Accuracy of Transient Elastography Data Combined With APRI in Detection and Staging of Liver Disease in Pediatric Patients With Cystic Fibrosis.

BACKGROUND & AIMS: Liver disease develops in 15%-72% of patients with cystic fibrosis, and 5%-10% develop cirrhosis or portal hypertension, usually during childhood. Transient elastography (TE) is a noninvasive method to measure liver stiffness. We aimed to validate its accuracy in detection of liver disease and assessment of fibrosis in children with cystic fibrosis. METHODS: We performed a cross-sectional study to evaluate the accuracy of TE in analysis of liver disease in 160 consecutive children who presented with cystic fibrosis (9.0 ± 0.4 years old, 53% male) at a tertiary referral pediatric center in Australia, from 2011 through 2016. Patients were classified as having cystic fibrosis-associated liver disease (CFLD) or cystic fibrosis without liver disease (CFnoLD) based on clinical, biochemical, and imaging features. Fibrosis severity was determined from histologic analysis of dual-pass liver biopsies from children with CFLD, as the reference standard. Data from healthy children without cystic fibrosis (n = 64, controls) were obtained from a separate study. Liver stiffness measurements (LSMs) were made by Fibroscan analysis, using the inter-quartile range/median ≤30% of 10 valid measurements. Children with macronodularity or portal hypertension with heterogeneous changes on ultrasound without available biopsy were assigned to the category of stage F3-F4 fibrosis. RESULTS: LSM was made reliably in 86% of children; accuracy increased with age. LSMs were significantly higher in children with CFLD (10.7 ± 2.4 kPa, n = 33) than with CFnoLD (4.6 ± 0.1 kPa, n = 105) (P < .0001) or controls (4.1 ± 0.1kPa) (P < .0001); LSMs were higher in children with CFnoLD than controls (P < .05). At a cut-off value of 5.55kPa, LSM identified children with CFLD with an area under the receiver operating characteristic (AUROC) curve of 0.82, 70% sensitivity, and 82% specificity (P < .0001). Classification and regression tree models that combined LSM and aspartate aminotransferase to platelet ratio index (APRI) identified children with CFLD with an AUROC curve of 0.89, 87% sensitivity, and 74% specificity (odds ratio, 18.6). LSMs correlated with fibrosis stage in patients with CFLD (r = 0.67, P = .0001). A cut-off value of 8.7kPa differentiated patients with stage F3-F4 fibrosis from patients with stage F1-F2 fibrosis (AUROC, 0.87; 75% sensitivity; 100% specificity, P=.0002). The combination of LSMs and APRI improved the differentiation of patients with F3-F4 fibrosis vs F1-F2 fibrosis (AUROC, 0.92; 83% sensitivity; and 100% specificity (P < .01). CONCLUSIONS: LSMs made by TE accurately detect liver disease in children with cystic fibrosis; diagnostic accuracy increases when LSMs are combined with APRI. LSMs also differentiate between children with cystic fibrosis with mild-moderate fibrosis vs advanced fibrosis.

Lung and liver growth and retinoic acid status in human fetuses with congenital diaphragmatic hernia.

BACKGROUND: Abnormal retinoic acid (RA) signalling is considered a major cause of congenital diaphragmatic hernia (CDH). Pulmonary hypoplasia and pulmonary hypertension are the major causes of morbidity and mortality in infants born with CDH. Experimental studies in animals have found that RA signalling is involved in lung and liver development, but animal models of CDH do not directly correlate with CDH in human fetuses. This study investigated if RA status is also linked to lung and liver growth in human fetuses with CDH. STUDY DESIGN AND PATIENTS: Hepatic stellate cells (HSC) in autopsy human fetal liver tissue were identified using cRBP-1 immunohistochemistry and the numbers of HSC manually counted. In mammals, RA is principally stored in HSC complexed to cRBP-1 and therefore cRBP-1+ HSC numbers were used as an indicator of fetal RA status. The number of HSCs was correlated with liver and lung weights, calculated relative to either normal biometric values or fetal body weight. RESULTS: The number of cRBP-1+ HSCs correlated with lung weight contralateral to the side of the diaphragmatic hernia (r=0.82, p=0.025) and combined lung weight (r=0.78, p=0.039) but not with ipsilateral lung weight (r=0.43, p=0.33), in fetuses with right and left CDH and a case of giant omphalocoele. Liver growth was influenced by contact with diaphragm but not significantly correlated with cRBP-1 expression (r=0.52, p=0.056). CONCLUSION: Fetal RA stores, reflected in the number of cRBP-1+ HSCs, influence lung growth as well as diaphragm development in human fetuses with CDH. Contact with diaphragm influenced liver growth.

