Injury to the Muscle Layer and Risk of Non-cardiac Chest Pain after Endoscopic Submucosal Dissection for Esophageal Cancer.

BACKGROUND AND AIMS: Non-cardiac chest pain (NCCP) is a frequent complication of endoscopic submucosal dissection (ESD) for early-stage esophageal cancer. However, little is known about relationships between ESD findings and NCCP. This study aims to evaluate the risk factors for NCCP, including ESD findings related to injury to the muscle layer. METHODS: We enrolled a total of 296 lesions from 270 patients with esophageal squamous cell carcinoma (ESCC), who underwent ESD in our center. The grade of injury to the muscle layer caused by ESD was categorized as follows: grade 0: no exposure of muscularis propria; grade 1: muscularis propria exposure and/or whitish color change by the electrocoagulation; grade 2: torn muscularis propria with whitish color change by the electrocoagulation; and grade 3, esophageal perforation. The risk factors for NCCP, including ESD findings, were analyzed by univariate and multivariate analyses. RESULTS: NCCP occurred in 89 patients (33.0%) after esophageal ESD. Multivariate analysis demonstrated that younger age [odds ratio (OR) 0.95, 95% confidence interval (95%CI) 0.92-0.98, p=0.003), postoperative fever (>= 38°C) (OR=25.9, 95%CI: 2.89-232.10, p=0.004), ESD findings (grade 1: OR=3.99, 95%CI: 1.63-9.75, p=0.003 and grade 2: OR=3.18, 95%CI: 1.54-6.57, p=0.002) were independently associated with the incidence of post ESD NCCP. CONCLUSIONS: ESD findings relate to slight Injury to the muscle layer, such as muscularis propria exposure and whitish color change by the electrocoagulation were identified as risk factor for post ESD NCCP. We should therefore perform esophageal ESD carefully to avoid injuring the muscle layers.

Microarchitectures of Barrett's esophagus associated with DNA methylation status.

Aim: DNA methylation is involved in esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE). Microarchitectures of on-neoplastic BE associated with DNA methylation status were examined using magnifying narrow-band imaging (NBI) endoscopy. Patients and methods: Using biopsies from non-neoplastic BE without cancer (n = 66; N group), with EAC (n = 27; ADJ group) and EAC tissue (n = 22; T group), methylation of N33, DPYS, SLC16A12, miR124a3 and miR34bc genes were quantified. Magnifying NBI features of non-neoplastic BE were classified according to their morphologies. Results: The ADJ and T groups presented higher DNA methylation compared with the N group. Magnifying NBI endoscopic features of non-neoplastic BE also correlated with DNA methylation as an independent factor. Conclusion: Microarchitectures of BE visualized by magnifying NBI endoscopy correlated with DNA methylation.

Telomere Shortening of Barrett's Esophagus and Esophageal Adenocarcinoma in Japanese Patients.

Telomere shortening is deeply involved in many types of cancer. Telomere length of esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE) was examined in Japanese patients. Among BE from cancer free patients (Cancer free), BE from patients with EAC (Adjacent) and EAC tissue (Cancer), Cancer free group presented the longest telomeres, while Cancer group presented the shortest telomeres and Adjacent group presented intermediate telomeres. Direction of endoscopic biopsy, 2 o'clock direction was also significantly associated with shorter telomere length in non-neoplastic BE (p = 0.027). Shortened telomere highlighted the impact of this molecular change in early carcinogenesis in EAC.

Intravenous injection of tumor extracellular vesicles suppresses tumor growth by reducing the regulatory T cell phenotype.

