Interferon γ and plasminogen activator inhibitor 1 regulate adhesion formation after partial hepatectomy.

BACKGROUND: The pathophysiology of intra-abdominal adhesions has not been studied extensively. The aim of this study was to elucidate the molecular mechanisms underlying adhesion formation in a murine model and in patients undergoing hepatectomy. METHODS: Partial hepatectomy was performed using bipolar forceps in mice. Wild-type mice, antibodies to CD4 and interferon (IFN) γ, IFN-γ, natural killer T (NKT) cells and plasminogen activator inhibitor (PAI) 1 knockout (KO) mice were used. Recombinant hepatocyte growth factor (HGF) was tested for its ability to prevent adhesions. Liver specimens were obtained during surgery from patients undergoing hepatectomy. Adhesion formation was evaluated using a scoring system that ranged from 0 (no adhesions) to 5 (severe adhesions). Levels of IFN-γ and PAI-1 mRNA, and protein concentration of PAI-I were measured, and fluorescence immunostaining was performed. RESULTS: Adhesion formation depended on IFN-γ produced by NKT cells, and NKT KO mice developed few adhesions (mean(s.d.) 1·7(0·3) versus 4·6(0·4) in wild-type mice; P = 0·037). In wild-type mice, the level of PAI-1 mRNA increased after hepatectomy, followed by a decrease in the tissue plasminogen activator (tPA) mRNA level. Adhesion formation was inhibited completely in PAI-1 KO mice (0(0) versus 4·1(0·8) in wild-type mice; P = 0·002). HGF inhibited formation of abdominal adhesions after hepatectomy by reducing IFN-γ and PAI-1 levels, and increasing tPA levels compared with those in mice treated with phosphate-buffered saline (P < 0·001, P = 0·002 and P = 0·035 respectively). In human liver specimens, NKT cells accumulated in the liver after hepatectomy, and PAI-1 expression was increased 5·25-fold (P = 0·030). CONCLUSION: IFN-γ is a key molecule for abdominal adhesion formation after hepatectomy, acting via the reciprocal balance of PAI-1 and tPA. This molecular mechanism may also regulate adhesion formation in patients following hepatectomy. HGF inhibited formation of adhesions by regulating IFN-γ and PAI-1, suggesting that it may be an important target for prevention of adhesions after hepatectomy. SURGICAL RELEVANCE: Postoperative intra-abdominal adhesions can be asymptomatic or cause significant morbidity and mortality. Adhesion formation after hepatectomy has not been studied extensively. In the present study, the molecular mechanisms underlying intra-abdominal adhesions after hepatectomy were investigated in a murine model and in patients. Interferon (IFN) γ produced by natural killer T cells is a key molecule for adhesion formation after hepatectomy in mice, acting via the reciprocal balance between plasminogen activator inhibitor (PAI) 1 and tissue plasminogen activator, the pivotal factors in fibrinolytic activity. This mechanism was also involved in the regulation of adhesions in human tissue samples. Hepatocyte growth factor (HGF) strongly inhibited adhesion formation by regulating IFN-γ and PAI-1. These results indicate that IFN-γ and PAI-1 are possible therapeutic targets, and HGF could prevent postoperative adhesion formation after hepatectomy.

NFkappaB prevents apoptosis and liver dysfunction during liver regeneration.

Although NFkappaB binding activity is induced during liver regeneration after partial hepatectomy, the physiological consequence of this induction is unknown. We have assessed the role of NFkappaB during liver regeneration by delivering to the liver a superrepressor of NFkappaB activity using an adenoviral vector expressing a mutated form of IkappaBalpha. This adenovirus (Ad5IkappaB) was almost exclusively expressed in the liver and inhibited NFkappaB DNA binding activity and transcriptional activity in cultured cells as well as in the liver in vivo. After partial hepatectomy, infection with Ad5IkappaB, but not a control adenovirus (Ad5LacZ), resulted in the induction of massive apoptosis and hepatocytes as demonstrated by histological staining and TUNEL analysis. In addition, infection with Ad5IkappaB but not Ad5LacZ decreased the mitotic index after partial hepatectomy. These two phenomena, increased apoptosis and failure to progress through the cell cycle, were associated with liver dysfunction in animals infected with the Ad5IkappaB but not Ad5LacZ, as demonstrated by elevated serum bilirubin and ammonia levels. Thus, the induction of NFkappaB during liver regeneration after partial hepatectomy appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression.

