Improving the function of liver grafts exposed to warm ischemia by the Leuven drug protocol: exploring the molecular basis by microarray.

Livers exposed to warm ischemia (WI) before transplantation are at risk for primary nonfunction (PNF), graft dysfunction, and ischemic biliary strictures, all associated with ischemia/reperfusion injury (IRI). Our multifactorial approach, Leuven drug protocol (LDP), has been shown to reduce these effects and increase recipient survival in WI/IRI-damaged porcine liver transplantation. The aim was the identification of the molecular mechanisms responsible for the hepatoprotective effects of the LDP. Porcine livers were exposed to 45 minutes of WI, cold-stored for 4 hours, transplanted, and either modulated (LDP group; n = 3) or not modulated (control group; n = 4). In the LDP group, the donor livers were flushed with streptokinase and epoprostenol before cold perfusion; the recipients received intravenous glycine, a-1-acid-glycoprotein, FR167653 (a mitogen-activated protein kinase inhibitor), a-tocopherol, glutathione, and apotransferrin. Liver samples were taken before WI and 1 hour after reperfusion. Gene expression was determined with microarrays and molecular pathways and key regulatory genes were identified. The number of genes changed between baseline and 1 hour after reperfusion was 686 in the LDP group and 325 in the control group. The extra genes in the LDP group belonged predominantly to pathways related to cytokine activity, apoptosis, and cell proliferation. We identified 7 genes that were suppressed in the LDP group. These genes could be linked in part to the administered drugs. New potential drug targets were identified on the basis of genes induced in the control group but unaffected in the LDP group and interactions predicted by the literature. In conclusion, the LDP primarily resulted in the suppression of inflammation-regulating genes in IRI. Furthermore, the microarray technique helped us to identify additional gene targets.

Multifactorial biological modulation of warm ischemia reperfusion injury in liver transplantation from non-heart-beating donors eliminates primary nonfunction and reduces bile salt toxicity.

OBJECTIVE: To design a multifactorial biological modulation approach targeting ischemia reperfusion injury to augment viability of porcine liver grafts from non-heart-beating donors (NHBD). BACKGROUND DATA: Liver Transplantation (LTx) from NHBD is associated with an increased risk of primary nonfunction (PNF) and biliary complications. In porcine NHBD-LTx, we previously reported a 50% risk of PNF and toxic bile formation in grafts exposed to > or =30' warm ischemia (WI). METHODS: Porcine livers exposed to 45' WI were cold stored, transplanted and either modulated (n = 6) or not (controls, n = 9). In the modulation group, donor livers were flushed with warm Ringers (avoiding cold-induced vasoconstriction), streptokinase (eliminating stagnating thrombi), and epoprostenol (vasodilator, platelet aggregation inhibitor) prior to cold storage. In recipients, glycine (Kupffer cell stabilizer), alpha1-acid-glycoprotein (anti-inflammatory protein), MAPKinase-inhibitor (pro-inflammatory cytokine generation inhibitor), alpha-tocopherol and glutathione (anti-oxidants), and apotransferrin (iron chelator) were administrated intravenously. PNF, survival, lactate, transaminase, TNF-alpha, redox-active iron, and biliary bile salt-to-phospholipid ratio were monitored. RESULTS: No PNF was observed in modulated versus 55% in control pigs (P = 0.025). Survival was 83% in modulated versus 22% in control pigs (P = 0.02). At 180' postreperfusion, lactate was lower in modulated (5.4 +/- 1.9 mmol/L) versus control pigs (9.4 +/- 2.2 mmol/L; P = 0.011). At 60' postreperfusion, there was a trend for lower AST in modulated versus control pigs at 60' (939 +/- 578 vs. 1683 +/- 873 IU/L; P = 0.089). Postreperfusion, TNF-alpha remained stable in modulated pigs (49 +/- 27 pg/mL at 15' and 85 +/- 26 pg/mL at 180'; P = 0.399) but increased in control pigs (107 +/- 36 pg/mL at 15' and 499 +/- 216 pg/mL at 180'; P = 0.023). At 180' postreperfusion, redox-active iron was higher in control pigs versus modulated pigs (0.21+/-0.18 vs. 0.042+/-0.062 mum; P = 0.038). Biliary bile salt-to-phospholipid ratio post-LTx was lower in modulated versus control pigs (1128 +/- 447 vs. 4836 +/- 4619; P = 0.05). CONCLUSIONS: A multifactorial biological modulation eliminates PNF, improves liver function and increases survival. Biochemically, TNF-alpha and redox-active iron are suppressed and biliary bile salt toxicity is reduced. Translating this strategy clinically may lead to wider and safer use of NHBD.

Preventive and therapeutic effects of alpha-acid glycoprotein in mice infected with B. anthracis.

We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha.

Alpha1-acid-glycoprotein protects against trauma-hemorrhagic shock.

BACKGROUND: Recent studies have shown that the acute phase protein alpha(1)-acid-glycoprotein (AAG) directly modifies endothelial cell responsiveness and is a crucial factor for maintaining endothelial barrier function. We hypothesized that the addition of AAG to the resuscitation fluid will prevent edema formation, increases circulating blood volume, and reduces tissue inflammation following soft tissue trauma and hemorrhagic shock. MATERIALS AND METHODS: Male Sprague-Dawley rats (338 +/- 28 g) underwent a 5-cm midline laparotomy (i.e., induction of soft tissue trauma) and were bled to and maintained at a mean arterial pressure of 35 mm Hg for 90 min. The rats were then resuscitated with four times the shed blood volume with Ringer's lactate containing 200 mg/kg AAG or the same amount of albumin. At 6 h after resuscitation, organ wet-to-dry weight ratios and circulating blood volume (Evans blue dilution) were determined. Neutrophil accumulation (myeloperoxidase activity, MPO) and tissue lipid peroxidation (thiobarbituric acid reactive substances) were also measured in the lungs, liver, and intestine. RESULTS: Administration of AAG during the resuscitation significantly increased circulating blood volume and reduced edema formation, neutrophil accumulation, and lipid peroxidation. Interestingly, concomitant plasma IL-6 levels increased while TNF-alpha levels were not significantly affected. CONCLUSIONS: Since addition of AAG to the resuscitation fluid increased circulating blood volume, reduced edema formation, and neutrophil accumulation following trauma and hemorrhagic shock, supplementation of this acute phase protein appears to be a potential adjunct to prevent capillary leakage in patients undergoing major traumatic injury.

