Reproduction and fertility in wild-type and transgenic mice after orthotopic transplantation of cryopreserved ovaries from 10-d-old mice.

Ovary cryopreservation and subsequent transplantation can enable researchers to preserve valuable transgenic animal strains. Some studies have indicated, however, that this process may impair ovary viability and recipient fertility. The authors investigated the effects of ovary vitrification followed by orthotopic transplantation in five strains of mice. They grafted fresh and frozen ovaries of 10-d-old mice into 4-week-old ovariectomized recipients. In addition to using wild-type strains (BALB/cAn and ICR/JCL), the authors used a transgene system that enabled them to identify whether offspring derived from the ovary of the recipient or that of the donor: they transplanted ovaries from one transgenic strain (LAP/rtTA) into wild-type C57BL/6J mice and into mice from a second transgenic strain (pTet/Cd226). The authors then determined the origin of the offspring born to these recipients using PCR. Ovary cryopreservation seemed to have no effect on the long-term fertility and reproductive characteristics of recipients and their offspring.

The intragenic epistatic association of ADD3 with biliary atresia in Southern Han Chinese population.

Biliary atresia (BA) is a multifactorial pathogenic disease with possible genetic components. As a member of membrane skeletal proteins in the liver and bile ducts, a haplotype composed by five single nucleotide polymorphisms (SNPs) on adducin 3 (ADD3) has been identified as associated with BA. However, limited study was designed to further elaborate the mutual relationship amongst those replicated SNPs to disease. We selected three susceptibility SNPs in ADD3 and conducted a replication study using 510 BA cases and 1473 controls to evaluate the individual function of the SNPs and further stratified the potential roles with disease and its subclinical features. Two SNPs in ADD3 were replicated as associated with BA (1.60E-04 ≤ P≤1.70E-04, 1.33 ≤ odds ratio (OR) ≤ 1.58 for rs17095355, 2.10E-04 ≤ P≤5.30E-04, 1.26 ≤ OR ≤ 1.57 for rs2501577). Though we failed to replicate the individual association of rs10509906 to disease, the intragenic epistatic effect between rs10509906 and rs2501577 was suggested as exhibiting susceptibility to BA, further cross-validated by multifactor dimensionality reduction (MDR) (P=0.068, OR = 1.37), which may explain extra hidden heritability of ADD3 to BA. Furthermore, through subclinical stratification, we also observed the association of risk to disease mainly came from the female patients.

Downregulation of Hes1 expression in experimental biliary atresia and its effects on bile duct structure.

AIM: To analyze the expression and function of the Notch signaling target gene Hes1 in a rhesus rotavirus-induced mouse biliary atresia model. METHODS: The morphologies of biliary epithelial cells in biliary atresia patients and in a mouse model were examined by immunohistochemical staining. Then, the differential expression of Notch signaling pathway-related molecules was investigated. Further, the effects of the siRNA-mediated inhibition of Hes1 expression were examined using a biliary epithelial cell 3D culture system. RESULTS: Both immature (EpCAM+) and mature (CK19+) biliary epithelial cells were detected in the livers of biliary atresia patients without a ductile structure and in the mouse model with a distorted bile duct structure. The hepatic expression of transcripts for most Notch signaling molecules were significantly reduced on day 7 but recovered to normal levels by day 14, except for the target molecule Hes1, which still exhibited lower mRNA and protein levels. Expression of the Hes1 transcriptional co-regulator, RBP-Jκ was also reduced. A 3D gel culture system promoted the maturation of immature biliary epithelial cells, with increased expression of CK19+ cells and the formation of a duct-like structure. The administration of Hes1 siRNA blocked this process. As a result, the cells remained in an immature state, and no duct-like structure was observed. CONCLUSION: Our data indicated that Hes1 might contribute to the maturation and the cellular structure organization of biliary epithelial cells, which provides new insight into understanding the pathology of biliary atresia.

