Biliary atresia (BA) is a progressive, inflammatory cholangiopathy of infancy that leads to fibrosis and obliteration of the extra and intrahepatic bile ducts. There are two forms of BA: perinatal or acquired and embryonic or fetal. In addition, ~8% cases have a cystic variant of BA characterized by presence of biliary cysts.[1] The existence of clinical forms and variants suggests that BA encompasses a spectrum of disease phenotypes.

 

The only effective treatment for BA is the surgical construction of a portoenterostomy i.e. Kasai’s procedure. Early diagnosis (<30-45 days of life) of BA is associated with improved post-surgical outcome and longer survival with the native liver.[2] However, establishing this diagnosis is problematic, largely due to lack of awareness and disease rarity in comparison to the much more common indirect hyperbilirubinemia in neonates.

 

Last few decades have witnessed multiple attempts to improve the outcome of BA patients which include, educating pediatricians to look for high colored urine in a jaundiced baby to identify those with conjugated hyperbilirubinemia, early measurement of serum conjugated bilirubin, looking for presence of pale stools to recognize those likely to have BA and thereafter prompt referral to centers with surgical expertise. The universal use of stool color cards improved the national rate of the Kasai operation before 60 days of age from 60% in 2004 to 74.3% in 2005 in Taiwan.[3] The yellow alert campaign is another effort to improve the timely diagnosis and surgery by eight weeks of age.[4]

 

The determinants of the outcome of portoenterostomy include age at surgery, center’s experience, presence of associated congenital anomalies, and postoperative occurrence of cholangitis. However, even with prompt surgical intervention at the best centers, ongoing injury to the intrahepatic bile ducts leads to end-stage cirrhosis. Nearly 80% of BA patients eventually require liver transplantation with approximately 50% needing it by 2 years of life.[5]

 

Factors known to play a role in BA pathogenesis include: exposure to environmental toxins, defect in fetal circulation, defect in biliary tract morphogenesis, viral infection and inflammation.[6,7] Histological grading of liver biopsy specimens and gene expression profiling has shown two main disease forms: inflammatory and fibrotic,[8] with subjects having an inflammatory signature being younger and those with fibrotic signature having decreased transplant free survival at 2 years of age. It may be speculated that these are two distinct but interrelated stages of the disease, with fibrosis following inflammation.

 

Major research in recent years is targeted to the viral and immune mediated disease pathogenesis. In this issue of the journal, El-Faramawy et al9 have evaluated the role of serum proinflammatory cytokines (IL6 and IL8) in pathogenesis and outcome of BA infants. They have compared IL6 and IL8 levels in BA with cases of intrahepatic cholestasis of mixed etiology and healthy controls. Higher IL6 and IL8 levels were found in BA as compared to both the control groups. IL6 levels were significantly higher in BA patients with severe fibrosis on liver histology at diagnosis than those with mild fibrosis. IL8 levels had a positive correlation with lymphocyte count and negative correlation with serum albumin and were higher in patients with persistent jaundice after portoenterostomy. The authors conclude that ongoing inflammation as evident by elevated IL6 and IL8 levels may serve to determine disease severity and may predict the progression to liver fibrosis.

 

Although the authors have taken care to exclude sepsis at time of cytokine estimation, but the study raises certain questions in the reader’s mind. Firstly, there was a gap of 9-12 months between liver histology and cytokine estimation, which questions the observed association between liver fibrosis and IL6 levels. The authors have cited ethical reasons for not doing a repeat biopsy at 9-12 months but they could have very easily estimated the cytokines at time of initial biopsy and then at a later date. Various factors like episodes of cholangitis, success of surgery and baseline disease severity, can all affect the liver histology in follow up at 9-12 months of age. In order to correlate the specific serum cytokine with disease pathogenesis, the target organ i.e. liver and cytokines should be studied concurrently and not sequentially.