Blockade of liver macrophages by gadolinium chloride reduces lethality in endotoxemic rats--analysis of mechanisms of lethality in endotoxemia.

We investigated the effects of gadolinium chloride (GdCl3.6H2O), which blocks phagocytosis by liver macrophages, on the mortality, blood tumor necrosis factor (TNF) levels, and hepatotoxicity in a lethal endotoxic shock rat model system [10 mg/kg body weight lipopolysaccharide (LPS) intravenously]. With administration of GdCl3, twice at 0.5 or 5 mg/kg, the survival rate 24 h after LPS injection was 56% and 100%, respectively, whereas the level of TNF in blood was not affected. Microscopic investigation of the liver revealed that the focal necrosis of hepatocytes under endotoxemia was completely protected by the administration of GdCl3 at 5 mg/kg. We then investigated the effects of GdCl3 on superoxide (O2-) production by isolated liver macrophages in vitro. The O2- production by liver macrophages isolated from control rats was suppressed by GdCl3 in a dose-dependent manner. GdCl3 also had a cytotoxic effect on these macrophages. The enhanced O2- production by liver macrophages isolated from sublethal endotoxemic (1 mg/kg) rats was suppressed by pretreatment with GdCl3 (5 mg/kg). It was suggested that lethality in endotoxemia cannot be explained only by the degree of increase in blood TNF levels and that the mechanism by which GdCl3 reduces mortality and hepatotoxicity in endotoxemia possibly includes suppression of superoxide production by liver macrophages.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

Mechanisms of alcohol-induced hepatotoxicity: studies in rats.

Alcohol treatment results in increases in the release of endotoxin from gut bacteria and membrane permeability of the gut to endotoxin, or both. Females are more sensitive to these changes. Elevated levels of endotoxin activate Kupffer cells to release substances such as eicosanoids, TNF-alpha and free radicals. Prostaglandins increase oxygen uptake and most likely are responsible for the hypermetabolic state in the liver. The increase in oxygen demand leads to hypoxia in the liver, and on reperfusion, alpha-hydroxyethyl free radicals are formed which lead to tissue damage in oxygen-poor pericentral regions of the liver lobule.

Clinicopathologic evaluation of hepatocellular carcinoma with bile duct thrombi.

BACKGROUND: The aim of this study was to evaluate the clinicopathologic characteristics of patients with hepatocellular carcinoma (HCC) and bile duct thrombi (BDT). PATIENTS: Seventeen patients with HCC and BDT among 671 patients with HCC who underwent hepatic resection were enrolled in this study. RESULTS: There were no significant differences in the survival rates between patients with and those without BDT, although the rate of stage IV or portal vein invasion was significantly higher in patients with HCC and BDT than in those with HCC but without BDT. In 9 of 17 patients with BDT, preoperative jaundice was observed. Five of the 17 patients underwent a bile duct resection combined with hepatic resection, and 12 patients underwent hepatic resection with removal of the BDT without bile duct resection. None of the patients had histopathologic evidence of direct tumor invasion into the bile duct wall or of any tumor recurrence related to the BDT. There were no significant differences in the survival rates between patients who underwent bile duct resection and those who did not. CONCLUSION: Hepatic resection and the removal of BDT without bile duct resection were sufficient surgical interventions to treat patients with HCC and BDT.

Adult mesenchymal hamartoma of the liver mimicking bile duct cystadenoma.