Taurocholate Induces Biliary Differentiation of Liver Progenitor Cells Causing Hepatic Stellate Cell Chemotaxis in the Ductular Reaction: Role in Pediatric Cystic Fibrosis Liver Disease.

Cystic fibrosis liver disease (CFLD) in children causes progressive fibrosis leading to biliary cirrhosis; however, its cause(s) and early pathogenesis are unclear. We hypothesized that a bile acid-induced ductular reaction (DR) drives fibrogenesis. The DR was evaluated by cytokeratin-7 immunohistochemistry in liver biopsies, staged for fibrosis, from 60 children with CFLD, and it demonstrated that the DR was significantly correlated with hepatic fibrosis stage and biliary taurocholate levels. To examine the mechanisms involved in DR induction, liver progenitor cells (LPCs) were treated with taurocholate, and key events in DR evolution were assessed: LPC proliferation, LPC biliary differentiation, and hepatic stellate cell (HSC) chemotaxis. Taurocholate induced a time-dependent increase in LPC proliferation and expression of genes associated with cholangiocyte differentiation (cytokeratin 19, connexin 43, integrin ß4, and γ-glutamyltranspeptidase), whereas the hepatocyte specification marker HNF4α was suppressed. Functional cholangiocyte differentiation was demonstrated via increased acetylated α-tubulin and SOX9 proteins, the number of primary cilia+ LPCs, and increased active γ-glutamyltranspeptidase enzyme secretion. Taurocholate induced LPCs to release MCP-1, MIP1α, and RANTES into conditioned medium causing HSC chemotaxis, which was inhibited by anti-MIP1α. Immunofluorescence confirmed chemokine expression localized to CK7+ DR and LPCs in CFLD liver biopsies. This study suggests that taurocholate is involved in initiating functional LPC biliary differentiation and the development of the DR, with subsequent induction of chemokines that drive HSC recruitment in CFLD.

Transient liver elastography in unsedated control children: Impact of age and intercurrent illness.

AIM: Transient elastography (TE) is a rapid, non-invasive, reproducible assessment of liver fibrosis by liver stiffness measurement (LSM). Uncertainty remains regarding utility in children, unsedated and <6 years of age. The importance of general health at the time of study has not been addressed. We report our experience of TE in unsedated control children, impact of intercurrent illness and using new published reliability criteria. METHODS: From April 2011 to March 2013, 173 studies were performed in unsedated, healthy control children and children with intercurrent illness without detectable liver disease presenting to the Royal Children's Hospital, Brisbane, Australia. LSM reliability was assessed using interquartile range/median (IQR/M ≤ 30%) of 10 valid measurements. RESULTS: A total of 123 (F:M, 52:71) of 173 studies (71.1%) gave reliable results. In children 0-2 years reliability was 36%, and >2 years reliable results were obtained in ~80%. LSM increased with age; 0-2 years (3.5 ± 0.5 kPa), 3-5 years (3.8 ± 0.3 kPa) and 6-11 years (4.1 ± 0.2 kPa) with healthy older children 12-18 years similar to adults (4.5 ± 0.2 kPa). LSM did not vary with gender (female, 4.5 ± 0.2 vs. male, 4.8 ± 0.2 kPa). Children with intercurrent, non-hepatological illnesses had higher LSM (5.2 ± 0.2 kPa (range, 2.8-11.1 kPa)) compared to healthy children ((4.1 ± 0.1 kPa, range, 2.1-6.3 kPa); P = 0.0001). CONCLUSIONS: TE in unsedated children is feasible from infancy but most reliable after 2 years. Intercurrent illness increases LSM; hence, study context is important when interpreting results.

