Ringer's ethyl pyruvate solution attenuates hypoperfusion and renal injury after multivisceral ischemia-reperfusion in rabbits.

Ringer's ethyl pyruvate solution (REPS) has been protective against experimental renal, intestinal, and spinal ischemia and may be useful for organ protection in major vascular surgery. The purpose of this study was to investigate whether REPS attenuates organ injury in a rabbit model of supraceliac aortic cross-clamp that simulates thoracoabdominal aortic surgery. Following the Institutional Animal Care and Use Committee's approval, 20 rabbits were undergone cross-clamping of the supraceliac thoracic aorta for 30 min, and observed for 180 min after reperfusion. Either REPS (33 mg/kg/h of ethyl pyruvate) or Ringer's lactate solution were infused throughout the study period. Arterial pressure and aortic blood flow were continuously monitored. Blood lactate concentration, serum transaminase levels, neutrophil activation, and urinary N-acetyl-beta-glucosaminidase (NAG) activity were evaluated. After reperfusion, supraceliac aortic blood flow was significantly higher, and urinary NAG was significantly lower in animals that received REPS, while the other parameters were not significantly different. In conclusion, REPS attenuated the reduction of aortic blood flow and urinary NAG elevation after the cross-clamp of supraceliac aorta.

Aberrant resting-state functional connectivity of the dorsolateral prefrontal cortex to the anterior insula and its association with fear avoidance belief in chronic neck pain patients.

Chronic neck pain (CNP), a global health problem, involves a large amount of psychological and socioeconomic burdens. Not only physical causes but also behavioral disorders such as a fear-avoidance belief (FAB) can associate with the chronicity of neck pain. However, functional brain mechanisms underlying CNP and its related behavioral disorders remain unknown. The aim of the current resting-state functional magnetic resonance imaging (fMRI) study was to explore how the functional brain networks differed between CNP patients and age- and sex-matched healthy, pain-free controls (HCs). We also investigated whether these possible brain network changes in CNP patients were associated with fear avoidance belief (FAB) and the intensity of pain. We analyzed the resting-state fMRI data of 20 CNP patients and 20 HCs. FAB and the intensity of pain were assessed by Tampa Scale for Kinesiophobia (TSK) and Visual Analog Scale (VAS) of pain. The whole brain analysis showed that CNP patients had significant different functional connectivity (FC) compared with HCs, and the right dorsolateral prefrontal cortex (DLPFC) was a core hub of these altered functional networks. Furthermore, general linear model analyses showed that, in CNP patients, the increased FC between the right DLPFC and the right anterior insular cortex (aIC) significantly associated with increased TSK (p = 0.01, statistical significance after Bonferroni correction: p<0.025), and the FC between the right DLPFC and dorsal posterior cingulate cortex had a trend of inverse association with VAS (p = 0.04). Our findings suggest that aberrant FCs between the right DLPFC and aIC associated with CNP and its related FAB.

Cesarean hysterectomy in a hybrid operating room for placenta percreta: a report of three cases.

BACKGROUND: Placenta percreta is the most severe abnormality in invasive placenta and often treated with cesarean hysterectomy. Endovascular embolization for placental abnormality is known to reduce bleeding from the placental bed and from the abnormal neovasculature surrounding the uterus. We describe three cases of placenta percreta treated with uninterrupted cesarean hysterectomy and embolization performed using a hybrid operating room (HOR). CASE DESCRIPTION: Cases were two placenta previa percretas and an impending uterine rupture with placenta percreta, treated with elective cesarean hysterectomy in HOR. Planned conversion of spinal to general anesthesia was performed after the fetal delivery. Immediate embolic devascularization of abnormal neovasculature was directly observed and facilitated adhesiolysis. Surgical blood losses were 1850 g, 2500 g, and 1180 g, respectively. CONCLUSION: Cesarean hysterectomy combined with endovascular embolization in the HOR for placenta percreta is an advantageous option to enhance patient safety by multidisciplinary approach without patient transfer.

