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Metabolic reprogramming is a hallmark of cancer, including hepatocellular carcinoma (HCC). However, its role in HCC remains to be elucidated. Herein, we identified GTP cyclohydrolase 1 (GCH1), the first rate-limiting enzyme in tetrahydrobiopterin (BH4) de novo biosynthesis, as a novel metabolic regulator of HCC. GCH1 was frequently down-regulated in HCC tissues and cell lines by promoter methylation. Low GCH1 expression was associated with larger tumor size, increased tumor number, and worse prognosis in two independent cohorts of HCC patients. Functionally, GCH1 silencing promoted HCC growth in vitro and in vivo, while GCH1 overexpression exerted an opposite effect. The metabolite BH4 inhibited HCC growth in vitro and in vivo. GCH1 silencing exerted its growth-promoting effect through directly inhibiting BH4 de novo biosynthesis. Mechanistically, GCH1 silencing activated ASK1/p38 signaling; pharmacological or genetic inhibition of ASK1 or p38 abolished GCH1 silencing-induced growth-promoting effect. Further mechanistic studies found that GCH1 silencing-induced BH4 reduction resulted in an increase of intracellular superoxide anion levels in a dose-dependent manner, which mediated the activation of ASK1/p38 signaling. Collectively, our study reveals that epigenetic silencing of GCH1 promotes HCC growth by activating superoxide anion-mediated ASK1/p38 signaling via inhibiting BH4 de novo biosynthesis, suggesting that targeting GCH1/BH4 pathway may be a promising therapeutic strategy to combat HCC.

Adenoid hypertrophy is a pathological hyperplasia of the adenoids, which may cause snoring and apnea, as well as impede breathing during sleep. The lateral cephalogram is commonly used by dentists to screen for adenoid hypertrophy, but it is tedious and time-consuming to measure the ratio of adenoid width to nasopharyngeal width for adenoid assessment. The purpose of this study was to develop a screening tool to automatically evaluate adenoid hypertrophy from lateral cephalograms using deep learning. We proposed the deep learning model VGG-Lite, using the largest data set (1,023 X-ray images) yet described to support the automatic detection of adenoid hypertrophy. We demonstrated that our model was able to automatically evaluate adenoid hypertrophy with a sensitivity of 0.898, a specificity of 0.882, positive predictive value of 0.880, negative predictive value of 0.900, and F1 score of 0.889. The comparison of model-only and expert-only detection performance showed that the fully automatic method (0.07 min) was about 522 times faster than the human expert (36.6 min). Comparison of human experts with or without deep learning assistance showed that model-assisted human experts spent an average of 23.3 min to evaluate adenoid hypertrophy using 100 radiographs, compared to an average of 36.6 min using an entirely manual procedure. We therefore concluded that deep learning could improve the accuracy, speed, and efficiency of evaluating adenoid hypertrophy from lateral cephalograms.

Multiple mechanisms are involved in regulating hepatic ischemia‑reperfusion injury (IRI), in which Kupffer cells (KCs), which are liver‑resident macrophages, play critical roles by regulating inflammation and the immune response. Suberoylanilide hydroxamic acid (SAHA), a pan‑histone deacetylase inhibitor, has anti‑inflammatory effects and induces autophagy. To investigate whether SAHA ameliorates IRI and the mechanisms by which SAHA exerts its effects, an orthotopic liver transplantation (OLT) rat model was established after treatment with SAHA. The results showed that SAHA effectively ameliorated OLT‑induced IRI by reducing M1 polarization of KCs through inhibition of the AKT/glycogen synthase kinase (GSK)3β/NF‑κB signaling pathway. Furthermore, the present study found that SAHA upregulates autophagy 5 protein (ATG5)/LC3B in KCs through the AKT/mTOR signaling pathway and inhibition of autophagy by knockdown of ATG5 in KCs partly impaired the protective effect of SAHA on IR‑injured liver. Therefore, the current study demonstrated that SAHA reduces M1 polarization of KCs by inhibiting the AKT/GSK3β/NF‑κB pathway and upregulates autophagy in KCs through the AKT/mTOR signaling pathway, which both alleviate OLT‑induced IRI. The present study revealed that SAHA may be a novel treatment for the amelioration of OLT‑induced IRI.

Insufficient high-intensity focused ultrasound (HIFU) can promote the rapid progression of the residual tumor through the hypoxia inducible factor-2α +(HIF-2α)/vascular endothelial growth factor A (VEGFA)/ephrin type-A receptor 2 (EphA2) pathway. Although sorafenib has been shown to significantly improve the survival of patients with advanced liver cancer, the use of sorafenib in residual tumor tissues following HIFU has rarely been elucidated. Thus, this study aimed to investigate the potential adjuvant therapeutic effects of sorafenib following HIFU in order to reduce the relapse rate following insufficient HIFU.Xenograft tumors were established using nude mice injected with liver cancer cells. At approximately 4 weeks after the inoculation of the tumor cells (tumors reached 1.3-1.5 cm), all mice were randomly divided into 3 groups as follows: i) The control group (no treatment); ii) the HIFU-alone group, and iii) the combination group (HIFU + sorafenib), with 6 mice per group. The residual tumor volume was determined among the different treatment groups. The protein expression levels of HIF-2α, VEGFA and EphA2 were determined by immunohistochemistry and western blotting, and the mRNA levels were detected by RT-qPCR. The microvessel density (MVD) was calculated by CD31 immunohistochemistry staining.The results revealed that by comparing the control group, insufficient HIFU promoted HIF-2α, VEGFA and EphA2 expression (P0.05). Compared with the HIFU-alone group, the protein and mRNA levels of HIF-2α, VEGFA and EphA2 were markedly decreased in the group that received combined treatment with HIFU and sorafenib (P0.05). Similar results were obtained for MVD expression. Synergistic tumor growth inhibitory effects were also observed between the control group and HIFU group (P0.05).The findings of this study demonstrate that the expression of HIF-2α, VEGFA and EphA2 can be inhibited by sorafenib, and that sorafenib is likely to provide an effective adjunct treatment for patients with HCC following HIFU ablation.

