Tetracyclines enhance antitumor T-cell immunity via the Zap70 signaling pathway.

BACKGROUND: Cancer immunotherapy including immune checkpoint inhibitors is only effective for a limited population of patients with cancer. Therefore, the development of novel cancer immunotherapy is anticipated. In preliminary studies, we demonstrated that tetracyclines enhanced T-cell responses. Therefore, we herein investigated the efficacy of tetracyclines on antitumor T-cell responses by human peripheral T cells, murine models, and the lung tumor tissues of patients with non-small cell lung cancer (NSCLC), with a focus on signaling pathways in T cells. METHODS: The cytotoxicity of peripheral and lung tumor-infiltrated human T cells against tumor cells was assessed by using bispecific T-cell engager (BiTE) technology (BiTE-assay system). The effects of tetracyclines on T cells in the peripheral blood of healthy donors and the tumor tissues of patients with NSCLC were examined using the BiTE-assay system in comparison with anti-programmed cell death-1 (PD-1) antibody, nivolumab. T-cell signaling molecules were analyzed by flow cytometry, ELISA, and qRT-PCR. To investigate the in vivo antitumor effects of tetracyclines, tetracyclines were administered orally to BALB/c mice engrafted with murine tumor cell lines, either in the presence or absence of anti-mouse CD8 inhibitors. RESULTS: The results obtained revealed that tetracyclines enhanced antitumor T-cell cytotoxicity with the upregulation of granzyme B and increased secretion of interferon-γ in human peripheral T cells and the lung tumor tissues of patients with NSCLC. The analysis of T-cell signaling showed that CD69 in both CD4+ and CD8+ T cells was upregulated by minocycline. Downstream of T-cell receptor signaling, Zap70 phosphorylation and Nur77 were also upregulated by minocycline in the early phase after T-cell activation. These changes were not observed in T cells treated with anti-PD-1 antibodies under the same conditions. The administration of tetracyclines exhibited antitumor efficacy with the upregulation of CD69 and increases in tumor antigen-specific T cells in murine tumor models. These changes were canceled by the administration of anti-mouse CD8 inhibitors. CONCLUSIONS: In conclusion, tetracyclines enhanced antitumor T-cell immunity via Zap70 signaling. These results will contribute to the development of novel cancer immunotherapy.

Risk factors for rebleeding in gastroduodenal ulcers.

BACKGROUND: Rebleeding after hemostasis of the gastroduodenal ulcer (GDU) is one of the indicators associated with death among GDU patients. However, there are few studies on risk score that contribute to rebleeding after endoscopic hemostasis of bleeding peptic ulcers. AIMS: The aim of this study was to identify factors associated with rebleeding, including patient factors, after endoscopic hemostasis of bleeding gastroduodenal ulcers and to stratify the risk of rebleeding. METHODS: We retrospectively enrolled 587 consecutive patients who were treated for Forrest Ia to IIa bleeding gastroduodenal ulcers with endoscopic hemostasis at three institutions. Risk factors associated with rebleeding were assessed using univariate and multivariate logistic regression analyses. The Rebleeding Nagoya University (Rebleeding-N) scoring system was developed based on the extracted factors. The Rebleeding-N score was internally validated using bootstrap resampling methods. RESULTS: Sixty-four patients (11%) had rebleeding after hemostasis of gastroduodenal ulcers. Multivariate logistic regression analysis revealed four independent rebleeding risk factors: blood transfusion, albumin <2.5, duodenal ulcer, and diameter of the exposed vessel ≧2 mm. Patients with 4 risk factors in the Rebleeding-N score had a 54% rebleeding rate, and patients with 3 risk factors had 44% and 25% rebleeding rates. In the internal validation, the mean area under the curve of the Rebleeding-N score was 0.830 (95% CI = 0.786-0.870). CONCLUSIONS: Rebleeding after clip hemostasis of bleeding gastroduodenal ulcers was associated with blood transfusion, albumin <2.5, diameter of the exposed vessel ≧2 mm, and duodenal ulcer. The Rebleeding-N score was able to stratify the risk of rebleeding.

Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Superrelaxed Myosin.

BACKGROUND: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. METHODS: We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). RESULTS: Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. CONCLUSIONS: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.

Scoring system for predicting the prognosis of elderly gastric cancer patients after endoscopic submucosal dissection.

