Standardization of preoperative stoma site marking and its utility for preventing stoma leakage: a retrospective study of 519 patients who underwent laparoscopic/robotic rectal cancer surgery.

PURPOSE: Stoma site marking is an important preoperative intervention for preventing various stoma-associated complications. In our institution, standardized stoma site marking is routinely performed before rectal cancer surgery with stoma creation, and various stoma-associated factors are recorded in the ostomy-record template. The present study investigated risk factors for stoma leakage. METHODS: Our stoma site marking is standardized so that it can be performed by non-stoma specialists. To identify risk factors of stoma leakage at 3 months after surgery, various preoperative factors associated with stoma site marking in our ostomy-record template were retrospectively analyzed in 519 patients who underwent rectal cancer surgery with stoma creation from 2015 to 2020. RESULTS: Stoma leakage was seen in 35 of the 519 patients (6.7%). The distance between the stoma site marking and the umbilicus was less than 60 mm in 27 of the 35 patients (77%) who experienced stoma leakage, so a distance of less than 60 mm was identified as an independent risk factor for stoma leakage. Aside from preoperative factors, stoma leakage was also caused by postoperative skin wrinkles or surgical scars near the stoma site in 8 of 35 patients (23%). CONCLUSION: Preoperative standardized stoma site marking is necessary to achieve reliable marking that is easy to perform. To reduce the risk of stoma leakage, a distance of 60 mm or more between the stoma site marking and the umbilicus is ideal, and surgeons need to contrive ways to keep surgical scars away from the stoma site.

Multicenter prospective trial of total gastrectomy versus proximal gastrectomy for upper third cT1 gastric cancer.

BACKGROUND: The appropriate surgical procedure for patients with upper third early gastric cancer is controversial. We compared total gastrectomy (TG) with proximal gastrectomy (PG) in this patient population. METHODS: A multicenter, non-randomized trial was conducted, with patients treated with PG or TG. We compared short- and long-term outcomes between these procedures. RESULTS: Between 2009 and 2014, we enrolled 254 patients from 22 institutions; data from 252 were included in the analysis. These 252 patients were assigned to either the PG (n = 159) or TG (n = 93) group. Percentage of body weight loss (%BWL) at 1 year after surgery, i.e., the primary endpoint, in the PG group was significantly less than that of the TG group (- 12.8% versus - 16.9%; p = 0.0001). For short-term outcomes, operation time was significantly shorter for PG than TG (252 min versus 303 min; p < 0.0001), but there were no group-dependent differences in blood loss and postoperative complications. For long-term outcomes, incidence of reflux esophagitis in the PG group was significantly higher than that of the TG group (14.5% versus 5.4%; p = 0.02), while there were no differences in the incidence of anastomotic stenosis between the two (5.7% versus 5.4%; p = 0.92). Overall patient survival rates were similar between the two groups (3-year survival rates: 96% versus 92% in the PG and TG groups, respectively; p = 0.49). CONCLUSIONS: Patients who underwent PG were better able to control weight loss without worsening the prognosis, relative to those in the TG group. Optimization of a reconstruction method to reduce reflux in PG patients will be important.

FUT2 non-secretor status is associated with Type 1 diabetes susceptibility in Japanese children.

AIM: To examine the contribution of the FUT2 gene and ABO blood type to the development of Type 1 diabetes in Japanese children. METHODS: We analysed FUT2 variants and ABO genotypes in a total of 531 Japanese children diagnosed with Type 1 diabetes and 448 control subjects. The possible association of FUT2 variants and ABO genotypes with the onset of Type 1 diabetes was statistically examined. RESULTS: The se2 genotype (c.385A>T) of the FUT2 gene was found to confer susceptibility to Type 1A diabetes in a recessive effects model [odds ratio for se2/se2, 1.68 (95% CI 1.20-2.35); corrected P value = 0.0075]. CONCLUSIONS: The FUT2 gene contributed to the development of Type 1 diabetes in the present cohort of Japanese children.

Highly purified eicosapentaenoic acid ameliorates cardiac injury and adipose tissue inflammation in a rat model of metabolic syndrome.

