Hepatology highlights
Authors: Harvey Alter
Journal: Hepatology (2006)

In an elegant study, von Hahn et al. examined the mechanisms of HCV cell entry, particularly as they relate to scavenger receptor class B type I (SR-BI), a receptor highly expressed in the liver and whose ligands are HDL, LDL and VLDL. A specific receptor for HCV has been sought ever since its discovery in 1989. No single receptor has been isolated, but CD-81, a tetraspanin, has been shown to be critical to, but not sufficient for the entry process. Lipoprotein receptors seemed an attractive target since HCV has been shown to associate with lipoproteins, particularly LDL accounting for its lower than expected buoyant density. The bottom line of the von Hahn study is that oxidized LDL (oxLDL), a high affinity ligand for SR-BI, potently inhibits the entry of HCV pseudoparticles (HCVpp) and HCV cell culture virus (HCVcc) into a variety of HCV susceptible cell lines. The authors reach this conclusion by a series of meticulous experiments that are very convincing and can be summarized as follows: (1) Incubation of HCVpp with oxLDL, but not native LDL reduced infectivity in Hep3B cells more than 100-fold and had no significant effect on control viruses; inhibition was dose dependent; (2) HDL was shown to specifically enhance HCVpp cell entry; when oxLDL was added this enhancement effect was abrogated by at least 95%; (3) by separating the LDL apolipoprotein from the lipid moiety, it was shown that both components had inhibitory capacity, but that the lipid moiety was most potent; (4) the inhibitory effect required that both oxLDL and HCVpp were on the cell surface at the same time; (5) competition studies on CHO cells expressing CD81 or SR-BI showed that the effect of oxLDL was not simply competition at the receptor site; (6) transfection with siRNAs against CD81 showed clear inhibition of HCVpp entry whereas siRNA against SR-BI only showed inhibition in the presence of HDL; whether this is the result of inadequate silencing or the fact that CD81 is a more important receptor is not clear from this study. The investigators conclude that an oxidized lipid contained in oxLDL is the active component inhibiting HCV cell entry and that inhibition involves a “tertiary interaction between virus, SR-BI, and oxLDL requiring the simultaneous presence of all 3 agents.” It appears that oxLDL interacts with both the virus and the cell surface. Thus, the long-sought receptor for HCV may not exist as a single entity, but requires interactions that involve oxidized lipids, the scavenger receptor BI, CD-81 and perhaps other as yet unrecognized molecules. As Neil Sedaka almost said, “breaking in is hard to do.” (See HEPATOLOGY 2006;43:932–942.) The scid-Alb-uPA mouse is a clever animal model wherein SCID mice are made transgenic for an array of murine urokinase genes under control of an albumin promoter. The over-expression of urokinase is lethal for murine hepatocytes. Fresh or cryopreserved, uninfected human hepatocytes are injected into the mouse spleen 7-14 days after birth, migrate to the liver, and selectively replace the dying murine hepatocytes. This humanized mouse liver has been shown to not only produce human proteins, but also to be susceptible to HCV infection. Although this is a very difficult model to produce and to maintain once established, such animals have great potential to be models for anti-viral therapy and to bridge the gap between replicons or other in-vitro systems and human trials. In this study, Kneteman et al. tested how the HCV-infected scid-Alb-uPA mouse responds to Interferon-α2b, an NS3-protease inhibitor (BILN-2061) and an NS5 polymerase inhibitor (HCV371), agents whose efficacy in humans is already known. Cohorts of mice (usually 4-7 per experiment) with HCV serum titers of >104 copies/ml were treated daily for short periods (7-28 days) and the change in HCV RNA level was compared to placebo treatment. The outcome in the mouse model was very similar to that observed in humans in that (1) IFN-α2b caused rapid and significant reduction in HCV RNA and resulted in a biphasic response curve with relapse occurring after the end of short term therapy; this immunodeficient model also tells us that interferon has significant antiviral effects that are independent of immune activation; (2) the response in genotype 3 infected animals was more rapid, more pronounced and more sustained than genotype 1 infected animals; (3) BILN2061 given orally reached levels similar to those achieved in humans and resulted in 1 to >2 log reductions in HCV RNA within 4 days; (4) the NS5 polymerase inhibitor that had failed human trials also failed to achieve significant reduction of viral load in chimeric mice. Thus, the SCID mouse with human hepatocytes appears to be a good model for antiviral therapies and may help determine which candidate drugs should come to human trials. However, these mice have high mortality and there are problems timing the acquisition of human liver cells to coincide with the age when animals can be inoculated. Thus, it is necessary to do experiments in small batches introducing an undesired variable. These problems also make the model expensive (despite their having human livers, insurance companies refuse to pay for these treatments). Nonetheless, this study proves that the scid-Alb-uPA mouse model is viable and an accurate predictor of treatment responses in humans. (See HEPATOLOGY 2006;43:1346–1353.) While much is known about the ability of interferons (IFNs) to activate a large repertoire of interferon regulatory genes (IRGs) and particularly to upregulate interferon sensitivity genes (ISGs), the kinetics and impact of