Tetsuji Fujita

Analyzing risk factors for intrahepatic cholangiocarcinoma.

1. Guillemot J, Canuel M, Essalmani R, Prat A, Seidah NG. Implication of the proprotein convertases in iron homeostasis: PC7 sheds human transferrin receptor 1 and furin activates hepcidin. HEPATOLOGY 2013;57: 2514-2524. 2. Gkouvatsos K, Papanikolaou G, Pantopoulos K. Regulation of iron transport and the role of transferrin. Biochim Biophys Acta 2012;1820: 188-202. 3. Ohnishi K, Torimoto Y, Ikuta K, Tanaka H, Hosoki T, Tanaka S, et al. Detection of soluble HFE associated with soluble transferrin receptor in human serum. Int J Mol Med 2011;27:435-439.

Endotoxemia in alcoholic liver disease

As described in an interesting review by Rao,1 much evidence derived from numerous experimental studies of alcoholic liver disease (ALD) support the association of endotoxemia with the initiation and progression of ALD. The review focuses on the role of endotoxemia and the mechanisms of gut barrier dysfunction in ALD. The author mentions that endotoxemia is conventionally believed to exist when the plasma endotoxin level rises higher than 2.5 endotoxin units (EU)/mL, and the endotoxin levels in ALD patients, ranging from 8.5 to 206 pg/mL, are 5to 20-fold higher than those in normal subjects. First, I would like to comment on the determination and interpretation of endotoxemia in the clinical setting. Historically, the rabbit pyrogenic test was introduced to detect the contamination of endotoxins and other toxins in biological preparations in 1940. In the early 1960s, dose-dependent endotoxin-induced coagulation of lysates from the horseshoe crab amoebocyte was found, leading to the development of a new method of quantifying the endotoxin in 1970s, which is now called the limulus amoebocyte lysate (LAL) assay. This assay was quickly approved by the US Food and Drug Administration (FDA) because the LAL test is more sensitive, less time-consuming, more costeffective, and better standardized than the rabbit test. For interpreting the results of the LAL assay, it should be noted that LAL is not prepared for the detection of endotoxemia but is intended only for in vitro diagnostic purposes. Endotoxin antagonists, antibiotics, plasma proteins, and unknown substances in the blood can interfere with or activate the LAL test. The term EU indicates the national standard biological activity in the LAL assay, which was determined by the FDA and other organizations on the basis of the relationship between the LAL assay and the rabbit pyrogenic test. Although the FDA initially defined 1 EU as the endotoxin activity of 200 pg of a reference standard endotoxin, the conversion ratio is variable because it is dependent on the source of the endotoxin used for each assay, ranging from 1 EU to 20 pg to 1 EU to 500 pg. Commercially available LAL kits use a control standard endotoxin because a reference standard endotoxin is very expensive. There are substantial variations in endotoxin potency among the control standard endotoxins. Additionally, substantial variation in the reactivity of lysates from different horseshoe crabs has been observed. Therefore, I would like to ask the author whether it was quite difficult to define endotoxemia and assess the differences in endotoxin levels among the studies. The author concludes that the evidence is clear that alcohol consumption leads to increased intestinal permeability and endotoxemia, which are involved in the pathogenesis of ALD. However, there is no definite clinical evidence showing that improvements in intestinal permeability or anti-endotoxin therapy can ameliorate liver injury in ALD. Also, in an experimental study, ethanol-induced liver damage was not markedly enhanced by the long-term infusion of endotoxin to levels that were 50to 100-fold higher than those achieved with ethanol alone.2 It is well known that a primary endotoxin challenge causes insensitivity to a secondary challenge, which is termed endotoxin tolerance, and this should be borne in mind when we try to explain the cause-effect relationship in ALD with endotoxemia.

