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BACKGROUND: Prostate cancer (PCa) is the most common type of cancer among men in Western countries. Despite numerous therapeutic options, few treatments are available for patients with end-stage disease. In the present study, different somatostatin receptors (SSTs) and the chemokine receptor CXCR4 were evaluated for their suitability as novel therapeutic targets in PCa. MATERIALS AND METHODS: The expression of SST subtypes 1, 2A, 3, and 5 and of CXCR4 was evaluated in 276 PCa tumor samples on a tissue microarray (TMA) in 23 whole-block tumor samples and in 3 PCa cell lines by immunohistochemistry using well-characterized monoclonal antibodies. RESULTS: Overall, the frequency and intensity of expression of SSTs and CXCR4 were very low among the PCa samples investigated. Specifically, SST5, SST2A, and SST3 were expressed, albeit at low intensity, in 10.5%, 9.1%, and 0.7% of the TMA samples, respectively. None of the TMA samples showed SST1 or CXCR4 expression. Only a single small-cell-type neuroendocrine carcinoma that was coincidentally included among the whole-block samples exhibited strong SST2A, SST5, and CXCR4 and moderate SST3 expression. Independent of the tumor cells, the tumor capillaries in many of the PCa samples were strongly positive for SST2A, SST3, SST5, or CXCR4 expression. SST expression in the tumor cells was associated with advanced tumor grade and stage. CONCLUSION: Overall, SST and CXCR4 expression levels are clearly of no therapeutic relevance in PCa. SST- or CXCR4-based therapy might be feasible, however, in rare cases of small-cell-type neuroendocrine carcinoma of the prostate.

Background Hepatocellular (HCC) and cholangiocellular carcinomas (CCC) display an exceptionally poor prognosis. Especially for advanced disease no efficient standard therapy is currently available. Recently, somatostatin analogs have been evaluated for the treatment of HCC, however, with contradictory results. Besides, for both malignancies the chemokine receptor CXCR4 has been discussed as a possible new target structure. Methods Expression of somatostatin receptor (SSTR) subtypes 1, 2A, 3, 4, and 5, and of CXCR4 was evaluated in a total of 71 HCCs and 27 CCCs by immunohistochemistry using well-characterized novel monoclonal antibodies. Results In HCC tumor cells, frequency and intensity of expression of SSTRs and CXCR4 were only low. CXCR4 was present in about 40% of the HCCs, although at a low intensity. SSTR5, SSTR2, and SSTR3 were detected in about 15%, 8%, and 5% of the HCC tumors, respectively. SSTR and CXCR4 expression was much higher in CCC than in HCC. CXCR4 and SSTR1 were present in 60% and 67% of the CCC samples, respectively, followed by SSTR2 and SSTR5, which were detected in 30% and 11% of the tumors, respectively. Most notably, CXCR4 was intensely expressed on the tumor capillaries in about 50% of the HCCs and CCCs. CXCR4 expression on tumor vessels was associated with poor patient outcomes. Conclusions CCC, but not HCC, may be suitable for SSTR-based treatments. Because of the predominant expression of SSTR1, pan-somatostatin analogs should be preferred. In both HCC and CCC, indirect targeting of tumors via the CXCR4-positive tumor capillaries may represent a promising additional therapeutic strategy. Electronic supplementary material The online version of this article (10.1186/s12885-017-3911-3) contains supplementary material, which is available to authorized users.

Carbon monoxide (CO) exerts protective effects on hepatic ischemia/reperfusion injury (IRI), but the underlying molecular mechanisms are not fully understood. High-mobility group box 1 (HMGB1) is an important mediator of injury and inflammation in hepatic IRI. Here, we investigated whether CO could attenuate hepatic IRI via inhibition of HMGB1 release, particularly through sirtuin 1 (SIRT1). CO was released by treatment with carbon monoxide-releasing molecule (CORM)-2. CORM-2-delivered CO ameliorated hepatic IRI, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory responses, and less severe ischemia/reperfusion-associated histopathologic changes. Treatment with CORM-2 significantly inhibited IRI-induced HMGB1 translocation and release. SIRT1 expression was increased by CORM-2 pretreatment. When CORM-2-induced SIRT1 expression was inhibited using EX527, HMGB1 translocation and release were increased and hepatic IRI was worsened, whereas SIRT1 activation by resveratrol reversed this trend. In vitro, CORM-2 reduced hypoxia/reoxygenation-induced HMGB1 translocation and release, these inhibitions were blocked by SIRT1 inhibition using EX527 or SIRT1 small interfering RNA both in alpha mouse liver 12 cells and RAW264.7 macrophages. Moreover, SIRT1 directly interacted with and deacetylated HMGB1. IRI increased HMGB1 acetylation, which was abolished by CORM-2 treatment via SIRT1. In conclusion, these results suggest that CO may increase SIRT1 expression, which may decrease HMGB1 acetylation and subsequently reduce its translocation and release, thereby protecting against hepatic IRI. Liver Transplantation 23 510-526 2017 AASLD.

// Anding Liu 1,2 , Enshuang Guo 3 , Jiankun Yang 1 , Renlong Li 3 , Yan Yang 1 , Shenpei Liu 1 , Jifa Hu 1 , Xiaojing Jiang 3 , Olaf Dirsch 2 , Uta Dahmen 2 , Jian Sun 4 and Mingwen Ouyang 5 1 Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2 Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Friedrich-Schiller-University Jena, Jena, Germany 3 Department of Infectious Diseases, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China 4 Department of Biliopancreatic Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China 5 Department of Anesthesiology, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, China Correspondence to: Mingwen Ouyang, email: // Keywords : ischemic preconditioning; steatosis; liver ischemia/reperfusion injury; autophagy; heme oxygenase-1, Pathology Section Received : April 25, 2016 Accepted : November 02, 2016 Published : November 10, 2016 Abstract Steatotic livers are more susceptible to ischemia/reperfusion (I/R) injury, which is ameliorated by ischemic preconditioning (IPC). Autophagy possesses protective action on liver I/R injury and declines in steatotic livers. The aim of this study was to test the hypothesis that the increased susceptibility of steatotic livers to I/R injury was associated with defective hepatic autophagy, which could be restored by IPC via heme oxygenase-1 (HO-1) signaling. Obesity and hepatic steatosis was induced using a high fat diet. Obesity impaired hepatic autophagy activity and decreased hepatic HO-1 expression. Induction of HO-1 restored autophagy activity and inhibited calpain 2 activity. Additionally, suppression of calpain 2 activity also restored autophagy activity. Mitochondrial dysfunction and hepatocellular injury were significantly increased in steatotic livers compared to lean livers in response to I/R injury. This increase in sensitivity to I/R injury was associated with defective hepatic autophagy activity in steatotic livers. IPC increased autophagy and reduced mitochondrial dysfunction and hepatocellular damage in steatotic livers following I/R injury. Furthermore, IPC increased HO-1 expression. Inhibition of HO-1 decreased the IPC-induced autophagy, increased calpain 2 activity and diminished the protective effect of IPC against I/R injury. Inhibition of calpain 2 restored autophagic defect and attenuated mitochondrial dysfunction in steatotic livers after I/R. Collectively, IPC might ameliorate steatotic liver damage and restore mitochondrial function via HO-1-mediated autophagy.

Aging is often associated with a decreased autophagic activity that contributes to the high sensitivity of aged livers to ischemia reperfusion injury (IRI). Blood from young animals can positively affect aged animals. This study was designed to evaluate the effect of young plasma in a model of liver IRI in aged rats. Aged rats were treated with pooled plasma collected from young rats before ischemia. Administration of young plasma restored aging-induced suppression in hepatic autophagic activity and reduced liver IRI. Inhibition of the young-plasma-restored autophagic activity abrogated the beneficial effect of young plasma against liver IRI. Similarly, young serum restored autophagic activity and reduced cellular injury after hypoxia/reoxygenation (H/R) in primary old rat hepatocytes. Mechanistic studies showed thatadministration of young plasma increased AMPK phosphorylation and led to unc-51-like autophagy activating kinase (ULK)1 activation. Furthermore, AMPK-inhibition abrogated the young serum-induced ULK1 activation and autophagic activity and diminished the protective action of young serum against H/R injury in primary old rat hepatocytes, whereas AMPK-activation potentiated the effects of young serum. Young plasma could restore age-impaired autophagy, at least in part, via AMPK/ULK1 signaling. Restoration of age-impaired autophagic activity may be a critical contributing mechanism to young-plasma-afforded protection against liver IRI in aged rats.-Liu, A., Yang, J., Hu, Q., Dirsch, O., Dahmen, U., Zhang, C., Gewirtz, D. A., Fang, H., Sun, J. Young plasma attenuates age-dependent liver ischemia reperfusion injury.

