Risk of metachronous recurrence after endoscopic submucosal dissection of esophageal squamous cell carcinoma.

Development of endoscopic submucosal dissection (ESD) improves the en bloc resection rate of superficial esophageal squamous cell carcinoma (SESCC). Although the background mucosa after ESD remains malignant potential, esophageal (sub)circumferential ESD, in cases where the mucosal defect is greater than three-fourths of the circumference, might induce refractory stricture, and it may disturb early detection of the recurrence. Therefore, we aimed to elucidate whether the patients treated by (sub)circumferential ESD for SESCC may remain at risk of metachronous recurrence. In a single-center retrospective study, we collected data from 154 consecutive patients who were treated with curative ESD for SESCC from 2002 to 2013 and followed by surveillance for longer than 12 months. Metachronous recurrence was defined as histologically proven SESCC at other site of the ESD scar or abnormal nodal swelling was detected later than 12 months after ESD. The primary endpoint was to identify the risk of metachronous recurrence using multivariate analyses. The secondary endpoint was to investigate difference in clinical pathological features between patients with and without the recurrence. The overall rate of metachronous recurrence was 14.9% during 40.5 median months after the initial ESD. 24.1% and 9.0% of overall metachronous recurrence were observed in patients treated with (sub)circumferential ESD and non-subcircumferential ESD, respectively, despite no significant difference in their observation duration. After the application of a stepwise regression model that included all variants, a Cox proportional hazards regression model identified (sub)circumferential ESD as the only risk for the recurrence (hazard ratio (HR): 1.48, 95% confidence intervals (CI): 1.04-2.08, P = 0.028). The cumulative recurrence rate revealed a significant difference between patients treated by (sub)circumferential ESD and those by nonsubcircumferential ESD (HR: 3.094, 95% CI: 1.33-7.52, P = 0.009), despite no significant difference in their cause-specific survival. Additionally, the session numbers of the follow-up endoscopy until the detection of metachronous recurrence after the non-subcircumferential ESD were significantly less than those after the (sub)circumferential ESD (7.8 ± 1.8 vs. 15.2 ± 1.5 P = 0. 005), despite no significant difference in their cancer-free duration. In conclusion, we demonstrated that patients treated by curative (sub)circumferential ESD for SESCC might be high risk for metachronous recurrence. Therefore, we should establish a risk-stratified surveillance program after (sub)circumferential ESD and preventive strategies for post-ESD stricture.

Western Diet-Induced Nonalcoholic Fatty Liver Disease Mice Mimic the Key Transcriptomic Signatures Observed in Humans.

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by the accumulation of fat in the liver in the absence of excessive alcohol consumption or a secondary cause of hepatic steatosis. The prevalence of NAFLD is increasing worldwide and its management has become a public health concern. Animal models are traditionally used to elucidate disease mechanisms and identify potential drug targets; however, their translational aspects in human diseases have not been fully established. This study aimed to clarify the utility of animal models for translational research by assessing their relevance to human diseases using gene expression analysis. Weighted gene co-expression network analysis of liver tissues from Western diet (WD)-induced NAFLD mice was performed to identify the modules associated with disease progression. Moreover, the similarity of the gene co-expression network across species was evaluated using module preservation analysis. Nineteen disease-associated modules were identified. The brown module was positively associated with disease severity, and functional analyses indicated that it may be involved in inflammatory responses in immune cells. Moreover, the gene co-expression network of the brown module was highly preserved in human NAFLD liver gene expression datasets. These results indicate that WD-induced NAFLD mice have similar gene co-expression networks (especially genes associated with inflammatory responses) to humans and are thought to be a useful experimental tool for preclinical research on NAFLD. Keywords: Nonalcoholic fatty liver disease (NAFLD), Weighted gene co-expression network analysis (WGCNA), Western diet (WD).

Serious laryngeal edema during endoscopic resection for squamous cell carcinoma of the esophagus.