Retinol‐binding protein 4 as a circulating marker of hepatic steatosis

2265.2008.03295.x. 6. Tacke F, Wustefeld T, Horn R, Luedde T, Srinivas RA, Manns MP, et al. High adiponectin in chronic liver disease and cholestasis suggests biliary route of adiponectin excretion in vivo. J Hepatol 2005;42:666673. 7. Yagmur E, Trautwein C, Gressner AM, Tacke F. Resistin serum levels are associated with insulin resistance, disease severity, clinical complications, and prognosis in patients with chronic liver diseases. Am J Gastroenterol 2006;101:1244-1252. 8. Seo JA, Kim NH, Park SY, Kim HY, Ryu OH, Lee KW, et al. Serum retinol-binding protein 4 levels are elevated in non-alcoholic fatty liver disease. Clin Endocrinol (Oxf) 2008;68:555-560.

Who is benefited by one daily glass of wine

We are grateful to Drs. Xin and Xuan for their interest in our study, in which the long-term outcome of 1965 hepatitis B surface antigen (HBsAg) carriers with antibody against hepatitis B e antigen (antiHBe) and normal alanine aminotransferase at baseline were studied.1 The authors had three comments or questions on our article: (1) As described in the section of Patients and Methods, all study patients did not receive antiviral or immunomodulatory therapy before entry and during follow-up, so all 24 patients with reactivation of hepatitis B had “spontaneous” HBsAg seroclearance 3-20 years after the onset of hepatitis B. (2) The number of patients with age at entry of 60-69 years and 70 years were very small, so they were grouped together with those with age at entry of 50-59 years as patients with age at entry 50 years. (3) Many authors have divided the natural course of perinatally acquired chronic hepatitis B virus infection into four phases: immune tolerance, immune clearance, inactive carrier state, and reactivation of hepatitis B. However, as seen in the current study, only 16% (314/ 1965) of anti-HBe–positive carriers had reactivation of hepatitis B during a long-term follow-up of more than 20 years. The cumulative probabilities of reactivation of hepatitis B in this series were 9.7%, 17.1%, 19.6%, and 21.5%, respectively, after 5, 10, 15, and 20 years.1 These findings indicated that reactivation of hepatitis B tended to occur much more frequently during the first 10 years of follow-up and became relatively uncommon 15 years later. Furthermore, the natural course following spontaneous hepatitis B e antigen (HBeAg) seroconversion has been studied in two different cohorts from our unit, one involving 283 patients with HBeAg-positive chronic hepatitis B2 and the other involving 240 HBeAg-positive carriers.3 The annual rates of reactivation of hepatitis B were 3.3% and 2.2% per year, respectively.2,3 The cumulative probability of HBeAg-negative chronic hepatitis B after 15 years was about 23%.2 Reactivation of hepatitis B occurred much more frequently during the first 10 years after HBeAg seroconversion.2 On the basis of the data of these studies, the overall incidence of reactivation of hepatitis B following HBeAg seroconversion was estimated to be as high as 25%. Accordingly, in Taiwan, following HBeAg seroconversion, only 25% of HBsAg carriers will go through the fourth phase, while the other 75% of HBsAg carriers will stop at the third phase as inactive carriers with lifelong sustained remission of hepatitis. Perhaps the incidence of reactivation of hepatitis B may be much lower for HBsAg carriers from low-endemic or intermediateendemic areas.4 Therefore, we preferred to divide the natural course of chronic hepatitis B virus infection into three phases, because the division of the natural course of chronic hepatitis B into four phases likely will give us a misimpression that all or the majority of HBsAg carriers will go through the four phases.

