Journal of Autoimmunity 73 (2016) 1e9

Contents lists available at ScienceDirect

Journal of Autoimmunity
journal homepage: www.elsevier.com/locate/jautimm

Review article

Biliary atresia: A comprehensive review

Bhanumathi Lakshminarayanan, Mark Davenport*
Department of Paediatric Surgery, King’s College Hospital, London, UK

a r t i c l e i n f o a b s t r a c t

Article history: Biliary atresia presents as an obliterative cholangiopathy with neonatal jaundice and pale stools. The
Received 12 June 2016 disease exhibits aetiological heterogeneity with a multiplicity of potential causative factors, both
Accepted 13 June 2016 developmental and environmental. A number of clinical variants making up a minority of all cases can be
Available online 23 June 2016
defined relatively precisely which match suggested aetiology better although in most it still remains
speculative. These include the syndromic form (BASM), the cystic form and those associated with CMV
Keywords:
IgM antibodies.
Biliary atresia
We review not only the clinical evidence for a developmental or an immune-mediated aetiology
Inflammation
Kasai portoenterostomy
perhaps triggered by perinatal viral exposure but also several other recently suggested concepts such as
Immune response microchimerism, gene susceptibility and environmental toxins.
Microchimerism © 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Historical context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Clinical context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3.1. Biliary Atresia Splenic Malformation (BASM) syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3.2. Cystic biliary atresia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3.3. CMV-IgM þ ve associated biliary atresia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3.4. Isolated BA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
4. Epidemiological context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
5. The developmental context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
6. Role of the virus in context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
7. The inflammatory response in context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
8. The regulatory T cell window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
9. The humoral response in context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
10. Sidelines, blind alleys or intriguing possibility? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
10.1. Maternal microchimerism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
10.2. Biliatresone & Burrinjuck (Fig. 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
10.3. Susceptibility genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
11. Diagnosis and management in practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
11.1. Key variables in outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
End note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Abbreviations: CMV, cytomegalovirus; T cells, T-regs regulatory; RT-PCR,

reverse-transcriptase polymerase chain reaction; IL, interleukin; FISH, fluorescent
in situ hybridization; ERCP, endoscopic retrograde cholangiopancreatography; MCT,
medium chain triglycerides. 1. Introduction
* Corresponding author. Dept. of Paediatric Surgery, King’s College Hospital,
Denmark Hill, London, SE5 9RS, UK. Biliary atresia (BA) comes to us as a peculiar, possibly unique,
E-mail address: Markdav2@ntlworld.com (M. Davenport).

