INVITED REVIEW
Biliary Atresia: Clinical Lessons Learned Amy G. Feldman and Cara L. Mack ABSTRACT Biliary atresia is a rare disease of unclear etiology, in which obstruction of the biliary tree causes severe cholestasis leading to cirrhosis and ultimately death if left untreated. Biliary atresia is the leading cause of neonatal cholestasis and the most frequent indication for pediatric liver transplantation. Any infant with persistent jaundice beyond 2 weeks of life needs to be evaluated for biliary atresia with fractionation of the bilirubin into conjugated and unconjugated portions. Early performance of a hepatoportoenterostomy in the first 45 days of life to restore bile flow and lessen further damage to the liver is thought to optimize outcome. Despite surgery, progressive liver scarring occurs, and 80% of patients with biliary atresia will require liver transplantation during childhood. Key Words: biliary atresia, liver transplant, neonatal cholestasis
(JPGN 2015;61: 167–175)
B
iliary atresia (BA) is the most common cause of cholestasis in the first 3 months of life and the most frequent pediatric indication for liver transplantation, accounting for up to 50% of pediatric liver transplants in the United States. The incidence in the United States is 1 in 12,000 live births and is the highest in Taiwan (1 in 5600) and the lowest in Europe (1 in 18,000). BA is more common in female infants, Asians, and African Americans (1); however, it can be seen in infants from all of the countries and racial populations. At the time of diagnosis, a Kasai hepatoportoenterostomy (HPE) is performed in an attempt to reestablish biliary flow. Unfortunately, this procedure fails to prevent the intrahepatic biliary cirrhosis from progressing, and the majority of children will need transplant for survival.
PATHOGENESIS The etiology of BA is unknown, and theories of pathogenesis include viral infection, autoimmune-mediated bile duct destruction, and abnormalities in bile duct development because of genetic mutations. With regard to a gene mutation association, a recent study from China analyzed single-nucleotide polymorphisms (SNPs) and susceptibility genes in BA through genome-wide association studies (2). The results revealed a strong association of BA with the SNP rs17095355 on chromosome 10q24. Two genes
Received September 17, 2014; accepted January 30, 2015. From the Section of Pediatric Gastroenterology, Hepatology and Nutrition, Digestive Health Institute, Children’s Hospital Colorado, University of Colorado School of Medicine, Aurora. Address correspondence and reprint requests to Amy G. Feldman, MD, Section of Pediatric Gastroenterology, Hepatology and Nutrition, Digestive Health Institute, Children’s Hospital Colorado, University of Colorado School of Medicine, 13123 East 16th Ave, B290, Aurora, CO 80045 (e-mail: [email protected]). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000755
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in the region of this SNP include X-prolyl aminopeptidase P1 (XPNPEP1) and adducin 3 (ADD3). XPNPEP1 is expressed in biliary epithelia and is involved in the metabolism of inflammatory mediators. ADD3 is expressed in hepatocytes and biliary epithelia and is involved in the assembly of spectrin-actin membrane protein networks at sites of cell-to-cell contact. Defective ADD3 could result in excessive deposition of actin and myosin, contributing to biliary fibrosis. A recent study from the United States tested the association of SNPs on chromosome 10q24 and BA and found the strongest signal to be at rs7099604 within the ADD3 gene (3). We refer the reader to a complete review of potential etiologies of BA (4), and will focus herein on recent immunopathogenesis studies. A plethora of data have been accumulated to support both an early virus infection and an aberrant immune response in the pathogenesis of disease. One theory that ties these 2 together is that the bile duct injury in BA may be initiated by a virus infection followed by a secondary autoimmune response targeting bile duct epithelia (5). The damaged bile duct cells may express self-proteins that are recognized as foreign, and elicit autoreactive T-cellmediated inflammation and B-cell production of autoantibodies. In 1974, Landing (6) first proposed that BA and other infantile obstructive cholangiopathies were caused by viral infection of the liver and the hepatobiliary tree. Candidate viruses that may trigger the bile duct injury include cytomegalovirus (CMV) (7), rotavirus (8), and reovirus (9). Controversy remains as to the detection of all of these viruses at the time of diagnosis, and it is possible that the virus infection of the biliary tree is short-lived. Rotavirus is an interesting candidate, because the rhesus rotavirusinduced mouse model of BA recapitulates the early events in the inflammatory biliary obstruction found in human BA (8,10). A recent study by Lin et al (10) found a significant decrease in BA incidence in Taiwan following introduction of the rotavirus vaccination. The authors proposed that although rotavirus vaccination is initiated at 2 months of age, later than the usual age of onset of BA, herd immunity may decrease rotavirus infection during pregnancy or the neonatal period. The authors also, however, showed that