Acute Herpes Simplex Viral Esophagitis Occurring in 5 Immunocompetent Individuals With Eosinophilic Esophagitis.

Herpes simplex esophagitis (HSE) is an acute, severe viral infection of the esophagus, rarely occurring in immunocompetent individuals. Eosinophilic esophagitis (EoE) is a rare immune-mediated esophageal disorder. We recently observed 5 severe HSE cases in diagnosed EoE patients. Four of the 5 patients had active, untreated EoE at the time of infection, so HSE is not likely a side effect of swallowed topical corticosteroids, the first-line medical treatment of EoE. However, this coincidence of these 2 rare conditions raises the question of a causal relationship between these 2 forms of esophagitis, and whether active EoE might predispose to HSE infection.

Outcome of patients with ischemic-like cholangiopathy with secondary sclerosing cholangitis after liver transplantation.

BACKGROUND AND AIMS: Sclerosing cholangitis in critically ill patients (SC-CIP) with sepsis and acute respiratory distress syndrome (ARDS) is a cholestatic liver disease with a rapid progression to liver cirrhosis and hepatic failure. Data on outcome of these patients after liver transplantation (LT) are sparse. PATIENTS AND METHODS: Eleven patients (46 ± 12 years; mean labMELD-score: 27 ± 7) with SC-CIP underwent LT. Six patients had severe polytrauma with multiple bone fractures, sepsis and ARDS. Five non-traumatic patients acquired SC-CIP during long-term intensive-care-unit stays due to sepsis and ARDS. Time to diagnosis, the microbiologic results and the survival rates after LT were evaluated. RESULTS: SC-CIP was diagnosed by endoscopic retrograde cholangiopancreatography (ERCP) within 3 ± 1 months after manifestation of cholestasis and histologically confirmed in explanted livers. The predominant microorganisms isolated in bile were: Enterococcus and Candida albicans. Mean follow-up after LT was 28 ± 20 months. One female patient (non-traumatic) died due to sepsis 26 days after LT. All other patients left the hospital alive, but two (non-traumatic) patients died from sepsis, and one (traumatic) patient died in a hemorrhagic shock, thereafter. Seven of 11 patients (5 with polytrauma) are still alive and have a good quality of life. The survival of the SC-CIP patients after LT was comparable with that of patients transplanted due to alcoholic liver cirrhosis. CONCLUSION: SC-CIP develops rapidly within several months. Enterococcus and C. albicans were the main isolated microorganisms in the bile. Sepsis was the main cause of death after LT. Overall, SC-CIP is a good indication for LT in selected patients.

Role of HSP-90 for increased nNOS-mediated vasodilation in mesenteric arteries in portal hypertension.

AIM: To explore the role of heat shock protein-90 (HSP-90) for nitrergic vasorelaxation in the splanchnic circulation in rats with and without portal hypertension. METHODS: Neuronal nitric oxide synthase (nNOS) and HSP-90 were analyzed by immunofluorescence, western blotting and co-immunoprecipitation in the mesenteric vasculature and isolated nerves of portal-vein-ligated (PVL) rats and sham operated rats. In vitro perfused de-endothelialized mesenteric arterial vasculature was preconstricted with norepinephrine (EC(80)) and tested for nNOS-mediated vasorelaxation by periarterial nerve stimulation (PNS, 2-12 Hz, 45V) before and after incubation with geldanamycin (specific inhibitor of HSP-90 signalling, 3 microg/mL) or L-NAME (non-specific NOS-blocker, 10(-4) mol/L). RESULTS: nNOS and HSP-90 expression was significantly increased in mesenteric nerves from PVL as compared to sham rats. Moreover, nNOS and HSP-90 were visualized in mesenteric nerves by immunofluorescence and immunoprecipitation of nNOS co-immunoprecipitated HSP-90 in sham and PVL rats. PNS induced a frequency-dependent vasorelaxation which was more pronounced in PVL as compared to sham rats. L-NAME and geldanamycin markedly reduced nNOS-mediated vasorelaxation abrogating differences between the study groups. The effect of L-NAME and geldanamycin on nNOS-mediated vasorelaxation was significantly greater in PVL than in sham animals. However, no difference in magnitude of effect between L-NAME and geldanamycin was noted. CONCLUSION: HSP-90 acts as a signalling mediator of nNOS-dependent nerve mediated vascular responses in mesenteric arteries, and the increased nitrergic vasorelaxation observed in portal hypertension is mediated largely by HSP-90.

