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We investigated the efficacy of a Wilms' tumor gene 1 (WT1) vaccine combined with gemcitabine (GEMWT1) and compared it with gemcitabine (GEM) monotherapy for advanced pancreatic ductal adenocarcinoma (PDAC) in a randomized phase II study. We randomly assigned HLA-A*02:01– or HLA-A*24:02–positive patients with advanced PDAC to receive GEMWT1 or GEM. We assessed WT1-specific immune responses via delayed-type hypersensitivity (DTH) to the WT1 peptide and a tetramer assay to detect WT1-specific cytotoxic T lymphocytes (WT1-CTL). Of 91 patients enrolled, 85 were evaluable (GEMWT1: n = 42; GEM: n = 43). GEMWT1 prolonged progression-free survival [PFS; hazard ratio (HR), 0.66; P = 0.084] and improved overall survival rate at 1 year (1-year OS%; GEMWT1: 35.7%; GEM: 20.9%). However, the difference in OS was not significant (HR: 0.82; P = 0.363). These effects were particularly evident in metastatic PDAC (PFS: HR 0.51, P = 0.0017; 1-year OS%: GEMWT1 27.3%; GEM 11.8%). The combination was well tolerated, with no unexpected serious adverse events. In patients with metastatic PDAC, PFS in the DTH-positive GEMWT1 group was significantly prolonged, with a better HR of 0.27 compared with the GEM group, whereas PFS in the DTH-negative GEMWT1 group was similar to that in the GEM group (HR 0.86; P = 0.001). DTH positivity was associated with an increase in WT1-CTLs induced by the WT1 vaccine. GEM plus the WT1 vaccine prolonged PFS and may improve 1-year OS% in advanced PDAC. These clinical effects were associated with the induction of WT1-specific immune responses. Cancer Immunol Res; 6(3); 320–31. ©2018 AACR.

FACT-G subscale by treatment group

The aim of this study was to determine the efficacy and the biomarkers of the CHP-NY-ESO-1 vaccine complexed with full-length NY-ESO-1 protein and a cholesteryl pullulan (CHP) in patients with esophageal squamous cell carcinoma (ESCC) after surgery. We conducted a randomized phase II trial. Fifty-four patients with NY-ESO-1-expressing ESCC who underwent radical surgery following cisplatin/5-fluorouracil-based neoadjuvant chemotherapy were assigned to receive either CHP-NY-ESO-1 vaccination or observation as control. Six doses of CHP-NY-ESO-1 were administered subcutaneously once every two weeks, followed by nine more doses once every four weeks. The endpoints were disease-free survival (DFS) and safety. Exploratory analysis of tumor tissues using gene-expression profiles was also performed to seek the biomarker. As there were no serious adverse events in 27 vaccinated patients, we verified the safety of the vaccine. DFS in 2 years were 56.0% and 58.3% in the vaccine arm and in the control, respectively. Twenty-four of 25 patients showed NY-ESO-1-specific IgG responses after vaccination. Analysis of intra-cohort correlations among vaccinated patients revealed that 5% or greater expression of NY-ESO-1 was a favorable factor. Comprehensive analysis of gene expression profiles revealed that the expression of the gene encoding polymeric immunoglobulin receptor (PIGR) in tumors had a significantly favorable impact on outcomes in the vaccinated cohort. The high PIGR-expressing tumors that had higher NY-ESO-1-specific IgA response tended to have favorable prognosis. These results suggest that PIGR would play a major role in tumor immunity in an antigen-specific manner during NY-ESO-1 vaccinations. The IgA response may be relevant.

Adoptive transfer of MAGE-A4 T-cell receptor gene−transduced lymphocytes in patients with recurrent esophageal cancer.

Disease progression and overall survival of 10 patients who received transfer of TCR-transduced lymphocytes.

Purpose: Preparative lymphodepletion, the temporal ablation of the immune system, has been reported to promote persistence of transferred cells along with increased rates of tumor regression in patients treated with adoptive T-cell therapy. However, it remains unclear whether lymphodepletion is indispensable for immunotherapy with T-cell receptor (TCR) gene–engineered T cells.Experimental Design: We conducted a first-in-man clinical trial of TCR gene-transduced T-cell transfer in patients with recurrent MAGE-A4–expressing esophageal cancer. The patients were given sequential MAGE-A4 peptide vaccinations. The regimen included neither lymphocyte-depleting conditioning nor administration of IL2. Ten patients, divided into 3 dose cohorts, received T-cell transfer.Results: TCR-transduced cells were detected in the peripheral blood for 1 month at levels proportional to the dose administered, and in 5 patients they persisted for more than 5 months. The persisting cells maintained ex vivo antigen-specific tumor reactivity. Despite the long persistence of the transferred T cells, 7 patients exhibited tumor progression within 2 months after the treatment. Three patients who had minimal tumor lesions at baseline survived for more than 27 months.Conclusions: These results suggest that TCR-engineered T cells created by relatively short-duration in vitro culture of polyclonal lymphocytes in peripheral blood retained the capacity to survive in a host. The discordance between T-cell survival and tumor regression suggests that multiple mechanisms underlie the benefits of preparative lymphodepletion in adoptive T-cell therapy. Clin Cancer Res; 21(10); 2268–77. ©2015 AACR.

Peptides which have homologue sequences with MAGE-A4143-151 peptide.

