Liver Viability Research Articles

564 TLR4 INDUCTION BY TUDCA IN EXPERIMENTAL MODELS OF SYNGENEIC AND ALLOGENEIC STEATOTIC LIVER TRANSPLANTATION

3. Antioxidant capability of rMnSOD as a supplement of a commercially available preservation solution was evaluated in hepatic biopsies cold stored for transplantation. Results: 1. Cold storage induced a marked increase in O−2 levels and a decrease in NO bioavailability in SEC, those detrimental effects were abolished in cells preserved with rMnSOD. 2. In rats, administration of rMnSOD ameliorated hepatic injury and endothelial dysfunction derived from cold storage and warm reperfusion injuries. The beneficial effects of rMnSOD were associated with a reduction in hepatic O−2, ONOO − and inflammation together with an improved antioxidant activity and nitric oxide bioavailability. 3. rMnSOD added to a conventional preservation solution maintains its marked antioxidant activity avoiding oxidative stress formation in hepatic tissue preserved for transplantation. Conclusions: This study demonstrates that rMnSOD markedly improves liver viability and endothelial function after cold ischemia and warm reperfusion. rMnSOD represents a new therapeutic strategy to protect liver grafts undergoing transplantation.

Determination and Extension of the Limits to Static Cold Storage using Subnormothermic Machine Perfusion

Static cold storage (SCS) of the liver for transplantation is limited by time. Continuation of metabolic activity leads to depletion of energy stores and loss of cellular function, which results in poor post-transplant function. Machine perfusion (MP) applied at the end of preservation may improve the viability of marginal liver grafts and provides information on the quality of the organ. We attempt to define the limits to SCS in terms of easily measurable perfusion parameters and investigate whether MP can improve liver viability. Rat livers were cold-stored for 0, 24, 48, 72, and 120 h, after which they were treated with subnormothermic machine perfusion (SNMP). Livers cold-stored for 48 and 72 h were transplanted orthotopically with or without SNMP. During SNMP easily measurable parameters were monitored and adenosine triphosphate (ATP) content was measured following preservation and SNMP. ATP increased significantly during SNMP, but the recovered ATP content deteriorated with increased duration of SCS, with minimal improvement after 72 h of SCS. Vascular resistance during SNMP increased with extended preservation. After 48 h of SCS, orthotopic transplantation survival increased significantly from 50% to 100% with SNMP, but did not improve after 72 h. Vascular resistance and ATP recovery suggest a decrease in viability after 48 h of SCS. Survival data confirms the loss of post-transplant graft function and supports the use of ATP and vascular resistance as useful indicators. Further, we show that the recoverability of a liver using SNMP is limited to 48 h of SCS.

Leptin attenuates ischemia-reperfusion injury in the rat liver

Leptin is an adipocytokine that reduces ischemic damage in several organs including brain and heart. STAT3 activation is a key step for the attainment of leptin effects in various tissues. We evaluated the possible effect of leptin on liver viability and STAT3 activation, in a rat model of ischemia-reperfusion injury. Rat livers, flushed and stored with Belzer solution (4° C for 24 h), were warmly reperfused (3.5 ml/min/g liver for 1 h at 37° C with O(2) ) with Krebs-Ringer bicarbonate. Treatment group underwent an identical protocol with the adjunct of Leptin (10 ng/ml). Liver effluent was harvested to assess LDH and AST output. Liver tissue was used for pSTAT3 expression (western blot and immunostaining), optical microscopy, TUNEL, and Cell Death Detection assays. The pSTAT3 expression was enhanced by administration of leptin. In parallel, LDH and AST output were reduced (P = 0.04 and P = 0.02 for LDH and AST, respectively). Optical microscopy, TUNEL, and Cell Death Detection assay results demonstrated increased viability in livers treated with leptin in comparison with others (Optical microscopy P = 0.02; TUNEL P = 0.01; Cell death Detection assay P = 0.003). In conclusion, cold storage and reperfusion with leptin reduce liver ischemia-reperfusion injury. This effect is associated with an increased expression of pSTAT-3.