INTRODUCTION: n-3 Polyunsaturated fatty acids such as eicosapentaenoic acid (EPA), which are abundant in fish oil, have been shown to delay the onset of cardiovascular events. We previously established DahlS.Z-Leprfa/Leprfa (DS/obese) rats, which are derived from a cross between Dahl salt-sensitive and Zucker rats, as a model of metabolic syndrome. This study has now explored the influence of highly purified EPA on cardiac and adipose tissue pathophysiology in this animal model. MATERIALS AND METHODS: DS/obese rats were administered EPA (300 or 1,000 mg kg-1 d-1, per os) or vehicle from age 9 to 13 weeks. Homozygous lean (DahlS.Z-Lepr+/Lepr+, or DS/lean) littermates were studied as controls. RESULTS: Whereas EPA had no effect on body weight, food intake or systolic blood pressure in DS/obese rats, it attenuated cardiac fibrosis, diastolic dysfunction, oxidative stress and inflammation in these animals. In addition, EPA did not affect insulin resistance but reduced adipocyte hypertrophy and inflammation in visceral fat of DS/obese rats. Moreover, EPA increased circulating levels of adiponectin as well as attenuated both the down-regulation of AMP-activated protein kinase phosphorylation and the up-regulation of phosphorylation of the p65 subunit of nuclear factor-kB in the heart of DS/obese rats. CONCLUSIONS: Treatment of DS/obese rats with EPA did not affect hypertension but reduced cardiac fibrosis and diastolic dysfunction, with the latter effects being accompanied by AMP-activated protein kinase activation and inactivation of nuclear factor-kB signalling in the heart, possibly as a result of an increase in adiponectin secretion. EPA may be suitable for the treatment of cardiac injury associated with metabolic syndrome.

Variants associated with autoimmune Type 1 diabetes in Japanese children: implications for age-specific effects of cis-regulatory haplotypes at 17q12-q21.

AIMS: The aim of this study was to clarify the significance of previously reported susceptibility variants in the development of autoimmune Type 1 diabetes in non-white children. Tested variants included rs2290400, which has been linked to Type 1 diabetes only in one study on white people. Haplotypes at 17q12-q21 encompassing rs2290400 are known to determine the susceptibility of early-onset asthma by affecting the expression of flanking genes. METHODS: We genotyped 63 variants in 428 Japanese people with childhood-onset autoimmune Type 1 diabetes and 457 individuals without diabetes. Possible association between variants and age at diabetes onset was examined using age-specific quantitative trait locus analysis and ordered-subset regression analysis. RESULTS: Ten variants, including rs2290400 in GSDMB, were more frequent among the people with Type 1 diabetes than those without diabetes. Of these, rs689 in INS and rs231775 in CTLA4 yielded particularly high odds ratios of 5.58 (corrected P value 0.001; 95% CI 2.15-14.47) and 1.64 (corrected P value 5.3 × 10-5 ; 95% CI 1.34-2.01), respectively. Age-specific effects on diabetes susceptibility were suggested for rs2290400; heterozygosity of the risk alleles was associated with relatively early onset of diabetes, and the allele was linked to the phenotype exclusively in the subgroup of age at onset ≤ 5.0 years. CONCLUSIONS: The results indicate that rs2290400 in GSDMB and polymorphisms in INS and CTLA4 are associated with the risk of Type 1 diabetes in Japanese children. Importantly, cis-regulatory haplotypes at 17q12-q21 encompassing rs2290400 probably determine the risk of autoimmune Type 1 diabetes predominantly in early childhood.

Attenuation of cold stress-induced exacerbation of cardiac and adipose tissue pathology and metabolic disorders in a rat model of metabolic syndrome by the glucocorticoid receptor antagonist RU486.

OBJECTIVES: Chronic stress affects the central nervous system as well as endocrine, metabolic and immune systems. However, the effects of cold stress on cardiovascular and metabolic disorders in metabolic syndrome (MetS) have remained unclear. We recently characterized DahlS.Z-Lepr(fa)/Lepr(fa) (DS/obese) rats, derived from a cross between Dahl salt-sensitive and Zucker rats, as a new animal model of MetS. We have now investigated the effects of chronic cold stress and glucocorticoid receptor (GR) blockade on cardiac and adipose tissue pathology as well as on metabolic parameters in this model. METHODS: DS/obese rats were exposed to cold stress (immersion in ice-cold water to a depth of 1-2 cm for 2 h per day) with or without subcutaneous injection of the GR antagonist RU486 (2 mg kg(-1)day(-1)) for 4 weeks beginning at 9 weeks of age. Age-matched homozygous lean (DahlS.Z-Lepr(+)/Lepr(+)) littermates served as a control. RESULTS: Chronic cold stress exacerbated hypertension as well as left ventricular (LV) hypertrophy, fibrosis and diastolic dysfunction in DS/obese rats in a manner sensitive to RU486 treatment. Cold stress with or without RU486 did not affect body weight or fat mass. In contrast, cold stress further increased cardiac oxidative stress as well as macrophage infiltration and proinflammatory gene expression in LV and visceral fat tissue, with all of these effects being attenuated by RU486. Cold stress also further increased GR and 11ß-hydroxysteroid dehydrogenase type 1 mRNA and protein abundance in LV and visceral adipose tissue, and these effects were again inhibited by RU486. In addition, RU486 ameliorated the stress-induced aggravation of dyslipidemia, glucose intolerance and insulin resistance in DS/obese rats. CONCLUSIONS: Our results implicate GR signaling in cold stress-induced exacerbation of cardiac and adipose tissue pathology as well as of abnormal glucose and lipid metabolism in a rat model of MetS.

