Jorge Luiz dos Santos

Th2 signals induce epithelial injury in mice and are compatible with the biliary atresia phenotype

Biliary atresia (BA) is a destructive cholangiopathy of childhood in which Th1 immunity has been mechanistically linked to the bile duct inflammation and obstruction that culminate in liver injury. Based on reports of decreased Th1 cytokines in some patients and the development of BA in mice lacking CD4+ T cells, we hypothesized that Th1-independent mechanisms can also activate effector cells and induce BA. Here, we tested this hypothesis using Stat1-/- mice, which lack the ability to mount Th1 immune responses. Infection of Stat1-/- mice with rhesus rotavirus type A (RRV) on postnatal day 1 induced a prominent Th2 response, duct epithelial injury and obstruction within 7 days, and atresia shortly thereafter. A high degree of phosphorylation of the Th2 transcription factor Stat6 was observed; however, concurrent inactivation of Stat1 and Stat6 in mice did not prevent BA after RRV infection. In contrast, depletion of macrophages or combined loss of Il13 and Stat1 reduced tissue infiltration by lymphocytes and myeloid cells, maintained epithelial integrity, and prevented duct obstruction. In concordance with our mouse model, humans at the time of BA diagnosis exhibited differential hepatic expression of Th2 genes and serum Th2 cytokines. These findings demonstrate compatibility between Th2 commitment and the pathogenesis of BA, and suggest that patient subgrouping in future clinical trials should account for differences in Th2 status.

The extent of biliary proliferation in liver biopsies from patients with biliary atresia at portoenterostomy is associated with the postoperative prognosis

BACKGROUND/PURPOSE In biliary atresia (BA), a derangement in the biliary system remains, despite portoenterostomy performance. Many factors can influence the disease progression rate. This study aimed to analyze the association between biliary proliferation extent in biopsies from BA patients and postoperative prognosis. METHODS Biliary proliferation was evaluated by a morphometric analysis of the cytokeratin 7 positivity percentage (PCK7) in wedge liver biopsies from 47 BA patients. The extent of fibrosis was evaluated by a fibrosis score (FS). The outcome 1-year native liver survival was correlated, using a multivariable regression analysis, with PCK7, FS, and age at portoenterostomy. RESULTS The PCK7 ranged between 0.80% and 14.79% (M +/- SD = 7.36% +/- 4.15%). Patients who died or underwent transplantation had higher PCK7 than survivors with their native livers (P < .001). The area under the receiver operating characteristic curve for PCK7 in relation to the outcome was 0.845 (P < .001). The cutoff point of PCK7 for the maximal effect on postoperative prognosis was 10.18% (sensitivity = 0.71, specificity = 0.88). The PCK7 was the only studied variable associated with 1-year native liver survival, independently of age and FS (P = .002). CONCLUSION The extent of biliary proliferation at portoenterostomy, evaluated by PCK7, was associated with 1-year native liver survival of BA patients.

Cholestatic liver disease in children.

Inherited syndromes of intrahepatic cholestasis and biliary atresia are the most common causes of chronic liver disease and the prime indication for liver transplantation in children. Our understanding of the pathogenesis of these diseases has increased substantially by the discovery of genetic mutations in children with intrahepatic cholestasis and the findings that inflammatory circuits are operative at the time of diagnosis of biliary atresia. Building on this solid foundation, recent studies provide new insight into genotype-phenotype relationships and how mutations produce altered bile composition and cholestasis. New evidence exists that although liver transplantation is curative for patients with end-stage liver disease owing to cholestasis, some patients may develop recurrence of cholestasis because of the emergence of autoantibodies that disrupt canalicular function in the new graft. Progress is also evident in biliary atresia, with recent studies identifying candidate modifier genes and directly implicating lymphocytes and inflammatory signals in the pathogenesis of bile duct injury and obstruction.

