肝周淋巴结的慢性乙肝急性发作的表现与HBeAg状态和HBeAg血清

StephenW

Manifestations of Perihepatic Lymph Nodes in Acute Flare of Chronic Hepatitis B: Association with HBeAg Status and with HBeAg Seroconversion

    Yen-Ling Ko ¶,

    Chi-Shu Sun ¶,

    Kun-Ming Chung,

    Yu-Min Lin,

    I-Che Feng,

    Ming-Jen Sheu,

    Lok-Beng Koay,

    Ching-Yih Lin,

    Chung-Han Ho,

    Hsing-Tao Kuo mail

    Published: February 17, 2015
    DOI: 10.1371/journal.pone.0117590
Chi-Shu Sun, Yu-Min Lin, I-Che Feng, Ming-Jen Sheu, Lok-Beng Koay, Ching-Yih Lin, Hsing-Tao Kuo
    Department of Internal Medicine, Division of Hepatogastroenterology, Chi-Mei Medical Center, Tainan, Taiwan
Hsing-Tao Kuo
    Department of Senior Citizen Service Management, Chia Nan University of Pharmacy & Science, Tainan, Taiwan
Yen-Ling Ko, Kun-Ming Chung
    Department of Internal Medicine, Division of General Internal Medicine, Chi-Mei Medical Center, Tainan, Taiwan
Chung-Han Ho
    Department of Medical Research, Chi Mei Medical Center, Yongkang, Tainan, Taiwan
Chung-Han Ho
    Department of Hospital and Health Care Administration, Chia Nan University of Pharmacy and Science, Tainan, Taiwan

Abstract

It has been observed that enlargement of perihepatic lymph nodes may be seen in patients with chronic hepatitis B, particularly during acute flares of CHB. We hypothesized that there may be a correlation between the nodal change patterns in CHB patients with acute flare and HBeAg status. Perihepatic lymph node sizes of 87 patients with acute flares of CHB were documented, with a median follow up of 43 months. Patients were separated into 3 groups, HBeAg-positive with HBe seroconversion (group 1), HBeAg-positive without HBe seroconversion (group 2), and HBeAg-negative (group 3). Group 1 has the highest incidence of enlarged lymph nodes (92.3%) compared with group 2 (75.8%) and group 3 (46.8%) (p = 0.003). And if nodal width at acute flare was > 8mm and interval change of nodal width was >3mm, the incidence of HBeAg seroconversion will be 75% (p<0.001).
Conclusion

Larger perihepatic lymph nodes are seen in CHB acute flare patients with positive HBeAg and the magnitude of nodal width change may predict HBeAg seroconversion at recovery.

StephenW

协会与HBeAg状态和HBeAg血清转换：肝周淋巴结的慢性乙肝急性发作的表现

    颜玲柯¶，

    志舒诉嗯¶，

    坤明涌，

    喻敏林

    I-澈峰，

    铭仁SHEU，

    乐崩Koay，

    清勤益，

    仲汉河，

    幸陶扩邮件

    发布时间：2015年2月17日
    DOI：10.1371 / journal.pone.0117590
智姝孙瑜民琳，I-澈峰，鸣仁SHEU，乐崩Koay，清勤益，幸陶扩
    内科，Hepatogastroenterology科，奇美医学中心，台南，台系
幸陶扩
    长者安居服务管理，药学与科学嘉暨南大学，台南，台系
颜玲柯坤明涌
    内科，普通内科部，奇美医学中心，台南，台系
仲汉豪
    医学研究，奇美医学中心，永康市，台系
仲汉豪
    医院和医疗管理，嘉南药理大学，台南，台系