Toxicological effects of cationic nanobubbles on the liver and kidneys: Biomarkers for predicting the risk

Nanobubbles with acoustical activity are used as both diagnostic and therapeutic carriers for detecting and treating diseases. We aimed to prepare nanobubbles and assess toxic responses to them in the liver and kidneys. The cytotoxicity of nanobubbles was determined by examining the viability of liver (HepG2) and kidney (293T) cell lines after a 24-h treatment at various concentrations (0.01–2%). Toxic effects of different formulations were compared by determining functional markers such as γ-glutamyl transferase (γ-GT) and blood urea nitrogen (BUN) after intravenous administration of nanobubbles. Cationic nanobubbles caused concentration-dependent cytotoxicity against cultured cells with a more significant effect in the liver than in the kidneys. A significant reduction of viability was revealed at a concentration as low as 0.1%. Cational systems with soyaethyl morpholinium ethosulfate (SME) exhibited the greatest γ-GT level at 6-fold higher than the control. Immunohistochemistry detected liver fibrosis and inflammation with nanobubbles treatment, especially SME-containing ones at higher doses. According to plasma proteomic profiles, gelsolin and fetuin-B were significantly downregulated 3-fold in the high-dose SME-treated group. Transthyretin decreased by 6-fold in this group. The fibrinogen gamma chain expression was highly elevated. The results suggest that these protein biomarkers are sensitive for assessing the risk of nanobubble exposure. This study is the first to systematically evaluate the possible toxicity of nanobubbles in the liver and kidneys.

Pretransplant Screening and Evaluation of Liver Graft Viability Using Machine Perfusion Preservation in Porcine Transplantation

A novel method using machine perfusion for pretransplant screening and evaluation of the viability of liver grafts has been proposed, seeking to prevent severe ischemia-reperfusion injury and to reduce the risk of primary graft nonfunction. This study sought to evaluate the viability of critical grafts, which were obtained from expanded criteria donors or donation after cardiac death donors during preservation with a new machine preservation perfusion system (NES-01). The normalized pressure transition in the hepatic artery was employed as an evaluation index for liver viability. As a result, the normalized pressure (p/p0) in the hepatic artery showed a distinctive transition under each experimental conditions controlled by warm ischemic time (WIT). The high viability graft, obtained under the condition of WIT as 0 minutes (WIT0), showed a quick response to hepatic artery pressure after initiating perfusion, whereas the normalized pressure showed a sudden decrease. In contrast, the normalized pressure among WIT60, which may cause the graft to lose viability, showed a poor hepatic artery response. These findings corresponded to the cumulative release of enzymes. The findings of our study suggest that monitoring of the pressure drop rate in the hepatic artery during machine perfusion can be used to evaluate liver graft viability.

Impact of venous systemic oxygen persufflation supplemented with nitric oxide gas on cold-stored, warm ischemia-damaged experimental liver grafts

The increasing shortage of donor organs has led to the increasing use of organs from non-heart-beating donors. We aimed to assess the impact of venous systemic oxygen persufflation (VSOP) supplemented with nitric oxide (NO) gas during the cold storage (CS) of warm ischemia (WI)-damaged experimental liver grafts. Rat livers (n = 5 per group) were retrieved after 30 minutes of WI induced by cardiac arrest (the WI group) and were thereafter preserved for 24 hours by CS in histidine tryptophan ketoglutarate solution. During CS, gaseous oxygen was insufflated via the caval vein with 40 ppm NO (the VSOP-NO group) or without NO (the VSOP group). Cold-stored livers without WI served as controls. Liver viability was assessed after the preservation period by normothermic isolated reperfusion for 45 minutes with oxygenated Krebs-Henseleit buffer. After 45 minutes of reperfusion, the VSOP-NO-treated livers showed significantly lower alanine aminotransferase values than the WI-damaged livers (10.2 ± 0.2 versus 78.2 ± 14.6 IU/L), whereas the control livers showed no differences from the VSOP-NO-treated livers. The mitochondrial enzyme release was lower in the VSOP-NO group (4.0 ± 0.7 IU/L) versus the WI group (18.2 ± 4.9 IU/L). An increased portal vein pressure was observed throughout reperfusion (45 minutes) in the WI group (21.7 ± 0.2 mm Hg) versus the VSOP-NO group (12.2 ± 0.8 mm Hg) and the control group (19.9 ± 0.4 mm Hg). Furthermore, the NO concentration in the perfusate after 5 minutes of reperfusion was highest in the VSOP-NO group. The release of malondialdehyde into the perfusate was significantly reduced in the VSOP-NO group (0.9 ± 0.1 nmol/mL) versus the WI group (31.3 ± 5.3 nmol/mL). In conclusion, the resuscitation of livers after 30 minutes of WI to a level comparable to that of nonischemically damaged livers is possible with VSOP supplemented with NO gas. Moreover, the application of VSOP with NO minimizes the extent of injuries caused by oxygen free radicals during preservation.