the incidence of BA was negatively correlated with the country’s gross domestic product, so further studies will be important to determine whether the rotavirus vaccine or overall improvement in socioeconomic status was truly responsible for the decreased incidence of BA. The greatest body of research entails investigations of CMV at the time of diagnosis of BA. A higher prevalence of CMV antibodies in the mothers of infants with BA, higher serum CMVimmunoglobulin (Ig) M levels, and greater amounts of Ig deposits on the canalicular membrane of the hepatocytes in infants with BA have been reported (11). Strong evidence for a perinatal CMV infection associated with BA was described by Xu et al (12). Liver tissue obtained at the time of portoenterostomy on 85 infants with BA was analyzed by real-time polymerase chain reaction (PCR) for the presence of multiple viruses, and the majority (60%) was positive for CMV. Studies confirming the presence of CMV in PCR-positive liver samples were performed with immunocytochemical detection of CMV-pp65 antigens within liver bile duct epithelia and hepatocytes. Brindley et al (13) analyzed the liver memory T-cell response to a variety of viruses from infants with BA
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JPGN
at the time of diagnosis. The majority (56%) of patients with BA had significant increase in interferon (IFN)-g-producing liver T cells in response to CMV homogenate and CMV-pp65 antigen, suggesting that perinatal CMV infection had occurred. An interesting observation from the past virus studies is the ability of all of the 2 abovementioned viruses to infect and damage bile duct epithelia, lending support to a primary cholangiotropic viral infection as the initiating event in the pathogenesis of BA. In the last decade, scientific investigations have concentrated on the role of the adaptive immune system in bile duct injury in BA. A plausible theory that could explain the progression of biliary tract injury that predominates in BA is that of an autoimmune-mediated attack on biliary epithelia. After the initial viral insult to the biliary tree, the damaged bile duct epithelial cells may express previously sequestered ‘‘self’’ antigens that are recognized as foreign and elicit autoreactive TH1-cell-mediated inflammation and B-cell production of autoantibodies directed at duct epithelia. The predominant cellular immune response in BA encompasses activated CD4þ and CD8þ T cells within portal tracts that produce TH1 cytokines (interleukin-2 and IFN-g) and macrophages secreting tumor necrosis factor (TNF)-a (14,15). These lymphocytes have been found invading between bile duct epithelia, leading to degeneration of intrahepatic bile ducts. The strongest evidence for the autoimmune theory has been gained from mouse studies, in which autoreactive T cells and autoantibodies targeting bile duct epithelia have been identified (16–18). In humans, only circumstantial evidence exists for the role of autoimmunity in BA pathogenesis. Human leukocyte antigen (HLA) associations with BA have been reported with conflicting results. European and American studies of HLA predominance in BA found no significant differences compared with controls (19). In contrast, a Japanese study found significant association between BA and HLA-DR2 as well as a linkage disequilibrium with a high frequency of HLA-A24-B52-DR2 (20). The presence of autoantibodies and periductal immune deposits, suggesting a humoral autoimmune response, has also been described (11,17). The potential contribution of adaptive immune and autoimmune responses to bile duct injury in BA is represented in Figure 1. Research pertaining to deciphering the etiology of BA is robust and is focusing on genetic and immunologic links to the pathogenesis of disease.
OPN OPN
Key Virus Ag Bile duct epithelial Ag Macrophage
IL-12
Apoptotic bile duct epithelia
CD4+
Volume 61, Number 2, August 2015
Another area of research in BA has focused on the extensive fibrosis that occurs with this disease. Portal bridging fibrosis and even cirrhosis are commonly seen at the time of diagnosis of BA, and the degree of fibrosis correlates with outcome. One mechanism of fibrogenesis that could explain the severity of fibrosis entails the Hedgehog (Hh) pathway. The Hh pathway is important in tissue remodeling and Hh activation, with subsequent epithelial-mesenchymal transition (EMT) of cells involved in fibrogenesis related to hepatobiliary diseases. It has been previously shown in BA that biliary epithelia undergo EMT, promoting fibrosis. BA patient livers demonstrated significant upregulation of Hh ligand within intra- and extrahepatic ductular cells, as well as increased expression of Hh target genes. In addition, immature ductular cells within BA livers showed a profibrotic mesenchymal phenotype that was Hh responsive (21). The authors concluded that excessive Hh activation impedes ductular morphogenesis and enhances fibrogenesis by promoting accumulation of immature ductular cells with a mesenchymal phenotype. Most searches for defects at the chromosomal level have produced negative results; however, recently, a genetic link to the Hh pathway has been described. Identified deletions at chromosome 2q37.3 in patients with BA results in deletion of 1 copy of glypican-1 (GPC1), a heparan sulfate proteoglycan that regulates Hh signaling and inflammation. Liver tissues of patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with controls. Cui et al (22) used a gpc1 knockdown zebrafish model to show developmental biliary defects and gallbladder atresia. Exposure of the gpc1 morphants to cyclopamine, an Hh antagonist, rescued the gpc1-knockdown phenotype. These studies not only identify GPC1 as a risk gene for BA but also offer mechanistic insight into the potential pathogenic role of GPC1 and Hh in BA. Another potential mechanism associated with activation of fibrosis involves the recent novel finding of prominin-1 (PROM-1) expressing stem cells adjacent to ductular reactions within the portal tracts of patients with BA and in murine BA (23). The PROM-1þ cells expressed both markers of epithelial and mesenchymal cells, produced collagen, and correlated with portal fibrosis and bilirubin levels. In addition, expansion of PROM-1þ cells was associated with the activation of fibroblast growth factor and transforming growth factor-b providing potential mechanisms of action of
Clonal expansion CXCR3
(•)
Dendritic cell
TNF-α iNOS
γ N-
IF
CD4+ (TH1)
CD8+
Perforin granzyme IFN-γ
CD8+
T-cell activation B cell
FIGURE 1. Contribution of adaptive immunity in the pathogenesis of BA. This figure summarizes human investigations pertaining to the role of adaptive immunity in bile duct injury and offers a theory on the role of autoimmune responses in the pathogenesis of disease. Bile duct injury is initiated by virus infection (ie, CMV) with clonal expansion of virus-specific CD4þ T cells. IL-12, osteopontin, and CXCR3 chemokine receptors all promote TH1 cell differentiation. These TH1 cells activate effector cells including macrophages (via CD4þ IFN-g stimulation) with subsequent generation of TNF-a and inducible nitric oxide synthase, cytotoxic CD8þ T cells that directly invade epithelia and release cytotoxic molecules, and B cells that mature into antibody-producing plasma cells with release of IgM and IgG targeting bile duct epithelia. Over time, proteins from apoptotic bile duct epithelia may be presented and seen as ‘‘foreign,’’ eliciting autoreactive T-cell-mediated inflammation that perpetuates the bile duct injury. BA ¼ biliary atresia; CMV ¼ cytomegalovirus; IFN ¼ interferon; Ig ¼ immunoglobulin; IL ¼ interleukin; TNF ¼ tumor necrosis factor.
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Biliary Atresia: Clinical Lessons Learned
fibrogenesis. These exciting studies of fibrogenesis in BA provide insight into potential future targets of antifibrotic therapy.
CLINICAL FEATURES There are 3 distinct clinical patterns of BA (24). The majority of infants with BA (84%) have the perinatal or acquired form of BA without associated major malformation. These babies are asymptomatic, often jaundice-free at birth and appear to be thriving until 2 to 6 weeks of life when they have persistent jaundice, acholic stools, dark urine, and hepatomegaly. On laboratory evaluation, they have elevated total and direct bilirubin levels, elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and elevated g-glutamyl transpeptidase (GGTP) levels >200 IU/L. Ascites and splenomegaly, results of portal hypertension, are late findings and not usually seen on initial presentation. In a second group (6%), infants have at least 1 major malformation but no laterality defects. Finally, in 10% of patients with BA, the infants are syndromic and have 1 or more laterality defects. These infants appear jaundiced from birth and do not have an asymptomatic period.
EVALUATION OF NEONATAL CHOLESTASIS Any infant who remains jaundiced at 2 weeks of age needs to be evaluated for cholestasis, with fractionation of the bilirubin into a conjugated (direct) and unconjugated (indirect) portion. Conjugated hyperbilirubinemia associated with cholestasis is defined as a direct/conjugated bilirubin of >1 to 2 mg/dL and >20% of the total bilirubin concentration. One must have a high suspicion for
hepatobiliary dysfunction and proceed with further workup in the setting of cholestasis, acholic stools, hepatomegaly, or splenomegaly. In an infant with cholestasis, further evaluation should be conducted with a sense of urgency to identify those causes that are amenable to medical and surgical correction (Fig. 2). Outcome of infants with BA is directly correlated to the timing of diagnosis and HPE. A comprehensive metabolic panel will likely show a total bilirubin of 5 to 12 mg/dL and an ALT and AST in the 100 to 200 U/ L, findings not unique to BA. It is rare for an infant with BA to have a GGTP level 1 or 2 patients of BA during their careers and as a result may dismiss jaundice at 2 weeks of age to be the result of breast-feeding without considering BA. Second, in the United States health care system, infants are routinely seen at 2 weeks of age and not again until 8 weeks of age. Without a routine 4-week visit, the crucial diagnostic period to identify and intervene for BA is often missed. Third, there are no pathognomonic lab findings that distinguish BA from other causes of neonatal cholestasis. Finally, at this time, there are no universal screening programs in place in the United States to help identify infants with BA within the first month of life.