抽象

已经观察到的肝周淋巴结肿大即可以看出在慢性乙型肝炎，尤其是在慢性乙型肝炎的急性发作。我们推测，有可能是节点变化模式在慢性乙型肝炎患者的急性耀斑和HBeAg状态之间的相关性。 87例慢性乙肝急性发作的肝周淋巴结大小均有详细记载，中位随访43个月。患者被分成3组，HBeAg阳性与HBe抗体的血清转化（第1组），没有HBe的血清转换HBeAg阳性（第2组），和阴性（组3）。组1具有与组2（75.8％）和组3（46.8％）（P = 0.003）相比，淋巴结肿大（92.3％）的发生率最高。如果在急性发作期结宽度>8毫米节点的宽区间变化> 3mm时，HBeAg血清学转换的发病率将是75％（P <0.001）。
结论

较大的肝周淋巴结可见于慢性乙肝急性发作患者HBeAg阳性和节点宽度变化的幅度可以预测HBeAg血清学转换的恢复。

StephenW

Introduction

In the natural history of hepatitis B virus (HBV) infection via perinatal transmission, the point of hepatitis B envelope antigen (HBeAg) seroconversion is an important milestone. Long term follow up show chronic hepatitis B (CHB) infected individuals with HBeAg seroconversion have longer survival and less incidences of cirrhosis and hepatoma occurrence[1–4]. The estimated annual HBeAg seroconversion rate is 2–15%[5]. Most spontaneous HBeAg seroconversion occurs before age 40[6], those older than 40 years old have HBeAg seroconversion rates lower than 10% and higher rates of progression to cirrhosis[7]. Thus, the current guidelines in treatment of CHB patients further separate patients into different groups according to the HBeAg status and the achievement of HBeAg seroconversion is one of the major goals in HBeAg positive patients[8].

In the 1980s, Realdi[9] and Hoofnagle[10] have found that spontaneous HBeAg seroconversion in CHB patients is accompanied by a decrease in HBV DNA titers and alanine aminotransferase (ALT) levels, sometimes even disappearance of hepatitis B surface antigen (HBsAg). Furthermore, study by Liaw et al found that the occurrence of acute exacerbation, defined as an elevation of ALT >300IU/L, is a key factor in precipitating HBeAg seroconversion and CHB with acute exacerbation has similar biochemical and histological presentations as acute hepatitis B but with milder clinical manifestations that last longer in duration[11]. Review summary of previously known facts include those younger than 40 years old, horizontal transmissions, individuals infected at a later age, specific HLA genotypes, immunocompetent individuals, those infected with genotypes B or D, high ALT during CHB with acute exacerbation, α-fetoprotein (AFP) level >100ng/ml, serum DNA level <107copies/mL, and presence of severe inflammation such as bridge hepatic necrosis during acute exacerbations are all factors favorable to HBeAg seroconversion[12–18].

Earlier studies demonstrated a high prevalence of perihepatic lymphadenopathy in patients with chronic hepatitis C (CHC) and other acute and chronic liver disease of viral-related or immune-related etiology. The accuracy of ultrasound detection of enlarged lymph nodes have been well correlated with concurrent examinations by CT and MRI. [19,20] The mechanism of perihepatic lymphadenopathy in CHC is unknown but appears to be related to viral replication within the liver and the host’s immune-mediated inflammatory response. Associations between perihepatic lymphadenopathy to serum parameters of cytolysis, severity of histologic damage, viremia, and a high CD8 lymphocyte level have been reported in some CHC studies[21–23]. There were few reports concerning perihepatic lymphadenopathy in patients with CHB. We had mentioned a correlation between the nodal size and ALT level in subjects of HBV infection[24]. The nodal change during CHB with acute exacerbation is interesting, and the relationship of the nodal size with HBeAg seroconversion has never been observed. We herein perform this prospective observational study in our clinical practice.

