Quantification of Serum Matrix Metallopeptide 7 Levels May Assist in the Diagnosis and Predict the Outcome for Patients with Biliary Atresia.

OBJECTIVE: To assess the diagnostic and prognostic usefulness of the serum matrix metallopeptidase-7 (MMP-7) level for biliary atresia in infants with cholestasis after hepatoportoenterostomy. STUDY DESIGN: We enrolled 100 infants with cholestasis (age, 43.56 ± 1.97 days; 62 males) with a direct bilirubin level of >1 mg/dL, of whom 36 (36%) were diagnosed with biliary atresisa. The MMP-7 levels in serum samples collected during the cholestasis workup and 6 months after hepatoportoenterostomy were assessed by enzyme-linked immunosorbent assay. We quantified liver fibrosis by Picro Sirius red staining of collagen in specimens from the 81 infants with cholestasis. RESULTS: Infants with biliary atresisa had a significantly higher serum MMP-7 level than that of non-biliary atresisa infants with cholestasis of equivalent age (P < .0001). Receiver operating characteristic analysis showed that a serum MMP-7 level of >1.43 ng/mL was predictive of biliary atresisa in infants with cholestasis (diagnostic accuracy, 88%). There was a positive correlation between the serum MMP-7 level and the severity of liver fibrosis (P = .0002). Survival analysis showed that the frequency of liver transplantation was significantly higher in infants with biliary atresisa with a serum MMP-7 level of >10.30 ng/mL compared with a serum MMP-7 level of ≤10.30 ng/mL after hepatoportoenterostomy (hazard ratio, 4.22; P = .02). CONCLUSIONS: The serum MMP-7 level, which reflects the severity of liver fibrosis and can be determined noninvasively, may facilitate the diagnosis of biliary atresisa among infants with cholestasis. Moreover, the serum MMP-7 level after hepatoportoenterostomy is associated with a need for liver transplantation in infants with biliary atresisa.

Hepatocytes buried in the cirrhotic livers of patients with biliary atresia proliferate and function in the livers of urokinase-type plasminogen activator-NOG mice.

The pathogenesis of biliary atresia (BA), which leads to end-stage cirrhosis in most patients, has been thought to inflame and obstruct the intrahepatic and extrahepatic bile ducts. BA is not believed to be caused by abnormalities in parenchymal hepatocytes. However, there has been no report of a detailed analysis of hepatocytes buried in the cirrhotic livers of patients with BA. Therefore, we evaluated the proliferative potential of these hepatocytes in immunodeficient, liver-injured mice [the urokinase-type plasminogen activator (uPA) transgenic NOD/Shi-scid IL2rγnull (NOG); uPA-NOG strain]. We succeeded in isolating viable hepatocytes from the livers of patients with BA who had various degrees of fibrosis. The isolated hepatocytes were intrasplenically transplanted into the livers of uPA-NOG mice. The hepatocytes of only 3 of the 9 BA patients secreted detectable amounts of human albumin in sera when they were transplanted into mice. However, human leukocyte antigen-positive hepatocyte colonies were detected in 7 of the 9 mice with hepatocyte transplants from patients with BA. We demonstrated that hepatocytes buried in the cirrhotic livers of patients with BA retained their proliferative potential. A liver that was reconstituted with hepatocytes from patients with BA was shown to be a functioning human liver with a drug-metabolizing enzyme gene expression pattern that was representative of mature human liver and biliary function, as ascertained by fluorescent dye excretion into the bile canaliculi. These results imply that removing the primary etiology via an earlier portoenterostomy may increase the quantity of functionally intact hepatocytes remaining in a cirrhotic liver and may contribute to improved outcomes.

Interleukin-33 overexpression is associated with gamma-glutamyl transferase in biliary atresia.