KASAI HEPATOPORTOENTEROSTOMY In 1959, Kasai developed the HPE procedure in which a Roux-en-Y loop of jejunum is anastomosed to the hilum of the liver, thus creating a new conduit for biliary drainage. The HPE has transformed BA from a universally fatal disease in early childhood to one in which restoration of bile flow and normalization of bilirubin may result in long-term survival with the native liver. HPE reestablishes short-term bile drainage in approximately twothirds of patients. Several factors have been reported to affect outcome after HPE. Nonmodifiable risk factors that predict poor outcome include obstruction proximal to the common bile duct, bridging fibrosis at the time of HPE, and polysplenia syndrome. www.jpgn.org
Prognostic factors related to care include age at Kasai, experience of the center in managing BA, and accessibility to liver transplant. Increased age at HPE has a progressive and lasting detrimental effect on outcome. If the HPE is performed within the first 60 days of life, 70% to 80% of patients show bile drainage. If performed between 60 and 90 days, 40% to 50% of patients show drainage; after 90 days of life only 25% of patients show drainage; if performed later than 120 days of life 5 HPEs per year (61.3% vs 13.7% and 91.2% vs 75%) (31). This has led to centralization of care in many European countries. These modifiable prognostic factors offer areas in which quality initiatives could drastically improve disease outcome. Success of the HPE can best be judged by restoration of bile flow and clearance of jaundice. By 3 months after HPE, a clear difference in the 2-year transplant-free survival can be seen between those children with total bilirubin 6 mg/dL (84% vs 16%; P < 0.0001) (28). If jaundice clears successfully by 3 months after HPE, the 10-year transplant-free survival rate ranges from 75% to 90%; conversely, if jaundice persists after HPE, the 3-year transplant-free survival rate is only 20%. There is no standard treatment regimen for patients with BA who are post-HPE. Regimens may include ursodeoxycholic acid, antibiotic prophylaxis against cholangitis, and a fatsoluble vitamin preparation. Steroids were previously believed to improve clinical outcomes secondary to their choleretic, antiinflammatory, and immunomodulatory properties. In the recent multicenter trial in which 140 infants with BA from 14 centers across the United States were, however, randomized to receive
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either high-dose steroids following HPE or placebo; there was no difference between groups in bile drainage at 6 months or 2 years post-HPE and no difference in transplant-free survival at 2 years (32).
COMPLICATIONS OF BA
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D, E, and/or K should be administered as needed (37) (Table 2). Nutrition remains a modifiable risk for outcome in BA. Prospective multicenter trials are needed to better understand the specific effects of different nutritional interventions on improving outcomes.
Cholangitis
Failure to Thrive and Fat-Soluble Vitamin Deficiencies Failure to thrive is a significant problem in patients with BA. In the Studies of Pediatric Liver Transplant registry, 755 patients with BA who underwent liver transplantation were analyzed, and 40% had growth failure (33). This growth failure was found to be an independent risk factor for pretransplant mortality, posttransplant mortality, longer hospital stays, and graft failure (33). The pathogenesis of growth failure and malnutrition in infants with BA is multifactorial. Infants with BA have poor bile flow resulting in reduced delivery of bile acids to the small intestine, decreased mixed micelle formation, and subsequent fat and fat-soluble vitamin malabsorption. In addition, infants with BA often have poor appetite and increased energy expenditure with a resting energy expenditure 29% greater than age-matched controls (34). One must be careful not to be reassured by a normal weight and height in an infant with BA. Weight can be falsely elevated secondary to ascites and organomegaly, and diminished height can be a late finding of poor nutrition. To get an accurate assessment of true nutritional status, one must check anthropometrics including triceps skin fold thickness and midarm circumference. Growth failure after HPE is associated with increased risk of transplantation or death by 24 months of age (35). To that end, the importance of growth is acknowledged in the present pediatric end-stage liver disease allocation system in the United States in which children receive higher scores when they have growth failure (36). Optimization of nutrition is a crucial part of the care of infants with BA. Infants should be started on a formula containing medium-chain triglycerides, which can be absorbed independently of bile salts. Caloric intake should be approximately 125% of the recommended dietary allowance based on ideal body weight. If patients are unable to ingest the needed calories orally, they should be started on nasogastric tube feeds. Fat-soluble vitamin levels need to be carefully monitored and treated because deficiencies can result in rickets, bone fractures, coagulopathy, cerebellar ataxia, and impaired vision. All of the infants with cholestasis should be on a fat-soluble vitamin supplement that takes advantage of the ability of tocopherol polyethylene glycol succinate (TPGS) to be absorbed independently of bile salts. A recent study showed that fat-soluble vitamin deficiencies often persist despite initiation of a TPGS containing liquid multiple fat-soluble vitamin preparation; therefore, additional individual vitamin supplementation with vitamin A,