TNFalpha is required for cholestasis-induced liver fibrosis in the mouse.

TNFalpha, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFalpha knockout mice (TNFalpha-/-). Survival after BDL was 60% in wild-type mice (TNFalpha+/+) and 90% in TNFalpha-/- mice. Body weight loss and liver to body weight ratios were reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFalpha+/+ mice (268.6+/-28.2U/L) compared to TNFalpha-/- mice (105.9U/L+/-24.4). TNFalpha-/- mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, alpha-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-beta mRNA, a profibrogenic cytokine, were markedly reduced in TNFalpha-/- mice compared to TNFalpha+/+ mice. Thus, our data indicate that TNFalpha induces hepatotoxicity and promotes fibrogenesis in the BDL model.

Dietary glycine blunts liver injury after bile duct ligation in rats.

AIM: To investigate the effects of (dietary) glycine against oxidant-induced injury caused by bile duct ligation (BDL). METHODS: Either a diet containing 5% glycine or a standard diet was fed to male Sprague-Dawley (SD) rats. Three days later, BDL or sham-operation was performed. Rats were sacrificed 1 to 3 d after BDL. The influence of deoxycholic acid (DCA) in the presence or absence of glycine on liver cells was determined by measurement of calcium and chloride influx in cultivated Kupffer cells and lactate dehydrogenase (LDH) activity was determined in the supernatant of cultivated hepatocytes. RESULTS: Serum alanine transaminase levels increased to about 600 U/L 1 d after BDL. However, enzyme release was blunted by about two third in rats receiving glycine. Release of the alkaline phosphatase and aspartate aminotransferase was also blocked significantly in the group fed glycine. Focal necrosis was observed 2 d after BDL. Glycine partially blocked the histopathological changes. Incubation of Kupffer cells with DCA led to increased intracellular calcium that could be blocked by incubation with glycine. However, systemic blockage of Kupffer cells with gadolinium chloride had no effects on transaminase release. Incubation of isolated hepatocytes with DCA led to a significant release of LDH after 4 h. This release was largely blocked when incubation with glycine was performed. CONCLUSION: These data indicate that glycine significantly decreased liver injury, most likely by a direct effect on hepatocytes. Kupffer cells do not appear to play an important role in the pathological changes caused by cholestasis.

Role of TLR9 in hepatic stellate cells and experimental liver fibrosis.

Accumulating evidence indicates that bacteria and bacterial products promote hepatic fibrogenesis. The activation of hepatic stellate cells (HSC) plays a central role in hepatic fibrosis. Here, we demonstrate that HSC express toll-like receptor 9 (TLR9), a pattern recognition receptor that is activated by CpG motifs present specifically in bacterial DNA. Upon CpG stimulation human as well as murine HSC isolated from wild-type (TLR9+/+) mice express increased levels of the profibrogenic chemokine monocyte chemotactic protein 1 (MCP-1). In contrast, HSC isolated from TLR9 deficient (TLR9-/-) mice lacked CpG motif induced MCP-1 expression indicating the functionality of TLR9 in HSC. Bile duct ligation revealed significantly lower hepatic MCP-1 and collagen expression and less hepatic fibrosis in TLR9-/- compared to TLR9+/+ mice. In addition, the expression of hepatic alpha-smooth-muscle actin, a known marker for HSC activation, was reduced in TLR9-/- mice indicating that bacterial DNA induces the activation of HSC and therefore promotes hepatic fibrosis.

A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation.

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

Heme oxygenase-1 overexpression increases liver injury after bile duct ligation in rats.