OBJECTIVES: Comprehensive assessments of the long-term outcomes of endoscopic submucosal dissection (ESD) for early gastric cancer (EGC) in the elderly are unavailable. We aimed to create a scoring system to predict the long-term prognosis after ESD for EGC among patients aged ≥75 years. METHODS: We conducted retrospective studies of two cohorts: a single-center cohort (2006-2011) for developing the scoring system, and a multicenter cohort for validating the developed system (2012-2016). In the development cohort, factors related to death after ESD were identified using multivariable Cox regression analysis, and a predictive scoring system was developed. In the validation cohort, the scoring system was validated in 295 patients. RESULTS: In the development cohort, Charlson comorbidity index (CCI) ≥3 (hazard ratio [HR] 3.017), high psoas muscle index (PMI) (HR 2.206), and age ≥80 years (HR 1.978) were significantly related to overall survival after ESD. Therefore, high CCI, low PMI, and age ≥80 years were assigned 1 point each. The patients were categorized into low (≤1 point) and high (≥2 points) score groups based on their total scores. In the validation cohort, 184 and 111 patients were assigned to the low- and high-score groups, respectively. In comparisons based on Kaplan-Meier curves, the 5-year survival rate was 91.5% in the low-score group and 57.8% in the high-score group (log-rank test; P < 0.001). CONCLUSION: Our scoring system including high CCI, low PMI, and age ≥80 years could stratify the long-term prognosis of elderly patients aged ≥75 years after ESD for EGC.

Modified N score is helpful for identifying patients who need endoscopic intervention among those with black stools without hematemesis.

OBJECTIVES: Although black stools are one of the signs of upper gastrointestinal bleeding, not all patients without hematemesis need endoscopic intervention. There is no apparent indicator to select who needs treatment thus far. The aim of this study was to establish a novel score that predicts the need for endoscopic intervention in patients with black stools without hematemesis. METHODS: We retrospectively enrolled 721 consecutive patients with black stools without hematemesis who underwent emergency endoscopy from two facilities. In the development stage (from January 2016 to December 2018), risk factors that predict the need for endoscopic intervention were determined from the data of 422 patients by multivariate logistic regression analysis, and a novel scoring system, named the modified Nagoya University score (modified N score), was developed. In the validation stage (from January 2019 to September 2020), we evaluated the diagnostic value of the modified N score for 299 patients. RESULTS: Multivariate logistic regression analysis revealed four predictive factors for endoscopic intervention: syncope, the blood urea nitrogen (BUN) level, and the BUN/creatinine ratio as positive indicators and anticoagulant drug use as a negative indicator. In the validation stage, the area under the curve of the modified N score was 0.731, and the modified N score showed a sensitivity of 82.0% and a specificity of 58.8%. CONCLUSIONS: Our modified N score, which consists of only four factors, can identify patients who need endoscopic intervention among those with black stools without hematemesis.

Effect of weight change and lifestyle modifications on the development or remission of nonalcoholic fatty liver disease: sex-specific analysis.

The effects of changes in various lifestyle habits on nonalcoholic fatty liver disease (NAFLD) have not been well elucidated. We aimed to clarify how weight change and lifestyle modifications were associated with the development or remission of NAFLD. In this longitudinal cohort study, we reviewed the periodic health checkup data of 1,421 subjects with no causes of liver disease besides NAFLD who had received at least two health checkups between 2009 and 2018. The prevalence of NAFLD at baseline was 34.1% (484/1,421). During follow-up period (4.6 ± 2.8 years), 104 subjects developed NAFLD and 127 subjects demonstrated NAFLD remission. The frequency of NAFLD development or that of NAFLD remission significantly increased as the larger weight gain or weight loss was, respectively (both, p < 0.001). Approximately 40% of the subjects who maintained ≥ 1%/year weight loss achieved NAFLD remission. By multivariate analysis, quitting smoking were independently associated with NAFLD development (adjusted odds ratio [AOR], 2.86; 95% CI, 1.24-6.62). Subjects who quit smoking demonstrated large weight gain (≥1%/year) significantly more frequently than the other subjects (p < 0.001). In sex-specific analysis, starting to exercise was independently associated with NAFLD remission in men (AOR, 2.38; 95% CI, 1.25-4.53).

A case of mucinous cystic neoplasm of the pancreas misdiagnosed as a pancreatic pseudocyst at the initial exam and resected after a 2-year follow-up.

A 44-year-old woman was referred to our hospital because of a cystic lesion in the pancreatic body that was found by computed tomography (CT) as a result of a screening for impaired liver function after the patient presented with a high fever in 2011. Trans-abdominal ultrasonography (US) revealed a 33-mm unilocular cyst within the pancreatic body and a 5-mm hypoechoic mass in the pancreatic neck. Contrast-enhanced CT showed a slight enhancement around the cyst and a mild dilation of the main pancreatic duct, but neither septum nor nodule was detected inside. Contrast-enhanced endoscopic ultrasonography (CE-EUS) revealed a hyperechoic elevated lesion inside the cystic lesion without enhancement in the pancreatic body; CE-EUS also revealed a 5-mm homogeneous hypoechoic mass with a remarkable enhancement in the pancreatic neck with the use of Sonazoid(®) as a contrast medium. These lesions were diagnosed as a pancreatic pseudocyst and a neuroendocrine tumor (NET), respectively, and were followed up with periodic examinations. The cystic lesion showed contraction 6 months after the initial exam. However, US revealed an enlargement of the cystic lesion to 40 mm in diameter 2 years after the initial exam, and EUS showed irregular thickening of the wall with a cyst-in-cyst appearance. The diagnoses of a mucinous cystic neoplasm (MCN) and a concomitant small NET were made after a distal pancreatectomy. We herein report a rare case of MCN that showed various morphological changes over 2 years of observation.

