Mutated GM-CSF-based CAR-T cells targeting CD116/CD131 complexes exhibit enhanced anti-tumor effects against acute myeloid leukaemia.

OBJECTIVES: As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)-T cells for AML are more challenging than those targeting CD19 in B-cell malignancies. We recently developed piggyBac-modified ligand-based CAR-T cells that target CD116/CD131 complexes, also known as the GM-CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. METHODS: To further improve the efficacy of the original GMR CAR-T cells, we have developed novel GMR CAR vectors incorporating a mutated GM-CSF for the antigen-binding domain and G4S spacer. All GMR CAR-T cells were generated using a piggyBac-based gene transfer system. The anti-tumor effect of GMR CAR-T cells was tested in mouse AML xenograft models. RESULTS: Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. Furthermore, GMR CAR-T cells incorporating a G4S spacer significantly improved long-term in vitro and in vivo anti-tumor effects. By employing a mutated GM-CSF at residue 21 (E21K), the anti-tumor effects of GMR CAR-T cells were also improved especially in long-term in vitro settings. Although GMR CAR-T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal. CONCLUSIONS: GMR CAR-T cell therapy represents a promising strategy for CD116+ R/R AML.

Repeated Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma.

PURPOSE: In clinical practice, whether cirrhotic livers in patients with hepatocellular carcinoma (HCC) can withstand repeated stereotactic body radiation therapy (SBRT) remains unclear. This study aimed to evaluate the outcomes and toxicities in these patients. METHODS AND MATERIALS: This retrospective study included patients with HCC who were treated with SBRT at least twice between January 2012 and June 2019. Local control and overall survival rates were calculated. Liver function before and after irradiation was evaluated using the Child-Pugh score and modified albumin-bilirubin grade. All toxicities were assessed using the Common Terminology Criteria for Adverse Events (version 4.0). RESULTS: Fifty-two patients underwent 136 courses (148 lesions) of SBRT, which was mostly performed for out-of-field tumors but 3 in-field recurrences. The median follow-up duration from the first SBRT was 52.6 months (range, 15.7-89.3 months). The median gross tumor volume was 4.6 cm3 (range, 0.8-55.2 cm3) at the second SBRT. The 3-year local control rate was 94.5% (95% confidence interval, 88.0%-97.5%). The 3-year overall survival rate after the second course was 62.8% (95% confidence interval, 45.1%-76.2%). Although the Child-Pugh score did not deteriorate after the second course, deterioration of the modified albumin-bilirubin grade at 6, 12, and 24 months was statistically significant compared with that before the second course. One patient (1.9%) experienced grade 3 hypoalbuminemia and 2 patients (3.8%) had grade 3 thrombocytopenia 6 months after the second course. Mild fatigue and nausea were reported in 9 (17.3%) and 6 (11.5%) patients, respectively. One instance of grade 5 toxicity was observed. Two patients (1.5%) had grade 2 gastric ulcers. No other grade ≥3 gastrointestinal toxicities occurred. CONCLUSIONS: Repeated SBRT is feasible and produces minimal toxicity in patients with HCC and Child-Pugh scores of ≤7 and a low normal liver dose.

Remogliflozin Etabonate Improves Fatty Liver Disease in Diet-Induced Obese Male Mice.

