Cytological scoring for pancreatic specimens obtained by endoscopic ultrasound-guided fine needle aspiration.

OBJECTIVE: Cytological diagnosis of pancreatic specimens obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is often challenging because of the small sample size or well-differentiated adenocarcinoma with weak cytological atypia. Therefore, the sensitivity and specificity of cytological diagnosis for pancreatic cancer should be improved. Hence, we aimed to clarify the utility of cytological scoring to distinguish malignant from benign lesions for cytological diagnosis of pancreatic EUS-FNA specimens. METHODS: Seven reviewers, including four cytotechnologists and three medical doctors, evaluated 20 morphological indices in pancreatic specimens obtained by EUS-FNA (malignant, n = 111; benign, n = 31). Statistical analyses were performed using Fisher's exact test, logistic regression analysis, the area under the receiver operating characteristic curve, and Youden index. RESULTS: Among the 20 indices, there was a high incidence rate (>40%) of the following 13 indices in malignant cases: irregular structure, hyperchromatic nucleus, irregular cell polarity, unclear cell boundaries, nuclear membrane thickening, anisonucleosis, overlapping, irregular nuclei, high nuclear-cytoplasmic ratio, binding decline, the simultaneous appearance of malignant and benign cells, enlarged nucleoli, and background necrosis. When we diagnosed pancreatic specimens using these 13 cytological indices, the cut-off value of 8/9 showed the highest Youden index (0.950) as well as high sensitivity and specificity in distinguishing malignant from benign specimens (98% and 97%, respectively). CONCLUSION: Thirteen cytological indices showed high sensitivity and specificity in differentiating malignant and benign lesions using pancreatic EUS-FNA samples. All 13 indices were important for diagnosing malignancy in the pancreatic cytology smear of EUS-FNA. Further validation studies are required.

Emergency lower gastrointestinal endoscopy performed safely in a COVID-19 patient on extracorporeal membrane oxygenation (ECMO) with hemorrhagic shock.

Emergency endoscopy in coronavirus disease 2019 (COVID-19) patients should be avoided whenever possible to ensure the safety of medical staff; however, it may be unavoidable in some cases. We report a case of emergency lower gastrointestinal endoscopy performed with full personal protective equipment in a patient on extracorporeal membrane oxygenation with severe COVID-19 pneumonia admitted in a restricted area under negative pressure in the intensive care unit. To avoid the risk of fecal-oral transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the procedure, the patient's lower body was covered with a 2 m2 vinyl sheet with an aperture (diameter, approximately 2 cm). None of the medical staff involved exhibited any signs of SARS-CoV-2 infection after the procedure. Although patients with severe COVID-19 pneumonia on extracorporeal membrane oxygenation have a high risk of bleeding, we believe that emergency lower endoscopy can be safely performed in such patients by reducing exposure to dispersed feces and using full personal protective equipment.

Overexpression of carbohydrate sulfotransferase 15 in pancreatic cancer stroma is associated with worse prognosis.

Carbohydrate sulfotransferase 15 (CHST15) synthesizes matrix proteoglycan that regulates various pathogenic mediators and contributes to tissue remodeling and fibrosis during injury. CHST15 has been reported to promote tumor growth and invasion in various types of cancer. Our previous study reported the safety and efficacy of EUS-guided fine-needle injection (EUS-FNI) of STNM01, a double-stranded RNA oligonucleotide that specifically represses CHST15, for use in patients with pancreatic cancer. The present study aimed to determine the expression and clinicopathological characteristics of CHST15 in pancreatic cancer. Immunohistochemical staining was performed for CHST15 using pancreatic tissues from 64 patients (28 males and 36 females; age, 69.0±9.6 years) with pancreatic cancer who underwent surgery. For the evaluation of fibrosis, two categories were defined (mature and immature), based on the existence of collagen, myxoid stroma and fibroblasts, using hematoxylin and eosin specimens. The positive percentage of CHST15 was quantified, patients were divided into two groups according to high and low CHST15 expression in both the cancer and stroma tissues, and the association between CHST15 expression in cancer cells and the stroma was analyzed. Additionally, the present study analyzed the association between CHST15 expression and clinicopathological information, including overall and disease-free survival. The expression levels of CHST15 were detected in the cytoplasm of pancreatic cancer cells and fibroblasts in the cancer stroma. CHST15 expression in cancer cells was not identified to be associated with overall survival (P=0.52). However, patients with high CHST15 expression in the stroma exhibited worse overall survival compared with patients with low CHST15 expression (P=0.02). CHST15 expression in the stroma exhibited a positive association with that in cancer cells (P=0.01). High CHST15 expression in the stroma group was associated with a higher incidence of immature fibrosis (P=0.02) compared with mature fibrosis. CHST15 expression in cancer cells was associated with Union for International Cancer Control stage (P=0.02) and invasive front. Age and sex were not associated with CHST15 expression. The present study revealed that overexpression of CHST15 in stroma was associated with worse overall survival and immature fibrosis. Overexpression of CHST15 in cancer cells was associated with tumor stage. These results suggested that targeting therapy for CHST15 may be useful for stroma fibroblasts and cancer cells.