Interleukin-33 (IL-33) plays a crucial role in inflammation. However, it is not clear whether IL-33 levels are of clinical significance for patients with biliary atresia (BA). The purpose of this study was to determine correlations between serum IL-33 levels and the clinicopathologic features of BA. Serum samples were collected from 18 BA infants, 12 nonicteric choledochal cyst (CC) infants with normal liver function, and 10 healthy controls (HCs). Serum IL-33 levels were measured with an enzyme-linked immunosorbent assay (ELISA). Routine liver function tests were performed on the serum samples. qRT-PCR and Western blot analysis were used to detect IL-33 expression in BA liver biopsy tissues. Hepatic lobule localization of IL-33 expression in the hepatic lobule was conducted by immunohistochemistry (IHC). IL-33 levels in serum collected from BA infants were significantly elevated in comparison with CC and HC patients. Furthermore, the elevated serum levels of IL-33 in BA infants were correlated with gamma-glutamyl transferase (GGT) levels. The expression of IL-33 mRNA and protein levels were up-regulated in BA liver biopsy tissues in comparison with CC patients. IHC analysis revealed increased positive immunostaining for IL-33 in BA liver tissues as compared to that in CC tissues. These results suggest that IL-33 may play an important role in the pathogenesis of BA. In addition, the correlation of serum IL-33 levels with GGT levels may provide a novel marker for the diagnosis of BA.

Relationship of non-visualization of the fetal gallbladder and amniotic fluid digestive enzymes analysis to outcome.

OBJECTIVE: The aims of this study were evaluate the significance of non-visualization of fetal gallbladder at routine ultrasound scan in a series of 102 cases and to determine the contribution of amniotic fluid digestive enzyme (AF-DE) analysis towards the outcome. METHOD: This is a multicenter retrospective study. Outcome of pregnancies, karyotype, and result of screening for CFTR gene mutations were known in all cases. Amniotic fluid gamma-glutamyl-transpeptidase and intestinal alkaline phosphatase isoenzyme were assayed. RESULTS: Non-visualization of the fetal gallbladder was associated with a severe disease in 25 cases (cystic fibrosis in ten, biliary duct atresia in eight, digestive tract anomalies in six, and chromosomal anomaly in one). In the remaining 77 cases, gallbladder agenesis was diagnosed in 22, and in 55, the gallbladder was subsequently demonstrated. Before 22 weeks of gestation (n=30), an abnormal AF-DE pattern had a 90% sensitivity and 80% specificity in detecting cystic fibrosis or biliary duct atresia. After 22 weeks, sensitivity fell to 53%. The AF-DE pattern was normal in 82% of gallbladder agenesis cases (benign) and in 91% of the cases where the gallbladder was subsequently detected. CONCLUSION: Non-visualization of the fetal gallbladder was associated with severe anomalies in 24% of cases. Prior to 22 weeks, determination of AF-DE contributes to the prediction of biliary atresia or the presence of cystic fibrosis.

α-enolase autoantibodies cross-reactive to viral proteins in a mouse model of biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is a neonatal cholangiopathy of unknown etiology. The bile duct injury that occurs in patients with BA might result from a hepatobiliary viral infection followed by an autoimmune response against the bile duct epithelia. We aimed to identify autoantigens recognized by serum antibodies in the Rhesus rotavirus (RRV)-induced mouse model of BA; findings were correlated with BA in humans. METHODS: Bile duct epithelial proteins were screened for their reactivity with serum antibodies from the mouse model of BA using immunoblot assays. Unique proteins that reacted with sera antibodies were identified by mass spectrometry and verified using enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. Candidate autoantibodies in BA patient sera were analyzed by ELISA. RESULTS: A bile duct epithelial antigen that reacted strongly with serum immunoglobulin (Ig) G from the mouse model of BA was identified as α-enolase. α-Enolase autoantibody specificity was confirmed by ELISA and immunoblot analyses. Anti-RRV and anti-enolase antibodies cross-reacted with enolase and RRV proteins; we identified regions of sequence homology between RRV and enolase. Serum samples from patients with BA had increased levels of anti-enolase IgM and IgG. CONCLUSIONS: We have identified autoantibodies against α-enolase in a mouse model of BA (infected with RRV) and in serum samples from patients, indicating a role of humoral autoimmunity in disease pathogenesis. The cross-reactivity between an anti-enolase antibody and RRV proteins indicates that molecular mimicry might activate humoral autoimmunity in BA patients; further studies are required.

High serum matrix metalloproteinase-3 and liver stiffness in postoperative biliary atresia.

