Hepatology Highlights (Oct AASLD)

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Hepatology Highlights Harvey J. Alter, Viral Hepatitis Editor The Clash of the Titers: HBV Meets HCV The study by Boni et al. is remarkable for the vast amount of information derived from the study of only 2 patients. The investigators comprehensively analyzed the T-cell and selected cytokine responses of 2 hepatitis C virus (HCV) carriers during the acute and postacute phases of superimposed hepatitis B virus (HBV) infection to address several important virological and immunological uncertainties. Five important points are made: (1) As previously reported, superimposed HBV infection markedly diminishes HCV replication, HCV RNA reaching undetectable levels in these 2 patients with rapid recovery after clearance of HBV; (2) HCV did not interfere with HBV-specific T-cell differentiation, proliferation, or function; the frequency of HBV tetramer+ CD8 cells, perforin expression, expansion capacity, interferon (IFN)-? expression, and cytolytic activity were the same in HBV-HCV coinfection as in HBV monoinfection, and these parameters were not influenced by HCV viral load; (3) marked suppression of HCV replication by HBV did not restore the impaired HCV-specific CD8 function generally seen in chronic HCV carriers, at least during the short duration of HCV growth suppression observed in this study; thus short-term control of HCV replication may not be sufficient to allow recovery of CD8 cells that have been in a state of chronic suppression and dysfunction; (4) HBV superinfection did not correlate with changes in IFN-a, interleukin (IL)-10, IL-12, or IL-18, suggesting that its effect on HCV replication was not mediated by the innate immune system; and (5) as a corollary to points 3 and 4, a change in HCV titer during HBV infection is more likely related to direct viral interactions than to immunologic factors. Five lessons from 2 patients is a nice learning ratio. We now need to find 2 equally well-studied HBV carriers superinfected with HCV to complete the story. (See HEPATOLOGY 2004;40:289–299.) Hip, Hip, Array for NS-5 Increasingly, the hepatitis C virus (HCV) NS5 protein has been reported to have downstream effects that might play an important role in HCV pathogenesis and in the defective immune regulation that leads to persistent infection. Additionally, NS5 has been reported to have an interferon (IFN) sensitivity region that might serve as an important determinant of IFN response. Such effects of viral proteins on host cell responses are not unique to NS5 and have also been shown for NS3 and HCV core. Girard et al. performed a comprehensive and sophisticated analysis of NS5-host interactions by transfecting Huh 7 cells with NS5 genes from the isolates of 2 patients who failed to respond to IFN, and of 2 responders. Differences were assessed by gene array analysis, and greater than 2-fold differences from control were considered significant. One hundred three genes were either upregulated (43) or downregulated (60). These genes coded for diverse functions, including cell adhesion and motility, calcium hemostasis, lipid metabolism, and immune response. The accompanying discussion hypothesized upon how each of these functions might relate to HCV pathogenesis and persistence. The extent of speculation is more than can be addressed in this highlight. However, I found two points of particular interest: (1) Despite published evidence that NS5A plays a role in resistance to IFN, there was no difference in the gene array pattern of patients who responded to IFN and those who did not; and (2) using computer-based approaches to assess transcription factor binding and regulation, Girard et al. found that 39 of the 43 upregulated gene promoter regions contained one or more binding sites for NF?B, suggesting that NF?B mediates NS5A effects on gene expression. This finding is consistent with prior reports of the central role of NF?B in NS5A-induced gene expression. So what can we make of these interesting data and the general field of gene array analysis? Using NS5A, the authors found 103 genes upregulated or downregulated. Had they used NS5B, NS3 or HCV core, they might have found an equal or greater number of affected genes for each. Hence, hundreds and perhaps thousands of genes may be affected by some sequence in the HCV genome using these increasingly sophisticated techniques. How does one determine which, if any, are most important? How does one establish causal relationships rather than simple transcriptional events? Do downregulations modulate upregulations, or are these events independent? Do these measurements really reflect elements of pathogenesis, immune response and treatment response or are they merely epiphenomenona? Would proteomics provide a more meaningful answer than gene array analysis? I