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Adult autologous human epidermal stem cells can be extensively expanded ex vivo for cell and gene therapy. Identifying the mechanisms involved in stem cell maintenance and defining culture conditions to maintain stemness is critical, because an inadequate environment can result in the rapid conversion of stem cells into progenitors/transient amplifying cells (clonal conversion), with deleterious consequences on the quality of the transplants and their ability to engraft. Here, we demonstrate that cultured human epidermal stem cells respond to a small drop in temperature through thermoTRP channels via mTOR signaling. Exposure of cells to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single-cell analysis that long-term inhibition of mTORC1 reduces clonal conversion and favors the maintenance of stemness. Taken together, our results demonstrate that human keratinocyte stem cells can adapt to environmental changes (e.g., small variations in temperature) through mTOR signaling and constant inhibition of mTORC1 favors stem cell maintenance, a finding of high importance for regenerative medicine applications., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Skin regenerative capacity declines with age, but the underlying mechanisms are largely unknown. Here we demonstrate a functional link between epidermal growth factor receptor (EGFR) signaling and type XVII collagen (COL17A1) proteolysis on age-associated alteration of keratinocyte stem cell dynamics in skin regeneration. Live-imaging and computer simulation experiments predicted that human keratinocyte stem cell motility is coupled with self-renewal and epidermal regeneration. Receptor tyrosine kinase array identified the age-associated decline of EGFR signaling in mouse skin wound healing. Culture experiments proved that EGFR activation drives human keratinocyte stem cell motility with increase of COL17A1 by inhibiting its proteolysis through the secretion of tissue inhibitor of metalloproteinases 1 (TIMP1). Intriguingly, COL17A1 directly regulated keratinocyte stem cell motility and collective cell migration by coordinating actin and keratin filament networks. We conclude that EGFR-COL17A1 axis-mediated keratinocyte stem cell motility drives epidermal regeneration, which provides a novel therapeutic approach for age-associated impaired skin regeneration., (© 2021 Nanba et al.)

Stem cell-based products have clinical and industrial applications. Thus, there is a need to develop quality control methods to standardize stem cell manufacturing. Here, we report a deep learning-based automated cell tracking (DeepACT) technology for noninvasive quality control and identification of cultured human stem cells. The combination of deep learning-based cascading cell detection and Kalman filter algorithm-based tracking successfully tracked the individual cells within the densely packed human epidermal keratinocyte colonies in the phase-contrast images of the culture. DeepACT rapidly analyzed the motion of individual keratinocytes, which enabled the quantitative evaluation of keratinocyte dynamics in response to changes in culture conditions. Furthermore, DeepACT can distinguish keratinocyte stem cell colonies from non-stem cell-derived colonies by analyzing the spatial and velocity information of cells. This system can be widely applied to stem cell cultures used in regenerative medicine and provides a platform for developing reliable and noninvasive quality control technology., (© 2021 The Authors. STEM CELLS published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Impaired wound healing in critical limb ischemia (CLI) results from multiple factors that affect many cell types and their behavior. Epidermal keratinocytes and dermal fibroblasts play crucial roles in wound healing. However, it remains unclear whether these cell types irreversibly convert into a non-proliferative phenotype and are involved in impaired wound healing in CLI. Here, we demonstrate that skin keratinocytes and fibroblasts isolated from CLI patients maintain their proliferative potentials. Epidermal keratinocytes and dermal fibroblasts were isolated from the surrounding skin of foot wounds in CLI patients with diabetic nephropathy on hemodialysis, and their growth potentials were evaluated. It was found that keratinocytes from lower limbs and trunk of patients can give rise to proliferative growing colonies and can be serially passaged. Fibroblasts can also form colonies with a proliferative phenotype. These results indicate that skin keratinocytes and fibroblasts maintain their proliferative capacity even in diabetic and ischemic microenvironments and can be reactivated under appropriate conditions. This study provides strong evidence that the improvement of the cellular microenvironments is a promising therapeutic approach for CLI and these cells can also be used for potential sources of skin reconstruction., (© 2020 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Identification and quality assurance of stem cells cultured in heterogeneous cell populations are indispensable for successful stem cell therapy. Here we present an image-processing pipeline for automated identification and quality assessment of human keratinocyte stem cells. When cultivated under appropriate conditions, human epidermal keratinocyte stem cells give rise to colonies and exhibit higher locomotive capacity as well as significant proliferative potential. Image processing and kernel density estimation were used to automatically extract the area of keratinocyte colonies from phase-contrast images of cultures containing feeder cells. The DeepFlow algorithm was then used to calculate locomotion speed of the colony area by analyzing serial images. This image-processing pipeline successfully identified keratinocyte stem cell colonies by measuring cell locomotion speed, and also assessed the effect of oligotrophic culture conditions and chemical inhibitors on keratinocyte behavior. Therefore, this study provides automated procedures for image-based quality control of stem cell cultures and high-throughput screening of small molecules targeting stem cells.