The nanoparticle complex of cholesteryl pullulan (CHP) and NY-ESO-1 antigen protein (CHP-NY-ESO-1) presents multiple epitope peptides to MHC class I and II pathways, leading to CD8+ and CD4+ T cell responses. Poly-ICLC is a synthetic, double-stranded RNA, an agonist of toll-like receptor (TLR)-3, and a cytoplasmic receptor of melanoma differentiation-associated gene (MDA)-5. It should be a suitable immune adjuvant of cancer vaccine to overcome the inhibitory tumor microenvironment. We conducted a phase 1 clinical trial of CHP-NY-ESO-1 with poly-ICLC in patients with advanced or recurrent esophageal cancer. CHP-NY-ESO-1/poly-ICLC (μg/mg) was administered at a dose of 200/0.5 or 200/1.0 (cohorts 1 and 2, respectively) every 2 weeks for a total of six doses. The primary endpoints were safety and immune response. The secondary endpoint was tumor response. In total, 16 patients were enrolled, and six patients in each cohort completed the trial. The most common adverse event (AE) was injection site skin reaction (86.7%). No grade 3 or higher drug-related AEs were observed. No tumor responses were observed, and three patients (30%) had stable disease. The immune response was comparable between the two cohorts, and all patients (100%) achieved antibody responses with a median of 2.5 vaccinations. Comparing CHP-NY-ESO-1 alone to the poly-ICLC combination, all patients in both groups exhibited antibody responses, but the titers were higher in the combination group. In a mouse model, adding anti-PD-1 antibody to the combination of CHP-NY-ESO-1/poly-ICLC suppressed the growth of NY-ESO-1-expressing tumors. Combining the vaccine with PD-1 blockade holds promise in human trials.

Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells without prior sensitization, and have the potential to exert antibody-dependent cellular cytotoxicity (ADCC). In this clinical trial, we examined the safety and efficacy of the use of NK cells, generated using a novel expansion system, in combination with IgG1 antibodies for the treatment of advanced gastric or colorectal cancers. Treatment consisted of trastuzumab- or cetuximab-based chemotherapy, plus adoptive NK cell therapy. For administration of expanded NK cells, dose escalation with a sequential 3 + 3 design was performed in three steps, at doses of 0.5 × 109 , 1.0 × 109 , and 2.0 × 109 cells/injection (N = 9). After 3 days of IgG1 antibody administration, patients were infused with expanded NK cells three times at triweekly intervals. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D (97.6%) and CD16 (69.6%). The combination therapy was very well tolerated with no severe adverse events. Among six evaluable patients, four presented stable disease (SD) and two presented progressive disease. Of the four SD patients, three showed an overall decrease in tumor size after combination therapy. Immune monitoring suggested that combination therapy enhanced whole blood IFN-γ production and reduced peripheral regulatory T cells (Tregs). In conclusion, this phase I trial provides evidence of good tolerability, induction of Th1 immune responses, and preliminary anti-tumor activity for this combination therapy, in patients with advanced gastric and colorectal cancer that have received previous therapy.

Chemo (chemoradio) therapy can induce oral mucositis and change body composition in patients with esophageal cancer. The impact of the amino acid-rich elemental diet Elental® on oral mucositis and changes in body composition during chemo (chemoradio) therapy is unclear. Thus, the purpose of the present study was to examine the preventive effects of Elental on oral mucositis and sarcopenia progression during chemo (chemoradio) therapy for esophageal cancer. Patients were randomized to receive either azulene oral rinse (Arm 1) or Elental (Arm 2) during the treatment cycle (4 weeks). The incidence of oral mucositis and other adverse events was evaluated weekly. Body composition pre- and post-treatment cycle was measured by bioelectric impedance analysis. Thirty‑three patients (17 azulene and 16 Elental) completed the study, and the groups were well matched. Elental tended to reduce the incidence of oral mucositis (Arm 1, 23.5% and Arm 2, 12.5%), but there was no statistically significant difference between the groups. The average body mass index (BMI) and body fat mass decreased significantly in both groups after the treatments. Lean body mass (LBM) was reduced in Arm 1, but was increased in Arm 2 after the treatment; the relative change of LBM after the treatment was significant between Arm 1 and Arm 2 (P=0.007). This study revealed that Elental nutrition could counteract sarcopenia development during chemoradiotherapy for esophageal cancer. These properties may lead to improvement of the quality of life and clinical outcome of esophageal cancer patients treated with chemo (chemoradio) therapy (Clinical Trial Registry ID: UMIN 000007960).

The phenotype and severity of cancer cachexia differ among tumor types and metastatic site in individual patients. In this study, we evaluated if differences in tumor microenvironment would affect the development of cancer cachexia in a murine model, and demonstrated that body weight, adipose tissue and gastrocnemius muscle decreased in tumor-bearing mice. Interestingly, a reduction in heart weight was observed in the intraperitoneal tumor group but not in the subcutaneous group. We evaluated 23 circulating cytokines and members of the TGF-β family, and found that levels of IL-6, TNF-α and activin A increased in both groups of tumor-bearing mice. Eotaxin and G-CSF levels in the intraperitoneal tumor group were higher than in the subcutaneous group. Atrogin 1 and MuRF1 mRNA expressions in the gastrocnemius muscle increased significantly in both groups of tumor-bearing mice, however, in the myocardium, expression of these mRNAs increased in the intraperitoneal group but not in subcutaneous group. Based on these results, we believe that differences in microenvironment where tumor cells develop can affect the progression and phenotype of cancer cachexia through alterations in various circulating factors derived from the tumor microenvironment.

Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells without prior sensitization, and have the potential to exert antibody-dependent cellular cytotoxicity (ADCC). In this clinical trial, we examined the safety and efficacy of the use of NK cells, generated using a novel expansion system, in combination with IgG1 antibodies for the treatment of advanced gastric or colorectal cancers. Treatment consisted of trastuzumab- or cetuximab-based chemotherapy, plus adoptive NK cell therapy. For administration of expanded NK cells, dose escalation with a sequential 3 + 3 design was performed in three steps, at doses of 0.5 × 10

We investigated the efficacy of a Wilms' tumor gene 1 (WT1) vaccine combined with gemcitabine (GEMWT1) and compared it with gemcitabine (GEM) monotherapy for advanced pancreatic ductal adenocarcinoma (PDAC) in a randomized phase II study. We randomly assigned HLA-A*02:01– or HLA-A*24:02–positive patients with advanced PDAC to receive GEMWT1 or GEM. We assessed WT1-specific immune responses via delayed-type hypersensitivity (DTH) to the WT1 peptide and a tetramer assay to detect WT1-specific cytotoxic T lymphocytes (WT1-CTL). Of 91 patients enrolled, 85 were evaluable (GEMWT1: n = 42; GEM: n = 43). GEMWT1 prolonged progression-free survival [PFS; hazard ratio (HR), 0.66; P = 0.084] and improved overall survival rate at 1 year (1-year OS%; GEMWT1: 35.7%; GEM: 20.9%). However, the difference in OS was not significant (HR: 0.82; P = 0.363). These effects were particularly evident in metastatic PDAC (PFS: HR 0.51, P = 0.0017; 1-year OS%: GEMWT1 27.3%; GEM 11.8%). The combination was well tolerated, with no unexpected serious adverse events. In patients with metastatic PDAC, PFS in the DTH-positive GEMWT1 group was significantly prolonged, with a better HR of 0.27 compared with the GEM group, whereas PFS in the DTH-negative GEMWT1 group was similar to that in the GEM group (HR 0.86; P = 0.001). DTH positivity was associated with an increase in WT1-CTLs induced by the WT1 vaccine. GEM plus the WT1 vaccine prolonged PFS and may improve 1-year OS% in advanced PDAC. These clinical effects were associated with the induction of WT1-specific immune responses. Cancer Immunol Res; 6(3); 320–31. ©2018 AACR.

Epithelial-mesenchymal transition (EMT) plays a crucial role in cancer metastasis. In this study, we evaluated the effect of heat treatment on tumor growth factor-β1 (TGF-β1)-induced EMT in pancreatic cancer cells and tried to ascertain the mechanism related to any observed effects. Human pancreatic cancer cell lines (BxPC-3, PANC-1 and MIAPaCa-2) were stimulated by TGF-β1, and evaluated for morphological changes using immunofluorescence and EMT-related factors (i.e., E-cadherin, Vimentin, Snail or ZEB-1) using RT-PCR. To examine the effect of heat on EMT, the cancer cells were heat-treated at 43°C for 1 h then stimulated with TGF-β1. We then evaluated whether or not heat treatment changed the expression of EMT-related factors and cell migration and also whether Smad activation was inhibited in TGF-β signaling. After being treated with TGF-β1, pancreatic cancer cells resulted in EMT and cell migration was enhanced. Heat treatment inhibited TGF-β1-induced changes in morphology, inhibited the expression of EMT-related factors, and attenuated TGF-β1-induced migration in pancreatic cancer cells. Additionally, we observed that heat treatment blocked TGF-β1-induced phosphorylation of Smad2 in PANC-1 cells. Our results suggest that heat treatment can suppress TGF-β1-induced EMT and opens the possibility of a new therapeutic use of hyperthermia as a potential treatment for cancer metastasis.

A core challenge in administering immune-based treatments for cancer is the establishment of easily accessible immunological assays that can predict patients' clinical responses to immunotherapy. In this study, our aim was to predict the therapeutic effects of adoptive T-cell therapy in patients with advanced pancreatic cancer. To do this, we evaluated whole blood cytokine levels and peripheral regulatory T cells (Tregs) in 46 patients with unresectable or recurrent pancreatic cancer who received adoptive T-cell therapy at 2-week intervals. To test immune function, venous blood was obtained from patients before the start of therapy and 2 weeks after the 4th treatment. Whole blood interferon (IFN)-α levels (after stimulation with the Sendai virus) were evaluated, as well as the levels of 9 cytokines stimulated with phytohemagglutinin [interleukin (IL)−2, IL-4, IL-5, IL-10, IL-12(p70), IL-13, tumor necrosis factor-α, IFN-γ, and granulocyte-monocyte colony-stimulating factor]. Peripheral Tregs were analyzed by flow cytometry. Using the obtained data, we then observed the relationship between these immunological parameters and clinical outcome of patients. We found that the whole blood production of IFN-γ, IL-2, IL-4, IL-5 and IL-13 significantly increased after adoptive T-cell therapy, whereas the number of peripheral Tregs did not change. Multivariate Cox proportional hazards analyses indicated that the number of peripheral Tregs before receiving adoptive T-cell therapy and the change in IFN-γ levels after adoptive T-cell therapy were independent variables predicting overall survival. The findings of this study indicate that the assay of whole blood IFN-γ production offers promise for evaluating the clinical response of patients to cancer immunotherapy.

The technological development in endoscopy is directed toward improved accuracy of the diagnoses of novel diseases. The capsule endoscope and balloon-assisted endoscope are examples of such technological development. By these novel technologies, the small intestine can be examined in more detail. Therefore, an increasing number of novel diseases have been discovered, requiring the establishment of diagnosis and treatment strategies for these unknown diseases. In particular, obscure gastrointestinal bleeding, Crohn's disease, and nonsteroidal anti-inflammatory drug-induced enteropathy are of great interest to endoscopists. The capsule endoscope is the best method for screening the small intestine; however, the development of supporting methods such as the patency capsule is eagerly desired.

In recent years, roles of the immune system in immune surveillance of cancer have been explored. And natural killer (NK) cells are considered to be critical players in anticancer immunity. However, cancers are able to develop mechanisms to escape NK cell attack or to induce defective NK cells.

In liver cancer, there are two kinds of liver cancer. One is a primary hepatocellular carcinoma and the other is metastatic liver cancer.