http://dx.doi.org/10.1016/j.jaut.2016.06.005
0896-8411/© 2016 Elsevier Ltd. All rights reserved.
2 B. Lakshminarayanan, M. Davenport / Journal of Autoimmunity 73 (2016) 1e9
disease presenting in infancy with established obliteration of their
biliary tree. The condition is rare and one of its peculiarities is that it
may have many parents, i.e. it may only be an end-stage phenotype
with a multiplicity of different aetiological mechanisms. There is in
some of these affected infants a pronounced inflammatory milieu
in which there are hallmarks of immune activation and as a result
organ damage. Much work has been done on trying to define the
nature of the offending trigger and its immunological consequence.
Observed evidence of what is happening inside their liver and
biliary tract and speculation as to its cause will form the backbone
of this article.
2. Historical context
The earliest reference to this condition appeared in a textbook
published in 1817 by Dr John Burns from the University of Glasgow,
who mentions this condition in passing as an “incurable state of the
biliary apparatus” [1,2]. A case report and review was published as a
thesis by John Thomson an Edinburgh physician in 1891 [3]. At the
time these were simply considered as a congenital obliteration of
the bile ducts with his example showing two small bile-containing
cysts in the porta hepatis unconnected to an otherwise normal
gallbladder and common bile duct.
Some notable surgical successes were reported by William Ladd
working in Boston, USA during the 1920’s but only because he
found proximal bile-containing ducts to anastomose with adjacent
intestinal loops [4]. Increasing surgical experience however became
more pessimistic as it was realised that the majority did not have
anything resembling proximal bile ducts, rather a solid extrahe-
patic biliary tree. The phrase “uncorrectable biliary atresia”
conveyed this rather brutal truth and there were few if any
survivors.
Morio Kasai described a more radical approach to surgery in the
1950s involving simple transection of an apparently solid proximal
biliary tract with Roux loop reconstruction anyway [5]. This did
lead, somewhat amazingly, to bile flow and resolution of jaundice
in some, due to the transection of microscopic bile ductules and has
remained the surgical procedure of choice in most to the present
day. The alternative is liver transplantation which although pio-
neered in the 1960s [6] by Thomas Starzl and a group in Denver, CO.
only truly became realistic in the 1980s. Currently we regard the
two options as complementary with an initial attempt at salvage of
the native liver, followed by liver transplant if this demonstrably
fails [7].
3. Clinical context
It is clear that BA is not a single disease with a defined aetiology
and predictable response to treatment. Rather it seems to a
phenotype characterised by extrahepatic biliary obliteration or
absence connected to an array of abnormal, intrahepatic ductules
often lacking the usual branching pattern of mature ducts. We have
tried to define three specific entities which seem to share enough
similarities to be considered homogenous.
3.1. Biliary Atresia Splenic Malformation (BASM) syndrome
These infants (usually female) are characterised by a constella-
tion of unusual anomalies including polysplenia (sometimes
asplenia), vascular anomalies (preduodenal portal vein, absence of
the cava), situs inversus and cardiac anomalies. There appears to be
a primary failure of extrahepatic bile duct development e the
gallbladder is invariably atrophic and the CBD absent. This devel-
opmental failure probably occurs early from 20 to 40 days gestation
to account for the other anomalies and maternal diabetes appears
to predispose [8,9].
There are other congenital anomalies such as oesophageal
atresia, jejunal atresia and the cat eye syndrome [10] which do
appear as rare associations but these appear to be different to those
within the BASM grouping.
3.2. Cystic biliary atresia
This can be defined as cystic change in an otherwise obliterated
biliary tract and is seen in about 5e10% of most large series [11]. The
cyst itself may contain bile (~20%) implying onset after establish-
ment of continuity between intra- and extra-hepatic bile ducts
(10e12 weeks gestation), or mucus [11]. Around half of these are
picked up antenatally on the maternal ultrasound scan and they
have a good prognosis post Kasai surgery.
3.3. CMV-IgM þ ve associated biliary atresia
Recently we have defined on a group of infants with BA who
have IgM antibodies to CMV [12]. These made up 10% of our clinical
series and were more commonly seen infants from non-caucasian
parents. Clinically they were older at diagnosis and came to Kasai
surgery later. Biochemically they had higher bilirubin and AST
levels, with larger spleens as measured on ultrasound than com-
parable IgM-ve BA infants, even when corrected for their older age.