Advances in biliary atresia: from patient care to research

Biliary atresia, the most common cause of liver transplantation in children, remains a challenge for clinicians and investigators. The development of new therapeutic options, besides the typical hepatoportoenterostomy, depends on a greater understanding of its pathogenesis and how it relates to the clinical phenotypes at diagnosis and the rate of disease progression. In this review, we present a perspective of how recent research has advanced the understanding of the disease and has improved clinical care protocols. Molecular and morphological analyses at diagnosis point to the potential contributions of polymorphism in the CFC1 and VEGF genes to the pathogenesis of the disease, and to an association between the degree of bile duct proliferation and long-term outcome. In experimental models, cholangiocytes do not appear to have antigen-presenting properties despite a substantial innate and adaptive immune response that targets the biliary epithelium and produces duct obstruction. Initial clinical trials assessing the efficacy of corticosteroids in decreasing the inflammation and improving outcome do not show a superior effect of corticosteroids as an adjuvant treatment following hepatoportoenterostomy. The best outcome still remains linked to an early diagnosis and surgical treatment. In this regard, the Yellow Alert campaign by the Sociedade Brasileira de Pediatria and the inclusion of the Stool Color Card in the health booklet given to every neonate in Brazil have the potential to decrease the age of diagnosis, shorten the time between diagnosis and surgical treatment, and improve the long-term outcome of children with this devastating disease.

Histopathological diagnosis of intra- and extrahepatic neonatal cholestasis

The histopathology of the liver is fundamental for the differential diagnosis between intra- and extrahepatic causes of neonatal cholestasis. However, histopathological findings may overlap and there is disagreement among authors concerning those which could discriminate between intra- and extrahepatic cholestasis. Forty-six liver biopsies (35 wedge biopsies and 11 percutaneous biopsies) and one specimen from a postmortem examination, all from patients hospitalized for neonatal cholestasis in the Pediatrics Service of Hospital de Clínicas de Porto Alegre, were prospectively studied using a specially designed histopathological protocol. At least 4 of 5 different stains were used, and 46 hepatic histopathological variables related to the differential diagnosis of neonatal cholestasis were studied. The findings were scored for severity on a scale from 0 to 4. Sections which showed less than 3 portal spaces were excluded from the study. Sections were examined by a pathologist who was unaware of the final diagnosis of each case. Bile tract permeability was defined by scintigraphy of the bile ducts and operative cholangiography. The F test and discriminant analysis were used as statistical methods for the study of the hepatic histopathological variables. The chi-square method with Yates correction was used to relate the age of the patients on the date of the histopathological study to the discriminatory variables between intra- and extrahepatic cholestasis selected by the discriminant function test. The most valuable hepatic histopathological variables for the discrimination between intra- and extrahepatic cholestasis, in decreasing order of importance, were periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, foci of myeloid metaplasia, and portal-portal bridges. The only variable which pointed to the diagnosis of intrahepatic cholestasis was myeloid metaplasia. Due to the small number of patients who were younger than 60 days on the date of the histopathological study (N = 6), no variable discriminated between intra- and extrahepatic cholestasis before the age of 2 months and all of them, except for the portal expansion, were discriminatory after this age. In infants with cholestasis, foci of myeloid metaplasia, whenever present in the liver biopsy, suggested intrahepatic cholestasis. Periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, portal cholestasis and portal-portal bridges suggested extrahepatic obstructive cholestasis.

Biliary atresia : we still operate too late

OBJECTIVE To analyze the age at surgery for children with biliary atresia and their survival periods without need for liver transplantation. The study was performed at Hospital de Clínicas de Porto Alegre, in Porto Alegre, state of Rio Grande do Sul, Brazil. METHODS The medical records of patients operated between 1982 and 2007 who were residents of the state of Rio Grande do Sul were reviewed. RESULTS Of the 112 cases of children with biliary atresia studied, 38 (33.9%) occurred between 1982 and 1989, 46 (41.1%) between 1990 and 1999 and 28 (25.0%) after 2000. Portoenterostomy was not performed for 12 cases (10.7%). Age at surgery ranged from 25 to 297 days (median: 80.5; IQR25-75: 61.3-109.0 days); for 20.5% of cases, the age was below 60 days. There was no age difference at diagnosis for the three decades in the study. Patients from the countryside (median: 87.0; IQR25-75: 69.0-115.0 days) were referred significantly later (p = 0.007) than those living in Porto Alegre and the metropolitan area (median: 68.0; IQR25-75: 55.5-98.0 days). The ratio of patients younger than 60 days was significantly lower (p = 0.013) for those from the countryside. Survival periods with native liver for all patients was 46.2% in 2 years, falling continuously until reaching 15.3% in 20 years. Patients operated before reaching 60 days of age had longer survival periods with native livers (log rank < 0.0001). CONCLUSIONS Late performance of portoenterostomy was a constant in the past 25 years, and this delay led to shorter survival periods with native livers for biliary atresia patients.