Excorporeal Normothermic Machine Perfusion Resuscitates Pig DCD Livers with Extended Warm Ischemia

The shortage in donor livers has led to increased use of allografts derived from donation after cardiac death (DCD). The compromised viability in these livers leads to inferior post-transplantation allograft function and survival compared with donation after brain death (DBD) donor grafts. In this study, we reconditioned DCD livers using an optimized normothermic machine perfusion system. Livers from 12 Yorkshire pigs (20-30 kg) were subjected to either 0 min (WI-0 group, n = 6) or 60 min (WI-60 group, n = 6) of warm ischemia and 2 h of cold storage in UW solution, followed by 4 h of oxygenated sanguineous normothermic machine perfusion. Liver viability and metabolic function were analyzed hourly. Warm ischemic livers showed elevated transaminase levels and reduced ATP concentration. After the start of machine perfusion, transaminase levels stabilized and there was recovery of tissue ATP, coinciding with an increase in bile production. These parameters reached comparable levels to the control group after 1 h of machine perfusion. Histology and gross morphology confirmed recovery of the ischemic allografts. Our data demonstrate that metabolic and functional parameters of livers with extended warm ischemic time (60 min) can be significantly improved using normothermic machine perfusion. We hereby compound the existing body of evidence that machine perfusion is a viable solution for reconditioning marginal organs.

Anticancer peptide from Chinese toad (Bufo Bufo Gargarizans) skin enhanced sensitivity to 5-Fu in hepatocarcinoma cells (HepG2)

OBJECTIVE To investigate the antiproliferative and apoptogenic activities of peptide extracted from the Chinese toad (Bufo bufogargarizans) skin (TSP) and its effects on hepatocarcinoma cell line. METHODS MTT assaywas used to detect the effects of TSP (50 mg/mL and 5 mg/mL) on the proliferation and viability of Hepatocarcinoma cell line (HepG2) and liver cell line (L-02); Flow cytometry was used in DNA content analysis to determine the cell distribution in different phases of cell cycle; Annexin V-FITC/PI stained fluorescence-activated cell sorter (FACS) and transmission electron microscope (TEM) were used to detect the apoptosis of the treated cells. RESULTS TSP could not suppress the proliferation and viability of normal liver L-02 cells, but strongly inhibited the proliferation and viability of HepG2 cells; TSP (50 mg/mL) primarily arrested the HepG2 cells at G1 phase of the cell cycle; TSP (50 mg/mL) induced apoptosis in HepG2 cells and enhanced the effects of 5-Fu. CONCLUSION TSP has potent antineoplastic activity against human hepatocarcinoma cells with little toxicity to normal liver cells and can enhance the effects of 5-Fu.