Cigarette smoke is a known risk factor for urothelial carcinoma (UC). However, there is limited information about the distributions and effects of volatile organic compounds (VOCs) on smoking-related UC risk. With this hospital-based case-control study, we explored the associations between urinary levels of cotinine and VOC metabolites (acrylamide, 1,3-butadiene, and benzene) and the risk of UC. Urological examinations and pathological verifications were used to confirm the diagnoses of UC. All study participants provided smoking-related information via questionnaires and face-to-face interviews; they also provided urine samples for the measurement of VOC metabolites, cotinine, and 8-hydroxydeoxyguanosine (8-OHdG), which was used as an indicator of oxidative stress. We applied multiple logistic regression analysis to estimate the risk of UC, and we found that levels of urinary cotinine and 8-OHdG were higher in the UC group than in the control group. Furthermore, urinary levels of VOC metabolites, including N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA), N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine, N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine-3, trans,trans-muconic acid (t,t-MA), and S-phenylmercapturic acid (SPMA), increased with increasing levels of urinary cotinine. After adjusting for potential risk factors, dose–response relationships were observed between UC risk and urinary levels of AAMA, t,t-MA, SPMA, and 8-OHdG. Participants with high urinary levels of cotinine, AAMA, t,t-MA, SPMA, and 8-OHdG had risks of UC that were 3.5- to 6-fold higher than those of participants with lower levels. Future, large-scale investigations of the risks of UC should be explored, and repeated measurement of VOC metabolites should be assessed.

AIM To evaluate the prognostic value of the number of retrieved lymph nodes (LNs) and other prognostic factors for patients with distal cholangiocarcinomas, and to determine the optimal retrieved LNs cut-off number. METHODS The Surveillance, Epidemiology and End Results database was used to screen for patients with distal cholangiocarcinoma. Patients with different numbers of retrieved LNs were divided into three groups by the X-tile program. X-tile from Yale University is a useful tool for outcome-based cut-point optimization. The Kaplan-Meier method and Cox regression analysis were utilized for survival analysis. RESULTS A total of 449 patients with distal cholangiocarcinoma met the inclusion criteria. The Kaplan-Meier survival analysis for all patients and for N1 patients revealed no significant differences among patients with different retrieved LN counts in terms of overall and cancer-specific survival. In patients with node-negative distal cholangiocarcinoma, patients with four to nine retrieved LNs had a significantly better overall (P = 0.026) and cancer-specific survival (P = 0.039) than others. In the subsequent multivariate analysis, the number of retrieved LNs was evaluated to be independently associated with survival. Additionally, patients with four to nine retrieved LNs had a significantly lower overall mortality risk [hazard ratio (HR) = 0.39; 95% confidence interval (CI): 0.20-0.74] and cancer cause-specific mortality risk (HR = 0.32; 95%CI: 0.15-0.66) than other patients. Additionally, stratified survival analyses showed persistently better overall and cancer-specific survival when retrieving four to nine LNs in patients with any T stage of tumor, a tumor between 20 and 50 mm in diameter, or a poorly differentiated or undifferentiated tumor, and in patients who were ≤ 70-years-old. CONCLUSION The number of retrieved LNs was an important independent prognostic factor for patients with node-negative distal cholangiocarcinoma. Additionally, patients with four to nine retrieved LNs had better overall and cancer-specific survival rates than others, but the reason and mechanism were unclear. This conclusion should be validated in future studies.