Continuous postoperative pain control using a multiple-hole catheter after iliac bone grafting: comparison between ropivacaine and levobupivacaine.

The aim of this study was to compare the analgesic effects of ropivacaine and levobupivacaine in continuous infiltration anaesthesia delivered via a multiple-hole catheter for the purpose of postoperative analgesia after iliac bone grafting. Thirty-four patients scheduled for iliac bone grafting in the maxillofacial region participated in this study. The patients were randomized to a ropivacaine group (Ropi group) and a levobupivacaine group (Levo group). After harvesting the iliac bone for grafting, a multiple-hole catheter was placed on the periosteum of the iliac bone. When surgery was completed, continuous administration was started at 4 ml/h of 0.2% ropivacaine (Ropi group) or 0.25% levobupivacaine (Levo group). Pain was evaluated in the recovery room and at 4h after surgery, as well as at 9:00 and 18:00 on postoperative days 1, 2, and 3, using a visual analogue scale. Side effects were also recorded. No significant difference in the visual analogue scale scores at rest or in motion was observed between the two groups. In addition, there were no side effects in the two groups. Both 0.2% ropivacaine and 0.25% levobupivacaine provided comparable analgesic effects in continuous infiltration anaesthesia delivered via a multiple-hole catheter after iliac bone grafting.

Interaction between gastrocnemius muscle weakness and moderate exercise deteriorates joint integrity in rat knee.

The aim of this study was to determine the effect on the knee joint of the interaction between ankle muscle weakness and moderate exercise. Gastrocnemius muscle weakness was induced by intramuscular injection of botulinum toxin type A (BTX) in rats. Low-speed treadmill running (12 m/min for 60 min) was applied for 6 weeks in rats with and without BTX. Untreated animals were used as controls. After BTX injection, the gastrocnemius muscle weakness was confirmed by 3-D motion analysis in kinematic features of the hindlimb during locomotion as an increased maximal dorsiflexion angle during the stance phase. Serum biomarker analysis by enzyme-linked immunosorbent assay revealed that low-speed running decreased the catabolic effect on type II collagen. However, the inhibition of catabolism induced by running exercise was significantly counteracted by BTX injection. In addition, thinning of the cartilage layer and a reduction in the chondrocyte density was also found in the tibial plateau of the knee in the BTX-injected rats after running for 6 weeks. These data suggest that moderate exercise have a positive effect on joint homeostasis. However, ankle muscle weakness may alter the mechanical environment of the knee and impair the integrity of joint cartilage with moderate exercise.

The combined effects of midazolam and propofol sedation on muscle power.

We performed a randomised, crossover study to investigate the effects of intravenous sedation on grip strength and bite force. Twenty male volunteers received a bolus intravenous injection of midazolam (0.02 mg.kg(-1)) together with a 30-min propofol infusion designed to achieve an effect-site concentration of 1.0 µg.ml(-1). Observed variables included bispectral index, observer's assessment of alertness/sedation, correct answer rate of Stroop colour-word test, grip strength and bite force. Grip strength decreased from a median (IQR [range]) of 483 (443-517 [380-586]) N to 358 (280-405 [108-580]) N (p < 0.001) during sedation and recovered following flumazenil administration, while bite force increased from 818 (593-1026 [405-1406]) N to 1377 (1243-1585 [836-2357]) N (p < 0.001) during sedation. Although bite force gradually returned to baseline following flumazenil administration, it remained increased throughout the experimental period. We conclude that bite force increased during intravenous sedation and that this may have clinical implications.

Role of multidrug resistance protein 2 (MRP2) in chemoresistance and clinical outcome in oesophageal squamous cell carcinoma.