AIM: To investigate the effects of heme oxygenase-1 (HO-1) against oxidant-induced injury caused by bile duct ligation (BDL). METHODS: Either cobalt protoporphyrin (CoPP), a HO-1 inducer, or saline were injected intraperitoneally in male SD-rats. Three days later, BDL or sham-operations were performed. Rats were sacrificed 3 wk after BDL and livers were harvested for histology. Fibrosis was evaluated by sirius red staining and image analysis. Alpha-smooth muscular actin, which indicates activation of stellate cells, was detected by immunohistochemical staining, and cytokine and collagen-Ialpha (Col-Ialpha) mRNA expression was detected using RNase protection assays. RESULTS: Serum alanine transaminase increased 8-fold above normal levels one day after BDL. Surprisingly, enzyme release was not reduced in rats receiving CoPP. Liver fibrosis was evaluated 3 wk after BDL and the sirius red-positive area was found to be increased to about 7.8%. However, in CoPP pretreated rats sirius red-positive areas were increased to about 11.7% after BDL. Collagen-Ialpha and TGF-beta mRNA increased significantly by BDL. Again, this effect was increased by HO-1 overexpression. CONCLUSION: Hepatic fibrosis due to BDL is not reduced by the HO-1 inducer CoPP. In contrast, HO-1 overexpression increases liver injury in rats under conditions of experimental chronic cholestasis.

Characterization of rat and human Kupffer cells after cryopreservation.

Kupffer cells (KC) are the resident macrophages of the liver and represent about 80% of the total fixed macrophage population. They are involved in disease states such as endotoxin shock, alcoholic liver diseases and other toxic-induced liver injury. They release physiologically active substances such as eicosanoids and inflammatory cytokines (IL-1, IL-6, TNFalpha), and produce free radical species. Thus, KC are attractive targets for anti-inflammatory therapies and potential candidates responsible for differences in inflammation in liver disease seen between different individuals. However, to perform parallel in vitro experiments with KC from different donors a suitable method for conservation of KC would be necessary. Therefore, the present study evaluated, whether rat and human KC can be frozen, stored and recovered without losing their functional integrity. Rat and human KC were isolated and either cultured under standard conditions (fresh KC) or cryopreserved in special freezing medium (cryopreserved KC). At least 24 h later, cryopreserved KC were thawed, brought into suspension and seeded in the same density as fresh cells for subsequent experiments. Viability of cultured KC was analyzed by trypan blue exclusion. LPS (or PBS as control) stimulation was performed at different time points and cytokine release was analyzed with IL-6 and TNFalpha ELISAs, respectively. Phagocytic capacity was investigated by using a specific phagocytosis assay and FACS analysis. The recovery rate after thawing was around 57% for rat and around 65% for human cryopreserved KC. The results indicate, that KC can successfully be cryopreserved with an adequate recovery rate of viable cells. The properties of fresh and frozen KC can also be compared after thawing. Freshly isolated and cryopreserved cultured KC showed near-normal morphology and did not differ in the cultivation profiles over a period of 72 h. One to three days after seeding, frozen rat or human KC also retained inducible functions such as the production of TNFalpha or IL-6 after LPS challenge. Finally, regardless if they were cryopreserved or not, no differences in the phagocytic activities of the cells were obtained. Taken together, it is concluded that cryopreservation of KC does not change the physiological characteristics of the cells in vitro. Therefore, the method used here for cryopreservation of especially human KC allows the accumulation of KC from several donors for parallel in vitro experiments.

PD-L1 is induced in hepatocytes by viral infection and by interferon-alpha and -gamma and mediates T cell apoptosis.

BACKGROUND/AIMS: B7-H1 (PD-L1) is a B7-family member that binds to programmed death-1 (PD-1). Recently, deficiency of PD-L1 has been demonstrated to result in accelerated hepatocyte damage in experimental autoimmune hepatitis, and PD-L1 was suggested to play a critical role in regulating T cell homeostasis. Absence of PD-1 enhanced proliferation of T cells in adenovirus-infected livers and resulted in a rapid clearance of the virus. Here, we aimed to get more insight into hepatic PD-L1 expression, regulation and function. METHODS: PD-L1 expression was analyzed by quantitative PCR and FACS-analysis in primary human liver cells and hepatoma cells. Furthermore, coculture experiments with primary human T cells or Jurkat T cells were established. RESULTS: In addition to nonparenchymal liver cells, also hepatocytes constitutively expressed low levels of PD-L1. PD-L1 expression in hepatocytes was strongly enhanced by activated T cells and viral infection, and markedly augmented by further stimulation with type I or type II interferons. Moreover, PD-L1 expression on hepatocytes induced apoptosis in T cells. CONCLUSIONS: Our results suggest a novel bidirectional interaction between hepatocytes and lymphocytes modulated by PD-L1 expression in hepatocytes, which may contribute to the unique immunological properties of the liver.

