TCF1highPD-1+Ly108+CD8+ T Cells Are Associated with Graft Preservation in Sensitized Mice Treated with Non-Fc Receptor-Binding CD3 Antibodies.

The non-Fc-binding anti-CD3 Ab [anti-CD3F(ab')2] can induce graft acceptance depending on the therapeutic window in a rodent heart transplant model. The delayed protocol allows for early graft infiltration of lymphocytes, which may behave in an inhibitory manner. We investigated the most effective protocol for anti-CD3F(ab')2 in sensitized conditions to confirm the evidence for clinical application. C57BL/6 mice were sensitized with BALB/c tail skin grafts and transplanted with BALB/c heart grafts at 8-12 wk after sensitization. Fifty micrograms of anti-CD3F(ab')2 was administered daily for 5 consecutive days on days 1-5 (day 1 protocol) or days 3-7 (delayed protocol). In nonsensitized mice, the delayed protocol significantly prolonged graft survival after transplantation from BALB/c to naive B6 (median survival time [MST], >100 d). In contrast, the delayed protocol was unable to prevent graft rejection in sensitized mice (MST, 5 d). A significantly increased percentage of granzyme B+ CD8+ T cells was observed in the graft on day 3 posttransplantation in sensitized conditions. Further, the day 1 protocol significantly prolonged graft survival (MST, 18 d), even in sensitized conditions. Day 1 treatment significantly increased the percentage of Foxp3+CD25+CD4+ T cells and phenotypically changed CD8+ T cells in the graft (i.e., caused a significant increase in the proportion of Ly108+TCF1highPD-1+CD8+ T cells). In conclusion, different timings of delayed anti-CD3F(ab')2 treatment promoted allograft preservation in association with phenotypic changes in CD4+ and CD8+ T cells in the graft under sensitized conditions.

Functional roles of graft-infiltrating lymphocytes during early-phase post-transplantation in mouse cardiac transplantation models.

Immunological behavior of graft-infiltrating lymphocytes (GILs) determines the graft fate (i.e., rejection or acceptance). Nevertheless, the functional alloreactivity and the phenotype of GILs at various times during the early post-transplantation phase have not been fully elucidated. We examined the immunological activities of early-phase GILs using a murine model of cardiac transplantation. GILs from 120-h allografts, but not 72-h allografts, showed robust activation and produced proinflammatory cytokines. In particular, a significant increase in CD69+ T-bet+ Nur77+ T cells was detected in 120-h allografts. Furthermore, isolated GILs were used to reconstitute BALB/c Rag2-/- γc-/- (BRG) mice. BRG mice reconstituted with 120-h GILs displayed donor-specific immune reactivity and rejected donor strain cardiac allografts; conversely, 72-h GILs exhibited weak anti-donor reactivity and did not reject allografts. These findings were confirmed by re-transplantation of cardiac allografts into BRG mice at 72-h post-transplantation. Re-transplanted allografts continued to function for >100 days, despite the presence of CD3+ GILs. In conclusion, the immunological behavior of GILs considerably differs over time during the early post-transplantation phase. A better understanding of the functional role of early-phase GILs may clarify the fate determination process in the graft-site microenvironment.

Best Procedure for the Management of Common Bile Duct Stones via the Papilla: Literature Review and Analysis of Procedural Efficacy and Safety.