// Kun He 1 , Xiwen Zhu 1 , Yan Liu 4 , Chunmu Miao 1 , Tao Wang 1 , Peizhi Li 1 , Lei Zhao 2 , Yaxi Chen 2 , Junhua Gong 1 , Can Cai 4 , Jinzheng Li 1 , Shengwei Li 1 , Xiong Z. Ruan 2, 3 , Jianping Gong 1 1 Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China 2 Centre for Lipid Research, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China 3 Centre for Nephrology, University College London (UCL) Medical School, Royal Free Campus, London, United Kingdom 4 Department of Gastroenterology and Hepatology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China Correspondence to: Jianping Gong, email: gongjianping11@126.com Shengwei Li, email: lishengwei11@163.com Keywords: NLRP3 inflammasome, Kupffer cell, non-alcoholic fatty liver disease, IL-1β Received: March 20, 2017 Accepted: April 17, 2017 Published: April 27, 2017 ABSTRACT NOD-like receptor (NLR) NLRP3 inflammasome activation has been implicated in the progression of non-alcoholic fatty liver disease (NAFLD) from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). It has been also shown that palmitic acid (PA) activates NLRP3 inflammasome and promotes interleukin-1β (IL-1β) secretion in Kupffer cells (KCs). However, the specific mechanism of the NLRP3 inflammasome activation is unclear. We studies the molecular mechanisms by investigating the roles of Thioredoxin-interacting protein (TXNIP) and NLRP3 on NAFLD development in patients, high-fat diet (HFD)-induced NAFL and methionine choline deficient (MCD) diet-induced NASH in wild type (WT), TXNIP −/− (thioredoxin-interacting protein) and NLRP3 −/− mice, and isolated KCs. We found that the expressions of NLRP3 and TXNIP in human liver tissues were higher in NASH group than in NAFL group. Furthermore, co-immunoprecipitation analyses show that activation of the TXNIP-NLRP3 inflammasome protein complex occurred in KCs of NASH WT mice rather than NAFL WT mice, thus suggesting that the formation and activation of this protein complex is mainly involved in the development of NASH. NLRP3 −/− mice exhibited less severe NASH than WT mice in MCD diet model, whereas TXNIP deficiency enhanced NLRP3 inflammasome activation and exacerbated liver injury. PA triggered the activation and co-localization of the NLRP3 inflammasome protein complex in KCs isolated from WT and TXNIP −/− but not NLRP3 −/− mice, and most of the complex co-localized with mitochondria of KCs following PA stimulation. Taken together, our novel findings indicate that TXNIP plays a protective and anti-inflammatory role in the development of NAFLD through binding and suppressing NLRP3.

Background Perivascular adipose tissue (PVAT) accelerates plaque progression and increases cardiovascular risk. We tested the hypothesis that PVAT contributed to plaque vulnerability and investigated whether endoplasmic reticulum stress (ER stress) in PVAT played an important role in vulnerable plaque. Methods We transplanted thoracic aortic PVAT or subcutaneous adipose tissue as a control, from donor mice to carotid arteries of recipient apolipoprotein E deficient (apoE−/−) mice after removing carotid artery collar placed for 6 weeks. Two weeks after transplantation, ER stress inhibitor 4-phenyl butyric acid (4-PBA) was locally administrated to the transplanted PVAT and then animals were euthanized after 4 weeks. Immunohistochemistry was performed to quantify plaque composition and neovascularization. Mouse angiogenesis antibody array kit was used to test the angiogenic factors produced by transplanted adipose tissue. In vitro tube formation assay, scratch wound migration assay and mouse aortic ring assay were used to assess the angiogenic capacity of supernatant of transplanted PVAT. Results Ultrastructural detection by transmission electron microscopy showed transplanted PVAT was a mixed population of white and brown adipocytes with abundant mitochondria. Transplanted PVAT increased the intraplaque macrophage infiltration, lipid core, intimal and vasa vasorum neovascularization and MMP2/9 expression in plaque while decreased smooth muscle cells and collagen in atherosclerotic plaque, which were restored by local 4-PBA-treatment. Antibody array analysis showed that 4-PBA reduced several angiogenic factors [Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), MCP-1, IL-6] secreted by PVAT. Besides, conditioned medium from 4-PBA treated-PVAT inhibited tube formation and migration capacity of endothelial cells and ex vivo mouse aortic ring angiogenesis compared to conditioned medium from transplanted PVAT. mRNA expression and protein levels of GM-CSF were markedly elevated in adipocytes under ER stress which would be suppressed by 4-PBA. In addition, ER stress enhanced NF-κB binding to the promoter of the mouse GM-CSF gene in adipocytes confirmed by Chromatin immunoprecipitation analyses. Conclusions Our findings demonstrate that ER stress in PVAT destabilizes atherosclerotic plaque, in part through increasing GM-CSF paracrine via transcription factor NF-κB. Electronic supplementary material The online version of this article (10.1186/s12967-018-1481-z) contains supplementary material, which is available to authorized users.

Rapid growth of residual tumors can occur as a result of their recurrence and progression. The present study aimed to investigate the expression of hypoxia inducible factor-2 subunit α (HIF-2α), vascular endothelial growth factor A (VEGFA), erythropoietin-producing hepatocellular A2 (EphA2) and angiogenesis in residual hepatocellular carcinoma (HCC), following treatment with high-intensity focused ultrasound (HIFU) ablation, in order to investigate the association between protein expression and tumor recurrence and growth. Athymic BALB/c (nu/nu) mice were subcutaneously inoculated with the HCC cell line HepG2, in order to create xenograft tumors. Approximately 30 days post-inoculation, eight mice were treated with HIFU, whereas eight mice received no treatment and acted as the control group. Residual tumor tissues were obtained from the experimental groups after one month. Levels of HIF-2α, VEGFA, EphA2 and cluster of differentiation 31 (CD31) expression was measured by immunohistochemical staining. CD31-positive vascular endothelial cells were counted to calculate microvascular density (MVD), and western blot analysis was performed to determine levels of HIF-2α, VEGFA, and EphA2 protein. It was found that the expression levels of HIF-2α, VEGFA, EphA2, and MVD proteins in residual HCC tissues were significantly higher than in the control group tissues (P