Their histological appearance of the extrahepatic duct was more
inflammatory with prominent nodes. This inflammatory appear-
ance carried over to the histological appearance within the liver
with an obvious mononuclear cell infiltrate consisting of largely
CD4þ Th1þ T cells [13]. Interestingly none of the liver biopsies in
our clinical series stained positive for actual presence of CMV. Of all
the clinical groups we have defined these appeared to have the
worse response to Kasai surgery and also appeared susceptible to
death during infancy.
Of all the clinical groups described this is the group that appears
to fulfil most of the requirements to support an immune-
destructive pathogenesis following viral triggering.
3.4. Isolated BA
In the absence of any distinguishing features this forms the
largest (70e80%) clinical grouping. Real evidence of what has
caused BA in this group is minimal and remains open to specula-
tion. Such infants could equally well have a developmental path-
ogenesis or alternatively a secondary obliterative cholangiopathy.
Some recent evidence towards the former was presented by a re-
view of early liver biochemistry (day 1 and day 2 of life) in about
half of a series of infants from Texas later shown to have BA [14].
This showed that all had elevated conjugated bilirubin by 24 h
implying biliary obstruction at the time of birth.
4. Epidemiological context
There is marked variation in incidence of BA ranging from about
1 in 5e10,000 live births in Taiwan [15,16] and Japan [17] to about 1
in 15e20,000 in mainland Europe [18], England & Wales [19] and
North America [20]. Furthermore there is marked geographical
variation among the specific variants mentioned above. The inci-
dence of BASM varies widely, and is rarely reported in Asian series
but accounts for about 10% of European and North American series.
The incidence of CMV IgM þ ve BA also varies from 10 to 20% in
European series [12,21,22] but up to half in series from China [23].
Some small studies have suggested a seasonal variability [24]
although not larger national studies [19].
 B. Lakshminarayanan, M. Davenport / Journal of Autoimmunity 73 (2016) 1e9
5. The developmental context
Bile ducts are derived in two distinct ways with a different
schedule.
Extrahepatic bile duct are the first visible structure in the em-
bryo and arise from an outpouching of foregut endoderm starting at
about 20 days of gestation and essentially complete with gall-
bladder, lumen and a funnel-shaped proximal segment in intimate
contact with the liver anlage at about 45 days. The cholangiocytes
within appear to arise from foregut endoderm. By contrast there is
no sign of an intrahepatic biliary network until at least 49 days. The
liver anlage up until this point is largely composed of hepatoblasts
and primitive haematopoietic cells. Biliary epithelial cells
(expressing SOX9 and CK19) differentiate from hepatoblasts and
begin to assume a uni-then bi-laminar appearance around
ingrowing portal venules e the ductal plate [25]. From around 8
week gestation and by a process of selection and deletion the bile
ductules form a radiating network from porta hepatis to periphery
with the smallest biliary units in contact with the canalicular aspect
of the hepatocytes. The porta hepatis is the interface between intra-
and extra-hepatic systems and successful union must happen to
allow bile to reach the developing intestine timed to begin at
around 11e12 weeks.
Of the clinical variants described above those with BASM
syndrome are most obviously developmental in origin. Their
associated features all have their origins in early embryonic life
from 20 to 50 days gestation with some earlier such as deter-
mination of abdominal situs to some at the end of this phase such
as the development of the intrahepatic vena cava. Although a
genetic cause appeared likely this hasn’t come to fruition as yet.
CFC-1 missense mutations have been shown in some but have
not been exclusive [26]. In our clinical series many infants with
BASM had abnormal “events” in the 1st trimester, were from
in vitro fertilisation pregnancies or who had mothers who were
diabetics. Some also had overlap anomalies such as sacral agen-
esis and double-outlet right ventricle - markers of a diabetic
embryopathy.
Obviously infants with cystic biliary atresia are also develop-
mental in origin e at least half can be detected on a maternal ul-
trasound scan. Whatever is causing the obstruction must happen
from at least 11e12 weeks as in about 20% bile can be shown to be
within the cyst, implying a degree of continuity down to, usually,
the common bile duct [11].
6. Role of the virus in context
The American paediatrician Benjamin Landing suggested that
there might be a common factor in the aetiology of choledochal
cyst, neonatal hepatitis and biliary atresia in the 1970s invoking the
influence of a hepatotropic virus capable of causing cholangiolar
