A Case of Progressive Cholestatic Drug-Induced Liver Injury Due to Terbinafine.

INTRODUCTION: Terbinafine is commonly prescribed for onychomycosis. It rarely leads to severe, prolonged cholestatic drug-induced liver injury. Clinicians should remain vigilant for this complication. CASE PRESENTATION: A 62-year-old woman was started on terbinafine and developed mixed hepatocellular and cholestatic drug-induced liver injury, confirmed on liver biopsy. The injury became predominantly cholestatic. Unfortunately, she developed coagulopathy with elevated international normalized ratio and progressive drug-induced liver injury with severely elevated alkaline phosphatase and total bilirubin, requiring repeat liver biopsy. Fortunately, she did not develop acute liver failure. DISCUSSION: Prior case reports and series have documented severe cholestatic drug-induced liver injury (although with lesser degree of bilirubin elevation) due to terbinafine, which has very rarely been associated with acute liver failure, need for liver transplantation, and/or death. CONCLUSIONS: Non-acetaminophen drug-induced liver injury is idiosyncratic. Complications including acute liver failure and vanishing bile duct syndrome can be slow to develop, so monitoring for them is important over longitudinal follow-up.

[Ligandrol-induced liver injury - Case Report]. / Ligandrol-induzierte toxische Hepatopathie ­ ein Fallbericht.

A 37-year-old man presented with painless jaundice and pruritus. Total Bilirubin was 30-fold the upper limit of normal (ULN), while ALT and further cholestasis parameters were found to be only slightly elevated. As comprehensive diagnostics showed no abnormal findings and ruled out frequent causes of elevated cholestasis markers, we performed a liver biopsy. The biopsy revealed canalicular cholestasis with ductopenia and periportal fibrosis. Only after a further and intensive anamnesis a ligandrol-abuse could be determined as cause for the symptoms. Ligandrol is misused as a selective androgen receptor modulator to promote muscle building. This case represents a typical case of abuse of anabolic substances in amateur sports.

Inhibition of Autotaxin Ameliorates LPA-Mediated Neuroinflammation and Alleviates Neurological Dysfunction in Acute Hepatic Encephalopathy.

Growing evidence suggests an essential role of neuroinflammation in behavioral abnormalities associated with hepatic encephalopathy (HE). Here, we report the involvement of autotaxin-lysophosphatidic acid (LPA) signaling in HE's pathogenesis. We demonstrate that the autotaxin (ATX) inhibitor PF-8380 attenuates neuroinflammation and improves neurological dysfunction in the mouse model of HE. In the thioacetamide (TAA)-induced model of HE, we found a twofold increase in the levels of ammonia in the brain and in plasma along with a significant change in HE-related behavioral parameters. Mice with HE show an increased brain weight, increased levels of tumor necrosis factor-α (TNF-α), IL-1ß (interleukin-1ß), interleukin-6 (IL-6), and LPA 18:0 in the cerebral cortex and hippocampus, and increased levels of LPA 18:0 in plasma. Treatment with the autotaxin inhibitor (ATXi) did not affect liver injury, as we observed no change in liver function markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBIL) and no change in ammonia levels in the brain and plasma. However, ATXi treatment significantly ameliorated the neuroinflammation, reduced the levels of LPA 18:0 in the cerebral cortex and hippocampus in the brain and plasma, and reduced brain edema and the levels of IL1ß, IL-6, and TNF-α. The neurobehavioral symptoms for HE such as the cognitive and motor function deficit and overall clinical grading score were significantly improved in ATXi-treated mice. Mouse astrocytes and microglia stimulated with NH4CL with or without ATXi showed significant attenuation of oxidative stress and the neuroinflammatory effect of NH4CL in ATXi-treated cells.

Identifying Potential Therapeutic Applications and Diagnostic Harms of Increased Bilirubin Concentrations: A Clinical and Genetic Approach.

Bilirubin has antioxidant and anti-inflammatory properties in vitro and in animal studies and protects against inflammatory, cardiovascular, and other diseases in observational studies; therefore, bilirubin has potential as a therapeutic agent. However, observational studies could be confounded by many factors. We used a genetic (n = 61,281) and clinical (n = 234,670) approach to define the association between bilirubin and 19 conditions with a putative protective signal in observational studies. We also tested if individuals with genetically higher bilirubin levels underwent more diagnostic tests. We used a common variant in UGT1A1 (rs6742078) associated with an 26% increase in bilirubin levels in the genetic studies. Carriers of the variant had higher bilirubin levels (P = 2.2 × 10-16 ) but there was no significant association with any of the 19 conditions. In a phenome-wide association study (pheWAS) to seek undiscovered genetic associations, the only significant finding was increased risk of "jaundice-not of newborn." Carriers of the variant allele were more likely to undergo an abdominal ultrasound (odds ratio = 1.04, [1.00-1.08], P = 0.03). In contrast, clinically measured bilirubin levels were significantly associated with 15 of the 19 conditions (P < 0.003) and with 431 clinical diagnoses in the pheWAS (P < 1 × 10-5 adjusted for sex, age, and follow-up). With additional adjustment for smoking and body mass index, 7 of 19 conditions and 260 pheWAS diagnoses remained significantly associated with bilirubin levels. In conclusion, bilirubin does not protect against inflammatory or other diseases using a genetic approach; the many putative beneficial associations reported clinically are likely due to confounding.