Mechanism of pancreatic and liver malformations in human fetuses with short-rib polydactyly syndrome.

BACKGROUND: The short-rib polydactyly (SRP) syndromes are rare skeletal dysplasias caused by abnormalities in primary cilia, sometimes associated with visceral malformations. METHODS: The pathogenesis of ductal plate malformation (DPM) varies in different syndromes and has not been investigated in SRP. We have studied liver development in five SRP fetuses and pancreatic development in one SRP fetus, with genetically confirmed mutations in cilia related genes, with and without DPMs, using the immunoperoxidase technique, and compared these to other syndromes with DPM. RESULTS: Acetylated tubulin expression was abnormal in DPM in SRP, Meckel syndrome, and autosomal recessive polycystic kidney disease (ARPKD), confirming ciliary anomalies. SDF-1 was abnormally expressed in SRP and two of three cases of autosomal dominant polycystic kidney disease (ADPKD) but not ARPKD or Meckel. Increased density of quiescent hepatic stellate cells was seen in SRP, Meckel, one of three cases of ARPKD, and two of three cases of ADPKD with aberrant hepatocyte expression of keratin 19 in SRP and ADPKD. Immunophenotypic abnormalities were present even in fetal liver without fully developed DPMs. The SRP case with DPM and pancreatic malformations showed abnormalities in the pancreatic head (influenced by mesenchyme from the septum transversum, similar to liver) but not pancreatic body (influenced by mesenchyme adjacent to the notochord). CONCLUSION: In SRP, there are differentiation defects of hepatocytes, cholangiocytes, and liver mesenchyme and, in rare cases, pancreatic mesenchymal anomalies. The morphological changes were subtle in early gestation but immunophenotypic abnormalities were present. Mesenchymal-epithelial interactions may contribute to the malformations. Birth Defects Research (Part A) 106:549-562, 2016. © 2016 Wiley Periodicals, Inc.

The development of hepatic stellate cells in normal and abnormal human fetuses - an immunohistochemical study.

The precise embryological origin and development of hepatic stellate cells is not established. Animal studies and observations on human fetuses suggest that they derive from posterior mesodermal cells that migrate via the septum transversum and developing diaphragm to form submesothelial cells beneath the liver capsule, which give rise to mesenchymal cells including hepatic stellate cells. However, it is unclear if these are similar to hepatic stellate cells in adults or if this is the only source of stellate cells. We have studied hepatic stellate cells by immunohistochemistry, in developing human liver from autopsies of fetuses with and without malformations and growth restriction, using cellular Retinol Binding Protein-1 (cRBP-1), Glial Fibrillary Acidic Protein (GFAP), and α-Smooth Muscle Actin (αSMA) antibodies, to identify factors that influence their development. We found that hepatic stellate cells expressing cRBP-1 are present from the end of the first trimester of gestation and reduce in density throughout gestation. They appear abnormally formed and variably reduced in number in fetuses with abnormal mesothelial Wilms Tumor 1 (WT1) function, diaphragmatic hernia and in ectopic liver nodules without mesothelium. Stellate cells showed similarities to intravascular cells and their presence in a fetus with diaphragm agenesis suggests they may be derived from circulating stem cells. Our observations suggest circulating stem cells as well as mesothelium can give rise to hepatic stellate cells, and that they require normal mesothelial function for their development.

Case Report: Fetal Bilateral Diaphragmatic Agenesis, Ectopic Liver and Abnormal Pancreas.

Congenital bilateral diaphragm agenesis is a very rare condition. We describe limited (abdomen only) autopsy findings of a case of bilateral diaphragm agenesis in a 27-week male fetus with unusual findings of fibrosis of the pancreatic head and ectopic liver nodules in a mass at the upper abdomen that may represent a possible diaphragm anlage. We have correlated our observations with data from experimental and embryological studies to suggest possible mechanisms for the malformations that were present and their implications for our understanding of pancreas, liver and diaphragm development in the human fetus.

Circulating microRNAs as noninvasive diagnostic biomarkers of liver disease in children with cystic fibrosis.