Successful traction-assisted endoscopic submucosal dissection using dental floss and a clip for a huge superficial nonampullary duodenal epithelial tumor with severe fibrosis (with video).

Recently, traction-assisted endoscopic submucosal dissection (ESD) using a clip and thread was reported as useful for treating lesions in the esophagus, stomach, and colorectum in terms of shortening the duration of the procedures and reducing the risk of intraoperative perforation. However, no traction method using the thread and clip for duodenal ESD as described in this article has been reported to date. We report a case in which traction-assisted ESD using dental floss and a clip was successfully performed on a huge superficial nonampullary duodenal epithelial tumor accompanied by severe fibrosis caused by preoperative biopsies. A 65-year-old woman had a 55-mm flat-elevated tumor in the second part of the duodenum. Severe fibrosis of the submucosal layer was expected due to repeated biopsies at the same site by the patient's previous endoscopist. We selected ESD for this lesion, and the initial incision was started from the side proximal to the lesion, but it was difficult to insert the scope under the submucosal layer directly beneath the biopsy scar. Therefore, traction with an endoclip and dental floss was performed to lift the lesion. Excellent traction allowed safe resection of the fibrotic part under accurate visual observation. Finally, the lesion was resected en bloc without adverse events. Traction-assisted ESD using dental floss and a clip is likely to be an effective adjunctive technique for quick, safe, and successful resection of lesions in the duodenum on which it is difficult to perform ordinary ESD and that have a high probability of intraoperative perforation and massive bleeding.

Abnormal immunolabelling of SMAD4 in cell block specimens to distinguish malignant and benign pancreatic cells.

BACKGROUND: Accurate diagnosis of malignant and benign pancreatic lesions can be challenging, especially with endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) samples that are small and/or degraded. In the present study, we determined how to best evaluate abnormal SMAD4 expression by immunohistochemical staining on cell block specimens from EUS-FNA samples. RESULTS: In surgically resected pancreas, when abnormal SMAD4 immunolabelling was evaluated as negative SMAD4 expression, the sensitivity was low (33%), but when it was evaluated as decreased SMAD4 expression, the sensitivity improved (53%). Specificity and positive predictive value were high for both evaluations. There were no false-positive cases. In cell block specimens, decreased SMAD4 expression showed 47% sensitivity and 72% specificity, while negative SMAD4 expression showed lower sensitivity (20%) and higher specificity (100%). Both evaluations in cell block specimens showed lower sensitivity and specificity compared to resected specimens. False-positive and -negative rates were higher for cell blocks than for resected specimens. CONCLUSIONS: Decreased SMAD4 immunolabelling provided improved sensitivity as compared to negative SMAD4 immunolabelling; therefore, it is important to compare SMAD4 expression in a sample to its expression in normal cells. Abnormal SMAD4 labelling showed low sensitivity and high specificity; therefore, SMAD4 staining using EUS-FNA samples might be helpful to detect malignancies that possess SMAD4 gene abnormalities.

Effects of EUS-guided intratumoral injection of oligonucleotide STNM01 on tumor growth, histology, and overall survival in patients with unresectable pancreatic cancer.

