New observations on the fine structure of the liver in goldfish (Carassius auratus)

Summary A re-examination of goldfish liver was made through the use of SEM of fractured samples and TEM of ultrathin-sections and freeze-etch replicas. Several new hepatic fine structures described in the present study are morphologically similar to those reported previously in many higher vertebrates including mammals. Hepatic sinusoids of goldfish contain fenestrations which are arranged into sieve plates. Although the hepatic plates are made up of two layers of hepatocytes, the parenchymal cells of goldfish liver are morphologically similar to mammalian hepatocytes, particularly with respect to the sinusoidal surfaces which are studded with numerous microvilli. The intercellular surfaces of hepatocytes have both nexus and desmosomal junctions, similar to those found in various epithelial cells of higher vertebrates, as cell attachments and communication foci. Tight junctions are found mainly between the openings of the intracellular bile canaliculi and the intralobular bile ductules which are situated in the center of the bicellular hepatic plate.Supported in part by Grants # GM92 and ES07017     

IGL-1 solution reduces endoplasmic reticulum stress and apoptosis in rat liver transplantation

Injury due to cold ischemia reperfusion (I/R) is a major cause of primary graft non-function following liver transplantation. We postulated that I/R-induced cellular damage during liver transplantation might affect the secretory pathway, particularly at the endoplasmic reticulum (ER). We examined the involvement of ER stress in organ preservation, and compared cold storage in University of Wisconsin (UW) solution and in Institute Georges Lopez-1 (IGL-1) solution. In one group of rats, livers were preserved in UW solution for 8 h at 4 °C, and then orthotopic liver transplantation was performed according to Kamada''s cuff technique. In another group, livers were preserved in IGL-1 solution. The effect of each preservation solution on the induction of ER stress, hepatic injury, mitochondrial damage and cell death was evaluated. As expected, we found increased ER stress after liver transplantation. IGL-1 solution significantly attenuated ER damage by reducing the activation of three pathways of unfolded protein response and their effector molecules caspase-12, C/EBP homologous protein-10, X-box-binding protein 1, tumor necrosis factor-associated factor 2 and eukaryotic translation initiation factor 2. This attenuation of ER stress was associated with a reduction in hepatic injury and cell death. Our results show that IGL-1 solution may be a useful means to circumvent excessive ER stress reactions associated with liver transplantation, and may optimize graft quality.     

Hepatic leukocyte recruitment in response to time-limited expression of TNF-alpha and IL-1beta

The development of chronic liver diseases is mediated by sustained hepatic inflammation. Our objective was to characterize the molecular mechanisms responsible for the hepatic inflammatory response to time-limited TNF-alpha and IL-1beta expression. C57Bl/6 mice were injected with 2 x 10(7) plaque forming units intraperitoneally of an adenoviral vector containing TNF-alpha or IL-1beta (AdTNF-alpha or AdIL-1beta). A nonreplicating adenoviral vector served as control. Four days later, under ketamine and xylazine anesthesia, the liver microvasculature was examined by intravital microscopy. In the postsinusoidal venules, leukocyte rolling increased significantly in response to both AdTNF-alpha and AdIL-1beta, compared with controls. This response was significantly reduced following injection of an anti-alpha4-integrin monoclonal antibody (MAb). Postsinusoidal rolling was further reduced to baseline following injection of an anti-P-selectin or anti-L-selectin MAb. Sinusoidal adhesion was greater in mice treated with AdIL-1beta than with AdTNF-alpha. Blocking alpha4-integrin, P-selectin, or L-selectin had no significant effect on sinusoidal or postsinusoidal adhesion. In separate experiments, we administered AdTNF-alpha or AdIL-1beta to mice deficient in ICAM-1. In ICAM-1-/- mice, postsinusoidal leukocyte rolling significantly increased following expression of IL-1beta but not TNF-alpha. AdIL-1beta- but not AdTNF-alpha-mediated sinusoidal adhesion was ICAM-1 dependent. AdTNF-alpha-induced sinusoidal adhesion was significantly reduced following 4 days of anti-MIP-2 MAb and anti-KC MAb. Prolonged expression of the cytokines TNF-alpha and IL-1beta increases hepatic leukocyte-endothelial cell interactions. Interestingly, the mechanisms through which these cytokines bring about adhesion within the sinusoids differ; AdIL-1beta sinusoidal adhesion uses an ICAM-1-dependent mechanism whereas AdTNF-alpha-mediated adhesion is ICAM-1 independent but CXC chemokine dependent.     