We report a rare case of mesenchymal hamartoma in the cirrhotic liver of a 52-year-old Japanese male. The tumor, 3.5 cm in diameter, contained a cystic lesion and was located in the lateral segment. Bile duct cystadenoma was considered most likely preoperatively because of the patient's age and the normal levels of tumor markers. However, since malignancy of the lesion could not be ruled out by preoperative imaging diagnosis, lateral segmentectomy was performed. Histological examination led to a diagnosis of mesenchymal hamartoma, since the lesion consisted of a multilocular abnormal bile duct accompanied by abundant myxomatous or loose collagen.

Management of adrenal metastasis of hepatocellular carcinoma by asynchronous resection of bilateral adrenal glands.

We report on a 65-year-old man who received asynchronous bilateral adrenalectomy for adrenal metastasis of hepatocellular carcinoma. Fifteen months after curative resection of right hepatic lobe for hepatocellular carcinoma, a metastatic lesion of the left adrenal gland was detected and left adrenalectomy was performed. Ten months after the second operation, a metastatic lesion in the right adrenal gland, associated with tumor thrombus in the inferior vena cava, was revealed. Transcatheter arterial embolization of the arteries feeding the metastatic tumor was performed, but its effects were incomplete. As there was the tumor thrombus in the inferior vena cava and no other intrahepatic recurrence or extrahepatic metastasis was found, resection of the right adrenal gland with tumor thrombus, without the employment of veno-venous bypass, was performed, followed by postoperative hormonal supplementation. Changes in the patient's alpha-fetoprotein level were clinically useful for the detection of the metastatic lesions and the evaluation of therapeutic effects. Metastasis to adrenal gland from hepatocellular carcinoma should be actively managed, and the appropriate surgical treatment selected, if intrahepatic recurrence and/or other extrahepatic metastasis are controlled. To achieve higher curability and better outcome in patients with bilateral adrenal metastasis of hepatocellular carcinoma, bilateral total adrenalectomy is indicated, accompanied by effective postoperative hormonal supplementation.

Nine-year survivor after resection of cholangiocellular carcinoma with tumor thrombi in the main portal trunk.

We report a patient with cholangiocellular carcinoma with tumor thrombi in the main portal trunk who has survived for 9.5 years after hepatic resection. A 57-year-old woman underwent an extended left lobectomy, and resection of the caudate lobe plus the main portal trunk for a liver tumor that had a portal tumor thrombus in the main portal trunk. The portal vein was reconstructed with an autologous vein graft obtained from the external iliac vein. Histological examination of the resected specimen revealed moderately differentiated tubular adenocarcinoma compatible with cholangiocellular carcinoma. Factors contributing to the patient's long-term survival are discussed. Aggressive surgical resection can be effective even for such an advanced case of cholangiocellular carcinoma.

[Analysis of mechanism of lung edema in E. coli injected septic rat model--in relation to tumor necrosis factor (TNF) produced from liver, spleen, and alveolar macrophages].

After injection of live E. coli, TNF in blood and culture supernatant of the isolated macrophages from the lung, liver, and spleen, were measured, and possible relationship between their TNF levels and lung edema was examined. The blood TNF activity increased significantly until 3h after the injection in the lethal group (p less than 0.01). The TNF activity of alveolar macrophages showed no changes even in the lethal group. The TNF activities of the liver and spleen macrophages decreased significantly in the lethal group (p less than 0.01-0.05), while those in the sublethal group that didn't induce lung edema and showed no significant decrease. The blood leukocyte count decreased until 6h after the injection in the both sublethal and lethal groups, but there was no significant difference between the two groups. The lung weight difference of the lethal group was significantly higher than that in the control group 12h after injection (p less than 0.05). Therefore, the elevated blood TNF activities in our study may be not elicited from alveolar macrophages but mainly from liver and spleen macrophages. There may be a relationship between the lung edema and the elevated blood TNF activity in the lethal groups.

[Recurrence of hepatocellular carcinoma after liver resection].