Growth differentiation factor 11 (GDF11) has been implicated in a variety of aging conditions and the regulation of organ regeneration after injury; however, the role of GDF11 in liver ischemia reperfusion injury (IRI) is unknown. The aim of the current study was to investigate the possible role of GDF11 in liver IRI. We investigated the effects of GDF11 in liver IRI in both young (3 mo) and old (22 mo) mice in vivo, and in primary young and old mouse hepatocytes in vitro. Both serum and hepatic GDF11 protein expression levels increased with age and after IRI. Treatment with recombinant GDF11 significantly increased IRI-induced elevations of serum aminotransferase levels, worsened the histologic status of livers, and impaired liver regeneration. In contrast, inhibition of GDF11 activity with neutralizing Abs significantly decreased liver injury and improved liver regeneration after IRI. In vitro, treatment with recombinant GDF11 significantly delayed cell proliferation in cultured hepatocytes that were subjected to hypoxia/reoxygenation insult. Moreover, suppression of cell-cycle progression may be a key mechanism by which GDF11 inhibited hepatocyte regeneration. Collectively, rather than acting as a rejuvenating agent, GDF11 worsens hepatocellular injury and impairs liver regeneration after IRI.-Liu, A., Dong, W., Peng, J., Dirsch, O., Dahmen, U., Fang, H., Zhang, C., Sun, J. Growth differentiation factor 11 worsens hepatocellular injury and liver regeneration after liver ischemia reperfusion injury.

Multimodal treatment concepts including liver transplantation for hepatocellular carcinoma (HCC), extended resection methods and neoadjuvant chemotherapy for colorectal liver metastasis significantly improve patients’ outcome. However, surgery-induced hepatic ischemia-reperfusion injury (IRI) and chemotherapy-associated hepatotoxicity result in hepatocellular damage and compromised liver function. Activation of common key pathways in ischemic liver and hepatotoxic injury results in oxidative stress, inflammatory responses and apoptosis causing organ damage. Controlling liver damage before and during surgery is essential for the postoperative outcome. Nigella sativa has a long tradition as a natural remedy. In the essential oil, Thymoquinone (TQ) was identified as the main component and responsible for most of the therapeutic effects. Therefore, this systematic review aimed to summarize the hepatoprotective effects of TQ and its potential suitability to improve surgical outcome by reducing surgical ischemic injury and hepatotoxicity of neoadjuvant chemotherapy. The key findings can be summarized as TQ having strong antioxidant, anti-inflammatory, antifibrotic, anti-/proapoptotic and anticarcinogenic effects. Almost no side effects were reported irrespective of a large dose range, suggesting a wide therapeutic window. These results give rise to the expectation that TQ could evolve to a novel powerful drug to reduce hepatic injury.

Cholestasis is a common complication in liver diseases that triggers a proliferative response of the biliary tree. Bile duct ligation (BDL) is a frequently used model of cholestasis in rodents. To determine which changes occur in the three‐dimensional (3D) architecture of the interlobular bile duct during cholestasis, we used 3D confocal imaging, surface reconstructions, and automated image quantification covering a period up to 28 days after BDL. We show a highly reproducible sequence of interlobular duct remodeling, where cholangiocyte proliferation initially causes corrugation of the luminal duct surface, leading to an approximately five‐fold increase in surface area. This is analogous to the function of villi in the intestine or sulci in the brain, where an expansion of area is achieved within a restricted volume. The increase in surface area is further enhanced by duct branching, branch elongation, and loop formation through self‐joining, whereby an initially relatively sparse mesh surrounding the portal vein becomes five‐fold denser through elongation, corrugation, and ramification. The number of connections between the bile duct and the lobular bile canalicular network by the canals of Hering decreases proportionally to the increase in bile duct length, suggesting that no novel connections are established. The diameter of the interlobular bile duct remains constant after BDL, a response that is qualitatively distinct from that of large bile ducts, which tend to enlarge their diameters. Therefore, volume enhancement is only due to net elongation of the ducts. Because curvature and tortuosity of the bile duct are unaltered, this enlargement of the biliary tree is caused by branching and not by convolution. Conclusion: BDL causes adaptive remodeling that aims at optimizing the intraluminal surface area by way of corrugation and branching. (Hepatology 2016;63:951–964)

Background/Aim: Liver size regulation is based on the balance between hepatic regeneration and atrophy. To achieve a better understanding of intrahepatic size regulation, we explored the size regulation of a portally deprived liver lobe on a liver subjected to concurrent portal vein ligation (PVL) and partial hepatectomy (PHx). Materials and Methods: Using a surgical rat model consisting of right PVL (rPVL) plus 70% PHx, we evaluated the size regulation of liver lobes 1, 2, 3, and 7 days after the operation in terms of liver weight and hepatocyte proliferation. Portal hyperperfusion was confirmed by measuring portal flow. The portal vascular tree was visualized by injection of a contrast agent followed by CT imaging of explanted livers. Control groups consisted of 70% PHx, rPVL, and sham operation. Results: The size of the ligated right lobe increased to 1.4-fold on postoperative day 7 when subjected to rPVL + 70% PHx. The right lobe increased to 3-fold when subjected to 70% PHx alone and decreased to 0.3-fold when subjected to rPVL only. The small but significant increase in liver weight after the combined procedure was accompanied by a low proliferative response. In contrast, hepatocyte proliferation was undetectable in the right lobe undergoing atrophy after PVL only. The caudate lobe in the rPVL + 70% PHx group increased to 4.6-fold, which is significantly more than in the other groups. This increase in liver weight was paralleled by persisting portal hyperperfusion and a prolonged proliferative phase of 3 days. Conclusions: A discontinued portal blood supply does not always result in atrophy of the ligated lobe. The concurrent regenerative stimulus induced by 70% PHx seemed to counteract the local atrophy after a simultaneously performed rPVL, leading to a low but prolonged regenerative response of the portally deprived liver lobe. This observation supports the conclusion that portal flow is not necessary for liver regeneration. The persisting portal hyperperfusion may be crucial for the specific kinetics of prolonged liver regeneration after rPVL + 70% PHx in the portally supplied caudate lobe. Both observations deserve more attention regarding the underlying mechanism in further studies.

HighlightsA new method for identifying fat droplets in histological images is presented.Adjacency statistics are utilized as shape features.Fat droplets are identified with high sensitivity and specificity.Adjacency statistics greatly improve the identification of clustered fat droplets.The method can be quickly executed on standard computers. Background and objectiveThe accurate identification of fat droplets is a prerequisite for the automatic quantification of steatosis in histological images. A major challenge in this regard is the distinction between clustered fat droplets and vessels or tissue cracks. MethodsWe present a new method for the identification of fat droplets that utilizes adjacency statistics as shape features. Adjacency statistics are simple statistics on neighbor pixels. ResultsThe method accurately identified fat droplets with sensitivity and specificity values above 90%. Compared with commonly-used shape features, adjacency statistics greatly improved the sensitivity toward clustered fat droplets by 29% and the specificity by 17%. On a standard personal computer, megapixel images were processed in less than 0.05s. ConclusionsThe presented method is simple to implement and can provide the basis for the fast and accurate quantification of steatosis.

Liver dysfunction has been known to occur frequently in cases of sepsis. Baicalein, the main active ingredient of the Scutellaria root, exerts anti-inflammatory and anti-apoptotic properties in endotoxic shock. However, the role of baicalein in polymicrobial sepsis-induced liver injury and its regulatory mechanisms remain unclear. In this study, we aimed to investigate the protective effects of baicalein on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Mice were treated with baicalein (100mg/kg, i.p) at 1h, 6h and 12h following CLP. Baicalein significantly improved the survival of septic mice. Treatment with baicalein ameliorated the CLP-induced liver injury, as indicated by the lower serum aminotransferase levels and the fewer histopathologic abnormalities. Baicalein reduced the neutrophil infiltration and the hepatic inflammatory cytokine expression and release. It also decreased the hepatic and the serum high-mobility group box 1 and macrophage migration inhibitory factor levels in septic mice. Moreover, baicalein significantly inhibited the mitogen-activated protein kinases (MAPKs) activation and suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB). In conclusion, these results suggest that baicalein treatment could protect against the sepsis-induced liver injury, and improve the survival of mice with polymicrobial sepsis. The mechanism of the protective action of baicalein seems to involve its ability to reduce inflammatory response, to inhibit hepatic apoptosis, and to suppress MAPKs and NF-κB activation.