Background: Skin fibroblast heterogeneity is of growing interest due to its relevance in not only skin development but also cutaneous wound healing. However, the characterization of human dermal fibroblasts at a clonal level has not been accomplished and their functional heterogeneity remains poorly understood., Objective: The aim of this study was to define the clonal heterogeneity of human dermal fibroblasts., Methods: Isolated human dermal fibroblasts were clonally expanded and categorized by comprehensive phenotypic and gene expression profiling., Results: Single fibroblasts were significantly multiplied and efficiently cloned without chromosomal abnormalities under hypoxic conditions. Individual clones were heterogeneous in their proliferative capacity, and gene expression profiling revealed differences in the expression of genes involved in extracellular matrix synthesis and degradation. Each cloned fibroblast also had different abilities in terms of collagen remodeling. All phenotypic and gene expression data were analyzed with Spearman's rank correlation, and fibroblasts were categorized into at least two functional clonal types. One was highly proliferative, while the other was less proliferative but had the ability to remodel the tissue architecture. The proliferative clones were predominant in infants, but decreased with physiological aging., Conclusion: This study provides strong evidence for the functional heterogeneity of human dermal fibroblasts at a clonal level, which has implications regarding skin repair and aging., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation., (© 2015 Nanba et al.)

Background/purpose: To clarify the role of interval appendectomy (IA) in pediatric patients with acute appendicitis with an appendiceal inflammatory mass or abscess, we histologically analyzed the appendices removed during IA., Patients and Methods: We treated 355 consecutive pediatric patients with acute appendicitis and reviewed the admission charts of patients who started conservative management (CM). The histology of the appendix removed during IA was also examined. The relationships among the clinical features, appendicolith formation at the time of IA and histological findings were analyzed by stepwise regression analyses., Results: (1) CM was started in 48 patients (13.5 %). Recurrence or a remaining abscess was observed in nine patients (18.8 %). (2) Histopathological changes, particularly foreign body reaction with fibrosis and infiltration of inflammatory cells, were observed in about half of the specimens. (3) In a stepwise regression analysis, the presence of an appendicolith at IA was correlated with an appendicolith at diagnosis, foreign body reaction in the appendix and a decrease in the inflammatory reaction at diagnosis., Conclusion: More than half the patients had strong histopathological changes in the appendix, suggesting a high possibility of recurrence. The presence of appendicolith formation at IA, which is a risk factor for recurrence, was influenced by the presence of an appendicolith at diagnosis, foreign body reaction in the appendix and the inflammatory status of patients at diagnosis. These clinical findings are indications for IA.

The epidermal growth factor (EGF) receptor/ligand system stimulates multiple pathways of signal transduction, and is activated by various extracellular stimuli and inter-receptor crosstalk signaling. Aberrant activation of EGF receptor (EGFR) signaling is found in many tumor cells, and humanized neutralizing antibodies and synthetic small compounds against EGFR are in clinical use today. However, these drugs are known to cause a variety of skin toxicities such as inflammatory rash, skin dryness, and hair abnormalities. These side effects demonstrate the multiple EGFR-dependent homeostatic functions in human skin. The epidermis and hair follicles are self-renewing tissues, and keratinocyte stem cells are crucial for maintaining these homeostasis. A variety of molecules associated with the EGF receptor/ligand system are involved in epidermal homeostasis and hair follicle development, and the modulation of EGFR signaling impacts the behavior of keratinocyte stem cells. Understanding the roles of the EGF receptor/ligand system in skin homeostasis is an emerging issue in dermatology to improve the current therapy for skin disorders, and the EGFR inhibitor-associated skin toxicities. Besides, controlling of keratinocyte stem cells by modulating the EGF receptor/ligand system assures advances in regenerative medicine of the skin. We present an overview of the recent progress in the field of the EGF receptor/ligand system on skin homeostasis and regulation of keratinocyte stem cells., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Introduction: The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment., Methods: In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry., Results: Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene., Conclusions: This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine.

Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Remodeling of collagen fibrils is involved in a variety of physiological and pathological processes including development, tissue repair, and metastasis. Fibroblast-populated collagen gel contraction has been employed as a model system to investigate the collagen fibril remodeling within three-dimensional collagen matrices. Research on collagen gel contraction is also important for understanding the mechanism underlying connective tissue repair, and for design considerations for engineered tissues in regenerative medicine. Second harmonic generation (SHG) is a non-linier optical effect by which well-ordered protein assemblies, including collagen fibrils, can be visualized without any labeling, and used for a noninvasive imaging of collagen fibrils in the skin. Here we demonstrate that the remodeling of collagen fibrils in the fibroblast-populated collagen gel can be analyzed by SHG imaging with a multiphoton microscope. Two models of collagen gel contraction (freely versus restrained contraction) were prepared, and orientation of fibroblasts, density, diameter, and distribution of collagen fibrils were examined by multiphoton fluorescent and SHG microscopy. Three-dimensional construction images revealed vertical and horizontal orientation of fibroblasts in freely and restrained gel contraction, respectively. Quantitative analysis indicated that collagen fibrils were accumulated within the gel and assembled into the thicker bundles in freely but not restrained collagen gel contraction. We also found that actomyosin contractility was involved in collagen fibril remodeling. This study elucidates how collagen fibrils are remodeled by fibroblasts in collagen gel contraction, and also proves that SHG microscopy can be used for the investigation of the fibroblast-populated collagen gel.