Neutrophil gelatinase-associated lipocalin regulates gut microbiota of mice.

BACKGROUND AND AIM: Because neutrophil gelatinase-associated lipocalin (NGAL) is known to provide significant bacteriostatic effects during infectious conditions, we tested the hypothesis that this protein is up-regulated and secreted into the intraluminal cavity of the gut under critically ill conditions and is thus responsible for the regulation of bacterial overgrowth. METHODS: With our institutional approval, male C57BL/6J mouse (6-7 weeks) were enrolled and applied for lipopolysaccharide or peritonitis model compared with naïve control. We assessed NGAL protein concentrations in intestinal lumen and up-regulation of NGAL expression in intestinal tissues in in vivo as well as ex vivo settings. Simultaneously, we examined the effects of NGAL protein administration on the growth of Escherichia coli (E. coli) in in vivo and in vitro experimental settings. The localization of NGAL in intestinal tissues and lumen was also assessed by immunohistological approach using NGAL antibody. RESULTS: Both lipopolysaccharide and peritonitis insults evoked the marked up-regulation of NGAL mRNA and protein levels in gut tissues such as crypt cells. In addition, the administration of NGAL protein significantly inhibited the outgrowth of enteric E. coli under both in vitro and in vivo conditions, accompanied by histological evidence. CONCLUSION: Neutrophil gelatinase-associated lipocalin protein accompanied by apparent bacteriostatic action accumulated in the intestinal wall and streamed into the mucosal layer during critically ill state, thereby possibly shaping microbiota homeostasis in the gut.

Preferential blockade of dioxin-induced activation of the aryl hydrocarbon receptor by Antrodia camphorata.

Halogenated and polycyclic aromatic hydrocarbons are widely distributed pollutants in environments. These toxic substances activate the aryl hydrocarbon receptor (AhR) and thereby cause a broad spectrum of pathological changes. Development of AhR inhibitors will be useful for prevention of diseases caused by AhR activation. Using the dioxin responsive element (DRE)-based sensing via secreted alkaline phosphatase (DRESSA), we examined effects of Antrodia camphorata, a mycerial extract, on the activation of AhR by halogenated and polycyclic aromatic hydrocarbons. We found that Antrodia camphorata markedly suppressed activation of AhR triggered by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, activation of AhR by polycyclic aromatic hydrocarbons (benzo[a]pyrene and 3-methylcholanthrene) was inhibited only modestly by this mycelium. Similarly, Antrodia camphorata only mildly attenuated activation of AhR by cigarette smoke that contains polycyclic aromatic hydrocarbons. Consistent with these results, Northern blot analysis revealed that DRE-driven exogenous and endogenous gene expression triggered by TCDD was abolished by Antrodia camphorata, whereas it did not substantially affect DRE-induced transcription triggered by benzo[a]pyrene, 3-methylcholanthrene or cigarette smoke. We also found that the inhibitory effect of Antrodia camphorata on TCDD-induced AhR activation was ascribed to neither down-regulation of AhR, down-regulation of the AhR nuclear translocator, nor up-regulation of the AhR repressor. These results suggest that Antrodia camphorata preferentially inhibits AhR activation and DRE-dependent gene expression triggered by dioxin.

Development of salmon milt DNA/salmon collagen composite for wound dressing.

