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Hepatitis B virus (HBV) infection is a major factor responsible for HBV+ hepatocellular carcinoma (HCC).An immunological classification of HBV+ HCC may provide both biological insights and clinical implications for this disease.Based on the enrichment of 23 immune signatures, we identified two immune-specific subtypes (Imm-H and Imm-L) of HBV+ HCC by unsupervised clustering. We showed that this subtyping method was reproducible and predictable by analyzing three different datasets.Compared to Imm-L, Imm-H displayed stronger immunity, more stromal components, lower tumor purity, lower stemness and intratumor heterogeneity, lower-level copy number alterations, higher global methylation level, and better overall and disease-free survival prognosis. Besides immune-related pathways, stromal pathways (ECM receptor interaction, focal adhesion, and regulation of actin cytoskeleton) and neuro-related pathways (neuroactive ligand-receptor interaction, and prion diseases) were more highly enriched in Imm-H than in Imm-L. We identified nine proteins differentially expressed between Imm-H and Imm-L, of which MYH11, PDCD4, Dvl3, and Syk were upregulated in Imm-H, while PCNA, Acetyl-a-Tubulin-Lys40, ER-α_pS118, Cyclin E2, and β-Catenin were upregulated in Imm-L.Our data suggest that "hot" tumors have a better prognosis than "cold" tumors in HBV+ HCC and that "hot" tumors respond better to immunotherapy.

The split-Hopkinson pressure bar (SHPB) and digital image correlation (DIC) techniques are combined to analyze the dynamic compressive failure process of coal samples, and the box fractal dimension is used to quantitatively analyze the dynamic changes in the coal sample cracks under impact load conditions with different loading rates. The experimental results show that the fractal dimension can quantitatively describe the evolution process of coal fractures under dynamic load. During the dynamic compression process, the evolution of the coal sample cracks presents distinct stages. In the crack propagation stage, the fractal dimension increases rapidly with the progress of loading, and in the crack widening stage, the fractal dimension increases slowly with the progress of loading. The initiation of the crack propagation phase of the coal samples gradually occurs more quickly with increasing loading rate; the initial cracks appear earlier. At the same loading time point, when the loading rate is greater, the fractal dimension of the cracks observed in the coal sample is greater.

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.

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.

Additional file 1. PVAT promotes destabilization of atherosclerotic plaque.

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

Chen, Y., Ye, Y., Wang, L., Liu, Y., Li, S. and Jiang, M. 2009. Cloning and sequence analysis of transferrin gene in Yak. J. Appl. Anim. Res., 36: 147–151. To study the transferrin (tf) gene in yak, cDNA sequence encoding TF from yak was cloned by RT-PCR method using gene specific PCR primers. The cloned cDNA fragment (2215 bp) contains a 2115 bp open reading frame, encodes 704 amino acids (AAs) with a molecular mass of 77.552 KDa. The deduced amino sequence shows a high level of sequence identity to Bos taurus (99.24%), Sus scrofa (80.19%), Equus caballus (79.33%), Homo sapiens (77.83%) and Mus musculus (72.19%), respectively. tf gene was expressed only in liver tissues. It also contained two conserved TF domains: one is in its N-terminal (25 AAs-351 AAs) and the other in its C-terminal (364AAs-689AAs). The phylogenetic analysis showed that yak and Bos taurus were the closest species. The results of this study suggested that tf gene of yak plays the same function as other species.

Li, S., Jiang, M., Liu, Y., Fu, X., Drackley, J.K., Chen, Y., Ye, Y., Wang, Y. and Zhong, J. 2009. Molecular cloning and sequence analysis of hepatic lipase gene in yak. J. Appl. Anim. Res., 36: 97–101. In order to study the biological function of hepatic lipase (hl) gene in yak, the cDNA sequence encoding hepatic lipase from yak was cloned by RT-PCR method using gene specific PCR primers. The cloned cDNA fragment (1540 bp) contained a 1503 bp open reading frame, encoded 500 amino acids (AAs) with a molecular mass of 56.734 kDa. It showed a high level of sequence identity to Bos Taurus (99.40%), Homo sapiens (77.64%), Oryctolagus cuniculus (76.20%), Mus musculus (68.88%), Xenopus laevis (55.27%) and Danio rerio (47.69%). The hl gene was expressed only in liver tissue and not in heart, spleen, kidney and brain tissues. Further analysis of yak HL amino acid sequence implied that it contained a complete lipase active site (VHLIGYSLGA) ranging from 162 to 171 amino acid residues. It also contained tw...