such gene perturbations are just beginning to emerge. Additionally, it has been unclear why HCV-infected chimpanzees are unresponsive or minimally responsive to α-IFNs that are potent in humans. Hence, Lanford and associates undertook IFN kinetic studies in HCV-naive chimps and compared chimp responses to both human and chimpanzee-derived α-IFNs. This is an elegant study, well worth reading. The current study did not focus on identifying the genes that are up or down-regulated during therapy, but rather on the temporal sequence of gene activations, the differences in expression profiles in the liver compared to PBMCs, the differential responses to IFN-α versus IFN-γ, and the comparative responses to human and chimpanzee interferons. One of the key questions was why high levels of exogenous or endogenous IFN-α in chimp liver failed to induce a reduction in viral load and whether this related to the species origin of the IFN. The study thus compared human Peg-IFN-α2 with chimpanzee IFN-α in their ability to induce IRGs in 3 uninfected chimps as measured by DNA microarray analysis and RT-PCR. The findings can be summarized as follows: (1) the level of ISG transcripts rose rapidly after IFN administration and some increased 400-700 fold by 4 hours; (2) the decline in transcript level was equally rapid and returned to near baseline by 8 hours even though levels of Peg-IFN were still rising in the circulation; this suggested that the IFN signal transduction was rapidly suppressed in vivo; (3) the IFN response was tissue specific; of 1778 IRGs that were significantly altered after IFN, 538 genes were unique to the liver, 950 unique to PBMCs, and 290 expressed in both tissues; (4) the chimpanzee responses to chimpanzee and human interferons were indistinguishable and thus IFN non-responsiveness in the chimp is not simply due to the use of a cross-species therapeutic product. Thus the cause of IFN non-responsiveness in the chimp is not due to species differences in the IFN used or to a failure to upregulate ISGs, but rather seems related either to an inability to respond to the active transcriptional events or to the rapid down-regulation of ISG transcripts that occurs despite the presence of high levels of circulating IFN. The rapid down-regulation remains unexplained. Interestingly, the authors suggest that the decreased slope in phase 2 of the IFN response curve may be due not only to the reduction in the number of infected cells, as previously hypothesized, but also to decreased IFN efficacy from the down-regulation of IFN gene response pathways. By whatever mechanism, failure to respond to IFN has diminished the role of the chimp as a therapeutic model. Fortunately replicons, pseudoparticles, humanized mice, and now tissue culture systems have come to fill the void. (See HEPATOLOGY 2006;43:961–972.) In Italy, Sangiovanni et al. conducted a careful prospective study of 214 HCV-infected patients who had compensated cirrhosis without HCC at the time of enrollment and were followed a mean of 114 months (range 1-199); 45% were followed >10 years and 20% >15 years. A negligible number received antiviral therapy, making this a true natural history study. The key findings were that HCC developed in 32%, ascites in 23%, jaundice in 17%, GI bleeding in 6% and encephalopathy in 1% with corresponding annual incidences of 3.9%, 2.9%, 2.0%, 0.7% and 0.1% respectively. The annual incidence of developing at least one of these negative events was 6.9%. Conversely, since many of these occurrences were overlapping, 49% of patients had none of these adverse events during the duration of the study. The clinical status as assessed by Child-Pugh score remained unchanged in 72%, whereas it progressed to class B in 21% and to class C in 7%. Of the 68 patients with HCC, 32 (47%) had a single node tumor that in 91% was <5 cm. By multivariate analysis, esophageal varices (HR=2.6) and elevated bilirubin (HR=2.6) were the only independent predictors of clinical decompensation, while AFP < 20ngml (HR 1.5), male sex (HR 2.7) and GGTP (HR 2.1) were the only independent predictors of HCC. HCC incidence was not increased in those with antibody to HBV core or those with mild or moderate alcohol intake. 75 (35%) patients died, with an annual mortality rate of 4%. Among the deceased, 44% died of tumor progression, 8% of GI bleeding, 20% of hepatic decompensation and 1% following liver transplantation; the remainder died of causes unrelated to their liver disease. Mortality was 31% per year once HCC was diagnosed. This study like an earlier comprehensive European study by Fattovich et al. (Gastroenterology 1997;112:463-472) shows that the evolution of compensated cirrhosis is relatively slow if not accelerated by other causes of liver disease. In the current study, HCC was the most frequent complication and the primary cause of death. This would certainly be true in Japan as well, but may not be true in the US where HCC is still less frequent. Overall, it appears that the slow evolution to cirrhosis that characterizes HCV infection in 20% to 30% of patients continues its slow evolution even after the onset of cirrhosis. In the absence of comorbid events such as alcohol abuse, hereditary or autoimmune liver disease, or severe immunodeficiency, patients with compensated cirrhosis have a near 50% chance to survive 10 years after cirrhosis is established. With careful and frequent monitoring for HCC and early cancer intervention, that survival rate can be further enhanced. Patients with HCV-associated cirrhosis are thus often better compensated then the physicians who treat them. (See HEPATOLOGY 2006;43:1303–1310.) Apoptosis, from the Greek word