Autoimmune liver disease-related autoantibodies in patients with biliary atresia.

AIM: To investigate the prevalence and clinical significance of autoimmune liver disease (ALD)-related autoantibodies in patients with biliary atresia (BA). METHODS: Sera of 124 BA patients and 140 age-matched non-BA controls were assayed for detection of the following autoantibodies: ALD profile and specific anti-nuclear antibodies (ANAs), by line-blot assay; ANA and anti-neutrophil cytoplasmic antibody (ANCA), by indirect immunofluorescence assay; specific ANCAs and anti-M2-3E, by enzyme linked immunosorbent assay. Associations of these autoantibodies with the clinical features of BA (i.e., cytomegalovirus infection, degree of liver fibrosis, and short-term prognosis of Kasai procedure) were evaluated by Spearman's correlation coefficient. RESULTS: The overall positive rate of serum autoantibodies in preoperative BA patients was 56.5%. ALD profile assay showed that the positive reaction to primary biliary cholangitis-related autoantibodies in BA patients was higher than that to autoimmune hepatitis-related autoantibodies. Among these autoantibodies, anti-BPO was detected more frequently in the BA patients than in the controls (14.8% vs 2.2%, P < 0.05). Accordingly, 32 (25.8%) of the 124 BA patients also showed a high positive reaction for anti-M2-3E. By comparison, the controls had a remarkably lower frequency of anti-M2-3E (P < 0.05), with 6/92 (8.6%) of patients with other liver diseases and 2/48 (4.2%) of healthy controls. The prevalence of ANA in BA patients was 11.3%, which was higher than that in disease controls (3.3%, P < 0.05), but the reactivity to specific ANAs was only 8.2%. The prevalence of ANCAs (ANCA or specific ANCAs) in BA patients was also remarkably higher than that in the healthy controls (37.9% vs 6.3%, P < 0.05), but showed no difference from that in patients with other cholestasis. ANCA positivity was closely associated with the occurrence of postoperative cholangitis (r = 0.61, P < 0.05), whereas none of the autoantibodies showed a correlation to cytomegalovirus infection or the stages of liver fibrosis. CONCLUSION: High prevalence of autoantibodies in the BA developmental process strongly reveals the autoimmune-mediated pathogenesis. Serological ANCA positivity may be a useful predictive biomarker of postoperative cholangitis.

Role of CD56-expressing immature biliary epithelial cells in biliary atresia.

AIM: To analyze the clinical and pathological parameters and expression of the neural cell adhesion molecule (CD56) in patients with biliary atresia (BA). METHODS: Established clinical laboratory markers of hepatic function, including enzyme activity, protein synthesis, and bilirubin metabolism, were evaluated in patients with BA and compared with those in patients with choledochal cysts and neonatal hepatitis. Pathological changes in tissue morphology and fibrosis were examined by histological and tissue collagen staining. Immunohistochemical staining for the biliary epithelial cell markers CD56 and CK19 together with the Notch signaling related molecules Notch1 and Notch2 was performed in the context of alterations in the structure of intrahepatic biliary ducts. RESULTS: Differences in some clinical laboratory parameters among the three diseases examined were observed, but they did not correlate with the pathological classification of fibrosis in BA. Immunohistochemical staining showed the presence of CD56-positive immature bile ducts in most patients (74.5%) with BA but not in patients with choledochal cysts or neonatal hepatitis. The number of CD56-expressing cells correlated with disease severity, with more positive cells present in the later stages of liver damage (81.8% vs 18.2%). Furthermore, bile plugs were mainly found in CD56-positive immature biliary ducts. Notch signaling was a key regulatory pathway in biliary duct formation and played a role in tissue fibrosis. Notch1 was co-expressed in CD56-positive cells, whereas Notch2 was found exclusively in blood vessels in the portal area of patients with BA. CONCLUSION: The maturation of biliary epithelial cells and the expression of Notch may play a role in the pathogenesis of BA.