Differences in LPS responsiveness influence the outcome of patients with sepsis. The intensity of the response is highly variable in patients and strain dependent in rodents. However, the role of the liver for initiating the LPS response remains ill defined. We hypothesize that hepatic LPS uptake is a key event for initiating the LPS response. In the present study, the severity of the LPS-induced inflammatory response and the hepatic LPS uptake was compared in two rat strains (Lewis (LEW) rats and Brown Norway (BN) rats). Using a transplantation model, we demonstrated the decisive role of the liver. The expression of hepatic TNF-α, IL-6, and IL-1β mRNA levels in BN rats was significantly lower than that in LEW rats. LEW rats were sensitized to LPS via G-CSF pretreatment. Sensitization caused by G-CSF pretreatment induced severe liver injury and mortality in LEW rats, but not in BN rats (survival rate: 0% (LEW) versus 100% (BN), p<0.01). LEW rats presented with higher liver enzymes, more alterations in histology, and higher expression of caspase 3 and higher cytokines levels. One of the reasons could be the increased hepatic LPS uptake, which was only observed in LEW but not in BN livers. Using the transplantation model revealed the decisive role of the LPS responsiveness of the liver. Injection of LPS to the high-responding LEW recipient before transplantation of a low-responder BN liver resulted in a 50% survival rate. In contrast, injecting the same dose of LPS into the high-responding LEW recipient after transplanting the low-responding BN liver resulted in a 100% survival rate. The severity of inflammatory response in different strains might be related to the differences in hepatic LPS uptake. This observation suggests that the liver plays a genetically defined decisive role in modulating the inflammatory severity.

Background: Automated image analysis enables quantitative measurement of steatosis in histological images. However, spatial heterogeneity of steatosis can make quantitative steatosis scores unreliable. To improve the reliability, we have developed novel scores that are ""focused"" on steatotic tissue areas. Methods: Focused scores use concepts of tile-based hotspot analysis in order to compute statistics about steatotic tissue areas in an objective way. We evaluated focused scores on three data sets of images of rodent liver sections exhibiting different amounts of dietary-induced steatosis. The same evaluation was conducted with the standard steatosis score computed by most image analysis methods. Results: The standard score reliably discriminated large differences in steatosis (intraclass correlation coefficient ICC = 0.86), but failed to discriminate small (ICC = 0.54) and very small (ICC = 0.14) differences. With an appropriate tile size, mean-based focused scores reliably discriminated large (ICC = 0.92), small (ICC = 0.86) and very small (ICC = 0.83) differences. Focused scores based on high percentiles showed promise in further improving the discrimination of very small differences (ICC = 0.93). Conclusions: Focused scores enable reliable discrimination of small differences in steatosis in histological images. They are conceptually simple and straightforward to use in research studies.

Introduction. Sepsis is the primary cause of death from infection. We wanted to improve the outcome of sepsis by stimulating innate immunity in combination with modulating the severity of inflammatory responses in rats.Method. Sepsis was induced by the injection of feces suspension (control). A 5-day course of G-CSF treatment was given before the septic insult (G-CSF). The inflammatory response was decreased using various doses of the LPS-blocking peptide LBPK95A (5 mg/kg=100%Combi group,0.5 mg/kg=10%Combi group, and0.05 mg/kg=1%Combi group). Survival rates were observed. Bacterial clearance, neutrophil infiltration, tissue damage, and the induction of hepatic and systemic inflammatory responses were determined 2 h and 12 h after the septic insult.Results. High-dose LBPK95A (100% Combi) reduced the survival rate to 10%, whereas low-dose LBPK95A (10% and 1% Combi) increased the survival rates to 50% and 80%, respectively. The survival rates inversely correlated with multiorgan damage as indicated by the serum levels of ALT and urea. G-CSF treatment increased the white blood cell counts, hepatic neutrophil infiltration, and bacterial clearance in the liver, lung, and blood. The blockade of the LPS-LBP interaction decreased neutrophil infiltration, led to increased white blood cell count, and decreased hepatic neutrophil infiltration, irrespective of dose. However, bacterial clearance improved in the 1% and 10% Combi groups but worsened in the 100% Combi group. G-CSF increased TNF-αand IL-6 levels. Irrespective of dose, the blockade of the LPS-LBP interaction was associated with low systemic cytokine levels and delayed increases in hepatic TNF-αand IL-6 mRNA expression. The delayed increase in cytokines was associated with the phosphorylation of STAT3 and AKT.Conclusion. Our results revealed that increasing innate immunity by G-CSF pretreatment and decreasing inflammatory responses using LBPK95A improved the survival rates in a rat sepsis model and could be a novel strategy to treat sepsis.

Sepsis and SIRS (systemic inflammatory response syndrome) belong to a severe disease complex characterized by infection and/or a whole-body inflammatory state. There is a growing body of evidence that neutrophils are actively involved in sepsis and are responsible for both release of cytokines and phagocytosis of pathogens. The neutrophil level is mainly regulated by G-CSF, a cytokine and drug, which is widely used in the septic patient with neutropenia. This review will briefly summarize the role of neutrophils and the therapeutic effect of G-CSF in sepsis. We further suggest that targeting neutrophil function to modulate the balance between innate immunity and inflammatory injury could be a worthwhile therapeutic strategy for sepsis.

Because of the lack of adequate organs, the number of patients with end-stage liver diseases, acute liver failure or hepatic malignancies waiting for liver transplantation is constantly increasing. Accepting aged liver grafts is one of the strategies expanding the donor pool to ease the discrepancy between the growing demand and the limited supply of donor organs. However, recipients of organs from old donors may show an increased posttransplantation morbidity and mortality due to enhanced ischemia-reperfusion injury. Energy metabolism, inflammatory response, and autophagy are 3 critical processes which are involved in the aging progress as well as in hepatic ischemia-reperfusion injury. Compared with young liver grafts, impairment of energy metabolism in aged liver grafts leads to lower adenosine triphosphate production and an enhanced generation of free radicals, both aggravating the inflammatory response. The aggravated inflammatory response determines the extent of hepatic ischemia-reperfusion injury and augments the liver damage. Autophagy protects cells by removal of damaged organelles, including dysfunctional mitochondria, a process impaired in aging and involved in ischemia-reperfusion-related apoptotic cell death. Furthermore, autophagic degradation of cellular compounds relieves intracellular adenosine triphosphate level for the energy depressed cells. Strategies targeting the mechanisms involved in energy metabolism, inflammatory response, and autophagy might be especially useful to prevent the increased risk for ischemia-reperfusion injury in aged livers after major hepatic surgery.

Ischemic preconditioning exerts a protective effect in hepatic ischemia/reperfusion injury. The exact mechanism of ischemic preconditioning action remains largely unknown. Recent studies suggest that autophagy plays an important role in protecting against ischemia/reperfusion injury. However, the role of autophagy in ischemic preconditioning-afforded protection and its regulatory mechanisms in liver ischemia/reperfusion injury remain poorly understood. This study was designed to determine whether ischemic preconditioning could protect against liver ischemia/reperfusion injury via heme oxygenase-1-mediated autophagy.Laboratory investigation.University animal research laboratory.Male inbred Lewis rats and C57BL/6 mice.Ischemic preconditioning was produced by 10 minutes of ischemia followed by 10 minutes of reperfusion prior to 60 minutes of ischemia. In a rat model of hepatic ischemia/reperfusion injury, rats were pretreated with wortmannin or rapamycin to evaluate the contribution of autophagy to the protective effects of ischemic preconditioning. Heme oxygenase-1 was inhibited with tin protoporphyrin IX. In a mouse model of hepatic ischemia/reperfusion injury, autophagy or heme oxygenase-1 was inhibited with vacuolar protein sorting 34 small interfering RNA or heme oxygenase-1 small interfering RNA, respectively.Ischemic preconditioning ameliorated liver ischemia/reperfusion injury, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory cytokines, and less severe ischemia/reperfusion-associated histopathologic changes. Ischemic preconditioning treatment induced autophagy activation, as indicated by an increase of LC3-II, degradation of p62, and accumulation of autophagic vacuoles in response to ischemia/reperfusion injury. When ischemic preconditioning-induced autophagy was inhibited with wortmannin in rats or vacuolar protein sorting 34-specific small interfering RNA in mice, liver ischemia/reperfusion injury was worsened, whereas rapamycin treatment increased autophagy and mimicked the protective effects of ischemic preconditioning. Furthermore, ischemic preconditioning increased heme oxygenase-1 expression. The inhibition of heme oxygenase-1 with tin protoporphyrin IX in rats or heme oxygenase-1-specific small interfering RNA in mice decreased ischemic preconditioning-induced autophagy and diminished the protective effects of ischemic preconditioning against ischemia/reperfusion injury.Ischemic preconditioning protects against liver ischemia/reperfusion injury, at least in part, via heme oxygenase-1-mediated autophagy.