BACKGROUND: Endoscopic management of common bile duct stones (CBDS) is standard; however, various techniques are performed via the papilla, and the best procedure in terms of both efficacy and safety has not been determined. METHODS: Endoscopic procedures were classified into five categories according to endoscopic sphincterotomy (EST) and balloon dilation (BD): (1) EST, (2) endoscopic papillary BD (≤10 mm) (EPBD), (3) EST followed by BD (≤10 mm) (ESBD), (4) endoscopic papillary large BD (≥12 mm) (EPLBD), and (5) EST followed by large BD (≥12 mm) (ESLBD). We performed a literature review of prospective and retrospective studies to compare efficacy and adverse events (AEs). Each procedure was associated with different efficacy and AE profiles. RESULTS: In total, 19 prospective and seven retrospective studies with a total of 3930 patients were included in this study. For EST, the complete stone removal rate at the first session, rate of mechanical lithotripsy (ML), and rate of overall AEs in EST were superior to EPBD, but a higher rate of bleeding was found for EST. Based on one retrospective study, complete stone removal rate at the first session, rate of ML, and rate of overall AEs were superior for ESBD vs. EST, and the rate of bleeding for the former was also lower. Complete stone removal rate at the first session and rate of ML for ESLBD were superior to those for EST, with no significant difference in rate of AEs. For EST vs. EPLBD, complete stone removal rate at the first session and rate of ML were superior for the latter. For EPLBD vs. ESLBD, the efficacy and safety were similar. CONCLUSIONS: ESBD is considered the best procedure for the management of small CBDS, but strong evidence is lacking. For large CBDS, both ESLBD and EPLBD are similar.

RACK1 regulates centriole duplication through promoting the activation of polo-like kinase 1 by Aurora A.

Breast cancer gene 1 (BRCA1) contributes to the regulation of centrosome number. We previously identified receptor for activated C kinase 1 (RACK1) as a BRCA1-interacting partner. RACK1, a scaffold protein that interacts with multiple proteins through its seven WD40 domains, directly binds to BRCA1 and localizes to centrosomes. RACK1 knockdown suppresses centriole duplication, whereas RACK1 overexpression causes centriole overduplication in a subset of mammary gland-derived cells. In this study, we showed that RACK1 binds directly to polo-like kinase 1 (PLK1) and Aurora A, and promotes the Aurora A-PLK1 interaction. RACK1 knockdown decreased phosphorylated PLK1 (p-PLK1) levels and the centrosomal localization of Aurora A and p-PLK1 in S phase, whereas RACK1 overexpression increased p-PLK1 level and the centrosomal localization of Aurora A and p-PLK1 in interphase, resulting in an increase of cells with abnormal centriole disengagement. Overexpression of cancer-derived RACK1 variants failed to enhance the Aurora A-PLK1 interaction, PLK1 phosphorylation and the centrosomal localization of p-PLK1. These results suggest that RACK1 functions as a scaffold protein that promotes the activation of PLK1 by Aurora A in order to promote centriole duplication.This article has an associated First Person interview with the first author of the paper.

Evaluation of the safety and efficacy of minimal endoscopic sphincterotomy followed by papillary balloon dilation for the removal of common bile duct stones.

BACKGROUND/AIM: A sufficiently open papilla is needed to remove common bile duct stones (CBDS) but endoscopic sphincterotomy (EST) requires a high level of skill and is difficult with endoscopic papillary balloon dilation (EPBD). The main adverse event of EST is bleeding and perforation and that of EPBD is post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis. To reduce these adverse events we employed minimal EST followed by papillary dilation (ESBD), and retrospectively evaluated its efficacy and safety compared with EST. PATIENTS AND METHODS: CBDS patients who underwent EST (n = 114) or ESBD (n = 321) at Juntendo University Hospital from January 2009 to December 2018 were consecutively enrolled, retrospectively. The exclusion criteria were large-balloon dilation (≥ 12 mm), large CBDS (>12 mm), and previous EST/EPBD. We compared the overall stone removal rate, incidence of adverse event, procedure time, number of ERCP procedures, and rate of mechanical lithotripsy (ML) between the two groups. RESULTS: Complete stone removal was successful in both ESBD and EST group. However, the rate of multiple ERCP sessions was significantly lower (35.1% vs. 12.8%, P < 0.001), procedure time was shorter (31.6 vs. 25.8 min, P = 0.01), and rate of ML was lower (16.7% vs. 7.8%, P = 0.01) in ESBD group. Bleeding was significantly more frequent in the EST group (9.6% vs. 1.2%, P < 0.001), particularly acute bleeding (7.9% vs. 0.9%, P < 0.001). CONCLUSIONS: ESBD is more efficient and safer in the management of CBD stones than EST. A prospective randomized study comparing ESBD with EST is needed to establish this combination technique.