 

It is mentioned that prophylactic antibiotics were given for a year to BA patients and these might have affected the cytokine profile. The disease control group is very heterogeneous and includes benign conditions like idiopathic neonatal hepatitis and aggressive conditions associated with early cirrhosis like progressive familial intrahepatic cholestasis. There is no logic of putting all of them together and then comparing their liver functions with that of BA patients. In addition, there is no information whether an attempt was made to correlate the histology (fibrosis and inflammation) in the disease control group with cytokine levels. Comparison of the cytokine profile in subjects with successful and unsuccessful portoenterostomy would have been useful to elaborate the role of cytokines in progressive intrahepatic biliary disease. Recently, Bezzera et al10 have suggested a likely pathogenetic mechanism for BA. The initial event may be a viral infection, which targets the biliary epithelium and primes macrophages and dendritic cells (initiating phase). This is followed by activation of NK cells that injure cholangiocytes and disrupt the epithelial continuity (epithelial injury phase). Thereafter, amplification of the adaptive immune response by CD4+ and CD8+ T cells and release of proinflammatory cytokines perpetuates the injury and causes biliary obstruction (phase of obstruction), which is followed by collagen deposition to produce the atresia phenotype.

 

Support for a viral etiology comes from the seasonal disease pattern, ability of viruses (reovirus and rotavirus) to induce cholangitis and obstruction of extrahepatic bile ducts in young mice[11] and demonstration of increased expression of TLR3 (toll like receptors) after stimulation with a pathogen associated molecular pattern (PAMP) analogue of viral RNA.[12]

 

There is increasing evidence from both human and animal model studies that inflammation plays the key role in bile duct and liver injury in BA. Evidence for a proinflammatory state came from a gene expression analysis of liver biopsy specimens which showed a coordinated activation of genes involved in Th1 response at early stages of BA.[13]

 

In keeping with a T helper mediated process, there is an abundance of CD4+ T lymphocytes in liver of BA patients, possibly due to the enhanced expression of adhesion molecules.[14] Further immunohistochemistry studies revealed increase in CD8+ and CD4+ T cells and Kupffer cells (CD68+) in the portal tracts of BA patients. Reverse transcription–PCR analysis of BA tissue confirmed a Th1-type cytokine profile with expression of IL-2, interferon-a, tumor necrosis factor-a and IL-12.[15] This process was not found in the liver of normal control subjects or choledochal cyst and neonatal hepatitis patients. This suggested that the observed immune repertoire is not merely a response to cholestasis but rather an indicator of specific immune processes involved in the pathogenesis of BA. We still do not know whether the lymphocytes are clonal or the specific antigen against which they are directed, e.g. viral or self-bile duct epithelial antigen.

 

Around the same time, two separate workers studied IL8 in BA patients. Nobili et al[16] reported presence of significantly higher  IL8 levels in patients with BA, PFIC and Alagille syndrome in comparison to neonatal hepatitis and healthy controls. In addition the IL8 levels correlated with the histological activity index (HAI) in the concurrent liver, suggesting that elevated IL8 leads to progressive inflammation and fibrosis in the liver. Honsawek et al[17] studied BA patients and healthy children at a later age of 6.3+ 0.6 and 6.7+1.1 years, respectively. BA patients were classified based on outcome, with jaundice and without jaundice. Serum IL-8 levels were significantly higher in those with jaundice (516.5+130.0 vs. 49.3+10.4 pg/ml). In the jaundicefree group, IL-8 levels were higher in patients with portal hypertension (PHT) than those without PHT. Both these studies and that of El-Faramawy9 suggest that elevated IL-8 levels may be markers of poor outcome in form of persistent jaundice and liver fibrosis with PHT.

 

A possible Th2 involvement in BA pathogenesis is suggested by the finding of intrahepatic-periductal IgG deposits and serum antibodies reactive to biliary epithelial cells in murine BA models.[18] The targets of the serum autoantibodies are unknown with alpha-enolase and vimentin being likely candidates.[19] The published literature suggests that the immune system plays a major role in the progressive fibrosis and obliteration  of bile ducts in BA. The best therapeutic approaches are thosewhich target the key mechanism- i.e. inflammation and fibrosis. We are still awaiting the results of a multicentre controlled trial looking at the role of steroids as an anti-inflammatory agent. The need of the hour is to clearly sort out this jigsaw puzzle of etiopathogenesis so as to develop tailor made therapy with agents like immunosuppressants, cytokine blockers, antioxidants, or anti-fibrotics depending on the disease stage.