Obstructive Jaundice Expands Intrahepatic Regulatory T Cells, Which Impair Liver T Lymphocyte Function but Modulate Liver Cholestasis and Fibrosis

Although obstructive jaundice has been associated with a predisposition toward infections, the effects of bile duct ligation (BDL) on bulk intrahepatic T cells have not been clearly defined. The aim of this study was to determine the consequences of BDL on liver T cell phenotype and function. After BDL in mice, we found that bulk liver T cells were less responsive to allogeneic or syngeneic Ag-loaded dendritic cells. Spleen T cell function was not affected, and the viability of liver T cells was preserved. BDL expanded the number of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), which were anergic to direct CD3 stimulation and mediated T cell suppression in vitro. Adoptively transferred CD4(+)CD25(-) T cells were converted into Treg within the liver after BDL. In vivo depletion of Treg after BDL restored bulk liver T cell function but exacerbated the degrees of inflammatory cytokine production, cholestasis, and hepatic fibrosis. Thus, BDL expands liver Treg, which reduce the function of bulk intrahepatic T cells yet limit liver injury.

Two Chinese Herbal Regimens Safe for the Elderly on Inhibiting Liver and Bladder Tumor Cell Growth and Regulating Gene Expression

Cancers have become one of the most lethal diseases in elders. In traditional Chinese medicine, Tan-Chih-Hsiao-Yao-San (TCHYS) and Long-Daan-Shiah-Gan-Tang (LDSGT) are used to treat cancers. However, the growth-inhibitory effects and gene expression profiles of these drugs on cancer cells are still unclear. This study assessed the effects of TCHYS and LDSGT on viability of liver and bladder tumor cells, and bladder TCCSUP cells were further subjected to profile gene expression patterns with microarray technology for identifying gene candidates that may be involved in the tumorigenesis. The results revealed that both drugs significantly eliminated the growth of Chang liver and three hepatoma cells. On the contrary, the embryonic liver WRL68 cells showed less response to the treatments, whereas the control agent genistein had much higher inhibitory effect in WRL68 cells than in the other hepatoma cells. Both TCHYS and LDSGT, as well as cisplatin and paclitaxel, exhibited dose-dependent suppression on the viability of all bladder cancer cells. To characterize the possible regulation for such effects, the profiling of gene expression was performed with complementary DNA chips. When bladder transitional cell carcinoma (TCC) TCCSUP cells were treated with TCHYS, 29 upregulated and 28 downregulated genes were detected; whereas 54 genes were upregulated in the same cells treated with LDSGT. Moreover, the detected gene expression patterns were also confirmed by using the reverse transcription-polymerase chain reaction assay. This study initiates the evaluations of drug efficacies and gene expression profiles of traditional Chinese medicines, which may provide important information and identify useful biomarkers for treating cancers.

Dopamine as additive to cold preservation solution improves postischemic integrity of the liver

Dopamine pretreatment has been used to confer protection against cellular injury following hypothermia or anoxia, especially in vascular endothelial cells. Ischemia/reperfusion-associated tissue alterations still represent a major drawback in liver transplantation. The present study was aimed to investigate the effect of dopamine as an ex vivo adjunct, added to the cold storage solution, on cold preservation of the liver. Rat livers were excised 30 min after cardiac arrest, flushed with preservation solution and cold stored for 18 h. Dopamine (10, 50 or 100 microM) was added to the preservation solution in other livers. Organ viability was evaluated by 120 min of warm reperfusion in vitro (n = 6, resp.). Dopamine induced a dose related up to fourfold (at 50 mum) reduction in parenchymal (ALT, LDH) and mitochondrial (GLDH) enzyme release and significantly reduced histologic signs of tissue injury. Bile production and tissue ATP was doubled by dopamine. On the molecular level, dopamine enhanced postischemic phosphorylation of protein kinase A and p42/44 MAP kinase. Inhibition of cAMP-PKA pathway by simultaneous application of RP-cAMPs had no effect on P42/44 phosphorylation, or functional recovery of dopamine-treated grafts. Dopamine supplementation of the flush-out solution appears as a simple way for ex vivo augmentation of liver viability during preservation, not mediated via the catecholamine-cAMP signal cascade.

