For each array hybridization, a dye-swap hybridization was performed, such that for each data point, two of the biological replicates were hybridized as test (Cy5, red) versus control (Cy3, green) and the other two biological samples were hybridized as control (Cy5) versus test (Cy3). The replicate dye-swap analysis reduces the impact of dye bias or other labeling artifacts on the ratio of gene expression at a given data point. The median intensities of each spot on the array were measured by an Agilent Scanner using GenePix version 5 software, and the ratio of expression for each element on the array was calculated in terms

of M (log2 (red/green)) and A ((log2 (red) + log2 (green))/2)). The data were normalized by the print tip lowess method using the Statistical learn more Microarray Analysis package in the software package R.21 For statistical analysis, the genes were identified as differentially expressed using Vismodegib the Patterns from Gene Expression package (PaGE version 5.0) as described.22

Two-class, unpaired data tests were also performed to specifically identify genes that were differentially expressed by more than 1.5-fold when comparing the different data points. Microarray data can be found on GEO, the public web site, at http://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?cc=GSE22977. The values of fold change across the three comparisons were used to perform a hierarchical clustering analysis using Euclidean distance and the average agglomeration method.23 This procedure assigned each expressed gene to a unique cluster; these clusters were then classified according to their dynamics of change over time. Each gene cluster was subjected to a core analysis via Ingenuity Pathway

Analysis (IPA, Ingenuity Systems), using the fold change difference between compensated and decompensated cirrhosis, for an assessment of the signaling pathways, molecular networks, and biological processes most significantly perturbed by the genes expressed per cluster with progression of cirrhosis to the decompensated state. IPA is based on a manually curated database of interactions among genes and gene products and can impute the presence of a given gene in a network from the expression pattern based on this interaction database. The gene networks generated by this analysis were scored by IPA to rank according to the degree of relevance to the set of genes present in our cluster. Additional MCE methods are presented in the Supporting Information. Hepatocytes were isolated from the livers of Lewis rats at two different stages of cirrhosis and from age-matched controls. Animals treated with 14 weeks of CCl4 had normal liver function (compensated cirrhosis) with bilirubin levels of <0.1 mg/dL, albumin levels of 3.4-3.6 g/dL, prothrombin times of 13-14 seconds, and hepatic encephalopathy scores of 15, representing normal behavior.24 Animals that received 26-28 weeks of CCl4, however, had decompensated liver function with bilirubin levels of 0.4 ± 0.2 mg/dL, albumin levels of 2.

Genetic ablation of Them1 in mice decreases hepatic FFA concentrations and improves glucose tolerance in high fat fed mice, putatively

by reducing endoplasmic reticulum Seliciclib manufacturer stress. However, because Them1-deficient mice also exhibit increased energy expenditure and resistance to diet-induced obesity, the contribution of Them1 expression in liver to NAFLD remains unclear. Aim: This study was designed to assess the specific contribution of hepatic Them1 expression to glucose metabolism in high fat fed mice. Methods: We designed conditional Them1 transgenic (c-Them1Tg) mice with Cre recombinase-dependent Them1 overexpression. Liver-specific Them1 transgenic (L-Them1Tg) mice were generated by infection with Cre recombinant adenovirus in c-Them1Tg mice. Control mice were infected with GFP recombinant adenovirus. Hepatic Them1 expression was assessed by immunoblot analysis. Mice were fed high fat (60% of kcal) diet for 2 w. Body composition was determined by magnetic resonance

spectroscopy. For tolerance tests (n=5 mice/group) to insulin (ITT), pyruvate (PTT) or glucose (GTT), beta-catenin signaling plasma glucose concentrations were determined in fasting mice and then periodically up to 90 min after i.p. injection of insulin (1 U/kg bw), pyruvate (2 mg/g bw) or glucose (1 mg/g bw) respectively. Mice were maintained on the high fat diet and allowed to recover for 7 days between each test. Results: L-Them1Tg mice exhibited a 3.4-fold increase