BACKGROUND: Biliary atresia (BA) is a neonatal liver disorder characterized by chronic inflammation and obliteration of extrahepatic bile ducts. The purpose of the study was to investigate serum matrix metalloproteinase-3 (MMP-3) in postoperative BA patients and the association of MMP-3 with clinical outcome and liver stiffness score. METHODS: Fifty-eight BA patients post-Kasai operation and 20 controls were enrolled. None of the patients had undergone liver transplantation. BA patients were classified into two groups according to their serum total bilirubin (TB) levels (TB < 2 mg/dL, no jaundice vs. TB ≥ 2 mg/dL, persistent jaundice) and alanine aminotransferase (ALT) levels (ALT < 45 IU/L, normal ALT vs. ALT ≥ 45 IU/L, elevated ALT). Serum MMP-3 levels were determined by enzyme-linked immunosorbent assay. Liver stiffness scores were measured by FibroScan. RESULTS: BA patients had greater MMP-3 levels (10.8 ± 1.0 vs. 7.9 ± 0.8 ng/mL, P = 0.02) and higher liver stiffness values than controls (29.7 ± 3.0 vs. 5.1 ± 0.5 kPa, P < 0.001). Serum MMP-3 levels were significantly elevated in BA patients with jaundice when compared with those without jaundice (15.3 ± 2.2 vs. 8.5 ± 0.8 ng/mL, P = 0.004). In addition, BA patients with elevated ALT had higher levels of serum MMP-3 than those with normal ALT (12.4 ± 1.5 vs. 8.3 ± 0.9 ng/mL, P = 0.02). Moreover, BA patients with portal hypertension displayed higher serum MMP-3 than those without portal hypertension (13.5 ± 1.5 vs. 7.4 ± 0.8 ng/mL, P = 0.001). There was also a correlation between serum MMP-3 and liver stiffness scores (r = 0.448, P ≤ 0.001). CONCLUSION: Serum MMP-3 was associated with hepatic dysfunction and liver stiffness in postoperative BA patients. Accordingly, MMP-3 could play a role in the pathophysiology of hepatic fibrosis in BA after Kasai operation.

Endotoxin and CD14 in the progression of biliary atresia.

BACKGROUND: Biliary atresia (BA) is a typical cholestatic neonatal disease, characterized by obliteration of intra- and/or extra-hepatic bile ducts. However, the mechanisms contributing to the pathogenesis of BA remain uncertain. Because of decreased bile flow, infectious complications and damaging endotoxemia occur frequently in patients with BA. The aim of this study was to investigate endotoxin levels in patients with BA and the relation of these levels with the expression of the endotoxin receptor, CD14. METHODS: The plasma levels of endotoxin and soluble CD14 were measured with a pyrochrome Limulus amebocyte lysate assay and enzyme-linked immunosorbent assay in patients with early-stage BA when they received the Kasai procedure (KP), in patients who were jaundice-free post-KP and followed-up at the outpatient department, in patients with late-stage BA when they received liver transplantation, and in patients with choledochal cysts. The correlation of CD14 expression with endotoxin levels in rats following common bile duct ligation was investigated. RESULTS: The results demonstrated a significantly higher hepatic CD14 mRNA and soluble CD14 plasma levels in patients with early-stage BA relative to those with late-stage BA. However, plasma endotoxin levels were significantly higher in both the early and late stages of BA relative to controls. In rat model, the results demonstrated that both endotoxin and CD14 levels were significantly increased in liver tissues of rats following bile duct ligation. CONCLUSIONS: The significant increase in plasma endotoxin and soluble CD14 levels during BA implies a possible involvement of endotoxin stimulated CD14 production by hepatocytes in the early stage of BA for removal of endotoxin; whereas, endotoxin signaling likely induced liver injury and impaired soluble CD14 synthesis in the late stages of BA.

Integrin alphavbeta6 and mediators of extracellular matrix deposition are up-regulated in experimental biliary atresia.

INTRODUCTION: Biliary atresia (BA) is a progressive obliteration of the extrahepatic bile ducts resulting in hepatic fibrosis. The underlying mechanisms have not been defined. We used an animal model of BA to evaluate mediators of extracellular matrix (ECM) processing to determine which factors may be involved. METHODS: Newborn BALB/c mice received an intraperitoneal injection with rhesus rotavirus or saline within 24 h of birth. Livers were harvested on days 7 and 14 for histology and immunohistochemistry (IHC). RNA expression was determined using quantitative real-time PCR. Human liver from patients with BA and those having a resection for nonfibrosing diseases was also evaluated. RESULTS: In experimental mice, mRNA expression for tissue inhibitor of metalloproteinase (TIMP)-1 and matrix metalloproteinase (MMP)-7 was increased 18-fold and 69-fold, respectively on day 7, with further increases on day 14. On day 14, mRNA expression for plasminogen activator inhibitor (PAI)-1 (38-fold), TIMP-4 (9.5-fold), and MMP-9 (5.5-fold) mRNA was also observed. Furthermore, integrin alpha(v) beta(6) mRNA expression was increased on days 7 (11-fold) and 14 (6-fold). Presence of integrin alpha(v) beta(6) protein was confirmed by IHC in both mouse and human specimens in the proliferating biliary epithelium. CONCLUSIONS: Our data suggest experimental BA is associated with increased mRNA expression of ECM degradation inhibitors, TIMP-1, PAI-1, and TIMP-4. MMP-7 and MMP-9 expression is also elevated in this model. Furthermore, increased gene expression of integrin alpha(v)beta(6) was demonstrated and IHC confirmed protein expression. Integrin alpha(v)beta(6) or the inhibitors of ECM breakdown may be attractive targets for future treatment strategies.