do not mean to imply that these carefully conducted studies are not important to perform or relevant to HCV infection, but it seems we need some new molecular Koch’s postulates to establish the virological, immunological, and clinical relevance of these observational events. We need to transcribe observation to causation and therapeutic intervention. I have no doubt this will occur. We’re just not there yet. (See HEPATOLOGY 2004;40:708–718.) HBV Assay Comes Full Circle Wong et al. describe a clever new signal amplification technology to detect covalently closed circular DNA (cccDNA) as well as whole hepatitis B virus (HBV) DNA in both liver and serum of chronic HBV carriers. The accurate and rapid detection of cccDNA has been a long-sought weapon in the study of persistent HBV infection. For those, like myself, whose minds have been covalently closed to understanding the complexities of HBV replication, cccDNA is generated by the repair of relaxed circular replicative HBV DNA (rcDNA) in hepatocyte nuclei, and it provides the template for viral and pregenomic messenger RNA. Of clinical note, lamivudine has profound effects on the more accessible rcDNA and little or no effect on cccDNA, possibly explaining HBV DNA rebound after cessation of lamivudine. The major portion of this paper describes the methodology (Invader, Third Wave Technologies) that employs primers that span the gap between minus and plus strands to amplify across noninterrupted cccDNA. The assay is quantitative and avoids the pitfalls of PCR assays for cccDNA that often result in nonspecific amplification of rcDNA. The principles of the assay are complex and will need to be read in the original. Simplistically, the assay depends on hybridization of the Invader probe followed by enzymatic cleavage to create a smaller oligonucleotide called a 5` flap. Cleavage can occur only if there is an existent covalent linkage of the 3` end of the plus strand to the 5` base of the minus strand and hence the assay’s specificity for cccDNA. Recycling of the primary probe on the target DNA creates more 5` flaps proportional to the concentration of the DNA target. I realize this is not a very lucid explanation, but no need to create a flap over it. Application of the assay to the liver and serum of 16 hepatitis B e antigen (HBeAg)-positive and 36 anti- HBze-positive patients revealed that anti-HBe+ had lower serum HBV DNA than HBeAg+ (7.8 × 106 vs. 24.8 × 107 copies/mL, P = .001), lower median total intrahepatic HBV DNA and lower cccDNA (0.75 vs. 3.2 copies/cell, P = .001), though the ratio of cccDNA to total DNA was the same in both groups. cccDNA was found in serum in 94% of HBeAg+ and in 67% of anti-HBe+, but it was not detectable if the total HBV DNA was >7.5 × 105 copies/mL. Although serum cccDNA significantly correlated with liver cccDNA, the coefficient of correlation was low (r = 0.48). The most interesting observation of this study was that when the total intrahepatic HBV DNA level was high, a large proportion was in the replicative rcDNA form, and when the level was low, the predominant form was nonreplicative cccDNA (Fig.). Thus, over the course of HBV infection as the HBeAg-to-anti-HBe switch occurs and as replication becomes less active, an increasing proportion of HBV DNA is in the nonreplicative cccDNA form. Nonetheless, this difficult-to-eradicate HBV DNA is sitting there capable of reactivation to a replicative form and to exacerbation of disease under certain conditions. This residual source of convertible HBV DNA accounts for the continuing risk of progressive liver disease and hepatocellular carcinoma even in those with anti-HBe and low viral loads. (See HEPATOLOGY 2004;40:727–737.) Dendritic Dysfunction Dynamically Displayed In attempting to explain persistent viral infection, attention rightfully has been focused on impaired function of CD4 Th1 helper cells and CD8 effector cells. However, the blunted immune response is multifactorial, and the study by van der Molen et al. addresses the functional impairment of myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) in chronic hepatitis B; mDc and pDC are the primary precursors of the antigen presenting cells vital to the antiviral immune response. Both mDC and pDC are characterized by the absence of lineage markers on their surface and the presence of blood dendritic cell antigens (BDCA1 for mDC and BDCA2/4 for pDC); mDC produce large amounts of interleukin (IL)-12 and require granulocyte-macrophage colony-stimulating factor for survival, whereas pDC produce type 1 interferons and require IL-3 for survival. The authors studied 30 patients with biopsy-proven chronic hepatitis B, 15 of whom were HBeAg positive and 19 healthy controls. The percentages of mDC and pDC in peripheral blood were similar in patients with hepatitis B and controls, and thus there