in the hepatic expression of Them1 compared with c-Them1Tg control. Total body weights, fat and lean masses of L-Them1Tg were similar to c-Them1Tg control mice, as were fasting plasma glucose concentrations. Indicative of decreased clearance rate of exogenous glucose, plasma glucose concentrations in L-Them1Tg mice were higher at each time point during MCE the GTT and the area under the curve was increased by 43% (c-Them1Tg, 16,532±1377, L-Them1Tg, 23,610±883; P=0.0025). By contrast, there were no differences in the ITT, which reflects whole body insulin sensitivity, or in the PTT, which estimates hepatic glucose production. Conclusion: Without altering body composition, liver-specific Them1 overexpression promotes glucose intolerance in high fat fed mice. These findings suggest a primary contribution of hepatic Them1 to the pathogenesis of NAFLD that is distinct from its function in suppressing energy expenditure. Disclosures: David E. Cohen – Advisory Committees or Review Panels: Merck, Genzyme; Consulting: Novartis, Aegerion, Dignity Sciences, Intercept; Speaking and Teaching: Merck The following people have nothing to disclose: Cafer Ozdemir Saturated fatty acids, such as palmitic acid (PA), play a key role in lipotoxicity and the pathogenesis of NASH. Sustained JNK activation is a key mechanism of lipotoxicity. Little is known about how JNK mediates the lethal lipotoxic effect of PA in hepatocytes.

Genetic ablation of Them1 in mice decreases hepatic FFA concentrations and improves glucose tolerance in high fat fed mice, putatively

by reducing endoplasmic reticulum CB-839 stress. However, because Them1-deficient mice also exhibit increased energy expenditure and resistance to diet-induced obesity, the contribution of Them1 expression in liver to NAFLD remains unclear. Aim: This study was designed to assess the specific contribution of hepatic Them1 expression to glucose metabolism in high fat fed mice. Methods: We designed conditional Them1 transgenic (c-Them1Tg) mice with Cre recombinase-dependent Them1 overexpression. Liver-specific Them1 transgenic (L-Them1Tg) mice were generated by infection with Cre recombinant adenovirus in c-Them1Tg mice. Control mice were infected with GFP recombinant adenovirus. Hepatic Them1 expression was assessed by immunoblot analysis. Mice were fed high fat (60% of kcal) diet for 2 w. Body composition was determined by magnetic resonance

spectroscopy. For tolerance tests (n=5 mice/group) to insulin (ITT), pyruvate (PTT) or glucose (GTT), R788 solubility dmso plasma glucose concentrations were determined in fasting mice and then periodically up to 90 min after i.p. injection of insulin (1 U/kg bw), pyruvate (2 mg/g bw) or glucose (1 mg/g bw) respectively. Mice were maintained on the high fat diet and allowed to recover for 7 days between each test. Results: L-Them1Tg mice exhibited a 3.4-fold increase

in the hepatic expression of Them1 compared with c-Them1Tg control. Total body weights, fat and lean masses of L-Them1Tg were similar to c-Them1Tg control mice, as were fasting plasma glucose concentrations. Indicative of decreased clearance rate of exogenous glucose, plasma glucose concentrations in L-Them1Tg mice were higher at each time point during MCE公司 the GTT and the area under the curve was increased by 43% (c-Them1Tg, 16,532±1377, L-Them1Tg, 23,610±883; P=0.0025). By contrast, there were no differences in the ITT, which reflects whole body insulin sensitivity, or in the PTT, which estimates hepatic glucose production. Conclusion: Without altering body composition, liver-specific Them1 overexpression promotes glucose intolerance in high fat fed mice. These findings suggest a primary contribution of hepatic Them1 to the pathogenesis of NAFLD that is distinct from its function in suppressing energy expenditure. Disclosures: David E. Cohen – Advisory Committees or Review Panels: Merck, Genzyme; Consulting: Novartis, Aegerion, Dignity Sciences, Intercept; Speaking and Teaching: Merck The following people have nothing to disclose: Cafer Ozdemir Saturated fatty acids, such as palmitic acid (PA), play a key role in lipotoxicity and the pathogenesis of NASH. Sustained JNK activation is a key mechanism of lipotoxicity. Little is known about how JNK mediates the lethal lipotoxic effect of PA in hepatocytes.

On a small number of occasions, we observed some bleeding around the dart wound. However, biopsy darts should cause less injury than immobilization darts, particularly rapid injection darts (Cattet et al. 2006). On average, biopsy darting took <7 min per bear, which is a considerable reduction in time spent disturbing animals compared to immobilization. Although it is possible to biopsy dart dependent cubs, we did not in this study because

of the challenges involved in keeping family groups together during darting runs. During capture of polar bears, mothers are typically sedated first and the dependent cubs typically stay near the sedated mother. Since there is no sedation involved in biopsy darting there is an increased risk of separation while attempting to sample dependent cubs. Remote biopsy darting provides an additional www.selleckchem.com/products/DMXAA(ASA404).html tool or an alternative to capturing polar bears and other wildlife, for the purpose RAD001 molecular weight of individual and sex identification and diet analysis. Although biopsy darting does not provide the detailed health and physiological information that can be attained

through capture, it is less invasive than immobilization and handling and may be more acceptable to local people who live in proximity to polar bears. Finally, biopsy darting can be used without the extensive equipment required for capture-based studies, and in some areas could be conducted on the ground with snowmachines to monitor remote subpopulations of polar bears that have limited research access