Effect of human interferon β gene transfer upon human glioma, transplanted into nude mouse brain, involves induced natural killer cells

We investigated the immunological responses induced by human interferon β (IFNβ) gene transfer in human gliomas produced in the brains of nude mice. A suspension of human glioma U251-SP cells was injected into the brains of nude mice. The IFNβ gene was transferred by intratumoral injection with cationic liposomes or cationic liposomes associated with anti-glioma monoclonal antibody (immunoliposomes). When intratumoral injection of liposomes or immunoliposomes containing the human IFNβ gene was performed every second day for a total of six injections, starting 7 days after tumor transplantation, complete disappearance of the tumor was observed in six of seven mice that had received liposomes and in all seven mice receiving immunoliposomes. In addition, experimental gliomas injected with immunoliposomes were much smaller than those injected with ordinary liposomes following delayed injections beginning 14 days after transplantation. An immunohistochemical study of the treated nude mouse brains revealed a remarkable induction of natural killer (NK) cells expressing asialoGM1 antigen. To investigate the significance of NK cells in the antitumor effect, we injected liposomes or immunoliposomes containing the human IFNβ gene into tumors in nude mice depleted of NK cells by irradiation and anti-asialoGM1 antibody administration. The antitumor effect of the liposomes or immunoliposomes was abolished. Subsequent subcutaneous glioma challenge of the nude mice after intracerebral tumor implantation and gene transfer resulted in no subcutaneous tumor growth. These results suggest that the induction of NK cells is important in the cytocidal effect of liposomes or immunoliposomes containing the human IFNβ gene upon experimental gliomas. Received: 10 February 1998 / Accepted: 1 September 1998     

Targeting of drug loaded immunoliposomes to herpes simplex virus infected corneal cells: an effective means of inhibiting virus replication in vitro

The goal of our studies was to develop liposomes containing antiviral drugs and targeted with antiviral antibody (immunoliposomes) that would be effective at inhibiting replication of herpes simplex virus (HSV) in vitro. To achieve this, a monoclonal antibody to glycoprotein D of HSV was derivatized with palmitic acid and was incorporated into the lamellae of dehydration-rehydration vesicles. The gD containing immunoliposomes were shown to bind specifically to HSV-infected rabbit corneal cells in vitro, whereas control immunoliposomes prepared with a monoclonal antibody of the same class as the anti-gD failed to preferentially bind to virus-infected cells. The gD immunoliposome binding was inhibitable by pretreatment with rabbit anti-HSV serum but not by aggregated normal serum. Thus liposome binding was judged to represent an antigen-antibody reaction not binding to Fc receptors expressed by cells infected with HSV. Immunoliposomes loaded with iododeoxyuridine (IUDR) leaked drug rapidly at 37 degrees C, whereas acyclovir (ACV)-loaded liposomes still contained 48% of drug after 24 hr at 37 degrees C. The ACV-liposomes retained 44% of drug after 14 days at 4 degrees C. The ability of immunoliposomes to inhibit virus replication was compared with that of untargeted and empty liposomes by means of virus yield assays in vitro, Immunoliposomes loaded with either IUDR or ACV inhibited virus replication, although ACV-containing immunoliposomes were the most efficacious. The implications of our in vitro results for the development of immunoliposomes suitable for the treatment of ocular herpes infection are briefly discussed.     

The Relationship between Fenestrations,Sieve Plates and Rafts in Liver Sinusoidal Endothelial Cells

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.     

Simple preparation and characterization of cationic liposomes associated with a monoclonal antibody against glioma-associated antigen (immunoliposomes)

Abstract

In this study we prepared and characterized monoclonal antibody associated cationic liposomes (immunoliposomes) to be used as a vehicle for human gene therapy of malignant glioma. This association method is especially amenable to mass production. The immunoliposomes consist of N-(a-trimethylammonio-acetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroyl phosphatidylcholine (DLPC), and dioleoyl phosphatidyl- ethanolamine (DOPE) in a molar ratio of 1:2:2 as TMAG:DLPC:DOPE. Their preparation required only the addition of a solution containing plasmid DNA and a monoclonal antibody against glioma-associated antigen to a lipid film of the above three lipids. The association of antibody on the surface of immunoliposomes was confirmed by an immunochemical procedure. Liposome-mediated LacZ gene transfection of human glioma cells resulted in p-galactosidase activity about 2- to 3-fold higher when immunoliposomes were used as compared to control liposomes that were not associated to antibody. Also, the production of human (3-interferon (HuIFN-P) into the medium was 2- to 7-fold higher when HuIFN-P gene was transfected. Based on the present results, the immunoliposomes associating a monoclonal antibody against glioma-associated antigen may become effective carriers for gene transfer to human glioma cells.     