BACKGROUND: Although multidrug resistance protein 2 (MRP2) confers chemoresistance in some cancer types, its implication on oesophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: We evaluated MRP2 expression by immunohistochemistry and RT-PCR using 81 resected specimens from ESCC patients who did or did not receive neo-adjuvant chemotherapy (NACT), including 5-fluorouracil, doxorubicin, and cisplatin (CDDP). Correlation between MRP2 expression and response to chemotherapy was also examined in 42 pre-therapeutic biopsy samples and eight ESCC cell lines. RESULTS: MRP2-positive immunostaining was more frequently observed in ESCCs with NACT than in those without NACT (27.3 vs 5.4%). The MRP2-positive patients showed poorer prognosis than MRP2-negative patients (5-year survival rate, 25.6 vs 55.7%). Concordantly, ESCC with NACT showed 2.1-fold higher mRNA expression of MRP2 than those without NACT (P=0.0350). In pre-therapeutic biopsy samples of patients with NACT, non-responders showed 2.9-fold higher mRNA expression of MRP2 than responders (P=0.0035). Among the panel of ESCC cell lines, TE14 showed the highest MRP2 mRNA expression along with the strongest resistance to CDDP. Inhibition of MRP2 expression by small-interfering RNA reduced chemoresistance to CDDP. CONCLUSION: Our data suggested that MRP2 is one of molecules, which regulate the sensitivity to chemotherapy including CDDP in advanced ESCC patients.

Closed DHS system to prevent dissolved methane emissions as greenhouse gas in anaerobic wastewater treatment by its recovery and biological oxidation.

Anaerobic wastewater treatment has been focused on its eco-friendly nature in terms of the improved energy conservation and reduction in carbon dioxide emissions. However, the anaerobic process discharges unrecovered methane as dissolved methane. In this study, to prevent the emission of dissolved methane from up-flow anaerobic sludge blanket (UASB) reactors used to treat sewage and to recover it as useful gas, we employed a two-stage down-flow hanging sponge (DHS) reactor as a post-treatment of the UASB reactor. The closed DHS reactor in the first stage was intended for the recovery of dissolved methane from the UASB reactor effluent; the reactor could successfully recover an average of 76.8% of the influent dissolved methane as useful gas (containing methane over 30%) with hydraulic retention time of 2 h. During the experimental period, it was possible to maintain the recovered methane concentrations greater than 30% by adjusting the air supply rate. The remaining dissolved methane after the first stage was treated by the next step. The second closed DHS reactor was operated for oxidation of the residual methane and polishing of the remaining organic carbons. The reactor had a high performance and the influent dissolved methane was mostly eliminated to approximately 0.01 mgCOD L(-1). The dissolved methane from the UASB reactor was completely eliminated--by more than 99%--by the post-treatment after the two-stage closed DHS system.

Cytokeratins 18 and 8 are poor prognostic markers in patients with squamous cell carcinoma of the oesophagus.

BACKGROUND: Cytokeratins (CKs) are structural marker proteins specific for epithelial cells. However, recent studies indicate their involvement in cancer progression. METHODS: We evaluated CK18 and its filament partner, CK8 expression, by immunohistochemistry in 210 resected specimens from patients with oesophageal squamous cell carcinoma (OSCC). We also analysed the relationship between their expression and various clinicopathological parameters including prognosis. RESULTS: Neither CK18 nor CK8 was expressed in non-cancerous squamous epithelium whereas proper oesophageal glands expressed both CKs. Ninety (42.9%) tumours were CK18 positive and 85 (40.5%) CK8 positive, and the concordance rate for immunohistochemical classification for CK18 and CK8 was 82.4%. CK18 expression correlated with poorly differentiated tumours, use of neo-adjuvant chemotherapy, and advanced stage. Prognosis of patients with CK18-positive tumours was poorer than that of patients with negative OSCC (P<0.001). A similar trend was noted for CK8 expression. Multivariate analysis identified pT (P=0.020), pN number (P=0.001), and CK18 expression (P=0.004) as independent prognostic factors. CK18 expression in 83 pretreatment biopsy specimens was detected in 47 cases (56.6%) and also correlated with prognosis (P=0.045). CONCLUSION: CK18/CK8 expression correlated with progression of OSCC. The significant correlation with prognosis and stable expression in biopsy specimen suggest usefulness of CK18 in selection of treatment strategies for OSCC.

Development of a method to quantitatively monitor the effect of inhibition of nitric oxide synthase on tumour vascular activity using dynamic contrast-enhanced computed tomography.