Copper impairs biliary epithelial cells and induces lipid peroxidation, protein oxidation and oxidative DNA damage in the isolated perfused rat liver

Event Abstract Back to Event Copper impairs biliary epithelial cells and induces lipid peroxidation, protein oxidation and oxidative DNA damage in the isolated perfused rat liver Albena Alexandrova1*, Lubomir Petrov1, Margarita Kirkova1, Elina Tzvetanova1, Almira Georgieva1, Galina Nenkova1 and Stefani Vircheva1 1 Bulgarian Academy of Sciences, Institute of Physiology, Bulgaria The liver can be damaged by a wide variety of toxicants, including heavy metals. Although copper is an essential micronutrient, it can be harmful in excess. The isolated perfused rat liver model is considered to be a sensitive system for studying chemically-induced hepatotoxicity. In literature, however, there are rather scarce data about copper effects on isolated perfused rat liver. The hepatic injury, caused by copper overload is hypothesized to result from its role in the generation of reactive oxygen species (ROS). Being highly reactive ROS are able to impair all cellular constituents – lipids, proteins and DNA. The aim of the present study was to investigate whether CuSO4 (0.01, 0.03, and 0.1 mM) in perfused rat liver (for 60 min) was associated with lipid peroxidation, protein oxidation, and oxidative DNA damage, as well as with biliary epithelial cell injury. The results regarding liver viability and function (copper decreased the portal flow and bile secretion and led to liver swelling) showed a concentration-response effect. The highest tested concentration of copper caused complete blockage of portal flow and bile production within 30 min of perfusion. In confirmation of copper-induced injury, increased leakage of lactate dehydrogenase, aspartate transaminase, and alanine transaminase (indexes of hepatocellular membrane integrity) into perfusate were registered. Compared to controls, 0.01 and 0.03mM concentrations of copper increased the amount of thiobarbituric acid reacting substances, a marker of lipid peroxidation, tissue protein carbonyl groups, an index of protein oxidation, and 8-oxo-7,8-dihydro-2-deoxyguanosine, a marker of oxidative DNA damage; the biliary levels of gamma-glutamyltransferase, an index of biliary epithelial cell integrity, were also increased. The results suggest that copper toxicity in the isolated perfused rat liver could be a consequence of its ability to impair the integrity of biliary epithelial cells and is associated with the enhanced lipid peroxidation, protein oxidation, and oxidative DNA damage. Keywords: Copper, Bile, Oxidative Stress Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Poster Topic: Xenobiotic toxicity Citation: Alexandrova A, Petrov L, Kirkova M, Tzvetanova E, Georgieva A, Nenkova G and Vircheva S (2010). Copper impairs biliary epithelial cells and induces lipid peroxidation, protein oxidation and oxidative DNA damage in the isolated perfused rat liver. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00099 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Albena Alexandrova, Bulgarian Academy of Sciences, Institute of Physiology, Sofia, Bulgaria, a_alexandrova_bas@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Google Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Google Scholar Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva PubMed Albena Alexandrova Lubomir Petrov Margarita Kirkova Elina Tzvetanova Almira Georgieva Galina Nenkova Stefani Vircheva Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Delisheng, a Chinese medicinal compound, exerts anti-proliferative and pro-apoptotic effects on HepG2 cells through extrinsic and intrinsic pathways

The anti-proliferative, cytotoxic and apoptogenic activities of delisheng, a Chinese medicinal compound, has been investigated. In this study, the hepatocarcinoma cell line (HepG2) and the liver cell line (L-02) were exposed to delisheng (6.25, 50 and 100 μl/ml). Delisheng suppressed the proliferation and viability of normal liver L-02 cells slightly, but strongly inhibited the proliferation and viability of hepatocarcinoma HepG2 cells. The flow cytometric analysis of HepG2 cells demonstrated that delisheng primarily arrested the HepG2 cells at the G1 phase of the cell cycle. Annexin V-FITC/PI staining corroborates the apoptogenic nature of delisheng on HepG2 cells. The anti-proliferative and pro-apoptotic effect of delisheng in HepG2 cells was associated with changes in the Bcl-2/Bax ratio and the induction of caspase-mediated apoptosis. Upregulation of DR5 expression was observed in HepG2 cells after treatment with delisheng. The findings from the present study suggest that delisheng has selective cytotoxic activities against HepG2 cells. Delisheng triggered time- and dose-dependent apoptosis in HepG2 cells by activating the mitochondria-mediated and death receptor-mediated apoptotic pathways.

