Maternal Microchimerism in Lymph Nodes of Infants With Biliary Atresia at Time of Kasai's Operation
Condition: Biliary Atresia
MeSH terms: Biliary Atresia
Study type: Observational

Summary: 
      Maternal microchimerism has been discussed as an etiological mechanism in infantile
      (perinatal) biliary atresia (BA). In Kasai's operation (resection of the liver hilum plaque
      followed by hepato-portoenterostomy) surgeons frequently encounter swollen portal and
      mesenteric lymph nodes.

      Lymph nodes were sampled during Kasai' s operation and examined for maternal DNA.
    

Detailed description: 
      Upon operation of infants with non-syndromatic biliary atresia (BA, commonest form), surgeons
      almost invariably note the prominent finding of multiple swollen lymph nodes of up to 2 cm in
      size in the porta hepatis and the small bowel mesentery where they may constitute grape like
      complexes. Same-age infants laparotomized for other pathology commonly do not present such
      nodes.

      Biliary atresia is a disease of the newborn which is fatal when left untreated.
      Histologically it corresponds to a degeneration of intra- und extra hepatic bile ducts within
      a in part specific inflammatory infiltrate and progressive cirrhosis.

      The etiology of this rare but constant disease (1:8-1:15.000) is unknown. Currently in BA
      etiology research two main avenues are pursued:

        1. Virally induced perinatal (auto)immune processes are suspected. An experimental form of
           BA can be provoked by infecting murine mothers with dsRNA-viruses e.g.
           Rhesus-rotaviruses or Reo-viruses while in mice, vaccination of mothers has prevented
           experimental BA . The importance of the γ/δ-T-cell and IL17/IL 23 pathway , for BA is
           currently probed. However, in groups of syngenic animals treated by the same batch of
           viruses BA tends to be inconstant and it is limited to extrahepatic bile ducts such as
           about the validity of the animal model is an issue.

        2. The other direction of research does not primarily rely on experiments involving newborn
           animals: There are indications that during pregnancy the placental barrier may become
           permeable for maternal lymphocytes (e.g. by a faulty silencing of T-cell attracting
           chemokines in decidual stroma cells) and already in 2008 maternal lymphocytes have been
           demonstrated in livers of BA infants . Subsequently the phenomenon of materno-fetal
           microchimerism has come into focus of BA ethiology research: in the context of mixed
           materno-fetal immune reactions cells of maternal origin could misdirect the immature
           immune system of the infant into auto-immunity or act directly as effectors in a graft
           vs. host like fashion.

      The importance of mesenteric and portal lymph nodes for maturation of the immune system
      during pregnancy has been reported recently . Moreover it has been demonstrated that flow the
      between liver and portal lymph nodes is bidirectional . In In mice the frequence of CD4+ and
      FoxP3+ regulatory T-cells was found to be linked to the mothers immune status. We thus
      hypothesized that the prominently swollen lymph nodes of BA infants which surgeons constantly
      encounter during Kasai's operation may hold a key to the better understanding of
      pathophysiological important lymphocyte trafficking mechanisms.

      In the standard operation, the Kasai-operation which in a race with increasing liver
      cirrhosis makes sense up to an age of 4 months, the degraded rests of the major bile ducts in
      the liver hilus (the so called "hilar plate") are resected, A Roux y-loop is sutured onto the
      resection site. It shall drain any contingent bile flow from remaining bile ducts. The long
      term success rate of this tentative of surgical cure which is of moderate technical
      complexity and invasiveness is estimated to ca. 15-20%. In the remaining a partly sucess may
      postpone the necessity of liver transplantation. We examined in seven consecutive patients
      with histologically proven biliary atresia whether in periportal lymph nodes there was
      evidence for maternal gentic material.

      Methods :

      7 consecutive patients with the preoperative diagnosis of BA were included in the study.
      Other causes of icterus prolongatus (α1 anti-trypsin deficiency, Alagilles syndrom, PFIC
      etc.) had been excluded amongst the methods technical successful ERCP (5/7 patients) which
      showed absence of intrahepatic bile ducts.

      After obtaining the parent's informed consent 2 ml EDTA whole blood were collected from
      mother and infant. During Kasai's operation one portal and one mesenterial lymphnode were
      sampled (Hannover Medical School ethical committee vote No.1650/11.12.2012. After bipolar
      dissection the mesenteric gaps were closed with 4-0 Vicryl sutures.

      Lymph nodes were disrupted mechanically and DNA was extracted enzymatically (QIAmp Kit,
      Qiagen, Hilden/Germany) as described previously and normalized to 30 ug/ml.

      Short tandem repeat analysis was realized with multiplex PCR for markers LIPOL, VWA, TH01,
      D19S253, CSF, PLA2A. FGA, D21S11, D18S51 as described. Briefly, fragment length analysis was
      performed an Abi Prism 310 System with GeneScan 3.11. software for comparison of mother's and
      infant's blood with lymph node DNA.

      Genetic single nucleotide polymorphisms (SNPs) were traced by quantitative real time PCR
      chimerism analyses. Reactions were executed on an iCycler iQ5 real-time PCR platform
      (Bio-Rad) using the ABsolute QPCR Rox Mix (Thermo scientific). Custom designed primers and
      probes were provided by Sigma-Aldrich. Control reactions and normalization of sample DNA were
      performed by amplifying the albumin gene.
    

Intervention name(s): Lymph node and blood sampling
Intervention type(s): Procedure
Intervention description(s): By bipolar dissection 1 portal and 1 mesenteric lymph node are removed from the infant.
Blood (1ml) is taken from mother and infant.
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Children with newly and preoperatively diagnosed infantile Biliary Atresia of the
        non-syndromatic form
      
Biospecimen description: 
      Lymph nodes from children Whole Blood from mother and infant