BACKGROUND: Colorectal cancer is a disease of unmet medical need. Although extracellular vesicles (EVs) have been implicated in anti-tumor responses, discrepancies were observed among studies. We analyzed the role of tumor-derived EVs (TEVs) in tumor progression in vivo by focusing on regulatory T (Treg) cells, which play essential roles in tumor development and progression. METHODS: A mouse model of colorectal cancer lung metastasis was generated using BALB/c mice by tail vein injection of the BALB/c colon adenocarcinoma cell line Colon-26. TEVs derived from Colon-26 and BALB/c lung squamous cell carcinoma ASB-XIV were retrieved from the culture media supernatants. A TEV equivalent to 10 µg protein was injected every other day for 2 weeks. RESULTS: Histology and immunohistochemistry studies revealed that lung tumors reduced in the Colon-26-EV group when compared to the phosphate-buffered saline (PBS) group. The population of CD4 + FoxP3 + cells in the lung was upregulated in the PBS group mice when compared to the healthy mice (P < 0.001), but was significantly downregulated in the Colon-26-EV group mice when compared to the PBS group mice (P < 0.01). Programmed cell death protein 1, glucocorticoid-induced TNFR-related protein, and CD69 expression in lung Treg cells were markedly upregulated in the PBS group when compared to the healthy mice, but downregulated in the Colon-26-EV group when compared to the PBS group. The changes in expression were dose-dependent for Colon-26-EVs. ASB-EVs also led to significantly downregulated Treg cell expression, although non-cancer line 3T3-derived EVs did not. CONCLUSION: Our study suggests that TEVs possess components for tumor suppression.

A Short-Term Model of Colitis-Associated Colorectal Cancer That Suggests Initial Tumor Development and the Characteristics of Cancer Stem Cells.

The mechanisms underlying the transition from colitis-associated inflammation to carcinogenesis and the cell origin of cancer formation are still unclear. The azoxymethane (AOM)/dextran sodium sulfate (DSS) mouse model reproduces human colitis-associated colorectal cancer. To elucidate the mechanisms of cancer development and dynamics of the linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr)-positive cells, we explored the early stages of colitis-associated colorectal cancer in AOM/DSS mice. The AOM/DSS mice were sacrificed at 4 to 6 weeks following AOM administration. To analyze the initial lesions, immunofluorescence staining for the following markers was performed: ß-catenin, Ki67, CDK4, Sox9, Bmi1, cyclin D1, and pSmad2/3L-Thr. Micro-neoplastic lesions were flat and unrecognizable, and the uni-cryptal ones were either open to the surfaces or hidden within the mucosae. These neoplastic cells overexpressed ß-catenin, Sox9, Ki67, and Cyclin D1 and had large basophilic nuclei in the immature atypical cells. In both the lesions, pSmad2/3L-Thr-positive cells were scattered and showed immunohistochemical co-localization with ß-catenin, CDK4, and Bmi1 but never with Ki67. More ß-catenin-positive neoplastic cells of both lesions were detected at the top compared to the base or center of the mucosae. We confirmed initial lesions in the colitis-associated colorectal cancer model mice and observed results that suggest that pSmad2/3L-Thr is a biomarker for tissue stem cells and cancer stem cells.

Comprehensive DNA methylation profiling of Barrett's esophagus and esophageal adenocarcinoma in Japanese patients.

Molecular mechanisms of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) remain unclear in Japanese patients. Japanese EACs frequently have underlying short length BE: short-segment BE (SSBE), for which, neoplastic potential remains unclear. We performed comprehensive methylation profiling of EAC and BE in Japanese patients, mostly comprised with SSBE. Using three different groups of biopsies obtained from non-neoplastic BE from patients without cancer (n = 50; N group), with EAC (n = 27; ADJ group) and EAC (n = 22; T group), methylation statuses of nine candidate genes (N33, DPYS, SLC16A12, CDH13, IGF2, MLF1, MYOD1, PRDM5, and P2RX7) were examined by the bisulfite pyrosequencing. Reduced representation bisulfite sequencing was performed to characterize the genome-wide methylation status in 32 samples (12 from N, 12 ADJ, and 8 from T groups). In the candidate approach, methylation levels of N33, DPYS, and SLC16A12 were higher in ADJ and T groups compared to that in N group. The ADJ group was an independent factor for higher DNA methylation in non-neoplastic BE. The genome-wide approach demonstrated an increase of hypermethylation from ADJ to T groups relative to N group near the transcription start sites. Among gene groups hypermethylated in ADJ and T groups (n = 645) and T group alone (n = 1438), 1/4 and 1/3 were overlapped with downregulated genes in the microarray data set, respectively. Accelerated DNA methylation is observed in EAC and underlying BE in Japanese patients, mostly comprised with SSBE, highlighting the potential impact of methylation in early carcinogenesis.