Gamma-glutamyl transferase in the diagnosis of biliary atresia.

BACKGROUND: Early diagnosis of biliary atresia (BA) is important because the prognosis is closely related to timing of Kasai operation. The aim of this study was to test the clinical application of serum gamma-glutamyl transferase (GGT) concentration and the ratio of serum GGT to aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in differentiating BA from neonatal hepatitis (NH). METHODS: Ninety-three (46 male and 47 female) cases of BA and 65 (45 male and 20 female) NH were included in this study. Serum concentrations of GGT, AST, and ALT were measured in all cholestatic infants. The results of peak GGT level, GGT/AST ratio, GGT/ALT ratio were compared between groups. RESULTS: The serum GGT levels were significantly higher in BA patients than those in NH patients (353.3 +/- 334.4 IU/L vs. 114.8 +/- 86 IU/L, P < 0.001). GGT/AST values were over 2 in 55/68 BA and 15/ 54 NH (OR = 11.0, 95% CI 4.7-25.7, P < 0.001). GGT/ALT values were over 2 in 54/65 BA and 19/50, NH respectively (OR = 8.0, 95% CI 3.4-19.0, P < 0.001). A GGT level greater than 300 IU/L had a sensitivity of 39.7% in the diagnosis of BA, GGT/AST over 2 was 80.9% and GGT/ALT over 2 was 83.1%; the specificities were 98.1%, 72.2% and 62.0%, respectively. The respective accuracies of the diagnosis of BA were 65.6%, 77.1% and 73.9%. CONCLUSIONS: GGT/AST ratio over 2 indicates high possibility of biliary atresia and should prompt further investigations to confirm the diagnosis.

Diagnostic value of anti-smooth muscle antibodies and liver enzymes in differentiation of extrahepatic biliary atresia and idiopathic neonatal hepatitis.

BACKGROUND: We aimed to evaluate the diagnostic value of anti-smooth muscle antibodies (ASMA) and two liver markers (gamma-glutamyl transpeptidase [GGT] and alkaline phosphatase [ALP]) for differentiating between patients with extrahepatic biliary atresia (EHBA) and idiopathic neonatal hepatitis (INH). MATERIALS AND METHODS: During April 2010-2011, all infants at 2 weeks of age who were diagnosed with cholestasis and admitted to Children's Hospital of Tabriz were enrolled. Based on the results of physical examination, laboratory, imaging and pathological studies, neonates were divided into two groups (EHBA and INH). Receiver operating characteristics analysis was used to define sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy for ASMA, GGT and ALP. RESULTS: Thirty neonates with cholestasis (18 with EHBA and 12 with INH) and mean age of 54.66 ΁ 25.86 days were enrolled. Total and direct bilirubin, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and ASMA titres were highly not significant (P > 0.05) in patients with INH. GGT (P = 0.008) and ALP (P = 0.01) had statistically significant differences that were higher in patients with EHBA. The sensitivity, specificity, PPV and NPV, accuracy, LR+ and LR- of SMA in differentiating cases with BA were 66.7%, 75%, 80% 60%, 70%, 2.68 and 0.44, respectively. For GGT, the values were 88.9%, 66.7%, 80%, 80%, 79.1%, 3.08 and 0.31, respectively. Finally, for ALP, the values were 77.8%, 75%, 82.4%, 69.2%, 80%, 2.66 and 0.24, respectively. CONCLUSION: Our study showed that ASMA may be a useful biomarker for differentiation of EHBA from INH. Further studies with larger samples are recommended for confirming the results of this study.