and actual structural bile duct damage (“infantile obstructive
cholangiopathy”) [27]. Initially, supporting evidence for this was
provided by Rachel Moreki et al. showing higher titres of antibodies
against REOvirus type 3 compared to jaundiced infants without BA
[28]. In a later study, Tyler et al. detected REOvirus RNA in liver and/
or biliary tissues by RT-PCT in 55% of infants with BA and almost
80% of patients with choledochal cysts [29]. By complete contrast,
Saito et al. could find no evidence of REOvirus RNA in over 60 in-
fants with BA, choledochal cyst and a variety of other hepatobiliary
pathologies using similar technology [30,31].
Other groups have looked for evidence of other candidate vi-
ruses, with initially group C rotavirus looking promising. Marie
Riepenhoff-Talty in a key paper published in 1996 identified, again
using RT-PCR, rotavirus DNA in half of 20 liver samples from infants
with BA or choledochal cyst [32]. However, more recently a large-
3
scale multicentre study from North America looked at group A
and C rotavirus antibodies in cholestatic infants, < 6 months old,
amongst which were 40 infants with BA [33]. The incidence of
group A rotavirus IgM þ ve positivity did not differ between infants
with (10%) or without BA (18%). Further group C rotavirus IgM was
never seen in BA. It is interesting to note that despite such un-
promising clinical evidence the rhesus rotavirus has become the
mainstay of inducing a BAelike condition in the mouse model [34].
Indeed an entire edifice of inflammatory response and key cellular
dynamics has subsequently been erected upon these rather
insubstantial clinical foundations.
Perhaps of all the candidates, cytomegalovirus, has attracted the
greatest clinical attention. This was first identified in a Swedish
study with CMV DNA being found in half of their BA infants,
exciting an IgM response evident on their hepatocyte canalicular
membranes [21,35]. Brindley et al. identified a CMV-specific liver T
cell response in just over half of a cohort of American BA infants and
a positive correlation of this particular laboratory finding with
plasma CMV IgM levels [36]. They also related this to lower levels of
circulating Tregs (see later). This appears to be a particularly high
incidence compared with European estimates of exposure such as
our own at ~10% and 11% in Germany [12, 22] and more like that
seen in Chinese series. For instance Xu et al. [23] identified viral
CMV-pp65 in 51 out of 85 (60%) BA livers, far higher than other
comparable viruses (e.g. REOvirus).
There is only one study which has looked at a panel of possible
hepatotropic viruses identifying viral genomic material (REOvirus
33%; cytomegalovirus 11%; adenovirus 1% and enterovirus 1.5%) in
less than half of a cohort of 74 infants. Indeed some infants had
multiple viruses making relevance perhaps less likely [22].
The weakness of the viral hypothesis has to be that so many
viruses or at least parts of their genome can be found later in infants
later shown to have BA. A recent systematic review identified 19
studies conducted on 16 different kinds of viruses using PCR
(REOvirus, n ¼ 5), rotavirus (n ¼ 3), CMV (n ¼ 10), Epstein-Barr
virus (n ¼ 5) and parvovirus B19 (n ¼ 4) [37].
The question, so far unanswered is how does transient viral
exposure initiate such a powerful inflammatory response that bile
ducts become obliterated, bile flow stops and liver fibrosis and
cirrhosis becomes inevitable. At the time of the Kasai operation at
least there is rarely any physical evidence for active viruses in the
form of particles or inclusion bodies etc. and the range of possible
viruses surely casts a degree of doubt upon a primary role sug-
gesting that their presence may be co-incidental e an innocent
bystander as it were. The faecal flora of infants with BA is certainly
different [38] and absence of bile salts may allow selection of a
whole host of enteroviruses.
7. The inflammatory response in context
Early studies published during the 1990s identified abnormal
expression of ICAM-1 and less commonly VCAM-1 in the livers of 7
infants with BA [39] suggesting the triggering phase of an inflam-
matory reaction was occurring. We followed this using a larger
cohort of 28 infants and confirmed significant abnormal expression
of ICAM-1, VCAM and E-selectin on sinusoidal and biliary epithe-
lium in 50%, 25% and 10% respectively [40]. We also noted that there
was abnormal class II antigen HLA-DR expression particularly on
Kupffer cells and proliferating bile ducts. More recently abnormal
P-selectin (CD62P) expression has been shown in platelets,
sinusoidal and biliary endothelium in about 70% of infants with
BA [41].
We measured the circulating fraction of soluble cellular adhe-
sion molecules in a large series of 61 infants at the time of Kasai
showing significantly elevated levels of s-ICAM and s-VCAM but not
4 B. Lakshminarayanan, M. Davenport / Journal of Autoimmunity 73 (2016) 1e9