The detection of early esophageal squamous cell carcinoma (ESCC) in patients following radiotherapy for squamous cell carcinoma of the head and neck (HNSCC) has increased with the development of endoscopic technologies. The aim of the current case - control study was to elucidate the risk factors of serious laryngeal edema, a lethal complication that occurs during endoscopic resection for ESCC. Among 184 consecutive patients who were treated by endoscopic resection for ESCC between January 2009 and May 2012, five of 22 patients with a history of radiotherapy for HNSCC suffered from serious laryngeal edema, which was not observed in patients who had not undergone radiotherapy. The susceptibility to serious laryngeal edema in patients with a history of radiotherapy followed by neck dissection for HNSCC was significantly greater than those without such histories. Despite the limited number of cases, we suggest that previous radiotherapy followed by neck dissection for HNSCC might be a predictive factor for serious laryngeal edema during endoscopic resection.

High-cholesterol diet in combination with hydroxypropyl-beta-cyclodextrin induces NASH-like disorders in the liver of rats.

Non-alcoholic fatty liver disease (NAFLD) is a general term for fatty liver disease not caused by viruses or alcohol. Fibrotic hepatitis, cirrhosis, and hepatocellular carcinoma can develop. The recent increase in NAFLD incidence worldwide has stimulated drug development efforts. However, there is still no approved treatment. This may be due in part to the fact that non-alcoholic steatohepatitis (NASH) pathogenesis is very complex, and its mechanisms are not well understood. Studies with animals are very important for understanding the pathogenesis. Due to the close association between the establishment of human NASH pathology and metabolic syndrome, several animal models have been reported, especially in the context of overnutrition. In this study, we investigated the induction of NASH-like pathology by enhancing cholesterol absorption through treatment with hydroxypropyl-beta-cyclodextrin (CDX). Female Sprague-Dawley rats were fed a normal diet with normal water (control group); a high-fat (60 kcal%), cholesterol (1.25 %), and cholic acid (0.5 %) diet with normal water (HFCC group); or HFCC diet with 2 % CDX water (HFCC+CDX group) for 16 weeks. Compared to the control group, the HFCC and HFCC+CDX groups showed increased blood levels of total cholesterol, aspartate aminotransferase, and alanine aminotransferase. At autopsy, parameters related to hepatic lipid synthesis, oxidative stress, inflammation, and fibrosis were elevated, suggesting the development of NAFLD/NASH. Elevated levels of endoplasmic reticulum stress-related genes were evident in the HFCC+CDX group. In the novel rat model, excessive cholesterol intake and accelerated absorption contributed to NAFLD/NASH pathogenesis.

Unilateral nephrectomized SHR/NDmcr-cp rat shows a progressive decline of glomerular filtration with tubular interstitial lesions.

In patients with diabetic kidney disease (DKD), the estimated glomerular filtration rate (eGFR) or creatinine clearance rate (Ccr) is always used as an index of decline in renal function. However, there are few animal models of DKD that could be used to evaluate renal function based on GFR or Ccr. For this reason, it is desirable to develop animal models to assess renal function, which could also be used for the evaluation of novel therapeutic agents for DKD. Therefore, we aimed to develop such animal model of DKD by using spontaneously hypertensive rat (SHR)/NDmcr-cp (cp/cp) rats with the characteristics of obese type 2 diabetes and metabolic syndrome. As a result, we have found that unilateral nephrectomy (UNx) caused a chronic Ccr decline, development of glomerular sclerosis, tubular lesions, and tubulointerstitial fibrosis, accompanied by renal anemia. Moreover, losartan-mixed diet suppressed the Ccr decline in UNx-performed SHR/NDmcr-cp rats (UNx-SHR/cp rats), with improvement in renal anemia and histopathological changes. These results suggest that UNx-SHR/cp rats could be used as a DKD model for evaluating the efficacy of therapeutic agents based on suppression of renal function decline.

Temporal and sequential changes of glial cells and cytokine expression during neuronal degeneration after transient global ischemia in rats.