Liver dysfunction is a serious complication in the early phase following major liver resection or liver transplantation and might be aggravated by the translocation of bacteria and lipopolysaccharide (LPS). As a preventive strategy, granulocyte colony-stimulating factor (G-CSF) is prophylactically applied in patients who are subjected to major surgery. However, we previously demonstrated that G-CSF can induce LPS sensitization. In this study, we aimed to evaluate the effects of G-CSF pretreatment on hepatic microcirculatory disturbances and postoperative liver dysfunction after 70 % partial hepatectomy (PH) in rats. PH alone was well tolerated by all animals (100 % survival rate, slight liver damage and inflammation). LPS application after 70 % PH caused moderate inflammation, microcirculatory disturbances and hepatic damage and led to a 24-h survival rate of 30 % after the operations. In the G-CSF-LPS-PH group, all of the rats died within 4 h with severe inflammatory responses and liver damage (i.e., pronounced erythrocyte congestion and neutrophil infiltration). Portal hypertension and microcirculatory disorders (i.e., inhomogeneous perfusion, sinusoidal dilatation and reductions on functional capillary density) were more pronounced in the G-CSF-LPS-PH group. In conclusion, increased circulating LPS levels were associated with an imbalanced inflammatory response and microcirculatory dysfunction that preceded liver damage and subsequent dysfunction following surgery. G-CSF-pretreatment aggravated microcirculatory disturbances and liver damage, which might have been related to G-CSF-induced LPS sensitization.

In this study we analysed ischemic rat liver lobes with Near Infrared Spectroscopy (NIRS) to evaluate the potential of NIRS as predictor of ischemic reperfusion injury (IRI). We were able to predict duration of ischemia using visible and near infrared light.

Background Steatotic livers are particularly vulnerable to ischemia/reperfusion injury (IRI). One of the reasons is an underlying impairment of autophagy. Autophagy is regulated by glycogen synthase kinase 3b (GSK3b) and extracellular signal-regulated kinases (ERK1/2) pathways. Both of them are target proteins of a cell-protective drug, lithium chloride. Lithium chloride treatment reduces IRI in many organs including liver. Therefore, we aimed to investigate the effect of lithium chloride treatment on autophagy induction in steatotic rat livers. We also wanted to evaluate the related cell-protective effects on the enhanced hepatic IRI. Materials and methods After inducing hepatic steatosis, rats were injected with lithium chloride or normal saline for 3 d before being subjected to 70% selective warm ischemia for 60 min. After reperfusion, rats were observed for 30 min, 6, 24, and 48 h. Results Lithium chloride appeared to protect hepatocytes from IRI via its ability to induce autophagy by modulation of both GSK3b and ERK1/2 pathways. Hepatic damage was significantly decreased in the treatment group as indicated by a reduced inflammatory response, less apoptosis, less necrosis, and lower liver enzyme levels. Conclusions Simultaneous modulation of GSK3b and ERK1/2 pathways might be an interesting strategy to reduce IRI in steatotic livers with an impairment of autophagy.

We previously demonstrated that baicalein could protect against liver ischemia/reperfusion (I/R) injury in mice. The exact mechanism of baicalein remains poorly understood. Autophagy plays an important role in protecting against I/R injury. This study was designed to determine whether baicalein could protect against liver I/R injury via induction of autophagy in rats. Baicalein was intraperitoneally injected 1 h before warm ischemia. Pretreatment with baicalein prior to I/R insult significantly blunted I/R-induced elevations of serum aminotransferase levels and significantly improved the histological status of livers. Electron microscopy and expression of the autophagic marker LC3B-II suggested induction of autophagy after baicalein treatment. Moreover, inhibition of the baicalein-induced autophagy using 3-methyladenine (3-MA) worsened liver injury. Furthermore, baicalein treatment increased heme oxygenase (HO)-1 expression and pharmacological inhibition of HO-1 with tin protoporphyrin IX (SnPP) abolished the baicalein-mediated autophagy and the hepatocellular protection. In primary rat hepatocytes, baicalein-induced autophagy also protected hepatocytes from hypoxia/reoxygenation injury in vitro and the beneficial effect was abrogated by 3-MA or Atg7 siRNA, respectively. Suppression of HO-1 activity by SnPP or HO-1 siRNA prevented the baicalein-mediated autophagy and resulted in increased hepatocellular injury. Collectively, these results suggest that baicalein prevents hepatocellular injury via induction of HO-1-mediated autophagy.

High mobility group box 1 (HMGB1) acts as an early mediator in inflammation and organ injury. Ischemia reperfusion (I/R) injury induces HMGB1 translocation and expression in ischemic areas. However, it is unknown whether selective warm liver I/R injury also induces the expression of HMGB1 in non-ischemic lobes. The present study aimed to test the hypothesis that selective liver I/R injury also causes HMGB1 translocation and up-regulates its expression in non-ischemic liver areas. In the present study, selective I/R injury was induced by clamping the median and left lateral liver lobes for 90 min followed by 0.5, 6 and 24 h reperfusion. We used male inbred Lewis rats; six animals for each point in time and six animals for the normal control group. Selective hepatic I/R injury induced morphological changes not only in ischemic lobes but also in non-ischemic lobes. HMGB1 translocation and expression was increased in a time-dependent manner in the ischemic lobes, and increased in with delayed onset in the non-ischemic lobes. Serum HMGB1 levels were increased after reperfusion. Furthermore, liver I/R injury up-regulated the expression of HMGB1 receptors (Toll-like receptor 4 and receptor for advanced glycation end products and pro-inflammatory cytokines (Tumor necrosis factor-alpha and interleukin-6) in both ischemic lobes, however, the up-regulation of these cytokines was more prominent in the ischemic lobes. In conclusion, selective warm I/R induces a substantial "sympathetic/bystander" effect on the non-ischemic lobes in terms of HMGB1 translocation and local cytokine production.

Many physiological processes and pathological conditions in livers are spatially heterogeneous, forming patterns at the lobular length scale or varying across the organ. Steatosis, a common liver disease characterized by lipids accumulating in hepatocytes, exhibits heterogeneity at both these spatial scales. The main goal of the present study was to provide a method for zonated quantification of the steatosis patterns found in an entire mouse liver. As an example application, the results were employed in a pharmacokinetics simulation.For the analysis, an automatic detection of the lipid vacuoles was used in multiple slides of histological serial sections covering an entire mouse liver. Lobuli were determined semi-automatically and zones were defined within the lobuli. Subsequently, the lipid content of each zone was computed. The steatosis patterns were found to be predominantly periportal, with a notable organ-scale heterogeneity.The analysis provides a quantitative description of the extent of steatosis in unprecedented detail. The resulting steatosis patterns were successfully used as a perturbation to the liver as part of an exemplary whole-body pharmacokinetics simulation for the antitussive drug dextromethorphan. The zonated quantification is also applicable to other pathological conditions that can be detected in histological images. Besides being a descriptive research tool, this quantification could perspectively complement diagnosis based on visual assessment of histological images. Graphical abstractDisplay Omitted HighlightsA new spatial quantification shows heterogeneity of liver biomarkers on two scales.Steatosis was assessed in histological serial sections covering an entire mouse liver.The steatosis is distributed periportally with a notable variation across the organ.Steatosis distributions influence results of a whole-body pharmacokinetics simulation.This quantification can complement visual evaluation of histological images.