Usefulness of texture and color enhancement imaging (TXI) in early gastric cancer found after Helicobacter pylori eradication.

Early-stage gastric cancer (EGC) found after Helicobacter pylori (Hp) eradication is often difficult to diagnose using conventional white light (WL) endoscopy. We aimed to evaluate whether Texture and Color Enhancement Imaging (TXI), a new image-enhanced endoscopy enhances the EGC lesions after Hp eradication. We also compared diagnostic accuracy and lesion detection time between WL and TXI in trainee endoscopists. 58 EGC lesions after successful Hp eradication were enrolled. Using endoscopic images in WLI, TXI mode 1 (TXI1), and TXI mode 2 (TXI2), visibility of EGC was assessed by six expert endoscopists using a subjective score. Mean color differences (ΔE) of four matched adjacent and intra-tumoral points were examined. Using randomly allocated images, diagnostic accuracy and lesion detection time were evaluated in three trainee endoscopists. Visibility score was unchanged (Score 0) in 20.7% (12/58) and 45.6% (26/57), slightly improved (Score 1) in 60.3% (35/58) and 52.6% (30/57), obviously improved (Score 2) in 45.6% (26/58) and 1.8% (1/57), in TXI1 and TXI2 compared to WL, respectively. Mean ΔE ± SEM in TXI1 (22.90 ± 0.96), and TXI2 (15.32 ± 0.71) were higher than that in WL (1.88 ± 0.26, both P < 0.0001). TXI1 presented higher diagnostic accuracy compared to WL, in two of three trainees (94.8% vs. 74.1%, 100% vs. 89.7%, P = 0.003; < 0.005, respectively). Lesion detection time was shorter in TXI1 in two of three trainees (P = 0.006, 0.004, respectively) compared to WL. TXI improves visibility of EGC after Hp eradication that may contribute to correct diagnosis.

Long-term Outcomes after Steroid Pulse Therapy in Patients with Type 1 Autoimmune Pancreatitis.

Objective Steroid pulse therapy is a regimen involving the intravenous administration of supra-pharmacological doses of corticosteroids in the short term. It is used to treat various inflammatory and autoimmune conditions. However, the strengths and limitations of steroid pulse therapy for induction of remission in type 1 autoimmune pancreatitis (AIP) are unknown. Methods Depending on the steroid therapy regimen administered, the 104 patients with type 1 AIP included in this retrospective study were divided into three groups: conventional oral prednisolone (PSL) regimen (PSL group), intravenous methylprednisolone (IVMP) pulse followed by oral PSL regimen (Pulse+PSL group), and IVMP pulse-alone regimen (Pulse-alone group). We then examined the relapse rate and adverse events among the three groups. Results The Kaplan-Meier estimates of the relapse rate at 36 months after steroid therapy were 13.6% in the PSL group, 13.3% in the Pulse+PSL group, and 46.2% in the Pulse-alone group. The log-rank test revealed that the relapse-free survival in the Pulse-alone group was significantly shorter than that in the PSL (p=0.024) and Pulse+PSL groups (p=0.014). The exacerbation of glucose tolerance after steroid therapy was less frequently observed in the Pulse-alone group (0%) than in the PSL group (17%, p=0.050) and Pulse+PSL groups (26%, p=0.011). Conclusion Although treatment with IVMP pulse alone resulted in unsatisfactory relapse prevention outcomes compared with conventional steroid therapy, the IVMP pulse-alone regimen might be an alternative treatment strategy for type 1 AIP from the perspective of avoiding adverse events from steroids.

Establishment of a Novel Colitis-Associated Cancer Mouse Model Showing Flat Invasive Neoplasia.