OBJECTIVES: Cystic fibrosis liver disease (CFLD), resulting from progressive hepatobiliary fibrosis, causes significant morbidity and mortality in up to 20% of children with cystic fibrosis (CF). Both pathogenesis and early detection of CFLD are elusive. Current diagnostic procedures to detect early CFLD and stage fibrosis severity are inadequate. Recent studies highlight a role for microRNAs (miRNAs) in the pathogenesis of many diseases and have suggested that serum miRNAs could be used as diagnostic biomarkers. METHODS: We profiled circulating serum miRNA levels in patients with CFLD (n = 52), patients with CF without liver disease (CFnoLD, n = 30), and non-CF pediatric controls (n = 20). Extracted RNA was subjected to polymerase chain reaction (PCR) array of 84 miRNAs detectable in human serum. Seven candidate miRNAs identified were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), normalizing data to geNorm-determined stable reference genes, miR-19b and miR-93. RESULTS: miR-122 was significantly elevated in patients with CFLD versus patients with CFnoLD and controls (P < 0.0001). miR-25 (P = 0.0011) and miR-21 (P = 0.0133) were elevated in patients with CFnoLD versus patients with CFLD and controls. CFLD was discriminated by both miR-122 (area under the curve [AUC] 0.71, P = 0.002) and miR-25 (AUC 0.65, P = 0.026). Logistic regression combining 3 miRNAs (-122, -25, -21) was greatly predictive of detecting CFLD (AUC 0.78, P < 0.0001). A combination of 6 miRNAs (-122, -21, -25, -210, -148a, -19a) distinguished F0 from F3-F4 fibrosis (AUC 0.73, P = 0.04), and miR-210 combined with miR-22 distinguished F0 fibrosis from any fibrosis, that is, F1-F4 (AUC 0.72, P = 0.02). CONCLUSIONS: These data provide the first evidence of changes to circulating miRNA levels in CF, suggesting that serum-based miRNA analysis may complement and extend current CFLD screening strategies with potential to predict early hepatic fibrosis.

Abnormal WT1 expression in human fetuses with bilateral renal agenesis and cardiac malformations.

BACKGROUND: Bilateral renal agenesis has multiple etiologies. Animal models have provided useful information on possible causes of this condition, but its etiology in humans is less clear. We recently described autopsy findings of two human fetuses with bilateral renal agenesis and abnormal expression of WT1 (Wilms tumor 1) in liver mesothelium. METHODS: We have identified 14 additional fetuses with bilateral renal agenesis from autopsies performed in our institution over the past 10 years and subjected archival liver biopsy specimens from these cases to immunohistochemistry for WT1, as well as α-smooth muscle actin (α-SMA) and desmin to assess liver mesenchymal abnormalities. RESULTS: Six of seven fetuses with combined bilateral renal agenesis and cardiac anomalies showed abnormalities of WT1 expression in liver mesothelial cells, which was not seen in other fetuses with bilateral renal agenesis. Except in one case, the fetuses with renal agenesis and cardiac defects also showed liver mesenchymal anomalies (assessed by increased α-SMA expression), which was not present in other renal agenesis fetuses. CONCLUSIONS: WT1 is widely expressed in mesothelial cells during development, and we hypothesized that some of the defects are caused by abnormal function of mesenchyme derived from mesothelial cells, similar to the mesothelium-derived defects proposed in animal models. The methods we used are available to many laboratories and can be applied to archival paraffin tissue blocks. We suggest that future similar studies could help to expand the understanding of renal agenesis in humans and could help to subclassify this condition. This would be useful in patient management and counseling.

Possible role of WT1 in a human fetus with evolving bronchial atresia, pulmonary malformation and renal agenesis.

