In Situ CD14 Expression in Biliary Atresia: Comparison Between Early and Late Stages By Abul Faiz Kabir Uddin Ahmed, Masaki Nip, Haruo Ohtani, Hiroshi Nagura, and Ryoji Ohi Sendai, Japan Purpose: The aim of this study was to compare the in situ expression of CD14 between early and late stage of biliary atresia (BA) to determine if a time course of CD14 expression exists in BA. Methods: Immunohistochemical analysis of membrane-bound CD14 expression was carried out in periodate-lysine-parafor- maldehyde (PLP)-fixed frozen sections from 9 early- (ob- tained during Kasai procedure) and 6 late- (obtained during liver transplantation) stage cases of BA. Normal liver (n = 3) and choledochal cysts (n = 5) served as normal controls and disease controls respectively. Results: In the early stage, 6 patients (66.66%) showed ex- tensive CD14 expression (grade 3 [G3], more than 50% pos- itive cells), whereas no CD14-positive cells could be detected in 4 patients (66.66%) in the late stage. In both stages, most of the positive cells were observed in the parenchyma of the hepatic Iobules where Kupffer cells and sinusoidal endothe- lial cells stained positive. Arterial and venous endothelium, bile duct cells, and hepatocytes were negative for CD14. Double immunohistochemistry in the early stage showed a higher colocalization rate of CD14 and CD68 in the sinusoidal locations (33.69 ± 9.270% [mean -+ SEM]) than in the portal tract (7.6 + 4.64% [mean -+ SEM]; P< .05). Similar pattern of colocalization also was observed in the late stage, In the normal controls no expression of CD14 could be detected, whereas in the disease controls only 1 case showed mild expression (grade 1 [G1], 1% to 10% positive cells) and the rest showed no expression of CD14. Conclusion: These results suggest that CD14 expression in BA is a dynamic phenomenon having time-related change with overexpression in the early stage and reduced expres- sion in the late stage. J Pediatr Surg 36:240-243. Copyright © 2001 by W.B. Saunders Company. INDEX WORDS: Biliary atresia, CD14, Kupffer cells, sinusoi- dal endothelial cells, immunohistochemistry. B ACTERIAL INVASION of liver or ascending cholan- gitis in biliary atresia (BA) after hepatic portoenter- ostomy (HPE) is associated with progressive liver fibrosis and cirrhosis in BA.~ Among the various factors that 19o - tentiate the deleterious effects of ascending cholangitis in BA, CD14, the 50- to 55-kDa glycoprotein lipopolysaccha- ride (LPS) receptor, is of prime importance because its binding of LPS through the facilitation of the lipopolysac- charide binding protein (LBP) 2 results in activation of the macrophages with consequent release of cytokines such as interleukin (IL)-I, IL-6, and tumor necrosis factor (TNF)-c~ and interferon (IFN)-%3 Although increased expression of CD14 in surgically biopsied BA specimens have been reported, 4 the exact mechanism of such overexpression in biliary atresia is From the Departments of Pediatric Surgery and Pathology, Tohoku University School of Medicine, Sendai, Japan. Presented at the 31st Annual Meeting of the American Pediatric Surgical Association, Orlando, Florida, May 25-29, 2000. Address reprint requests to Ryoji Ohi, MD, FAAP, Professor and Chairman, Department of Pediatric Surgery, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3601-0045503.00/0 doi:10.1053/jpsu.2 O01.20063 yet to be elucidated. In addition to causing activation of macrophages on binding LPS, CD14 also confers to the macrophages an ability to phagocytose the invading bacteria. 5 These dual roles of CD 14 make it an important molecule in the context of biliary atresia pathology. However, there is no report as to whether the expression of CD14 changes as the disease progresses. This study was, therefore, designed to examine if the expression of CD 14 in the early stage of biliary atresia differs from that in the late stage. MATERIALS AND METHODS Tissue Specimens The Ethical Committee of Tohoku University School of Medicine approved the current study, Frozen sections were obtained from 15 patients with biliary atresia. Table I shows the patients' age, sex, and type of biliary atresia. Specimens in 9 cases (2 boys and 7 girls) were obtained by wedge biopsy during Kasai's operation (designated as the early stage group). Specimens from another 6 cases (3 boys and 3 girls) were obtained during the living-related liver transplantation operation (designated as the late stage group), The age ranges of the early and late stage were 25 days to 78 days (58.88 + 18.69 days [mean ± 1 SD]) and 0.4 years to 21.9 years (7.6 + 9.4 years [mean ± 1 SD]), respectively. All patients in the late-stage group were treated initially by Kasai's operation, and each of them received prednisolone for variable duration as part of treatment protocol for cholestasis. Liver specimens (n = 3, 1 sample obtained from a 8-month-old patient who underwent operation for neuroblastoma and 2 samples obtained from autopsy of 2 stillborn 240 Journal of Pediatric Surgery, Vol 36, No 1 (January), 2001: pp 240-243