In order to study the biological function of gapdh gene in yak, and prove whether the gapdh gene was a useful intra-reference gene that can be given an important role in molecular biology research of yak, the cDNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase from yak was cloned by the RT-PCR method using gene specific PCR primers. The sequence results indicated that the cloned cDNA fragment (1,008 bp) contained a 1,002 bp open reading frame, encoding 333 amino acids (AAs) with a molecular mass of 35.753 kDa. The deduced amino acids sequence showed a high level of sequence identity to Bos Taurus (99.70%), Xenopus laevis (94.29%), Homo sapiens (97.01%), Mus musculus (97.90%) and Sus scrofa (98.20%). The expression of yak's gapdh gene in heart, spleen, kidney and brain tissues was also detected; the results showed that the gapdh gene was expressed in all these tissues. Further analysis of yak GAPDH amino acid sequence implied that it contained a complete glyceraldehyde-3-phosphate dehydrogenase active site (ASCTTNCL) which ranged from 148 to 155 amino acid residues. It also contained two conserved domains, a NAD binding domain in its N-terminal and a complete catalytic domain of sugar transport in its C-terminal. The phylogenetic analysis showed that yak and Bos taurus were the closest species. The prediction of secondary structures indicated that GAPDH of yak had a similar secondary structure to other isolated GAPDH. The results of this study suggested that the gapdh gene of yak was similar to other species and could be used as the intra-reference to analyze the expression of other genes in yak.

FcγRIIIa (CD16) is a low-affinity Fc receptor of IgG. As the idio-binding receptor of IgG Fc, it plays an important role in the antibody-dependent cellular cytotoxicity of natural killer cells. The aim of the present study was to investigate the distribution of Kupffer cells (KCs) and the expression of their surface receptor FcγRIIIa in hepatocellular carcinoma. Furthermore, we also aimed to observe the functional mechanism of FcγRIIIa. Immunohistochemical analysis was employed to study KCs and FcγRIIIa. In order to explore the role of FcγRIIIa in the growth of cancer cells, KCs and H22 tumor cells were co-cultured in different serum. The mRNA expression levels of tumor necrosis factor (TNF)-α and FcγRIIIa were analyzed by RT-qPCR; the TNF-α and FcγRIIIa protein expression levels were examined by enzyme‑linked immunosorbent assay and western blot analysis, respectively. Our results showed that the number of Kuppfer cells in cancerous tissues (21.6±7.8) was lower than those in para-cancerous (68.8±9.1) tissues and adjacent normal hepatic tissues (62.0±1.9) (P

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

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

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.

Since the widespread adoption of laparoscopic cholecystectomy (LC) in the late 1980s, a rise in common bile duct (CBD) injury has been reported. We analyzed the factors contributing to a record of zero CBD injuries in 10 000 consecutive LCs.The retrospective investigation included 10 000 patients who underwent LC from July 1992 to June 2007. LC was performed by 4 teams of surgeons. The chief main surgeon of each team has had over 10 years of experience in hepatobiliary surgery. Calot's triangle was carefully dissected, and the relationship of the cystic duct to the CBD and common hepatic duct was clearly identified. A clip was applied to the cystic duct at the neck of the gallbladder and the duct was incised with scissors proximal to the clip. The cystic artery was dissected by the same method. Then, the gallbladder was dissected from its liver bed. A drain was routinely left at the gallbladder bed for 1-2 days postoperatively.No CBD injuries occurred in 10 000 consecutive LCs, and there were 16 duct leaks (0.16%). Among these, there were 10 Luschka duct leaks (0.1%) and 6 cystic duct leaks (0.06%). Four hundred thirty cases were converted to open cholecystectomy (OC), giving a conversion rate of 4.3%. After a mean follow-up of 17.5 months (range 6-24 months), no postoperative death due to LC occurred, and good results were observed in 95% of the patients.In our 10 000 LCs with zero CBD injuries, the techniques used and practices at our department have been successful. Surgeon's expertise in biliary surgery, preoperative imaging, precise operative procedures, and conversion from LC to OC when needed are important measures to prevent CBD injuries.