BACKGROUND: How glial cells and cytokines are associated with the progression of delayed neuronal death induced by transient global ischemia is still unclear. To further clarify this point, we studied morphological changes in glial cells (microglial cells and astrocytes), and cytokine protein levels, during the progression of neuronal cell loss in CA1 (Cornu Ammonis 1) of the hippocampus after transient global ischemia. METHODS: Morphological changes in glial cells were studied immuno-histochemically. Nine cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and TNF-α) were simultaneously measured by a multiplexed bead-based immunoassay from 6 h to day21 after transient four vessel occlusion (4VO) in rats. RESULTS: During the process of neuronal loss, we observed four distinct phases: (1) lag phase day0-2 (no NeuN+ cell loss observed), (2) exponential phase day2-7 (NeuN+ cells reduced in number exponentially), (3) deceleration phase day7-14 (reduction rate of NeuN+ cells became low), (4) stationary phase day14 onward (NeuN+ cell loss progressed no longer). In the lag phase, activated glial cells were observed in the entire hippocampus but later were gradually restricted to CA1. Cytokine protein levels in the lag and exponential phases were lower than in the deceleration and stationary phases. IL-1α, IL-1ß, IL-4, IL-6 and IFN-γ in 4VO were significantly higher in all four phases than in sham. Compared with sham level, GM-CSF was significantly high in the deceleration phase. TNF-α was significantly high in both the deceleration and stationary phases. CONCLUSION: Ischemic stress in 4VO activated glial cells in areas beyond CA1 in the lag phase. Pyramidal neurons were injured in CA1 from the end of the lag phase and then neuronal cells reduced in CA1 in the exponential phase. After neuronal death began, the influence of dead cells on glial cells and cytokine expression gradually became stronger than the influence by ischemic stress. Therefore, from the deceleration phase, changes in glial cells and cytokine production were likely caused by dead cells. Cytokine interaction in the microenvironment may determine the functions of IL-1α, IL-1ß, IL-4, IL-6 and IFN-γ in all four phases. The function of GM-CSF and TNF-α in the deceleration phase may be neurotrophic.

Intracellular glutathione redox status in human dendritic cells regulates IL-27 production and T-cell polarization.

BACKGROUND: Glutathione redox status, changes in intracellular reduced (GSH) or oxidized (GSSG) glutathione, plays a significant role in various aspects of cellular function. In this study, we examined whether intracellular glutathione redox status in human dendritic cells (DCs) regulates the polarization of Th1/Th2 balance. METHODS: Human monocyte-derived DCs (MD-DCs) treated with glutathione reduced form ethyl ester (GSH-OEt) or L-buthionine-(S,R)-sulfoximine (BSO) were stimulated by lipopolysaccharide (LPS), and the levels of polarization cytokines were measured. Next, DCs matured by LPS or thymic stromal lymphopoietin (TSLP) were cocultured with allogeneic CD4(+) naive T cells and Th1/Th2 balance was evaluated by cytokine production from the primed T cells. RESULTS: Monocyte-derived DCs exposed to GSH-OEt and BSO had increased and decreased intracellular GSH contents, respectively. Lipopolysaccharide-induced interleukin (IL)-27 production was enhanced by GSH-OEt and suppressed by BSO, but neither GSH-OEt nor BSO affected the expression of HLA-DR, CD80, CD83, or CD86. Mature GSH-OEt-treated MD-DCs enhanced interferon (IFN)-γ production from CD4(+) T cells compared with nontreated MD-DCs, and small interfering RNA (siRNA) against IL-27 suppressed the effect of GSH-OEt on IFN-γ production. Additionally, although human myeloid DCs activated by TSLP (TSLP-DCs) prime naïve CD4(+) T cells to differentiate into Th2 cells, treatment of TSLP-DCs with GSH-OEt reduced IL-13 production and enhanced IFN-γ production by CD4(+) T cells. Interleukin-27 siRNA attenuated the inhibitory effect of GSH-OEt on Th2 polarization. CONCLUSION: Our results reveal that Th1 and Th2 responses are controlled by intracellular glutathione redox status in DCs through IL-27 production.

Gastric hyposecretion in esophageal squamous-cell carcinomas.