Since many of the patients who receive adoptive immunotherapy also receive chemotherapy and/or radiation therapy, evaluation of the curative effect of adoptive immunotherapy itself is difficult. To overcome this, I perform immune monitoring to examine how much individual immunotherapies contribute to the curative effects. As a result, I found that higher whole blood levels of Th1 cytokines prodicing potential may be used to predict better clinical responses. In addition, I showed that the naive T-cell adoptive therapy, which we developed, has superior clinical efficacy when compared to other immunotherapies. This chapter also describes the results of our clinical trial. In addition, I describe the application of hyperthermia, which can influence the immune-response of a tumor tissue. Basic experiments showed that hyperthermia could be applied to strengthen a patient’s immune system. Next, we performed naive T-cell adoptive immunotherapy combined with hyperthermia. Finally, I will describe a case of advanced gastric cancer that showed complete remission after treatment with naive T-cell adoptive immunotherapy combined with hyperthermia.

Background The relationship between Helicobacter pylori infection and metabolic syndrome is not well understood. Adiponectin is an adipose-derived protein considered to play a significant role in the development of metabolic syndrome. The aim of this study was to clarify the influence of H. pylori infection on circulating adiponectin in humans. Methods In a prospective study, 456 patients underwent endoscopy and H. pylori testing. All of the 338 H. pylori -positive patients received eradication therapy. Treatment was successful in 241 patients. Circulating adiponectin and other metabolic parameters were measured at baseline in all patients and 12 weeks after eradication therapy in those initially positive for H. pylori. Results Circulating adiponectin levels were not different between H. pylori -positive and H. pylori -negative patients. In the group with successful eradication, levels of total adiponectin and each multimer form were significantly increased after therapy. Conversely, the levels of total adiponectin and high-molecular-weight adiponectin, but not middle-molecular-weight and low-molecular-weight adiponectin, were increased in the group with unsuccessful eradication after the therapy. Conclusions Eradication therapy of H. pylori increased circulating adiponectin levels in Japanese individuals and could be beneficial for preventing metabolic syndrome conditions.

We investigated the effects of anticancer agents on peripheral blood mononuclear cells for the purpose of providing data to support new translational chemoimmunotherapy regimens. Peripheral-blood mononuclear cells were treated with one of four anticancer agents (5-fluorouracil, irinotecan, cisplatin, and gemcitabine) for 2 h, after which cell viability was determined. For assessment of effects of each drug on proliferation and cytokine production, cells were stimulated with phytohemagglutinin for 48 h. As a result, the anticancer agents did not affect cell viability. Cell proliferation was unaffected by 5-fluorouracil and irinotecan but inhibited by cisplatin and gemcitabine. Treatment with gemcitabine enhanced the production of IFN-γ and decreased the number of regulatory T cells. gemcitabine treatment increased IFN-γ production among CD4 T cells but not among CD8 T cells. The results indicated that GEM had immunoregulatory properties that might support immune response against cancer. This finding has implications for designing chemoimmunotherapy strategies.

Acetyl salicylic acid (ASA) is one of the most frequently prescribed medications for the secondary prevention of cardiovascular and cerebrovascular events. It has recently been reported to cause small intestinal mucosal injury at a considerably higher rate than previously believed. The aim of this study is to investigate the mechanism by which this occurs using an in vitro small intestine model focusing on the role of oxidative stress and cell permeability. Differentiated Caco-2 exhibits a phenotype similar to human small intestinal epithelium. We measured whether ASA induced the increase of differentiated Caco-2 permeability, the decrease of tight junction protein expression, the production of reactive oxygen species (ROS), and the expression of ROS-modified zonula occludens-1 (ZO-1) protein. In some experiments, Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, a superoxide dismutase mimetic) was used. The nontoxic concentration of ASA decreased transepithelial electrical resistance and increased the flux of fluorescein isothiocyanate-conjugated dextran across Caco-2 in a time-dependent manner. The same concentration of ASA significantly decreased ZO-1 expression among TJ proteins as assessed by Western blot and immunocytochemistry and increased ROS production and the expression of oxidative stress-modified ZO-1 protein. However, MnTMPyP suppressed the ASA-induced increased intercellular permeability and the ASA-induced ROS-modified ZO-1 expression. Our findings indicate that ASA-induced ROS production can specifically modify the expression of ZO-1 protein and induce increased cell permeability, which may ultimately cause small intestinal mucosal injury.

Objective Several epidemiological studies have shown that regular exercise can prevent the onset of colon cancer, although the underlying mechanism is unclear. Myokines are secreted skeletal muscle proteins responsible for some exercise-induced health benefits including metabolic improvement and anti-inflammatory effects in organs. The purpose of this study was to identify new myokines that contribute to the prevention of colon tumorigenesis. Methods To identify novel secreted muscle-derived proteins, DNA microarrays were used to compare the transcriptome of muscle tissue in sedentary and exercised young and old mice. The level of circulating secreted protein acidic and rich in cysteine (SPARC) was measured in mice and humans that performed a single bout of exercise. The effect of SPARC on colon tumorigenesis was examined using SPARC-null mice. The secretion and function of SPARC was examined in culture experiments. Results A single bout of exercise increased the expression and secretion of SPARC in skeletal muscle in both mice and humans. In addition, in an azoxymethane-induced colon cancer mouse model, regular low-intensity exercise significantly reduced the formation of aberrant crypt foci in wild-type mice but not in SPARC-null mice. Furthermore, regular exercise enhanced apoptosis in colon mucosal cells and increased the cleaved forms of caspase-3 and caspase-8 in wild-type mice but not in SPARC-null mice. Culture experiments showed that SPARC secretion from myocytes was induced by cyclic stretch and inhibited proliferation with apoptotic effect of colon cancer cells. Conclusions These findings suggest that exercise stimulates SPARC secretion from muscle tissues and that SPARC inhibits colon tumorigenesis by increasing apoptosis.