Enhanced Y1-receptor-mediated vasoconstrictive action of neuropeptide Y (NPY) in superior mesenteric arteries in portal hypertension.

BACKGROUND/AIMS: Vascular hyporeactivity to catecholamines contributes to arterial vasodilation and hemodynamic dysregulation in portal hypertension. Neuropeptide Y (NPY) is a sympathetic neurotransmitter facilitating adrenergic vasoconstriction via Y1-receptors on the vascular smooth muscle. Therefore, we investigated its role for vascular reactivity in the superior mesenteric artery (SMA) of portal vein ligated (PVL) and sham operated rats. METHODS: In vitro perfused SMA vascular beds of rats were tested for the cumulative dose-response to NPY dependent on the presence and level of alpha1-adrenergic vascular tone (methoxamine MT: 0.3-10 microM). Moreover, the effect of NPY (50 nM) on vascular responsiveness to alpha1-adrenergic stimulation (MT: 0.3-300 microM) was evaluated. Y1-receptor function was tested by Y1-selective inhibition using BIBP-3226 (1 microM). RESULTS: NPY dose-dependently and endothelium-independently enhanced MT-pre-constriction in SMA. This potentiation was increasingly effective with increasing adrenergic pre-stimulation and being more pronounced in PVL rats as compared to sham rats at high MT concentrations. NPY enhanced vascular contractility only in PVL rats correcting the adrenergic vascular hyporeactivity. Y1-receptor inhibition completely abolished NPY-evoked vasoconstrictive effects. CONCLUSIONS: NPY endothelium-independently potentiates adrenergic vasoconstriction via Y1-receptors being more pronounced in portal hypertension improving mesenteric vascular contractility and thereby correcting the splanchnic vascular hyporeactivity. This makes NPY a superior vasoconstrictor counterbalancing arterial vasodilation in portal hypertension.

Up-regulation of nNOS and associated increase in nitrergic vasodilation in superior mesenteric arteries in pre-hepatic portal hypertension.

BACKGROUND/AIMS: Splanchnic arterial vasodilation in portal hypertension has been attributed largely to vascular NO overproduction. Three NO-synthase (NOS) isoforms have been identified of which e(ndothelial)-NOS has been found up-regulated and i(nducible)-NOS not expressed in the splanchnic circulation in portal hypertension. So far, n(euronal)-NOS has not been investigated and hence, the current study evaluates nNOS-expression and nNOS-mediated vasorelaxation in a model of portal vein-ligated rats (PVL). METHODS: Mesenteric vasculature of PVL and sham rats was evaluated for nNOS-protein (immunohistochemically and Western blotting). In vitro perfused de-endothelialized mesenteric arterial vasculature was pre-constricted with norepinephrine (EC(80)) and tested for nNOS-mediated vasorelaxation by periarterial nerve stimulation (PNS, 2-12 Hz, 45V) before and after incubation with the NOS-inhibitor L-NAME (10(-4)M). RESULTS: nNOS was localized to the adventitia of the mesenteric arterial tree showing more intense staining and increased protein expression in PVL as compared to sham rats. PNS induced a frequency-dependent vasorelaxation, which was more pronounced in PVL rats. L-NAME abolished this difference in nerval-mediated vasorelaxation, the effect being significantly greater in PVL than in sham animals. CONCLUSIONS: Perivascular nNOS-protein expression is enhanced in mesenteric arteries in portal hypertension mediating an increased nerval NO-mediated vasorelaxation. This nNOS-derived NO overproduction may play an important role in the pathogenesis of arterial vasodilation in portal hypertension.

Characterization of the human zinc finger protein 267 promoter: essential role of nuclear factor Y.