Plasma concentration and efficacy of tolvaptan in cirrhotic patients with refractory ascites.

To investigate the relationship between the exposure and efficacy of tolvaptan, we measured pharmacokinetics of total drug at 7 days after repeated doses of 3.75 mg/day tolvaptan in 16 patients with hepatic edema. Nine patients (56.3%) were responders, which were defined as those with body weight reduction of >1.5 kg/week. Serum albumin levels were significantly lower in responders than in non-responders (P = 0.031). However, the pharmacokinetics varied greatly among individuals and was not relevant to the clinical response.

Efficacy and Safety of Deep Sedation in Percutaneous Radiofrequency Ablation for Hepatocellular Carcinoma.

INTRODUCTION: Radiofrequency ablation (RFA) has been accepted as safe and effective for treating early-stage hepatocellular carcinoma (HCC). However, it often causes severe pain. Therefore, in this study, we performed RFA under deep sedation and investigated its efficacy and safety. METHODS: We conducted a retrospective study including 511 HCC patients who received approximately 886 RFA treatments between December 2014 and November 2016 at our institution. Respiratory depression was defined as oxygen saturation of below 90%; and severe body movement was defined as movement caused by pain, which was managed by lowering the power of the generator. Factors associated with respiratory depression and severe body movement were examined via univariate and multivariate regression analyses. RESULTS: Respiratory depression occurred in 15.3% of the patients and severe body movement in 26.5% of the patients. In the multivariate analysis, BMI (≥ 25 kg/m2, odds ratio [OR] = 1.75, P = 0.035) and longer ablation (≥ 10 min, OR = 2.59, P = 0.002) were significant respiratory depression-related factors. Male sex (OR = 2.02, P = 0.005), Child-Pugh class A (odds ratio = 1.96, P = 0.018), and longer ablation (≥ 10 min, OR = 3.03, P < 0.001) were significant factors related to severe body movement. CONCLUSION: Deep sedation for RFA can be performed safely and effectively. Higher BMI and longer ablation were risk factors for respiratory depression and male sex, Child-Pugh class A, and longer ablation were independent predictors of severe body movement during RFA under deep sedation.

RACK1 regulates centriole duplication by controlling localization of BRCA1 to the centrosome in mammary tissue-derived cells.

Breast cancer gene 1 (BRCA1) is a tumor suppressor that is associated with hereditary breast and ovarian cancer. BRCA1 functions in DNA repair and centrosome regulation together with BRCA1-associated RING domain protein (BARD1), a heterodimer partner of BRCA1. Obg-like ATPase 1 (OLA1) was identified as a protein that interacts with BARD1. OLA1 regulates the centrosome by binding to and collaborating with BRCA1 and BARD1. We identified receptor for activated C kinase (RACK1) as a protein that interacts with OLA1. RACK1 directly bound to OLA1, the N-terminal region of BRCA1, and γ-tubulin, associated with BARD1, and localized the centrosomes throughout the cell cycle. Knockdown of RACK1 caused abnormal centrosomal localization of BRCA1 and abrogated centriole duplication. Overexpression of RACK1 increased the centrosomal localization of BRCA1 and caused centrosome amplification due to centriole overduplication. The number of centrioles in cells with two γ-tubulin spots was higher in cell lines derived from mammary tissue compared to those derived from other tissues. The effects of aberrant RACK1 expression level on centriole duplication were observed in cell lines derived from mammary tissue, but not in those derived from other tissues. Two BRCA1 variants, R133H and E143K, and a RACK1 variant, K280E, associated with cancer, which weakened the BRCA1-RACK1 interaction, interfered with the centrosomal localization of BRCA1 and reduced centrosome amplification induced by overexpression of RACK1. These results suggest that RACK1 regulates centriole duplication by controlling the centrosomal localization of BRCA1 in mammary tissue-derived cells and that this is dependent on the BRCA1-RACK1 interaction.