(Vongraven et al. 2012). The type of biopsy dart to use will depend on the type of habitat and season of the study. We thank K. Simac, P. Hessing, M. St. Martin, G. Durner, and M. Lockhart for field and logistical support. We also thank T. and P. Austin with Paxarms N.Z., Ltd. and T. Taylor with Palmer Cap-Chur Equipment, Inc. for their help in developing these biopsy darts. We thank S. Iverson (Dalhousie 上海皓元医药股份有限公司 University) for support with the lipid and fatty acid analysis. We thank L. Pagano for creating dart images and S. Bee for help testing dyes. The U.S. Geological Survey (USGS) Ecosystem Mission’s Changing Arctic Ecosystems Initiative, USGS’ Climate and Land Use Change Research and Development Program, and the Bureau of Ocean Energy Management provided funding for biopsy darting field efforts, genetic, lipid, and fatty acid analyses. Biopsy darting of polar bears was made possible under U.S. Fish and Wildlife marine mammal research permit 690038 granted to the USGS, Alaska Science Center. Biopsy darting procedures were conducted under the approval of the Alaska Science Center Institutional Animal Care and Use Committee (IACUC) protocols (assurance no. 2010-14). We thank the U.

On a small number of occasions, we observed some bleeding around the dart wound. However, biopsy darts should cause less injury than immobilization darts, particularly rapid injection darts (Cattet et al. 2006). On average, biopsy darting took <7 min per bear, which is a considerable reduction in time spent disturbing animals compared to immobilization. Although it is possible to biopsy dart dependent cubs, we did not in this study because

of the challenges involved in keeping family groups together during darting runs. During capture of polar bears, mothers are typically sedated first and the dependent cubs typically stay near the sedated mother. Since there is no sedation involved in biopsy darting there is an increased risk of separation while attempting to sample dependent cubs. Remote biopsy darting provides an additional check details tool or an alternative to capturing polar bears and other wildlife, for the purpose selleck screening library of individual and sex identification and diet analysis. Although biopsy darting does not provide the detailed health and physiological information that can be attained

through capture, it is less invasive than immobilization and handling and may be more acceptable to local people who live in proximity to polar bears. Finally, biopsy darting can be used without the extensive equipment required for capture-based studies, and in some areas could be conducted on the ground with snowmachines to monitor remote subpopulations of polar bears that have limited research access

(Vongraven et al. 2012). The type of biopsy dart to use will depend on the type of habitat and season of the study. We thank K. Simac, P. Hessing, M. St. Martin, G. Durner, and M. Lockhart for field and logistical support. We also thank T. and P. Austin with Paxarms N.Z., Ltd. and T. Taylor with Palmer Cap-Chur Equipment, Inc. for their help in developing these biopsy darts. We thank S. Iverson (Dalhousie 上海皓元 University) for support with the lipid and fatty acid analysis. We thank L. Pagano for creating dart images and S. Bee for help testing dyes. The U.S. Geological Survey (USGS) Ecosystem Mission’s Changing Arctic Ecosystems Initiative, USGS’ Climate and Land Use Change Research and Development Program, and the Bureau of Ocean Energy Management provided funding for biopsy darting field efforts, genetic, lipid, and fatty acid analyses. Biopsy darting of polar bears was made possible under U.S. Fish and Wildlife marine mammal research permit 690038 granted to the USGS, Alaska Science Center. Biopsy darting procedures were conducted under the approval of the Alaska Science Center Institutional Animal Care and Use Committee (IACUC) protocols (assurance no. 2010-14). We thank the U.