e-selectin [42]. Furthermore there was modest correlation of

increasing age at surgery with levels of sICAM-1 (r ¼ 0.33, P ¼ 0.01),
but not sVCAM or e-selectin. However, levels didn’t really seem to
matter in general when we looked at outcome. Only high sVCAM
levels approached prognostic significance.
We investigated the characteristics of this circulating in-
flammatory milieu beyond the Kasai operation by serial esti-
mation of not only the CAMs measured in the previous study but
a range of soluble cytokines. These included proinflammatory
mediators such as Th1 markers (IL-2 and Interferon-g), Th2
markers (IL-4 and IL-10) and macrophage markers (TNF-a and
IL-18) as representative of the inflammatory process [43]. This
showed a fairly non-polarized increase in CAMs and cytokines
generally but with 100-fold increases in IL-2, TNF-a and IL-18 in
particular. What was interesting was just how long this increase
lasted even in those who were clearing their jaundice. We also
Fig. 2. Quantification Study of Th-1 T cells in Variants of Biliary Atresia. Th-1 (Tbetþ)
identified that sICAM-1 levels at 1 month post-operatively was a
subset quantification according to perceived aetiology: CMV IgM þ ve biliary atresia
good prognostic test for need for transplantation at 1 year (n ¼ 9); BASM (n ¼ 9); isolated biliary atresia (n ¼ 19). [Reproduced with permission
(cut-off of 1779 ng/ml gave a 92% specificity and 87% from #13].
sensitivity).
Abnormal CAM expression, activation of antigen-presenting
cells such as dendritic cells and Kupffer cells by expression of
costimulatory factors B7-1, B7-2 and CD40 [44] appear to be the
earliest phase of response and are followed sinusoidal infiltration
by activated mononuclear cells. Initially using immunohistochem-
ical staining we identified a pronounced infiltrate in about 30% of
infants with otherwise isolated BA which and appeared to be pre-
dominantly NK cells (CD56þ) and CD4þ T cells [40]. Later work in
the murine model has suggested that NK cells might be immature
in the newborn and are not able to kill virus-infected chol-
angiocytes [45].
Later work by Cara Mack et al. [46,47] showed that the T cell
infiltrate is oligoclonal in nature, and therefore suggestive of a
specific provocation. We have quantified infiltrating T cell subsets
in BA livers and showed that there is a clear Th-1 and Th-17 pre-
dominant profile for BA in general, compared to cholestatic controls
[13]. Th-17 T cells appear to play a significant role in mucosal host
defence and, at least in adults, have been implicated in some
Fig. 3. Effect of Degree of Th-17 Cellular Infiltration on Outcome. Clearance of jaundice
autoimmune cholangiopathies such as primary biliary cirrhosis. (bilirubin <20 mmol/L) in infants with biliary atresia (n ¼ 37) according to number of
Such Th17 þ cells, can be shown to accumulate at the bile duct cells/hpf. [Reproduced with permission from #13].
epithelial interface and infiltration of this subset in portal tracts
also appears to be a characteristic of BA (Fig. 1) and may have an
effect on prognosis (Fig. 2) [48]. More specifically, we observed that 8. The regulatory T cell window
those with CMV IgM þ ve BA have a population predominantly of
Th-1 cells (Fig. 3). Overall there were very low numbers of both Th- The current standard animal model of BA uses a particular strain
2 and T-regs cells [13]. of mice pups (Balb/c) and a particular strain of rhesus rotavirus
Other clinical studies have suggested that CD8þ T cells are the inoculated intraperitoneally within 12 h of birth. This produces an
predominant cell line in the infiltrate [49] although whether they inflammatory reaction causing BA-like lesions in the extrahepatic
are appropriately cytotoxic has been questioned [50]. ducts and intrahepatic ductular obliteration [34,51].

