AIM OF STUDY: Lymphocyte-mediated inflammation within the liver and bile ducts has been proposed as a potential mechanism in the pathogenesis of biliary atresia (BA). Recent reports have suggested that BA is associated with upregulation of TH1-type inflammatory cytokines. Quantitative real-time polymerase chain reaction (qrt-PCR) is a useful tool to assess immune cell density and cytokine gene expression in tissues. The aim of this study was to characterize the inflammation in BA by quantifying lymphocyte density and expression of TH1 cytokines such as IL2 and tumor necrosis factor alpha (TNF-alpha) in liver biopsies using qrt-PCR. METHODS: Total RNA was extracted from archival formalin-fixed paraffin-embedded liver biopsy specimens collected from three age-matched groups of patients: group I, nonsyndromic BA at the time of Kasai portoenterostomy (n = 10); group II, choledochal dilatation at the time of surgery (n = 2); group III, other cholestatic diseases including biliary hypoplasia and parenteral nutrition-associated cholestasis (n = 3). Qrt reverse transcription-PCR was performed to measure the relative levels of messenger RNA (mRNA) expression of CD4, CD8, IL2, and TNF-alpha. Their levels were normalized to the level of beta-actin expression. The numbers of CD4+ and CD8+ lymphocytes infiltrating portal tracts were also measured using immunohistochemistry. Data were expressed as median and ranges, and statistical comparison and correlation was by nonparametric test (Kruskal-Wallis and Spearman rank). RESULTS: CD8 mRNA expression was significantly increased in group I (median 0.26, range 0.11-6.29) compared with groups II and III (0.002, 0-0.004 and 0.02, 0-0.12) (P < .05). Levels of IL2 mRNA expression were also significantly increased in group I (1.22, 0.05-7.54) compared with groups II (0.58, 0-1.17) and III (0, 0-0.03) (P < .05). In contrast, there was no significant increase in CD4 and TNF-alpha expression in group I compared with groups II and III. There was a significant correlation between the number of CD8+ lymphocytes in portal tracts and the levels of CD8 mRNA expression. CONCLUSION: Increased expression of IL2 and increased CD8 T-cell infiltration in liver biopsy specimens from infants with BA suggest the possibility that IL2 plays a role in the pathogenesis of this condition.
AIM OF STUDY: Lymphocyte-mediated inflammation within the liver and bile ducts has been proposed as a potential mechanism in the pathogenesis of biliary atresia (BA). Recent reports have suggested that BA is associated with upregulation of TH1-type inflammatory cytokines. Quantitative real-time polymerase chain reaction (qrt-PCR) is a useful tool to assess immune cell density and cytokine gene expression in tissues. The aim of this study was to characterize the inflammation in BA by quantifying lymphocyte density and expression of TH1 cytokines such as IL2 and tumor necrosis factor alpha (TNF-alpha) in liver biopsies using qrt-PCR. METHODS: Total RNA was extracted from archival formalin-fixed paraffin-embedded liver biopsy specimens collected from three age-matched groups of patients: group I, nonsyndromic BA at the time of Kasai portoenterostomy (n = 10); group II, choledochal dilatation at the time of surgery (n = 2); group III, other cholestatic diseases including biliary hypoplasia and parenteral nutrition-associated cholestasis (n = 3). Qrt reverse transcription-PCR was performed to measure the relative levels of messenger RNA (mRNA) expression of CD4, CD8, IL2, and TNF-alpha. Their levels were normalized to the level of beta-actin expression. The numbers of CD4+ and CD8+ lymphocytes infiltrating portal tracts were also measured using immunohistochemistry. Data were expressed as median and ranges, and statistical comparison and correlation was by nonparametric test (Kruskal-Wallis and Spearman rank). RESULTS:CD8 mRNA expression was significantly increased in group I (median 0.26, range 0.11-6.29) compared with groups II and III (0.002, 0-0.004 and 0.02, 0-0.12) (P < .05). Levels of IL2 mRNA expression were also significantly increased in group I (1.22, 0.05-7.54) compared with groups II (0.58, 0-1.17) and III (0, 0-0.03) (P < .05). In contrast, there was no significant increase in CD4 and TNF-alpha expression in group I compared with groups II and III. There was a significant correlation between the number of CD8+ lymphocytes in portal tracts and the levels of CD8 mRNA expression. CONCLUSION: Increased expression of IL2 and increased CD8 T-cell infiltration in liver biopsy specimens from infants with BA suggest the possibility that IL2 plays a role in the pathogenesis of this condition.