meaning, “I can get another publication,” has become an increasingly important concept, though no two people pronounce the word exactly the same way. I jest, but programmed cell death is indeed a very important mechanism in both the natural senescence of cells and in cell injury from a variety of assaults, including hepatitis virus infection. Volkmann et al. now examine the role of apoptosis, as measured by caspase activation, in the response to anti-viral therapy for hepatitis C. Caspases are a family of intracellular cysteine proteases that cleave a variety of substrates ranging from those involved in DNA replication to those involved in cell structure, including the cytokeratin type 1 family. Prior studies have shown that caspases are upregulated in HCV-infected liver and correlate with inflammatory grade and early fibrosis. A primary mechanism of apoptosis involves the death receptors CD95 and TNF-related apoptosis inducing ligand (TRAIL). When these receptors are engaged, they begin an intracellular cascade of death that terminally leads to caspase activation. It has now been shown that interferons induce the expression of death receptors, death ligands and certain caspases. To study whether apoptosis is involved in the IFN response, Volkman et al. used an ELISA assay to measure the serum level of a caspase-generated neoepitope of cytokeratin-18 known to be abundantly expressed in hepatocytes. They also employed a chemiluminescence assay for active caspase-3 and 7. The results of the two assays showed very strong correlation. In this rapid communication, only 23 patients were studied including 11 treatment responders, 7 nonresponders, and 5 who relapsed. Patients who responded showed significantly higher levels of the CK-18 fragment at baseline (471±95 U/L) compared to non-responders (171±24 U/L) and relapsers (195±65 U/L). Most interestingly, these differences were apparent before treatment and within the first 3 days of treatment, but not thereafter. In those who responded, the decline in caspase activity closely coincided with the decline in viral load (r=0.81).There is a suggestion in this study that pre-treatment caspase activity could be used as a predictor of treatment response. This may be a correct inference, but the conclusion is premature based on this small number of subjects. More intriguing to me is the question of how active pre-treatment apoptosis primes for a favorable treatment response. As in hepatitis B where de novo high-level inflammation presages a better treatment response, it seems that IFN requires a host capable of meeting the drug half-way by initiating its own reasonable, albeit incomplete, anti-viral response. This study suggests that apoptosis plays an important role in viral clearance after therapy and probably also during spontaneous recovery. Virus specific, intrahepatic CTLs may be the central element in this process by several mechanisms including perforin-related cell membrane destruction, γ-IFN induced viral clearance without cell destruction and by inducing apoptosis through a receptor or perforin/granzyme B pathway. Lastly, the authors suggest that the role of caspases may not be restricted to apoptosis, but might also cleave or activate critical cytokines. One such established effect is caspase activation of IL-18 (IFN-γ inducing factor), thus linking caspases to the critical role of γ-IFN in intrahepatic viral clearance. It is all coming together nicely. I would like to continue this discussion, but this Highlight has been programmed to die imminently, albeit not eminently…(See HEPATOLOGY 2006;43:1311–1316.) After 52 weeks of adefovir (ADV) monotherapy in 480 HBeAg+ Chinese subjects, the median HBV DNA reduction was 4.5 log copies/ml; 67% had <105cop./ml and 28% had undetectable HBV DNA; ALT normalized in 79%. Patients with YMDD mutations responded as well as wild-type infection. No ADV resistance was observed over 1 year. (See HEPATOLOGY 2006;44:108–116.) In contrast, Lee et al. found that 18% of 57 lamivudine-resistant patients developed ADV resistant mutations within 48 weeks compared to none of 38 treatment-naïve patients who were treated with ADV. (See HEPATOLOGY 2006;43:1385-1391.) It may now be possible to clear HCV while simultaneously lowering your cholesterol. Ikeda et al. used an HCV replicon system to measure the inhibitory effects of 5 statin drugs alone or in combination with IFN. Some statins, particularly Fluvistatin, efficiently inhibited HCV replication when used alone, and synergistically enhanced the efficacy IFN such that combined therapy decreased HCV replication to near baseline in their reporter assay. The anti-viral effect of statins is independent of the cholesterol effect and thought due to inhibition of geranylgeranylation of cellular proteins. Higher statin doses than used for cholesterol lowering will be needed to achieve therapeutic levels. Given the safety and multiple benefits of statins, a clinical trail seems warranted. The ability to eat all the eggs you want may offset the personal trauma of interferon therapy. (See HEPATOLOGY 2006;44:117–125.) A 12 week course of 30mg or 50 mg clevudine resulted in 4.49 and 4.45 log10 reductions in HBV DNA, respectively compared to 0.2 logs in placebo-treated subjects. Most importantly, 24 weeks off therapy there was still a 2.28 and 1.40 log10reduction, respectively and median ALT levels fell to normal and then remained in the normal range for the full 24 weeks off therapy. This durable post-treatment response may be due to a reduction in cccDNA through elimination of HBV infected hepatocytes during early treatment, as has been observed in woodchucks. (See HEPATOLOGY 2006;43:982–988.)