Background We adapted the anatomically oriented parenchyma-preserving resection technique for associating liver partition with portal vein ligation (PVL) for staged hepatectomy (ALPPS) in rats and examined the role of revascularization in intrahepatic size regulation. Methods We performed the procedures based on anatomic study. The ALPPS procedure consisted of a 70% PVL (occluding the left median, left lateral, and right lobes), parenchymal transection (median lobe) and partial (10%) hepatectomy (PHx; caudate lobe). The transection effect was evaluated by measuring the extent of hepatic atrophy or regeneration of individual liver lobes in the ALPPS and control groups (70% PVL and 10% PHx without transection). The survival rates after stage II resection and collateral formation within the portal vein system was examined. Results Anatomic study revealed a close spatial relationship between the demarcation line and the middle median hepatic vein. This enabled placing the transection plane without injuring the hepatic vein. Transection was achieved via stepwise clamping, followed by 2–3 parenchyma-preserving piercing sutures on both sides of the clamp. Ligated liver lobes atrophy was significantly enhanced after ALPPS compared with the control group. In contrast, both a significantly greater relative weight of the regenerated lobe and proliferation index on the first postoperative day were observed. All animals tolerated stage II-resection without complications. Portoportal collaterals were only observed in the control group. Conclusion We developed an anatomically precise technique for parenchymal transection. The lack of a dense vascular network between the portalized and deportalized lobes may play an important role in accelerating regeneration and atrophy augmentation.

The effect of granulocyte colony-stimulating factor (G-CSF) on sepsis is discussed controversially in clinical studies. We previously demonstrated that G-CSF treatment induced lipopolysaccharide (LPS) sensitization via up-regulation of LPS-binding protein (LBP). We hypothesized that the futile effect of G-CSF-treatment in sepsis might be due to its ability to up-regulate LBP. Therefore, blockade of LBP may attenuate the G-CSF-induced LPS sensitization and protect animals from polymicrobial sepsis. Endogenous LBP levels were up-regulated by pretreatment with G-CSF, and the LBP protein was blocked by administration of a specific blocking peptide-LBPK95A. Polymicrobial sepsis was induced by intraperitoneal injection of feces slurry. Rats were monitored every 3 up to 72 h to observe the survival rate. Tissue injury, bacterial infiltration, local inflammatory response, and neutrophil infiltration at 0, 2, and 12 h after the septic insult were analyzed. The survival benefit of G-CSF pretreatment was improved when combined with LBPK95A treatment (control vs. G-CSF vs. combi: 36% vs. 56% vs. 93%; P < 0.05). Combined treatment of G-CSF and LBPK95A was associated with the minimal tissue damage. Treatment with LBPK95A significantly inhibited the neutrophil infiltration without interfering with the bacterial clearance. The G-CSF-induced inflammatory sensitization effect was inhibited by LBPK95A, indicated by the decrease of cytokines expression, and the activation of nuclear factor kappa B and signal transducer and activator of transcription 3 signaling pathway. In conclusion, these results suggested that the effect of prophylactic augmentation of the host's response via G-CSF pretreatment was further enhanced by inhibition of the up-regulation of LBP.

In this experimental study we investigated the potential of near infrared (NIR) spectroscopy to support intra-operative assessment of the fat content of the liver, as needed in the context of liver transplantation. Rats with hepatic steatosis of different severities were subjected to the intra-operative assessment using a medical device to record NIR spectra. We observed that the changes in the optical properties of the liver, at 1210 nm using the second derivative, corresponded to the severity of hepatic steatosis as assessed by histology. Further studies have to be undertaken to validate the results, to add more parameters, and to further develop the technology for easy and safe use in the clinic.

Background. Reinfection of liver grafts with hepatitis B virus (HBV) is a pertinent problem in clinical liver transplantation, requiring the development of new treatment strategies. However, no animal model is currently available to study the course and mechanism of hepatitis B reinfection. This was the reason to establish the technique of liver transplantation in the woodchuck, which is a widely used animal model for HBV infection. Material and Methods. For the reinfection study, woodchuck hepatitis virus (WHV)-negative animals were selected as donors, whereas chronic carriers served as recipients (n=3). Immunosuppression consisted of cyclosporine in a daily dose of 5 mglkg. Blood and liver samples were obtained before and 8 hr, 3 weeks, and 10 weeks after transplantation. Virological markers included serological testing for WHV DNA, WHV surface antigen (WHsAg), core antigen (WHcAg), and their antibodies (anti-WHs and anti-WHc). WHV DNA replication intermediates and viral RNA were detected by Southern blot hybridization and Northern blot, respectively. Viral proteins in the liver were visualized by immunohistochemistry for WHsAg and WHcAg. Results. Early after transplantation membranous but no intracytoplasmic staining for WHsAg was detected in the liver graft, which was negative for WHcAg as well as WHV-DNA and RNA. Nearly all hepatocytes in the liver grafts of animals killed at 3 weeks and 10 weeks posttransplant showed strong membranous (WHsAg) and intracytoplasmic (WHsAg and WHcAg) staining, which was higher in frequency and intensity than in carriers before transplantation. The apparently reduced level of WHV replication intermediates and viral RNA in the reinfected liver grafts compared with the carrier animals was caused by the severe morphological changes leading to a replacement of hepatocytes by extended portal infiltrates. Conclusion. The woodchuck proved to be a suitable model to study WHV reinfection after liver transplantation, because the operative procedure was well tolerated. The first sign of viral presence in the graft was WHsAg detected exclusively in the sinusoids. Reinfection was proven by heavy intracytoplasmic staining for WHsAg and WHcAg in the majority of hepatocytes and detection of viral DNA and RNA in the graft.

Summary Recent studies showing the therapeutic effect of young blood on aging-associated deterioration of organs point to young blood as the solution for clinical problems related to old age. Given that defective autophagy has been implicated in aging and aging-associated organ injuries, this study was designed to determine the effect of young blood on aging-induced alterations in hepatic function and underlying mechanisms, with a focus on autophagy. Aged rats (22 months) were treated with pooled plasma (1 ml, intravenously) collected from young (3 months) or aged rats three times per week for 4 weeks, and 3-methyladenine or wortmannin was used to inhibit young blood-induced autophagy. Aging was associated with elevated levels of alanine transaminase and aspartate aminotransferase, lipofuscin accumulation, steatosis, fibrosis, and defective liver regeneration after partial hepatectomy, which were significantly attenuated by young plasma injections. Young plasma could also restore aging-impaired autophagy activity. Inhibition of the young plasma-restored autophagic activity abrogated the beneficial effect of young plasma against hepatic injury with aging. In vitro, young serum could protect old hepatocytes from senescence, and the antisenescence effect of young serum was abrogated by 3-methyladenine, wortmannin, or small interfering RNA to autophagy-related protein 7. Collectively, our data indicate that young plasma could ameliorate age-dependent alterations in hepatic function partially via the restoration of autophagy.

Intrinsic of non-alcoholic fatty liver diseases is an aberrant accumulation of triglycerides (steatosis), which occurs inhomogeneously within lobules. To improve our understanding of the mechanisms involved in this zonation patterning, we developed a mathematical multicompartment model of hepatic fatty acid metabolism accompanied by blood flow simulations. A model analysis determines the influence of the uptake process of fatty acids, the porto-central gradient of plasma fatty acid concentration, and the oxygen supply via blood on the zonation of triglyceride accumulation. From this theoretical perspective, the plasma oxygen gradient, but not the fatty acid gradient, leads the way to a zonated triglyceride accumulation by its decisive role in oxidative processes. In addition, the uptake mechanism of fatty acids seems to be fundamental for a pericentral dominance of steatosis. However, the mechanism of cellular fatty acid uptake from the blood is still under debate. Our theoretical approach supports the transporter-mediated uptake mechanism and reveals that the maximal velocity of fatty acid uptake affects the switching between a periportal and a pericentral triglyceride accumulation. Further research on hepatic fatty acid uptake is needed to push forward our understanding of aberrant triglyceride accumulation in diet-induced steatosis.

Ischemia/reperfusion (I/R) is a pathophysiologic process that occurs during hemorrhagic shock, liver resection and liver transplantation. Baicalein, the main active ingredient of the Scutellaria root, exerts anti-inflammatory and anti-apoptotic properties in the setting of I/R injury in the heart and brain. However, the role of baicalein in liver I/R injury and its regulatory mechanisms remain poorly understood. This study was designed to evaluate the effects of baicalein in a model of liver I/R in mice and to explore the possible mechanisms. Baicalein (100mg/kg) was intraperitoneally injected 1h before warm ischemia. Pretreatment with baicalein protected against liver I/R injury, as indicated by the decreased serum aminotransferase levels and the reduced histopathologic abnormalities. Baicalein also significantly reduced cellular hepatic apoptosis in response to I/R injury. Moreover, pretreatment with baicalein significantly inhibited nuclear factor-kappa B (NF-κB) activation and the subsequent proinflammatory cytokine production, and decreased leukocyte infiltration. In vitro studies, baicalein treatment inhibited the proinflammatory cytokine production via the modulation of NF-κB signaling pathway in lipopolysaccharide-stimulated macrophages. Taken together, these results suggest that baicalein could protect against liver I/R injury via inhibition of inflammation by down-regulating NF-κB activity, and suppression of cellular hepatic apoptosis.