BACKGROUND: Chronic inflammation, such as ulcerative colitis, increases the risk of developing colitis-associated cancers. Currently, mice administered with azoxymethane/dextran sodium sulfate are well-known models for colitis-associated cancers. Although human colitis-associated cancers are often flat lesions, most azoxymethane/dextran sodium sulfate mouse cancers are raised lesions. AIMS: To establish a novel mouse model for colitis-associated cancers and evaluate its characteristics. METHODS: A single dose of azoxymethane was intraperitoneally administered to CD4-dnTGFßRII mice, which are genetically modified mice that spontaneously develop inflammatory bowel disease at different doses and timings. The morphological and biological characteristics of cancers was assessed in these mice. RESULTS: Colorectal cancer developed with different proportions in each group. In particular, a high rate of cancer was observed at 10 and 20 weeks after administration in 12-week-old CD4-dnTGFßRII mice dosed at 15 mg/kg. Immunohistochemical staining of tumors was positive for ß-catenin, ki67, and Sox9 but not for p53. Grade of inflammation was significantly higher in mice with cancer than in those without cancer (p < 0.001). In CD4-dnTGFßRII/azoxymethane mice, adenocarcinomas with flat lesions were observed, with moderate-to-severe inflammation in the non-tumor area. In comparison, non-tumor areas of azoxymethane/dextran sodium sulfate mice had less inflammation than those of CD4-dnTGFßRII/azoxymethane mice, and most macroscopic characteristics of tumors were pedunculated or sessile lesions in azoxymethane/dextran sodium sulfate mice. CONCLUSIONS: Although feasibility and reproducibility of azoxymethane/CD4-dbTGFßRII appear to be disadvantages compared to the azoxymethane/dextran sodium sulfate model, this is the first report to demonstrate that the chronic inflammatory colitis model, CD4-dnTGFßRII also develops colitis-related colorectal cancer.

Decreased CD57 expression of natural killer cells enhanced cytotoxicity in patients with primary sclerosing cholangitis.

Background/aims: Primary sclerosing cholangitis (PSC) is a chronic inflammatory biliary disease for which the immunopathological basis remains an enigma. Natural killer (NK) cells are key components of innate immunity and seemingly play diversified roles in different autoimmune disorders (AIDs). The aim of this study was to determine the role of NK cells in the pathogenesis of PSC. Methods: The frequency and phenotype of circulating NK cells in a large cohort of patients with PSC and healthy controls (HCs) were systematically examined. In addition, the functional capacity of NK cells including cytotoxicity and cytokine production was studied. Results: The frequency of CD3-CD56dimCD16+ (defined as CD56dim) NK cells in PSC patients was significantly lower in comparison to HCs. CD56dim NK cells from PSC displayed a more immature phenotype including high expression of the natural killing receptor NKp46 and downregulation of the highly differentiated NK cell marker CD57. Interestingly, the reduction of CD57 expression of NK cells was associated with the disease severity of PSC. In addition, PSC CD56dim NK cells exhibited increased CD107a degranulation and cytolytic activity toward target cells compared with HCs. Further analysis demonstrated that CD57-CD56dim NK cells from PSC had elevated expression of NKp46, NKp30, IL-2 receptor, and KLRG1 and higher cytotoxic capacity as compared to CD57+CD56dim NK cells. Conclusions: Our data demonstrate that the differentiation of PSC NK cells is dysregulated with enhanced cytotoxic activity. This change is likely to be functionally involved in pathogenesis and disease progression, deducing the potential of NK-directed immunotherapy for PSC.

Analysis of the initial dose and reduction rate of corticosteroid for ulcerative colitis in clinical practice.

Background and Aim: Trends in steroid use and the effects of the initial dose, duration of use, and tapering schedule on clinical efficacy were assessed in Japanese patients with ulcerative colitis (UC) undergoing steroid treatment. Methods: We enrolled 191 cases with UC who underwent steroid treatment between 2006 and 2020. We assessed the difference in clinical remission rates in cases with different initial doses of steroid. Clinical factors for clinical remission at week 4 and discontinuation of corticosteroid within 12 weeks were also assessed. Results: Clinical remission and response at week 4 were obtained in 107 (56.0%) and 58 cases (30.4%), respectively. In hospitalized patients, male sex (odds ratio [OR], 0.373; 95% confidence interval [CI], 0.146-0.956) and younger age (OR, 0.974; 95% CI, 0.951-0.998) were associated with clinical remission at week 4. Partial Mayo score (OR, 0.643; 95% CI, 0.451-0.918) and initial steroid dose of ≥30 mg (OR, 3.278; 95% CI, 1.274-8.435) were associated with clinical remission at week 4 in outpatients. Clinical remission at week 4 (OR, 0.300; (95% CI, 0.126-0.718)) and the steroid dose reduction rate at week 4 (OR, 0.092; 95% CI, 0.036-0.234) were associated with treatment discontinuation within 12 weeks. The proportion of patients in whom corticosteroids were discontinued at week 12 was significantly higher (P = 0.006) in 2016-2020 (28/52; 53.8%) than in 2006-2010 (15/54; 27.8%). Conclusion: The steroid reduction rate at week 4 may be critical for discontinuation within 12 weeks. Withdrawal of corticosteroids has been becoming more appropriate in the last 5 years than before.

