; (2) altered expression of regulatory genes involved in global gene expression, such as DNA methyltransferase and histone acetyltransferase; (3) increased expression of oncogenes and growth promoting genes; (4) overexpression of genes involved in placental growth, such as Plac1; and (5) identification of many novel genes overexpressed in ES-derived NT mouse placentas, including Pitrm1, a new member of the metalloprotease family. The results indicate that placentomegaly in ES-derived NT mice is associated with large-scale dysregulation of normal gene expression patterns. The study also suggests the presence of two regulatory pathways that may lead to histologically discernable placentomegaly. The discovery of groups of genes with altered expression may provide potential targets for intervention to mimic natural regulation more faithfully in NT mice.

Aquaporins in rat pancreatic interlobular ducts.

The aquaporin (AQP) family of water channels is distributed ubiquitously in many epithelia and plays a fundamental role in transmembrane water transport. The aim of this study is to identify the water transport pathway in pancreatic duct cells where most of the HCO-rich fluid originates. Using digital videomicroscopy, we measured the osmotic water permeability (P(f)) of pancreatic duct epithelium by exposing isolated rat interlobular ducts to the hypotonic solution (145 mosM). To identify mRNA and protein of AQPs expressed in duct cells, we conducted RT-PCR analysis and immunohistochemistry of the isolated duct and pancreas. The calculated P(f) (160-230 microm/s) of the isolated ducts was significantly reduced to 16-35 microm/s by 80-90% with either basolateral or luminal applications of HgCl(2). Fluid secretion evoked by secretin was almost completely abolished by a basolateral or luminal application of HgCl(2). A large amount of AQP1 and small amounts of AQP5 transcripts were detected in the isolated duct cells by RT-PCR. AQP1, but not AQP5, immunoreactivity was present in both luminal and basolateral membranes of the interlobular duct cells. Mercury-sensitive water channels are present in both luminal and basolateral membranes of rat pancreatic ducts. AQP1 of the known AQPs appears to be the main water pathway in interlobular ducts.

A finger sweat chloride test for the detection of a high-risk group of chronic pancreatitis.

OBJECTIVES: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are associated with chronic pancreatitis in Caucasians. We developed a simple method for measuring finger sweat chloride concentration to test whether CFTR dysfunction underlies chronic pancreatitis in Japan where cystic fibrosis (CF) is rare. METHODS: We studied 25 patients with chronic (21 alcoholic and 4 idiopathic) pancreatitis and 25 healthy volunteers. Sweat chloride concentrations were measured by a finger sweat chloride test. We analyzed DNA for 20 common CFTR mutations in Europeans, 9 CF-causing mutations in Japanese, and 2 polymorphic loci, a poly-T tract and (TG) repeats, at intron 8. RESULTS: Thirteen patients (52%) had sweat chloride levels >60 mmol/L, a level consistent with CF, while only 4 (16%) healthy subjects exceeded this level. The 29 CF mutations and the 5T allele were detected in neither the patients nor controls. The (TG) 12 allele was common in both the patients (58%) and controls (48%). The (TG) 12/12 genotype was common in alcoholic pancreatitis (29%) compared with the (TG) 11/11 (10%). Patients with the (TG) 12/12 genotype had significantly higher sweat chloride concentrations than the controls. CONCLUSION: CFTR dysfunction as evidenced by a finger sweat chloride test is present in about half of Japanese patients with chronic pancreatitis, suggesting that this test may be useful for detecting the high-risk group. A higher proportion of the (TG) 12 allele may be a genetic background for elevated sweat chloride concentrations in Japanese patients.