Computer-assisted automatic quantification (CAQ) was developed as an alternative method for the diagnosis of hepatic steatosis in order to compensate for observer-dependent bias. Here, we aim to demonstrate that CAQ can provide an accurate and precise result in analysis of fatty content, but that it is inappropriate to validate CAQ by comparison with conventional pathologist estimation (PE). Male rats were fed with a methionine-choline-deficient plus high-fat diet for three days, one week, or two weeks to induce mild, moderate, or severe steatosis. Samples were collected from all liver lobes. Severity of hepatic steatosis was assessed by an experienced pathologist who estimated the percentage of hepatocytes containing lipid droplets. Fatty content was quantified by PE, CAQ, and biochemical analysis (BA). CAQ, PE, and BA can correctly reflect severe fatty change. However, in the case of mild and moderate steatosis, PE could not reflect the true fatty content ( r between PE and BA was

Liver dysfunction has been known to occur frequently in cases of sepsis. Excessive inflammation and apoptosis are pathological features of acute liver failure. Recent studies suggest that activation of glycogen synthase kinase- (GSK-) 3βis involved in inflammation and apoptosis. We aimed to investigate the protective effects of GSK-3βinhibition on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP), and SB216763 was used to inhibit GSK-3βin C57BL/6 mice. GSK-3βwas activated following CLP. Administration of SB216763 decreased mortality, ameliorated liver injury, and reduced hepatic apoptosis. The inhibition of GSK-3βalso reduced leukocyte infiltration and hepatic inflammatory cytokine expression and release. Moreover, GSK-3βinhibition suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB) but enhanced the transcriptional activity of cAMP response element binding protein (CREB) in the liver. In in vitro studies, GSK-3βinhibition reduced inflammatory cytokine production via modulation of NF-κB and CREB signaling pathways in lipopolysaccharide-stimulated macrophages. In conclusion, these findings suggest that GSK-3βblockade protects against CLP-induced liver via inhibition of inflammation by modulating NF-κB and CREB activity and suppression of hepatic apoptosis.

We aimed to evaluate whether using sodium hyaluronate carboxymethylcellulose membrane (Seprafilm™) can facilitate assessment of hepatic microcirculation via orthogonal polarization spectroscopy (OPS) by preventing intra-abdominal adhesions and whether Seprafilm™ as a foreign material can evoke local or systemic inflammatory reactions. After the right median hepatic vein was ligated, rats received either placement of Seprafilm™ or untreated with observation of 1 or 4 weeks (n = 6/group). Hepatic microcirculation was visualized. Systemic and local inflammatory reactions were evaluated by blood count, histology and immunohistochemical staining for CD68. Seprafilm™ significantly (P

A hallmark of the nonalcoholic fatty liver disease is the accumulation of lipids. We developed a mathematical model of the hepatic lipid dynamics to simulate the fate of fatty acids in hepatocytes. Our model involves fatty acid uptake, lipid oxidation, and lipid export. It takes into account that storage of triacylglycerol within hepatocytes leads to cell enlargement reducing the sinusoids radius and impairing hepatic microcirculation. Thus oxygen supply is reduced, which impairs lipid oxidation. The analysis of our model revealed a bistable behavior (two stable steady states) of the system, in agreement with histological observations showing distinct areas of lipid accumulation in lobules. The first (healthy) state is characterized by intact lipid oxidation and a low amount of stored lipids. The second state in our model may correspond to the steatotic cell; it is marked by a high amount of stored lipids and a reduced lipid oxidation caused by impaired oxygen supply. Our model stresses the role of insufficient oxygen supply for the development of steatosis. We discuss implications of our results in regard to the experimental design aimed at exploring lipid metabolism reactions under steatotic conditions. Moreover, the model helps to understand the reversibility of lipid accumulation and predicts the reversible switch to show hysteresis. The system can switch from the steatotic state back to the healthy state by reduction of fatty acid uptake below the threshold at which steatosis started. The reversibility corresponds to the observation that caloric restriction can reduce the lipid content in the liver.

The inflammatory response after liver ischemia/reperfusion (I/R) contributes to increased risk of liver failure after liver surgery. Strategies aimed to preventing inflammation could be beneficial in reducing liver I/R injury. Recent studies have demonstrated that peptide B"15-42 is able to decrease the injury of I/R in heart and kidney by inhibition of leukocyte migration and preserving endothelial barrier function. Prompted by these results, we hypothesized that B"15-42 could also possess anti-inflammatory abilities to protect from or reduce hepatic I/R injury. Therefore, in this study, we aimed to evaluate the effects of B"15-42 in a model of liver I/R injury in rats. Rats were treated with B"15-42 at initiation of reperfusion and 2 h thereafter. Rats were killed at 0.5, 6, 24, and 48 h after reperfusion. Hepatic mRNA levels of fibrinogen-! (Fg!), Fg" ,F g+ were significantly increased after I/R. Treatment with Fg-derived B"15-42 ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels and fewer I/R-associated histopathologic changes. B"15-42 treatment decreased leukocyte infiltration and expression of hepatic inflammatory cytokines. Moreover, B"15-42 significantly reduced high-mobility group box 1 release and altered mitogen-activated protein kinase activation. In conclusion, B"15-42 treatment protected against liver warm I/R injury. The mechanism of protective action of B"15-42 seemed to involve its ability to reduce hepatic inflammatory response through preventing high-mobility group box 1 release and altering mitogen-activated protein kinase activation. KEYWORDS—Liver ischemia/reperfusion, fibrinogen, B"15-42, inflammatory response, HMGB1, MAPK

Background Lipopolysaccharide (LPS) binding protein (LBP) is an acute phase protein, which upregulated in response to surgical interventions. LBP plays an important role in modulating LPS-induced inflammatory response. We investigated the expression of LBP and the translocation of LPS in rat models of hepatic ischemia reperfusion injury and liver transplantation (LTx). We also elucidated the effect of LBP on the inflammatory response. Methods In this study, cold ischemia (CI), warm ischemia/reperfusion (WI/R), and LTx models were used to model relevant physiologic situations during LTx. Serum and effluent protein levels as well as hepatic-mRNA and protein levels of LBP were examined. LBP released into the effluent during CI was used in a macrophage-LPS-stimulation assay to investigate the role of LBP in modulating the LPS-induced inflammatory response. Blocking experiments using an LBP-inhibitory peptide were performed to confirm the relevance of LPS/LBP for the induction of the inflammatory response. Impairment of the intestinal barrier and translocation of LPS into the liver was visualized by immunohistochemistry. Induction of tumor necrosis factor-alpha (TNF-α) mRNA expression in the liver was taken as indicator of the inflammatory response. Results Upregulation of LBP in serum and/or liver tissue was observed after WI/R, CI and LTx, respectively. The LBP released during CI enhanced the LPS induced inflammatory response in vitro as indicated by an induction of TNF-α. On the other hand, blocking LBP using LBP inhibitory peptide, suppressed the induction of TNF-α in vitro markedly. After LTx, elevated serum LBP levels were associated with post-operative LPS translocation and production of inflammatory cytokines. Conclusions Our findings suggest that translocation of LPS occurs after LTx and that LBP is mediating the LPS-induced inflammatory response after LTx. Blocking LBP using LBP-inhibitory peptide might represent a novel strategy to reduce the I/R-induced inflammatory response.