Large Metastatic Cardiac Tumor from Ascending Colon Cancer with Autopsy.

A metastatic cardiac tumor from colon cancer is an exceedingly rare clinical feature associated with a poor prognosis without therapeutic intervention; however, such cases may be frequently encountered in clinical practice, especially among the elderly. We report a case of synchronous double cancer of the prostate and ascending colon with metastases to multiple organs, including a large cardiac tumor. A 71-year-old Japanese man had prostate cancer with neck and para-aortic metastasis. He visited our hospital with complaints of fatigue and a positive fecal occult blood test result. Colonoscopy findings revealed the presence of a tumor in the ascending colon, and contrast-enhanced CT revealed a tumor in the heart, which was possibly due to metastasis from the ascending colon. The patient received palliative care and declined anticancer treatment. He died due to respiratory failure 3 months after the first diagnosis but did not show critical arrhythmia until death. Autopsy revealed the presence of a large mass in the right ventricle with tumor embolism of the right coronary artery. The cardiac mass was pathologically consistent with metastasis from the colon. In case of colorectal cancer with cardiac metastasis involving poor prognosis or performance status, best supportive care without any therapeutic intervention could be the optimal treatment for the quality of the remaining time.

Linker Threonine-phosphorylated Smad2/3 Is a Biomarker of Colorectal Neoplastic Stem-like Cells that Correlates With Carcinogenesis.

BACKGROUND/AIM: This study analysed threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr) expression and investigated whether pSmad2/3L-Thr is related to the transition from human colorectal adenoma (CRA) to carcinoma (CRC). MATERIALS AND METHODS: Immunofluorescent staining was performed forß-catenin, p53, CDK4, Ki67, Sox9, aldehyde dehydrogenase (ALDH) 1, and pSmad2/3L-Thr. RESULTS: We analysed specimens of diffuse p53-positive CRCs arising from p53-negative CRAs. Percentage of p53, nuclear ß-catenin, Ki67, CDK4, and pSmad2/3L-Thr-positive cells at the site of CRCs was significantly higher than that at the site of CRAs. At the site of normal colorectal mucosae, few epithelial cells were stained positively for pSmad2/3L-Thr. At the site of CRCs, pSmad2/3L-Thr-positive cells showed co-localization with p53, nuclear ß-catenin, and ALDH1. At any site, pSmad2/3L-Thr-positive cells showed co-localization with CDK4. CONCLUSION: pSmad2/3L-Thr correlates with human CRC carcinogenesis, and pSmad2/3L-Thr-positive cells show human colorectal stem cell-like and cancer stem cell characteristics.

Serum free light chain assessment in type 1 autoimmune pancreatitis.