Rare case of idiopathic sclerosing cholangitis, which was difficult to distinguish from cholangiocarcinoma: A case report.

It is often difficult to correctly diagnose patients who present with dilation of the bile duct. Cholangiocarcinoma, primary sclerosing cholangitis (PSC) and immunoglobulin (Ig)G4-related sclerosing cholangitis must be considered as potential diagnoses for these cases. The current study presents a 73-year-old female patient who presented with a high fever and abdominal pain. Contrast-enhanced computed tomography and magnetic resonance cholangiopancreatography revealed stenosis and dilation of the intrahepatic bile duct without solid components. It was suspected that the patient had intrahepatic cholangiocarcinoma. A left liver lobectomy, cholecystectomy and distal gastrectomy combined with a D2 lymph node dissection were performed. A pathological examination of the liver revealed increased fibrosis in the stroma, irregular bile duct dilation and clusters of inflamed lymph cells. No carcinoma or IgG4-positive plasma cells were observed and the typical findings of PSC were not detected. Based on these clinical and pathological results, the diagnosis was idiopathic sclerosing cholangitis, which is particularly rare. It is often difficult to preoperatively differentiate between cholangiocarcinoma and benign bile duct stenosis.

L-Carnitine supplementation reduces the general fatigue of cancer patients during chemotherapy.

L-Carnitine (LC) plays an important role in the metabolism of fatty acids, and LC deficiency is associated with a feeling of weakness or general fatigue. Cancer patients receiving chemotherapy often develop LC deficiency, which is considered to be a factor contributing to general fatigue. The aim of the present study was to evaluate the efficacy of LC supplementation as a treatment for general fatigue in cancer patients during chemotherapy. A total of 11 cancer patients who were suffering from general fatigue during chemotherapy in our hospital between September 2014 and December 2015 were examined (6 cases involved adjuvant chemotherapy and 5 cases involved chemotherapy for unresectable or recurrent disease). The patients were administered 1,500 mg/day of levocarnitine per os, and the change in mean daily fatigue from the baseline to 8 weeks was assessed using the Brief Fatigue Inventory. The change in the plasma levels of albumin and the lymphocyte counts from the baseline to 8 weeks were also assessed. LC supplementation reduced general fatigue in all cases. Moreover, LC supplementation maintained the plasma levels of albumin and lymphocyte counts during chemotherapy, and enabled patients to continue chemotherapy sequentially without dose reduction. Therefore, LC supplementation improved general fatigue in all the examined cancer patients during chemotherapy. This treatment may make improve the tolerability of chemotherapy in cancer patients by reducing general fatigue and improving the nutritional status.

Impact of MUC1 Expression on the Progression of Intraductal Papillary Mucinous Neoplasm With Worrisome Features During Follow-up.

OBJECTIVES: The aim of this study was to investigate whether MUC1 expression is associated with progression of intraductal papillary mucinous neoplasms with worrisome features during follow-up. METHODS: Fifteen patients positive for MUC1 and negative for MUC2 (MUC1 group) and 16 patients negative for MUC1 and MUC2 (control group) were followed up and examined for changes in diameters of the main and ectatic branches of pancreatic ducts, enlargement of mural nodules, and appearance of a solid mass, by imaging studies. All of them presented worrisome features, and none had "high-risk stigmata." RESULTS: The sizes of the main and ectatic branches of pancreatic ducts increased in 8 (53.3%) and 8 (53.3%) patients, respectively, of the MUC1 group and in 1 (6.3%) and 1 (6.3%) patients, respectively, of the control group (P = 0.0059 and 0.0059, respectively). A solid mass developed in 6 patients (33.3%) of the MUC1 group but in none of the control group patients (P = 0.0373). CONCLUSIONS: Positive MUC1 expression in cell block cytology specimens may be associated with progressive dilation of the main and ectatic branches of pancreatic ducts and appearance of a solid mass in patients with intraductal papillary mucinous neoplasm with worrisome features during follow-up.