The association of peripheral bronchial atresia and congenital pulmonary airway malformation (CPAM) has recently been recognised, but the pathology of the lesions evolving together has not been described. We present autopsy findings in a 20 week fetus showing areas of peripheral bronchial destruction and airway malformation consistent with developing CPAM in the right lung supporting a causal relationship between these lesions. This fetus also had congenital heart defect, bilateral renal agenesis and syndactyly. We identified another fetus from our autopsy files, with bilateral renal agenesis, similar right sided pulmonary malformation and cardiac defects. Similar bilateral renal agenesis and defects of the heart and lungs are found in wt1(-/-) mice and we have investigated the expression of WT1 in these fetuses. We hypothesise that the cardiac, liver, renal and possibly lung lesions in these two cases may arise due to mesenchymal defects consequent to WT1 misexpression and discuss evidence for this from the scientific literature. We used immunoperoxidase stains to analyse WT1 expression in autopsy hepatic tissue in both fetuses. We also investigated the expression of α-smooth muscle actin (α-SMA), a marker of activated hepatic stellate cells/myofibroblasts, and desmin in hepatic mesenchyme and compare these findings with control fetuses, without congenital malformations. We found reduced WT1 expression in hepatic mesothelium in both fetuses with malformations. There was also increased expression of α-SMA in liver perisinusoidal cells, as seen in the wt1(-/-) mouse model. We therefore propose that abnormality of WT1 signalling may be an underlying factor, as WT1 is expressed in coelomic lining cells from which mesenchyme is derived in many organs.

Transcriptional basis for hepatic fibrosis in cystic fibrosis-associated liver disease.

OBJECTIVES: Liver disease contributes to significant morbidity and mortality in cystic fibrosis (CF). Although all patients with CF express the defective CF transmembrane conductance regulator in cholangiocytes, many develop asymptomatic fibrosing liver disease. Only some develop cirrhosis, with pathogenesis remaining enigmatic. Available noninvasive diagnostic tools do not identify patients at risk before development of advanced fibrosis. We conducted a pilot study to identify genes associated with hepatic injury and fibrosis on liver biopsy that may help elucidate determinants of CF-associated liver disease (CFLD). METHODS: Liver tissue from children with CFLD with various stages of hepatic fibrosis was compared with pediatric controls using cDNA array analysis. Differential expression of genes of interest was then assessed relative to pediatric control liver and non-CF cholestatic disease control liver from patients with biliary atresia, using both real-time reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: cDNA array demonstrated differential expression of numerous genes associated with hepatic fibrogenesis including collagens, matrix metalloproteinases, and chemokines in CFLD versus normal controls, particularly decreased expression in tissue remodeling genes including plasminogen activator inhibitor-1 (PAI-1, up to 25-fold) and tissue inhibitor of metalloproteinase-1 (TIMP-1); this was validated by real-time reverse transcription-polymerase chain reaction (PAI-1, P = 0.004; TIMP-1, P = 0.019). No significant decrease in PAI-1 or TIMP-1 mRNA was observed in biliary atresia versus normal control. Immunohistochemistry confirmed the decreased expression of hepatic PAI-1 and TIMP-1 protein in CFLD versus both normal and biliary atresia disease controls. CONCLUSIONS: The coordinated differential expression of these genes associated with liver fibrosis provides evidence for a transcriptional basis for the pathogenesis of CFLD and provides avenues for further study. Clarifying the pathogenesis of CFLD will facilitate techniques for early, precirrhotic detection and targeted interventions.

Importance of hepatic fibrosis in cystic fibrosis and the predictive value of liver biopsy.

UNLABELLED: Cystic fibrosis liver disease (CFLD), which results from progressive hepatobiliary fibrosis, is an important cause of morbidity and mortality, but it is difficult to identify before portal hypertension (PHT) ensues. Clinical signs, serum alanine aminotransferase (ALT) levels, and ultrasound (US) are widely applied, but their value in predicting the presence of cirrhosis, the development of PHT, or adverse outcomes is undetermined. The potential gold standard, liver biopsy, is not standard practice and, notwithstanding sampling error considerations, has not been systematically evaluated. Forty patients with cystic fibrosis (median age = 10.6 years) with abnormal clinical, biochemical, and US findings were subjected to dual-pass percutaneous liver biopsy. Clinical outcomes were recorded over 12 years of follow-up (median = 9.5 years for survivors). Logistic regression and receiver operating characteristic analyses were applied to predict hepatic fibrosis (which was assessed by fibrosis staging and quantitative immunohistochemistry) and the occurrence of PHT. PHT occurred in 17 of 40 patients (42%), including 6 of 7 (17%) who died during follow-up. Clinical examination, serum ALT levels, and US findings failed to predict either the presence of liver fibrosis or the development of PHT. Fibrosis staging on liver biopsy, where the accuracy was improved by dual passes (P = 0.002, nonconcordance = 38%), predicted the development of PHT (P < 0.001), which occurred more frequently and at a younger age in those with severe fibrosis. CONCLUSION: Clinical modalities currently employed to evaluate suspected CFLD help to identify a cohort of children at risk for liver disease and adverse outcomes but do not predict an individual's risk of liver fibrosis or PHT development. Liver fibrosis on biopsy predicts the development of clinically significant liver disease. Dual passes help to address sampling concerns. Liver biopsy has a relevant role in the management of patients with suspected CFLD and deserves more widespread application.