Background and Aim: Adequate mucosal elevation by submucosal injection is important for definitive en bloc resection and prevention of perforation during endoscopic mucosal resection (EMR). The objective of this study is to determine the efficacy of 0.13% hyaluronic acid (HA) solution for high and sustained mucosal elevation during colorectal EMR. Methods: The study was a prospective randomized controlled trial; a total of 196 patients with colon polyps of

Serpin B1 is a monocyte neutrophil elastase (NE) inhibitor and is one of the most efficient inhibitors of NE. In the present study, we investigated the role of serpin B1 in the pathogenesis of ulcerative colitis by using clinical samples and an experimental model. The colonic expression of serpin B1 was determined by real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistological studies in both normal and inflamed mucosa from patients with ulcerative colitis. Serpin B1 mRNA expression was determined by real-time PCR in the mouse dextran sodium sulfate (DSS)-induced colitis model. Young adult mouse colonic epithelial (YAMC) cells were used to determine the role of serpin B1. Serpin B1 gene transfected YAMC cells were treated with H2O2to measure cell viability. The expression of NE was determined in YAMC cells treated with H2O2. NE-silenced YAMC cells were also treated with H2O2and then measured for viability. Upregulated expression of serpin B1 in colonic mucosa was confirmed from patients with active ulcerative colitis. Immunohistochemical studies showed that serpin B1 expression was localized not only in inflammatory infiltration cells but also in epithelial cells. Serpin B1 mRNA expression was also increased in colonic mucosa of mouse DSS-induced colitis. Serpin B1-transfected YAMC cells were resistant against the treatment of H2O2. H2O2treatment significantly induced NE in YAMC cells, and NE-silenced YAMC cells were also resistant against the treatment of H2O2. These results suggest that serpin B1 may be a novel marker of active ulcerative colitis and may play an important role in the pathogenesis of inflammatory bowel disease.

Background and Aims: Recent advancements in capsule endoscopy and double-balloon endoscopy have revealed that non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, can induce small intestinal mucosal damage. However, the precise pathogenesis and therapeutic strategy have not been fully revealed. The aim of the present study was to determine the upregulated proteins in the small intestine exposed to indomethacin. Methods: Indomethacin (10 mg/kg) was administered subcutaneously to male Wistar rats to induce small intestinal damage and the severity of the intestinal injury was evaluated by measuring the area of visible ulcerative lesions. The intestinal mucosal tissue samples were collected and then analyzed by two-dimensional gel electrophoresis, with matrix-assisted laser desorption/ionization time-of-flight spectrometer peptide mass fingerprinting being used to determine the differentially expressed proteins between normal and injured intestinal mucosa. Results: Among several protein spots showing differential expression, one, hemopexin (HPX), was identified as upregulated in indomethacin-induced injured intestinal mucosa using the MASCOT search engine. Conclusion: HPX was identified as upregulated protein in the small intestine exposed to indomethacin. HPX may be responsible for the development of the intestinal inflammation induced by NSAIDs.

A 50-year-old female became aware of skin yellowing and consulted another hospital where she was diagnosed intraoperatively with duodenal cancer because of lymph node metastases around the aorta. Endoscopy revealed type IIa + IIc cancer distal to the duodenal papilla, and biopsy allowed a diagnosis of well-differentiated adenocarcinoma. Computed tomography revealed a large number of lymph node metastases around the aorta and in the left supraclavicular cavity. The patient was given many regimens of chemotherapy, mainly containing S-1, and multidisciplinary treatment, and achieved long-term survival for 6 years and 1 month. This is a valuable case suggesting the usefulness of this therapeutic approach. In view of the fact that duodenal cancer is a relatively rare disease and the possibility that the incidence of this disease may increase in the future, it seems essential to collect additional data from multicenter prospective studies towards the goal of establishing a standard method of treatment for this disease.

Objective To investigate the relation between functional impairments of cancer patients and circulating cytokines using a multiplex technique. Design and methods 50 patients with cancer were assessed using the quality of life (QOL) questionnaire. 27 plasma cytokine levels were determined by using the Bio-Plex array system. The relation to QOL scores was assessed using Chi-square test for categorical variables and univariate linear regression analysis for cytokine levels. Results Multivariate analysis showed that interleukin-6 (IL-6) level is a significant independent determinant of physical (β = − 0.238, P = 0.0126) and cognitive functioning (β = − 0.462, P = 0.0006) and that vascular endothelial growth factor (VEGF) level is a significant independent determinant of emotional functioning (β = − 0.414, P = 0.039). Conclusion This study, in which 27 cytokines are simultaneously tested with cutting edge technology, demonstrates that plasma IL-6 and VEGF are significant independent determinants of functional impairments in patients with cancer.

We examined the effects of adoptive T-cell transfer (ACT) on the population of regulatory T cells (Tregs) in a mouse colorectal cancer transplant model. In an in vivo study, Treg populations in Balb/c mice colon26 transplant model after ACT were analyzed in peripheral blood, local lymph node, and tumor. In an in vitro study CD4+ cells were cultured in medium containing TGF-beta to induce Tregs. LAK cells were added or not in this Treg induction system. Treg induction after coculture with LAK was investigated. We also studied the role of IFN-gamma in the mechanism of Treg induction. Tregs in the draining lymph nodes and tumor were significantly suppressed by ACT. The induction of Tregs in vitro was inhibited by coculture with LAK cells. Furthermore, Tregs in the cultured cells were significantly inhibited by addition of exogenous IFN-gamma. Moreover, Tregs were increased by addition of IFN-gamma mAb. ACT may decrease Tregs in tumor-bearing hosts. One of the mechanisms is considered to be IFN-gamma inhibiting the induction of Tregs.