Ultrastructural changes in the histohematic barrier of the liver in the early period after endotoxin administration

Investigation of the changes of the mouse liver cells revealed early reaction of endothelial sinusoid cells and Kupffer cells after endotoxin application. During first 60 min. after endotoxin injection there were certain bulge of nuclear zone, changes of nuclear shape, appearance of microfilaments in the cytoplasm of endothelial cells. An increase in the number of the pores and fenestrae grouped in sieve plates was discovered in the endothelial cells. Their luminal surface formed the bubbles. Such changes of the endothelial cells can be described as their activation. Reaction of the endothelial cells of hepatic sinusoids and activation of the Kupffer cells were revealed at the same time.     

Role of ICAM-1 in chronic ethanol consumption-enhanced liver injury after gut ischemia-reperfusion in rats

Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the hepatic microvascular dysfunction elicited by gut ischemia-reperfusion (I/R). Although the effects of chronic ethanol (EtOH) consumption on the liver are well known, it remains unclear whether this condition renders the hepatic microcirculation more vulnerable to the deleterious effects of gut and/or hepatic I/R. The objectives of this study were to determine whether chronic EtOH consumption alters the severity of gut I/R-induced hepatic microvascular dysfunction and hepatocellular injury and to determine whether ICAM-1 contributes to this response. Male Wistar rats, pair fed for 6 wk a liquid diet containing EtOH or an isocaloric control diet, were exposed to gut I/R. Intravital video microscopy was used to monitor leukocyte recruitment in the hepatic microcirculation, the number of nonperfused sinusoids (NPS), and plasma concentrations of endotoxin and tumor necrosis factor-alpha. Plasma alanine aminotransferase (ALT) levels were measured 6 h after the onset of reperfusion. In control rats, gut I/R elicited increases in the number of stationary leukocytes, NPS, and plasma endotoxin, tumor necrosis factor-alpha, and ALT. In EtOH-fed rats, the gut I/R-induced increases in NPS and leukostasis were blunted in the midzonal region, while exaggerated leukostasis was noted in the pericentral region and terminal hepatic venules. Chronic EtOH consumption also enhanced the gut I/R-induced increase in plasma endotoxin and ALT. The exaggerated responses to gut I/R normally seen in EtOH-fed rats were largely prevented by pretreatment with a blocking anti-ICAM-1 monoclonal antibody. In conclusion, these results suggest that chronic EtOH consumption enhances gut I/R-induced hepatic microvascular dysfunction and hepatocellular injury in the pericentral region and terminal hepatic venules via an enhanced hepatic expression of ICAM-1.     

Three-dimensional structure of endothelial cells in hepatic sinusoids of the rat as revealed by the Golgi method

Summary The three-dimensional structure of endothelial cells in the hepatic sinusoids of the rat was studied by application of light- and electron microscopy on Golgi-impregnated specimens. A number of endothelial cells could thus be individually delineated throughout the hepatic lobules. The cytoplasm, showing heavy silver deposits, consists of two distinct areas, a thick and thin portion. The thick portion, issuing from the region of the perikaryon, branches and tapers toward the cell periphery. The thin portion, occupying the remainder of the cytoplasm, consists largely of highly fenestrated sieve plates. Some intralobular variation can be noted; the thick portion of the endothelial cells is well developed in the periportal zone, while the cells in the centrilobular zone are relatively rich in thin portions. In addition, the area of distribution of an individual endothelial cell is larger in the centrilobular sinusoids than in the periportal zone. Some endothelial cells also possess unique cytoplasmic processes projecting into the intercellular space between hepatocytes and connecting the sinusoidal walls of neighboring sinusoids. These processes may anchor the endothelial cells to the hepatic plates.     

Cellular uptake of liposomes targeted to intercellular adhesion molecule-1 (ICAM-1) on bronchial epithelial cells.