Recurrence of hepatocellular carcinoma after liver resection is usually observed in the remnant liver, and includes metachronous multicentric occurrence and intrahepatic metastasis. In stage I and II, disease-free survival rates of clinical stage I patients are significantly better than those of clinical stage II patients, although there are no differences in the disease-free survival rates of patients with advanced disease. Disease-free survival rates in long-term survivors decreased at a constant rate due to metachronous multicentric recurrence. Therefore it is important to follow postoperative patients as long as possible. In the treatment of recurrent tumors, every effort should be made to resect the tumor in the liver. Then, other regional therapies, such as percutaneous ethanol injection therapy, microwave coagulation therapy, and transcatheter arterial chemoembolization, are indicated for patients for whom re-resection is not indicated. To prevent recurrence of hepatocellular carcinoma, it is also important to suppress the hepatic necroinflammatory process due to viral hepatitis.

[Usefulness and problems of total hepatic vascular exclusion in liver surgery].

Total hepatic vascular exclusion (THVE) is an useful method enabling safe and sure hepatic resection in patients with liver tumors adjacent to the large hepatic veins or inferior vena cava (IVC), tumor thrombi, invasion of the IVC, etc. To avoid serious hypotension during THVE, test clamping of the IVC prior to the procedure is indispensable. Hemodynamics should be carefully maintained by blood transfusion and sufficient infusion of colloidal and electrolyte solutions during THVE. The veno-venous bypass method which shunts blood from the IVC and portal vein to the superior vena cava enables prolongation of the period of THVE and is useful to avoid postoperative renal dysfunction. In situ liver perfusion with cold solution during THVE is an additional modality by which the liver is protected from warm ischemic injury and the duration of THVE can be further prolonged. However, the maximum duration of THVE is still controversial, especially in patients with chronic liver damage. The most appropriate method for THVE should be carefully chosen in each case by considering the type of lesion, liver function, and the goal of the surgery.

Nuclear factor {kappa}B inactivation in the rat liver ameliorates short term total warm ischaemia/reperfusion injury.

BACKGROUND: In hepatic ischaemia/reperfusion injury, activated liver macrophages (Kupffer cells) are dominantly regulated by a transcription factor, nuclear factor kappaB (NFkappaB), with respect to expression of inflammatory cytokines, acute phase response proteins, and cell adhesion molecules. AIMS: We assessed whether inactivation of NFkappaB in the liver could attenuate total hepatic warm ischaemia/reperfusion injury. METHODS: We studied rats with hepatic overexpression of inhibitor kappaBalpha super-repressor (IkappaBalpha SR) caused by a transgene introduced using an adenoviral vector. Hepatic ischaemia/reperfusion injury was induced under warm conditions by total occlusion of hepatoduodenal ligament structures for 20 minutes, followed by reperfusion. Controls included uninfected and control virus (AdLacZ) infected rats. RESULTS: IkappaBalpha SR was overexpressed in Kupffer cells as well as in hepatocytes, blocking nuclear translocation of NFkappaB (p65) into the nucleus after reperfusion. Gene transfection with IkappaBalpha SR, but not with LacZ, markedly attenuated ischaemia/reperfusion injury, suppressing inducible nitric oxide synthase and nitrotyrosine expression in the liver. Moreover, no remarkable hepatocyte apoptosis was detected under IkappaBalpha SR overexpression. CONCLUSIONS: Adenoviral transfer of the IkappaBalpha SR gene in the liver ameliorates short term warm ischaemia/reperfusion injury, possibly through attenuation of hepatic macrophage activation.

Lung water accumulation in rats after repeated challenges of a sublethal dose of E. coli and its relation to the hepatic energy charge.