Reactive Oxygen Species-Responsive Celastrol-Loaded : Bilirubin Nanoparticles for the Treatment of Rheumatoid Arthritis.

Celastrol (CLT) has shown anti-rheumatic activity against rheumatoid arthritis, while its poor water solubility and high organ toxicity restrict its further therapeutic applications. To mitigate these challenges, a reactive oxygen species (ROS)-responsive nanoparticle was developed for celastrol delivery based on the excessive ROS at the pathologic sites, which was synthesized by conjugating bilirubin to a polyethylene glycol (PEG) chain. The PEGylated bilirubin self-assembled into nanoparticle (BRNP) in aqueous solution had a hydrodynamic diameter of around 68.6 nm, and celastrol was loaded into BRNP (CLT/BRNP) with a drug encapsulation efficiency of 72.6% and a loading capacity of 6.6%. In vitro study revealed that CLT/BRNP exhibited the capacity of scavenging intracellular ROS and down-regulating the level of nitric oxide after it was effectively internalized by activated macrophages. Furthermore, in adjuvant-induced arthritis rats, BRNP was accumulated preferentially at inflamed joints, alleviating the joint swelling and bone erosion, which significantly decreased the secretion of pro-inflammatory cytokines to suppress the RA progression. Importantly, CLT/BRNP markedly enhanced its anti-arthritic effect and attenuated the toxic effect compared with free celastrol. Taken together, our results suggested that CLT/BRNP could be used for targeted drug delivery in rheumatoid arthritis.

Circulating bilirubin levels and risk of colorectal cancer: serological and Mendelian randomization analyses.

BACKGROUND: Bilirubin, a byproduct of hemoglobin breakdown and purported anti-oxidant, is thought to be cancer preventive. We conducted complementary serological and Mendelian randomization (MR) analyses to investigate whether alterations in circulating levels of bilirubin are associated with risk of colorectal cancer (CRC). We decided a priori to perform analyses separately in men and women based on suggestive evidence that associations may differ by sex. METHODS: In a case-control study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC), pre-diagnostic unconjugated bilirubin (UCB, the main component of total bilirubin) concentrations were measured by high-performance liquid chromatography in plasma samples of 1386 CRC cases and their individually matched controls. Additionally, 115 single-nucleotide polymorphisms (SNPs) robustly associated (P < 5 × 10-8) with circulating total bilirubin were instrumented in a 2-sample MR to test for a potential causal effect of bilirubin on CRC risk in 52,775 CRC cases and 45,940 matched controls in the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), the Colon Cancer Family Registry (CCFR), and the Colorectal Transdisciplinary (CORECT) study. RESULTS: The associations between circulating UCB levels and CRC risk differed by sex (Pheterogeneity = 0.008). Among men, higher levels of UCB were positively associated with CRC risk (odds ratio [OR] = 1.19, 95% confidence interval [CI] = 1.04-1.36; per 1-SD increment of log-UCB). In women, an inverse association was observed (OR = 0.86 (0.76-0.97)). In the MR analysis of the main UGT1A1 SNP (rs6431625), genetically predicted higher levels of total bilirubin were associated with a 7% increase in CRC risk in men (OR = 1.07 (1.02-1.12); P = 0.006; per 1-SD increment of total bilirubin), while there was no association in women (OR = 1.01 (0.96-1.06); P = 0.73). Raised bilirubin levels, predicted by instrumental variables excluding rs6431625, were suggestive of an inverse association with CRC in men, but not in women. These differences by sex did not reach formal statistical significance (Pheterogeneity ≥ 0.2). CONCLUSIONS: Additional insight into the relationship between circulating bilirubin and CRC is needed in order to conclude on a potential causal role of bilirubin in CRC development.