Pancreatic encephalopathy (PE), an unfamiliar complication of severe acute pancreatitis (SAP), is difficult to diagnose and treat, and it has a high mortality. The aim of this study was to investigate the manifestation, classification, mechanism and therapy of PE.Of 132 patients with SAP treated at our hospital from March 1994 to March 2004, 24 patients complicated by PE were analyzed retrospectively.The causes of SAP were mostly biliary and alcoholic. Twenty-four patients (18.2%) were complicated by PE within 3 hours-38 days(average 6.6 days)[21(87.5%) within 2 weeks, and 3(12.5%) after 2 weeks] . Eleven patients were male and 13 female, with an average of 47 years (range 25-72 years). Excitement or restrain was the main manifestation. Nine patients (37.5%) received surgery and 15(62.5%) conservative treatment, with a mortality of 11.1%(1/9) and 66.7%(10/15), respectively.PE occurs 2 weeks after SAP and is part of multiple organ failure (MOF). Some patients have PE in the late stage of SAP because of lack of VitB1 and nutrition. But PE can be prevented by prescribing adequate nutrition and VitB1 in the early stage of SAP.

To explore the changes of plasma endothelin (ET) and nitric oxide (NO) levels in patients with acute pancreatitis.The level of plasma ET was measured by radioactive-immunoassay, and NO by spectrophotometry.The levels of ET, NO and the ET/NO ratio in patients with severe acute pancreatitis (SAP) within 24 hours in hospital were all significantly higher than those in other groups of patients [(176+/-8) pg/ml, (97+/-11) micromol/L, and 1.83+/-0.12, P0.01]. Compared to healthy controls (N), the levels of ET and NO in patients without pancreatitis acute abdomen (NAP) and patients with mild acute pancreatitis (MAP) increased significantly (P0.01). After appropriate treatment, the levels of ET and NO in the MAP group were lower (P0.01). Compared with those before treatment, the levels of ET and NO in the SAP group on the 3rd and 7th day in hospital dropped significantly (P0.01). The ET/NO ratio on the 7th day was also lower than that on admission (P0.01).The malfunction of endothelial cells and the increased ET/NO ratio may be related to the mechanism of pancreatic microcirculatory disturbance in patients with SAP; early dynamic determination of these parameters may help predict the prognosis of SAP.

To determine the role of mononuclear macrophages in the pathogenesis of acute lung injury during acute obstructive cholangitis.Sixty Wistar rats were used to study the correlation between the behavior of mononuclear macrophages and acute pulmonary injury during acute obstructive cholangitis (AOC). Animal model of AOC was made according to the method that the common bile duct was injected with Escherichia coli and ligated. The rats were killed at 6 h, 12 h, 24 h and 48 h after operation. The phagocytic function of Kupffer cells (KCs), the number of alveolar macrophages (AMs) in bronchoalveolar lavage liquid, and the extravascular water content of lung tissue were measured. The levels of lipid peroxide (LPO) and supperoxide dismutase (SOD) were determined too. Pathological alterations of liver and lung tissue were observed under light and electron microscopes.KCs phagocytic function was significantly elevated at the 6th hour but markedly decreased from the 24th hour to the 48th hour in the AOC group as compared with the control (P0.05). From the 12th to the 48th hour, the number of AMs, the extravascular water content of lung tissue, and the content of LPO significantly increased, but the SOD level of lung tissue decreased greatly (P0.05). Morphologically, KCs proliferated diffusely in the early period in livers of the AOC group, but decreased markedly in the late period. Mitochondria of KCs were swollen or even vacuolated; focal cytoplasmic degeneration and many myeli like figures could be seen in the cytoplasm. The changes of injury such as disturbance of pulmonary capillary blood circulation, degeneration and/or necrosis of the lung tissue and endothelium, and inflammatory reactions could be observed. In other two groups, no evident morphological changes were observed.KCs phagocytic function is decreased, whereas AM is activated by the invading bacteria to release such inflammatory mediators as free radicals, resulting in acute pulmonary injury. It seems that there is a close relationship between the functional status of mononuclear macrophages and the development of acute lung injury. The dysfunction of mononuclear macrophages may play an important role in the pathogenesis of multiple organ damage, especially acute pulmonary injury.

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.