Forty percent to 60% of infants with BA develop cholangitis in the first 2 years following HPE (28). Because cholangitis is presumed to be an ascending infection, it only occurs in those children who have some degree of bile flow. Symptoms may be nonspecific, and cholangitis should be considered whenever a child who is post-HPE presents with fever, vomiting, decreased oral intake, pale stools, worsening jaundice, right upper quadrant abdominal or shoulder pain, or worsening laboratory values (rising ALT, AST, GGTP, bilirubin, or white blood cell count). A blood culture may be positive and common infectious agents include Escherichia coli, Enterobacter, or Klebsiella; however,
Biliary Atresia: Clinical Lessons Learned Amy G. Feldman and Cara L. Mack ABSTRACT Biliary atresia is a rare disease of unclear etiology, in which obstruction of the biliary tree causes severe cholestasis leading to cirrhosis and ultimately death if left untreated. Biliary atresia is the leading cause of neonatal cholestasis and the most frequent indication for pediatric liver transplantation. Any infant with persistent jaundice beyond 2 weeks of life needs to be evaluated for biliary atresia with fractionation of the bilirubin into conjugated and unconjugated portions. Early performance of a hepatoportoenterostomy in the first 45 days of life to restore bile flow and lessen further damage to the liver is thought to optimize outcome. Despite surgery, progressive liver scarring occurs, and 80% of patients with biliary atresia will require liver transplantation during childhood. Key Words: biliary atresia, liver transplant, neonatal cholestasis
(JPGN 2015;61: 167–175)
B
iliary atresia (BA) is the most common cause of cholestasis in the first 3 months of life and the most frequent pediatric indication for liver transplantation, accounting for up to 50% of pediatric liver transplants in the United States. The incidence in the United States is 1 in 12,000 live births and is the highest in Taiwan (1 in 5600) and the lowest in Europe (1 in 18,000). BA is more common in female infants, Asians, and African Americans (1); however, it can be seen in infants from all of the countries and racial populations. At the time of diagnosis, a Kasai hepatoportoenterostomy (HPE) is performed in an attempt to reestablish biliary flow. Unfortunately, this procedure fails to prevent the intrahepatic biliary cirrhosis from progressing, and the majority of children will need transplant for survival.
PATHOGENESIS The etiology of BA is unknown, and theories of pathogenesis include viral infection, autoimmune-mediated bile duct destruction, and abnormalities in bile duct development because of genetic mutations. With regard to a gene mutation association, a recent study from China analyzed single-nucleotide polymorphisms (SNPs) and susceptibility genes in BA through genome-wide association studies (2). The results revealed a strong association of BA with the SNP rs17095355 on chromosome 10q24. Two genes
Received September 17, 2014; accepted January 30, 2015. From the Section of Pediatric Gastroenterology, Hepatology and Nutrition, Digestive Health Institute, Children’s Hospital Colorado, University of Colorado School of Medicine, Aurora. Address correspondence and reprint requests to Amy G. Feldman, MD, Section of Pediatric Gastroenterology, Hepatology and Nutrition, Digestive Health Institute, Children’s Hospital Colorado, University of Colorado School of Medicine, 13123 East 16th Ave, B290, Aurora, CO 80045 (e-mail: [email protected]). The authors report no conflicts of interest. Copyright # 2015 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000755
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in the region of this SNP include X-prolyl aminopeptidase P1 (XPNPEP1) and adducin 3 (ADD3). XPNPEP1 is expressed in biliary epithelia and is involved in the metabolism of inflammatory mediators. ADD3 is expressed in hepatocytes and biliary epithelia and is involved in the assembly of spectrin-actin membrane protein networks at sites of cell-to-cell contact. Defective ADD3 could result in excessive deposition of actin and myosin, contributing to biliary fibrosis. A recent study from the United States tested the association of SNPs on chromosome 10q24 and BA and found the strongest signal to be at rs7099604 within the ADD3 gene (3). We refer the reader to a complete review of potential etiologies of BA (4), and will focus herein on recent immunopathogenesis studies. A plethora of data have been accumulated to support both an early virus infection and an aberrant immune response in the pathogenesis of disease. One theory that ties these 2 together is that the bile duct injury in BA may be initiated by a virus infection followed by a secondary autoimmune response targeting bile duct epithelia (5). The damaged bile duct cells may express self-proteins that are recognized as foreign, and elicit autoreactive T-cellmediated inflammation and B-cell production of autoantibodies. In 1974, Landing (6) first proposed that BA and other infantile obstructive cholangiopathies were caused by viral infection of the liver and the hepatobiliary tree. Candidate viruses that may trigger the bile duct injury include cytomegalovirus (CMV) (7), rotavirus (8), and reovirus (9). Controversy remains as to the detection of all of these viruses at the time of diagnosis, and it is possible that the virus infection of the biliary tree is short-lived. Rotavirus is an interesting