We produced a septic model of rats in which lung water accumulation was developed. The degree of lung water accumulation was then compared with the hepatic energy status because the liver is not only a metabolic central organ but also one of the central organs of the reticulo-endothelial system (RES), and clinically, lung edema in sepsis seems to relate to failure of the RES. Three experimental models were examined to form lung water accumulation, namely: the lethal model, given E. coli 6.0 X 10(6) CFU/g BW, the sublethal model, given E. coli 2.0 X 10(6) CFU/g BW, and the repeated sublethal dose injection model, given E. coli 2.0 X 10(6) X 2 at 12 hour intervals. In the lethal models, the energy charge (EC) of the liver decreased markedly (p less than 0.001) without recovery and the lung water accumulated (p less than 0.05). In the sublethal models, EC decreased transiently (p less than 0.05) and the lung water did not increase. However, when the microbial challenge with a sublethal dose was repeated, the decreases in EC were prolonged and the lung water increased significantly after the second injection (p less than 0.001) despite a 4.9 per cent mortality during the subsequent 24 hours. It is suggested that when the decrease in liver EC is prolonged, even if it is not fatal, an accumulation of lung water is possible in septic states. This finding may help further analyses of the interrelationship between the lung and the liver in severely infected patients.

Prediction of recurrence after HCC resection. Faint oily deposits on preoperative Lipiodol-CT of remnant liver tissue.

In trying to clarify the high recurrence rate after removal of small hepatocellular carcinoma (HCC), we assessed the postoperative evolution of minute hepatic Lipiodol deposits which had been diagnosed as artifacts on the preoperative Lipiodol-CT. Of 27 patients with solitary HCC less than 5 cm in diameter, 14 had such Lipiodol deposits in the preoperative CT and 9 of them (64%) developed recurrent tumors. On the other hand, 6 of the 13 patients without deposits (46%) suffered recurrence, but in 5 of these 6 patients the HCC was metachronous multicentric. The cumulative survival rate of the non-deposit group was better than that of the deposit group (p < 0.1). The present study suggested that, even in patients with small HCC, minute concomitant tumors invisible by conventional imaging techniques may exist at the time of surgery. Some of these lesions without sufficient tumor vasculature showing a hypervascular blush on angiography appear to retain small, vague Lipiodol deposits.

Intrahepatic distant metastasis and metachronous multicentric occurrence in solitary hepatocellular carcinoma of less than five centimeters in diameter.

During the 7 years from 1984 to 1990, 36 patients underwent liver resection for solitary hepatocellular carcinoma (HCC) measuring less than 5 cm in diameter, with no intrahepatic vascular invasion on imaging diagnoses and no macroscopic infiltration into the tumor capsule or surrounding tissues. Although HCC is less likely to cause intrahepatic adjacent metastasis to the cut liver surface, an analysis revealed the possibility of intrahepatic distant metastasis and metachronous multicentric occurrences, even after complete removal of the primary tumor. The 5-year cumulative survival rate was 53%, while the 5-year cumulative recurrence-free survival rate was 19%. Of the 36 patients, 18 (50%) had suffered a recurrence by April, 1992, one with extrahepatic metastasis. Recurrence of intrahepatic metastasis was multifocal in 5 patients, single and adjacent in 1, and single (or a few) and distant in 11. Multifocal recurrence was observed within 1 year after liver resection. The sole single and adjacent metastatic case occurred in one of eight patients in the recurrent group in whom distance of the surgical margin was less than 1 cm [TW(+)]. Multicentric occurrence was found in 6 of 13 patients (46%) whose recurrent tumors were examined histologically, and all belonged to the "single (or a few) and distant" type of recurrence. In this report, we also present two typical cases of metastasis, one being multifocal metastasis occurring within 3 months after liver resection and the other being intrahepatic metastasis occurring after a 4-year-dormant state, to demonstrate the complicated nature of the intrahepatic metastatic pattern.

Superoxide production by liver macrophages in a septic rat model--relation to arterial ketone body ratio.