Cyclooxygenase-2 Inhibitor Reduces Hepatic Stiffness in Pediatric Chronic Liver Disease Patients Following Kasai Portoenterostomy.

PURPOSE: The purpose of this study was to define the role of cyclooxygenase-2 inhibitors (COX-2i) in reducing hepatic fibrosis in pediatric patients with chronic liver disease. MATERIALS AND METHODS: From September 2009 to September 2010, patients over 2 years old who visited our outpatient clinic for follow-up to manage their chronic liver disease after Kasai portoenterostomy for biliary atresia, were included in this study. Volunteers were assigned to the study or control groups, according to their preference. A COX-2i was given to only the study group after obtaining consent. The degree of hepatic fibrosis (liver stiffness score, LSS) was prospectively measured using FibroScan, and liver function was examined using serum analysis before and after treatment. After 1 year, changes in LSSs and liver function were compared between the two groups. RESULTS: Twenty-five patients (18 females and 7 males) were enrolled in the study group. The control group included 44 patients (26 females and 18 males). After 1 year, the least square mean values for the LSSs were significantly decreased by 3.91±0.98 kPa (p=0.004) only in the study group. Serum total bilirubin did not decrease significantly in either group. CONCLUSION: COX-2i treatment improved the LSS in patients with chronic liver disease after Kasai portoenterostomy for biliary atresia.

Rhesus rotavirus VP6 regulates ERK-dependent calcium influx in cholangiocytes.

The Rhesus rotavirus (RRV) induced murine model of biliary atresia (BA) is a useful tool in studying the pathogenesis of this neonatal biliary obstructive disease. In this model, the mitogen associated protein kinase pathway is involved in RRV infection of biliary epithelial cells (cholangiocytes). We hypothesized that extracellular signal-related kinase (ERK) phosphorylation is integral to calcium influx, allowing for viral replication within the cholangiocyte. Utilizing ERK and calcium inhibitors in immortalized cholangiocytes and BALB/c pups, we determined that ERK inhibition resulted in reduced viral yield and subsequent decreased symptomatology in mice. In vitro, the RRV VP6 protein induced ERK phosphorylation, leading to cellular calcium influx. Pre-treatment with an ERK inhibitor or Verapamil resulted in lower viral yields. We conclude that the pathogenesis of RRV-induced murine BA is dependent on the VP6 protein causing ERK phosphorylation and triggering calcium influx allowing replication in cholangiocytes.

Validation of aspartate aminotransferase to platelet ratio for diagnosis of liver fibrosis and prediction of postoperative prognosis in infants with biliary atresia.

AIM: To validate the value of aspartate aminotransferase to platelet ratio index (APRI) in assessment of liver fibrosis and prediction of postoperative prognosis of biliary atresia (BA) infants from Mainland China. METHODS: Medical records of 153 BA infants who were hospitalized from January 2010 to June 2013 were reviewed. The efficacy of APRI for diagnosis of liver fibrosis was assessed using the receiver operator characteristic (ROC) curve compared to the pathological Metavir fibrosis score of the liver wedge specimens of 91 BA infants. The prognostic value of preoperative APRI for jaundice persistence, liver injury, and occurrence of cholangitis within 6 mo after KP was studied based on the follow-up data of 48 BA infants. RESULTS: APRI was significantly correlated with Metavir scores (rs = 0.433; P < 0.05). The mean APRI value was 0.76 in no/mild fibrosis group (Metavir score F0-F1), 1.29 in significant fibrosis group (F2-F3), and 2.51 in cirrhosis group (F4) (P < 0.001). The area under the ROC curve (AUC) of APRI for diagnosing significant fibrosis and cirrhosis was 0.75 (P < 0.001) and 0.81 (P = 0.001), respectively. The APRI cut-off of 0.95 was 60.6% sensitive and 76.0% specific for significant fibrosis diagnosis, and a threshold of 1.66 was 70.6% sensitive and 82.7% specific for cirrhosis. The preoperative APRI in infants who maintained jaundice around 6 mo after KP was higher than that in those who did not (1.86 ± 2.13 vs 0.87 ± 0.48, P < 0.05). The AUC of APRI for prediction of postoperative jaundice occurrence was 0.67. A cut-off value of 0.60 showed a sensitivity of 66.7% and a specificity of 83.3% for the prediction of jaundice persistence. Preoperative APRI had no significant association with later liver injury or occurrence of cholangitis. CONCLUSION: Our study demonstrated that APRI could diagnose significant liver fibrosis, especially cirrhosis in BA infants, and the elevated preoperative APRI predicts jaundice persistence after KP.