Liver fibrosis results from an excessive deposition of extracellular matrix proteins secreted by activated hepatic stellate cells (HSCs). The activation process is accompanied by an increased activity of various transcription factors, including zinc finger protein 267 (ZNF267). Recently, ZNF267 has been shown to modulate gene expression and to function as a transcriptional repressor. MMP-10 was identified as a target gene; its gene expression and promoter activity are inhibited by ZNF267, which might promote liver fibrogenesis through diminished matrix degradation. However, the transcriptional regulation of the ZNF267 gene is unknown. In the present study, we have cloned and characterized the human ZNF267 promoter containing a 1.5 kb fragment of the 5'-flanking region (-1414/+173). The ZNF267 gene has a TATA-less promoter with multiple transcription initiation sites. Analysis of serial 5'-deletions of luciferase reporter constructs revealed a minimal promoter between -72 and +173 bp. Mutational analysis of putative regulatory elements indicated that a CCAAT box within this region was essential for ZNF267 promoter activity. Electrophoretic mobility shift assays demonstrated that transcription factor nuclear factor Y (NF-Y) bound to the CCAAT box. In co-transfection experiments, NF-YA increased the promoter activity of ZNF267. In conclusion, our results suggest that the binding site for NF-Y is critical for ZNF267 gene regulation and, herewith, the activation of this transcriptional factor may play an important role in the activation process of HSCs and in liver fibrosis.

Contribution of angiotensin II to alcohol-induced pancreatic fibrosis in rats.

The mechanisms by which alcohol causes pancreatic fibrosis remain unknown. Recent studies have demonstrated that angiotensin II contributes to the development of fibrosis in liver, kidney, and heart injury. Here, the effects of angiotensin-converting enzyme inhibitor (captopril) and angiotensin II receptor antagonist (losartan) on alcohol-induced pancreatic fibrosis were examined in an intragastric ethanol-feeding model. Male rats were fed a high-fat liquid diet with either ethanol (16-20 g/kg/day) or isocaloric maltose-dextrin (control) for 4 weeks. Subgroups daily received captopril (60 mg/kg/day), losartan (3 mg/kg/day), or no additional agent included in liquid diets. Mean urine alcohol concentrations in all groups fed ethanol were more than 270 mg/dl and not significantly different. Dietary alcohol caused diffuse gland atrophy and interlobular and intralobular fibrosis with mild structural distortion in the pancreas, an effect that was blunted by captopril or losartan treatment. Alcohol also increased the number of alpha-smooth muscle actin-positive cells and transforming growth factor-beta mRNA expression in the pancreas. These increases were blunted significantly by captopril or losartan treatment. These data suggest that angiotensin II contributes to the development of chronic alcohol-induced pancreatic fibrosis through its stimulation of transforming growth factor-beta expression.

TNF alpha-induced Ras activation due to ethanol promotes hepatocyte proliferation independently of liver injury in the mouse.

Tumor necrosis factor alpha (TNFalpha) has been shown to be both proapoptotic and mitogenic for hepatocytes and necessary for alcohol-induced liver injury. Ras, a known proto-oncogene, is very important in the regulation of cellular responses to TNFalpha. Therefore, the purpose of this study was to investigate the role of Ras in alcohol-induced pathogenesis. Male C57Bl/6 mice were fed ethanol or high-fat control diet via intragastric cannulation for 4 weeks. Ras activity was increased significantly after 4 weeks of ethanol and correlated with an increase in pathologic features. However, in mice deficient in the receptor-type 1 for TNFalpha (TNFR1(-/-)), ethanol-induced liver injury and the increase in Ras activity were significantly blunted compared with wild-type mice. Furthermore, it was demonstrated that H-, K-, and R-Ras isoforms were increased after ethanol exposure in wild-type mice. In TNFR1(-/-) mice, R-Ras activity remained elevated by ethanol, whereas H-Ras and K-Ras activity was blunted significantly under these conditions. Interestingly, hepatocellular proliferation, which was elevated approximately fivefold after 4 weeks of chronic ethanol in wild-type mice, was also blunted in TNFR1(-/-) mice given ethanol. Inhibition of Ras with adenovirus containing a dominant-negative Ras had no effect on ethanol-induced liver injury, but significantly blunted ethanol-induced hepatocyte proliferation by more than 50%. Overexpression of mitochondrial superoxide dismutase using recombinant adenovirus blunted lipid peroxidation and attenuated hepatic injury resulting from ethanol, but had no effect on Ras activation and hepatocyte proliferation caused by ethanol. In conclusion, these data support the hypotheses that hepatocellular oxidative stress leads to cell death and that TNFalpha-induced Ras activation is important in hepatic proliferation in response to ethanol-induced liver injury.