Assessing a microrNA panel in diagnosing early cholangiocarcinoma

1. Galbraith JW, Franco RA, Donnelly JP, Rodgers JB, Morgan JM, Viles AF, et al. Unrecognized chronic hepatitis C virus infection among baby boomers in the emergency department. HEPATOLOGY 2015;61:776-782. 2. Kazmierczak J, Pawełczyk A, Cortes KC, Radkowski M. Seronegative hepatitis C virus infection. Arch Immunol Ther Exp (Warsz) 2014; 62(2):145-151. 3. Carreno V. Occult hepatitis C virus infection: a new form of hepatitis C. World J Gastroenterol 2006;12(43):6922-6925. 4. Keyvani H, Bokharaei-Salim F, Monavari SH, Esghaei M, Nassiri Toosi M, Fakhim S, et al. Occult hepatitis C virus infection in candidates for liver transplant with cryptogenic cirrhosis. Hepat Mon 2013;13(8): e11290. 5. Castillo I, Bartolom e J, Quiroga JA, Barril G, Carre~ no V. Diagnosis of occult hepatitis C without the need for a liver biopsy. J Med Virol 2010;82(9):1554-1559. 6. Welker MW, Zeuzem S. Occult hepatitis C: how convincing are the current data? HEPATOLOGY 2009;49(2):665-675. 7. Carre~ no V, Bartolom e J, Castillo I, Quiroga JA. New perspectives in occult hepatitis C virus infection. World J Gastroenterol 2012;18(23): 2887-2894. 8. Shazly YE, Hemida K, Rafik M, Swaff RA, Ali-Eldin ZA, GadAllah S. Detection of occult hepatitis C virus among healthy spouses of patients with HCV infection. J Med Virol 2014; doi:10.1002/jmv.24074. 9. Rezaee-Zavareh MS, Einollahi B. Treatment of occult hepatitis C virus infection: does it need special attention? Hepat Mon 2014;14(7): e16665.

Role of retinol binding protein 4 in hepatic steatosis.

Using HepG2 cell, Xia et al. revealed that retinol binding protein 4 (RBP4) activated critical lipogenic transcription factors of sterol regulatory element-binding protein-1c (SREBP-1c) and liver X receptor through the stimulation of peroxisome proliferator-activated receptor-c coactivator 1b (PGC-1b). The stimulatory effect of RBP4 on SREBP-1c activity associated with hepatic lipogenesis was ensured also in vivo. Xia et al. conclude that the activation of SREBP-1c by RBP4 is fully dependent on up-regulation of PGC1b, because RBP4 did not exhibit lipogenic effect in PGC-1bknockout mice. In the associated editorial, Dr. Maher raises an important question of whether RBP4 is directly linked to hepatic lipogenesis, but neither Xia et al. or Maher address the issue about the unidentified hepatic receptor of RBP4. The only known receptor for RBP4 is a receptor stimulated by retinoic acid-6 (STRA6), which is expressed in several tissues, including retina, brain, spleen, thymus, female genital tract, testis, muscle, and placental endothelial cells (and in small quantities in heart and lung), whereas STRA6 has not been identified in the liver. Until the discovery of the hepatic receptor of RBP4, the metabolic link between RBP4 and fat is not confirmed. Recently, Alapatt et al. reported that RBP4 receptor-2 (RBPR2), which is primarily expressed in the liver and intestine and induced in adipose tissue in obese mice, is likely to mediate effects of RBP4 on insulin action and glucose homeostasis in the capacity of an RBP4 receptor. To define the role of RBP4 in lipid metabolism, it would be necessary to investigate the effect of RBP4 also under the condition of knockdown of RBPR2. PGC-1b has been thought to be a coactivator of lipogenic transcription factors, leading to lipogenesis. However, a recent study suggests that PGC-1b plays dual roles in regulating hepatic fatty acid metabolism by controlling the expression of programs of genes involved in both fatty acid oxidation and de novo fatty acid synthesis, because PGC-1b-deficient mice were outwardly normal, but exhibited a significant increase in hepatic triglyceride content at 6 weeks of age. The lipogenic effects of RBP4 shown by Xia et al. might be time-dependent.

Role of interleukin-30 as a modulator of transcription signaling in liver injury.