BACKGROUND: /Object: Some patients with type 1 autoimmune pancreatitis (AIP), the pancreatic manifestation of IgG4-related disease, have normal serum IgG4. The aim of this study is to investigate the diagnostic value of measuring serum free light chains (FLCs) in type 1 AIP. MATERIALS AND METHODS: Thirty-seven patients with type 1 AIP, and 21 healthy, 17 alcoholic chronic pancreatitis (ACP), 21 idiopathic chronic pancreatitis (ICP) and 20 pancreatic cancer (PC) patients were enrolled. Serum IgG4 and FLC concentrations were measured using sFLC Freelite assays on a nephelometric analyzer. RESULTS: Active AIP patients have significantly higher serum levels of κ (median 30.97 (12.3-227.0) mg/L) and λFLC (median 20.53 (12.36-102.7) mg/L)) than healthy controls (κFLC; median 12.5 (3.1-52.1) mg/L), λFLC: median 12.45 (5.4-39.5) mg/L) (p < 0.05) correlating with raised serum IgG4, and significantly higher summated FLCs (∑) (median 53.09 (25.0-218.0) mg/L) than ICP patients (median 26.77 (15.0-89.2) mg/L) and healthy controls (median 24.43 (8.5-91.6) mg/L) (p < 0.05). AIP patients (median 1.43 (0.84-3.24)) showed significantly higher κ/λ ratios than ACP (median 0.83 (0.42-1.18)), ICP (median 0.87 (0.47-2.16)), PC patients (median 0.90 (0.48-1.27)) and healthy controls (median 0.963 (0.51-1.32)). There was a correlation between increased κ and λ FLCs levels and the number of affected organs involved in IgG4 related disease. CONCLUSION: Patients with type 1 AIP have increased serum k and λ FLC concentrations, Σ FLC, and κ/λ ratios. These novel biomarkers may be useful in the diagnosis of type 1 AIP and in monitoring disease activity.

Specific Smad2/3 Linker Phosphorylation Indicates Esophageal Non-neoplastic and Neoplastic Stem-Like Cells and Neoplastic Development.

BACKGROUND: There is little known about stem cells in human non-neoplastic and neoplastic esophageal epithelia. We have demonstrated expression of linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr), suggesting presence of stem-like cells in mouse esophageal epithelium, and identified presence of pSmad2/3L-Thr-positive cells that might function as cancer stem cells in mouse model of colorectal carcinoma. AIMS: We explore whether pSmad2/3L-Thr can be used as a biomarker for stem cells of human esophageal epithelia and/or neoplasms. METHODS: We have used esophageal tissues from inpatients undergoing endoscopic submucosal dissection and performed double immunofluorescent staining of pSmad2/3L-Thr and Ki67, CDK4, p63, Sox2, CK14, p53, ALDH1, CD44 or D2-40 after which the sections were stained with hematoxylin and eosin. RESULTS: pSmad2/3L-Thr-positive cells showed immunohistochemical co-localization with CDK4, p63, CD44 and Sox2 in the basal and parabasal layers of non-neoplastic esophageal epithelia. In esophageal neoplasms, they showed immunohistochemical co-localization with p53, CDK4, ALDH1 and CD44. There was a significant increase in the percentage of pSmad2/3L-Thr-positive cells in the p53-positive neoplastic cell population with development of esophageal neoplasia. pSmad2/3L-Thr-positive cells localized to the lower section of low-grade intraepithelial neoplasia and were observed up to the upper section in carcinoma in situ. In invasive squamous cell carcinoma, they were scattered throughout the tumor with disappearance of polarity and were found in intraepithelial primary lesions and sites of submucosal and vessel invasion. CONCLUSIONS: We determined significant expression of pSmad2/3L-Thr in human esophageal non-neoplastic and neoplastic epithelia, indicating that these are epithelial stem-like cells and cancer stem cells, respectively, that correlate with developing esophageal neoplasms.

Long-term model of colitis-associated colorectal cancer suggests tumor spread mechanism and nature of cancer stem cells.