BACKGROUND: Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH) are serious conditions and are being diagnosed at an increased rate. The etiology of these hepatic disorders is not clear but involves insulin resistance and oxidative stress. Remogliflozin etabonate (Remo) is an inhibitor of the sodium glucose-dependent renal transporter 2 (SGLT2), and improves insulin sensitivity in type 2 diabetics. In the current study, we examined the effects of Remo in a diet-induced obese mouse model of NAFLD. METHODS: After 11-weeks on High-Fat-Diet 32 (HFD32), C57BL/6J mice were obese and displayed characteristics consistent with NAFLD. Cohorts of obese animals were continued on HFD32 for an additional 4-week treatment period with or without Remo. RESULTS: Treatment with Remo for 4 weeks markedly lowered both plasma alanine aminotransferase (76%) and aspartate aminotransferase (48%), and reduced both liver weight and hepatic triglyceride content by 42% and 40%, respectively. Remo also reduced hepatic mRNA content for tumor necrosis factor (TNF)-α (69%), and monocyte chemoattractant protein (MCP)-1 (69%). The diet-induced increase in thiobarbituric acid-reactive substances, a marker of oxidative stress, was reduced following treatment with Remo, as measured in both liver homogenates (22%) and serum (37%). Finally, the oxygen radical absorbance capacity (ORAC) in three different SGLT2 inhibitors was determined: remogliflozin, canagliflozin and dapagliflozin. Only remogliflozin had any significant ORAC activity. CONCLUSIONS: Remo significantly improved markers associated with NAFLD in this animal model, and may be an effective compound for the treatment of NASH and NAFLD due to its insulin-sensitizing and antioxidant properties.

Arrival time parametric imaging of the hemodynamic balance changes between the hepatic artery and the portal vein during deep inspiration, using Sonazoid-enhanced ultrasonography: A case of Budd-Chiari syndrome.

This case report concerns a 40-year-old male who had previously been treated for an esophageal varix rupture, at the age of 30 years. The medical examination at that time revealed occlusion of the inferior vena cava in the proximity of the liver, leading to the diagnosis of the patient with Budd-Chiari syndrome. The progress of the patient was therefore monitored in an outpatient clinic. The patient had no history of drinking or smoking, but had suffered an epileptic seizure in 2004. The patient's family history revealed nothing of note. In February 2012, color Doppler ultrasonography (US) revealed a change in the blood flow in the right portal vein branch, from hepatopetal to hepatofugal, during deep inspiration. Arrival time parametric imaging (At-PI), using Sonazoid-enhanced US, was subsequently performed to examine the deep respiration-induced changes observed in the hepatic parenchymal perfusion. US images captured during deep inspiration demonstrated hepatic parenchymal perfusion predominantly in red, indicating that the major blood supply was the hepatic artery. During deep expiration, the portal venous blood flow remained hepatopetal, and hepatic parenchymal perfusion was displayed predominantly in yellow, indicating that the portal vein was the major source of the blood flow. The original diagnostic imaging results were reproduced one month subsequently by an identical procedure. At-PI enabled an investigation into the changes that were induced in the hepatic parenchymal perfusion by a compensatory mechanism involving the hepatic artery. These changes occurred in response to a reduction in the portal venous blood flow, as is observed in the arterialization of hepatic blood flow that is correlated with the progression of chronic hepatitis C. It has been established that the peribiliary capillary plexus is important in the regulation of hepatic arterial blood flow. However, this case demonstrated that the peribiliary capillary plexus also regulates acute changes in portal venous blood flow, in addition to the chronic reduction in blood flow that is observed in patients with chronic hepatitis C.

Heterotopic Pancreas of the Jejunum Incidentally Detected by Preoperative Abdominal CT: Report of Two Cases and Review of the Literature.

Heterotopic pancreas (HP) is typically an asymptomatic malformation that can present anywhere along the gastrointestinal tract. It is often detected incidentally on surgery for other diseases or autopsy. We encountered 2 patients with jejunal HP incidentally detected by computed tomography (CT) performed for close evaluation of other diseases. In a 57-year-old woman diagnosed with reactive lymphoid hyperplasia on the dorsal portion of the pancreas head, CT detected a 15 mm oval-shaped submucosal lesion at the jejunum. In an 87-year-old woman diagnosed with type 2 adenocarcinoma occupying the sigmoid colon, CT detected a round-shaped submucosal tumor 15 mm in diameter in the jejunum. Both cases were histologically diagnosed as type 1 HP according to the classification by Heinrich. Contrast-enhanced CT revealed that the CT analyses of HP and pancreatic parenchyma were nearly identical in the arterial phase, but in the equilibrium phase, contrast enhancement persisted longer in HP than in the pancreatic parenchyma. There has been no report of asymptomatic jejunal HP preoperatively diagnosed by CT. These cases are presented with a review of the literature, particularly focusing on CT findings.

