Update on Etiology and Pathogenesis of Biliary Atresia.

Biliary atresia is a rare inflammatory sclerosing obstructive cholangiopathy that initiates in infancy as complete choledochal blockage and progresses to the involvement of intrahepatic biliary epithelium. Growing evidence shows that biliary atresia is not a single entity with a single etiology but a phenotype resulting from multifactorial events whose common path is obliterative cholangiopathy. The etiology of biliary atresia has been explained as resulting from genetic variants, toxins, viral infection, chronic inflammation or bile duct lesions mediated by autoimmunity, abnormalities in the development of the bile ducts, and defects in embryogenesis, abnormal fetal or prenatal circulation and susceptibility factors. It is increasingly evident that the genetic and epigenetic predisposition combined with the environmental factors to which the mother is exposed are potential triggers for biliary atresia. There is also an indication that a progressive thickening of the arterial middle layer occurs in this disease, suggestive of vascular remodeling and disappearance of the interlobular bile ducts. It is suggested that the hypoxia/ischemia process can affect portal structures in biliary atresia and is associated with both the extent of biliary proliferation and the thickening of the medial layer.

Herb-Induced Liver Injury-A Challenging Diagnosis.

Herb-induced liver injury (HILI) can be caused by supplements containing herbs, natural products, and products used in traditional medicine. Herbal products' most common adverse reaction is hepatotoxicity. Almost every plant part can be used to make herbal products, and these products can come in many different forms, such as teas, powders, oils, creams, capsules, and injectables. HILI incidence and prevalence are hard to estimate and vary from study to study because of insufficient large-scale prospective studies. The diagnosis of HILI is a challenging process that requires not only insight but also a high degree of suspicion by the clinician. HILI presents with unspecific symptoms and is a diagnosis of exclusion. For diagnosis, it is necessary to make a causality assessment; the Council for International Organizations of Medical Sciences assessment is the preferred method worldwide. The most effective treatment is the suspension of the use of the suspected herbal product and close monitoring of liver function. The objective of this review is to highlight the necessary steps for the clinician to follow to reach a correct diagnosis of herb-induced liver injury. Further studies of HILI are needed to better understand its complexity and prevent increased morbidity and mortality.

Drug Induced Liver Injury: Perspective of the Adverse Drug Reaction Reports to the Portuguese Pharmacovigilance System from 2010 to 2019.

BACKGROUND: Drug induced liver injury (DILI) is an adverse drug reaction that causes liver damage in a predictable (dose-dependent) or an unpredictable (idiosyncratic) fashion. We performed an assessment of DILI in Portugal, by analyzing the reports, sent to the Portuguese Pharmacovigilance System (PPS). METHODS: A search was performed on the PPS database, in a 10-year time frame, from 1 January 2010 to 31 December 2019. RESULTS: There was not a prevalence of either sex in any age group. Most reports (n = 1120, 55.0%) belonged to patients in the age group 19-64 years old. Hepatitis (n = 626, 26.7%) was the most common adverse drug reaction in our study. Hepatotoxicity (n = 362, 15.5%) and hepatitis (n = 333, 14.2%) were more frequent in age group 19-64 years old. Cholestasis was more prevalent in adults independently of age. Hepatic fibrosis and encephalopathy were more common in the elderly. Most patients consumed between one and four suspected drugs (n = 1867, 92%). Most patients in our study evolved to "cure" (n = 796; 39%). Hepatotoxicity (n = 23; 13.8%) and hepatitis (n = 610; 25.9%) had a female predominancy while choluria (n = 8; 4.8%) and splenomegaly (n = 8; 4.8%) were of male predominance. CONCLUSIONS: DILI is rare but can be fatal. As such, an active search of DILI is necessary.

Ductular reaction, cytokeratin 7 positivity, and gamma-glutamyl transferase in multistage hepatocarcinogenesis in rats.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is characterized by multistage formation. The presence of ductular reaction, cytokeratin 7 positivity (PCK7), and increased levels of gamma glutamyltransferase (γGT) has been observed during liver carcinogenesis and contribute to tumor progression. Our goal was to evaluate the ductular reaction in multistage carcinogenesis and to correlate PCK7 and γGT levels with tumor incidence, histological characteristics, liver DNA damage index, and the expression of oxidative stress proteins. HCC was induced in 24 male Wistar rats weighing 145-150 g by chronic and intermittent exposure to 50 or 100 mg/kg diethylnitrosamine (DEN). Six control animals received only vehicle. Blood was collected to determine hepatic enzyme levels. Animals were divided into three groups: control (CO), precancerous lesions (PL), and advanced HCC. Liver samples were obtained for immunohistochemical analyses and the measurement of protein expression. Statistical analyses included Tukey's test and Pearson's correlation analyses. We observed an extensive ductular reaction in advanced HCC and a strong correlation between PCK7 and levels of γGT and the poor prognosis and aggressiveness of HCC. The extent of PCK7 and high γGT levels were associated with overexpression of inducible nitric oxide synthase (iNOS) and heat shock factor protein 1 (HSF-1). However, PCK7 and γGT levels were negatively correlated with protein expression of nuclear factor erythroid 2-related factor 2 (NRF2) and inducible heat shock protein 70 (iHSP70). These findings suggest that ductular reaction is involved in the progression of multistage hepatocarcinogenesis.