In their relevant study, Zweers et al. demonstrate that fibroblast growth factor (FGF19) is secreted by human gallbladder epithelial cells. This novel intestinal hormone is also released by ileal enterocytes into the portal circulation in response to bile salt absorption. In target organs, FGF19 binds to FGF receptor 4 (FGFR4) and its coreceptor Klotho-b (KLB), which results in feedback inhibition of hepatic bile salt synthesis and might also stimulate mucin expression. Zweers et al. point out that it is unexplored whether genetic variation within the FGF19-FGFR4-KLB axis contributes to cholelithiasis. Recently, functional FGFR4-KLB variants have been identified. To investigate their relevance for gallstone disease, we genotyped common FGFR4 (rs351855, rs376618) and KLB (rs17618244) variants in a cohort of 239 gallstone patients from 107 families (age range, 24-80 years; 86% women) and 248 stone-free controls (age range, 21-78 years; 93% women); patient characteristics of the incipient cohort were reported in HEPATOLOGY. Table 1 shows that the KLB genotype [AA] is more prevalent in cases than controls. Therefore, we tested for associations between genotypes and gallstone disease using contingency tables (allele frequency difference/positivity, heterozygous/homozygous carriers). Individuals who are homozygous for the minor allele [A] are at increased risk of developing gallstones (odds ratio, 3.23; 95% confidence interval, 1.32-7.92; P 1⁄4 0.007) as compared to carriers of genotype [GG]. Departure of the KLB genotype distribution from Hardy-Weinberg equilibrium in cases (exact test, P < 0.001; Supplementary Fig. 1) but not in controls supports the association of the KLB polymorphism with gallstones. However, nonparametric linkage analysis in affected sibs was negative (P > 0.05). In contrast to the KLB variant, both FGFR4 variants are not associated with gallstones in our cohort (data not shown). In conclusion, this study supports the functional link between KLB and gallstone disease, as suggested by Zweers et al. Interestingly, carriers of the KLB risk allele [A] display longer small intestinal transit times as compared to homozygous carriers of the common allele. Because slow intestinal transit increases the cholelithogenic state due to hyperabsorption of cholesterol and synthesis of secondary hydrophobic bile salts such as deoxycholate, we speculate that intestinal hypomotility contributes to gallstone susceptibility in carriers of the KLB risk variant. MARCIN KRAWCZYK, M.D. MONICA ACALOVSCHI, M.D. FRANK LAMMERT, M.D. Department of Medicine II Saarland University Medical Center Hamburg, Germany Department of Medicine III Iuliu Hatieganu University of Medicine and Pharmacy Cluj-Napoca, Romania

How does bacterial DNA induce liver failure in cirrhosis

In their prospective study of 156 patients with cirrhosis with ascites, Dr. Zapater and colleagues1 found a significant difference in 1-year mortality rate between patients with bacterial DNA in ascitic fluid and serum (38%) and those without bacterial DNA (15%). The most frequent cause of death was acute-on-chronic liver failure, and approximately 40% of deaths in patients with bacterial DNA occurred in the early course of the study. In the discussion of the article, the authors present two possible reasons why the presence of bacterial DNA resulted in earlier and more frequent deaths. One is interferon-gamma–induced liver damage referring to a study in a mouse model of chronic liver injury.2 Another is the assumption that bacterial DNA–induced NO can provoke hemodynamic instability, reflecting a significantly lower mean arterial pressure in patients with bacterial DNA compared with those without bacterial DNA, and NO-induced hemodynamic embarrassment could lead to lethal acute-on-chronic liver failure. Currently, publications reveal a more direct cause-effect relationship. Bacterial DNA is characterized by the prevalence of unmethylated cytosine-phosphate-guanine dinucleotides, termed CpG motifs. CpG DNA is known to induce inflammatory mediators such as tumor necrosis factor-alpha and interferon-gamma via Toll-like receptor 9. In a rat model of endotoxemia, CpG DNA application induced early hepatic microcirculatory deterioration and liver dysfunction.3 In D-galactosamine–sensitized mice, CpG DNA induced acute liver injury through the mitochondrial apoptotic pathway.4 Also, in a rat model of hemorrhagic shock, CpG DNA increased apoptosis in the liver and decreased expression of the hepatoprotective protein A-20 binding inhibitor of NF-kappa B-1 (ABIN-1).5 I would like to ask the authors whether increased early death associated with acute-on-chronic liver failure in patients with bacterial DNA were explained by these pathways.