Previously, it was demonstrated that immunoliposomes, bearing anti-intercellular adhesion molecule-1 (ICAM-1) antibodies (mAb F10.2), can specifically bind to different cell types expressing ICAM-1. In this study, we have quantified the amount of immunoliposomes binding to IFN-gamma activated human bronchial epithelial cells (BEAS-2B) in vitro and studied the subsequent fate of cell-bound anti-ICAM-1 immunoliposomes. We demonstrate that binding of the immunoliposomes to the epithelial cells depends on the liposome concentration used. After binding to the cell surface, the anti-ICAM-1 immunoliposomes are rapidly internalised by the epithelial cells. Sixty percent of cell-bound immunoliposomes were internalised by the epithelial cells within 1 h of incubation at 37 degrees C. The results indicate that ICAM-1 targeted immunoliposomes may be used as carriers for the intracellular delivery of anti-inflammatory drugs to sites of inflammation characterised by an increased expression of ICAM-1.     

Hypothermic storage of coronary endothelial cells reduces nitric oxide synthase activity and expression

Preservation with University of Wisconsin (UW) solution has been implicated in coronary artery endothelial damage and loss of endothelium-dependent vasodilatation. Therefore, the objective of this study was to investigate the effect of this solution on basal nitric oxide (NO) release from porcine coronary endothelial cells (CEC). Cultures were exposed to cold (4 degrees C) storage in UW solution for 6, 8 and 12 h. Parallel cultures were incubated with control medium at 37 degrees C. After treatment, NO release was evaluated by nitrite production, a stable metabolite of NO. Activity of the constitutive endothelial nitric oxide synthase (eNOS) was measured by the conversion [3H]-l-arginine to [3H]-l-citrulline and eNOS protein expression by Western blotting. Nitrite production by control cells was augmented with increasing times of incubation, whereas no change was observed in those cultures preserved with UW solution. Activity of eNOS was significantly decreased compared to the respective control group by cold storage of cells for longer periods than 6 h. Such decrease was correlated with a diminished eNOS protein expression in CEC preserved with UW solution after 8- and 12-h storage. These results suggest that prolonged hypothermic storage of CEC with UW solution does not preserve basal NO release because of a certain loss of eNOS protein, which may contribute to the reported injury of heart transplants after long-term preservation.     

MONONUCLEAR PHAGOCYTES (KUPFFER CELLS) AND ENDOTHELIAL CELLS : Identification of Two Functional Cell Types in Rat Liver Sinusoids by Endogenous Peroxidase Activity

The fine structural characteristics and phagocytic properties of peroxidase-positive and peroxidase-negative cells in rat hepatic sinusoids were investigated. Cells with a positive peroxidase reaction in the endoplasmic reticulum and the nuclear envelope make up approximately 40% of cells in rat hepatic sinusoids and have abundant cytoplasm containing numerous granules and vacuoles, and occasional tubular, vermiform invaginations. After intravenous injection of colloidal carbon, the luminal plasma membrane of these cells shows continuous sticking of carbon, and there is evidence of avid phagocytosis of colloidal carbon particles. Peroxidase-positive cells are the only cells in hepatic sinusoids which phagocytize large (0.8 µ in diameter) latex particles. In contrast, the peroxidase-negative endothelial cells, which make up 48% of cells, have scanty perinuclear cytoplasm and organelles, and their long cytoplasmic extensions that form the lining of the hepatic sinusoids have fenestrations; these cells ingest small amounts of colloidal carbon, principally by micropinocytosis, exhibit no sticking of carbon particles to their plasma membranes, and do not ingest the larger (latex) particles. The so-called fat-storing cells are peroxidase negative and totally nonphagocytic. The peroxidase reaction thus distinguishes the typical mononuclear phagocytes or Kupffer cells of rat liver from the endothelial-lining cells.     

Protective effects of a carbon monoxide-releasing molecule (CORM-3) during hepatic cold preservation