candidate, because the rhesus rotavirusinduced mouse model of BA recapitulates the early events in the inflammatory biliary obstruction found in human BA (8,10). A recent study by Lin et al (10) found a significant decrease in BA incidence in Taiwan following introduction of the rotavirus vaccination. The authors proposed that although rotavirus vaccination is initiated at 2 months of age, later than the usual age of onset of BA, herd immunity may decrease rotavirus infection during pregnancy or the neonatal period. The authors also, however, showed that the incidence of BA was negatively correlated with the country’s gross domestic product, so further studies will be important to determine whether the rotavirus vaccine or overall improvement in socioeconomic status was truly responsible for the decreased incidence of BA. The greatest body of research entails investigations of CMV at the time of diagnosis of BA. A higher prevalence of CMV antibodies in the mothers of infants with BA, higher serum CMVimmunoglobulin (Ig) M levels, and greater amounts of Ig deposits on the canalicular membrane of the hepatocytes in infants with BA have been reported (11). Strong evidence for a perinatal CMV infection associated with BA was described by Xu et al (12). Liver tissue obtained at the time of portoenterostomy on 85 infants with BA was analyzed by real-time polymerase chain reaction (PCR) for the presence of multiple viruses, and the majority (60%) was positive for CMV. Studies confirming the presence of CMV in PCR-positive liver samples were performed with immunocytochemical detection of CMV-pp65 antigens within liver bile duct epithelia and hepatocytes. Brindley et al (13) analyzed the liver memory T-cell response to a variety of viruses from infants with BA
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JPGN
at the time of diagnosis. The majority (56%) of patients with BA had significant increase in interferon (IFN)-g-producing liver T cells in response to CMV homogenate and CMV-pp65 antigen, suggesting that perinatal CMV infection had occurred. An interesting observation from the past virus studies is the ability of all of the 2 abovementioned viruses to infect and damage bile duct epithelia, lending support to a primary cholangiotropic viral infection as the initiating event in the pathogenesis of BA. In the last decade, scientific investigations have concentrated on the role of the adaptive immune system in bile duct injury in BA. A plausible theory that could explain the progression of biliary tract injury that predominates in BA is that of an autoimmune-mediated attack on biliary epithelia. After the initial viral insult to the biliary tree, the damaged bile duct epithelial cells may express previously sequestered ‘‘self’’ antigens that are recognized as foreign and elicit autoreactive TH1-cell-mediated inflammation and B-cell production of autoantibodies directed at duct epithelia. The predominant cellular immune response in BA encompasses activated CD4þ and CD8þ T cells within portal tracts that produce TH1 cytokines (interleukin-2 and IFN-g) and macrophages secreting tumor necrosis factor (TNF)-a (14,15). These lymphocytes have been found invading between bile duct epithelia, leading to degeneration of intrahepatic bile ducts. The strongest evidence for the autoimmune theory has been gained from mouse studies, in which autoreactive T cells and autoantibodies targeting bile duct epithelia have been identified (16–18). In humans, only circumstantial evidence exists for the role of autoimmunity in BA pathogenesis. Human leukocyte antigen (HLA) associations with BA have been reported with conflicting results. European and American studies of HLA predominance in BA found no significant differences compared with controls (19). In contrast, a Japanese study found significant association between BA and HLA-DR2 as well as a linkage disequilibrium with a high frequency of HLA-A24-B52-DR2 (20). The presence of autoantibodies and periductal immune deposits, suggesting a humoral autoimmune response, has also been described (11,17). The potential contribution of adaptive immune and autoimmune responses to bile duct injury in BA is represented in Figure 1. Research pertaining to deciphering the etiology of BA is robust and is focusing on genetic and immunologic links to the pathogenesis of disease.
OPN OPN
Key Virus Ag Bile duct epithelial Ag Macrophage
IL-12
Apoptotic bile duct epithelia
CD4+
Volume 61, Number 2, August 2015
Another area of research in BA has focused on the extensive fibrosis that occurs with this disease. Portal bridging fibrosis and even cirrhosis are commonly seen at the time of diagnosis of BA, and the degree of fibrosis correlates with outcome. One mechanism of fibrogenesis that could explain the severity of fibrosis entails the Hedgehog (Hh) pathway. The Hh pathway is important in tissue remodeling and Hh activation, with subsequent epithelial-mesenchymal transition (EMT) of cells involved in fibrogenesis related to hepatobiliary diseases. It has been previously shown in BA that biliary epithelia undergo EMT, promoting fibrosis. BA patient livers demonstrated significant upregulation of Hh ligand within intra- and extrahepatic ductular cells, as well as increased expression of Hh target genes. In addition, immature ductular cells within BA livers showed a profibrotic mesenchymal phenotype that was Hh responsive (21). The authors concluded that excessive Hh activation impedes ductular morphogenesis and enhances fibrogenesis by promoting accumulation of immature ductular cells with a mesenchymal