Background To explore the anti-tumor effect of high-intensity focused ultrasound (HIFU) combined with herpes simplex virus thymidine kinase (HSV-TK) gene-loaded ultrasound-targeted microbubbles on VX2 rabbit liver tumors. Methods Seventy-five New Zealand white rabbits were randomly divided into five groups after the models of VX2 rabbit liver tumors were established: (a) HIFU group; (b) HIFU and HSV-TK group (HIFU + HSV-TK); (c) HIFU, HSV-TK and ultrasound group (HIFU + HSV-TK + US); (d) HIFU, HSV-TK gene-loaded microbubbles and ultrasound group (HIFU + HSV-TK-MBs + US); and (e) HSV-TK gene-loaded microbubbles and ultrasound group (HSV-TK-MBs + US). After 2 weeks of VX2 liver tumor implantation, rabbits in groups (a), (b), (c) and (d) received HIFU to establish rabbit models of residual tumor by ablating 80% of the tumor volume. After HIFU ablation, rabbits in different groups received MBs wrapped around HSV-TK or HSV-TK solution via marginal ear veins and/or local ultrasonic irradiation to the tumor. Six rabbits in each group were sacrificed 48 h after the corresponding treatment, and tumors were extracted for in vitro experiments. Thymidine kinase mRNA was detected by the real-time polymerase chain reaction. The green fluorescent protein expression in liver tumor was detected by western blotting and immunohistochemistry. Tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling. The growth curves of VX2 liver tumors and survival curves of rabbits were compared. Results Forty-eight hours after treatment, TK mRNA and protein were the highest in the HIFU + HSV-TK + US + MBs group and the HSV-TK + US + MBs group (p

A cDNA encoding a novel cysteine proteinase inhibitor (CPI) was isolated from a gland mutant Xiangmian-18 of upland cotton during the pigments gland forming stage. The cDNA comprises 378 bp and encodes 125 amino acid residues with molecular mass of 13.8 kDa. It contains the conserved motif of cysteine protease inhibitors and belongs to the cystatin superfamily (Gln-Val-Val-Ala-Gly). The deduced amino acid sequences of the domains are highly similar to the normal upland cotton (96.8%). SDS-PAGE and western hybridization analysis showed that the expressed recombinant protein was recombinant CPI. The inhibitory activity of recombinant CPI was 46 u/μg which was measured by inhibiting the protease activity of papain. RT-PCR results indicated that the expression level of developing gland stage was higher than that of undeveloped gland stage.

The cartilage tissue engineering is an inspiring and profitable way for the reconstruction of cartilage defects, but it has been hampered by two large obstacles: how to get qualified seed cells and credible scaffold. This study aimed to evaluate the chondrogenic potential of rat bone marrow stromal cells (BMSCs) by loading them on alginate gel. In this study, the compounds of SD rat BMSCs and alginate gel were injected on the dorsum of rats subcutaneously. The implantations were harvested and examined by histological and immunohistochemical examination, in situ hybridization and transmission electron microscopy at different time points after the operations. The results showed that the compounds of BMSCs and alginate gel are promising for cartilage tissue engineering applications.

Normal hepatocytes express connexin32 (Cx32), which forms gap junctions at cell‑cell contact areas. The aim of the present study was to investigate whether Cx32 mediates the cell death‑inducing effects of ultrasound microbubbles carrying the herpes simplex virus thymidine kinase (HSV‑TK) suicide gene against hepatocellular carcinoma cells in vitro and in vivo. HepG2 cells were exposed to different concentrations of trans‑retinoic acid (ATRA) in culture, to evaluate the intrinsic antitumor effect of ATRA. Detailed in‑vitro and in‑vivo investigations on the antitumor effects of ATRA via Cx32 mediation were performed, and the possible underlying mechanisms of action of the compound were then examined. The gene expression of HSV‑TK transfected by ultrasound wave irradiation in the HepG2 cells was quantified using reverse transcription‑quantitative polymerase chain reaction analysis. The effects on cell death were assessed using an MTT assay. The protein expression levels of Cx32 in ATRA‑untreated or ATRA‑treated tissues were quantified by immunohistochemical analysis and Western blot assays. The HSV‑TK gene was successfully transfected into the HepG2 cell using ultrasound wave irradiation, and was stably expressed. Compared with the other groups, the HSV‑TK gene group treated with ATRA exhibited an increased number of apoptotic cells (P

Hydroxyapatite-tricalcium phosphate (HA-TCP) is a new kind of material which shows good biocompatibility, biological degradability, and porosity. This study aimed to determine the effectiveness of HA-TCP as a bone tissue engineering scaffold. In this study, critical size cranial defects were reconstructed with compounds of autogenous bone marrow stromal cells (BMSCs) and HA-TCP. The resulting grafts were examined by X-ray, histological examination, semi-quantitative analysis of osteogenesis, immunochemical examination (collagen type I and III), scanning electron microscopy and transmission electron microscopy. The results showed that HA-TCP is a good bone tissue engineering scaffold and BMSCs/HA-TCP is a promising technique for reconstruction of bone defects.