StephenW

介绍

在乙型肝炎病毒（HBV）感染通过母婴传播的自然史，乙肝包膜抗原（HBeAg）血清转换点是一个重要的里程碑。长期随访显示慢性乙型肝炎（CHB）感染HBeAg血清学转换的个体有较长的生存期，肝硬化和肝癌的发生[1-4]的发病率较低。预计年HBeAg血清学转换率2-15％[5]。最自发的HBeAg血清学转换40岁之前发生[6]，那些年龄超过40岁的有HBeAg血清转换率低于10％或更高速率发展成肝硬化[7]。因此，在治疗慢性乙型肝炎患者的现行准则进一步单独患者分为按照HBeAg状态和实现HBeAg血清学转换的不同群体，是在大三阳的主要目标之一阳性患者[8]。

在20世纪80年代，Realdi [9]和胡夫纳格尔[10]发现，在慢性乙肝患者自发HBeAg血清学转换是伴随着HBV DNA滴度和丙氨酸氨基转移酶（ALT）水平，乙肝表面抗原，有时甚至消失的下降（乙肝表面抗原） 。此外，研究廖等人发现，急性加重的发生，定义为ALT> 300IU / L的高度，是沉淀HBeAg血清转换和乙肝急性发作的主要因素有相似的生化和组织学介绍为急性乙肝，但与持续时间更长的时间[11]温和的临床表现。先前已知的事实审查汇总包括那些年龄小于40岁，水平传输，感染在以后的年龄人群，特定的HLA基因型，免疫功能正常的人，那些在慢性乙型肝炎感染基因型B或D，ALT高的急性发作期，α胎蛋白（AFP）水平> 100ng / ml激活，血清DNA水平<107copies /毫升，和严重的炎症的存在，如在急性发作桥肝坏死有利于血清转换[12-18]中的所有因素。

早期的研究表明肝周淋巴结的高患病率慢性丙型肝炎（CHC）和病毒相关的或免疫相关的病因其他急性和慢性肝病。超声检测淋巴结肿大的准确性得到了很好的相关性与CT和MRI检查并发。 [19,20]肝周淋巴结中CHC的机理是未知的，但出现在肝和宿主的免疫介导的炎性反应中是相关的病毒复制。肝周淋巴结肿大之间的关联，以细胞溶解的血清参数，病理损伤，病毒血症和高CD8淋巴细胞级别严重程度已经报道了一些CHC研究[21-23]。有关于慢性乙肝患者肝周淋巴结肿大的报道很少。我们已经在HBV感染[24]的受试者中提到的节点尺寸和ALT水平之间的相关性。 CHB中节点的变化与急性发作有趣的是，与HBeAg血清学转换的节点规模的关系从未被观察到。在此，我们进行这项前瞻性观察研究我们的临床实践。

StephenW

Discussion

To our knowledge, this is the first published study on the manifestations of perihepatic lymph node in chronic hepatitis B with acute exacerbation. We found that most of the lymph nodes in CHB patient will enlarge in the acute exacerbation phase with significant difference in various clinical situations. Firstly, HBeAg positive group has larger nodal size than HBeAg negative group; secondly, HBeAg positive with seroconversion group has larger nodal size than those without seroconversion.

According to Sherlock et al in 1972, the inflammatory reaction of hepatitis B resulted from the host immune response[26] and HBeAg seroconversion often occurs in the immune clearance phase in the natural course of the virus [11,12]. Further study in CHB with acute exacerbation found that hepatitis B core antigen (HBcAg) might be a target of cytotoxic T lymphocytes, and expression of liver cell membrane HLA-1 also plays a role in promoting hepatocytolysis [27,28]. Tsai et al applied peripheral blood mononuclear cells (PBMC) to perform immunological test before and after acute exacerbation and found that increased T cell responses to HBcAg/HBeAg occurred before HBeAg seroconversion, but no changes were seen in HBsAg carriers[29]. Numerous studies had established the theory of HLA-1 restricted and cytotoxic T lymphocytes mediated hepatocytolysis. Recent immunological study also found the relationship between HBeAg seroconversion and IL-10/ IL-12[30] and IL-21[31]; chemokine (C-X-C motif) ligand CXCL-9 and CXCL-10 also play a major role in the development of CHB with acute exacerbation [32]. Those immunological responses had been identified by hepatic histological studies, serological studies and blood cellular studies. However, lymph nodal reaction, a symbol of immune response, in relation to HBeAg seroconversion, has not been studied before. Moreover, the celiac nodes were proven by studies to be an important lymphatic drainage path of the liver tissues by using purified dendritic cells and orally administered antigens able to trigger antigen-specific regulatory T-cells in the celiac lymph node [33–35]. Then, Zheng et al provided evidence via hydrodynamic HBV plasmid injection that liver-draining lymph nodes induce an anti-HBV specific immune response responsible for HBV clearance [36]. Our study found that perihepatic lymph nodes in CHB with acute exacerbation will increase in sizes(70%), indicating a robust immune reaction in the liver, which in turn support the importance of immune response in CHB with acute exacerbation.