Functional protection by acute phase proteins alpha(1)-acid glycoprotein and alpha(1)-antitrypsin against ischemia/reperfusion injury by preventing apoptosis and inflammation.

BACKGROUND: Ischemia followed by reperfusion (I/R) causes apoptosis, inflammation, and tissue damage leading to organ malfunction. Ischemic preconditioning can protect against such injury. This study investigates the contribution of the acute phase proteins alpha(1)-acid glycoprotein (AGP) and alpha(1)-antitrypsin (AAT) to the protective effect of ischemic preconditioning in the kidney. METHODS AND RESULTS: Exogenous AGP and AAT inhibited apoptosis and inflammation after 45 minutes of renal I/R in a murine model. AGP and AAT administered at reperfusion prevented apoptosis at 2 hours and 24 hours, as evaluated by the presence of internucleosomal DNA cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and the determination of renal caspase-1- and caspase-3-like activity. AGP and AAT exerted anti-inflammatory effects, as reflected by reduced renal tumor necrosis factor-alpha expression and neutrophil influx after 24 hours. In general, these agents improved renal function. Similar effects were observed when AGP and AAT were administered 2 hours after reperfusion but to a lesser extent and without functional improvement. Moreover, I/R elicited an acute phase response, as reflected by elevated serum AGP and serum amyloid P (SAP) levels after 24 hours, and increased hepatic acute phase protein mRNA levels after 18 hours of renal reperfusion. CONCLUSIONS: We propose that the antiapoptotic and anti-inflammatory effects of AGP and AAT contribute to the delayed type of protection associated with ischemic preconditioning and other insults. This mechanism is potentially involved in the course of many clinical conditions associated with I/R injury. Moreover, exogenous administration of these proteins may provide new therapeutic means of treatment.

Murine response to DNA encoding herpes simplex virus type-1 glycoprotein D targeted to the liver.

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cells expressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectable through 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with influx of T cells, predominantly CD4(+). The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may be useful in vaccination against herpes simplex virus type-1.

Targeted delivery of DNA encoding herpes simplex virus type-1 glycoprotein D enhances the cellular response to primary viral challenge.

Intravenous injection of plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) complexed with asialoorosomucoid-poly-L-lysine (gD-ASOR) targets foreign DNA to the liver, leading to hepatic expression of gD-1. BALB/c mice were given two intravenous injections of gD-ASOR, pBK-ASOR (plasmid lacking the gD-1 gene but complexed with ASOR), or PBS. The skin was inoculated with 1 x 10(4) PFU of HSV-1 or sham-inoculated, and analyzed for infectious virus and cellular infiltration 1, 3, and 5 days after inoculation. Prior immunization with gD-ASOR led to significantly lower (P < 0.05) viral titers in the skin 5 days after inoculation compared with controls. Infiltration of the skin at the site of inoculation by polymorphonuclear neutrophils (PMNs), T cells, B cells, dendritic cells, and macrophages was monitored immunohistochemically. Significantly higher numbers (P < 0.05) of CD4+ and CD8+ T cells, dendritic cells, and macrophages responded to HSV-1 challenge in mice immunized with gD-ASOR than in mice immunized with pBK-ASOR or PBS. The response by PMNs and B cells was indistinguishable among the treatment groups. These results suggest that BALB/c mice sensitized to gD-1 following gD-ASOR immunization develop an enhanced T-cell response to primary HSV-1 infection.

Prospects for gene therapy of familial hypercholesterolemia.

Familial hypercholesterolemia is an inherited disease in humans that is caused by a deficiency in the receptor that mediates the internalization and degradation of low density lipoprotein. Patients that inherit two abnormal low density lipoprotein receptor alleles have severe hypercholesterolemia, advanced atherosclerosis, and life-threatening coronary artery disease that is refractory to conventional therapies. In this review, we discuss the prospects for gene therapy in the treatment of familial hypercholesterolemia.

The mechanism of biliary excretion of alpha 1-acid glycoprotein in the rat: evidence for a molecular weight-dependent, nonreceptor-mediated pathway.

The transport of human alpha 1-acid glycoprotein from the circulation to the bile has been studied in the rat. Biliary excretion was proportional to the i.v. injected dose, and the percentage excreted remained constant. The amount excreted in the bile (over 4 hr) was inversely related to the rate of hepatic (hepatocyte) uptake and the galactose receptor which is specific for asialo glycoproteins was not involved. Reinjection of the glycoprotein excreted in bile resulted in a similar proportion of the dose being reexcreted, suggesting that a subset of the glycoprotein is not selected for excretion in bile. Transit times from blood to bile for glucagon, insulin, alpha 1-acid glycoprotein, fetuin, albumin, and carcinoembryonic antigen were directly related to their molecular weights. Removal of sialic acid from the asialo glycoproteins did not affect these transit times. Possible mechanisms for the biliary excretion of alpha 1-acid glycoprotein are discussed.