Reply
Authors: Reena Mourya, Kazuhiko Bessho, Pranavkumar Shivakumar, Jorge A. Bezerra
Journal: Hepatology (2014)

We welcome the insightful comments by Dong et al., focusing on the relationship between the expression of interleukin-8 (IL-8) and the diagnosis of biliary atresia (BA). They raise relevant points related to the gene expression signature in subjects in the non-BA group, the biological variables that may influence the expression of IL8, and interpretation of the findings vis-à-vis previous reports. From the 14 subjects that served as diseased controls (non-BA), the diagnosis of the nine subjects with cholestasis of unknown etiology was based on a comprehensive clinical, biochemical, histological, and infectious evaluation by experienced hepatologists, which failed to reveal an etiology for intrahepatic cholestasis. Thus, the expression levels of individual subjects cannot be linked to infection by common viruses. The authors are correct that the expression of IL8 and LAMC2 misclassified 3 of 14 subjects in the non-BA group as having BA. As described in the original publication, this misclassification resulted in a specificity of 85.7%. It will be important that follow-up validation studies examine potential changes in the specificity and sensitivity of hepatic mRNA expression of IL8 and LAMC2 in the diagnosis of biliary atresia, especially in studies that include a larger population of non-BA patients as diseased controls. We agree that the analysis of IL-8 expression should take into account its relationship with age and staging of fibrosis of children with biliary atresia. Although data on histological fibrosis were not available for all subjects, we were able to perform new analyses of IL-8 expression data (both mRNA and protein levels) to directly examine its relationship with age. To this end, we subdivided the subjects and dataset based on the three suggested age subgroups of <60 days, 60-90 days, and >90 days. We found that IL8 mRNA was significantly increased in all three subgroups in BA (Fig. 1A) above non-BA and normal controls (NC). At the protein level, the concentration of serum IL-8 did not differ between BA and non-BA subjects, regardless of the age groups (Fig. 1B). These results are similar to those reported in the original publication when the subjects were analyzed as single groups of BA and non-BA.1 (A) The hepatic mRNA expression of IL8 in patients with biliary atresia (BA) based on age groups and non-biliary atresia (non-BA) normalized to normal controls (NC) at the time of diagnosis. Non-BA subjects were kept as one group due to the low number of subjects (N = 14). (B) BA and non-BA subjects are depicted based on similar age groups. *P < 0.05; ***P < 0.001; NS: not significant. As discussed,1 the lack of differences in serum IL-8 contrasts with previous studies that reported increased concentrations of serum IL-8 in subjects with BA.2, 3 Although the reasons for the discrepancies are not obvious, one likely possibility is that our patients were analyzed at the time of diagnosis, with an average age of 2.01 ± 0.7 months (mean ± SD) in subjects with BA and 1.8 ± 0.7 months with non-BA, which contrasts with subjects at later stages of disease, with an average of 8.7 ± 2.1 months in reference2 and 6.3 ± 0.6 years in reference.3 Future studies investigating the use of IL8, LAMC2, and other genes and proteins in aiding the diagnosis of BA may provide insight into the mechanisms of the disease. They may also uncover biomarkers of stages of disease at diagnosis, allow for monitoring of progression of disease, and design individualization of treatment protocols to block progression of liver disease. Reena Mourya, M.Pharm. Kazuhiko Bessho, M.D., Ph.D. Pranavkumar Shivakumar, Ph.D. Jorge A. Bezerra, M.D. Division of Gastroenterology Hepatology and Nutrition Cincinnati Children's Hospital Medical Center Cincinnati, OH