Hypoxic-ischemic gene expression profile in the isolated variant of biliary atresia.

BACKGROUND: Biliary atresia (BA) includes a sclerosing cholangiopathy whose nature is not fully deciphered. Aiming to evaluate the role of an arteriopathy as an etiologic factor in BA, we investigated hypoxia and the correlated angiogenic response in livers from affected patients. METHODS: Gene expression of the molecular axis: hypoxia-inducible factor (HIF)1a, HIF2a and vascular endothelial growth factor A (VEGFA)/VEGFR1, VEGFR2. Liver biopsy specimens collected at exploratory laparotomy of age-matched patients with isolated, cytomegalovirus IgM-negative BA (n = 32) and intrahepatic cholestasis (IHC, n = 9) were evaluated. RESULTS: We observed higher HIF1a and HIF2a expression in BA than in IHC. Paradoxically, VEGFR2, the main target of VEGFA-induced angiogenesis, was underexpressed in BA, and VEGFA was decreased in most BA patients. Patients with the highest expression of HIFs and the lowest VEGFA and VEGFR2 were essentially the same, indicating hypoxia without the necessary angiogenesis. This group included most BA patients and, except for HIF2a, they were older and presented increased bilirubin serum levels. In the highest HIF2a/lowest VEGFR2 subsets, gene expression of the cytokeratin 19, marker of cholangiocyte phenotype, was decreased. CONCLUSION: This study suggests that hypoxia-ischemia is present in the livers of patients with BA, progresses over time and leads to a decreased cholangiocyte mass.

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-1ß (IL-1ß), 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.

The challenges for solid waste management in accordance with Agenda 21: a Brazilian case review.

This paper aims to evaluate the suitability of the Brazilian solid waste policy (BSWP) with global Agenda 21 and the challenges of implementing the BSWP in municipalities. For this, a review of the principles that guided the creation of this policy was performed to demonstrate that international pressures were important in determining its effectiveness. The contradictory relationship between the satisfactory legal framework that established the Brazilian waste management policy and its weakened implementation in the municipalities is also examined . To illustrate the difficulties faced at the local level, a case study involving municipalities that compose the state of Espírito Santowe was undertaken. In this state, the municipalities signed terms of environmental commitment with supervisory agencies who undertook, within a pre-established schedule, to implement a set of actions to shape the proper management of solid waste, adapted to the requirements of national policy and the guidelines of Agenda 21. Finally, the various difficulties in meeting the requirements are discussed. It is necessary and urgent that Brazil finds a way to coordinate the mechanisms of an innovative and well formulated legal instrument to ensure the successful implementation of solid waste management at the local level to achieve the environmental, economic and social objectives.

Atresia biliar: a experiência brasileira / Biliary atresia: the Brazilian experience

Objetivo: Avaliar as características epidemiológicas, clínicas e prognósticas de crianças com atresia biliar. Método: Dados sobre portoenterostomia, transplante hepático (TxH), idade no último seguimento e sobrevida foram coletados dos prontuários de pacientes acompanhados em seis centros no Brasil (1982-2008) e comparados em relação às décadas do procedimento cirúrgico. Resultados: Dos 513 pacientes, 76,4% foram submetidos a portoenterostomia [idade: 60,0-94,7 (82,6±32,8) dias] e 46,6% foram submetidos a TxH. Em 69% dos casos, o TxH foi realizado após a portoenterostomia, enquanto em 31% dos casos o TxH foi realizado como cirurgia primária. Os pacientes da região Nordeste foram submetidos a portoenterostomia mais tardiamente do que as crianças das regiões Sul (p = 0,008) e Sudeste (p = 0,0012), embora, mesmo nas duas últimas regiões, a idade no momento da portoenterostomia tenha sido superior ao desejável. Ao longo das décadas, houve aumento progres si vo do número de TxH realizados. A sobrevida global foi de 67,6%. A sobrevida aumentou nas últimas décadas (anos 1980 versus 90, p = 0,002; anos 1980 versus 2000, p < 0,001; anos 1990 versus 2000, p < 0,001). A sobrevida de 4 anos pós-portoenterostomia, com ou sem TxH, foi de 73,4%, inversamente correlacionada à idade no momento da portoenterostomia (80,77,7,60,5% para < 60,61-90, > 90 dias, respectivamente). Os pacientes transplantados apresentaram taxas de sobrevida mais elevadas (88,3%). A sobrevida de 4 anos com fígado nativo foi de 36,8%, inversamente correlacionada à idade no momento da portoenterostomia (54, 33,3, 26,6% para < 60,61-90, > 90 dias, respectivamente). Conclusões: Este estudo multicêntrico demonstrou que o encaminhamento tardio das crianças portadoras de atresia biliar ainda é um problema no Brasil, influenciando a sobrevida destes pacientes. Estratégias que proporcionam o encaminhamento precoce estão sendo desenvolvidas com o objetivo de reduzir a necessidade de transplante hepático nos primeiros anos de vida.