Clinical evaluation of hemorrhagic gastroduodenal ulcer in the elderly: is Helicobacter pylori infection a risk factor for hemorrhage?

AIM: In this study, the aim was to determine the demographic characteristics of elderly patients with gastroduodenal ulcer who had undergone endoscopic hemostasis by comparing them with younger patients. METHODS: A total of 353 patients with Forrest class I-IIa hemorrhagic gastroduodenal ulcer who underwent endoscopic hemostasis at our hospital between December 2004 and May 2010 were divided into two groups: one for those 75 years or older (old-old group; n = 71; age ≥75 years) and one for those younger than 75 years (younger group; n = 282; age <75 years). Then, their demographic characteristics were compared. RESULTS: There were significantly more female patients, patients with underlying chronic renal failure and patients using non-steroidal anti-inflammatory drugs in the old-old group than in the younger group. In addition, the prevalence of open-type atrophy in the background gastric mucosa was significantly higher in the old-old group. Although more than half the patients in each group were infected with Helicobacter pylori, the prevalence was significantly higher in the younger group. Of the patients who underwent endoscopic hemostasis only once, those in the old-old group constituted a significantly higher medical cost than those in the younger group. Comparison of deaths between the two groups revealed that the old-old patients were more likely to develop severe complications associated with hematemesis, such as aspiration pneumonia. CONCLUSIONS: The observed lower prevalence of Helicobacter pylori infection among the elderly patients compared to the younger patients with hemorrhagic gastroduodenal ulcer suggests that other factors, such as non-steroidal anti-inflammatory drugs use and chronic renal failure, predispose the elderly to hemorrhagic ulcer.

Short-term effects and early complications of balloon-occluded retrograde transvenous obliteration for gastric varices.

The short-term effects of balloon-occluded retrograde transvenous obliteration (BRTO) to treat gastric varices were evaluated by using computed tomography (CT) and gastroscopy (GF). The subjects were 77 patients who underwent BRTO to treat gastric varices. The short-term effects of BRTO were investigated with regard to ascites, pleural effusion, venous thrombus, and esophageal varices by comparing the findings of CT and GF performed within one month before and after BRTO. The mean duration of followup was 960.1 days. Ascites and pleural effusion were exacerbated after BRTO in 26 (33.8%) and 31 (40.3%), respectively. A significant difference in ascites exacerbation was noted in patients with hypoalbuminemia and a high Child-Pugh score, and a significant difference in exacerbation of pleural effusion was noted in patients with hypoalbuminemia. Venous thrombus was noted in 7 patients (9.1%). Esophageal varices were exacerbated in 14 (21.2%) of the 66 patients. The 2-year survival rate was 720 days, and significant differences were noted in the Child-Pugh classification and the concomitance of hepatocellular carcinoma (HCC) on multivariate analysis of prognosis-related factors. Conclusion. The frequencies of exacerbation of ascites, pleural effusion, and esophageal varices after BRTO were high but these may not be related to survival.

Gastrointestinal stromal tumor mimicking arteriovenous malformation of the jejunum.

There have been case reports of small intestinal gastrointestinal stromal tumors (GISTs) complicated with arteriovenous malformation (AVM) and angiodysplasia and exhibiting intense tumor staining. Herein we report a GIST of the small intestine that showed tumor staining and early venous return on imaging studies, and so the patient was suspected to have AVM. A 62-year-old male presented with intermittent pain in the left abdominal region. Contrast-enhanced computed tomography revealed a 15-mm-long spindle-shaped mass showing intense tumor staining and early venous return through the jejunal vein. In the arterial phase, the attenuation value of the tumor was 250 Hounsfield units. Color Doppler ultrasonography simultaneously delineated vessels extending from the serosal side and turbulent signals showing a mosaic pattern in the tumor. On angiography, intense staining was observed in the peripheral part of the second branch of the jejunal artery. Although these findings suggested AVM, the tumor was diagnosed as a GIST based on pathological examination of the resected specimens. In this case, no AVM or change in vascular density was noted despite the careful examination of pathological specimens, and the cause of the tumor staining remained unknown.