Human esophageal epithelium is continuously exposed to physical stimuli or to gastric acid that sometimes causes inflammation of the mucosa. Transient receptor potential vanilloid 1 (TRPV1) is a nociceptive, Ca2+-selective ion channel activated by capsaicin, heat, and protons. It has been reported that activation of TRPV1 expressed in esophageal mucosa is involved in gastroesophageal reflux disease (GERD) or in nonerosive GERD symptoms. In this study, we examined the expression and function of TRPV1 in the human esophageal epithelial cell line Het1A, focusing in particular on the role of oxidative stress. Interleukin-8 (IL-8) secreted by Het1A cells upon stimulation by capsaicin or acid with/without 4-hydroxy-2-nonenal (HNE) was measured by ELISA. Following capsaicin stimulation, the intracellular production of reactive oxygen species (ROS) was determined using a redox-sensitive fluorogenic probe, and ROS- and HNE-modified proteins were determined by Western blotting using biotinylated cysteine and anti-HNE antibody, respectively. HNE modification of TRPV1 proteins was further investigated by immunoprecipitation after treatment with synthetic HNE. Capsaicin and acid induced IL-8 production in Het1A cells, and this production was diminished by antagonists of TRPV1. Capsaicin also significantly increased the production of intracellular ROS and ROS- or HNE-modified proteins in Het1A cells. Moreover, IL-8 production in capsaicin-stimulated Het1A cells was enhanced by synthetic HNE treatment. Immunoprecipitation studies revealed that TRPV1 was modified by HNE in synthetic HNE-stimulated Het1A cells. We concluded that TRPV1 functions in chemokine production in esophageal epithelial cells, and this function may be regulated by ROS via posttranslational modification of TRPV1.

BTB and CNC homologue 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1). This study hypothesized that Bach1 plays an important role in the indomethacin-induced apoptosis in the case of small-intestinal mucosal injury. Eight-week-old male C57BL/6 (wild-type) and homozygous Bach1-deficient C57BL/6 mice were included in this study. Mucosal injuries induced by subcutaneously administering indomethacin were evaluated macroscopically, histologically and biochemically. Indomethacin-induced injuries were improved in Bach1-deficient mice. Immunohistochemistry showed an increase in the number of HO-1-positive cells, which were mainly F4/80 positive macrophages, in Bach1-deficient mice. Indomethacin administration increased the expression of HO-1 mRNA and protein in the small intestine in Bach1-deficient mice. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining showed that the extent of apoptosis was suppressed in Bach1-deficent mice. In conclusion, deficiency of the Bach1 gene inhibited apoptosis and thus suppressed mucosal injury, indicating that Bach1 is a novel therapeutic target for indomethacin-induced intestinal injury.

Background and Aim: Endoscopic mucosal resection (EMR) is the standard procedure for colorectal tumors. High mucosal elevation by submucosal injection is important for definite en bloc resection and the prevention of perforation. Hyaluronic acid (HA) is a reportedly useful injection solution for high and long-lasting mucosal elevation, but the ideal HA concentration for optimization of mucosal elevation maintenance, injection pres- sure, and cost is unknown. In the present study, we assessed the appropriate concentration of HA for EMR. Methods: A resected porcine colon and esophagus were used. The injection solutions examined were 0.9% normal saline (NS) and four concentrations of an 800-KDa HA preparation (0.4%, 0.2%, 0.13%, and 0.1%). Each solution (2 mL) was injected into the submucosa; injection pressure was calculated, and elevation was measured. The durations of mucosal elevation and EMR were additionally assessed in the living minipig colon. Results: In the resected porcine colon, the mucosal elevation was measured 0, 2, 4, and 6 min after the submucosal injection.All concentrations of HAsolution maintained greater mucosal elevation at all times than NS (P < 0.05).An almost similar result was obtained in the resected porcine esophagus. The injection pressure correlated with the HA concentra- tion. In the living minipig colon, mucosal elevation diminished 2 min after the submucosal injection with NS, but was maintained 2 min after injection with 0.4%, 0.2%, and 0.13% HA. The average duration of EMR was 139 s. Conclusions: Mucosal elevation by HAwas greater than that by NS in resected and living animal models. We recommend 0.13% HA for maintaining mucosal elevation, injection pressure, and cost.

Background and Aims: The pathogenesis of enteropathy induced by non-steroidal anti-inflammatory drugs (NSAIDs) is still unclear, and there are no established treatments. Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has been associated with the development of chronic inflammatory diseases, including autoimmune diseases. To define the role of IL-17A in small intestinal injury and inflammation, we studied the effects of indomethacin administration in mice with targeted deletions of the IL-17A gene. Methods: Male C57BL/6 (wild-type) and homozygous IL-17A-/- C57BL/6 mice were subjected to this study. Indomethacin (10 mg/kg) was subcutaneously administered to induce small-intestinal damage. Indomethacin-induced lesions in the small intestine were evaluated by measuring the injured area and by histopathology. Also assessed were myeloperoxidase (MPO) activity, as an index of neutrophil accumulation, and intestinal mRNA expression for inflammatory cytokines. Results: The area of macroscopic ulcerative lesions, the MPO activity and the mRNA expression of inflammatory-associated chemokines, such as keratinocyte chemoattractant (KC), monocyte chemotactic protein-1 (MCP-1), and granulocyte-colony stimulating factor (G-CSF), were significantly increased in indomethacin-treated groups compared with the sham groups. The development of intestinal lesions by indomethacin was inhibited in IL-17A-/- mice compared with wild-type mice, together with significant suppression of the increased levels of MPO activities and KC, MCP-1, and G-CSF levels. Conclusion: These findings demonstrate that IL-17A contributes to the development of indomethacin-induced small intestinal injury through upregulation of G-CSF, KC, and MCP-1. IL-17A might be a promising new therapeutic target to treat NSAID-induced enteritis.