Ischemia/reperfusion injury (IRI) occurs inevitably in liver transplantations and frequently during major resections, and can lead to liver dysfunction as well as systemic disorders. High-mobility group box 1 (HMGB1) plays a pathogenic role in hepatic IRI. In the normal liver, HMGB1 is located in the nucleus of hepatocytes; after ischemia reperfusion, it translocates to the cytoplasm and it is further released to the extracellular space. Unlike the well-explored functions of nuclear and extracellular HMGB1, the role of cytoplasmic HMGB1 in hepatic IRI remains elusive. We hypothesized that cytoplasmic HMGB1 interacts with binding proteins involved in the hepatocellular response to IRI. In this study, binding proteins of cytoplasmic HMGB1 during hepatic IRI were identified. Liver tissues from rats with warm ischemia reperfusion (WI/R) injury and from normal rats were subjected to cytoplasmic protein extraction. Co-immunoprecipitation using these protein extracts was performed to enrich HMGB1-protein complexes. To separate and identify the immunoprecipitated proteins in eluates, 2-dimensional electrophoresis and subsequent mass spectrometry detection were performed. Two of the identified proteins were verified using Western blotting: betaine–homocysteine S-methyltransferase 1 (BHMT) and cystathionine γ-lyase (CTH). Therefore, our results revealed the binding of HMGB1 to BHMT and CTH in cytoplasm during hepatic WI/R. This finding may help to better understand the cellular response to IRI in the liver and to identify novel molecular targets for reducing ischemic injury.

Aim To analyze time intervals of inflammation and regeneration in a cholestatic rat liver model. Methods In 36 Lewis rats, divided into six groups of 6 animals (postoperative observation periods: 1, 2, 3, 4, 6, 8 wk), the main bile duct was ligated with two ligatures and observed for the periods mentioned above. For laboratory evaluation, cholestasis parameters (bilirubin, γ-GT), liver cell parameters (ASAT, ALAT) and liver synthesis parameters (quick, albumin) were determined. For histological analysis, HE, EvG, ASDCL and HMGB-1 stainings were performed. Furthermore, we used the mRNA of IL-33, GADD45a and p-21 for analyzing cellular stress and regeneration in cholestatic rats. Results In chemical laboratory and histological evaluation, a distinction between acute and chronic cholestatic liver injury with identification of inflammation and regeneration could be demonstrated by an increase in cholestasis (bilirubin: 1-wk group, 156.83 ± 34.12 μmol/L, P = 0.004) and liver cell parameters (ASAT: 2-wk group, 2.1 ± 2.19 μmol/L.s, P = 0.03; ALAT: 2-wk group, 1.03 ± 0.38 μmol/L.s, P = 0.03) after bile duct ligation (BDL). Histological evaluation showed an increase of bile ducts per portal field (3-wk group, 48 ± 6.13, P = 0.004) during the first four weeks after bile duct ligation. In addition to inflammation, which is an expression of acute cholestasis, there was an increase of necrotic areas in the histological sections (2-wk group, 1.38% ± 2.28% per slide, P = 0.002). Furthermore, the inflammation could be verified by ASDCL (4-wk group, 22 ± 5.93 positive cells per portal field, P = 0.041) and HMGB-1 [2-wk group, 13 ± 8.18 positive cells per field of view (FoV), P = 0.065] staining. Therefore, in summary of the laboratory evaluation and histological studies, acute cholestasis could be found during the first four weeks after bile duct ligation. Subsequently, the described parameters declined so that chronic cholestasis could be assumed. For quantification of secondary biliary cirrhosis, eosin staining was performed, which did not reveal any signs of liver remodeling, thus precluding the development of a chronic cholestasis model. Additionally, to establish the chronic cholestasis model, we evaluated liver regeneration capacity through measurements of IL-33, p-21 and GADD45a mRNA. Conclusion We created a chronic cholestasis model. The point of inflammatory and regenerative balance was reached after four weeks. This finding should be used for experimental approaches dealing with chronic cholestatic liver damage.

Background Liver regeneration consists of cellular proliferation leading to parenchymal and vascular growth. This study complements previous studies on cellular proliferation and weight recovery by (1) quantitatively describing parenchymal and vascular regeneration, and (2) determining their relationship. Both together are needed to (3) characterize the underlying growth pattern. Methods Specimens were created by injecting a polymerizing contrast agent in either portal or hepatic vein in normal or regenerating livers after 70% partial hepatectomy. 3D image data were obtained through micro-CT scanning. Parenchymal growth was assessed by determining weight and volume of the regenerating liver. Vascular growth was described by manually determined circumscribed parameters (maximal vessel length and radius of right inferior portal/hepatic vein), automatically determined cumulative parameters (total edge length and total vascular volume), and parameters describing vascular density (total edge length/volume, vascular volume fraction). The growth pattern was explored by comparing the relative increase of these parameters to the increase expected in case of isotropic expansion. Results Liver volume recovery paralleled weight recovery and reached 90% of the original liver volume within 7 days. Comparing radius-related vascular parameters immediately after surgical resection and after virtual resection in-silico revealed a slight increase, possibly reflecting the effect of resection-induced portal hyperperfusion. Comparing length-related parameters between post-operative day 7 and after virtual resection showed similar vascular growth in both vascular systems investigated. In contrast, radius-related parameters increased slightly more in the portal vein. Despite the seemingly homogeneous 3D growth, the observed vascular parameters were not compatible with the hypothesis of isotropic expansion of liver parenchyma and vascular structures. Conclusion We present an approach for the quantitative analysis of the vascular systems of regenerating mouse livers. We applied this technique for assessing the hepatic growth pattern. Prospectively, this approach can be used to investigate hepatic vascular regeneration under different conditions.

Liver is the main organ for lipopolysaccharide (LPS) clearance. Sensitization to LPS is associated with the upregulation of LPS-binding protein (LBP) in animal models. Therefore, we hypothesized that LBP could induce LPS sensitization through enhancing hepatic uptake of LPS. In this study, we examined the role of LBP in pathogenesis of LPS induced systemic inflammatory response syndrome (SIRS). LBP expression was upregulated after granulocyte colony stimulating (G-CSF) pretreatment. The effect of LBP was further confirmed by blockade of LBP using LBP blocking peptide – LBPK95A. After G-CSF pretreatment, upregulation of LBP was observed in bone marrow cells and liver. The G-CSF induced LBP upregulation caused LPS hypersensitization in rats as indicated by higher mortality and severer liver damage. Of note, LBP blockade increased the survival rate and attenuated the liver injury. The LBP induced LPS hypersensitization was associated with increased hepatic uptake of LPS and augmented hepatic expression of LPS receptors, such as toll-like receptor (TLR)-4. Furthermore, LBP mediated early neutrophil infiltration, which led to increased monocyte recruitment in liver after LPS administration. In conclusion, G-CSF induced LBP expression could serve as a new model for investigation of LPS sensitization. We demonstrated the crucial role of LBP upregulation in pathogenesis of LPS induced SIRS. peerReviewed

Lipopolysaccharide (LPS) binding protein (LBP) is up-regulated in inflammation and infection. Its use as a biomarker of severe infection and sepsis is currently discussed controversially. Here a novel LBP-ELISA was established to assess serum LBP-levels after various degree of in a rat partial hepatectomy (PH) model. The LBP enzyme-linked immunosorbent assay (ELISA) was designed based on the binding between LPS and LBP. LPS was employed as capture molecule. An anti-LBP antibody was used as detection antibody. Serum LBP levels were measured in serum obtained 24h after 30% PH, 70% PH and 90% PH in rats using newly established LBP ELISA method. Expression of hepatic LBP mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). The detection range of the ELISA was from 0.1 μg/ml to 60 μg/ml. The correlation between ELISA and western blotting was strong (r=0.885, p0.0001). Hepatic LBP mRNA expression was upregulated after PH. Of note, elevations of serum LBP protein levels were positively correlated to the remnant liver mass (R=0.821, p0.0001), serum albumin levels (R=0.532, p0.05) and total protein levels(R=0.813, p0.0001). In conclusion, we established an economic and rapid ELISA assay for rat serum LBP quantification. Our results speak against using LBP as diagnostic marker of the acute phase response after liver resection as its elevation is related to the size and thereby the synthetic capacity of the small remnant liver.

Macrophage migration inhibitory factor (MIF) is an important mediator of ischemia/reperfusion (I/R) injury in heart, brain and intestine. We previously demonstrated that MIF was released during warm/cold ischemia in vitro. However, the role of MIF in liver I/R injury remains unclear. We aimed to test the hypothesis that MIF acts as an early proinflammatory cytokine and could mediate the inflammatory injury in liver I/R. Rats (n=6 per group) were subjected to 90 min warm ischemia followed by 0.5h, 6h and 24h reperfusion, respectively to liver transplantation (LTx) after 6h of cold ischemia followed by 24h of reperfusion. The expression of MIF, its receptor (cluster of differentiation 74 (CD74)) and the downstream inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)) were analyzed. Peritoneal macrophages were cultured for 6h alone or in the presence of effluent from cold-preserved livers or effluent depleted of MIF. Warm I/R increased hepatic MIF-mRNA and protein expression. MIF-protein was released into peripheral circulation in vivo with a maximum at 0.5h after reperfusion. Induction of MIF-expression was associated with the expression of proinflammatory cytokines and its receptor in both models. MIF released by isolated cold preserved livers, induced TNF-α and IL-1β production by cultured peritoneal macrophages. Intrahepatic upregulation of MIF, release into systemic circulation and the associated upregulation of the proinflammatory mediators suggest a role of MIF in mediating the inflammatory response to I/R injury. Blocking experiments will help to elucidate its role as potential molecular target for preventing hepatic I/R injury.