Paediatric cholestatic liver disease: Diagnosis, assessment of disease progression and mechanisms of fibrogenesis.

Cholestatic liver disease causes significant morbidity and mortality in children. The diagnosis and management of these diseases can be complicated by an inability to detect early stages of fibrosis and a lack of adequate interventional therapy. There is no single gold standard test that accurately reflects the presence of liver disease, or that can be used to monitor fibrosis progression, particularly in conditions such as cystic fibrosis. This has lead to controversy over how suspected liver disease in children is detected and diagnosed. This review discusses the challenges in using commonly available methods to diagnose hepatic fibrosis and monitor disease progression in children with cholestatic liver disease. In addition, the review examines the mechanisms hypothesised to be involved in the development of hepatic fibrogenesis in paediatric cholestatic liver injury which may ultimately aid in identifying new modalities to assist in both disease detection and therapeutic intervention.

Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.

UNLABELLED: Cholestatic liver diseases, such as cystic fibrosis (CF) liver disease and biliary atresia, predominate as causes of childhood cirrhosis. Despite diverse etiologies, the stereotypic final pathway involves fibrogenesis where hepatic stellate cells (HSCs) are recruited, producing excess collagen which initiates biliary fibrosis. A possible molecular determinant of this recruitment, monocyte chemotaxis protein-1 (MCP-1), an HSC-responsive chemokine, was investigated in CF liver disease and biliary atresia. The bile-duct-ligated rat and in vitro coculture models of cholestatic liver injury were used to further explore the role of MCP-1 in HSC recruitment and proposed mechanism of induction via bile acids. In both CF liver disease and biliary atresia, elevated hepatic MCP-1 expression predominated in scar margin hepatocytes, closely associated with activated HSCs, and was also expressed in cholangiocytes. Serum MCP-1 was elevated during early fibrogenesis. Similar observations were made in bile-duct-ligated rat liver and serum. Hepatocytes isolated from cholestatic rats secreted increased MCP-1 which avidly recruited HSCs in coculture. This HSC chemotaxis was markedly inhibited in interventional studies using anti-MCP-1 neutralizing antibody. In CF liver disease, biliary MCP-1 was increased, positively correlating with levels of the hydrophobic bile acid, taurocholate. In cholestatic rats, increased MCP-1 positively correlated with taurocholate in serum and liver, and negatively correlated in bile. In normal human and rat hepatocytes, taurocholate induced MCP-1 expression. CONCLUSION: These observations support the hypothesis that up-regulation of hepatocyte-derived MCP-1, induced by bile acids, results in HSC recruitment in diverse causes of cholestatic liver injury, and is a key early event in liver fibrogenesis in these conditions. Therapies aimed at neutralizing MCP-1 or bile acids may help reduce fibro-obliterative liver injury in childhood cholestatic diseases.

Serum markers of hepatic fibrogenesis in cystic fibrosis liver disease.