Background High-intensity focused ultrasound (HIFU) is a widely applied to treatment for unresectable hepatocellular carcinoma. However, insufficient HIFU can result in rapid progression of the residual tumor. The mechanism of such rapid growth of the residual tumor after HIFU ablation is poorly understood. Objective The aim of this study was to investigate the dynamic angiogenesis of residual tumor, and the temporal effect and mechanism of the HIF-1, 2α in the residual tumor angiogenesis. Methods Xenograft tumors of HepG2 cells were created by subcutaneously inoculating nude mice (athymic BALB/c nu/nu mice) with hepatoma cells. About thirty days after inoculation, all mice (except control group) were treated by HIFU and assigned randomly to 7 groups according to various time intervals (1st, 3rd, 5th day (d) and 1st, 2nd, 3rd, 4th week (w)). The residual tumor tissues were obtained from the experimental groups at various time points. Protein levels of HIF-1α, HIF-2α, VEGF-A, and EphA2 were quantified by immunohistochemistry analysis and Western Blot assays, and mRNA levels measured by Q-PCR. Microvascular density was calculated with counting of CD31 positive vascular endothelial cells by immunohistochemical staining. Results Compared with the control group, protein and mRNA levels of HIF-1α reached their highest levels on the 3rd day (P

Systemic inflammation affects hemostasis during severe acute pancreatitis (SAP). A hypercoagulable state occurs more frequently in SAP, which is not fully detected by traditional coagulation testing.The aim of this study was to evaluate the contribution of clot formation and lysis (CloFAL) assay to improve monitoring of global coagulation in patients with SAP.Twenty-five patients with SAP who were treated from December 2009 to April 2011 were studied. Plasma was collected at the time of admission, and CloFAL was measured using the CloFAL analyzer. The parameters evaluated include coagulation time (CT), fibrinolysis time (FT), and maximum amplitude (MA), from which the accelerating coagulation extent (ACE, MA/CT), accelerating fibrinolytic extent (AFE, MA/FT), and balance level exponent (BLE, ACE/AFE) were calculated. In addition, laboratory values for the traditional coagulation testing were measured. Values were compared to a control group of 20 healthy subjects.The MA, FT, ACE, and BLE values of the CloFAL assay were significantly increased in the SAP group compared to the control group (p\0.05 for all measurements). For the traditional coagulation testing, fibrinogen, plasminogen, and D-dimer levels were higher in patients in the SAP group compared to the control group (p\0.05).Our findings using the CloFAL analyzer indicate that the hypercoagulable state was due to increased fibrin generation and invariable fibrinolysis in patients with SAP. CloFAL assay is a simple and useful global coagulation assay to monitor hypercoagulable states during SAP.

Objective The purpose of the study was to explore the anti-tumor effect of ultrasound -targeted microbubble destruction mediated herpes simplex virus thymidine kinase (HSV-TK) suicide gene system on mice hepatoma. Methods Forty mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were estabilished: (1) PBS; (2) HSV-TK (3) HSV-TK+ ultrasound (HSV-TK+US); (4) HSV-TK+ultrasound+microbubbles (HSV-TK+US+MB). The TK protein expression in liver cancer was detected by western-blot. Applying TUNEL staining detected tumor cell apoptosis. At last, the inhibition rates and survival time of the animals were compared among all groups. Results The TK protein expression of HSV-TK+MB+US group in tumor-bearing mice tissues were significantly higher than those in other groups. The tumor inhibitory effect of ultrasound-targeted microbubble destruction mediated HSV-TK on mice transplantable tumor was significantly higher than those in other groups (p < 0.05), and can significantly improve the survival time of tumor-bearing mice. Conclusion Ultrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on tumors, which provides a new strategy for gene therapy in liver cancer.

Van der Woude syndrome (VWS) (OMIM 119300) is a dominantly inherited, developmental disorder that is characterized by pits and/or sinuses of the lower lip and a cleft lip and/or cleft palate. Mutations in the interferon regulatory factor 6 gene ( IRF6) have been recently identified in patients with VWS, with more than 60 mutations reported. However, the VWS phenotype, IRF6 mutation genotypes, and their interrelationships in Chinese VWS patients have not been studied. Here, we report 11 Chinese families with variable clinical phenotypes of VWS and identified mutations in all patients. Of the 11 mutations, 8 appeared to be novel: CC5.6GT, T342A, 566delA, C748T, C756A, C989A, C1209G, and 1316delT. Seven mutations caused a change or loss of the IRF6 domain. The marked phenotypic variation may be caused by the action of certain modifier genes on IRF6 function. Abbreviations: VWS, Van der Woude syndrome; IRF6, interferon regulatory factor 6; CL/P, cleft lip and/or cleft palate; DBD, DNA-binding domain; SMIR, Smad-interferon regulatory factor-binding domain; Kb, kilobase; PCR, polymerase chain-reaction.

Purpose This study aimed to investigate the odontogenic potential of bone marrow mesenchymal stem cells (BM-MSCs) for seeding in tooth regeneration. Materials and Methods In this study, BM-MSCs were co-cultured with oral epithelial cells derived from rat embryos. Expression of the odontogenic genes Pax9 , DMP1 , and DSPP was detected by the reverse-transcription polymerase chain reaction (RT-PCR) technique. To further characterize the odontogenic potential of BM-MSCs, the gold standard in vivo transplantation system was used. Results The results revealed that Pax9 , DMP1 , and DSPP expression was detected by RT-PCR only after co-culture of BM-MSCs and oral epithelial cells derived from embryos age E11.5. Histological analyses of the BM-MSCs/epithelial cell mass demonstrated the presence of tooth-like structures. Conclusions The series of experiments both in vitro and in vivo demonstrated that BM-MSCs can differentiate into functional odontoblast-like cells. This implies that BM-MSCs may become a novel source of cells for seeding in tooth regeneration research.