The CYP inhibitor 1-aminobenzotriazole does not prevent oxidative stress associated with alcohol-induced liver injury in rats and mice.

Cytochrome P450 (CYP) 2E1 is induced by ethanol and is postulated to be a source of reactive oxygen species during alcoholic liver disease. However, there was no difference in liver pathology and radical formation between wild-type and CYP2E1 knockout mice fed ethanol. Other CYP isoforms may contribute these effects if CYP2E1 is inhibited or absent. The purpose of this study was, therefore, to determine if blocking most of the P450 isoforms with 1-aminobenzotriazole (ABT; 100 mg/kg i.g.), has any effect on liver damage and oxidative stress due to alcohol in rats and mice. Male C57BL/6 mice and Wistar rats were fed either high-fat control or ethanol-containing enteral diet for 4 weeks. ABT had a significant inhibitory effect on many P450 isoforms independent of concomitant alcohol administration. However, ABT did not protect against liver damage due to alcohol in either species. Indices of oxidative stress and inflammation were also similar in livers from vehicle-treated and ABT-treated animals fed ethanol. In summary, suppression of P450 activity with ABT had no apparent effect on oxidative stress caused by alcohol in both rats and mice. These data support the hypothesis that oxidative stress and liver damage can occur independently of CYP activities in both rats and mice during early alcohol-induced liver injury.

Effects of three superoxide dismutase genes delivered with an adenovirus on graft function after transplantation of fatty livers in the rat.

BACKGROUND: Oxygen-derived free radicals play a central role in ischemia/reperfusion injury after organ transplantation and are degraded by endogenous radical scavengers such as superoxide dismutase (SOD). Overexpression of SOD by delivery of the cytosolic SOD gene with an adenovirus (Ad.SOD1) decreases organ injury and increases survival in a rat model of liver transplantation. However, it is unclear which of the three isoforms of SOD provides the most protective effect. The purpose of this study was to identify the isoform with the highest effectiveness against ischemia/reperfusion injury after transplantation of fatty livers, which are particularly susceptible. METHODS: Donor rats were given ethanol by gavage before harvest to induce steatotic livers. Some of the donors were infected with adenoviruses expressing either the gene lacZ encoding bacterial beta-galactosidase (Ad.lacZ), Ad.SOD1, Ad.SOD2 (mitochondrial isoform), or Ad.SOD3 (extracellular isoform). After transplantation, SOD activity in liver, survival, histopathology, transaminases, and activation of nuclear factor (NF)-kappaB, IkappaB kinase, Jun-N-terminal kinase (JNK), and tumor necrosis factor (TNF)-alpha were evaluated. RESULTS: Ad.SOD1 treatment increased survival, blunted transaminase release, and reduced necrosis, whereas Ad.SOD3 had no protective effect. Ad.SOD2 was not as protective as Ad.SOD1. Ad.SOD1 reduced the activation of NF-kappaB, blunted JNK activity, and reduced TNF-alpha activity. Ad.SOD2 treatment resulted in lower kinase, TNF-alpha, and NF-kappaB activities but was not as effective as Ad.SOD1. IkappaB kinase activity was not affected. CONCLUSION: This study demonstrates that cytosolic SOD represents the most effective isoform of SOD to protect transplanted livers from failure; this may be related to lowered NF-kappaB and JNK activities because of reduced oxygen-derived radical production.

Polyphenols from Camellia sinenesis attenuate experimental cholestasis-induced liver fibrosis in rats.