High mobility group box 1 (HMGB1) is a nuclear transcription factor. Once HMGB1 is released by damaged cells or activated immune cells, it acts as danger molecule and triggers the inflammatory signaling cascade. Currently, evidence is accumulating that posttranslational modifications such as oxidation may modulate the pro-inflammatory potential of danger signals. We hypothesized that oxidation of HMGB1 may reduce its pro-inflammatory potential and could take place during prolonged ischemia and upon reperfusion. Liver grafts were cold preserved for 24 h and flushed with saline in hourly intervals to collect the effluent. Liver grafts, cold-preserved for 6 h, were transplanted into syngeneic recipients to obtain serum and liver samples 24 h after initiation of reperfusion. Addition of the effluent to a macrophage culture induced the synthesis of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6. The stimulatory activity of graft effluent was reduced after depletion of HMGB1 via immunoprecipitation. Oxidation of the effluent HMGB1 using H 2O 2 attenuated its stimulatory activity as well. Liver transplantation of cold preserved grafts caused HMGB1 translocation and release as determined by immunohistochemistry and ELISA-assay, respectively. Using Western blot with non-reducing conditions revealed the presence of oxidized HMGB1 in liver samples obtained after 12 h and in effluent samples after 16 h of cold preservation as well as in liver and serum samples obtained 24 h after reperfusion. These observations confirm that post-translational oxidation of HMGB1 attenuates its pro-inflammatory activity. Oxidation of HMGB1 as induced during prolonged ischemia and by reoxygenation during reperfusion in vivo might also attenuate its pro-inflammatory activity. Our findings also call for future studies to investigate the mechanism of the inhibitory effect of oxidized HMGB1 on the pro-inflammatory potential. © 2012 Liu et al. OA gold

Mechanical injury or ischemia/reperfusion (I/R) injury induces high mobility of group box 1 (HMGB1) translocation and release. However, the surgical procedure itself can initiate pathophysiologic processes causing damage to the respective organ. A liver resection, as an example, leads to portal hyperperfusion injury of the remnant liver. Therefore, we aimed to elucidate the impact of different hepatic surgical injury models on cellular localization and expression of HMGB1. Focal warm I/R injury was induced by clamping the vascular blood supply to the median and left lateral liver lobes for 90 min followed by 0.5 h, 6 h and 24 h reperfusion, as reported previously. Liver injury by PH was induced by subjecting rats to 30%, 70% or 90% partial hepatectomy (PH) followed by a 24 h observation period. Additional 12 rats were subjected to 90% PH and sacrificed at 1 h and 6 h to investigate the expression and release pattern of HMGB1. Elevation of serum liver enzymes indicating hepatic injury peaked at 6 h and recovered thereafter in models, warm I/R injury and PH. Liver injury was confirmed by liver histology. HMGB1 was translocated from the nucleus to the cytoplasm in livers subjected to warm I/R; but not in livers subjected to PH. Both protein and mRNA expression of HMGB1 were significantly up-regulated in livers subjected to warm I/R. In contrast, neither 30% PH, 70% PH nor 90% PH caused an elevation of hepatic HMGB1 mRNA and protein expression. High serum levels of HMGB1 (30 ng/ml) were measured at 0.5 h reperfusion period after warm I/R, much lower levels thereafter (

Fatty liver is a common disease in the western society. Different stimuli, such as obesity or abuse of alcohol, cause the liver to store fat within liver cells, the so called hepatocytes. This growth induced by storage of fat influences the perfusion of the overlying structures, i. e. sinusoids, liver lobules, liver lobes, and consequently the liver itself. The scales of these structures reach from few µm (size of a hepatocyte) to several cm (size of liver). In view of finite element (FE) simulations, the necessity of introducing different scales becomes obvious. For the description of growth effects in the liver the relevant quantities are: perfusion of the liver and amount of substances that are needed for production of fat (glycogen and fatty acids). In this work we present a multiphasic continuum mechanical model for the description of micro-perfusion in the liver lobule using a homogenized multiphasic approach based on the theory of porous media (TPM). Into this model we inserted a zero-dimensional (0D) kinetic model calculating rates of all relevant substances. So far, the model focuses on the time dependent and spatial zonation of glycogen, since production of fat heavily depends on the stored glycogen. Additionally, a possible method for incorporating fatty acids and therefor also growth will be presented. (© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)

Liver grafts suffer from unavoidable injury due to ischemia and manipulation before implantation. Danger signals such as high-mobility group box -1(HMGB1) and macrophage migration inhibitory factor (MIF) play a pivotal role in the immune response. We characterized the kinetics of their release into the effluent during cold/warm ischemia and additional manipulation-induced mechanical damage. Furthermore, we evaluated the relationship between HMGB1/MIF release and ischemic/mechanical damage. Liver enzymes and protein in the effluent increased with increasing ischemia time. HMGB1/MIF- release correlated with the extent of hepatocellular injury. With increasing ischemia time and damage, HMGB1 was translocated from the nucleus to the cytoplasma as indicated by weak nuclear and strong cytoplasmic staining. Enhancement of liver injury by mechanical damage was indicated by an earlier HMGB1 translocation into the cytoplasm and earlier release of danger signals into the effluent. Our results suggest that determination of HMGB1 and MIF reflects the extent of ischemic injury. Furthermore, HMGB1and MIF are more sensitive than liver enzymes to detect the additional mechanical damage inflicted on the organ graft during surgical manipulation.

BACKGROUND: Liver transplantation is often the ultimate option of therapy for chronically hepatitis B virus (HBV) infected patients. Prevention of reinfection is therapy intensive and cost-effective. Adoptive transfer of HBV-specific immunity with the liver from an immune living liver donor (LLD) could be a new approach to prevent reinfection. METHODS: Forty-six potential LLDs were vaccinated against HBV. Humoral (antibodies to hepatitis B virus surface antigen [anti-HBs]-titer) and cellular (IFN-gamma-ELISpot and proliferation-assay) immune responses were examined in donors after immunization and in recipients before and after transplantation. RESULTS: Anti-HBs-titers of up to 50,000 IU/L were detected in LLDs. Fourteen recipients received livers from these donors. We detected humoral immunity in one HBV-naive recipient and in one chronically HBV-infected recipient after transplantation. A transfer of cellular immunity (SI>3) was seen in three recipients. These patients received livers from donors with high anti-HBs-titers of more than 9000 IU/L. Cellular immunity was also detected in the corresponding donors (SI >3 and spots >22). CONCLUSIONS: Our study demonstrates that HBV-specific humoral and cellular immunity can be transferred by liver transplantation after vaccination of the donors. The transfer of B-cell and T-cell immunity correlates with the magnitude of immune responses in the donor.

This study compared measurement of urinary ethyl glucuronide (EtG), a conjugated minor ethanol metabolite with a longer detection window than ethanol itself, with breath alcohol testing and self-report as ways to disclose recent drinking by 18 liver transplant candidates with an alcoholic liver disease diagnosis that underwent an addiction group therapy program. At each therapy session, patients were questioned about any alcohol consumption in the intervening time, and they also performed a mandatory breath alcohol test, while observed urine samples for measurement of EtG were delivered on a voluntary basis. None of the patients ever admitted to intake of alcohol, and only 1 of 127 breath alcohol tests turned out positive. However, 9 patients showed positive EtG results in 24 (49%) of 49 urine samples. The individual frequency of urine samples being positive for EtG ranged from 22% to 100% with a mean value of 57%. Because 6 patients refused to provide urine on a total of 18 occasions, alcohol use might have been even more common. These results underscore the uncertainty of self-report data and the low sensitivity of breath alcohol testing as ways to disclose recent drinking, and underline the necessity of introducing sensitive and specific objective measures of recent alcohol consumption, such as EtG, in the transplantation setting.