Background: The esophagus is physiologically devoid of eosinophils, so their presence would suggest some underlying pathology. The prevalence of eosinophilic esophagitis (EoE) has steadily increased in Western countries. Previous studies have described EoE in association with congenital esophageal atresia (CEA), which is the most common congenital anomaly of the esophagus. However, the association remains unclear., Methods: We performed a retrospective histological analysis examining for eosinophil infiltration in the esophagus of patients with CEA following surgical repair or congenital esophageal stenosis (CES) who underwent esophageal biopsy or surgical resection in our hospital between 2005 and 2012., Results: There were 6 patients with CEA following surgical repair or CES who had eosinophil-dominant infiltration in the esophagus. All had associated allergic disorders, including food allergies in 4. Moreover, all except for one fulfilled the histological criteria of EoE. Impairment of eosinophil infiltration and symptomatic improvement were observed in those treated with a proton pump inhibitor (PPI), either alone or in combination with steroids after esophageal dilatation., Conclusions: These findings suggest that CEA repair or CES in conjunction with allergic conditions and coexisting gastroesophageal reflux disease (GERD) may induce greater esophageal eosinophilic inflammation. In addition, esophageal dilatation followed by PPI treatment, alone or with steroids, may be a therapeutic strategy that can provide symptomatic relief by reducing eosinophilic inflammation in esophageal strictures or GERD associated with CEA or CES., (Copyright © 2013 S. Karger AG, Basel.)

Background: The timing of herniotomy in premature infants is controversial., Methods: Outcomes of herniotomy in 47 premature infants admitted to the neonatal intensive care unit (NICU) were retrospectively reviewed for preoperative clinical features, respiratory interventions, and anesthetic and surgical complications. The data were compared with those of full-term infants (n = 52). Fourteen of the premature infants underwent herniotomy before NICU discharge and 33 after discharge. The predictive factors for anesthetic and surgical complications were also investigated via multiple regression analysis., Results: Mean post-conceptional age at surgery in premature infants and full-term infants was 47 weeks and 50 weeks, respectively. Mean bodyweight at surgery in those infants was 4087 g and 5454 g, respectively. The rate of incarcerated hernia and emergency surgery was lower in premature infants. Delayed extubation of the tracheal tube after surgery was noted in four premature infants, but not in full-term infants. Two cases of cryptorchidism in premature infants and one recurrence in a full-term infant that required reoperation were noted. On multiple regression analysis no factor (including respiratory interventions) was found to be capable of predicting complications., Conclusion: Although no predictive factor for complications was identified, there were some anesthetic and surgical complications in premature infants. If there is no risk of incarceration, herniotomy in premature infants should be performed at a time when the risk of anesthetic complications is decreased. If there is a risk of incarcerated hernia, herniotomy should be performed carefully in order to avoid occurrence of anesthetic and surgical complications., (© 2012 The Authors. Pediatrics International © 2012 Japan Pediatric Society.)

Feeding dysfunction (FD) has recently been considered to comprise a prevalent set of symptoms in eosinophilic gastrointestinal disorders (EGIDs) in young children. We report the case of an 8-month-old girl with an EGID who visited our hospital due to vomiting, poor weight gain and feeding difficulties; her condition was discovered during the examination of the symptoms including FD. Tracheal aspiration and reduced esophageal clearance showed up in a barium swallow test and upper gastrointestinal contrast radiography, respectively. Delayed clearance from the stomach was also detected on gastrointestinal scintigraphy. Gastrointestinal endoscopy and biopsies revealed esophagitis with some eosinophils and duodenitis with eosinophilic inflammation. She was not a likely candidate for eosinophilic esophagitis. On administration of an elemental diet, the patient gained weight. Esophageal and stomach clearance subsequently improved, although the vomiting and FD persisted to some extent. We conclude that it is important to consider other EGIDs as well as eosinophilic esophagitis in the differential diagnosis of FD., (Copyright © 2012 S. Karger AG, Basel.)