Fig. 1. Th-17 infiltration in Biliary Atresia. Th-17 cells heavily concentrated in the portal tracts of a liver from 54 day old infant with isolated biliary atresia. A (10) B (20) H&E with
Avidin-biotin immunoperoxidase technique used to detect Il-17.
 B. Lakshminarayanan, M. Davenport / Journal of Autoimmunity 73 (2016) 1e9
Timing is critical in this model suggesting there is only a brief
window of viral opportunity just after the time of birth. One hy-
pothesis for this immaturity is that there is an absence of regulatory
T cells (T-regs) which are believed essential for suppressing and
inhibiting NK cell expansion [52,53]. In the murine model, T-regs
modified the expression of costimulatory region CD86 on dendritic
cells to reduce expansion of CD8þ cells, but when infected with
rhesus rotavirus, the region upregulated to cause BA [54].
The deficiency in T-regs seems to be more pronounced in CMV
compared to other viral infection [36]. Low dose CMV infection in
the absence of Tregs produced marked intra and extrahepatic
biliary inflammation in one mouse model [55]. This was associated
with increased infiltrate of CD8þ T cells and humoral response
including generation of autoantibodies such as a-enolase.
9. The humoral response in context
While much of the clinical and certainly experimental studies
have concentrated on the T cell response it is appreciated that there
is also a humoral response as well. Furthermore, B lymphocytes can
act as antigen presenting cells that on their own activate T cells and
the Th-1 pathway [56].
a-enolase is an enzyme involved in glycolysis and is ubiqui-
tously expressed in a variety of cells, including biliary epithelial
cells and hepatocytes. Anti-a-enolase antibodies have been detec-
ted in about 30% of those with auto-immune liver diseases (e.g.
autoimmune hepatitis, primary biliary cirrhosis). Lu et al. [57]
identified both anti-a-enolase IgG and IgM antibodies in sera
from both infants with BA and older children with their native
livers. Levels were higher as compared to certain cholestatic con-
trols (e.g. cystic fibrosis, parenteral nutrition associated) but lower
than children with autoimmune hepatitis.
Even though we can associate the development of BA with viral
infection, not all of them with the infection develop the disease.
This could be due to underlying genetic factors. The CD4þ T cells
from infants thought to have a virus-induced immune reaction
have hypomethylated DNA with the expression for the enzyme
DNA methyltransferase being very low [58]. But in the promoter
CD11a region of CD4þ T cells, there is hypermethylation which leads
to decreased expression of CD11a and promotes BA [59].
10. Sidelines, blind alleys or intriguing possibility?
10.1. Maternal microchimerism
Maternal microchimerism (Fig. 4) is the intriguing concept
whereby there is transplacental passage of immune active cells into
the fetal circulation and localizing within liver sinusoids to initiate
an immune reaction analogous to a graft-versus-host reaction.
Suskind et al. [60] first showed that using fluorescent in situ hy-
bridization (FISH) there was a consistent presence of double X
chromosomes in male infant livers with BA but not in male livers
with neonatal hepatitis used here as controls (Fig. 1). They esti-
mated that there were 2e4 maternal cells per slide and further
quantification showed that in native livers at the time of transplant
there was a range of 1e142 copies of maternal DNA detected per
25,000 copies of patients’ DNA. The maternal cells appear to be
CD8þ CD45þ and therefore stem cells with the potential to differ-
entiate into progenitor lymphocytes, effector lymphocytes or
differentiated biliary epithelial cells [61]. However, it is still not
clear what precise role they might have e possibly a target for
immune attack or possibly functioning as actual immune effector
cells mediating the immune reaction [61,62].
One side-effect of this phenomenon is that it might influence
the degree of tolerance of a donor liver graft [63]. Sanada et al. [64]
5
compared the acute cellular rejection rate after living donor
transplant from either the mother or father. The donor mother to
daughter recipient had the lowest rejection rate compared to donor
father to daughter recipient.
10.2. Biliatresone & Burrinjuck (Fig. 5)
Biliary atresia in animals is exceptional outside of the laboratory
and is poorly described. However an outbreak of BA occurred in
New South Wales, Australia in 1964 [65]. The summer before had
been hot and drought-like conditions around a dam in Burrinjuck
had exposed the foreshore due to falling water levels. On this new
ground there had been overgrowth of weeds of the genus dys-
phania glomulifera (colloquially known as red crumbweed or
pigweed). Sheep were allowed to graze on the overgrowth and
subsequently numerous lambs born during the next lambing sea-
son developed jaundice and died with their autopsies showing
biliary atresia. Remarkably this also happened to calves, whose
mothers had been allowed to graze as well and also recurred in
subsequent seasons where the same factors re-occurred. While the
connection between the plants, presumably acting as an in eutero
toxin to the developing bile ducts, had been known for a long time,
the actual toxin remained obscure [65]. Recently, four potentially
toxic compounds (isoflavonoids) were isolated from extracts of
dysphania spp. and tested in a Zebrafish system designed to model
early bile duct development [66]. One of these, subsequently
named Bilatresone, caused obliteration of fully developed gall-
bladders and bile ducts in the Zebrafish and was also show to set up
bile duct inflammation in a mouse model.
10.3. Susceptibility genes
There may be a genetic susceptibility to develop BA in humans,
outside of the peculiar genetic straitjacket of the murine model.
Largescale genetic screening studies in Chinese populations
have shown that there are two particular single nucleotide poly-
morphisms (SNPs) (rs17095355 C/T and rs10509906 G/C) in the
ADD3 gene, located in the 10q24.2 region which appear to be more
frequent in infants with BA (n ¼ 134) versus controls [67]. Further
genetic probing of the region was performed in 171 Caucasian in-
fants with BA confirming the association outside of China [68] and
identified the strongest signal at rs7099604 in intron 1 of the ADD3
gene.
11. Diagnosis and management in practice
BA is progressive and presents as persisting jaundice, pale stools
and dark urine in an otherwise healthy infant. There may be other
uncommon modes of presentation such a vitamin K induced
bleeding tendency but actual signs of chronic liver disease such as
portal hypertension, ascites and splenomegaly take time to develop
(>3 months).
Key diagnostic tests include ultrasonography [69], biochemical
liver function tests, viral serology, and, at least in Kings College
Hospital, a percutaneous liver biopsy. Key histological features
might include bile duct proliferation, a small cell infiltrate, portal
fibrosis, and absence of sinusoidal fibrosis [70]. Some centres rely
on more functional tests looking for an absence of bile in the in-
testine such as duodenal intubation and aspiration or hepatobiliary
radionuclide scans using a variety of technetium-labelled imino-
diacetic acid derivatives (HIDA) [71]. Use of endoscopic retrograde
cholangiopancreatography (ERCP) is possible in neonates but its
use is confined to specialist centres [72]. Furthermore there is
currently a dearth of appropriately-sized endoscopes available with
manufacturers pulling out of production and servicing and this
6 B. Lakshminarayanan, M. Davenport / Journal of Autoimmunity 73 (2016) 1e9