Retinyl palmitate hydrolase activity in human liver.

Retinyl palmitate hydrolase (RPH) activity was studied in human liver after the optimal conditions of the assay in normal human liver homogenates were determined. The mean activity was 118 +/- 66 nmol.min-1.g-1 (mean +/- SD) protein in liver homogenates from six children and seven adults; no correlation was found between liver hydrolase activity and the enzyme endogenous substrate, ie, liver vitamin A concentrations. RPH activity was also studied in a human model of vitamin A deficiency represented by 22 children with biliary atresia: 11 patients with vitamin A deficiency (liver vitamin A concentration less than 70 nmol/g wet wt of liver) and 11 patients with normal vitamin A status after vitamin A treatment. The enzymatic assay had to be conducted after the removal of endogenous bile salts by cholestyramine because bile salts are accumulated in the liver of children with biliary atresia. No correlation was found between RPH activity and vitamin A status.

Glutathione S-transferases in neonatal liver disease.

AIMS: To investigate the distribution of alpha and pi class glutathione S-transferases (GST) in normal fetal, neonatal, and adult liver; and to examine changes in GST expression in neonatal liver disease. METHODS: alpha and pi class GST were immunolocalised in sections of formalin fixed liver tissue obtained from human fetuses (n = 21), neonates (n = 8), young children (n = 9) and adults (n = 10), and from neonates with extrahepatic biliary atresia (n = 15) and neonatal hepatitis (n = 12). Monospecific rabbit polyclonal antibodies were used with a peroxidase-antiperoxidase method. RESULTS: Expression of pi GST was localised predominantly within biliary epithelial cells of developing and mature bile ducts of all sizes from 16 weeks' gestation until term and in neonatal and adult liver. Coexpression of pi and alpha GST was seen in hepatocytes of developing fetal liver between 16 and 34 weeks' gestation. Although pi GST was seen in occasional hepatocytes up to six months of life, this isoenzyme was not expressed by hepatocytes in adult liver. By contrast, alpha GST continued to be expressed by hepatocytes in adult liver; this isoenzyme was also seen in some epithelial cells of large bile ducts in adult liver. No change was observed in the distribution of alpha GST in either neonatal hepatitis or extrahepatic biliary atresia. However, aberrant expression of pi GST was identified in hepatocytes of all but one case of extrahepatic biliary atresia but in only two cases of neonatal hepatitis. CONCLUSIONS: The phenotypic alterations noted in extrahepatic biliary atresia may result from the effect of cholate stasis. Evaluation of the pattern of pi and alpha GST distribution by immunohistochemical staining may provide valuable information in distinguishing between these two forms of neonatal liver disease.

Differentiating biliary atresia from other causes of cholestatic jaundice.

Diagnosis of biliary atresia (BA) in infants presenting with cholestatic jaundice (CJ) requires exploratory surgery with cholangiography. However, the lack of a standardized approach to preoperative evaluation of infants with CJ can lead to a high number of negative surgical explorations. We reviewed our experience with CJ and BA to determine preoperative variables that might reliably identify BA. Infants explored for possible BA over a 5-year period were retrospectively reviewed. Preoperative clinical indices and liver biopsy results were reviewed. Statistical analysis was conducted by Student's t test and Fisher's exact test (P < 0.05). Twenty patients were identified, 10 with BA and 10 without (50% negative exploration rate). Nuclear cholescintigraphy (HIDA) excretion into the gastrointestinal tract was absent in all BA and in 8 of 10 without BA. Hepatomegaly was more common in the BA group (OR = 9.3, P = 0.07). BA had higher mean (± standard error) serum gamma-glutamyl transpeptidase levels (542 ± 130 vs 139 ± 25.8 U/L in non-BA, P = 0.03). There were insignificant differences in sex, type of feeding, TPN exposure and sepsis between the two groups. Although our small sample size limits conclusions, we suggest screening infants with CJ by measuring GGT levels, absence of hepatomegaly, presence of cholic stools and/or excretion on HIDA scan to undergo pecutaneous liver biopsy given the lower likelihood of BA necessary.

Antioxidant enzyme activities in hepatic tissue from children with chronic cholestatic liver disease.