Background: We aimed to develop experimental models of hypoxia/ischemia-induced cholestasis using neonatal and infantile rats., Methods: Hypoxia/ischemia was induced in the bile duct (BD) by injecting prostaglandin (PG) at birth and/or by coagulation of the hepatic artery (CHA) at about 3 weeks after birth. The rats were divided into 6 groups: control; PG-injected; sham-operated with or without PG; CHA; and CHA + PG. CHA was also performed in adult rats. Liver specimens and blood samples were obtained at 5 weeks after birth, and immunohistochemical and biochemical examinations were performed., Results: (1) BD proliferation with fibrosis (BDPF) was found in the intrahepatic portal tract in the CHA and CHA + PG groups. Low-grade BDPF was observed in the PG group. (2) Cyst formation in the extrahepatic BD (EBD) was observed in the porta hepatis of some rats in the CHA and CHA + PG groups. In these groups, the number of peribiliary vascular plexuses (PVPs) decreased. BD proliferation and infiltration of inflammatory cells were observed in the EBD wall in the CHA + PG group. (3) Ki-67 was expressed in BD and EBD cells in the CHA + PG group. (4) BDPF was not detected in adult rats with CHA. (5) Serum liver function tests indicated obstructive changes in the EBD in the CHA and CHA + PG groups., Conclusion: Reduced blood flow in the EBD during infancy induced BDPF and obstructive changes in the EBD, which may, along with immature PVP and inflammatory changes in the EBD, contribute to hypoxia/ischemia of the EBD.

Objective: We have reviewed the endoscopic retrograde cholangiopancreatography (ERCP) images of patients with autoimmune pancreatitis (AIP) and pancreatic carcinoma (Pca) in an attempt to identify findings that would facilitate making a differential diagnosis between AIP and Pca., Methods: The study cohort consisted of 39 patients diagnosed with AIP and 62 patients diagnosed with Pca. The ERCP findings in the pancreatic duct and biliary tract were compared between the two groups., Results: The ERCP images revealed that AIP patients had a higher prevalence of narrowing of the main pancreatic duct (MPD) for ≥ 3 cm of its length and a higher prevalence for the presence of side branches in the narrowed portion of the MPD than Pca patients (p < 0.001 and p < 0.001, respectively). In contrast, the prevalence of an upstream MPD having a maximal diameter ≥ 4 cm was significantly higher in the Pca patient group (p < 0.001). The discriminant analysis identified three significant factors: (1) whether or not side branches were present; (2) total length of the narrowed portion of the MPD; (3) maximal diameter of the upstream MPD. It was impossible to differentiate Pca from AIP in the two Pca patients in whom ERCP revealed both narrowing of the MPD for > 5 cm of its length and the presence of side branches., Conclusions: Among our patient cohort, the ERCP findings in terms of the length of the narrowed portion of the MPD, the presence of side branches, and maximal diameter of the upstream MPD enabled differential diagnosis between AIP and Pca in most of the cases. However, it must be borne in mind that some Pca patients have ERCP findings similar to those of AIP patients.

Aim: To investigate a relationship between the clinicopathological features and mucin phenotypes in advanced gastric adenocarcinoma (AGA)., Methods: Immunohistochemical staining was performed to determine the mucin phenotypes in 38 patients with differentiated adenocarcinomas (DACs), 9 with signet-ring cell carcinomas (SIGs), and 48 with other diffuse-type adenocarcinomas (non-SIGs) of AGA. The mucin phenotypes were classified into 4 types: gastric (G), gastrointestinal (GI), intestinal, and unclassified., Results: The G-related mucin phenotypes were highly expressed in all the histological subtypes of AGA. The expression of the GI phenotype in SIG patients was lower than that in DAC patients (P = 0.02), and this phenotype was observed in 56% of the non-SIG patients in the intramucosal layer. Among non-SIG cases, the expression of the GI phenotype was significantly higher in patients with extended adenocarcinomas and those with positive rates of lymph node metastasis. There was no difference between the expressions of the G and other GI phenotypes factors. Among DAC and non-SIG patients, there were no differences between the survival rates of the corresponding patient groups., Conclusion: The GI phenotype might possess more invasive characteristics than the G phenotype in non-SIG. Neither of the phenotypes indicated a poor prognosis of DAC and non-SIG.

Background: Using magnetic resonance imaging (MRI), changes in the livers of postoperative biliary atresia (BA) patients were investigated., Methods: Periodic MRI was performed in 32 postoperative BA patients. The findings were evaluated by calculating the near-normal liver tissue area that corresponded with normal- or high-signal regions on T1-weighted imaging. The patients were divided into three groups based on the extent of near-normal liver tissue on the final MRI: group A, n = 14; group B, n = 13; and group C, n = 5, included patients with >40%, 20-40%, and <20% area of near-normal liver tissue, respectively. The relationship among the macroscopic and histological findings in the liver at orthotopic living donor liver transplantation (OLDLT), patient outcomes, and MRI findings were investigated., Results: In group A, 11 patients had no evidence of liver dysfunction. In group B, six patients either had undergone or were awaiting OLDLT. In group C, all patients had undergone OLDLT. All patients had either adequate or impaired bile drainage in each liver segment. The segmental changes corresponded with the liver architecture at OLDLT. The changes could be evaluated on MRI at 1-2 years after surgery., Conclusions: Adequate and restricted areas of liver tissue with near-normal structure were indicative of good and poor prognoses, respectively. Shortly after portoenterostomy, these segmental changes occurred and/or developed in each liver segment and could be detected on MRI. It is emphasized that patients with >40% area of near-normal liver architecture at the initial stages did not require OLDLT, while those with <20% area did require OLDLT.