This study aims to develop a novel wound dressing comprising salmon milt DNA (sDNA) and salmon collagen (SC). The sDNA/SC composites were prepared by incubating a mixture of an acidic SC solution, an sDNA solution, and a collagen fibrillogenesis inducing buffer (pH 6.8) containing a crosslinking agent (water-soluble carbodiimide) for gelation, and a subsequent ventilation-drying process to give sDNA/SC films. The conjugation between sDNA and SC were confirmed by sDNA-elution assay and fluorescence microscopy. The sDNA/SC films with various doses of sDNA (sDNA/SC weight ratios of 1:5, 1:10, and 1:20) were used for in vitro cell cultures to evaluate their growth potentials of normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK). It was found that NHDF proliferation was increased by sDNA conjugation, whereas NHEK proliferation was dose-dependently inhibited. In light of the in vitro results, the appropriate dose of sDNA for in vivo study was determined to be the ratio of 1:10. For the implantation in full-thickness skin defects in rat dorsal region, the sDNA/SC films were reinforced by incorporating them on a porous SC sponge, because the sDNA/SC films exhibited early contraction and inadequate morphologic stability when implanted in vivo. The regenerated tissue in the sDNA/SC sponge group showed similar morphology to native dermis, while the SC sponge group without sDNA showed epithelial overgrowth, indicating that additional sDNA could reduce epidermal overgrowth. Furthermore, blood capillary formation was significantly enhanced in the sDNA/SC sponge group when compared to the SC sponge group. In conclusion, the results suggest that the sDNA/SC composite could be a potential wound dressing for clinical applications.

Tum-1, a tumstatin fragment, gene delivery into hepatocellular carcinoma suppresses tumor growth through inhibiting angiogenesis.

Since hepatocellular carcinoma (HCC) is a hypervascular cancer, anti-angiogenic therapy is a promising approach to treat HCC. In the present study, we investigated the antiangiogenic and antitumor effects of tum-1, a fragment of tumstatin, gene transduction into HCC in vitro and in vivo. Tum-1 gene was cloned into a pSecTag2B mammalian expression vehicle to construct pSecTag2B-tum-1. pSecTag2B-tum-1 or vehicle were transfected into human HCC cells, PLC/PRF/5 cells stably and Huh-7 cells tran-siently. pSecTag2B-tum-1 transfection slightly repressed the proliferation of both PLC/PRF/5 and Huh-7 cells in vitro. Addition of conditioned media (CM) from tum-1 expressing PLC/PRF/5 cells significantly inhibited the spontaneous and vascular endothelial growth factor (VEGF)-induced proliferation and migration of human umbilical vein endothelial cells (HUVEC) in vitro with diminishing the VEGF-induced phosphorylation of both Akt and extracellular signal-regulated kinase (ERK) that are known to mediate VEGF-induced proliferation and migration of endothelial cells. In in vivo experiments, intratumoral injection of pSecTag2B-tum-1 significantly repressed the growth of pre-established Huh-7 tumors in athymic mouse models accompanying the decreased density of CD34 positive vessels in tumors. In conclusion, our results suggest that antiangiogenic gene therapy using tum-1 gene may be an efficient strategy for the treatment of HCC.

Abrogation of constitutive STAT3 activity sensitizes human hepatoma cells to TRAIL-mediated apoptosis.

BACKGROUND/AIMS: Signal transducer and activator of transcription 3 (STAT3) is constitutively activated and regulates cell growth and survival of various cancer cells. We investigated the anti-tumor effect of AG490, a Janus kinase 2 specific inhibitor, inhuman hepatoma cells. METHODS: Effects of AG490 on STAT3 activation, on cell-growth and survival, and on the expression of cell-cycle- and apoptosis-related proteins were evaluated in Huh-1, Huh-7, HepG2 and Hep3B cells. Next, whether AG490 renders hepatoma cells susceptible to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was examined in vitro and in vivo. RESULTS: Constitutively activated STAT3 through tyrosine phosphorylation was detected in all hepatoma cells. AG490 inhibited the phosphorylation of STAT3 and its activity. AG490 induced cell cycle arrest in Huh-1, Huh-7 and HepG2 through cyclin D1 downregulation, and induced marked apoptosis in Hep3B. AG490 downregulated at least one of the anti-apoptotic proteins, Bcl-xL, survivin or XIAP in all hepatoma cells. AG490 sensitized Huh-1, Huh-7 and HepG2 to TRAIL-induced apoptosis in vitro. Intraperitoneal injection of AG490, the combination of AG490 and TRAIL more greatly, repressed the growth of subcutaneous Huh-7 tumors in athymic mice. CONCLUSIONS: Abrogation of constitutive activation of STAT3 by AG490 enhances the anti-tumor activity of TRAIL against human hepatoma cells.