Accumulation of hydrophobic bile acids during cholestasis leads to generation of oxygen free radicals in the liver. Accordingly, this study investigated whether polyphenols from green tea Camellia sinenesis, which are potent free radical scavengers, decrease hepatic injury caused by experimental cholestasis. Rats were fed a standard chow or a diet containing 0.1% polyphenolic extracts from C. sinenesis starting 3 days before bile duct ligation. After bile duct ligation, serum alanine transaminase increased to 760 U/l after 1 day in rats fed a control diet. Focal necrosis and bile duct proliferation were also observed after 1-2 days, and fibrosis developed 2-3 wk after bile duct ligation. Additionally, procollagen-alpha1(I) mRNA increased 30-fold 3 wk after bile duct ligation, accompanied by increased expression of alpha-smooth muscle actin and transforming growth factor-beta and the accumulation of 4-hydroxynenonal, an end product of lipid peroxidation. Polyphenol feeding blocked or blunted all of these bile duct ligation-dependent changes by 45-73%. Together, the results indicate that cholestasis due to bile duct ligation causes liver injury by mechanisms involving oxidative stress. Polyphenols from C. sinenesis scavenge oxygen radicals and prevent activation of stellate cells, thereby minimizing liver fibrosis.

L-Glycine: a novel antiinflammatory, immunomodulatory, and cytoprotective agent.

PURPOSE OF REVIEW: In recent years, evidence has mounted in favor of the antiinflammatory, immunomodulatory and cytoprotective effects of the simplest amino acid L-glycine. This article will focus on the recent findings about the responsible mechanisms of protection and review the beneficial effects of glycine in different disease states. RECENT FINDINGS: Glycine protects against shock caused by hemorrhage, endotoxin and sepsis, prevents ischemia/reperfusion and cold storage/reperfusion injury to a variety of tissues and organs including liver, kidney, heart, intestine and skeletal muscle, and diminishes liver and renal injury caused by hepatic and renal toxicants and drugs. Glycine also protects against peptidoglycan polysaccharide-induced arthritis and inhibits gastric secretion and protects the gastric mucosa against chemically and stress-induced ulcers. Glycine appears to exert several protective effects, including antiinflammatory, immunomodulatory and direct cytoprotective actions. Glycine acts on inflammatory cells such as macrophages to suppress activation of transcription factors and the formation of free radicals and inflammatory cytokines. In the plasma membrane, glycine appears to activate a chloride channel that stabilizes or hyperpolarizes the plasma membrane potential. As a consequence, agonist-induced opening of L-type voltage-dependent calcium channels and the resulting increases in intracellular calcium ions are suppressed, which may account for the immunomodulatory and antiinflammatory effects of glycine. Lastly, glycine blocks the opening of relatively non-specific pores in the plasma membrane that occurs as the penultimate event leading to necrotic cell death. SUMMARY: Multiple protective effects make glycine a promising treatment strategy for inflammatory diseases.

New method of delivering gene-altered Kupffer cells to rat liver: studies in an ischemia-reperfusion model.

BACKGROUND & AIMS: Kupffer cells play a major role in the pathogenesis of several diseases. They release physiologically active substances that often lead to localized tissue injury. Therefore, the aim of this study was to establish a model to protect the liver through supplementation of Kupffer cells that have been transduced by recombinant adenovirus. METHODS: Optimal conditions for intravenous injection in rats were established using carbon-labeled Kupffer cells. Adenoviral-transduced Kupffer cells encoding the Cu/Zn-SOD gene (Ad.SOD1) or beta-galactosidase reporter gene (Ad.LacZ) were transplanted into recipient rats. Twenty-four hours after transplantation, 70% hepatic ischemia-reperfusion was used to induce hepatic oxidative stress, and liver injury was determined 8 or 24 hours later. RESULTS: In initial experiments, 10%-20% of the injected carbon-labeled cells were localized in the host liver after 24 hours, representing approximately 1% of the total population of Kupffer cells. Pretreatment of the recipient with a single dose of cyclosporin A maximized Kupffer cell reseeding up to 4%-10% of the total Kupffer cell population, suggesting that efficiency is limited by host immune response. Moreover, reseeded Kupffer cells were retained in host livers for up to 14 days after transplant. In livers of animals injected with Kupffer cells transduced with Ad.LacZ, transgene expression was observed, indicating Kupffer cell functional integrity. Injection of Kupffer cells transduced with Ad.SOD1 significantly blunted the increase in serum transaminases and liver injury because of ischemia-reperfusion compared with controls. CONCLUSIONS: This novel approach allows delivery of transduced Kupffer cells in rats, which can be used as an investigative tool as well as a therapeutic strategy against inflammatory liver diseases.