[A case of type 2 autoimmune pancreatitis associated with Crohn disease].

A 45-year-old woman visited our hospital due to upper left quadrant pain and melena. Colonoscopy revealed longitudinal ulcers in the transverse colon. The endoscopic findings and pathological examination of a biopsy specimen led to diagnosis of Crohn disease, and mesalazine was administered. Although the colorectal lesions showed improvement with mesalazine, a blood test revealed elevation of biliary enzymes. Endoscopic retrograde cholangiopancreatography showed diffuse narrowing of the main pancreatic duct and smooth stricture of the distal bile duct. Steroid therapy improved the pancreatic lesion, which was diagnosed as type 2 autoimmune pancreatitis.

Importance of appropriate pharmaceutical management in pregnant women with ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) often occurs in women of childbearing age. Compared to Western countries, however, few studies have investigated the impact of UC on the progress of pregnancy in Asian populations. METHODS: We retrospectively examined 91 pregnancies in 64 patients with UC experienced at our hospital and related institutions from 1991 to 2011, focusing on the relationship between the progression of UC during pregnancy, progress of the pregnancy itself, and the treatment of UC. RESULTS: In 80 of 91 pregnancies the patient had already been diagnosed with UC at the time she became pregnant, of whom 31 (38.8%) experienced exacerbation during pregnancy. Regarding severity, moderate or severe active-stage disease during pregnancy was seen in 13.7% of those who had been in remission at the onset of pregnancy versus 58.6% of those who had been in the active stage at onset (OR 8.9: 95%CI 3.0~26.4; P<0.01). The incidence of miscarriage or abortion was 9.8% in pregnancies in which UC was in remission at onset versus 31% in those in which it was in the active stage at onset (OR 4.1: 95%CI 1.2~13.9; P=0.02). Among patients, 62.5% were receiving pharmaceutical treatment at onset of pregnancy. Exacerbation during pregnancy occurred in 26.5% of the group who continued to receive the same treatment during pregnancy versus 56.3% of those with a dose decrease or discontinuation after onset (OR 3.6: 95%CI 1.0~12.4; P=0.04). CONCLUSIONS: UC patients wishing to conceive should do so when in remission and continue appropriate pharmaceutical treatment during pregnancy.

New class of corticotropin-releasing factor (CRF) antagonists: small peptides having high binding affinity for CRF receptor.

The discovery of small and potent peptide antagonists of the corticotropin-releasing factor (CRF) receptor is described. Through the structure-activity relationship studies of 12-amino acid peptide corresponding to the C-terminal residues of astressin, we assumed that a particular surface of the alpha-helix was important for binding to the receptor. The small peptide containing d-Ala31 and cyclohexylalanine38 on that surface was as potent as astressin in binding to the CRF receptor and showed significant ACTH suppression when administered to rats.

4-(4-cycloalkyl/aryl-oxazol-5-yl)benzenesulfonamides as selective cyclooxygenase-2 inhibitors: enhancement of the selectivity by introduction of a fluorine atom and identification of a potent, highly selective, and orally active COX-2 inhibitor JTE-522(1).

A series of 4-(4-cycloalkyl/aryl-oxazol-5-yl)benzenesulfonamide derivatives were synthesized and evaluated for their abilities to inhibit cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) enzymes. In this series, substituent effects at the ortho position to the sulfonamide group on the phenyl ring were examined. Most substituents reduced or lost both COX-2 and COX-1 activities. In contrast, introduction of a fluorine atom preserved COX-2 potency and notably increased COX1/COX-2 selectivity. This work led to the identification of a potent, highly selective, and orally active COX-2 inhibitor JTE-522 [9d, 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide], which is currently in phase II clinical trials for the treatment of rheumatoid arthritis, osteoarthritis, and acute pain.

4-Aryl/cycloalkyl-5-phenyloxazole derivatives as selective COX-2 inhibitors.

A series of 4-aryl/cycloalkyl-5-phenyloxazole derivatives was synthesized and evaluated for their ability to inhibit cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1). These compounds were found to be potent and selective COX-2 inhibitors.