Fig. 4. Maternal Microchimerism. A male cell, depicted by a red signal, the X chromosome, and green signal, a Y chromosome within the blue nuclear material, can be seen in both
male BA liver specimens (A&B). In the same specimen, one can also see a female cell, depicted by two red signals, both X-chromosomes with no green signal within the blue nuclear
material (arrow). [used with permission from Suskind DL, Rosenthal P, Heyman MB et al. Maternal microchimerism in the livers of patients with biliary atresia. BMC Gastroenterol
2004; 4: 14. Published online 2004 Jul 31. http://dx.doi.org/10.1186/1471-230X-4-14.

Fig. 5. The Bilatresone Story. Sheep grazing on the newly exposed foreshore of the Burrinjuck Dam (A). Dysphania dysphania glomulifera subspecies glomulifera (B). Zebra fish (C) and
the isoflavonoid bilatresone (D).

doesn’t bode well for being able to continue with this method in the
future. The “acid test” for many remains operative visualisation
(which could be laparoscopic) supplemented by on-table cholan-
giography where possible.
The Kasai operation is a reasonable option in over 95% of infants
diagnosed with BA. Although many will fail and there are some
prognostic features which will guide us; none is perfect and only
overt end-stage liver disease really precludes an attempt. Primary
liver transplantation was used in only 2% of our national series [73].
During the early years of the new century there was a brief
flirtation with the concept of the laparoscopic Kasai option. It was
reported first by a Brazilian team in 2002 [74] and there have been
small case series since [75]. However one prospective trial from
Hannover, Germany and comparative experience from Hong Kong
[76,77] showed that results were not as good and so for the fore-
seeable future the open version will remain the cornerstone of
surgical endeavour.
Perhaps the real question should not be how the Kasai is done
but who should do it. It is an uncommon operation in general
paediatric surgical practice and many countries have now central-
ised their management. So not only do the larger centres manage
the diagnostic and therapy side but that they do it exclusively. We
in the UK led this change and centralised care of the infant with BA
into one of only 3 centres in England and Wales e all of them
dealing with >10/year. We reported our first 4 years outcomes in
2002 [7] and our 10 year outcomes in 2011 [73]. Both papers
showed conclusively that our national performance in terms of
time to the Kasai operation, subsequent clearance of jaundice,
native liver survival and overall survival had clearly changed and
become the leading benchmark figures thus far reported. This
centralisation policy has also been adopted in medium-sized
countries in Europe such as Finland [79], Denmark [80],
Switzerland, Holland and Norway but would clearly be a major
challenge in other health systems where the state does not already
have much central control.
Our current expectation is that we clear jaundice in 55e60% of
 B. Lakshminarayanan, M. Davenport / Journal of Autoimmunity 73 (2016) 1e9
infants coming to Kasai at a median now of <50 days. This will
allow a 5 year native liver survival of about 50% and overall survival
of 90% (Fig. 6).
11.1. Key variables in outcome
Biliary atresia should not be thought of as a single disease entity
with a predictable natural history and stereotypical response to
surgery. This aetiological heterogeneity is complex and even our
broad classification which seeks to categorize syndromic BA and
cystic BA as examples of developmental BA; and CMV IgM þ ve BA
as a clinically defined virus-associated BA still leaves many cases of
isolated BA with no obvious definable aetiology [81]. However,
infants with syndromic BASM respond less well to Kasai surgery
and have a poorer overall outcome with a higher risk of death and
infants with cystic BA usually clear their jaundice and have a better
long-term outcome [73]. Those with the poorest outcome and
highest risk of death are infants with CMV IgM þ ve associated BA
[12]. They are usually older at presentation (median age at Kasai 70
days) have a pronounced hepatic inflammatory reaction with a
higher degree of fibrosis. As a consequence, their chance of clear-
ance of jaundice following KPE is much lower and their need for