A 14-year-old girl was referred for a large tumor of the left kidney, with intraspinal and vertebral metastases. Left nephrectomy and intraspinal tumor resection were performed. The histology of both tumors was nephroblastoma with no anaplasia and favorable histology, and they were diagnosed as stage IV. The tumor bed and vertebras were irradiated. We started chemotherapy according to the DD-4A regimen of Japanese Wilms' Tumor Study Group. The vertebral metastasis was additionally irradiated. The patient has remained in disease-free remission for 45 months after the surgical resection. Intensive multimodality therapy including DD-4A regimen of National Wilms' Tumor Study can result in long-term disease-free remission.

Background: The present study aimed to examine whether and how colonic motility is affected by mild ischemia-induced intestinal injury in conscious rats through in vivo monitoring of colonic contractions, specifically with regard to the interstitial cells of Cajal (ICC) and the effect of nitric oxide (NO)., Methods: Using miniature strain-gauge transducers, colonic motility with or without ischemia was recorded in conscious rats on the 4th, 7th, and 14th days after surgery. Histological examination for c-kit-positive cells was performed., Results: In control nonischemic rats, the number and duration of contractions (NC and DC, respectively) decreased gradually, but the mean amplitude of contractions (MC) and motility index (MI) did not change. On the 7th day, the NC in the ischemic group increased significantly when compared with that in the control group (P = 0.037). The DC in the ischemic group was lower than that in the control group; the difference was significant on the 4th day (P = 0.008). The MIs in the ischemic group were lower than those in the control group. In both groups, administration of NGnitro-L: -arginine methyl ester on the 7th day increased only the resting cecal motility. Pathological examinations revealed c-kit-positive cells in both groups., Conclusions: Changes such as increased NC with shortened DC accompanied with decreased MI must have occurred at the ischemic site and might have been induced by an ischemic event. However, there exists a possibility that ICC and NO do not play a role in mild ischemia-induced dysmotility.

Background: The present study was undertaken to identify the clinicopathological differences between sclerosing cholangitis with autoimmune pancreatitis (SC-AIP) and primary sclerosing cholangitis (PSC)., Methods: We retrospectively compared the clinical, cholangiographic, and liver biopsy findings between 24 cases of PSC and 24 cases of SC-AIP., Results: Patient age at the time of diagnosis was significantly lower in the PSC group than in the SC-AIP group. The peripheral blood eosinophil count was significantly higher in the PSC group than in the SC-AIP group, but the serum IgG4 level was significantly higher in the SC-AIP group. Cholangiography revealed band-like strictures, beaded appearance, and pruned-tree appearance significantly more frequently in PSC, whereas segmental strictures and strictures of the distal third of the common bile duct were significantly more common in SC-AIP. Liver biopsy revealed fibrous obliterative cholangitis only in the PSC specimens. No advanced fibrous change corresponding to Ludwig's stages 3 and 4 was observed in any of the SC-AIP specimens. IgG4-positive plasma cell infiltration of the liver was significantly more severe in SC-AIP than in PSC. Subsequent cholangiography showed no improvement in any of the PSC cases, but all SC-AIP patients responded to steroid therapy, and improvement in the strictures was observed cholangio-graphically., Conclusions: Based on the differences between the patients' ages and blood chemistry, cholangiographic, and liver biopsy findings, SC-AIP should be differentiated from PSC.

A 3-year-old girl with recurrent chylous ascites was successfully treated by laparoscopic ligation of the ruptured lymphatic trunk. She was referred to our hospital at 16 days of age because of marked abdominal distension. Imaging methods showed massive ascites of unknown origin, and analysis of the ascites revealed its chylous nature. Conservative treatments were started. Her condition improved to some extent, and she was discharged. Two years later, she was readmitted with abdominal distension and loss of appetite. Laparoscopic surgery was planned to clarify the etiology and to treat intractable ascites. Sudan black B was orally administered, and laparoscopy revealed the presence of a whitish-gray fluid in the abdominal cavity, and a dark-blue stream of the dye was noticed. The responsible lesion of the chylous ascites was detected by tracking the stream. The lesion was ligated twice with an endoloop. She has been completely free from the symptoms for 3 years and 9 months. This experience indicates the usefulness of laparoscopic surgery in investigating the etiology of chylous ascites and treating it. The concomitant use of a lipophilic dye is mandatory to find the responsible lesion at surgery. Laparoscopic surgery, instead of open surgery, should be considered as a treatment of choice for intractable chylous ascites.