Recently, gastric fundic atrophy is reported to be an independent risk factor for esophageal squamous-cell carcinoma (ESCC). The aim of this study is to investigate the acid secretory level in ESCC in a case-control study. From April 2004 to March 2008, 100 consecutive subjects with early ESCC and 100 age- and sex-matched asymptomatic controls were prospectively enrolled. Gastrin-stimulated acid output was assessed by endoscopic gastrin test. Conditional regression analyses were used to adjust for other potential confounders. Multivariate analyses revealed a strong association between profound hypochlorhydria and ESCC with odds ratio (95% confidence interval): 6.0 (1.9-18.4). The association remained significant after adjusting for the effect of gastric atrophy as a covariate. The association became stronger as the ESCC developed more distal site of the esophagus. This study indicates that profound hypochlorhydria is a strong independent risk factor for ESCC even after adjusting for the influence of gastric atrophy.

Tbx5 and the retinotectum projection.

Dorsal and ventral aspects of the eye are distinct from the early stages of development. The developing eye cup grows dorsally, and the choroidal fissure is formed on its ventral side. Retinal axons from the dorsal and ventral retina project to the ventral and dorsal tectum, respectively. Misexpression of the Tbx5 gene induced dorsalization of the ventral side of the eye and altered projections of retinal ganglion cell axons. Thus, Tbx5 is involved in eye morphogenesis and is a topographic determinant of the visual projections between retina and tectum.

Silent venous thromboembolism before treatment in endometrial cancer and the risk factors.

Venous thromboembolism (VTE) often occurs after surgery and can even occur before surgery in patients with gynaecological malignancies. We investigated the incidence of VTE before treatment of endometrial cancer and associated risk factors. Plasma D-dimer (DD) levels before initial treatment were examined in 171 consecutive patients with endometrial cancer. Venous ultrasound imaging (VUI) of the lower extremities was performed in patients with DD >or=1.5 microg ml(-1), as the negative predictive value of DD for VTE is extremely high. For patients with deep vein thrombosis (DVT), pulmonary scintigraphy was performed to ascertain the presence of pulmonary thromboembolism (PTE). Risk factors for VTE were analysed using univariate and multivariate analyses for 171 patients. Of these, 37 patients (21.6%) showed DD >or=1.5 microg ml(-1), 17 (9.9%) displayed DVT by VUI and 8 (4.7%) showed PTE on pulmonary scintigraphy. All patients with VTE were asymptomatic. Univariate analysis for various risk factors revealed older age, non-endometrioid histology and several variables of advanced disease as significantly associated with VTE before treatment. Obesity, smoking and diabetes mellitus were not risk factors. Multivariate analysis confirmed extrauterine spread and non-endometrioid histology as independently and significantly associated with risk of VTE. These data suggest that silent or subclinical VTE occurs before treatment in at least around 10% of patients with endometrial cancer. Risk factors for VTE before treatment might not be identical to those after starting treatment.

The effects of MPTP on the activation of microglia/astrocytes and cytokine/chemokine levels in different mice strains.

The effects of MPTP on two mouse strains with different MPTP sensitivities and immunological backgrounds were compared: MPTP-sensitive C57BL/6 mice (B6) with a propensity for Th1 and less MPTP-sensitive BALB/c mice (BALB) with a propensity for Th2. It was found that acute MPTP treatment induced behavioral dysfunction, activated microglia/astrocytes, and increased the levels of IL-10, IL-12(p40) IL-13, IFN-gamma, and MCP-1 in CSF in B6, but not in BALB. This suggests that variances in immunological backgrounds might be a major contributing factor in sensitivity differences to MPTP.

A comparative study of the diagnostic accuracy of ELISA systems for the detection of anti-neutrophil cytoplasm antibodies available in Japan and Europe.