Immunolocalization of VEGF A and its receptors, VEGFR1 and VEGFR2, in the liver from patients with biliary atresia.

In biliary atresia (BA), a cholangiopathy of elusive etiology invariably leads to cirrhosis, and a disturbed angiogenesis may be involved. We evaluated the hepatobiliary immunolocalization of vascular endothelial growth factor (VEGF) A, VEGF receptor 1 (R1), and R2 in BA. We analyzed biopsies obtained at portoenterostomy from infants with BA (n=52), including embryonic (n=14) and perinatal (n=38) types. Controls were infants with intrahepatic cholestasis (IC; n=7). In BA, VEGF A immunolocalization was also evaluated in explants (n=33) and at the porta hepatis (n=16). We morphometrically assessed the percentage of CK7 (PCK7) positivity in BA and the ratio medial layer thickness/luminal diameter in hepatic artery branches in BA and IC. We found that arteries were more frequently positive for VEGF A in BA at portoenterostomy (P=0.006) than in other groups. In explants, VEGF A immunolocalization was mainly lobular (P<0.001). VEGFR2 was less frequently positive in BA than IC in bile ducts (P=0.023) and hepatocytes (P=0.011). A higher PCK7 positivity was associated with arterial (P<0.001) and biliary (P=0.040) VEGF A positivity. PCK7 was correlated with biliary (P=0.031), arterial (P=0.031), and hepatocytic (P=0.032) VEGF A positivity in BA at portoenterostomy. VEGF A was positive in arteries and bile ducts at the porta hepatis mainly in the perinatal BA type (P=0.013). Biliary (P=0.016) and arterial (P=0.044) VEGF A positivity were associated with higher ratio medial layer thickness/luminal diameter values. Our findings suggest that hypoxia/ischemia affects the portal structures in BA at portoenterostomy, beginning at the porta hepatis, and it is associated both with the extent of biliary proliferation and medial layer thickening.

Serum and tissue tumor growth factor β1 in children with biliary atresia

BACKGROUND Biliary atresia (BA) is an infantile disorder characterized by the obstruction of a portion or the entirety of the extrahepatic bile ducts, leading to hepatic fibrosis and loss of liver function. The gold standard for diagnosing and grading fibrosis is liver biopsy, but there are many groups searching for noninvasive biomarkers that could replace and/or complement this procedure. METHODS AND MATERIALS In this study, we evaluated serum and tissue transforming growth factor β1 (TGFβ1) and aspartate aminotransferase [AST]-to-platelet ratio index (APRI) in patients with BA at the time of diagnosis and at liver transplantation and correlated these data with tissue collagen density, to verify if they could act as biomarkers for BA. RESULTS At the time of diagnosis, TGFβ1 levels were highly variable in BA patients. However, serum values at transplantation were significantly decreased (13.75 ± 3.68 ng/mL) as compared to controls (34.36 ± 9.35 ng/mL) (P = .01). No correlation was found between serum TGF1β1 and collagen density in both groups analyzed. Serum TGFβ1 showed no correlation with APRI at diagnosis. At the time of liver transplantation, all patients had low serum TGFβ1 and variable APRI, although all higher than 2.0. However, when platelet count was used, an inverse correlation with serum TGFβ1 was observed at the time of diagnostics (r(2) = 0.749; P = .03). CONCLUSIONS Our findings suggest that at the time of diagnosis the fibrogenic process is active, with higher levels of TGFβ1, whereas later on, there is scar tissue, with reduced TGFβ1 expression. Although our results should be confirmed in larger sets of patients with BA, the lack of TGFβ1 at the time of liver transplantation may have important consequences for the patient because it is a pleiotropic molecule, responsible for many functions in the body, mainly those related to immune response and cell growth.