Little research is found on relationship of hepatitis B status and lymph node size. In 2001 Choi first reported 96%[37] CHB had a lymph node enlargement phenomenon related to the degree of liver inflammation, but not to the degree of liver fibrosis and hepatitis B viral load. In 2006, we noticed that about 60% [24] HBsAg carriers have enlarged lymph nodes which was linearly related to ALT values. Recently in 2013, Shu found that enlarged lymph node could be found in about 90% of CHB patients by using MRI and also used at least two lymph nodes with short diameter> 5mm and nodal size index (product of the long and short axes) > 180 mm2 to predict the degree of liver inflammation (≧grading 2) [38], which further support that nodal size and numbers are related to the degree of liver inflammation (grading). Our previous studies comparing various methods found that short diameter> 5mm is the simplest method for measuring lymph node size and also has its clinical significance[24,39]. In this study, we found that short diameter and volume can both represent reactive lymph nodes (Table 1). Therefore, we believed that lymph nodes around the common hepatic artery that have a short diameter> 5mm represent liver inflammation in hepatitis C or hepatitis B patients; and an enlarged perihepatic lymph node depicted during routine abdominal ultrasonography in a healthy or acutely ill individual should prone one to survey possible acute hepatitis flare.

In our study, the incidence of enlarged lymph nodes (width > = 5mm) and the nodal size were higher in HBeAg positive group than HBeAg negative group. The immunopathogenic mechanisms of HBeAg negative CHB have not been elucidated with studies supporting same immune responses in HBeAg positive and negative patients[40] and those favoring different serum cytokine expressions in the two subsets[41]. Our sonographic finding might also indicate a different immune response in the two important clinical subsets, which warrant further study. In addition, we also found that in HBeAg positive patients, lymph node enlargement is more common in spontaneous flare group than in antiviral withdrawal group (p<0.05) (Table 6). This result may partly explain the higher durability of HBeAg seroconversion after spontaneous flare when compared to that after antiviral withdrawal flare[42].

It would be clinically useful to predict HBeAg seroconversion, because antiviral treatments can be withheld in the patients in whom HBeAg disappears and anti-HBe develops spontaneously. As mentioned before, many indicators can predict HBeAg seroconversion. However, most studies rely on performance of viral protein in liver cells, antigen reaction in peripheral blood mononuclear cells (PBMC) and changes in serum cytokines/chemokines and single nucleotide polymorphism genotyping. Most of these indicators are processed in the laboratory and more time consuming than a quick clinical sonography exam. Through the observation of nodal size during acute flare stage, we can speculate the strength of immune response and predict the occurrence of HBeAg seroconversion, and save the expense of antiviral therapy. Besides, interval nodal size change between acute flare phase and recovery phase further enhance the predictive effect. If setting the acute flare stage lymph node width≧8mm as standard alone, there was 72% of positive prediction rate of HBeAg seroconversion; if setting interval nodal change ≧3mm as standard, there was 68% of positive prediction rate of HBeAg seroconversion; if setting both as standard, there was 75% of positive prediction rate of HBeAg seroconversion. If neither standards were met, only 22% will proceed to HBeAg seroconversion (p<0.001) (Table 6). Changes in nodal size during acute flare phase and recovery phase may provide a reference for clinicians in decision making of oral antiviral prescription.