BACKGROUND/AIMS: Hepatic fibrosis contributes to adverse outcome in cystic fibrosis (CF). Early detection of CF liver disease (CFLD) may identify patients at risk of significant complications. To evaluate the utility of serum markers to detect hepatic fibrosis in children with CFLD vs. CF patients without liver disease (CFnoLD) and controls. METHODS: Sera from 36 CFLD, 30 CFnoLD and 39 controls were assessed for tissue inhibitor of matrix metalloproteinase (MMP) (TIMP)-1, collagen (CL)-IV, MMP-2, hyaluronic acid (HA) and prolyl hydroxylase (PH) by enzyme immunoassay and were correlated with hepatic fibrosis score in CFLD. RESULTS: TIMP-1, PH and CL-IV were increased in CFLD vs. CFnoLD and controls. Fibrosis score was negatively correlated with TIMP-1 (r=-0.34, P=0.06) and PH (r=-0.48, P=0.008). Receiver-operating characteristics analysis showed CL-IV (AUC 0.785, P<0.0001) and TIMP-1 (AUC 0.765, P<0.0001) differentiated CFLD from CFnoLD and controls, while PH (AUC 0.814, P<0.0001) predicted early fibrogenesis. Diagnostic accuracy improved using logistic regression combining (i) CL-IV, TIMP-1, PH to identify CFLD (AUC 0.831, P<0.0001) and (ii) TIMP-1, PH to identify CFLD patients with no fibrosis (AUC 0.852, P<0.02). CONCLUSIONS: Elevated TIMP-1, CL-IV, PH may be indicators of hepatic fibrogenesis in CF. Increased TIMP-1, PH may be early markers of CFLD.

Endogenous ursodeoxycholic acid and cholic acid in liver disease due to cystic fibrosis.

Focal biliary cirrhosis causes significant morbidity and mortality in cystic fibrosis (CF). Although the mechanisms of pathogenesis remain unclear, bile acids have been proposed as potential mediators of liver injury. This study examined bile acid composition in CF and assessed altered bile acid profiles to determine if they are associated with incidence and progression of liver injury in CF-associated liver disease (CFLD). Bile acid composition was determined by gas-liquid chromatography/mass spectrometry in bile, urine, and serum samples from 30 children with CFLD, 15 children with CF but without liver disease (CFnoLD), and 43 controls. Liver biopsies from 29 CFLD subjects were assessed histologically by grading for fibrosis stage, inflammation, and disruption of the limiting plate. A significantly greater proportion of endogenous biliary ursodeoxycholic acid (UDCA) was demonstrated in CFnoLD subjects vs. both CFLD subjects and controls (2.4- and 2.2-fold, respectively; ANOVA, P =.04), and a 3-4 fold elevation in endogenous serum UDCA concentration was observed in both CFLD subjects and CFnoLD subjects vs. controls (ANOVA, P <.05). In CFLD, there were significant correlations between serum cholic acid and hepatic fibrosis, inflammation, and limiting plate disruption as well as the ratio of serum cholic acid/chenodeoxycholic acid to hepatic fibrosis, inflammation, and limiting plate disruption. In conclusion, elevated endogenous UDCA in CFnoLD suggests a possible protective role against liver injury in these patients. The correlation between both cholic acid and cholic acid/chenodeoxycholic acid levels with histological liver injury and fibrosis progression suggests a potential monitoring role for these bile acids in CFLD.

The role of hepatic stellate cells and transforming growth factor-beta(1) in cystic fibrosis liver disease.

Liver disease causes significant morbidity and mortality from multilobular cirrhosis in patients with cystic fibrosis. Abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cystic fibrosis-associated liver disease (CFLD), yet the mediators linking biliary events to fibrosis remain unknown. Activated hepatic stellate cells (HSCs) are the pre-eminent mediators of fibrosis in a range of hepatic disorders. The dominant stimulus for matrix production by HSCs is the cytokine transforming growth factor (TGF)-beta(1). In CFLD, the role of HSCs and the source of TGF-beta(1) have not been evaluated. Liver biopsy tissue obtained from 38 children with CFLD was analyzed. Activated HSCs, identified by co-localization of procollagen alpha(1)(I) mRNA and alpha-smooth muscle actin, were demonstrated as the cellular source of excess collagen production in the fibrosis surrounding the bile ducts and the advancing edge of scar tissue. TGF-beta protein and TGF-beta(1) mRNA expression were shown to be predominantly expressed by bile duct epithelial cells. TGF-beta(1) expression was significantly correlated with both hepatic fibrosis and the percentage of portal tracts showing histological abnormalities associated with CFLD. This study demonstrates a definitive role for HSCs in fibrogenesis associated with CFLD and establishes a potential mechanism for the induction of HSC collagen gene expression through the production of TGF-beta(1) by bile duct epithelial cells.