This study was undertaken to develop a method to quantitatively monitor the effect of inhibition of nitric oxide synthase (NOS) on tumour vascular activity using dynamic contrast-enhanced computed tomography (DCE-CT). The DCE-CT studies were performed in 13 anaesthetized rats bearing tumours. To investigate the effect of NOS inhibition, N-nitro-L-arginine (L-NNA) was intravenously administered in eight rats, while only the vehicle was administered in five rats. The contrast enhancement (CE) images were generated by subtracting the CT images before and after the administration of contrast agent. The tumour blood volume (TBV) images were also generated. The CE significantly decreased after L-NNA administration, while there were no significant changes when only the vehicle was administered. There was a good correlation between CE and TBV, suggesting that CE mainly reflects TBV. In conclusion, the present method appears to be useful for monitoring the effect of NOS inhibition on tumour vascular activity.

Activation of Wnt/beta-catenin signalling pathway induces chemoresistance to interferon-alpha/5-fluorouracil combination therapy for hepatocellular carcinoma.

Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to interferon (IFN)-alpha/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-alpha/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-alpha/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/beta-catenin signalling pathway contributed to resistance to IFN-alpha/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/beta-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/beta-catenin signalling by glycogen synthesis kinase-3 inhibitor (6-bromoindirubin-3'-oxime (BIO)) induced chemoresistance to IFN-alpha/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-alpha/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/beta-catenin signalling pathway induces chemoresistance to IFN-alpha/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.

Autosomal dominant moyamoya disease maps to chromosome 17q25.3.

BACKGROUND: Moyamoya disease (MMD) is an idiopathic steno-occlusive cerebrovascular disease that represents an important cause of stroke. However, etiology of the disease has remained largely unknown. METHODS: We previously showed that the inheritance pattern of MMD is autosomal dominant with incomplete penetrance. Here, we report the genome-wide parametric linkage analysis for MMD in 15 extended Japanese families. We conducted linkage analyses under two diagnostic classifications: narrow and broad. Affected member-only analysis was applied due to incomplete and age-dependent penetrance of the disease. RESULTS: Under both classifications, significant evidence of linkage was only observed on chromosome 17q25.3, with maximum multipoint logarithm of odds (lod) scores of 6.57 (under the narrow classification) and 8.07 (under the broad classification) at D17S704. Haplotype analysis revealed segregation of a disease haplotype in all families but one, and informative crossovers enabled mapping of the MMD locus to a 3.5-Mb region between D17S1806 and the telomere of 17q, encompassing 94 annotated genes. CONCLUSIONS: Our data suggest that there is a major gene locus for autosomal dominant moyamoya disease on chromosome 17q25.3.

Morphological and biochemical signs of age-related neurodegenerative changes in klotho mutant mice.

Klotho mutant mice, defective in the klotho gene, develop multiple age-related disorders with very short lifespans. Introduction of the exogenous klotho gene into these mutant mice leads to an improvement in their phenotypes, while overexpression of this gene in wild-type mice significantly extends their lifespan. These observations suggest that the klotho gene/protein has an anti-aging function. Since there have been only a few reports with some disagreement about results on the CNS of the mutant mice, we tried to clarify whether the CNS neurons generate aging-like features, even in premature stages, using biochemical and morphological approaches. Results obtained from the mutant mice, when compared with wild-type mice, were as follows. Neurofilaments (NFs) were increased significantly in axons, with the subunit proteins showing a significant enhancement in phosphorylation or expression of NF-H or NF-L, respectively. Microtubules in Purkinje cell dendrites were closer to each other, and in the CNS tissue tubulin was unaltered, but microtubule-associated protein (MAP) 2 was significantly reduced in expression. Neuronal cellular organelles were morphologically disordered. Lysosomes, cathepsin D and light chain 3 of MAP1A/B (LC3) were augmented with the appearance of putative autophagy-related structures. Antiapoptotic Bcl-xL and proapoptotic Bax were reduced and enhanced, respectively, and mitogen-activated protein kinase was reduced. Synapse-related proteins and structures were decreased. Neuronal degeneration was evident in hippocampal pyramidal cells, and possibly in Purkinje cells. Astrocytic glial filaments and glial fibrillary acidic protein were increased in density and expression, respectively. Together, the CNS neuronal alterations in klotho mutant mice were quite similar to those found in aged animals, including even premature death, so this mouse should be a more appropriate animal model for CNS aging than those previously reported.