Association between liver fibrosis and insulin sensitivity in chronic hepatitis C patients.

BACKGROUND: Several clinical studies have suggested a possible link between chronic hepatitis caused by hepatitis C virus (HCV) and the development of diabetes mellitus. We investigated the association between liver fibrosis and glucose intolerance in HCV-infected patients by measuring insulin sensitivity and beta-cell function. METHOD: A total of 83 chronic HCV-infected patients were recruited into this study. We evaluated insulin sensitivity and beta-cell function of all patients in a fasting state (homeostasis model assessment of insulin resistance [HOMA-R] and homeostasis model assessment of beta-cell function [HOMA-beta]) and after an oral load of 75 g glucose (whole-body insulin sensitivity index [WBISI] and Delta-insulin/Delta-glucose 30). RESULTS: In a multivariate analysis, severe fibrosis was the only independent factor associated with insulin resistance. There were significant differences in both HOMA-R (P= 0.0063) and WBISI (P= 0.0159) between patients with mild fibrosis (N = 34) and those with severe fibrosis (N = 49). Although HOMA-beta was increased significantly in the subjects with severe fibrosis compared with those with mild fibrosis (P= 0.0169), Delta-insulin/Delta-glucose 30 showed no significant difference in stage of liver fibrosis, suggesting an uncertain association between liver fibrosis and beta-cell function. CONCLUSION: Our findings suggest that the development of liver fibrosis is associated with insulin resistance in HCV-infected patients.

Aging of patients with hepatitis C virus-associated hepatocellular carcinoma: long-term trends in Japan.

The incidence of hepatocellular carcinoma (HCC) in Japan has been increasing. The aim of the present study was to analyze epidemiological changes in Japanese HCC patients. A total of 463 patients with HCC diagnosed at our hospital between 1982 and 2001 were recruited for this study. Cohorts of patients with HCC were categorized into intervals of five years. The number of HBV- and HCV-associated HCC cases had decreased and increased in 1987-1991, respectively, and thereafter reached a plateau. The mean age of patients at diagnosis of HCV-associated HCC showed a steady significant increase from 60 to 68 years of age during the period, suggesting that these findings were associated with a shift toward an older-age group that had the highest rate of HCV infection. The mean age of patients with other types of HCC did not significantly change during the period. Since it is known that the prevalence of HCV infection in young Japanese persons is low and that the incidence of HCV infection is very low at present, our findings may indicate that the prevalence of HCC will decline in Japan, an advanced country with regard to HCV-associated HCC, in the near future.

The impact of newer treatment modalities on survival in patients with hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. However, although the therapeutic approaches for HCC have progressed rapidly, it remains unknown whether the current management of patients with HCC has reduced its mortality. We analyzed changes of survival rate in patients with HCC over a 20-year period. METHODS: Between 1982 and 2001, 463 patients were diagnosed with HCC at our hospital. Subjects were enrolled in the current cohort according to the following inclusion criteria: HCC lesion measuring less than 3 cm in diameter, no evidence of extrahepatic metastasis, and no evidence of main portal vein infiltration/thrombosis. A total of 257 patients with HCC were recruited for this study, and categorized into 5-year intervals. RESULTS: The survival rates improved significantly during the study period. When the patients were stratified according to Child-Pugh score, only patients with Child's B showed improved survival rates. Furthermore, patients with surgical resection or transarterial chemoembolization during the latter period had a better prognosis than those during the early period. CONCLUSIONS: Our findings suggest that the development of therapeutic interventions for HCC have led to improvements in the prognosis for HCC patients.