Background: The present study was undertaken to evaluate the prevalence of pancreatic and biliary tract tumors in pancreas divisum (PD)., Methods: A retrospective single-center study was performed, and a total of 118 cases of complete PD and 7850 cases of fused pancreas were identified among the 8537 consecutive new endoscopic retrograde cholangiopancreatography (ERCP) examinations performed between 1980 and 2002. The prevalence of pancreatic cancer (PCA), intraductal papillary mucinous neoplasms (IPMNs), other pancreatic tumors, and biliary tract cancer in the patients with PD and the patients with a fused pancreas were compared., Results: The prevalence of the pancreatic tumors in the PD patients was: PCA, 10%; IPMN, 5.1%; other pancreatic tumors, 2.5%. The prevalence of pancreatic tumors in the patients with a fused pancreas was: PCA, 4.8%; IPMN, 2.6%; and other pancreatic tumors, 1.1%. The prevalence of PCA was significantly higher in the patients with PD than in those with a fused pancreas (P = 0.008; OR, 2.24). The percentages of PD patients with PCA who had pancreatic-type pain and a serum pancreatic enzyme elevation were significantly higher than among the PD patients without PCA. The prevalence of biliary tract cancer was 0.8% in the PD group and 5.3% in the fused pancreas group, and it was significantly lower in PD than in fused pancreas (P = 0.031)., Conclusions: The results of this study showed a significantly higher prevalence of PCA in PD than in fused pancreas. We concluded that patients with PD, especially patients presenting with pancreatic-type pain and pancreatic enzyme elevation, should be carefully followed up because of the risk of developing PCA.

Objective: We investigated the long-term outcome of autoimmune pancreatitis (AIP) including morphological changes in the pancreas, pancreatic duct, biliary tract, pancreatic function, and changes in the clinical manifestations after oral prednisolone (PSL) therapy., Patients and Methods: We prospectively followed 12 patients for a period of over 12 months (median follow-up period: 41 months; range: from 13 to 133 months). All twelve patients were treated with PSL. The morphological findings consisted of pancreatic enlargement (n=12), an irregularly narrowed main pancreatic duct (n=12), and bile duct stricture (n=10), and salivary gland swelling was observed in six patients. The initial dose of PSL was 30-40 mg/day, and it was subsequently tapered., Results: All 12 patients responded to PSL therapy. The enlargement of the pancreas and the irregularly narrowed main pancreatic duct improved to almost normal. Pancreatic atrophy developed in four of them (4/12, 33%), but no pancreatic calcification was observed in any of the patients. The bile duct stricture improved to various degrees in all 10 patients , but it persisted in the lower part of the bile duct in four of them (4/10, 40%). The salivary gland swelling also improved after PSL therapy. There was no recurrence of enlargement of the pancreas or irregularly narrowed main pancreatic duct after PSL therapy, but the bile duct stricture recurred in one case, and in three cases there was a relapse of salivary gland swelling that required a temporary increase in PSL dose during tapering. No deterioration of pancreatic exocrine function was detected in any of the patients. A malignant tumor was diagnosed in two patients during PSL therapy: early gastric cancer in one and rectal cancer in the other. All patients are alive., Conclusions: AIP treated with PSL has a favorable long-term outcome based on the morphological findings and assessments of pancreatic function. However, since two of the twelve patients developed a malignancy during PSL therapy, strict follow up should be part of the management of AIP.

Heparin-binding EGF-like growth factor (HB-EGF) is initially synthesized as a type I transmembrane protein (proHB-EGF). The proHB-EGF is shed by specific metalloproteases, releasing the N-terminal fragment into the extracellular space as a soluble growth factor (HB-EGF) and the C-terminal fragment (HB-EGF-C) into the intracellular space, where it prevents transcriptional repression by the promyelocytic leukemia zinc finger protein (PLZF). The goal of the present study was to characterize regulation of proHB-EGF shedding and study its temporal variations in HB-EGF-C localization throughout the cell cycle. Quantitative combination analyses of cell surface proHB-EGF and HB-EGF in conditioned medium showed that proHB-EGF shedding occurred during the G(1) cell cycle phase. Laser scanning cytometry (LSC) revealed that HB-EGF-C was internalized into the cytoplasm during the late G1 phase and accumulated in the nucleus beginning in the S phase. Subsequent nuclear export of PLZF occurred during the late S phase. Further, HB-EGF-C was localized around the centrosome following breakdown of the nuclear envelope and was localized to the interzonal space with chromosome segregation in the late M phase. Temporal variations in HB-EGF localization throughout the cell cycle were also characterized by time-lapse imaging of cells expressing YFP-tagged proHB-EGF, and these results were consistent with those obtained in cytometry studies. These results indicate that proHB-EGF shedding and subsequent HB-EGF-C signaling are related with progression of the cell cycle and may provide a clue to understand the unique biological significance of non-receptor-mediated signaling of proHB-EGF in cell growth., (2004 Wiley-Liss, Inc.)