was no quantitative disparity. However, DC in hepatitis B virus (HBV) patients demonstrated functional impairments, including their inability to express costimulatory molecules (CD80, CD86) upon in vitro maturation, impaired ability of mDC, but not pDC, to induce T-cell proliferation in a mixed lymphocyte culture, and impaired production of tumor necrosis factora by mDC and interferon(IFN)-? by pDC. To demonstrate that these impairments were virus specific, a separate group of patients with nonviral chronic inflammatory liver disease were studied and shown to have normal dendritic cell function. Lastly, HBV DNA was detected in the mDC of 15/30 chronic HBV patients and in pDC of 12/30. The level of HBV DNA was about a log higher in pDC than mDC, and the level for both cells correlated with serum HBV DNA. The finding of HBV DNA, as well as replicative intermediates, within DC suggests that DC dysfunction may be a direct effect of this viral invasion, though indirect effects cannot be excluded. Where do these observations fit in the grand scheme of the anti-viral immune response? For one, the production of IFN-a was severely reduced, and the critical role of this cytokine in modulating host resistance to viruses is well established. Further, the reduction in IFN-a was greatest in patients with higher alanine aminotransferase levels and high viral loads, providing a clinical correlate to the in vitro observation. It is possible, but not tested in this study, that HBV-infected patients with the lowest levels of endogenous IFN-a would be the most likely to respond to exogenous therapy. The key underlying question is whether DC dysfunction plays a causal role in viral persistence or, conversely, is the consequence of long-standing HBV infection. It is the classic chicken-or-egg dilemma that plagues observational studies performed at a single point in time after disease is already established. Prospective studies before and after HBV infection could help determine whether these DC eggs are scrambled or if the observation is sunny-side up. (See HEPATOLOGY 2004;40:738–746.) Return of the Living Dead Liver transplantation has been a wonderful advance in the management of virus-related end-stage liver disease (ESLD), but it is plagued by a shortage of cadaveric livers and, in hepatitis C virus infection, by recurrent disease that is more aggressive and accelerated than the initial infection. The former problem has been addressed by the increasing use living donor liver transplantation (LDLT). More than 3,000 LDLTs have been performed worldwide, usually using the right hepatic lobe. This undertaking has been difficult with increased surgical morbidity and rare but concerning donor mortality. This prospective study compares the outcome of cadaveric liver transplantation (CLT) versus living donor transplants for 116 patients with HCV-related ESLD (81% CLT, 19% LDLT). After a median follow-up of 22 months, a severe recurrence (SR), defined as cirrhosis by biopsy or clinical decompensation, occurred in 41% of LDLT, compared to 18% of CLT. LDLT was the only baseline variable predictive of SR by univariate analysis (P = .019) and the only independent predictor of SR in a multivariate analysis (OR = 2.5, P = .025). The 2-year probability of SR was significantly higher in LDLT compared to CLT (45% vs. 22%, P =.019) (Fig.). Although biliary complications were more common in LDLT, they did not correlate significantly with SR. There was no evidence of severe liver disease in 10 control patients who underwent LDLT during the same time period for conditions other than HCV. The authors state that the strengths of their study were its prospective design, its performance in a single center such that care was standardized, the rigid criteria for defining SR, and the comparability of the 2 patient groups prior to transplantation. Although their study was of too brief to fully assess graft and patient survival, it is known that patients who develop cirrhosis in this setting decompensate rapidly and have markedly impaired survival. The authors infer but do not state that they have ceased performing LDLT for HCV-infected patients and suggest that LDLT is not cost-effective in this setting. Clearly this issue is controversial and important. Why living donor transplants should do worse than cadaveric transplants remains an enigma, but the greatest enigma to me is why such aggressive disease occurs in the transplanted liver, whatever its source. One would think that the immunosuppressed transplant patient would have milder recurrent disease given that HCV-related liver damage is supposed to be immune mediated. Understanding how immunosuppression or other transplant-related events accelerate liver disease would shed considerable light on the pathogenesis of natural HCV infection. (See HEPATOLOGY 2004;40:699–707.)