DHMEQ, a novel NF-kappaB inhibitor, induces apoptosis and cell-cycle arrest in human hepatoma cells.

Several reports have indicated that nuclear factor-kappaB (NF-kappaB) is constitutively activated in a variety of cancer cells including hepatoma cells and plays a key role in their growth and survival. Dehydroxymethylepoxyquinomicin (DHMEQ) derived from the structure of an antibiotic epoxyquinomicin C is a novel NF-kappaB inhibitor. In the present study, we evaluated the effect of DHMEQ on the NF-kappaB activity in human hepatoma cells, Huh-7, HepG2 and Hep3B, and the anti-tumor effect of DHMEQ on these cells in vitro and in vivo. DHMEQ inhibited the steady-state transcriptional activity of NF-kappaB in all hepatoma cells. DHMEQ blocked the constitutive DNA-binding activity and TNF-alpha-mediated nuclear translocation of NF-kappaB in Huh-7 cells. DHMEQ (5-20 microg/ml) dose-dependently reduced the viable cell number of all hepatoma cells. DHMEQ (20 microg/ml) induced apoptosis in all hepatoma cells, especially in Hep3B cells, and cell-cycle arrest in Huh-7 and HepG2 cells. These effects were accompanied by downregulation of proteins involved in anti-apoptosis (Bcl-xL, XIAP or c-IAP2) and cell-cycle progression (cyclin D1), and induction of proteins involved in pro-apoptosis (Bax) and cell-cycle retardation (p21Waf1/Cip1), although the degree of changes by DHMEQ was different in each hepatoma cell type. Moreover, intraperitoneal administration of DHMEQ (8 mg/kg) significantly repressed the growth of Huh-7 tumor subcutaneously transplanted into BALB/c nu/nu athymic mice. Our results suggest that DHMEQ could qualify as a candidate for a new chemotherapeutic agent against human hepatoma.

Clinical benefits of hepatocellular carcinoma surveillance: a single-center, hospital-based study.

Although there is no definitive evidence that hepatocellular carcinoma (HCC) screening in high-risk groups improves survival, many physicians screen high-risk populations with various tools such as alpha-fetoprotein (AFP) and ultrasonography (USG). The aim of this study was to clarify clinical differences between HCC patients diagnosed by surveillance and those with incidentally detected HCC. Two hundred and seventy-one Japanese patients with HCC diagnosed between January 1991 and December 2001 were recruited. They were categorized into two groups: 178 patients (group 1) had subclinical HCC diagnosed by surveillance and 93 patients (group 2) presented with incidentally detected HCC. The tumor size was significantly smaller in group 1 compared to that of group 2 (2.8 vs. 5.6 cm; P<0.0001). A significantly higher proportion of patients in group 2 had multiple HCC and portal vein infiltration when compared to group 1. Eighty-six (48.3%) group 1 patients and 16 (17.2%) group 2 patients underwent local ablation treatment, which is a curative treatment available for small HCCs (P<0.0001). The cumulative actuarial survival rate was significantly higher in group 1 than in group 2 (P=0.0091). Early detection of HCC by surveillance may contribute to a greater chance of receiving effective treatment and prolonged survival, although a further prospective, randomized study is needed.

Comprehensive analysis of the effect of phytoestrogen, daidzein, on a testicular cell line, using mRNA and protein expression profile.