The relationship between superoxide production by liver macrophages and arterial ketone body ratio (AKBR), which reflects the oxidation-reduction state in the mitochondrial compartment of hepatocytes, was studied in rats with lethal and sublethal septicemia induced by intravenous injection of live Escherichia coli 014. In the sublethal model, AKBR decreased transiently (p < 0.01) and superoxide production by isolated liver macrophages increased significantly after opsonized zymosan (OZ) stimulation (p < 0.05). On the other hand, in the lethal model, AKBR decreased markedly (p < 0.01) to below 0.4 without recovery, and superoxide production was not activated by OZ stimulation. Thus, when AKBR decreases to an irreversible level, below about 0.4, superoxide production by liver macrophages is impaired, while as long as AKBR remains reversible, more than about 0.4, it is enhanced. It is suggested that superoxide production by the Kupffer cells is related to the intrahepatic oxidation-reduction state in the septic model.

Gadolinium chloride blocks alcohol-dependent liver toxicity in rats treated chronically with intragastric alcohol despite the induction of CYP2E1.

Hepatic CYP2E1 is induced in several models of alcohol administration, but clinically relevant pathology is only observed in rats in a model involving the continuous intragastric administration of an ethanol-containing, corn oil-based, high-fat diet. The level of CYP2E1 correlates with the degree of liver pathology in the intragastric feeding model, which leads to the hypothesis that radical production by CYP2E1 is responsible for the pathology. Destruction of the Kupffer cells with gadolinium chloride (GdCl3) prevented the development of ethanol-dependent pathology and decreased the production of radicals that appeared in the bile of intragastrically alcohol-fed rats. If the induction of CYP2E1 and subsequent formation of oxidant species by the enzyme is causative in the ethanol-dependent hepatic pathology, then protection by GdCl3 could be due an inhibition of CYP2E1 induction. In the current study, ethanol-administration for 4 wk produced marked steatosis, necrosis, and inflammation not seen in control rats. Immunochemically, CYP2E1 was induced 5- to 6-fold in microsomes from the ethanol-treated animals. Rates of p-nitrophenol and chlorzoxazone hydroxylation were elevated approximately 3-fold, consistent with CYP2E1 induction. When GdCl3 was administered with ethanol, there was a decrease of approximately 80% in Kupffer cell receptor expression, and there was a significant decrease in hepatic pathology, which confirms previous studies. However, in the ethanol and GdCl3-treated animals, there was no significant decrease in the induction of CYP2E1. CYP2E1 was elevated approximately 5-fold, as estimated by immunoblot analysis, and rates of p-nitrophenol and chlorzoxazone hydroxylation were elevated 3- to 4-fold in ethanol + GdCl3-treated rats. Thus, these results clearly dissociate the induction of CYP2E1 by intragastric infusion of ethanol from the generation of early alcohol-induced liver disease. It is concluded that Kupffer cells rather than CYP2E1 play the major role in the initiation of hepatocyte damage caused by alcohol.

The role of splenic macrophages in plasma tumor necrosis factor levels in endotoxemia.

We investigated the function of splenic macrophages (M phi s) with respect to changes in plasma tumor necrosis factor (TNF) in lethally endotoxemic rats treated with gadolinium chloride (GdCl3), which blocks the phagocytosis of large liver M phi s. We also carried out an immunohistochemical study to investigate the change of populations of liver and splenic M phi s under the condition of dysfunction of liver M phi s with or without splenectomy. Twenty-four-hour survival rates were 100% in the GdCl3-treated group (n = 6) and 0% in the nontreated group (n = 6). These rates did not change with splenectomy. Immunohistochemical examination with the primary monoclonal antibodies ED1, ED2 and ED3 revealed that large liver M phi s were eliminated after GdCl3 injection, and that this was not related to splenectomy. The splenic M phi s were not affected by GdCl3 treatment. Plasma TNF levels did not differ between the GdCl3-treated and the nontreated groups, irrespective of whether splenectomy was performed. It was suggested that plasma TNF levels are not affected by the splenic M phi s and that they do not compensate for dysfunction of liver M phi s after GdCl3 treatment.