Structure-function relationships in human d-aspartate oxidase: characterisation of variants corresponding to known single nucleotide polymorphisms.

d-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for the acidic amino acid d-aspartate, an endogenous agonist of the N-methyl-d-aspartate (NMDA) receptor. Dysregulation of NMDA receptor-mediated neurotransmission has been implicated in the onset of various neuropsychiatric disorders including schizophrenia and in chronic pain. Thus, appropriate regulation of the amount of d-aspartate is believed to be important for maintaining proper neural activity in the nervous system. Herein, the effects of the non-synonymous single nucleotide polymorphisms (SNPs) R216Q and S308N on several properties of human DDO were examined. Analysis of the purified recombinant enzyme showed that the R216Q and S308N substitutions reduce enzyme activity towards acidic d-amino acids, decrease the binding affinity for the coenzyme flavin adenine dinucleotide and decrease the temperature stability. Consistent with these findings, further experiments using cultured mammalian cells revealed elevated d-aspartate in cultures of R216Q and S308N cells compared with cells expressing wild-type DDO. Furthermore, accumulation of several amino acids other than d-aspartate also differed between these cultures. Thus, expression of DDO genes carrying the R216Q or S308N SNP substitutions may increase the d-aspartate content in humans and alter homeostasis of several other amino acids. This work may aid in understanding the correlation between DDO activity and the risk of onset of NMDA receptor-related diseases.

Long-term survival and prognosis associated with conversion surgery in patients with metastatic gastric cancer.

In gastric cancer, primary systemic chemotherapy is the standard approach for the management of patients with initially unresectable metastasis, and it occasionally leads to a reduction in the size of the lesion, which facilitates surgical resection. The aim of this study was to examine the prognosis of patients who were able to undergo complete resection following chemotherapy. A total of 10 patients who underwent radical surgery for stage IV primary gastric cancer after chemotherapy between 2009 and 2015 at the Department of Surgery of Hokkaido Social Work Association Obihiro Hospital (Obihiro, Japan) were retrospectively investigated. Three regimens were used (S-1, n=1; S-1 + cisplatin, n=8; and S-1 + docetaxel, n=1). The mean time from chemotherapy to surgery was 210 days. One total gastrectomy + splenectomy + colectomy, one total gastrectomy + splenectomy, four total gastrectomies and three distal gastrectomies were performed. There were two cases of pancreatic fistula formation postoperatively. All the patients survived for >1 year. Of the 10 patients, 5 survived without recurrence. The median survival time was 871.1 days after diagnosis. Therefore, curative resection after chemotherapy is associated with a better prognosis in stage IV gastric cancer patients.

Prior minimal endoscopic sphincterotomy to prevent pancreatitis related to endoscopic balloon sphincteroplasty.

AIM: To investigate the efficacy of prior minimal endoscopic sphincterotomy (EST) to prevent pancreatitis related to endoscopic balloon sphincteroplasty (EBS). METHODS: After bile duct access was gained and cholangiogram confirmed the presence of stones < 8 mm in the common bile duct at endoscopic retrograde cholangiography, patients were subjected to minimal EST (up to one-third of the size the papilla) plus 8 mm EBS (EST-EBS group). The incidence of pancreatitis and the difference in serum amylase level after the procedure were examined and compared with those associated with 8-mm EBS alone in 32 patients of historical control (control group). RESULTS: One hundred and five patients were included in the EST-EBS group, and complete stone removal was accomplished in all of them. The difference in serum amylase level after the procedure was - 25.0 (217.9) IU/L in the EST-EBS group and this value was significantly lower than the 365.5 (576.3) IU/L observed in the control group (P < 0.001). The incidence of post-procedure pancreatitis was 0% (0/105) in the EST-EBS group and 15.6% (5/32) in the control group (P < 0.001). CONCLUSION: Prior minimal EST might be useful to prevent the elevation of serum amylase level and the occurrence of pancreatitis related to EBS.