In this study, we examined the effects of exposure to phytoestrogen (daidzein), 17beta-estradiol (E2), diethylstilbestrol (DES) and staurosporin on the TM4 testicular cell line, using comprehensive analysis, such as cDNA microarray and two-dimension polyacrylamide gel electropholesis (2D-PAGE) analysis, and we demonstrated if these toxicogenomic analyses could classify the chemical compounds. First, RNA was extracted from TM4 cells that had been treated with daidzein (80 microM), DES, E2 (40 microM) and stauroporin (100 nM) for 30 min. We performed cDNA microarray analysis, and the expression ratio data thus obtained were then analyzed using hierarchical clustering. This hierarchical clustering showed that daidzein exposure induced a different effect on gene expression change from that of E2, DES and staurosporin. Next, protein extracted from TM4 cells also underwent cDNA microarray analysis for 3 h. We performed 2D-PAGE analysis, and the spot intensity ratio data thus obtained were analyzed using hierarchical clustering. As with cDNA microarray, the hierarchical clustering of protein spot ratios showed that daidzein exposure induced a different effect on gene expression change from that of the other substances. In conclusion, we have demonstrated for the first time that classification of these chemicals can be performed by clustering analysis, using data from cDNA microarray and 2D-PAGE analyses, and that exposure to daidzein induces effects different from those of E2, DES and staurosporin.

Antiangiogenic property of pigment epithelium-derived factor in hepatocellular carcinoma.

Pigment epithelium-derived factor (PEDF) is one of the most powerful endogenous antiangiogenic reagents discovered to date. Its antiangiogenic potential in neoplastic disease remains unclear. In this study, we investigated antiangiogenic property of PEDF in hepatocellular carcinoma (HCC), a typical hypervascular tumor. In HCC cell lines, constitutive messenger RNA and protein expression of PEDF varied. Genomic DNA encoding the PEDF gene was the same in the cell lines examined by Southern blotting. In chemically induced hypoxic conditions, secreted PEDF protein was suppressed in contrast to elevation of vascular endothelial growth factor protein. When PEDF was overexpressed by gene transfer, proliferation and migration of endothelial cells were inhibited in conditioned media derived from all HCC cell lines. However, the serum concentration of PEDF, as measured by enzyme-linked immunosorbent assay, was decreased in patients with cirrhosis or HCC complicated by cirrhosis compared to healthy volunteers and patients with chronic hepatitis. According to the endothelial cell proliferation assay, the serum PEDF of patients with HCC had antiangiogenic activity. Moreover, intratumoral injection of a PEDF-expressing plasmid in athymic mouse models caused significant inhibition of preestablished tumor growth. In conclusion, PEDF plays a role in the angiogenic properties of HCC. Reduction of serum PEDF concentration associated with the development of chronic liver diseases may contribute to the progression of HCC. In addition, gene therapy using PEDF may provide an efficient treatment for HCC.

A ruthenium-grafted hydrotalcite as a multifunctional catalyst for direct alpha-alkylation of nitriles with primary alcohols.

Treatment of a hydrotacite, Mg6Al2(OH)16CO3, with an aqueous solution of RuCl3.nH2O afforded a monomeric Ru(IV) species on the surface of the hydrotalcite. This novel Ru-grafted hydrotalcite (Ru/HT) efficiently catalyzed alpha-alkylation of nitriles with primary alcohols through the cooperative catalysis between the Ru species and the surface base sites. The catalyst system could be further extended for the one-pot synthesis of alpha,alpha-dialkylated phenylacetonitriles via the base-catalyzed Michael reaction of alpha-alkylated phenylacetonitrile with activate olefins.

Interferon alpha inhibits the nuclear factor kappa B activation triggered by X gene product of hepatitis B virus in human hepatoma cells.

X gene product of hepatitis B virus (HBV) (HBx) regulates many transcription factors including nuclear factor kappa B (NF-kappaB) and plays a key role in hepatocarcinogenesis. In this study, we demonstrated that the expression of full HBV genome and HBx gene similarly stimulated the transcriptional activity of NF-kappaB in HuH-7 human hepatoma cells, and that interferon (IFN)-alpha as well as dominant negative mutant of IkappaB kinase-alpha effectively inhibited the HBx-mediated NF-kappaB activation, but IFN-gamma did not. These results suggest that IFN-alpha may have a function to block the NF-kappaB activating pathway triggered by HBx in HBV hepatocytes.