BACKGROUND AND AIMS: Carbohydrate sulfotransferase 15 (CHST15) promotes tumor growth and invasion and is considered to be an emergent therapeutic target for pancreatic cancer. The aim of this study was to evaluate the safety and efficacy of EUS-guided fine-needle injection (EUS-FNI) of STNM01, the double-stranded RNA oligonucleotide that specifically represses CHST15, for use in patients with pancreatic cancer. METHODS: Six patients with unresectable pancreatic cancer, treated at Tokyo Metropolitan Geriatric Hospital, were used in this open-labeled, investigator-initiated trial. A total of 16 mL STNM01 (250 nM) was injected into the tumor through EUS-FNI. The study's primary endpoint was safety, with a secondary endpoint of tumor response 4 weeks after the initial injection. Some patients received a series of infusions as extensions. The local expression of CHST15 and overall survival (OS) were also evaluated. RESULTS: There were no adverse events. The mean tumor diameter changed from 30.7 to 29.3 mm 4 weeks after injection. Four patients exhibited necrosis of tumor in biopsy specimens. CHST15 was highly expressed at baseline, with 2 patients showing large reductions of CHST15 at week 4. The mean OS of these 2 patients was 15 months, whereas it was 5.7 months for the other 4 patients. CONCLUSIONS: EUS-FNI of STNM01 in pancreatic cancer is safe and feasible. The CHST15 reduction could predict tumor progression and OS. Injections of STNM01 during the beginning of treatment may reduce CHST15 and warrants further investigation.

Leptin inhibits amyloid ß-protein degradation through decrease of neprilysin expression in primary cultured astrocytes.

Pathogenesis of Alzheimer's disease (AD) is characterized by accumulation of extracellular deposits of amyloid ß-protein (Aß) in the brain. The steady state level of Aß in the brain is determined by the balance between its production and removal; the latter occurring through egress across blood and CSF barriers as well as Aß degradation. The major Aß-degrading enzymes in the brain are neprilysin (NEP) and insulin-degrading enzyme (IDE), which may promote Aß deposition in patients with sporadic late-onset AD. Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin levels and the onset of AD. However, the mechanisms underlying the relationship remain uncertain. We investigated whether leptin is associated with Aß degradation by inducing NEP and IDE expression within primary cultured astrocytes. Leptin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner through the activation of extracellular signal-regulated kinase (ERK) in cultured rat astrocytes. Furthermore, leptin inhibited the degradation of exogenous Aß in primary cultured astrocytes. These results suggest that leptin suppresses Aß degradation by NEP through activation of ERK.

Development of a new free radical absorption capacity assay method for antioxidants: aroxyl radical absorption capacity (ARAC).

A new free radical absorption capacity assay method is proposed with use of an aroxyl radical (2,6-di-tert-butyl-4-(4'-methoxyphenyl)phenoxyl radical) and stopped-flow spectroscopy and is named the aroxyl radical absorption capacity (ARAC) assay method. The free radical absorption capacity (ARAC value) of each tocopherol was determined through measurement of the radical-scavenging rate constant in ethanol. The ARAC value could also be evaluated through measurement of the half-life of the aroxyl radical during the scavenging reaction. For the estimation of the free radical absorption capacity, the aroxyl radical was more suitable than the DPPH radical, galvinoxyl, and p-nitrophenyl nitronyl nitroxide. The ARAC value in tocopherols showed the same tendency as the free radical absorption capacities reported previously, and the tendency was independent of an oxygen radical participating in the scavenging reaction and of a medium surrounding the tocopherol and oxygen radical. The ARAC value can be directly connected to the free radical-scavenging rate constant, and the ARAC method has the advantage of treating a stable and isolable radical (aroxyl radical) in a user-friendly organic solvent (ethanol). The ARAC method was also successfully applied to a palm oil extract. Accordingly, the ARAC method would be useful in free radical absorption capacity assay of antioxidative reagents and foods.

Surgical treatment of simultaneous rupture of the anterior cruciate ligament and the patellar tendon.

Although the rupture of the anterior cruciate ligament (ACL) is a common sports injury, a simultaneous rupture of the patellar tendon (PT) is relatively rare. We experienced a case in which a patient simultaneously ruptured the ACL, the medial collateral ligament (MCL), and the PT while sliding during a baseball game. We sutured the PT and MCL during the acute stage, and 7 months later we conducted a double-bundle reconstruction of the ACL. To our knowledge, this is the first report of PT repair using only fiber wire thread, and two-phase double-bundle ACL reconstruction.