 

The study by El-Faramawy is just another piece of this jigsaw puzzle. Our target is to have a good long term transplant free survival for BA patients. And only when we achieve this, which still seems like a wish, would the benchside research translate into useful bedside patient management.

 

References

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2.     Sokol RJ, Shepherd RW, Superina R, Bezerra JA, Robuck P, Hoofnagle JH. Screening and outcomes in biliary atresia: summary of a National Institutes of Health workshop. Hepatology. 2007;46:566–81.

3.     Hsiao CH, Chang MH, Chen HL, Lee HC, Wu TC, Lin CC, et al. Taiwan Infant Stool Color Card Study Group. Universal screening for biliary atresia using an infant stool color card in Taiwan. Hepatology. 2008;47:1233–40.

4.     Yachha SK, Mohindra S. Neonatal cholestasis syndrome: Indian scene. Indian J Pediatr. 1999;66:S94–6.

5.     Shneider BL, Brown MB, Haber B, Whitington PF, Schwarz K, Squires R. A multicenter study of the outcome of biliary atresia in the United States, 1997 to 2000. J Pediatr. 2006;148:467–74.

6.     Balistreri WF, Grand R, Hoofnagle JH, Suchy FJ, Ryckman FC, Perlmutter DH, et al. Biliary atresia: current concepts and research directions. Summary of a symposium. Hepatology. 1996;23:1682–92.

7.     Bezerra JA. The next challenge in pediatric cholestasis: deciphering the pathogenesis of biliary atresia. J Pediatr Gastroenterol Nutr. 2006;43:S23–9.

8.     Moyer K, Kaimal V, Pacheco C, Mourya R, Xu H, Shivakumar P, et al. Staging of biliary atresia at diagnosis by molecular profiling of the liver. Genome Med. 2010;2:33.

9.     El-Faramawy Amel AM, El-Shazly LBE, Abbas AA, Ismail HAB. Serum IL-6 and IL-8 in infants with biliary atresia in comparison to intrahepatic cholestasis. Trop Gastroenterol. 2011;32:48–53.

10.   Bessho K, Bezerra JA. Biliary atresia: will blocking inflammation tame the disease? Annu Rev Med. 2011;62:171–85.

11.   Petersen C, Biermanns D, Kuske M, Schäkel K, Meyer-Junghänel L, Mildenberger H. New aspects in a murine model for extrahepatic biliary atresia. J Pediatr Surg. 1997;32:1190–5.

12.   Harada K, Sato Y, Itatsu K, Isse K, Ikeda H, Yasoshima M, et al. Innate immune response to double-stranded RNA in biliary epithelial cells is associated with the pathogenesis of biliary atresia. Hepatology. 2007;46:1146–54.

13.   Bezzerra JA, Tiao G, Ryckman FC, Alonso M, Sabla GE, Shneider B, et al. Genetic induction of proinflammatory immunity in children with biliary atresia. Lancet. 2002;360:1653–9.

14.   Davenport M, Gonde C, Redkar R, Koukoulis G, Tredger M, Mieli-Vergani G, et al. Immunohistochemistry of the liver and biliary tree in extrahepatic biliary atresia. J Pediatr Surg. 2001;36:1017–25.

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16.   Nobili V, Marcellini M, Giovannelli L, Girolami E, Muratori F, Giannone G, et al. Association of serum interleukin-8 levels with the degree of fibrosis in infants with chronic liver disease. J Pediatr Gastroenterol Nutr. 2004;39:540–4.

17.   Honsawek S, Chongsrisawat V, Vejchapipat P, Thawornsuk N, Tangkijvanich P, Poovorawan Y. Serum interleukin-8 in children with biliary atresia: relationship with disease stage and biochemical parameters. Pediatr Surg Int. 2005;21:73–7.

18.   Mack CL, Tucker RM, Lu BR, Sokol RJ, Fontenot AP, Ueno Y, et al. Cellular and humoral autoimmunity directed at bile duct epithelia in murine biliary atresia. Hepatology. 2006;44:1231–9.

19.   Lu BR, Brindely SM, Tucker RM, Lambert C, Mack C. Serum autoantibodies reactive to alpha-enolase and vimentin in murine and human biliary atresia. Hepatology. 2008;48:412A.