4A-C). Similar to rodent hepatocytes,28 rhodamine fluorescence peaked in HepaRG cells (12 hours) after the peak of Mitosox Red fluorescence (Fig. 4A-C). HepaRG cells are bipotent progenitors that differentiate into two morphologically distinct cell populations.23, 24 The hepatocyte-like cells have a characteristic granular appearance and grow in small clusters or “hepatocyte islands” (Figs. 3, 4D). Surrounding these islands are flatter, clearer biliary epithelial-like cells (Figs. 3, 4D). To assess the contribution of each cell type to our data showing APAP toxicity, CHIR-99021 in vitro APAP-treated cells were exposed to PI, which stains nuclei of necrotic cells red (Fig. 4E,F). At 24 hours

the majority of the PI staining was seen in the hepatocyte-like cells, with very little among the biliary epithelial-like cells (Fig. 4E,F). The distribution was similar at 48 hours (data not shown). This suggests that APAP mainly affects the hepatocytes. Together, these data indicate that—similar to rodent hepatocytes—cell death in human HepaRG cells is preceded by GSH depletion, protein binding, formation of reactive

Alisertib cost oxygen and peroxynitrite, and mitochondrial dysfunction. To compare HepaRG cells with other hepatoma cell lines, APAP toxicity was evaluated in HepG2 cells. HepG2 cells treated with 20 mM APAP for 24 hours showed no evidence of GSH depletion, mitochondrial 上海皓元 dysfunction (JC-1 assay), or cell injury (LDH release) in response to the toxic dose of APAP (Table 1). However, low levels of protein adducts were identified despite the absence of toxicity (Table 1). Thus, the near absence of drug-metabolizing enzymes drastically reduced the metabolic activation of APAP and prevented any toxicity in HepG2

cells. Loss of mitochondrial membrane integrity can result in the release of proapoptotic proteins, including the caspase activator cytochrome c, into the cytosol. To determine whether or not APAP toxicity in HepaRG cells involves apoptosis, caspase-3 activity was measured in lysates of cells treated for 24 hours with APAP. There was no significant increase in caspase activity over control with 20 (Fig. 5A), 5, or 10 mM APAP (data not shown). In addition, the potent pan-caspase inhibitor Z-VD-fmk had no effect on APAP-induced LDH release at 24 hours (Fig. 5B), suggesting that APAP did not cause apoptosis in HepaRG cells. In contrast, caspase activity was significantly increased when cells were exposed to 100 ng/mL human TNF alpha (rhTNFα) and 5 mM galactosamine for 16.5 hours as a positive control (Fig. 5A). The caspase inhibitor prevented the increase in caspase activity after G/TNF. This indicates that HepaRG cells do have the capacity to undergo apoptotic cell death in response to an appropriate stimulus.

4A-C). Similar to rodent hepatocytes,28 rhodamine fluorescence peaked in HepaRG cells (12 hours) after the peak of Mitosox Red fluorescence (Fig. 4A-C). HepaRG cells are bipotent progenitors that differentiate into two morphologically distinct cell populations.23, 24 The hepatocyte-like cells have a characteristic granular appearance and grow in small clusters or “hepatocyte islands” (Figs. 3, 4D). Surrounding these islands are flatter, clearer biliary epithelial-like cells (Figs. 3, 4D). To assess the contribution of each cell type to our data showing APAP toxicity, Vadimezan mw APAP-treated cells were exposed to PI, which stains nuclei of necrotic cells red (Fig. 4E,F). At 24 hours

the majority of the PI staining was seen in the hepatocyte-like cells, with very little among the biliary epithelial-like cells (Fig. 4E,F). The distribution was similar at 48 hours (data not shown). This suggests that APAP mainly affects the hepatocytes. Together, these data indicate that—similar to rodent hepatocytes—cell death in human HepaRG cells is preceded by GSH depletion, protein binding, formation of reactive

find more oxygen and peroxynitrite, and mitochondrial dysfunction. To compare HepaRG cells with other hepatoma cell lines, APAP toxicity was evaluated in HepG2 cells. HepG2 cells treated with 20 mM APAP for 24 hours showed no evidence of GSH depletion, mitochondrial 上海皓元 dysfunction (JC-1 assay), or cell injury (LDH release) in response to the toxic dose of APAP (Table 1). However, low levels of protein adducts were identified despite the absence of toxicity (Table 1). Thus, the near absence of drug-metabolizing enzymes drastically reduced the metabolic activation of APAP and prevented any toxicity in HepG2

cells. Loss of mitochondrial membrane integrity can result in the release of proapoptotic proteins, including the caspase activator cytochrome c, into the cytosol. To determine whether or not APAP toxicity in HepaRG cells involves apoptosis, caspase-3 activity was measured in lysates of cells treated for 24 hours with APAP. There was no significant increase in caspase activity over control with 20 (Fig. 5A), 5, or 10 mM APAP (data not shown). In addition, the potent pan-caspase inhibitor Z-VD-fmk had no effect on APAP-induced LDH release at 24 hours (Fig. 5B), suggesting that APAP did not cause apoptosis in HepaRG cells. In contrast, caspase activity was significantly increased when cells were exposed to 100 ng/mL human TNF alpha (rhTNFα) and 5 mM galactosamine for 16.5 hours as a positive control (Fig. 5A). The caspase inhibitor prevented the increase in caspase activity after G/TNF. This indicates that HepaRG cells do have the capacity to undergo apoptotic cell death in response to an appropriate stimulus.