Objectives: Autoimmune pancreatitis (AIP) is a unique clinical entity that has been recently proposed, and it is frequently associated with bile duct stricture. The aim of this study was to investigate the pathophysiology of the biliary tract involvement in patients with AIP., Methods: We evaluated the clinicopathologic findings in 16 patients with AIP. Surgical resection was performed in 7 of the patients because of suspicion of a pancreatic tumor; 8 of the other patients were treated with oral prednisolone (PSL) therapy, and the remaining patient was observed clinically and not treated. The pancreas, bile duct, and gallbladder in the surgical cases were examined histologically and immunohistochemically. We also assessed the clinical manifestations and diagnostic imaging findings before and after oral PSL therapy in the 8 patients treated with PSL., Results: Stricture of the extrahepatic bile duct was detected in 88% (14/16) of the patients. Thickening of the bile duct wall was detected in 94% (15/16), and thickening of the gallbladder wall was observed in 56% (9/16). Histologically, the bile duct and gallbladder wall were characterized by diffuse lymphoplasmacytic infiltration and marked interstitial fibrosis. Immunohistochemically, the diffusely infiltrating cells consisted of predominantly CD8- or CD4-positive T lymphocytes and IgG4-positive plasma cells. These findings were the same as in the inflammatory process that was observed in the pancreas. After oral PSL therapy, the pancreatic enlargement and irregular narrowing of the main pancreatic duct improved to almost their normal size in all 8 patients; however, stricture of the extrahepatic bile duct persisted in 4 of the patients (57%, 4/7) in whom it was detected before PSL therapy., Conclusions: Based on the pathophysiologic and histologic findings and the response to PSL therapy, the biliary involvement in AIP developed by the same mechanism as the pancreatitis. CD8- and CD4-positive lymphocytes and IgG4-positive plasma cells may play an important role in the pathogenesis of AIP.

Heparin-binding EGF-like growth factor (HB-EGF) is initially synthesized as a type I transmembrane precursor (proHB-EGF). Proteolytic cleavage of proHB-EGF yields amino- and carboxy-terminal fragments (HB-EGF and HB-EGF-C, respectively). We have previously shown that HB-EGF-C is translocated from the plasma membrane into the nucleus, where it interacts with the transcription repressor, PLZF. Here we characterize the amino acid residues of the cytoplasmic domain of proHB-EGF on cell surface distribution and the interaction of HB-EGF-C with PLZF. The cytoplasmic domain contains three characteristic clusters with charged amino acids. Generation of various mutants of proHB-EGF showed that the arrangement of the charged amino acids in the cytoplasmic domain regulates the distribution of proHB-EGF at the plasma membrane but does not regulate proHB-EGF processing and internalization of HB-EGF-C. Further, the charged amino acids are also required for HB-EGF-C-PLZF interaction. These results indicate that the cytoplasmic domain of proHB-EGF is a multifunctional domain.

Objective: Ultrasound (US) has been used as a tool to determine the indication for surgery for neonatal ovarian cysts. The purpose of this study was to investigate whether magnetic resonance imaging (MRI) contributes to optimal management., Methods: Between 1993 and 2001, US and MRI studies were simultaneously performed on 13 consecutive infants younger than 2 months of age with ovarian cysts. The US Patterns were classified as complex or simple. Signal intensity (SI) of the cysts on MRI was compared with that of the liver on T1-weighted images (T1WI) and with urine on T2-weighted images (T2WI). We assumed that high SI on T1WI and iso or low SI on T2WI indicated complications., Results: There were 10 complex and three simple cysts on US. Of the 10 complex cysts, two had no complications at surgery or resolved spontaneously. These two cysts showed low SI on T1WI. Eight complex cysts showed high SI on T1WI and all were haemorrhagic. The US diagnosis corresponded to the MRI findings in three simple cysts. The sensitivity of US for haemorrhage was 80%, and that of MRI was 100%., Conclusions: We found that MRI was a more reliable diagnostic modality than US for diagnosing neonatal ovarian cysts.

The authors report two cases of the rare concurrence of intestinal aganglionosis and Waardenburg syndrome in Japanese infants. The patients were a 1-month-old girl and a 3-month-old boy at diagnosis, and both of them had either short segment or ultra-short segment aganglionosis. A review of 48 cases in the literature showed that the extent of the aganglionic segment is quite variable, from nearly total to ultra-short. The clinical features of aganglionosis in Waardenburg syndrome would appear to bear similarity in sex ratio and the extent of aganglionosis with those of Hirschsprung's disease associated with Ondine's curse, another type of neurocristopathy.