Use of hyperthermia as cancer treatment has often been impossible to continue at once following exacerbation of general condition due to cancer growth. However, no formal consensus has yet been defined regarding the acceptability of hyperthermia, so selection of patients for hyperthermia is not based on agreed clinical criteria. Aiming at the establishment of agreed clinical criteria concerning the selection of patients for hyperthermia, this report examined the treatment acceptability of hyperthermia based on patient backgrounds before advanced cancer treatment. Subjects comprised 45 patients with various advanced cancers treated with regional hyperthermia combined with chemo- or immunotherapy who visited our clinic between July 2008 and May 2009. Group A (n=24) underwent hyperthermia ≥ 8 times, while Group B (n=21) underwent hyperthermia ≤ 7 times (mean, 4.19 times). We investigated pretreatment laboratory data, body mass index, performance status (PS), Glasgow prognostic score (GPS), and quality of life (QOL). Patients with poor scores for both PS and GPS dropped out early. In these patients, elevations in both lactate dehydrogenase (LDH) and C-reactive protein (CRP) levels tended to be present in addition to hypoalbuminemia. QOL in Group B was already impaired before initiation of hyperthermia. PS and GPS appear to represent the most important factors when judging the acceptability of hyperthermia, while LDH, CRP, and albumin levels may help such judgments. The acceptability of hyperthermia can be predicted using patient background as evidenced by laboratory data and general conditions, including QOL, before cancer treatment. The results justified further examination in a large number of patients to aim the establishment of agreed clinical criteria concerning the selection of patients for hyperthermia.

The beneficial effects by peroxisome proliferator-activated receptor-γ (PPAR-γ) on gastric injury induced by ischemia-reperfusion have been confirmed, however, the precise mechanism of its cytoprotection is not elucidated thoroughly. The aim of the present study was to determine the gastric localization of PPAR-γ expression in the rat gastric mucosa, and to clarify the mechanism of its cytoprotective properties. The gastric expression of PPAR-γ was confirmed by RT-PCR and western blot, and localized on gastric epithelial cells. The protective effect of PPAR-γ ligands, pioglitazone or 15-deoxy-Δ(12,14)-prostaglandin J(2), on gastric ischemia-reperfusion injury was reversed by the co-administration with PPAR-γ antagonist. The gastric expression of tumor necrosis factor-α and cytokine-induced neutrophil chemoattractant-1 increased significantly in rats treated ischemia-reperfusion, and these increases were significantly inhibited by treatment with pioglitazone. Among the 1,032 probes, 18 probes were up-regulated at least 1.5-fold, 17 were down-regulated at least 1.5-fold by pioglitazone. The network including calnexin, endoplasmic reticulum stress protein, heat shock proteins, and proteasome genes was induced by pioglitazone treatment. In conclusion, activation of gastric epithelial PPAR-γ receptor by its ligands may represent a novel therapeutic approach for gastric inflammation via up-regulation of heat shock proteins and endoplasmic reticulum-related proteins.

Heat shock protein (HSP) 47 may play an important role in the pathogenesis of intestinal fibrosis. Daikenchuto (DKT), a traditional Japanese herbal (Kampo) medicine, has been reported to ameliorate intestinal inflammation. The aims of this study were to determine time-course profiles of several parameters of fibrosis in a rat model, to confirm the HSP47-expressing cells in the colon, and finally to evaluate DKT's effects on intestinal fibrosis. Colitis was induced in male Wistar rats weighing 200 g using an enema of trinitrobenzene sulfonic acid (TNBS). HSP47 localization was determined by immunohistochemistry. Colonic inflammation and fibrosis were assessed by macroscopic, histological, morphometric, and immunohistochemical analyses. Colonic mRNA expression of transforming growth factor β1 (TGF-β1), HSP47, and collagen type I were assessed by real time-polymerase chain reaction (PCR). DKT was administered orally once a day from 8 to 14 d after TNBS administration. The colon was removed on the 15th day. HSP47 immunoreactivity was coexpressed with α-smooth muscle actin-positive cells located in the subepithelial space. Intracolonic administration of TNBS resulted in grossly visible ulcers. Colonic inflammation persisted for 6 weeks, and fibrosis persisted for 4 weeks after cessation of TNBS treatment. The expression levels of mRNA and proteins for TGF-β1, HSP47, and collagen I were elevated in colonic mucosa treated with TNBS. These fibrosis markers indicated that DKT treatment significantly inhibited TNBS-induced fibrosis. These findings suggest that DKT reduces intestinal fibrosis associated with decreasing expression of HSP47 and collagen content in the intestine.

A 75-year-old man with general malaise and appetite loss was transferred to our hospital for assessment and treatment of liver failure. Laboratory findings on admission showed anemia, and gastroduodenoscopy (GDS) revealed linear esophageal varices and tensive duodenal varices (DV) in the second portion of the duodenum. Systemic examinations did not reveal any significant lesion capable of explaining his anemia, except for DV. Balloon-occluded retrograde transvenous obliteration was carried out to prevent DV bleeding. Good pooling of sclerosant was observed using two balloon catheters. However, contrast-enhanced computed tomography after the procedure revealed no thrombosis in DV, and the patient complained of tarry stools before additional therapy. Emergent GDS revealed ruptured DV with fresh blood and erosions on the surface. Emergent endoscopic obliteration using the tissue adhesive N-butyl-2-cyanoacrylate was carried out and complete hemostasis was achieved. Although no rebleeding episodes were observed after emergent obliteration, the patient died of sepsis following spontaneous bacterial peritonitis 53 days after admission. Autopsy revealed that DV dropped out, and the deep vein was replaced by granulation tissue. No signs of thrombi were detected, except varices. This autopsy case revealed the difficulty in DV management.