AIM: To observe expression of CD14 protein and CD14 gene in rat liver sinusoidal endothelial cells (LSECs) during endotoxemia, and the role of CD14 protein in the activation of lipopolysaccharide (LPS)-induced LSECs. METHODS: Wistar rat endotoxemia model was established first by injection of a dose of LPS (5 mg/kg, Escherichia coli O111:B4) via the tail vein, then sacrificed after 0 h, 3 h, 6 h, 12 h, and 24 h, respectively. LSECs were isolated from normal and LPS-injected rats by an in situ collagenase perfusion technique. The isolated LSECs were incubated with rabbit anti-rat CD14 polyclonal antibody, then stained with goat anti rabbit IgG conjugated fluorescein isothiocyanate (FITC) and flow cytometric analysis (FCM) was performed. The percentage and mean fluorescence intensity (MFI) of CD14-positive cells were taken as the indexes. LSECs were collected to measure the expression of CD14 mRNA by in situ hybridization analysis. The isolated LSECs from normal rats were incubated firstly with anti-CD14 antibody, then stimulated with different concentrations of LPS, and the supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)-α and Interleukin (IL)-6 with ELISA. RESULTS: In rats with endotoxemia, LSECs displayed a strong MFI distinct from that of control rats. CD14 positive cells in rats with endotoxemia were 54.32%, 65.83%, 85.64%, and 45.65% at 3 h, 6 h, 12 h, and 24 h respectively, there was significant difference when compared to normal group of animals (4.45%) (P < 0.01). The expression of CD14 mRNA in isolated LSECs was stronger than that in control rats. In LPS group, the levels of TNF-α and IL-6 were 54 ± 6 ng·L-1, 85 ± 9 ng·L-1, 206 ± 22 ng·L-1, 350 ± 41 ng·L-1, 366 ± 42 ng. L-1 and 103 ± 11 ng·L-1, 187 ± 20 ng·L-1, 244 ± 26 ng·L-1, 290 ± 31 ng·L-1, and 299 ± 34 ng·L-1, respectively at different concentration points. In anti-CD14 group, the levels of TNF-α and IL-6 were 56 ± 5 ng·L-1, 67 ± 8 ng·L-1, 85 ± 10 ng·L-1, 113 ± 12 ng·L-1, 199 ± 22 ng·L-1 and 104 ± 12 ng·L-1, 125 ± 12 ng·L-1, 165 ± 19 ng·L-1, 185 ± 21 ng·L-1, and 222 ± 23 ng·L-1, respectively at different concentration points. There was significant difference between the two groups (P < 0.01). CONCLUSION: LSECs can synthesize CD14 protein and express CD14 gene during endotoxemia. CD14 protein plays an important role in the activation of LPS-induced LSECs. This finding has important implications for the understanding of the mechanisms by which LPS may injure liver sinusoidal endothelial cells during sepsis.

Objective: To summarize the diagnosis and treatment of juxta-ampullary duodenal diverticulum (JAD) in our hospital. Methods: Of 5000 consecutive endoscopic retrograde cholangiopancreatography (ERCP) performed in our department, 225 patients were diagnosed with JAD and treated. All patients were classified based on the location of Ampullae of Vater in relation to the duodenal diverticulum. Of the 225 JAD patients, 96 patients (43%) required surgery. Results: The 225 patients with JAD were divided into Type A (146 cases, 65%) or Type B (79 cases, 35%). Type A patients presented with papillae near the diverticulum or in its margin. In this type, 36 patients (25%) presented with diverticulitis, bleeding, perforation or cholelithiasis, and were treated surgically. Type B patients presented with papillae inside the diverticulum. Among them, 60 patients (76%) had complications requiring surgery. Conclusions: JAD can be divided into two types based on location of the papillae. ERCP was the primary method of diagnosing JAD and patients with severe complications required surgical intervention.

AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD). METHODS: Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group was fed ethanol (dose of 5-12g·kg·d-1) and control group received dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM) or the reverse transcription polymerase chain reaction (RT-PCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-α and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy. RESULTS: In ethanol-fed group, the percentages of FITC-CD14 positive cells were 76.23% and 89.42% at 4 wk and 8 wk, respectively. Compared with control group (4.45% and 5.38%), the difference was significant (P < 0.05). The expressions of CD14 mRNA were 7.56 ± 1.02 and 8.74 ± 1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77 ± 0.21 and 1.98 ± 0.23) (P < 0.05). Plasma endotoxin levels at 4 wk and 8 wk increased significantly in ethanol-fed group (129 ± 21 ng·L-1 and 187 ± 35 ng·L-1) than those in control rats (48 ± 9 ng·L-1 and 53 ± 11 ng·L-1)(P < 0.05). Mean values of plasma ALT levels increased dramatically in ethanol-fed rats (112 ± 15 IU/L and 147 ± 22 IU/L) than those in the control animals (31 ± 12 IU/L and 33 ± 9 IU/L) (P < 0.05). In ethanol-fed rats, the levels of TNF-α were 326 ± 42 ng·L-1 and 402 ± 51 ng·l-1 at 4 wk and 8 wk, respectively which were significantly higher than those in control group (86 ± 12 ng·L-1 and 97 ± 13 ng·L-1) (P < 0.05). The levels of IL-6 were 387 ± 46 ng·L- 1 and 413 ± 51 ng·L-1, which were also higher than control group (78 ± 11 ng·L-1 and 73 ± 10 ng·L-1) (P < 0.05). In liver section from ethanol-fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group. CONCLUSION: Ethanol administration led to a significant synthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hepatic functional damages.