TRP channels and ASICs mediate mechanical hyperalgesia in models of inflammatory muscle pain and delayed onset muscle soreness.

The roles of ion channels in sensory neurons were examined in experimental models of muscle pain in the rat. Rats were injected with 50 microl of 4% carrageenan or subjected to an eccentric exercise (ECC) of the gastrocnemius muscle (GM). The Randall-Selitto and von Frey tests were performed on the calves to evaluate mechanical hyperalgesia of the muscle. The changes in expression of four genes and proteins of ion channels in dorsal root ganglia were examined using quantitative PCR and immunohistochemistry, respectively. Effects of antagonists to transient receptor potential (TRP) channels and acid sensing ion channels (ASICs) on the mechanical hyperalgesia induced by carrageenan injection or ECC were evaluated. The mechanical hyperalgesia was observed 6-24h after carrageenan injection and 1-3 days after ECC in the Randall-Selitto test. Infiltrations of the inflammatory cells in the GM were seen in carrageenan-injected animals but not in those subjected to ECC. Expressions of genes and proteins in sensory neurons showed no changes. Intramuscular injection of antagonists to TRPV1 showed an almost complete suppressive effect on ECC-induced muscle hyperalgesia but not a carrageenan-induced one. Antagonists to TRP channels and ASICs showed suppressive effects for both carrageenan- and ECC-induced muscle hyperalgesia. The carrageenan injection and ECC models are useful models of acute inflammatory pain and delayed onset muscle soreness (DOMS), respectively, and the time course and underlying etiology might be different. TRP channels and ASICs are closely related to the development of muscle mechanical hyperalgesia, and TRPV1 is involved in ECC-induced DOMS.

Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products.

Biological functions of globo-series glycosphingolipids are not well understood. In this study, murine cDNAs of two glycosyltransferases responsible for the synthesis of globo-series glycolipids and mRNA expression of those genes were analyzed. Distribution of their products was also analyzed. Murine cDNAs for Gb3/CD77 synthase and Gb4 synthase predicted that both of them are type II membrane proteins with 348 and 331 amino acids, respectively. In northern blotting, Gb3/CD77 synthase gene was mainly expressed in kidney and lung but also detected in many other tissues. Gb4 synthase was expressed in brain, heart, kidney, liver, skin, and testis. In the immunohistological analysis, Gb3/CD77 was mainly expressed in the proximal tubules as revealed with coincidental expression with angiotensin-converting enzyme (ACE). In spleen, it was detected in pre-B cells in the peripheral region of the white pulp, as suggested with coincidental expression with CD10. It was also expressed on the endothelia of the alveolar capillaries in lung and on the sebaceous ducts aside of the hair follicles. Gb4 was also detected mainly on the proximal tubules in kidney and on the endothelia of the alveolar capillaries in lung as Gb3/CD77. But it was also detected on the epithelium of the bronchus, seminiferous tubules and tails of spermatozoa in testis, blood vessels of choroids plexus and endothelial cells in brain, and central and hepatoportal veins in liver. The expression patterns of two genes and their products almost corresponded with some exception. The results would provide essential information for the functional studies of globo-series glycolipids.

Crosslinking of CD44 on human osteoblastic cells upregulates ICAM-1 and VCAM-1.

Cell adhesion of osteoblasts and osteoclastic precursors of hematopoietic origin is a prerequisite for osteoclast maturation. We have investigated the relevance of osteoblast-matrix binding and regulation of adhesion molecules to this process. Human osteoblastic cells highly expressed CD44, a major receptor for hyaluronan present in the surrounding bone matrix. Crosslinking of CD44 on osteoblastic cells with specific antibodies augmented the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. Hyaluronan, the major ligand of CD44, also up-regulated ICAM-1 expression. Stimulation of CD44 on osteoblastic cells amplified their adhesion to monocytic cells through ICAM-1 and VCAM-1. These results suggest that such crosstalk among distinct adhesion molecules may be relevant to bone metabolism, including osteoclastogenesis.