There is increasing evidence that carbon monoxide (CO), a signaling molecule generated during the degradation of heme by heme oxygenase-1 (HO-1) in biological systems, has a variety of cytoprotective actions, including anti-hypoxic effects at low temperatures. However, during liver cold preservation, a direct effect needs to be established. Here, we designed a study to analyze the role of CO, delivered via a carbon monoxide-releasing molecule (CO-RM) in the maintenance of liver function, and integrity in rats during cold ischemia/reperfusion (CI/R) injury. We used an isolated normothermic perfused liver system (INPL) following a clinically relevant model of ex vivo 48 h cold ischemia stored in a modified University of Wisconsin (UW) solution, to determine the specific effects of CO in a rat model. CO was generated from 50 μM tricarbonylchloro ruthenium-glycinato (CORM-3), a water-soluble transition metal carbonyl that exerts pharmacological activities via the liberation of controlled amounts of CO in biological systems. The physiological effects of CORM-3 were confirmed by the parallel use of a specific inactive compound (iCORM-3), which does not liberate CO in the cellular environment.CORM-3 addition was found to prevent the injury caused by cold storage by improving significantly the perfusion flow during reperfusion (by almost 90%), and by decreasing the intrahepatic resistance (by 88%) when compared with livers cold preserved in UW alone. Also, CORM-3 supplementation preserved good metabolic capacity as indicated by hepatic oxygen consumption, glycogen content, and release of lactate dehydrogenase. Liver histology was also partially preserved by CORM-3 treatment.

Conclusions

These findings suggest that CO-RM could be utilized as adjuvant therapeutics in UW solutions to limit the injury sustained by donor livers during cold storage prior to transplantation, as has been similarly proposed for the heart, and kidney.     

Adrenergic mechanisms in the hepatic microcirculation in the rat

The in vivo hepatic microvascular bed of the rat was observed microscopically in the transilluminated liver and the diameter of the hepatic sinusoids was measured by serial photomicrography. Intraportal infusion of tyramine induced concentration-dependent constriction of the hepatic sinusoids, but also dilatation of the sinusoids when the dose was small. These effects were attributed to the release of endogenous noradrenaline which activated either alpha- or beta-adrenergic receptors and caused constriction, or dilatation, of the sinusoids respectively. Adrenaline and noradrenaline induced similar changes in the hepatic sinusoids as tyramine, while phenoxybenzamine induced dilatation, and propranolol constriction, of the sinusoids. All the above responses were abolished by pretreatment with reserpine. A possible noradrenaline-mediated basal vasomotor tone in the hepatic sinusoids for autonomic control of the blood flow in the sinusoids was postulated.     

Gene expression and activity of urea cycle enzymes of rat hepatocytes cold stored up to 120h in University of Wisconsin solution

Urea cycle (UC) is the main pathway of ammonium removal. A deficiency in any of the five classical enzymes of the pathway causes a urea cycle disorder. Hepatocellular transplantation is one of the techniques applicable to treat this disorder. In the present work, we investigated the activities and the relative expression levels of two of the UC enzymes: Carbamyl phosphate synthetase I (CPSI) and ornithine transcarbamylase (OTC), in isolated hepatocytes preserved up to 120 h in University of Wisconsin (UW) solution at 0 degrees C, and during the rewarming of these suspensions. During preservation, CPSI showed differences in mRNA levels respect to time 0, while ornithine transcarbamylase remained unchanged. At the end of the rewarming, CPSI showed values of enzymatic activity and relative mRNA level comparable with the control, meanwhile, there was an increment in OTC activity. In line with these results, we found that hepatocytes cold preserved up to 120h in UW solution maintained their ability to remove an ammonium load comparable to freshly isolated hepatocytes. These data indicated that our preservation conditions up to 120h in UW solution followed by rewarming, preserves UC enzymes at levels similar to freshly isolated hepatocytes, allowing the use of these cells in bioartificial liver devices or hepatocellular transplantation.     

Molecular cloning of murine intercellular adhesion molecule (ICAM-1).

We have previously reported a murine lymphocyte surface antigen MALA-2 of approximately 95,000 Mr which is expressed mainly on activated lymphocytes. The rat monoclonal antibody YN1/1 that detects this antigen profoundly inhibits mixed lymphocyte response. We have now purified MALA-2 and determined its partial amino acid sequence. By using non-redundant synthetic oligonucleotides as probes, based on the amino acid sequence, we have isolated two full length cDNA clones encoding MALA-2. The two clones are identical except for the 5' end sequence. Expression of MALA-2 on transfected COS cells is only achieved with one of the two cDNA clones. The nucleotide sequence as well as the deduced amino acid sequence of MALA-2 display striking homology with those of the recently reported human intercellular adhesion molecule ICAM-1. All the unique features of the human ICAM-1, including its homology with the neural adhesion molecule NCAM, its internal repeat structure and the immunoglobulin-like structure, are found in MALA-2. Furthermore, purified MALA-2 crosslinked to a solid support binds Con A blasts that express LFA-1, the putative receptor for ICAM-1, and the binding can be blocked by YN1/1 antibody or antimurine LFA-1 antibody indicating a direct interaction of these molecules in cell adhesion. Therefore, we consider MALA-2 to be the murine homolog of human ICAM-1. Since ICAM-1 is known to be of primary importance in immune responses and inflammatory reactions, having a monoclonal antibody and a mouse model will provide the opportunity to study the functional role of ICAM-1 in vivo.     