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HBV 化验的完善

Wong et al. 描述了一种更加聪敏的新信号放大技术.该技术可以用于检测携带者血清中和肝细胞中的共价闭环DNA (cccDNA). 这种准确而且快速检测cccDNA的方法已经成为HBV长期携带者努力寻找的一种武器. 对于那些,就像我一样, 脑海里已经几乎完全理解HBV复制过程的人们, cccDNA 是由肝细胞核中松环复制中的HBV DNA (rcDNA)闭合产生并为病毒和信使RNA提供复制的模版. 临床学显示, 拉米夫丁对大多数的HBV DNA (rcDNA) 有显著作用,而对cccDNA没有作用,或许可以解释拉米停用之后HBVDNA 反弹的机理.

本论文的大部分描述的是方法.(Invader, Third Wave Technologies) 采用了"雷管,不知道是何物",该技术跨越了正股和负股之间的缺口,从而放大了十字非交叉cccDNA. 该化验是定量且避免了经常导致rcDNA不精确放大的PCR化验cccDNA的缺陷. 该化验技术的原理很复杂,必须从头仔细阅读. 简单来说, 该化验决定于 酶分解产生的一小段叫做5' flag 的寡核苷酸后Invader probe 的杂交. 分解过程只有在存在一种3'终端正股到5'负股基端的共价连接时才会发生,这样化验结果就准确的表达了cccDNA. 目标DNA的primary probe的循环产生了更多5` flaps, 这和DNA的浓度呈正比. 我意识到这种解释不是很明晰, 但已经没有必要再多创造一个flap 在上面.

16名(HBeAg)-阳性患者和 36 名E抗体阳性患者的肝细胞和血清化验结果表明E抗体阳性患者体内的HBV DNA 血清含量要低于HBeAg+ 患者(7.8 × 106 vs. 24.8 × 107 copies/mL, P = .001), 而且肝内平均HBV DNA 和cccDNA也要更低 (0.75 vs. 3.2 copies/cell, P = .001), 尽管 cccDNA对总共 DNA 的比值差不多.   HBeAg+ 和anti-HBe+的cccDNA 的血清含量分别是94% 和67% , 但是当所有HBV DNA的数量>7.5 × 105 copies/mL时, 仍然可以从体内发现. 尽管血清cccDNA 含量和肝细胞内cccDNA相关, 相关系数很低(r = 0.48). 该研究最有意思的观察结果是当肝内HBV DNA含量很高的时候, 大多数的病毒形式是rcDNA 复制模式,而当含量很低的时候, 绝大多数却是不再复制的cccDNA (Fig.). 因此, 对于 HBV 中发现HBeAg-to-anti-HBe 转换时并且复制水平也很低的时候, HBV DNA 更多则是存在于非复制状态的 cccDNA 模式. 并且, 这种非常难消灭的 HBV DNA 继续存在在那里,随时准备激活成为复制形态并在某种情况下造成病情的恶化. 这种残存的可转变的HBV DNA 将会导致肝病的不断恶化, 甚至在那些E抗体阳性低病毒载量的患者当中出现HCC的可能. (See HEPATOLOGY 2004;40:727–737.)