5A). Nevertheless, direct Mϕ/NK interaction provided a stronger NK cell activation, indicating additional involvement of Mϕ surface molecules. Eventually, blocking experiments confirmed IL12 or IL18 as sorafenib-triggered NK cell stimulus (Fig. 5B), whereas IL15 neutralization and isotype antibodies did not affect NK cell activity. IL12 and IL18 acted synergistically on NK cells, as reduction in killing efficacy was more pronounced if both cytokines were blocked simultaneously (IL12 versus IL12/IL18; K562: P = 0.0012; Raji: P = 0.0001) (Fig. www.selleckchem.com/products/NVP-AUY922.html 5B). In conclusion, NK cell activation was cytokine-dependent and was partially enhanced by direct contact

between Mϕ and NK cells. NF-κB regulates Mϕ activation and Selleck SAHA HDAC promotes cytokine expression. We therefore

analyzed sorafenib-triggered NF-κB activation in Mϕ cultures (Fig. 6A). Sorafenib activated the canonical and noncanonical NF-κB pathway in polarized Mϕ cultures in a dose- and LPS-dependent fashion, as shown by p100/p52 processing and RelA phosphorylation (Fig. 6A). Celastrol, an inhibitor of both NF-κB pathways, and TPCA-1, specifically subverting the canonical NF-κB pathway (Fig. 6A), were employed for NF-κB blocking experiments. Both compounds coadministered with sorafenib reduced NK cell killing (Fig. 6B) as well as NK cell degranulation (Fig. 6C). We next investigated if sorafenib sensitizes polarized Mϕ to apoptotic cells, as this reflects the constellation during cytotoxic HCC treatment in vivo. In fact, sorafenib-treated Mϕ provided a stronger stimulus on NK cells in the presence of ultraviolet (UV)-irradiated apoptotic HepG2 cells. Control experiments showed that this was not the case after addition

上海皓元医药股份有限公司 of untreated HepG2 cells and that caspase-3 cleavage distinguished UV-irradiated from untreated HepG2 (Fig. 6D-F). On the other hand, sorafenib did not induce apoptosis in Mϕ (Fig. S3A) and NK cell activation was not abolished by a caspase inhibitor during Mϕ/NK coculture experiments (Fig. S3B,C), indicating that apoptotic Mϕ did not contribute substantially to NK cell activation in our model. Complex TAM polarization is not completely resembled by in vitro models. We therefore isolated macrophages from freshly resected HCC tissue. Primary human TAM displayed a bipolar morphology in contrast to spherical monocytes derived from peripheral blood (Fig. 7A). CD68 and CD163 mRNA expression confirmed TAM identity, whereas AFP, albumin, and L-SIGN transcripts indicating tumor cells, hepatocytes, and endothelial cells were barely detectable (Fig. 7A). Sorafenib treatment triggered a stronger IL12 and IL18 mRNA expression in isolated TAM under LPS stimulation compared to untreated controls (Fig. 7B). Homologous TAM/NK cocultures derived from the same donor were used to confirm an interaction between both cell types. Upon coculture with sorafenib-treated TAM, NK cells showed increased IFN-γ expression, degranulation, and killing capacity (Fig. 7C-E).