There are some limitations in the present study. First, in this retrospective analysis, data of HBV genotype, a well-established predictor of HBeAg seroconversion are inadequate. Only 34 (39%) subjects have data of HBV genotype (B:C = 18:16). Most of the group 2 patients are genotype B and all subjects of group 3 are genotype C. Second, there are no data to correlate the nodal sizes and serum immune profiles, which will be our next objective.
Conclusion

We herein first present the ultrasonographic sign of chronic hepatitis B with acute exacerbation by detection of perihepatic lymph nodes. The nodal sizes are more prominent on HBeAg- positive subjects than those of HBeAg- negative subjects. Those with HBeAg seroconversion had larger lymph node then those without HBeAg seroconversion. And the nodal size change between acute flare phase and recovery phase also helps predict HBeAg seroconversion, which is a critical point in both the natural course and in antiviral therapy. The nodal reactions correlate with the host immune response on chronic B hepatitis and are worth further exploration.

StephenW

讨论

据我们所知，这是在急性发作的慢性乙型肝炎肝周淋巴结的表现第一次发表的研究报告。我们发现大多数在慢性乙型肝炎患者的淋巴结将结合在各种临床情况显著差异的急性发作阶段放大。首先，HBeAg阳性组比HBeAg阴性组较大结节的大小;其次，大三阳转阴有积极组比那些没有血清学转换节点较大规模。

根据1972年夏洛特等人，乙型肝炎炎症反应是由于宿主免疫应答[26]和血清转换经常发生在病毒[11,12]的自然过程的免疫清除期。进一步研究慢性乙型肝炎急性发作发现，乙肝核心抗原（HBcAg的）可能是细胞毒性T淋巴细胞的目标，以及肝细胞膜HLA-1的表达也起着促进hepatocytolysis [27,28]的作用。蔡等人采用外周血单个核细胞（PBMC）执行前，急性发作期和发现，增加T细胞对HBcAg的后HBeAg血清转换前/ e抗原发生免疫学试验，但没有变化主要出现在HBsAg携带者[29]。许多研究已经建立的HLA-1限制性和细胞毒性T淋巴细胞介导hepatocytolysis的理论。最近免疫学研究还发现血清转换和IL-10 / IL-12 [30]和IL-21 [31]之间的关系;趋化因子（CXC母题）配体CXCL-9，CXCL-10在CHB的发展与急性发作[32]也发挥了重要作用。这些免疫反应已经确定肝脏组织学研究，血清学研究和血细胞的研究。然而，淋巴结反应，免疫反应的象征，就HBeAg血清学转换，未曾研究。此外，腹腔节点通过研究证明了使用纯化的树突状细胞和口服施用抗原能够触发抗原特异性的调节性T细胞在腹腔淋巴结[33-35]是肝组织的重要淋巴引流路径。然后，Zheng等人提供通过流体动力学的HBV质粒注射的证据表明，肝引流淋巴结诱导抗HBV特异性免疫负责病毒清除[36]反应。我们的研究发现，在慢性乙型肝炎肝周淋巴结急性加重会在大小（70％）增加，表明肝脏有强大的免疫反应，这反过来又支持CHB免疫反应急性加重的重要性。