Endoscopic snare excision of a major duodenal papillary tumor.

BACKGROUND/AIMS: Tumors of the duodenal papilla include hyperplasia, adenoma, carcinoma in adenoma, and carcinoma. Since the duodenal papilla has special anatomical characteristics and treatment involves major intervention, a correct preoperative diagnosis is essential for successful treatment. METHODS: In patients with adenoma or early carcinoma of the papilla, endoscopic snare excision is indicated for complete removal of the tumor. Postoperative pancreatitis and cholangitis are major complications of endoscopic techniques, and we describe here in detail our procedure aiming to reduce the incidence of such complications. RESULTS: Endoscopic snare excision of a tumor of the major duodenal papilla was carried out in 36 patients. Bleeding after endoscopic excision occurred in 6 cases (17%), postoperative pancreatitis in 11 cases (30%), and postoperative cholecystitis in 1 case (3%). All patients recovered from the complications within 1 week. CONCLUSION: Our results suggest that the procedure for endoscopic snare excision used to resect major papillary tumors is safe and helps to prevent serious complications.

Carotid atherosclerosis mediated by visceral adiposity and adipocytokines in type 2 diabetic subjects.

The aim of this study was to verify the possible association of visceral fat accumulation with carotid atherosclerosis in order to identify the practical and feasible determinants for each parameter of atherosclerosis in type 2 diabetic subjects. The subjects were 151 diabetic (DM) and age-matched 83 nondiabetic subjects (C), without atherosclerotic disease. Visceral fat area (VFA) on a CT scan at the umbilicus level was measured. Ambulatory 24-h blood pressure (BP) was recorded. Stiffness index beta, intima-media thickness (IMT) and plaque formation of carotid arteries were measured by ultrasonography. Insulin sensitivity was estimated by homeostasis model assessment (HOMA). Serum levels of adiponectin and tumor necrosis factor (TNF)-alpha were determined. Male gender, HOMA, serum non-HDL-Cholesterol (Chol) and TNF-alpha/adiponectin ratio were higher, and VFA was larger in DM than in C. The IMT, stiffness index beta and plaque formation in DM were more pronounced than in C, even after adjusting for age, sex and 24-h systolic BP (sBP). VFA was positively correlated with TNF-alpha/adiponectin ratio and serum non-HDL-Chol in DM. Furthermore, multiple regression analysis revealed that, in DM, serum non-HDL-Chol was associated with IMT, VFA probably via an increase in TNF-alpha/adiponectin ratio was associated with stiffness index beta, and 24-h sBP, HOMA and VFA were associated with plaque formation independently of age and sex, respectively, although any association was not observed in C. Thus, we conclude that visceral fat-associated alterations in adipokines may be mediating the development and progression of atherosclerosis in type 2 diabetic subjects, compared with nondiabetic subjects.

Identification of tumor promotion marker genes for predicting tumor promoting potential of chemicals in BALB/c 3T3 cells.

Tumor promoters can cause development of tumors in initiated cells and the majority of them are non-genotoxic carcinogens. The detection of tumor promoters is important for the prevention of cancer. The in vitro two-stage transformation assay, using BALB/c 3T3 cells, is a useful system, and benefits from a convenient protocol and high predictability of mammalian carcinogenicity. But these assays are time-consuming and often require expertise for microscopic observation. To construct an in vitro tumor promoting activity test system, we performed large-scale gene expression analyses, using DNA microarrays, of BALB/c 3T3 cells following treatment with nine chemicals that are known to induce tumor promotion: TPA, zinc chloride, sodium orthovanadate, okadaic acid, insulin, lithocolic acid, phenobarbital sodium, sodium saccharide, sodium arsenite. As a result of DNA microarray and real time PCR analyses, 22 marker genes were identified. These consisted of genes related to cell cycle, regulation of transcription, anti-apoptosis, and positive regulation of cell proliferation. There was a correlation between these 22 marker genes and the cell transformation assay results in BALB/c 3T3 cells. These results suggest that this tumor promoting activity test system, based on 22 marker genes, can become a valuable tool for screening potential tumor promoters.