transplantation increased accordingly.
There is an effect of when infants come to surgery e the age
effect. This was often alluded to by Kasai himself but shown clearly
in one of our early reports from Kings written by the young Gior-
gina Mieli-Vergani in 1989 [82]. Subsequently when we re-
analysed outcomes from infants treated in the 1990s and early
2000s the effect proved more subtle. Age cohort analysis showed
clearly that up to about 90 days of age we could not predict
outcome (by clearance of jaundice or need for transplant by 2 years)
simply on the grounds of age for those with otherwise isolated BA
[83]. Certainly there were no “cut-offs” at 6, 8, or 10 weeks as had
been suggested before. Where there was a marked effect of age was
in the two developmental cohorts e those with BASM and partic-
ularly those with cystic BA.
Each centre treating infants with BA will have its own post-
operative regimen to try and maximise the restoration of bile
flow beyond that achieved by surgery. Most will include a pro-
longed period of oral antibiotic to prevent cholangitis; ursodeox-
ycholic acid to improve established bile flow; fat-soluble vitamin
supplementation to reverse deficit (perceived or measured);
medium-chain triglyceride (MCT) based formula milk to maintain
calorie intake and lastly a course of steroids to combat the in-
flammatory element. In truth little in each regimen will be based on
actual evidence of effect and the last of this list has produced much
controversy despite actual trial data. So according to a survey
published in 2010, about 50% of infants with BA treated in the USA
receive post-operative steroids [84]. There have been two
Fig. 6. Outcome of Biliary Atresia in England and Wales since Centralisation
(1999e2009). Actuarial true and native liver survival curves [median (±95% CI)] for
biliary atresia (n ¼ 443).
7
randomized, placebo controlled trials of steroids [low dose, starting
2 mg/kg/day [83] and high dose starting 5 mg/kg/day [85]. The first
showed biochemical effects on lowering bilirubin levels post-
operatively but no change in clinical outcome while the second
showed a non-significant increase in jaundice clearance (~15%) but
again no change in need for transplant. Further trial data from our
centre using a comparable high-dose regimen showed the same
degree of increase (~15%) in jaundice clearance but because of a
larger cohort this difference proved statistically significant [83]. The
other difference is that North American trial [85] had a much higher
proportion of older infants than the UK trials [86,87] and it does
appear that steroids are more efficacious in the younger livers,
presumably as there is less established fibrosis and scarring. A
meta-analysis recently published concludes that steroids are
beneficial [88].
Biliary atresia has proved to be a quixotic, unpredictable disease
with many immunological facets formerly unappreciated. Murine
models have shed some light on cellular mechanisms of disease but
it is only by considered and detailed examination of the laboratory
which is the human condition which will really allow progress and
thereby hope for future therapy.
End note
Sic transit gloria mundi
The senior author first moved to the surgical service under Ted
Howard at Kings College Hospital in 1989. It quickly became
apparent that to get on you had to team up and work closely with
the paediatric hepatologists there led by the venerable Scot Alex
ge
Mowat, but increasingly also by his Italian prote  Giorgina Mieli-
Vergani. These were exciting times for children with biliary atresia
at Kings. There were significant numbers of children now surviving
from the 1980s and transplants for some had started there only
recently under Mr KC Tan. Still not much was really known about
the disease itself. We tried to rectify this and from about 1998
onwards embarked upon laboratory-based work into its inflam-
matory and immunological characteristics as described in the text
above. This whole programme was performed, encouraged, su-
pervised and directed by Team Mieli-Vergani.
Acknowledgments
Much of the work, both clinical and experimental described
above has been made possible by grants from the Childrens Liver
Disease Foundation (registered charity).
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