To elucidate the role of NF-kB activation in the development of multiple organ dysfunction (MOD) during acute obstructive cholangitis (AOC) in rats.Forty-two Wistar rats were divided into three groups: the AOC group, the group of bile duct ligation (BDL group), and the sham operation group (SO group). All the animals in the three groups were killed in the 6th and 48th hour after operation. Morphological changes of vital organs were observed under light and electron microscopy. NF-kB activation was determined with Electrophoretic Mobility Shift Assay (EMSA). Arterial blood gas analyses and the serum levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine were performed. The concentrations of TNF-alpha and IL-6 in plasma were also measured.The significant changes of histology and ultrastructure of vital organs were observed in AOC group. By contrast, in BDL group, all the features of organs damage were greatly reduced. Expression of NF-kB activation in various tissues increased in AOC group when compared to other two groups. At 6 h, the arterial pH in three groups was 7.52+/-0.01, 7.46+/-0.02, and 7.45+/-0.02, and the blood pCO(2) was 33.9+/-0.95 mmHg, 38.1+/-0.89 mmHg, 38.9+/-0.94 mmHg, there was difference in three groups (P0.05). At 48 h, the blood pH values in three groups was 7.33+/-0.07, 7.67+/-0.04, and 7.46+/-0.03, and blood HCO(3)(-) was 20.1+/-1.29 mmol x L(-1), 26.7+/-1.45 mmol x L(-1) and 27.4+/-0.35 mmol x L(-1), there was also difference in three groups (P0.05). In AOC group, Levels of LDH, ALT, BUN and creatinine were 1,6359.9+/-2,278.8 nkat x L(-1), 5,796.2+/-941.9 nkat.L(-1), 55.7+/-15.3 mg/dl, and 0.72+/-0.06 mg/dl, which were higher than in SO group (3,739.1+/- 570.1 nkat x L(-1), 288.4+/-71.7 nkat x L(-1), 12.5+/-2.14 mg/dl, and 0.47+/-0.03 mg/dl) (P0.05). Levels of plasma TNF-alpha and IL-6 in AOC at 48 h were 429+/-56.62 ng x L(-1) and 562+/-57 ng x L(-1), which increased greatly when compared to BDL group (139+/-16 ng x L(-1), 227+/-43 ng x L(-1)) and SO group (74+/-10 ng x L(-1), 113+/-19 ng x L(-1)) (P0.05).The pathological damages and the NF-kB activation of many vital organs excised during AOC. These findings have an important implication for the role of NF-kB activation in MOD during AOC.

The objective of this study is to elucidate the potential role of poly-morphonuclear neutrophils (PMN) in the development of such a sinusoidal endothelial cell (SEC) injury during early acute obstructive cholangitis (AOC) in rats.Twenty one Wistar rats were divided into three groups: the AOC group, the bile duct ligated group (BDL group), and the sham operation group (SO group). The common bile duct (CBD) of rats in AOC group was dually ligated and 0.2 ml of the E. Coli O(111) B(4) (5 X 10(9)cfu/ml) suspension was injected into the upper segment, in BDL group, only the CBD was ligated and in SO group, neither injection of E. Coli suspension nor CBD ligation was done, but the same operative procedure. Such group consisted of seven rats, all animals were killed 6h after the operation. Morphological changes of the liver were observed under light and electron microscope. Expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in hepatic tissue was determined with reverse transcription polymerase chain reaction (RT-PCR). The serum levels of alanine aminotransferase (ALT) were determined with an autoanalyger and cytokine-induced neutrophil chemoattractant (CINC) was determined by enzyme-linked immunosorbent assay (ELISA).Neutrophils was accumulated in the hepatic sinusoids and sinusoidal endothelial cell injury existed in AOC group. In contrast, in rats of BDL group, all the features of SEC damage were greatly reduced. Expression of ICAM-1 mRNA in hepatic tissue in three groups were 7.54 +/- 0.82, 2.87 +/- 0.34, and 1.01 +/- 0.12, respectively. There were significant differences among three groups (P0.05). The serum CINC levels in the three groups were 188 +/- 21 ng.L(-1), 94+/-11 ng.L(-1), and 57+/-8 ng.L(-1), respectively. There were also significant differences among the three groups (P0.05). Activity of the serum ALT was 917 +/- 167 nkat.L(-1), 901 +/- 171 nkat.L(-1), and 908 +/- 164 nkat.L(-1), respectively, (P0.05).Hepatic SEC injury occurs earlier than hepatic parenchymal cells during AOC. Recruitments of circulating neutrophils in the hepatic sinusoidal space might mediate the SEC injury, and ICAM-1 in the liver may modulate the PMN of accumulation.