Background: A pathway consisting of bombesin, G-protein coupling receptors (GPCRs), metalloproteases, pro-heparin-binding epidermal growth factor (proHB-EGF), and epidermal growth factor receptor (EGFR) has been reported in prostate cancer cells. The occurrence of HB-EGF shedding from proHB-EGF in this pathway, however, has not been proven directly. In addition, it is still unclear how much this pathway contributes to the migration of prostate cancer cells. In this study, we tried to directly elucidate HB-EGF shedding in this pathway and to determine its contribution to the migration of prostate cancer cells., Methods: RT-PCR and indirect immunofluorescence staining for HB-EGF and its receptors, such as EGFR and HER4/erbB4, were performed on PC-3 cells. The influences of bombesin, anti-EGFR neutralizing monoclonal antibody, HB-EGF, and HB-EGF shedding inhibitor on the migration of PC-3 cells were studied by means of in vitro wound assays. The amount of HB-EGF shed from PC-3 cells with alkaline phosphatase-tagged HB-EGF in the presence of bombesin was determined by measuring AP activity. Immunoprecipitations and phosphotyrosine Western blotting were performed to detect EGFR transactivated by bombesin., Results: PC-3 expressed HB-EGF and EGFR, but not HER4/erbB4. PC-3 migrated in the presence of bombesin, but its migration was partly inhibited by the neutralizing antibody against EGFR. PC-3 also migrated in the presence of HB-EGF, but HB-EGF shedding inhibitor partly inhibited this phenomenon. HB-EGF was shed from PC-3 cells in the presence of bombesin, and this shedding was inhibited by HB-EGF shedding inhibitor. In addition, the EGFR on PC-3 was activated in the presence of bombesin and inactivated in the presence of HB-EGF shedding inhibitor., Conclusions: These results indicated that HB-EGF shedding and the following transactivation of EGFR occurs in this pathway and that this pathway partly contributes to the migration of prostate cancer cells., (Copyright 2003 Wiley-Liss, Inc.)

Objectives: The development of intrahepatic biliary cysts (IBC) after Kasai operation in patients with biliary atresia (BA) is recognized as an important problem; however, management strategy for IBC has not been clarified, particularly in the light of the increased use of liver transplantation., Methods: Forty consecutive BA patients underwent hepatic portoenterostomy during 18 years from 1983 to 2000. We compared the clinical course and prognosis of the patients who developed IBC with those who did not., Results: Seven of the 40 patients developed IBC. Three patients had type A (non-communicating cyst) and three patients had type C (multiple cystic dilation) IBC, and the remaining patients had type B (communicating cyst). Of the 7 patients, one patient underwent successful internal intestinal drainage, and one patient died of complications at the time of internal intestinal drainage. Three patients underwent liver transplantation due to either hepato-pulmonary syndrome (one case) or liver failure (two cases). One patient with IBC with liver failure was judged to require transplant, but was found to have pulmonary hypertension and was thus not a candidate. The remaining patient has survived without jaundice for 21 months postoperatively. Two of 21 patients with good initial bile drainage and without IBC underwent liver transplantation. The percentage of patients undergoing transplant was significantly higher in the group with IBC than in the group without IBC (P < 0.05)., Conclusions: IBC was associated with worsening liver function. Previously, IBC was treated using internal/external drainage, or the patients were observed without treatment, with limited success. We now consider it reasonable to carry out liver transplantation in patients with long-standing IBC.

Background: The incidence of intrahepatic cholelithiasis and cholangitis has not yet been well studied postoperatively in patients with choledochal cysts., Methods: One hundred three patients with choledochal cysts had operative cholangiography, underwent standard excision of a choledochal cyst with Roux-en-Y hepatico-jejunal anastomosis, and were at a mean follow-up of 12 1/2 years. The incidence of intrahepatic bile duct stones was analyzed according to the 3 morphologic types of intrahepatic bile duct observed at initial operative cholangiography: type 1, no dilatation of the intrahepatic bile ducts; type 2, dilatation of the intrahepatic bile ducts but without any downstream stenosis; and type 3, dilatation of the intrahepatic bile ducts associated with downstream stenosis. Initially, there was no evidence of intrahepatic bile duct stones in any of the 103 patients., Results: Among 50 type 1 patients, intrahepatic cholelithiasis developed in only 1 patient (2%). Among 43 type 2 patients, 1 patient (2%) had intrahepatic cholelithiasis, and 2 (5%) had postoperative cholangitis. Among 10 type 3 patients, 4 (40%) had intrahepatic cholelithiasis (P <.01), and 3 (30%) had postoperative cholangitis. Time intervals between the initial surgery and the first identification of intrahepatic stones ranged from 3 to 22 years., Conclusions: One of the major causes of formation of intrahepatic cholelithiasis has been clarified; patients with intrahepatic biliary dilatation with downstream stenosis can get intrahepatic bile duct stones long after excision of a choledochal cyst.

Three new carotenoids with a 3-methoxy-5-keto-5,6-seco-4,6-cyclo-beta end group (1-3) have been isolated from the seeds of Pittosporum tobira. Their structures were elucidated by detailed analyses of nuclear magnetic resonance and UV data.

Dermoid cysts are benign cystic teratomas lined by skin and epidermal appendages. We report a dermoid cyst occurring in a 26-year-old female whose chief complaint was irregular vaginal bleeding. Abdominal magnetic resonance image demonstrated a space-occupying lesion in the right lower abdomen. The mass showed hyperintensity on the T2 image and the signal was homogeneous for the interior. During abdominal surgery we made the diagnosis of subserous tumor of the colon and resected the ileocecal portion of the colon. The tumor measured 5.4 x 4.8 x 3.5 cm and was soft and elastic. On cross section, a unilocular cyst filled with atheromatous material was found. Pathological examination revealed a dermoid cyst. In the view of this diagnosis, a simple excision would have been an adequate treatment., (Copyright 2001 S. Karger AG, Basel)