Although chemical-induced animal models of colorectal cancer (CRC) suggest a lot about the disease, more efforts are required to establish metastasis models. Azoxymethane (AOM) and dextran sodium sulfate (DSS)-treated (AOM/DSS) Crl:CD-1 mice were sacrificed after 10 or 20 weeks in our previous study, and most colon tumors exhibited intramucosal adenocarcinomas. Our observations were extended until 30 weeks to study a colitis-associated advanced CRC mouse model, and explore whether linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr) immunostaining-positive cells were involved in the progressive course of colitis-associated CRC as cancer stem cells. AOM/DSS mice were sacrificed at 10, 20 and 30 weeks after AOM administration. Following the histopathological analysis, immunohistochemical staining was performed for the following markers: CD34, podoplanin, ß-catenin, E-cadherin, Ki67, Bmi1 and pSmad2/3L-Thr. Compared with AOM/DSS mice at 10 and 20 weeks, submucosal tumor infiltration and tumor invasion into vessels were markedly increased at 30 weeks. In the parts of colon tumors from AOM/DSS mice, particularly in mice at 30 weeks, the positive signal of E-cadherin was clearly reduced in the cell membranes. The percentage of Ki67-positive tumor cells in mucosal areas of AOM/DSS mice was higher than that in the sites of submucosal infiltration. In mucosal areas of colon tumors, pSmad2/3L-Thr-positive cells were scattered among tumor cells. At sites of submucosal infiltration and vessel invasion of these tumors, pSmad2/3L-Thr-positive cells were also observed among tumor cells. In colon tumors from AOM/DSS mice at 30 weeks, the percentage of pSmad2/3L-Thr-positive cells among the nuclear ß-catenin-positive tumor cells was higher than that among the cytoplasmic ß-catenin-positive tumor cells. For both non-neoplastic and neoplastic epithelial cells, pSmad2/3L-Thr-positive cells exhibited immunohistochemical co-localization with Bmi1. The present study developed an advanced CRC mouse model that exhibited tumor infiltration into the submucosa and invasion into vessels. The present study re-confirmed the theory that pSmad2/3L-Thr-positive cells may be cancer stem cells.

Interleukin-35 promotes the differentiation of regulatory T cells and suppresses Th2 response in IgG4-related type 1 autoimmune pancreatitis.

BACKGROUND: IgG4-related disease (IgG4-RD) is a systemic inflammatory disease, which includes type 1 autoimmune pancreatitis (AIP). Interleukin-35 (IL-35) exhibits immunosuppressive effects in several autoimmune diseases. However, the expression of IL-35 had not been reported so far in type 1 AIP. We evaluated the association between IL-35 and several cytokines, which mediate the function of Tregs in type 1 AIP. METHODS: Plasma was collected from patients with type 1 AIP, alcoholic chronic pancreatitis (ACP), and healthy controls (HC) and assayed for cytokine expression. Total mRNA separated from peripheral blood was isolated from naïve Tregs (nTregs) and effector Tregs (eTregs). EBI3 and IL-12p35 gene expressions were tested in these cells by quantitative PCR. In addition, expression of IL-35 subunits in the pancreatic tissues of patients with type 1 AIP and ACP was analyzed by immunohistochemistry. RESULTS: IL-35 was significantly elevated in type 1 AIP (n = 32) plasma compared with ACP (n = 16) and HC (n = 22), but IL-27 was not. We also detected many cells expressing both EBI3 and IL-12p35 in type 1 AIP tissues. Moreover, in peripheral blood lymphocyte, the percentage of nTregs and eTregs of CD4+ T cells in patients with type 1 AIP (n = 14) compared with HC (n = 15) was significantly decreased and increased, respectively. There were no significant differences of gene expression in patients with type 1 AIP and HC. CONCLUSIONS: This study identified elevated expression of plasma IL-35 and tissue IL-35 subunits in patients with type 1 AIP. This might lead to inflammation suppression via activated eTregs. IL-35 might be associated with this anti-inflammatory role, especially against the Th2 response through several cytokines and the differentiation of Tregs in type 1 AIP.

Extracellular vesicles microRNA analysis in type 1 autoimmune pancreatitis: Increased expression of microRNA-21.

BACKGROUND: The molecular basis of type 1 autoimmune pancreatitis (AIP) remains unclear. Recent attention on the role of extracellular vesicles microRNA (EV miRNA) in immune homeostasis has prompted us to perform an extensive miRNA screening of serum-derived EV in AIP. METHODS: EV miRNA expression was analyzed using microarrays in AIP, chronic pancreatitis (CP), and healthy adult (HC) samples (n = 10 from each group). Differences in signals, > 3 or <1/3 times, represented significant differences in expression. Another cohort of AIP (n = 14), CP (n = 10), and HC (n = 10) samples of EV miRNA was analyzed using reverse-transcription polymerase chain reaction (RT-PCR). miRNA expression in pancreatic tissues was evaluated using in situ hybridization (ISH) in three additional subjects from each group. RESULTS: Signals of eight miRNAs (miR-659-3p, -27a-3p, -99a-5p, -21-5p, -205-5p, -100-5p, -29c-3p, and -125b-1-3p) were significantly higher, while those of two miRNAs (miR-4252 and -5004-5p) were significantly lower in AIP than in HC. EV miR-21-5p was significantly up-regulated in AIP than in HC (P = 0.035) and CP (P = 0.048). The number of miR-21-5p positive inflammatory cells was significantly elevated in AIP than in CP (P = 0.014). CONCLUSIONS: Circulating EVs exhibited altered miRNA expression patterns with elevated miR-21-5p in AIP when compared with those in HC and CP. miR-21-5p was highly expressed in pancreatic inflammatory cells in AIP. Our data suggests that miR-21-5p may be involved in the regulation of effector pathways in the pathophysiology of AIP, thus differentiating AIP from CP.