Safety of immediate endoscopic sphincterotomy in acute suppurative cholangitis caused by choledocholithiasis.

AIM: To examine the safety of immediate endoscopic sphincterotomy (EST) in patients with acute suppurative cholangitis (ASC) caused by choledocholithiasis, as compared with elective EST. METHODS: Patients with ASC due to choledocholithiasis were allocated to two groups: Those who underwent EST immediately and those who underwent EBD followed by EST 1 wk later because they were under anticoagulant therapy, had a coagulopathy (international normalized ratio > 1.3, partial thromboplastin time greater than twice that of control), or had a platelet count < 50000 × 10(3)/µL. One of four trainees [200-400 cases of endoscopic retrograde cholangiopancreatography (ERCP)] supervised by a specialist (> 10000 cases of ERCP) performed the procedures. The success and complication rates associated with EST in each group were examined. RESULTS: Of the 87 patients with ASC, 59 were in the immediate EST group and 28 in the elective EST group. EST was successful in all patients in both groups. There were no complications associated with EST in either group of patients, although white blood cell count, C-reactive protein, total bilirubin, and serum concentrations of liver enzymes just before EST were significantly higher in the immediate EST group than in the elective EST group. CONCLUSION: Immediate EST can be as safe as elective EST for patients with ASC associated with choledocholithiasis provided they are not under anticoagulant therapy, or do not have a coagulopathy or a platelet count < 50000 × 10(3)/µL. Moreover, the procedure was safely performed by a trainee under the supervision of an experienced specialist.

Efficacy of recombinant human soluble thrombomodulin in patients with sepsis and disseminated intravascular coagulation in the gastroenterology field.

Recombinant human soluble thrombomodulin (rTM) was approved recently and has been used for treatment of disseminated intravascular coagulation (DIC). The aim of the present study was to evaluate the efficacy of rTM for DIC. The data of 53 inpatients with sepsis-induced DIC were retrospectively analyzed. Patients were classified into the rTM treatment group (n=25) and conventional treatment group (rTM not used) was the control group (n=28). Diagnosis of DIC was made according to the criteria for acute DIC of the Japan Association of Acute Medicine. Platelet count, prothrombin time-international normalized ratio, levels of fibrin/fibrinogen degradation products (FDP), C-reactive protein and DIC scores were measured on days 0, 3 and 7. Furthermore, the DIC resolution rate was assessed on days 3 and 7. Prior to treatment, DIC scores were 5.0±1.0 in the rTM group and 5.9±1.3 in the control group (P<0.05). Significant intra-group improvements were observed in all the parameters, except for FDP in the two groups. Significant improvements were observed in the DIC scores in the rTM group (Δ2.0±1.9 vs. Δ1.5±1.9, P=0.001). Therefore, the results suggest that rTM would be a useful medicine for treatment of DIC in the gastroenterology field.

Efficient synthesis of N-alkylated α,ß-unsaturated ketonitrones via Cu-catalyzed rearrangement.

N-Alkylated unsaturated ketonitrones were efficiently synthesized from propargyloxyamines using Cu catalysts. Mechanistic studies suggest that the rearrangement reaction proceeds via Cu-catalyzed intramolecular hydroamination, followed by thermally induced electrocyclic ring opening.

Copper-catalyzed rearrangement of (Z)-propynal hydrazones via N-N bond cleavage.

Propynal hydrazones are successfully converted to the corresponding 3-aminoacrylonitriles in the presence of copper catalysts in good to high yields. As an example, (Z)-N-(hex-2-ynylidene)morpholin-4-amine reacted in the presence of 10 mol % Cu(OAc)(2) in acetonitrile at 25 °C to afford (E)-3-morpholinohex-2-enenitrile ((E)-2 h) in 77% yield via C-N bond formation and subsequent ß-elimination involving cleavage of N-N and C-H bonds.