Insulin responses to selective arterial calcium infusion under hyperinsulinemic euglycemic glucose clamps: case studies in adult nesidioblastosis and childhood insulinoma.

Selective arterial calcium stimulation and hepatic venous sampling (ASVS) for insulin secretion is used as a diagnostic procedure in patients with insulinomas or adult nesidioblastosis. In some of those patients, severe hypoglycemia requiring urgent glucose administration occurs during the procedure. Such glucose administration, however, may affect the results and damage the validity of the test. We report two cases of hyperinsulinemic hypoglycemia, in which ASVS tests were successfully performed under hyperinsulinemic euglycemic glucose clamps. A 40-year-old male with nesidioblastosis developed continual severe hypoglycemia several years after a Billroth II-Braun gastrectomy, and continuous glucose infusion could not be stopped even during ASVS tests. A 9-year-old girl with an insulinoma that showed atypical hypovascularity on imaging examinations had ASVS tests under a glucose clamp for safety. Hyperinsulinemic (approximately 100 microU/ml) euglycemic (approximately 90 mg/dl) clamps were achieved by an artificial endocrine pancreas. The insulin analogue lispro was utilized for clamps and endogenous insulin was measured with an assay that does not cross-react with the analogue. Diagnostically significant responses (more than twofold) of insulin secretion were observed under hyperinsulinemic clamps in both cases. The use of the hyperinsulinemic glucose clamp technique during the ASVS test should be considered for maintaining the safety of some hypoglycemic patients.

Effects of bezafibrate, PPAR pan-agonist, and GW501516, PPARdelta agonist, on development of steatohepatitis in mice fed a methionine- and choline-deficient diet.

We evaluated the effects of bezafibrate, a peroxisome proliferator-activated receptor (PPAR) pan-agonist, and GW501516, a PPARdelta agonist, on mice fed a methionine- and choline-deficient (MCD) diet, a model of non-alcholic steatohepatitis (NASH), to investigate (a) the efficacy of bezafibrate against non-alcholic steatohepatitis and (b) the relation between non-alcholic steatohepatitis and the functional role of PPARdelta. Bezafibrate (50 or 100 mg/kg/day) and GW501516 (10 mg/kg/day) were administered by gavage once a day for 5 weeks. Hepatic lipid contents, plasma triglyceride, high density lipoprotein (HDL)-cholesterol and alanine aminotransferase (ALT) concentrations were evaluated, as were histopathological changes in the liver and hepatic mRNA expression levels. Bezafibrate and GW501516 inhibited the MCD-diet-induced elevations of hepatic triglyceride and thiobarbituric acid-reactants contents and the histopathological increases in fatty droplets within hepatocytes, liver inflammation and number of activated hepatic stellate cells. In this model, bezafibrate and GW501516 increased the levels of hepatic mRNAs associated with fatty acid beta-oxidation [acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-1 (CPT-1), liver-fatty acid binding protein (L-FABP) and peroxisomal ketothiolase], and reduced the levels of those associated with inflammatory cytokines or chemokine [transforming growth factor (TGF)-beta1, interleukin (IL)-6, IL-1beta, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) alpha and nuclear factor (NF)-kappaB1]. In addition, bezafibrate characteristically reduced the elevation in the level of plasma ALT, but enhanced that in plasma adiponectin and increased the mRNA expression levels of its receptors (adiponectin receptors 1 and 2). These results suggest that (a) bezafibrate (especially) and GW501516 might improve hepatic steatosis via an improvement in fatty acid beta-oxidation and a direct prevention of inflammation, (b) treatment with a PPARdelta agonist might improve non-alcholic steatohepatitis, (c) bezafibrate may improve non-alcholic steatohepatitis via activation not only of PPARalpha but also of PPARdelta, because bezafibrate is a PPAR pan-agonist.