Discriminate Liver Warm Ischemic Injury During Hypothermic Machine Perfusion by Proton Magnetic Resonance Spectroscopy: A Study in a Porcine Model

Objective Proton magnetic resonance spectroscopy ( 1H MRS) is a technique to identify and quantify the composition of biofluids. Hypothermic machine perfusion (HMP) is an organ preservation method that has the potential to evaluate organ viability prior to transplantation by analyzing the composition of the perfusate. The aim of this study was to use 1H MRS to examine the perfusate during HMP of porcine livers exposed to warm ischemia (WI) and to identify potential biomarkers of liver viability. Materials and Methods Porcine livers underwent 4 hours of HMP using kidney perfusion solution-1 (KPS-1) as perfusate after exposure to in situ WI of 0 (n = 6) or 2 hours (n = 5). Samples of the perfusate were taken at the start/end of HMP. Lactate and aspartate aminotransferase (AST) in the samples were measured biochemically as surrogates of the WI-induced damage. MRS acquisition was conducted on a 9.4 Tesla (400 MHz) high-resolution system. Results AST increased significantly during 4 hours of HMP within groups ( P < .02) and discriminated WI injury significantly from the start to the end of HMP ( P < .03). 1H MRS showed significantly elevated signal intensity of lactate, alanine, and histidine during HMP within both groups ( P < .02). Furthermore, alanine and histidine were significantly higher in the WI group than in the control group at the end of HMP ( P = .011 and P = .038, respectively). Conclusion AST, alanine, and histidine in HMP perfusate discriminated WI injury on porcine liver grafts and might be potential biomarkers of liver viability.

Effects of uranium on viability of human liver and kidney cells

Effects of uranium on viability of human liver and kidney cells

Mining metastasis related genes by primary-secondary tumor comparisons from large-scale databases

BackgroundMetastasis is the most dangerous step in cancer progression and causes more than 90% of cancer death. Although many researchers have been working on biological features and characteristics of metastasis, most of its genetic level processes remain uncertain. Some studies succeeded in elucidating metastasis related genes and pathways, followed by predicting prognosis of cancer patients, but there still is a question whether the result genes or pathways contain enough information and noise features have been controlled appropriately.MethodsWe set four tumor type classes composed of various tumor characteristics such as tissue origin, cellular environment, and metastatic ability. We conducted a set of comparisons among the four tumor classes followed by searching for genes that are consistently up or down regulated through the whole comparisons.ResultsWe identified four sets of genes that are consistently differently expressed in the comparisons, each of which denotes one of four cellular characteristics respectively – liver tissue, colon tissue, liver viability and metastasis characteristics. We found that our candidate genes for tissue specificity are consistent with the TiGER database. And we also found that the metastasis candidate genes from our method were more consistent with the known biological background and independent from other noise features.ConclusionWe suggested a new method for identifying metastasis related genes from a large-scale database. The proposed method attempts to minimize the influences from other factors except metastatic ability including tissue originality and tissue viability by confining the result of metastasis unrelated test combinations.

Rad50 Is Dispensable for the Maintenance and Viability of Postmitotic Tissues

The majority of spontaneous chromosome breakage occurs during the process of DNA replication. Homologous recombination is the primary mechanism of repair of such damage, which probably accounts for the fact that it is essential for genome integrity and viability in mammalian cells. The Mre11 complex plays diverse roles in the maintenance of genomic integrity, influencing homologous recombination, checkpoint activation, and telomere maintenance. The complex is essential for cellular viability, but given its myriad influences on genomic integrity, the mechanistic basis for the nonviability of Mre11 complex-deficient cells has not been defined. In this study we generated mice carrying a conditional allele of Rad50 and examined the effects of Rad50 deficiency in proliferative and nonproliferative settings. Depletion of Rad50 in cultured cells caused extensive DNA damage and death within 3 to 5 days of Rad50 deletion. This was not associated with gross telomere dysfunction, suggesting that the telomeric functions of the Mre11 complex are not required for viability. Rad50 was also dispensable for the viability of quiescent liver and postmitotic Purkinje cells of the cerebellum. These findings support the idea that the essential functions of the Mre11 complex are associated with DNA replication and further suggest that homologous recombination is not essential in nondividing cells.