OBJECTIVES: Primary systemic vasculitis associated with anti-neutrophil cytoplasm antibodies (ANCA) differs in its frequency and clinical expression between Japan and Europe. We sought to ascertain whether such differences arise from the performance of enzyme-linked immunosorbent assays (ELISAs) for ANCA. METHODS: Plasma samples from 64 consecutive Japanese patients with a clinical and histological diagnosis of primary systemic vasculitis including microscopic polyangiitis (MPA; n=52), Churg-Strauss syndrome (CSS; n=1), and Wegener's granulomatosis (WG; n=11), or those from disease controls with non-vasculitic glomerulonephritis (n=54) and healthy controls (n=55) were tested for the presence of myeloperoxidase (MPO) by ELISAs available in Japan (Nipro and MBL) and compared with those in Europe (Wieslab). The sensitivity and specificity were calculated for each ELISA, and its diagnostic performance was assessed by receiver operating characteristic curve analysis. RESULTS: The sensitivity and specificity of either MPO-ANCA assays for a diagnosis of MPA were 90.4% and 98.2% (Nipro), 88.2% and 96.3% (MBL), and 86.5% and 99.1% (Wieslab). The overall diagnostic performance, assessed as the area under curve of the MPO-ANCA ELISAs for MPA were 0.946+/-0.022 (Nipro), 0.970+/-0.017 (MBL), and 0.971+/-0.017 (Wieslab), while that of PR3-ANCA ELISAs for WG were 0.986+/-0.025 (Nipro), 0.993+/-0.017 (MBL), and 0.916+/-0.059 (Wieslab). CONCLUSIONS: The MPO-ANCA ELISAs commercially available in Japan exhibited high sensitivity and specificity for the diagnosis of ANCA-associated vasculitides and provided similar diagnostic value to those in Europe. These results facilitate further international comparison of ANCA-associated vasculitides between Japanese and European populations.

Resistance of infant leukemia with MLL rearrangement to tumor necrosis factor-related apoptosis-inducing ligand: a possible mechanism for poor sensitivity to antitumor immunity.

Malignant cells generally acquire some immune escape mechanisms for clonal expansion. Immune escape mechanisms also contribute to the failure of graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (allo-SCT). Infant leukemias with mixed-lineage leukemia (MLL) rearrangement have a remarkably short latency, and GVL effect after allo-SCT has not been clearly evidenced in these leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and FasL-mediated cytotoxic pathways play important roles in cytotoxic T-lymphocyte- and natural killer cell-mediated antitumor immunity and optimal GVL activity. We investigated the in vitro sensitivity of MLL-rearranged acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells to TRAIL- and FasL-mediated cytotoxicity. Most of cell lines and primary leukemia cells were highly resistant to TRAIL primarily owing to low cell-surface expression of death receptors in ALL and simultaneous expression of decoy receptors in AML. Nearly half of cell lines and majority of primary leukemia cells showed low sensitivity to FasL. These results suggest that resistance to death-inducing ligands, particularly to TRAIL, could be one of the mechanisms for a rapid clonal expansion and a poor sensitivity to the GVL effect in infant leukemias with MLL rearrangement.

Note: Simple 100 Hz N2 laser with longitudinal discharge tube and high-voltage power supply using neon sign transformer.

We developed a longitudinally excited N2 laser with a simple driver circuit and a simple power supply. The N2 laser consisted of a 20 cm-long glass tube with an inner diameter of 2.5 mm, a normal stable resonator formed by flat mirrors, a variable transformer, a neon sign transformer, a spark gap, and a 200 pF capacitance. The N2 laser produced a laser pulse with an energy of 379 nJ and a pulse width of 7.5 ns at a repetition rate of 100 Hz. The laser beam was circular and had a Gaussian profile with a correlation factor of 0.992 93.

Impaired expression of thrombospondin-1 in eyes with age related macular degeneration.

AIMS: This study investigated the expression and localisation of thrombospondin-1 (TSP-1), a known anti-angiogenic extracellular matrix protein, in normal aged control human eyes and eyes with age related macular degeneration (AMD). METHODS: Immunohistochemical analysis with mouse anti-human TSP-1 antibody and mouse anti-human CD 34 antibody, as a blood vessel marker, was performed on frozen sections from macular and peripheral blocks of aged control donor eyes (n = 12; mean age 78.8 years), and eyes with AMD (n = 12; mean age 83.9 years). Pigment in retinal pigment epithelium (RPE) and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In the macular region, TSP-1 expression was observed intensely in Bruch's membrane and weakly in RPE basement membrane, choriocapillaris, and the wall of large choroidal blood vessels in the aged control eyes. In eyes with AMD, TSP-1 immunoreactivity was significantly lower in all structures except RPE basement membrane (p<0.01). There was significantly lower TSP-1 in the far periphery than the equator and submacular regions in all eyes. TSP-1 immunoreactivity was low in choroidal neovascularisation (CNV), but it was high and diffuse in adjacent scar tissue. CONCLUSION: These findings suggest that decreased TSP-1 in Bruch's membrane and choroidal vessels during AMD may permit the formation of CNV.