Hematopoietic stem cells colonize the liver bud, emitting growth signals for liver. Consequently, hepatoblasts continue to proliferate and express placental

alkaline phosphatase, intermediate filament proteins (CK14, CK8 and CK18), γ-glutamyltransferase, α1-antitrypsin, glutathione S-transferase P, C/EBPα, lactate dehydrogenase and muscle pyruvate EGFR inhibitor kinase.3,17,18 As the commitment progresses, three distinct cell populations emerged: (i) hepatocyte-committed cells that express AFP and albumin; (ii) cholangiocyte-committed progenitor cells that express CK 19; and (iii) a bipotential hepatoblast population that express both hepatic and biliary markers. Differentiation along the cholangiocytic lineage is promoted by Notch signaling pathways.16,17,19 Hepatocyte growth factor (HGF), secreted by mesenchymal cells or non-parenchymal cells, antagonizes the cholangiocytic process, resulting in support of growth and differentiation of the fetal hepatocytes.3 Subsequently, cooperative action of Oncostatin M (OSM) and glucocorticoids induces partial hepatic maturation and suppression of embryonic hematopoiesis.3,16,17,20 Complete functional hepatic maturation ultimately takes place after birth upon co-assistance of HGF, produced by the surrounding nonparenchymal liver cells.3,20 IT IS GENERALLY believed that pluripotent stem cells can differentiate into BAY 57-1293 in vitro adult stem

cells such as neural, hematopoietic, mesenchymal and hepatic stem cells, and then they can differentiate into neuron, blood cells, bone and muscle cells, and hepatocytes/cholangiocytes, respectively (Fig. 1). Indeed, the pluripotent stem cells are reported to differentiate into cardiomyocytes, chondrocytes, pancreatic islet cells and hepatocytes.21–24 There are mainly two methods of differentiation from ES cells to hepatocytes,

one is three-dimensional culture using embryoid MCE body (EB) formation,25–27 and the other is monolayer culture.28–30 Generally, the former has higher efficiency of differentiation than the latter; however, the former in EB formation obtains lower numbers of differentiated cells. Therefore, it is expected that more hepatocytes can be obtained by monolayer culture. From the liver developmental studies, it is suggested that the differentiation protocols into hepatocytes are composed of three steps: (i) differentiation into the endoderm; (ii) differentiation into hepatobalsts; and (iii) differentiation into mature hepatocytes (Fig. 2).3 The first step of differentiation into the endoderm is confirmed by expression of Sox17, HNF3β, GSC and CXCR4. The second step of differentiation into hepatoblasts is confirmed by expression of AFP, transthyretin (TTR), albumin, CK18, AAT, HNF4α and HNF1. The third step of differentiation into mature hepatocytes is confirmed by expression of TAT, G6P, tryptophan oxygenase (TO), cytochrome P450 and C/EBPα.

17 In the present study, we selected eligible patients from the previous study (n = 81) and added new ones treated in recent years (n = 20). Patients who had previously received other treatments were excluded, since the other treatments could have affected and complicated the clinical outcome. The balloon or stent diameter used in this study

was 30 mm in order to compare the efficiency of both therapies using the same condition. All patients in this study had a complete follow up more than 12 months post-procedure, which was also useful to determine the long-term clinical outcome. However, the patients’ selection might have been a confounding factor, since people who receive graded balloon dilation can have a more favorable clinical outcome compared to those who need the single dilation. Moreover, patients with no clinical remission after a single or graded pneumatic dilation often apply the stent insertion, and LBH589 some could still achieve good results. These patients were excluded from this study. Thus, the present study only partially reflects the clinical effects of both treatments, and a better clinical outcome of stent insertion might have been underestimated because of the patients’ selection bias. In the present study, both Groups A and B acquired

symptom remission, significant esophageal manometry, and barium esophageal improvement immediately after the procedures, which 上海皓元医药股份有限公司 indicate that both balloons and stents effectively relieved Selleckchem A-769662 the symptoms in most achalasia patients. Moreover, symptom remission and esophageal manometry or barium esophageal improvement in Group B was significantly more conspicuous than in Group A. This probably occurred because the tearing of the cardia sphincter by stents was more symmetrical and sufficient than balloon dilations because of a longer and more durable dilation by stents. The cardia diameter after stent dilation might be larger and have less recoil than balloon dilation. This hypothesis can also explain why complications, such as pain

and bleeding, occurred more frequently in the stent group compared to the balloon group (42.9% vs 23.6% and 15.9% vs 8%). After more than 10 years of follow up, the total symptom scores in Group B was statistically different compared to Group A (P = 0.0096), but the esophageal manometry was not (P = 0.1687). We believe this difference was not very accurate because only five patients in Group A and three patients in Group B had more than 10 years of follow up, indicating that the sample was too small. After 8–10 years of follow up, TSS and esophageal manometry both had statistically significant differences (P < 0.0001). At more than 10 years’ follow up, the Kaplan–Meier method revealed better symptom remission in Group B than in Group A. The recurrence rate in Group A was 50% (19 out of 38) at 8–10 years’ follow up and 57.