Role of chemokines and their receptors in viral persistence and liver damage during chronic hepatitis C virus infection

Chemokines produced in the liver during hepatitis C virus (HCV) infection induce migration of activated T cells from the periphery to infected parenchyma. The milieu of chemokines secreted by infected hepatocytes is predominantly associated with the T-helper cell/Tc1 T cell (Th1/Tc1) response. These chemokines consist of CCL3 (macrophage inflammatory protein-1 alpha; MIP-1 alpha), CCL4 (MIP-1 beta), CCL5 (regulated on activation normal T cell expressed and secreted; RANTES), CXCL10 (interferon-gamma-inducible protein-10; IP-10), CXCL11 (interferon-inducible T-cell alpha chemoattractant; I-TAC), and CXCL9 (monokine induced by interferon gamma; Mig) and they recruit T cells expressing either CCR5 or CXCR3 chemokine receptors. Intrahepatic and peripheral blood levels of these chemokines are increased during chronic hepatitis C. The interaction between chemokines and their receptors is essential in recruiting HCV-specific T cells to control the infection. When the adaptive immune response fails in this task, non-specific T cells without the capacity to control the infection are also recruited to the liver, and these are ultimately responsible for the persistent hepatic damage. The modulation of chemokine receptor expression and chemokine secretion could be a viral escape mechanism to avoid specific T cell migration to the liver during the early phase of infection, and to maintain liver viability during the chronic phase, by impairing non-specific T cell migration. Some chemokines and their receptors correlate with liver damage, and CXCL10 (IP-10) and CXCR3 levels have shown a clinical utility as predictors of treatment response outcome. The regulation of chemokines and their receptors could be a future potential therapeutic target to decrease liver inflammation and to increase specific T cell migration to the infected liver.

Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia–warm reperfusion injury

Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia-warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after 2 conditions of CI (4 degrees C for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, whereas gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10 microM) was added to the University of Wisconsin solution, and livers were preserved for 42 hours (4 degrees C), then reperfused for 1 hour in Krebs solution (37 degrees C), containing 20% erythrocytes. Liver viability parameters were recorded, and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2%-60% of liver biopsies; mean, 26% +/- 9%) to isolated necrotic hepatocytes as found in treated livers (0.2%-12%; mean, 3% +/- 2%) (P < 0.05). These data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions.

Liver integrity after warm ischemia in situ and brief preservation ex vivo: The value of aerobic post-conditioning

We evaluated the respective effects of warm ischemic injury in non-heart-beating donor (NHBD) grafts and/or cold ischemia time on liver viability. Eventually, the restorative potential of oxygenated hypothermic perfusion after cold storage should be investigated. Livers were retrieved from male Wistar rats and preserved with HTK-solution for 6 h or 18 h by cold storage (CS). Organ retrieval took place either prior to (ctrl.) or 30 min after cardiac arrest (NHBD). Compared to 6 h CS of ctrl. livers, enzyme leakage and functional recovery (oxygen consumption, ammonia clearance, bile production) upon warm reperfusion were massively deteriorated after 18 h CS in NHBD-livers. By contrast, 6 h CS of NHBD resulted in an only limited impairment of all parameters, which was found quite similar to the results in ctrl. after 18 h CS. Induction of cellular apoptosis (cleavage PARP) was found equally influenced by preceding warm ischemia (NHBD) or extended times of CS, but significantly triggered only by the combination of both events. After 6 h of CS, 1 h of oxygenated hypothermic machine perfusion (‘post-conditioning’) was able to bring the performance of NHBD-liver into line with the controls. Based on this work, we concluded that a limited time of warm ischemia in the donor only multiplied graft injury after long-term CS, but does not need to preclude acceptable results if reperfusion is initiated after short periods of CS. Moreover, conditioning of those grafts is effective even 1 h prior to implantation and may help to judge liver viability according to adequate parameters after hypothermic machine perfusion has been established.