Receptor gene expression of glucagon-like peptide-1, but not glucose-dependent insulinotropic polypeptide, in rat nodose ganglion cells.

We previously reported that afferent signals of the rat hepatic vagus increased upon intraportal appearance of insulinotropic hormone glucagon-like peptide-1(7-36) amide (GLP-1), but not glucose-dependent insulinotropic polypeptide (GIP). To obtain molecular evidence for the vagal chemoreception of GLP-1, the concept derived from those electrophysiological observations, receptor gene expressions of GLP-1 and GIP in the rat nodose ganglion were examined by means of reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and Northern blot analysis. Gene expression of the GLP-1 receptor was clearly detected by both RT-PCR and Northern blot analysis. In situ hybridization study confirmed that the expression occurs in neuronal cells of the ganglion. As to the GIP receptor, RT-PCR amplified the gene transcript faintly though Northern blot analysis failed to detect any messages. However, semi-quantitative RT-PCR revealed that the ratio of the gene expression level of the GIP receptor to that of the GLP-1 receptor was less than 1:250, indicating that receptor gene expression of GIP is practically negligible in the ganglion. Additionally, an equal level of GLP-1 receptor gene expressions between left- and right-side ganglia was evidenced by semi-quantitative RT-PCR, implying possible extrahepatic occurrence of vagal GLP-1 reception in addition to the reception through the hepatic vagus (originating from the left-side ganglion). The present results offer, for the first time, the molecular basis for the vagal chemoreception of GLP-1 via its specific receptor.

Testosterone inhibits tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in human aortic endothelial cells.

We investigated the effect of testosterone (T) on tumor necrosis factor-alpha (TNF-alpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells. Incubation of these cells with T resulted in a dose-dependent reduction in the expression, with this reduction completely abolished by a selective androgen receptor blocker. Electrophoretic mobility shift assay demonstrated that T inhibited TNF-alpha-induced activation of the transcriptional nuclear factor-kappaB, which is critical for the inducible expression of VCAM-1, probably through the suppression of the nuclear translocation. Our results may suggest an inhibitory effect of T on atherogenesis, providing a novel insight into the consideration of the pathogenesis of atherosclerosis.

Thrombospondin expression in aldosterone-producing adenomas.

Thrombospondin (TSP) 1 and 2 are extracellular matrix proteins that appear to play a role in cell adhesion and cell migration. It has been demonstrated that the pattern of TSP expression is shifted from TSP1 to TSP2 under adrenocorticotrophic hormone treatment in bovine adrenocortical cells. We investigated the expression in human adrenal tissues by Northern blot analysis and correlated these data with the expression of the adrenocorticotrophic hormone-receptor (ACTH-R). All adrenal tissues (control adrenals, nonfunctional adenomas and ACTH-dependent aldosterone-producing adenomas (APA)) expressed both TSP1 and TSP2 mRNAs. Compared to control adrenals (TSP1 and TSP2 expression = 100 +/- 12%, respectively), TSP1 expression was negatively (51 +/- 10%, p < 0.01) and TSP2 expression was positively (289 +/- 36%, p < 0.01) regulated in APA. No significant differences in TSP1 and TSP2 expressions were found between control adrenals and nonfunctional adenomas. In APA, TSP1 (r = -0.86, p<0.01) and TSP2 (r = 0.88, p < 0.01) expressions correlated closely with the expression of ACTH-R. These results suggest that ACTH activity plays an important role in regulating the expression of TSPs in human adrenal tissues. We speculate that the shift of expression observed in APA may be associated with the phenotype of the tumors.