Cell adhesion molecules involved in the leukocyte recruitment induced by venom of the snake Bothrops jararaca

It has been shown that Bothrops jararaca venom (BjV) induces a significant leukocyte accumulation, mainly neutrophils, at the local of tissue damage. Therefore, the role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), LECAM-1, CD18, leukocyte function-associated antigen-1 (LFA-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) on the BjV-induced neutrophil accumulation and the correlation with release of LTB4, TXA2, tumor necrosis factor-alpha, interleukin (IL)-1 and IL-6 have been investigated. Anti-mouse LECAM-1, LFA-1, ICAM-1 and PECAM-1 monoclonal antibody injection resulted in a reduction of 42%, 80%, 66% and 67%, respectively, of neutrophil accumulation induced by BjV (250 microg/kg, intraperitoneal) injection in male mice compared with isotype-matched control injected animals. The anti-mouse CD18 monoclonal antibody had no significant effect on venom-induced neutrophil accumulation. Concentrations of LTB(4), TXA(2), IL-6 and TNF-alpha were significant increased in the peritoneal exudates of animals injected with venom, whereas no increment in IL-1 was detected. This results suggest that ICAM-1, LECAM-1, LFA-1 and PECAM-1, but not CD18, adhesion molecules are involved in the recruitment of neutrophils into the inflammatory site induced by BjV. This is the first in vivo evidence that snake venom is able to up-regulate the expression of adhesion molecules by both leukocytes and endothelial cells. This venom effect may be indirect, probably through the release of the inflammatory mediators evidenced in the present study.     

Microvascular changes of the liver preserved in UW solution. Pathological and immunohistochemical examination.

Rat livers preserved in University of Wisconsin (UW) solution for 24 h were compared with those preserved in Euro-Collins (EC) solution before and after liver transplantation using an immunohistochemical method. Tissue ATP and total tissue adenine nucleotide (TAN) were measured using HPLC. The levels of TAN in the UW group or the EC group were significantly low compared with the control group (no preservation) after 24-h storage. In the EC group, the levels of tissue adenine nucleotides (TAN) decreased 1 h after reperfusion and never reached control levels. In the UW group, the levels of TAN increased a little 1 h after reperfusion and increased more 3 h after reperfusion. After 24-h preservation, the expression of factor VIII-related antigen (FRA) in endothelial cells of central veins was weak in the EC group; in the UW group, FRA was clearly detected in these cells. After reperfusion, although severe endothelial cell damage to the central veins and numerous FRA-positive substances were observed in EC group, endothelial cells of central veins retained their normal structure and FRA-positive substances were rarely noted in the UW group. In both groups, no endothelial changes were detected in portal veins. From these results, it is concluded that UW solution prevents endothelial cell damage and microcirculatory injury in zone III during the preservation period resulting in prevention of initial graft nonfunction. Also, measurement of the TAN level after reperfusion is useful to predict the function of the graft.     

Targeted accumulation of polyethylene glycol-coated immunoliposomes in infarcted rabbit myocardium.

The less than optimal accumulation of immunoliposome-associated reagents at target sites has often been attributed to the rapid in vivo clearance of immunoliposomes from the blood. In an attempt to overcome the drawback of rapid clearance and use the targeting potential of immunoliposomes, we have prepared long-circulating, 111In-labeled immunoliposomes. Targeting properties and enhanced circulation times were demonstrated in a rabbit model of acute experimental myocardial infarct. The specificity of liposomes for newly exposed intracellular cardiac myosin at the necrotic sites was achieved by incorporating monoclonal antimyosin antibody. Extended circulation times were achieved by cocoating the antimyosin-liposomes with polyethylene glycol (PEG). The half-life of the immunoliposomes was 40 min, which increased to 200 min with 4% mol PEG and to approximately 1000 min with 10% mol PEG. The degree of binding of modified immunoliposomes at the target sites was also dependent on the concentration of PEG incorporated at the liposome surface. This study demonstrates the accumulation of long-circulating targeted liposomes at the area of acute rabbit experimental myocardial infarction.