Effects of recombinant alpha-interferon-gelatin conjugate on in vivo murine tumor cell growth.

A recombinant human alpha-interferon A/D (IFN), also known to be effective in mice, was conjugated to gelatin with a water-soluble carbodiimide. The IFN-gelatin conjugate was much more efficient than free IFN in activating mouse peritoneal macrophages (Mø) in an in vitro experiment to inhibit the growth of IFN-resistant subline cells (RR1) of murine fibrosarcoma. A single i.p. injection of the conjugate administered to normal mice was also more effective than one of free IFN in activating peritoneal Mø and natural killer cells in peritoneal exudate cell and spleen cell populations. In the investigation on body distribution of the IFN-gelatin conjugate, an enhanced affinity to Mø as well as a prolonged retention were observed in comparison with free IFN. An injection of the IFN-gelatin conjugate i.p. was more effective than one of free IFN in suppressing the in vivo growth of not only IFN-sensitive SS2 cells but also RR1 cells in the peritoneal cavity of mice, although RR1 cells were only susceptible to the indirect effect of IFN via host cells, in contrast to SS2 cells. In addition to an increased recruitment of Mø to the peritoneal cavity in RR1-bearing mice receiving i.p. injection of the IFN-gelatin conjugate, these Mø were activated to inhibit the in vitro growth of RR1 cells. These results indicate that the IFN-gelatin conjugate is a promising antitumor agent that is much more effective than free IFN. The dose of IFN in the conjugate required for exerting the antitumor effects is much lower than that of free IFN, which leads to a reduction of adverse effects of IFN.

Direct and indirect effects of interferon on in vivo murine tumor cell growth.

We cloned two sublines (S1 and R1) of murine Meth A fibrosarcoma cells with respect to their sensitivity to a murine alpha/beta-interferon (IFN) preparation. The growth of S1 cells was suppressed and that of R1 cells was hardly affected by IFN in vitro. This was also the case with cells enclosed in cell-impermeable diffusion chambers in peritoneal cavities. Nevertheless, IFN suppressed the growth of not only S1 cells but also R1 cells in mice inoculated i.p. with these cells, and the survival rates of both S1 cell recipients and R1 cell recipients were markedly improved. S1 cells were observed microscopically to be injured by the direct effect of IFN in vitro and in vivo, but R1 cells in in vitro culture with IFN and those surviving in vivo in the presence of IFN appeared to proliferate well. In the peritoneal cavity of R1 recipients treated daily with IFN, the recruitment of macrophages was enhanced in comparison with untreated R1 recipients. Adherent peritoneal exudate cells obtained from IFN-treated, R1-bearing mice were highly suppressive for the in vitro growth of not only R1 cells but also allogeneic and human cells. The role of macrophages in the indirect effect of IFN on tumor cell growth is discussed.

Effect of recombinant human interferon-alpha A/D on in vivo murine tumor cell growth.

We investigated the effect of human recombinant interferon-alpha A/D A/D-IFN), which is known to delay the growth of murine tumor cells, on the growth of S1 and R1 subline cells of murine Meth A fibrosarcoma in the peritoneal cavity of mice. In vitro growth of S1 cells was sensitive to, and that of R1 cells was resistant to, the direct effect of A/D-IFN, as with murine natural IFN-alpha/beta, which was used originally to isolate these sublines. In vivo, however, the growth of not only S1 cells but also R1 cells was suppressed by the administration of A/D-IFN, and the survival time of tumor-bearing mice was prolonged. Although A/D-IFN had a direct effect on S1 cells in vivo, R1 cells were susceptible only to the indirect effect via the host cells. Macrophages (M phi) harvested from the peritoneal cavity of A/D-IFN-treated mice bearing ascitic R1 cells were very effective in suppressing the in vitro growth of R1 cells; those from non-R1-bearing A/D-IFN-treated mice were less effective. The results of in vitro experiments indicate that M phi are very probably activated by the synergism of A/D-IFN and M phi diameter-activating factor(s) produced by lymphoid cells in tumor-bearing mice.