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 < 0.001; 1990s vs. 2000s, p < 0.001). The 4-year post portoenterostomy survival, with or without LTx, was 73.4%, inversely correlated with age at portoenterostomy (80, 77.7, 60.5% for < 60, 61-90, > 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 < 60, 61-90, > 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.

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 transforming [corrected] growth factor ß1 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 ß1 (TGFß1) 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, TGFß1 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 TGF1ß1 and collagen density in both groups analyzed. Serum TGFß1 showed no correlation with APRI at diagnosis. At the time of liver transplantation, all patients had low serum TGFß1 and variable APRI, although all higher than 2.0. However, when platelet count was used, an inverse correlation with serum TGFß1 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 TGFß1, whereas later on, there is scar tissue, with reduced TGFß1 expression. Although our results should be confirmed in larger sets of patients with BA, the lack of TGFß1 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.

Medial thickening of hepatic artery branches in biliary atresia. A morphometric study.

BACKGROUND/PURPOSE: Medial layer hypertrophy of hepatic arterial branches may be associated with biliary atresia (BA) pathogenesis. This study aimed at evaluating medial layer thickness in hepatic arterial branches at portoenterostomy and liver transplantation. METHODS: The authors evaluated 1274 arterial branches both in BA cases and in control subjects involving a total of 1108 arterioles and 166 arteries. Arterial branch characteristics were morphometrically evaluated in 47 BA patients at the time of portoenterostomy. Controls were patients with intrahepatic cholestasis (n = 3), immature neonates (n = 7), and infants (n = 7) without liver disease. Progression of medial layer thickening between the time of portoenterostomy and transplantation was evaluated in 7 BA patients. Biliary atresia patients at the time of transplantation were compared with non-BA-transplanted patients (n = 4). RESULTS: The arterial medial layer of BA cases at portoenterostomy was thicker than that of infants without liver disease ( P = .03). The arterial medial thickness increased during the interval between portoenterostomy and transplantation ( P = .05). Arterioles and arteries with thickened medial layers were found in transplanted BA patients but not in patients transplanted for other liver diseases (P = .05 and P = .01). Thickening of the medial layer of the hepatic arteries was associated with focal distribution of interlobular bile ducts in portal spaces in BA ( P = .02). CONCLUSIONS: In BA, there is a progressive thickening of the arterial medial layer, suggestive of vascular remodeling, which is associated to the disappearance of interlobular bile ducts.

[Factors related to the post-portoenterostomy prognosis of biliary atresia]. / Fatores relacionados ao prognóstico da atresia biliar pós-portoenterostomia.

OBJECTIVE: This study considered the presence of congenital anomalies, ductal plate malformation, area of fibrosis and, mainly, the patient's age in cases of biliary atresia submitted to surgery. The present study verified the influence of these factors on the follow-up of a biliary atresia sample. METHODS: A sample of 47 patients with biliary atresia was evaluated in a cross-sectional study. Their histologic specimens were stained for antibody anticytokeratin 19 and CAM 5.2 through immunohistochemistry in order to study biliary structures, and for picrosirius red to evaluate the area of fibrosis. The study of biliary structures was performed by two pathologists and the first author of the study. They were "blind" with regard to the clinical follow-up. The area of fibrosis was quantitatively evaluated. Data on the patients with regard to age, death and occurrence of liver transplantation were searched on the patients records. RESULTS: Age at portoenterostomy varied between 24 and 251 days of life (90.4 +/- 44.8 days) and follow-up was available in 32 cases (72%). The nine cases (19%) with extrahepatic congenital anomalies associated to biliary atresia did not present different prognosis from the remaining patients. Age at portoenterostomy influenced the prognosis (p=0.016). The area of fibrosis was different on patients aged less than 60 days and those aged more than 90 days at portoenterostomy (p=0.023), but did not influence the prognosis. The presence of ductal plate malformation, as well, did not influence the follow-up. CONCLUSIONS: Age at portoenterostomy was the only factor that influenced prognosis on this sample of biliary atresia. It is necessary to increase the biliary atresia sample to check the influence of congenital extrahepatic anomalies on the follow-up post-portoenterostomy.