Atresia biliar: a experiência Brasileira

OBJECTIVE: To evaluate epidemiological, clinical and prognostic characteristics of children with biliary atresia. METHODS: Data regarding portoenterostomy, liver transplantation (LTx), age at last follow-up and survival were collected from the records of patients followed up in six Brazilian centers (1982-2008) and compared regarding decades of surgery. RESULTS: Of 513 patients, 76.4% underwent portoenterostomy [age: 60-94.7 (82.6±32.8) days] and 46.6% underwent LTx. In 69% of cases, LTx followed portoenterostomy, whereas in 31% of cases LTx was performed as the primary surgery. Patients from the Northeast region underwent portoenterostomy later than infants from Southern (p = 0.008) and Southeastern (p = 0.0012) Brazil, although even in the latter two regions age at portoenterostomy was higher than desirable. Over the decades, LTx was increasingly performed. Overall survival was 67.6%. Survival increased over the decades (1980s vs. 1990s, p = 0.002; 1980s vs. 2000s, p 90 days, respectively). Higher survival rates were observed among transplanted patients (88.3%). The 4-year native liver survival was 36.8%, inversely correlated with age at portoenterostomy (54, 33.3, 26.6% for 90 days, respectively). CONCLUSIONS: This multicenter study showed that late referral for biliary atresia is still a problem in Brazil, affecting patient survival. Strategies to enhance earlier referral are currently being developed aiming to decrease the need for liver transplantation in the first years of life.

Serum proinflammatory cytokines and nutritional status in pediatric chronic liver disease.

AIM To evaluate the nutritional status and its association with proinflammatory cytokines in children with chronic liver disease. METHODS We performed a cross-sectional study with 43 children and adolescents, aged 0 to 17 years, diagnosed with chronic liver disease. All patients regularly attended the Pediatric Hepatology Unit and were under nutritional follow up. The exclusion criteria were fever from any etiology at the time of enrollment, inborn errors of the metabolism and any chronic illness. The severity of liver disease was assessed by Child-Pugh, Model for End-stage Liver Disease (MELD) and Pediatric End Stage Liver Disease (PELD) scores. Anthropometric parameters were height/age, body mass index/age and triceps skinfold/age according to World Health Organization standards. The cutoff points for nutritional status were risk of malnutrition (Z-score < -1.00) and malnutrition (Z-score < -2.00). Interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α levels were assessed by commercial ELISA kits. For multivariate analysis, linear regression was applied to assess the association between cytokine levels, disease severity and nutritional status. RESULTS The median (25(th)-75(th) centile) age of the study population was 60 (17-116)-mo-old, and 53.5% were female. Biliary atresia was the main cause of chronic liver disease (72%). With respect to Child-Pugh score, cirrhotic patients were distributed as follows: 57.1% Child-Pugh A, a mild presentation of the disease, 34.3% Child-Pugh B, a moderate stage of cirrhosis and 8.6% Child-Pugh C, were considered severe cases. PELD and MELD scores were only above the cutoff point in 5 cases. IL-6 values ​​were increased in patients at nutritional risk (34.9%) compared with those who were well-nourished [7.12 (0.58-34.23) pg/mL vs 1.63 (0.53-3.43) pg/mL; P = 0.02], correlating inversely with triceps skinfold-for-age z-score (rs = -0.61; P < 0.001). IL-6 levels were associated with liver disease severity assessed by Child-Pugh score (P = 0.001). This association remained significant after adjusting for nutritional status in a linear regression model. CONCLUSION High IL-6 levels were found in children with chronic liver disease at nutritional risk. Inflammatory activity may be related to nutritional status deterioration in these patients.