很少研究对乙肝的地位和淋巴结的大小关系找到。在2001年财首先报道96％[37]慢性乙型肝炎有淋巴结肿大现象有关的肝脏炎症的程度，但不能肝纤维化和乙肝病毒载量的程度。在2006年，我们注意到，约60％[24] HBsAg携带者已经扩大了其线性相关ALT值淋巴结。最近在2013年，舒发现淋巴结肿大可在约90％的慢性乙型肝炎患者的发现通过使用磁共振成像，也可用于与短径> 5mm至淋巴结大小索引（长轴和短轴的产品）>的至少两个淋巴结180平方毫米预测肝脏炎症的程度（≧分级2）[38]，这进一步支持了节点的规模和数量都与肝脏炎症（分级）的程度。我们以前比较各种方法的研究发现，短径>5毫米是衡量淋巴结大小的最简单的方法也有它的临床意义[24,39]。在这项研究中，我们发现，短径和体积可均代表反应性淋巴结（表1）。因此，我们认为，肝总动脉周围具有短径>5毫米淋巴结表示在丙型肝炎或乙型肝炎患者的肝脏炎症;而在常规腹部超声检查在健康或患急病个人描绘放大肝周淋巴结应俯卧一个调查可能的急性肝炎发作。

在我们的研究中，淋巴结肿大（宽度> = 5毫米）和节点规模的发生率在HBeAg阳性组较HBeAg阴性组高。对HBeAg阴性慢性乙型肝炎的免疫病理机制尚未阐明，在两个子集[41]的研究支持在HBeAg阳性和阴性的患者[40]相同的免疫反应而那些有利于不同血清细胞因子的表达。我们的超声发现也许能够说明两个重要的临床亚群，这值得进一步研究不同的免疫反应。此外，我们还发现，在HBeAg阳性患者中，淋巴结肿大是自发的耀斑组更常见较抗病毒停药组（p <0.05）（表6）。时相比，抗病毒药戒断耀斑[42]在此之后的结果可以部分解释自发火炬后HBeAg血清转换的较高的耐久性。

这将是临床上有用的预测HBeAg血清学转换，因为抗病毒治疗可以在病人谁大三阳消失，抗-HBe自发发展是功不可没的。正如前面提到的，很多指标可以预测HBeAg血清学转换。然而，大多数研究依赖于病毒蛋白的肝细胞，外周血单核细胞（PBMC），并改变在血清细胞因子/趋化因子和单核苷酸多态性基因分型抗原的反应性能。其中大部分指标都在实验室进行处理，而且比快速的临床超声检查耗费更多的时间。通过结尺寸的急性耀斑阶段的观察，我们可以推测免疫应答的强度和预测HBeAg血清转换的发生，并保存抗病毒治疗的费用。此外，急性发作期，恢复期之间的间隔结尺寸变化进一步提高预测效果。如果设置的急性发作期淋巴结宽度≧8毫米标准孤单，HBeAg血清转换阳性预测率为72％;如果设定的时间间隔节点变化≧3毫米作为标准配置，有HBeAg血清学转换的阳性预测率68％;如果同时设置为标准，有HBeAg血清的阳性预测率的75％。如果满足了既没有标准，只有22％会继续HBeAg血清学转换（P <0.001）（表6）。在急性发作期，恢复期变化节点的大小可为在口服抗病毒药物的处方决策的临床医生参考。

还有，在本研究的一些局限性。首先，在这个回顾性分析，HBV基因型数据，一套行之有效的HBeAg血清学转换的预测是不够的。只有34（39％）患者有HBV基因型（B：C = 18:16）的数据。大多数组2例患者是B型和3组所有受试者都是基因型C.第二，有没有数据关联的节点尺寸和血清免疫档案，这将是我们的下一个目标。
结论

我们在此先介绍治疗慢性乙型肝炎的超声迹象急性发作被检出肝周淋巴结。节点尺寸上比个HBeAg-阴性者的个HBeAg-阳性者更为突出。那些HBeAg血清转换有较大的淋巴结于是那些没有HBeAg血清学转换。和急性发作阶段和恢复阶段之间的节点尺寸变化也有助于预测HBeAg血清转换，这是在两者的自然过程，并在抗病毒治疗的一个关键点。节点相关的反应与慢性乙型肝炎的宿主免疫反应，值得进一步探讨。