phenotype. Most searches for defects at the chromosomal level have produced negative results; however, recently, a genetic link to the Hh pathway has been described. Identified deletions at chromosome 2q37.3 in patients with BA results in deletion of 1 copy of glypican-1 (GPC1), a heparan sulfate proteoglycan that regulates Hh signaling and inflammation. Liver tissues of patients with BA had reduced levels of apical GPC1 in cholangiocytes compared with controls. Cui et al (22) used a gpc1 knockdown zebrafish model to show developmental biliary defects and gallbladder atresia. Exposure of the gpc1 morphants to cyclopamine, an Hh antagonist, rescued the gpc1-knockdown phenotype. These studies not only identify GPC1 as a risk gene for BA but also offer mechanistic insight into the potential pathogenic role of GPC1 and Hh in BA. Another potential mechanism associated with activation of fibrosis involves the recent novel finding of prominin-1 (PROM-1) expressing stem cells adjacent to ductular reactions within the portal tracts of patients with BA and in murine BA (23). The PROM-1þ cells expressed both markers of epithelial and mesenchymal cells, produced collagen, and correlated with portal fibrosis and bilirubin levels. In addition, expansion of PROM-1þ cells was associated with the activation of fibroblast growth factor and transforming growth factor-b providing potential mechanisms of action of
Clonal expansion CXCR3
(•)
Dendritic cell
TNF-α iNOS
γ N-
IF
CD4+ (TH1)
CD8+
Perforin granzyme IFN-γ
CD8+
T-cell activation B cell
FIGURE 1. Contribution of adaptive immunity in the pathogenesis of BA. This figure summarizes human investigations pertaining to the role of adaptive immunity in bile duct injury and offers a theory on the role of autoimmune responses in the pathogenesis of disease. Bile duct injury is initiated by virus infection (ie, CMV) with clonal expansion of virus-specific CD4þ T cells. IL-12, osteopontin, and CXCR3 chemokine receptors all promote TH1 cell differentiation. These TH1 cells activate effector cells including macrophages (via CD4þ IFN-g stimulation) with subsequent generation of TNF-a and inducible nitric oxide synthase, cytotoxic CD8þ T cells that directly invade epithelia and release cytotoxic molecules, and B cells that mature into antibody-producing plasma cells with release of IgM and IgG targeting bile duct epithelia. Over time, proteins from apoptotic bile duct epithelia may be presented and seen as ‘‘foreign,’’ eliciting autoreactive T-cell-mediated inflammation that perpetuates the bile duct injury. BA ¼ biliary atresia; CMV ¼ cytomegalovirus; IFN ¼ interferon; Ig ¼ immunoglobulin; IL ¼ interleukin; TNF ¼ tumor necrosis factor.
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Biliary Atresia: Clinical Lessons Learned
fibrogenesis. These exciting studies of fibrogenesis in BA provide insight into potential future targets of antifibrotic therapy.
CLINICAL FEATURES There are 3 distinct clinical patterns of BA (24). The majority of infants with BA (84%) have the perinatal or acquired form of BA without associated major malformation. These babies are asymptomatic, often jaundice-free at birth and appear to be thriving until 2 to 6 weeks of life when they have persistent jaundice, acholic stools, dark urine, and hepatomegaly. On laboratory evaluation, they have elevated total and direct bilirubin levels, elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and elevated g-glutamyl transpeptidase (GGTP) levels >200 IU/L. Ascites and splenomegaly, results of portal hypertension, are late findings and not usually seen on initial presentation. In a second group (6%), infants have at least 1 major malformation but no laterality defects. Finally, in 10% of patients with BA, the infants are syndromic and have 1 or more laterality defects. These infants appear jaundiced from birth and do not have an asymptomatic period.
EVALUATION OF NEONATAL CHOLESTASIS Any infant who remains jaundiced at 2 weeks of age needs to be evaluated for cholestasis, with fractionation of the bilirubin into a conjugated (direct) and unconjugated (indirect) portion. Conjugated hyperbilirubinemia associated with cholestasis is defined as a direct/conjugated bilirubin of >1 to 2 mg/dL and >20% of the total bilirubin concentration. One must have a high suspicion for
hepatobiliary dysfunction and proceed with further workup in the setting of cholestasis, acholic stools, hepatomegaly, or splenomegaly. In an infant with cholestasis, further evaluation should be conducted with a sense of urgency to identify those causes that are amenable to medical and surgical correction (Fig. 2). Outcome of infants with BA is directly correlated to the timing of diagnosis and HPE. A comprehensive metabolic panel will likely show a total bilirubin of 5 to 12 mg/dL and an ALT and AST in the 100 to 200 U/ L, findings not unique to BA. It is rare for an infant with BA to have a GGTP level 1 or 2 patients of BA during their careers and as a result may dismiss jaundice at 2 weeks of age to be the result of breast-feeding without considering BA. Second, in the United States health care system, infants are routinely seen at 2 weeks of age and not again until 8 weeks of age. Without a routine 4-week visit, the crucial diagnostic period to identify and intervene for BA is often missed. Third, there are no pathognomonic lab findings that distinguish BA from other causes of neonatal cholestasis. Finally, at this time, there are no universal screening programs in place in the United States to help identify infants with BA within the first month of life.