To determine the NF-kB activity in peripheral blood mononuclear cells (PBMC) in patients with acute cholangitis of severe type (ACST) and correlate the degree of NF-kB activation with severity of biliary tract infection and clinical outcome.Twenty patients with ACST were divided into survivor group (13 cases) and nonsurvivor group (7 cases). Other ten patients undergoing elective gastrectomy or inguinal hernia repair were selected as control group. Peripheral blood samples were taken 24 hours postoperatively. PBMC were separated by density gradient centrifugation, then nuclear proteins were isolated from PBMC, and Electrophoretic Mobility Shift Assay (EMSA) used determined. The results were quantified by scanning densitometer of a Bio-Image Analysis System and expressed as relative optical density (ROD). The levels of TNF-alpha, IL-6, and IL-10 in the plasma of patients with ACST and healthy control subjects were determined by using an enzyme-linked immunoassay (ELISA).The NF-kB activity was 5.02 +/- 1.03 in nonsurvivor group, 2.98 +/- 0.51 in survivor group and 1.06 +/- 0.34 in control group. There were statistical differences in three groups (P0.05). The levels of TNF-alpha and IL-6 in plasma were (498 +/- 53)ng.L(-1)and (587 +/- 64)ng.L(-1)in nonsurvivor group, (284 +/- 32)ng.L(-1) and (318 +/- 49)ng.L(-1)in survivor group and (89 +/- 11)ng.L(-1) and (102 +/-13)ng.L(-1)in control group. All patients with ACST had increased levels of TNF-alpha and IL-6, which were many-fold greater than those of control group, and there was an evidence of significantly higher levels in those of nonsurvivor group than that in survivor group (P0.05). The levels of IL-10 in plasma were (378+/-32)ng.L(-1), (384+/-37)ng.L(-1) and (68+/-11)ng.L(-1) in three groups, respectively. All patients had also increased levels of IL-10 when compared with control group (P0.05), but the IL-10 levels were not significantly higher in nonsurvivors than in survivors (P0.05).NF-kB activity in PBMC in patients with ACST increases markedly and the degree of NF-kB activation is correlated with severity of biliary tract infection and clinical outcome.

AIM To determine the in vivo effects of phagocytic blockade of Kupffer cell (KC) on the release of proinflammatory cytokines in small intestinal lesion and on the integrity of intestinal tract by using gadolinium chloride (GdCl(3)) during early endotoxemia. METHODS Wistar rats were divided into three groups: Group A, rats were injected with endotoxin (E. coli O111:B(4), a dose of 12 mg x kg(-1)) only; Group B, rats were pretreated intravenously with 25 mg of GdCl(3) per kg 24 h are given endotoxin; and Group C, sham operation only. All animals were sacrificed 4 h after endotoxin injection. In portion of the rats of three groups, bile duct was cannulated, which the bile was collected externally. Morphological changes of ileum were observed under light microscopy and electronic microscopy. The KC were isolated from rats by collagenase perfusion and in KC, expression of TNF-alpha and IL-6 mRNA were determined by RT-PCR analysis. Plasma and bile TNF-alpha and IL-6 Levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS In group A, there were neutrophil infiltration and superficial epithelial necrosis of the ileal villi, sloughing of mucosal epithelium, and disappearance of some villi. In group B, the ileal mucosal damage was much reduced. which in group C, no significant morphological changes were seen. GdCl(3) pretreatment decreased significantly the expression of TNF-alpha and IL-6 mRNA in group B (4.32+/-0.47 and 4.05+/-0.43) when compared to group A (9.46+/-1.21 and 9.04+/-1.09) (P

AIM: To observe synthesis of CD14 protein and expression of CD14 mRNA in hepatic tissue and hepatocytes of rats during endotoxemia. METHODS: The endotoxemia model of Wistar rat was established by injection of a dose of lipopolysaccharide (LPS) (5 mg·kg-1, Escherichia coli O111:B4) via the tail vein, and then the rats were sacrificed after 3, 6, 12 and 24 h in batches. Hepatocytes were isolated from normal and LPS-injected rats by in situ collagenase perfusion technique and were collected to measure the expression of CD14 mRNA and synthesis of CD14 protein by reverse transcript-polymerase chain reaction (RT-PCR) or Western blot analysis. The binding of fluorescein isothiocyanate (FITC)-CD14 polyclonal antibody to isolated hepatocytes was also assessed by flow cytometric analysis (FCM). RESULTS: In the rats with endotoxemia, the expressions of CD14 mRNA in hepatic tissue and isolated hepatocytes were stronger at 3, 6, and 12 h than that in control rats (3.48 ± 0.15, 5.89 ± 0.62, 4.33 ± 0.18, vs 1.35 ± 0.14 in hepatic tissue, P < 0.01; 4.12 ± 0.17, 6.24 ± 0.64, 4.35 ± 0.18, vs 1.87 ± 0.15 in hepatocytoes, P < 0.01).The synthesis of CD14 protein in hepatic tissue and isolated hepatocytes increases also obviously in 6 and 12 h when compared to that in control rats (13.27 ± 1.27, 17.32 ± 1.35, 11.42 ± 1.20,vs 7.34 ± 0.72 in hepatic tissue, P < 0.01; 14.68 ± 1.30, 17.95 ± 1.34, 11.65 ± 1.19, vs 7.91 ± 0.70 in hepatocytes, P < 0.01). FCM showed that mean fluorescence intensity (MFI) and numbers of FITC-CD14 positive cells in the rats with endotoxemia increased obviously at 3, 6, 12 and 24 h when compared with normal control group (43.4%, 70.2%, 91.4%, 32.6% vs 4.5%, P < 0.01). CONCLUSION: LPS can markedly promote the synthesis of CD14 protein and up-regulate the expression of CD14 mRNA in isolated hepatocytes and hepatic tissue. Liver might be a main source for soluble CD14 production during endotoxemia.