BACKGROUND/AIM: To study the oxidative stress status in children with cholestatic chronic liver disease by determining activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) in liver tissue. MATERIALS AND METHODS: A total of 34 children suffering from cholestatic chronic liver disease were studied. They were selected from the Hepatology Clinic, Cairo University, and compared with seven children who happened to have incidental normal liver biopsy. The patients were divided into three groups: extrahepatic biliary atresia (n=13), neonatal hepatitis (n=15) and paucity of intrahepatic bile ducts (n=6); GPx, SOD and CAT levels were measured in fresh liver tissue using ELISA. RESULTS: In the cholestatic patients, a significant increase was found in mean levels of SOD, GPx and CAT in hepatic tissue compared to control children. The three enzymes significantly increased in the extrahepatic biliary atresia group, whereas in the groups of neonatal hepatitis and paucity of intrahepatic bile ducts, only GPx and CAT enzymes were significantly increased. CONCLUSION: Oxidative stress could play a role in the pathogenesis of cholestatic chronic liver diseases. These preliminary results are encouraging to conduct more extensive clinical studies using adjuvant antioxidant therapy.

Positive association of macrophage migration inhibitory factor gene-173G/C polymorphism with biliary atresia.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotrophic lymphocyte and macrophage cytokine; it is likely to play an important role in innate immunity. Its expression was increased in several inflammatory diseases, and MIF gene polymorphisms have an effect on disease outcome and response to glucocorticoid treatment. AIM: To investigate the role of the 173G/C polymorphism of the MIF gene for susceptibility to biliary atresia (BA). METHOD: Between February 2002 and November 2004, 18 patients (mean age 1 +/- 0.4 years) diagnosed as having BA were studied. After informed consent, blood was collected, and DNA was obtained. MIF 173C/G polymorphism was detected using the polymerase chain reaction-restriction fragment length polymorphism based method. BA patients were compared with a group of chronic liver disease patients (CLD) (n = 36) and a group of unrelated healthy controls (n = 103). RESULTS: MIF-173C allele frequency was significantly higher than both the CLD and healthy control groups (P = 0.03, odds ratio [OR] 4.4, 95% confidence interval [CI] 1.3-15.1; P = 0.000, OR 4.1, 95% CI 2.3-7.6, respectively). Univariate analysis showed that MIF-173G/C polymorphism was significantly associated with BA (for GC genotype, OR = 6, 95 % CI 2.8-11.5, P = 0.000). There was no significant correlation between pediatric end stage liver disease score and MIF genotypes both in BA and CLD groups. CONCLUSION: Our results suggest that the -173C allele of the MIF gene might be associated with the susceptibility to BA.

Evaluation of matrix metalloproteinases and their endogenous tissue inhibitors in biliary atresia-associated liver fibrosis.

BACKGROUND/PURPOSE: Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are major proteases responsible for remodeling the liver tissue, but their roles in biliary atresia (BA)--associated liver fibrosis are not clear. METHODS: A DNA microarray containing complementary DNA clones of 10 MMPs and 4 TIMPs was used to compare the expression profiles of the liver cytokines among 3 patients with BA at the time of Kasai procedure (KP) with 3 at the time of liver transplantation (LT). Liver samples from 2 children without liver fibrosis were used as normal controls. Those genes that were differentially expressed by more than 2-fold between groups were further quantified with real time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and validated with gel electrophoresis. RESULTS: In normal human liver, messenger RNAs (mRNAs) of TIMP-1, -2, and -3, but not of TIMP-4 and none of the 10 MMPs studied, were expressed in DNA microarray. With progression of liver fibrosis, only mRNA of MMP-7, but not other MMPs, was induced to express at a significantly higher level in the array. Despite its low level of expression, MMP-9 mRNA was significantly upregulated in KP but downregulated in LT, whereas MMP-2, which was not showed in the array, was significantly upregulated in LT than in KP and control in real time QRT-PCR. There was a more than 2-fold increase in TIMP-1 and TIMP-2 mRNA expression in LT over control in the array, which was confirmed in subsequent real time QRT-PCR. The expression of TIMP-3 mRNA was significantly downregulated in KP than in control. CONCLUSIONS: This study verified differential expression of MMPs and TIMPs in different stages of BA, with emphasis on the role of TIMP-1, -2, and -3 as well as MMP-2, -7, and -9 transcripts in remodeling of liver tissue during the progress of BA-associated liver fibrosis.