KASAI HEPATOPORTOENTEROSTOMY In 1959, Kasai developed the HPE procedure in which a Roux-en-Y loop of jejunum is anastomosed to the hilum of the liver, thus creating a new conduit for biliary drainage. The HPE has transformed BA from a universally fatal disease in early childhood to one in which restoration of bile flow and normalization of bilirubin may result in long-term survival with the native liver. HPE reestablishes short-term bile drainage in approximately twothirds of patients. Several factors have been reported to affect outcome after HPE. Nonmodifiable risk factors that predict poor outcome include obstruction proximal to the common bile duct, bridging fibrosis at the time of HPE, and polysplenia syndrome. www.jpgn.org
Prognostic factors related to care include age at Kasai, experience of the center in managing BA, and accessibility to liver transplant. Increased age at HPE has a progressive and lasting detrimental effect on outcome. If the HPE is performed within the first 60 days of life, 70% to 80% of patients show bile drainage. If performed between 60 and 90 days, 40% to 50% of patients show drainage; after 90 days of life only 25% of patients show drainage; if performed later than 120 days of life 5 HPEs per year (61.3% vs 13.7% and 91.2% vs 75%) (31). This has led to centralization of care in many European countries. These modifiable prognostic factors offer areas in which quality initiatives could drastically improve disease outcome. Success of the HPE can best be judged by restoration of bile flow and clearance of jaundice. By 3 months after HPE, a clear difference in the 2-year transplant-free survival can be seen between those children with total bilirubin 6 mg/dL (84% vs 16%; P < 0.0001) (28). If jaundice clears successfully by 3 months after HPE, the 10-year transplant-free survival rate ranges from 75% to 90%; conversely, if jaundice persists after HPE, the 3-year transplant-free survival rate is only 20%. There is no standard treatment regimen for patients with BA who are post-HPE. Regimens may include ursodeoxycholic acid, antibiotic prophylaxis against cholangitis, and a fatsoluble vitamin preparation. Steroids were previously believed to improve clinical outcomes secondary to their choleretic, antiinflammatory, and immunomodulatory properties. In the recent multicenter trial in which 140 infants with BA from 14 centers across the United States were, however, randomized to receive
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either high-dose steroids following HPE or placebo; there was no difference between groups in bile drainage at 6 months or 2 years post-HPE and no difference in transplant-free survival at 2 years (32).
COMPLICATIONS OF BA
Volume 61, Number 2, August 2015
D, E, and/or K should be administered as needed (37) (Table 2). Nutrition remains a modifiable risk for outcome in BA. Prospective multicenter trials are needed to better understand the specific effects of different nutritional interventions on improving outcomes.
Cholangitis
Failure to Thrive and Fat-Soluble Vitamin Deficiencies Failure to thrive is a significant problem in patients with BA. In the Studies of Pediatric Liver Transplant registry, 755 patients with BA who underwent liver transplantation were analyzed, and 40% had growth failure (33). This growth failure was found to be an independent risk factor for pretransplant mortality, posttransplant mortality, longer hospital stays, and graft failure (33). The pathogenesis of growth failure and malnutrition in infants with BA is multifactorial. Infants with BA have poor bile flow resulting in reduced delivery of bile acids to the small intestine, decreased mixed micelle formation, and subsequent fat and fat-soluble vitamin malabsorption. In addition, infants with BA often have poor appetite and increased energy expenditure with a resting energy expenditure 29% greater than age-matched controls (34). One must be careful not to be reassured by a normal weight and height in an infant with BA. Weight can be falsely elevated secondary to ascites and organomegaly, and diminished height can be a late finding of poor nutrition. To get an accurate assessment of true nutritional status, one must check anthropometrics including triceps skin fold thickness and midarm circumference. Growth failure after HPE is associated with increased risk of transplantation or death by 24 months of age (35). To that end, the importance of growth is acknowledged in the present pediatric end-stage liver disease allocation system in the United States in which children receive higher scores when they have growth failure (36). Optimization of nutrition is a crucial part of the care of infants with BA. Infants should be started on a formula containing medium-chain triglycerides, which can be absorbed independently of bile salts. Caloric intake should be approximately 125% of the recommended dietary allowance based on ideal body weight. If patients are unable to ingest the needed calories orally, they should be started on nasogastric tube feeds. Fat-soluble vitamin levels need to be carefully monitored and treated because deficiencies can result in rickets, bone fractures, coagulopathy, cerebellar ataxia, and impaired vision. All of the infants with cholestasis should be on a fat-soluble vitamin supplement that takes advantage of the ability of tocopherol polyethylene glycol succinate (TPGS) to be absorbed independently of bile salts. A recent study showed that fat-soluble vitamin deficiencies often persist despite initiation of a TPGS containing liquid multiple fat-soluble vitamin preparation; therefore, additional individual vitamin supplementation with vitamin A,
Forty percent to 60% of infants with BA develop cholangitis in the first 2 years following HPE (28). Because cholangitis is presumed to be an ascending infection, it only occurs in those children who have some degree of bile flow. Symptoms may be nonspecific, and cholangitis should be considered whenever a child who is post-HPE presents with fever, vomiting, decreased oral intake, pale stools, worsening jaundice, right upper quadrant abdominal or shoulder pain, or worsening laboratory values (rising ALT, AST, GGTP, bilirubin, or white blood cell count). A blood culture may be positive and common infectious agents include Escherichia coli, Enterobacter, or Klebsiella; however,