Clinical implications of elevated serum interleukin-6 in IgG4-related disease.

INTRODUCTION: Some patients with IgG4-related disease (IgG4-RD) exhibit elevated serum interleukin (IL)-6 with excessive inflammatory reactions or with repeating relapse. To date few reports pertaining to clinical implications of elevated serum IL-6 in IgG4-RD patients have been published. The aims of the current retrospective study were to investigate the clinical implications of elevated serum IL-6 in IgG4-RD patients, and to examine whether IL-6 can predict the activity and/or relapse of the disease. MATERIALS AND METHODS: We examined the clinical picture at the onset of 43 patients who were diagnosed with IgG4-RD in our hospital and were able to measure serum IL-6 before steroid treatment. RESULTS: The median level of serum IL-6 was 2.2 pg/mL. There was a significant correlation between IL-6 and C-reactive protein (CRP) level (r = 0.397, p = 0.008), hemoglobin level (r = -0.390, p = 0.010) and albumin level (r = -0.556, p < 0.001). When 43 patients were divided into two groups by using a cut-off IL-6 of 4 pg/mL, the high IL-6 group showed higher age, lower albumin, higher CRP and higher aspartate aminotransferase (AST) (age p = 0.014, albumin p = 0.006, CRP p <0.001, AST p = 0.009). Hepatic swelling and splenomegaly were significantly more prevalent in the high IL-6 group than it was in the low IL-6 group (liver p < 0.001, spleen p = 0.020). Biliary tract involvement tended to admit more in the high IL-6 group (p = 0.060). CONCLUSION: Serum IL-6 level at the onset of IgG4-RD may be significantly correlated with clinical inflammatory parameters and it may also be associated with involvement of the bile duct, liver, and spleen.

Esophageal Large-Cell Neuroendocrine Carcinoma with Inconsistent Response to Treatment in the Primary and Metastatic Lesions.

Esophageal large-cell neuroendocrine carcinoma (NEC) is a rare malignant tumor that is characterized by high-grade malignancy and a poor prognosis. However, the rarity of esophageal NEC has prevented the development of an established treatment, and no reports have described a discrepancy in the effectiveness of cisplatin plus irinotecan between primary and metastatic lesions. A 43-year-old Japanese man was referred to our hospital with refractory epigastralgia. A previous gastrointestinal endoscopy had revealed a 50-mm type 2 tumor in the abdominal esophagus. The pathological findings indicated poorly differentiated squamous cell carcinoma. Contrast-enhanced computed tomography revealed a metastatic liver tumor. One cycle of fluorouracil and cisplatin was not effective, and endoscopy was repeatedly performed. The pathological findings indicated a large-cell malignant tumor with tumor cells that were positive for CD56, synaptophysin, and Ki-67 (> 80%). Based on a diagnosis of esophageal large-cell NEC with a metastatic liver tumor, the patient received cisplatin plus irinotecan biweekly. After 4 months, computed tomography revealed marked shrinkage of the metastatic tumor, but the patient complained of dysphagia. Endoscopy revealed enlargement of the primary tumor, which was then treated using radiotherapy plus fluorouracil and cisplatin. The primary tumor subsequently shrank, and the patient's symptoms were relieved, but the metastatic tumor grew. Thus, chemoradiotherapy could be an option for managing a primary esophageal large-cell NEC that does not respond to chemotherapy alone. However, the possibility of an inconsistent response to therapy in primary and metastatic lesions should be considered.