Several epidemiological studies have shown that regular exercise can prevent the onset of colon cancer, although the mechanism involved is unclear. Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) is often elevated in an initial step of tumorigenesis and promotes colorectal cancer. We investigated the effect of exercise on colon tumorigenesis associated with iNOS and COX-2 in azoxymethan (AOM)-injected mice. Balb/c mice (8 weeks old) were divided into three groups of 20 animals each, consisting of a sedentary control group, an AOM group, and an exercise plus AOM group. Mice in the groups receiving AOM were injected intraperitoneally with AOM weekly for 2 weeks. Six weeks of regular exercise suppressed the generation of aberrant crypt foci (ACF) in the colon by AOM. Expression of iNOS was decreased by exercise compared with that in sedentary mice along with lower nitrotyrosine level while COX-2 was not changed by either AOM or exercise. Additionally, tumor necrosis factor alpha (TNFalpha) was decreased by exercise in the colon and plasma. There was no effect of exercise on the expression of antioxidant enzymes and chaperon protein in the colon. Our results suggest that regular exercise prevents colon tumorigenesis, at least partly via the suppression of iNOS expression associated with anti-inflammation.

Background; Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells, and produce numerous cytokines resulting in the activation of the adoptive immune systems. Thus, NK cell is considered ideal cell for adoptive cancer immunotherapy. But the difficulty of obtaining large numbers of NK cells that are safe to administer deters its clinical use. We successfully generated large numbers of activated NK cells from small quantities of blood and also determined that the expanded cells were safe to administer in a monotherapy (J Transl Med (2015) 13:277). Since NK cells play a key role in antibody-dependent cellular cytotoxicity (ADCC), administration of NK cells would be expected to enhance the efficacy of IgG1 monoclonal antibodies by augmenting ADCC. This phase I clinical trial evaluated the safety, toxicity, and immunological response of the adoptive NK therapy in combination with IgG1 antibodies. Patients and Methods; Patients with unresectable advanced gastric or colorectal cancer who have administered or have planned to administer IgG1 antibody (i.e. trastuzumab for HER2 positive gastric cancer or cetuximab for RAS-wild colorectal cancer). NK cells were expanded from PBMCs with the same method in previous clinical trial, briefly ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296-induced T cells. Expanded NK cells were intravenously injected 3 days after IgG1 antibody administration in a dose-escalating manner (dose 0.5 × 109, 1.0 × 109, 2.0 × 109 cells/injection, three patients/one cohort), every 3 weeks for 3 cycles. We evaluated the safety and efficacy of the combination therapy. To assess the immunological response, immunomonitoring was performed. Results; Nine eligible patients were enrolled. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D(97.6%) and CD16(69.6%). This combination therapy was well tolerated with no severe adverse events. Among six evaluable patients, 4 presented SD, 2 presented PD. Of the four SD patients, 3 showed an overall decrease in tumor size after combination therapy. In immunomonitoring analysis, whole blood IFN-gamma production level was increased and the proportion of Treg (Treg/CD4) in peripheral blood was decreased after the combination therapy. Conclusions; This is the first clinical trial of the combination of adoptive NK cell therapy and IgG1 monoclonal antibodies. Our data provide evidence of good tolerability and preliminary anti-tumor activity for this combination therapy. Moreover, immunological monitoring revealed that the combination therapy induced Th1-type immune response and reduced peripheral Tregs. However, given the limited activity observed in this clinical trial, additional studies are necessary to develop these combination approaches. Citation Format: Takeshi Ishikawa, Tetsuya Okayama, Naoyuki Sakamoto, Mitsuko Ideno, Kaname Oka, Tatsuji Enoki, Junichi Mineno, Hideyuki Konishi, Satoshi Kokura, Yuji Naito, Yoshito Itoh. Safety and efficacy of high purity and activity NK cells therapy in combination with IgG1 antibody in patients with gastric or colorectal cancer: A phase I clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT147.

Background and Aims: Ecabet sodium (ES) is a gastric mucosal protective and ulcer-healing agent. Recently enema therapy with ES was found to be effective for the treatment of human ulcerative colitis as well as experimental colitis in an animal model. Whereas ES possesses potential as a novel treatment for ulcerative colitis, its precise mechanism of action remains to be elucidated. In this study, we investigated the therapeutic efficacy of ES in an experimental rat model of colitis, and evaluated the restitution of intestinal epithelial cells treated with ES in vitro. Methods: Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. Rats received intrarectal treatment with ES daily starting on day 7 and were sacrificed on day 14 after the administration of TNBS. The distal colon was removed to evaluate various parameters of inflammation. Moreover, wound-healing assays were used to determine the enhanced restitution of rat intestinal epithelial (RIE) cells treated with ES. Results: Intracolonic administration of ES accelerated TNBS-induced ulcer healing. Increases in the wet weight of the colon after TNBS administration were significantly inhibited by ES treatment. The wound assay revealed ES enhancement of the migration of RIE cells migration through the phosphorylation of extracellular signal-regulated kinase. Conclusion: Daily administration of an ES enema promoted the healing of intestinal mucosal injury, in part by the enhanced restitution of intestinal epithelial cells via extracellular signal-regulated kinase activation. ES may thus represent a novel therapeutic approach for the treatment of inflammatory bowel disease.

Background and Aims: The aim of this study was to identify new intestinal proteins potentially associated with acute inflammation using proteomic profiling of an in vivo mice model of ulcerative colitis. Methods: 2D fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight spectrometer (MALDI-TOF) peptide mass fingerprinting were used to determine differentially expressed proteins between normal and inflamed intestinal mucosa. Acute colitis was induced by 8.0% dextran sodium sulfate (DSS) given p.o. for 7 days. Results: Among a total of seven protein spots showing differential expression, we identified five different proteins, of which two were upregulated and three downregulated in colitis in comparison to normal mucosa, using the MASCOT search engine. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase 2 and serpin b1a were upregulated proteins, and protein disulfide-isomerase A3, peroxiredoxin-6 and vimentin were identified as downregulated proteins. Conclusion: These identified proteins may be responsible for the development of the intestinal inflammation. 2D-DIGE and MALDI-TOF mass spectrometry are useful in the search for the differentially expressed proteins.