Targeted delivery of ribavirin improves outcome of murine viral fulminant hepatitis via enhanced anti-viral activity.

Side effects of interferon-ribavirin combination therapy limit the sustained viral response achievable in hepatitis C virus (HCV) patients. Coupling ribavirin to macromolecular carriers that target the drug to the liver would reduce systemic complications. The aim of this study was to evaluate the efficacy of a hemoglobin-ribavirin conjugate (HRC 203) in murine hepatitis virus strain 3 (MHV-3) induced viral hepatitis. HRC 203 had greater anti-viral activity on both isolated hepatocytes and macrophages, whereas both ribavirin and HRC 203 inhibited production of the pro-inflammatory cytokines interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) by macrophages. In vivo, untreated MHV-3-infected mice all developed clinical and biochemical signs of acute viral hepatitis and died by day 4 post infection. Livers recovered from untreated infected mice showed greater than 90% necrosis. In contrast, survival was enhanced in both ribavirin- and HRC 203-treated mice with a marked reduction in biochemical [ALT(max) 964 +/- 128 IU/L (ribavirin); 848 +/- 212 IU/L (HRC 203)] and histological evidence of hepatic necrosis (<10% in ribavirin/HRC 203 vs. 90% in untreated controls). Clinically, HRC 203-treated mice behaved normally, in contrast to ribavirin-treated mice, which developed lethargy and abnormal fur texture. In conclusion, targeted delivery of ribavirin to the liver alters the course of MHV-3 infection as demonstrated by prolonged survival, improved behavior, and reduced signs of histologically evident disease, as well as inhibition of viral replication and production of inflammatory cytokines in vitro.

Targeted deletion of Fgl-2/fibroleukin in the donor modulates immunologic response and acute vascular rejection in cardiac xenografts.

BACKGROUND: Xenografts ultimately fail as a result of acute vascular rejection (AVR), a process characterized by intravascular thrombosis, fibrin deposition, and endothelial cell activation. METHODS AND RESULTS: We studied whether targeted deletion of Fgl-2, an inducible endothelial cell procoagulant, (Fgl-2-/-) in the donor prevents AVR in a mouse-to-rat cardiac xenotransplantation model. By 3 days after transplant, Fgl-2+/+ grafts developed typical features of AVR associated with increased levels of donor Fgl-2 mRNA. Grafts from Fgl-2-/- mice had reduced fibrin deposition but developed cellular rejection. Treatment with a short course of cobra venom factor and maintenance cyclosporine resulted in long-term acceptance of both Fgl-2+/+ and Fgl-2-/- grafts. On withdrawal of cyclosporine, Fgl-2+/+ grafts developed features of AVR; in contrast, Fgl-2-/- grafts again developed acute cellular rejection. Rejecting Fgl-2+/+ hearts stained positively for IgG, IgM, C3, and C5b-9, whereas rejecting Fgl-2-/- hearts had minimal Ig and complement deposition despite xenoantibodies in the serum. Furthermore, serum containing xenoantibodies failed to stain Fgl-2-/- long-term treated hearts but did stain wild-type heart tissues. Treatment of Fgl-2-/- xenografts with mycophenolate mofetil and tacrolimus, a clinically relevant immune suppression protocol, led to long-term graft acceptance. CONCLUSIONS: Deletion of Fgl-2 ameliorates AVR by downregulation of xenoantigens and may facilitate successful clinical heart xenotransplantation.

The fgl2 prothrombinase/fibroleukin gene is required for lipopolysaccharide-triggered abortions and for normal mouse reproduction.

Increased fgl2 prothrombinase activity in maternal decidua and fetal trophoblasts may trigger abortions by proinflammatory cytokines induced by bacterial lipopolysaccharide (LPS) in mice and is implicated in human recurrent miscarriages and pre-eclampsia. Defining the physiological and pathological role of the fgl2/fibroleukin gene required an fgl2-knockout mouse and data on normal pattern of fgl2 expression during pregnancy. Expression of fgl2 protein was determined by immunostaining with specific antibody. Fgl2 knockout mice were generated and typed by PCR for presence of the altered gene. Immunostaining of timed CBAxDBA/2 mouse matings in a low-abortion-rate colony showed a distinct pattern of development of fgl2 protein expression in maternal decidua, and in embryonic tissues in early pregnancy. Outbred (mixed background) heterozygous fgl2 +/-x+/- matings with a similar low abortion rate showed selective occult loss of both +/- and, to a greater extent, -/- embryos prior to gestation day 11.5, in association with haemorrhage at the anti-mesometrial pole of fgl2-deficient embryo. LPS injected on day 6.5 caused classical abortions at mid-pregnancy in fgl2 +/+x+/+ matings, but not -/-x-/- matings. Physiological expression of fgl2 in fetal trophoblast may prevent occult loss in early pregnancy, along with other coagulation factors, but fgl2 expression is required for LPS to induce abortion pathology.

The Fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis.

Fibrin deposition and thrombosis within the microvasculature is now appreciated to play a pivotal role in the hepatocellular injury observed in experimental and human viral hepatitis. Importantly, the pathways by which fibrin generation is elicited in viral hepatitis may be mechanistically distinct from the classical pathways of coagulation induced by mechanical trauma or bacterial lipopolysaccharide (LPS). In the setting of murine hepatitis virus strain-3 (MHV-3) infection, a member of the Coronaviridae, activated endothelial cells and macrophages express distinct cell-surface procoagulants, including a novel prothrombinase, Fgl2/fibroleukin, which are important for both the initiation and localization of fibrin deposition. To assess the role of Fgl2/fibroleukin in murine viral hepatitis we generated a Fgl2/fibroleukin-deficient mouse. Peritoneal macrophages isolated from Fgl2/fibroleukin-/- mice did not generate a procoagulant response when infected with MHV-3. Fibrin deposition and liver necrosis were markedly reduced, and survival was increased in mice infected with MHV-3. To address the relevance of Fgl2/fibroleukin in human chronic viral hepatitis we studied patients with minimal and marked chronic hepatitis B. We detected robust expression of Fgl2/fibroleukin mRNA transcripts and protein in liver tissue isolated from patients with marked chronic hepatitis B. Fibrin deposition was strongly associated with Fgl2/fibroleukin expression. Collectively, these data indicate a critical role for Fgl2/fibroleukin in the pathophysiology of experimental and human viral hepatitis.

Gene transcription of fgl2 in endothelial cells is controlled by Ets-1 and Oct-1 and requires the presence of both Sp1 and Sp3.

The immune coagulant fgl2/fibroleukin has been previously shown to play a pivotal role in the pathogenesis of murine and human fulminant hepatitis and fetal loss syndrome. Constitutive expression of fgl2 transcripts at low levels are seen in cytotoxic T cells, endothelial, intestinal and trophoblast cells, while specific factors (such as virus and cytokines) are required to induce high levels of fgl2 expression in other cell types including monocytes/macrophages. To address the transcriptional mechanisms that regulate constitutive expression of fgl2, murine genomic clones were characterized and the transcription start site was defined by 5'-RACE and primer extension. A comprehensive assessment of basal fgl2 promoter activity in murine vascular endothelial cells defined a minimal 119 bp region responsible for constitutive fgl2 transcription. A complex positive regulatory domain (PRD) spanning a 39-bp sequence from -87 to -49 (relative to the transcription start site) was identified. Electrophoretic mobility shift assay studies in vascular endothelial cells revealed that the nucleoprotein complexes that form on this positive regulatory domain (PRD) contain Sp1/Sp3 family members, Oct-1, and Ets-1. Heterologous expression studies in Drosophila Schneider cells confirmed that the constitutive expression of this gene is controlled by Ets-1 and requires the presence both of the Sp1 and Sp3 transcription factors. The presence of this complex multicomponent PRD in the fgl2 proximal promoter is consistent with the observation that, in vivo, fgl2 expression is tightly regulated. Moreover, viral induced fgl2 expression also requires the presence of this PRD. These results clearly demonstrate that multiple cis DNA elements in a clustered region work cooperatively to regulate constitutive fgl2 expression and interact with inducible elements to regulate viral-induced fgl2 expression in endothelial cells.

Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity.

fgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-L-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent V(max) value of 6 mol/min/mol fgl2 and an apparent K(m) value for prothrombin of 8.3 microM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent V(max) value to 3670 mol/min/mol fgl2 and decreasing the apparent K(m) value for prothrombin to 7.2 microM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser(89) was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.