Beneficial effects of high‐density lipoprotein administration on endotoxemia in an animal model of biliary cirrhosis

icant rise in International Normalized Ratio (INR 3.45 for acetaminophen versus 2.66 for placebo at 14 days).3 Because this complication does not technically fall under the category of liver injury or liver failure, AASLD will revisit its decision to include warfarin among the list of agents that can potentiate acetaminophen-induced liver injury. Instead, a separate warning criterion will be proposed to indicate that acetaminophen can increase the likelihood of an adverse event (bleeding) in patients taking warfarin. (3) I stand by the statement that “most hepatologists might dispute” advertising touting acetaminophen as the safest over-thecounter analgesic. Each month the Acute Liver Failure Study Group identifies 4-6 patients with acetaminophen-related ALF, 30% of whom die. For each one of these cases, we typically discuss on our conference call two other “near misses,” patients admitted with severe acetaminophen-induced liver injury and coagulopathy who do not become encephalopathic. Fortunately, most of these latter patients recover, but they still undergo a near-fatal experience and represent a huge burden to the health care system. That amounts to 150 or more cases of severe acetaminophen hepatotoxicity each year from our own practices—hard to ignore! In summary, Drs. Kuffner and Baggish raise questions around the edges of the problem, while missing the larger issue: “What can we do, together, to stop the large number of needless acetaminophen deaths that occur annually?”

Adoptive transfer of regulatory T cells in an animal model of a diet‐induced fatty liver

1. Leung PS, Rossaro L, Davis PA, Park O, Tanaka A, Kikuchi K, et al. Antimitochondrial antibodies in acute liver failure: implications for primary biliary cirrhosis. HEPATOLOGY 2007;46:1436-1442. 2. Ferret PJ, Hammoud R, Tulliez M, Tran A, Trebeden H, Jaffray P, et al. Detoxification of reactive oxygen species by a nonpeptidyl mimic of superoxide dismutase cures acetaminophen-induced acute liver failure in the mouse. HEPATOLOGY 2001;33:1173-1180. 3. Wu CT, Eiserich JP, Ansari AA, Coppel RL, Balasubramanian S, Bowlus CL, et al. Myeloperoxidase-positive inflammatory cells participate in bile duct damage in primary biliary cirrhosis through nitric oxide-mediated reactions. HEPATOLOGY 2003;38:1018-1025. 4. Bernal W, Ma Y, Smith HM, Portmann B, Wendon J, Vergani D. The significance of autoantibodies and immunoglobulins in acute liver failure: a cohort study. J Hepatol 2007;47:664-670. 5. Ma Y, Okamoto M, Thomas MG, Bogdanos DP, Lopes AR, Portmann B, et al. Antibodies to conformational epitopes of soluble liver antigen define a severe form of autoimmune liver disease. HEPATOLOGY 2002;35:658-664. 6. Longhi MS, Ma Y, Bogdanos DP, Cheeseman P, Mieli-Vergani G, Vergani D. Impairment of CD4( )CD25( ) regulatory T-cells in autoimmune liver disease. J Hepatol 2004;41:31-37. 7. Longhi MS, Ma Y, Mitry RR, Bogdanos DP, Heneghan M, Cheeseman P, et al. Effect of CD4 CD25 regulatory T-cells on CD8 T-cell function in patients with autoimmune hepatitis. J Autoimmun 2005;25:63-71. 8. Longhi MS, Hussain MJ, Mitry RR, Arora SK, Mieli-Vergani G, Vergani D, et al. Functional study of CD4 CD25 regulatory T cells in health and autoimmune hepatitis. J Immunol 2006;176:4484-4491. Copyright