Niveles de proteína C reactiva, bilirrubina y leucocitos como predictores de evolución anatomopatológica apendicular / Levels of C-reactive Protein, Bilirubin and Leucocytes as Predictors of Anotomopathological Appendicular Evolution

RESUMEN Introducción: La apendicitis aguda es la urgencia quirúrgica más frecuente en cualquier hospital del mundo. Aunque la mayoría de las veces se trata de un proceso intrabdominal banal, en ocasiones presenta una no desdeñable morbilidad y todavía en la época actual. Esta morbimortalidad se asocia, en la mayoría de los casos, a estados avanzados de afección apendicular. Objetivo: Predecir, con la cifra de bilirrubina, la proteína C reactiva y el recuento leucocitario, el estado del proceso apendicular agudo que presentaban los pacientes. Métodos: Se realizó un estudio observacional descriptivo en el que se han incluido aquellos pacientes intervenidos por sospecha de apendicitis aguda durante un periodo de 3 años (2017-2019) que cumplían los criterios de inclusión. Se analizó, como datos de laboratorio, la cifra de leucocitos, proteína C reactiva y bilirrubina. Resultados: Se observó un aumento de las cifras de proteína C reactiva y bilirrubina en los casos apendiculares avanzados, al igual que otros autores han evidenciado en la literatura. Así mismo, estos dos valores han resultado ser un factor de riesgo para presentar formas graves. El nivel de leucocitos sin embargo no ha demostrado relacionarse con la gravedad del proceso. Conclusiones: Vemos relevante el uso de los biomarcadores estudiados para predecir la gravedad apendicular con el objetivo de mejorar la asistencia en estos enfermos y disminuir las complicaciones derivadas del retraso terapéutico(AU)

ABSTRACT Introduction: Acute appendicitis is the most frequent surgical emergency in any hospital worldwide. Although most of the time it is a trivial intraabdominal process, sometimes it presents an unneglectable morbidity. This morbidity and the subsequent mortality are associated, in most cases, with advanced stages of an appendicular disease. Objective: To predict, using the value corresponding to bilirubin, C-reactive protein and leukocyte count, the state of acute appendicular process presented by patients. Methods: A descriptive observational study was carried out, including patients operated on for suspected acute appendicitis during a period of three years (2017-2019) and who met the inclusion criteria. The values for leukocyte count, C-reactive protein, and bilirubin were analyzed as laboratory data. Results: An increase in the values of C-reactive protein and bilirubin levels was observed in advanced appendicular cases, as other authors have shown in the medical literature. Likewise, these two values ​​have turned out to be a risk factor for presenting severe forms. However, the level of leukocytes has not been shown to be related to the severity of the process. Conclusions: We consider the use of the biomarkers studied as relevant to predict appendicular severity in view of improving care of these patients and reducing complications derived from therapeutic delay(AU)

Baicalin Suppresses Bilirubin-Induced Apoptosis and Inflammation by Regulating p38 Mitogen-Activated Protein Kinases (MAPK) Signaling in Neonatal Neurons.

BACKGROUND Hyperbilirubinemia is associated with central nervous system damage in preterm neonates due to the neurotoxicity of bilirubin. This study explored the possible mechanisms of bilirubin's neurotoxicity, and the protective effect of baicalin (BAI) was also investigated. MATERIAL AND METHODS Isolated neonatal rat hippocampal neurons were exposed to free bilirubin (Bf). BAI was used to treat these neurons. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cell viability. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay was used to detect apoptosis. Contents of inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein expression and phosphorylation levels were assessed by Western blotting. Nuclear translocation was observed by immunofluorescent staining. RESULTS Bf incubation significantly induced apoptosis and decreased viabilities of neurons. The phosphorylation levels of MAP kinase kinase (MKK)3, MKK6, p38 mitogen- activated protein kinases (MAPK), nuclear translocation level of p65, and the expression levels of cleaved caspase3 and tumor necrosis factor (TNF)alpha were found to be dramatically higher in Bf-incubated neurons. BAI pre-treatment, however, increased cell viability by reducing cell apoptosis. BAI pre-treatment also reduced phosphorylation levels of MKK3, MKK6, p38 MAPK, and nuclear translocation level of p65, as well as the expression levels of cleaved caspase3 and TNFalpha, in Bf- incubated neurons. CONCLUSIONS BAI suppressed bilirubin-induced neuron apoptosis and inflammation by deactivating p38 MAPK signaling.

Bilirubin Nanomedicines for the Treatment of Reactive Oxygen Species (ROS)-Mediated Diseases.

Reactive oxygen species (ROS) are chemically reactive species that are produced in cellular aerobic metabolism. They mainly include superoxide anion, hydrogen peroxide, hydroxyl radicals, singlet oxygen, ozone, and nitric oxide and are implicated in many physiological and pathological processes. Bilirubin, a cardinal pigment in the bile, has been increasingly investigated to treat cancer, diabetes, ischemia-reperfusion injury, asthma, and inflammatory bowel diseases (IBD). Indeed, bilirubin has been shown to eliminate ROS production, so it is now considered as a promising therapeutic agent for ROS-mediated diseases and can be used for the development of antioxidative nanomedicines. This review summarizes the current knowledge of the physiological mechanisms of ROS production and its role in pathological changes and focuses on discussing the antioxidative effects of bilirubin and its application in the experimental studies of nanomedicines. Previous studies have shown that bilirubin was mainly used as a responsive molecule in the microenvironment of ROS overproduction in neoplastic tissues for the development of anticancer nanodrugs; however, it could also exert powerful ROS scavenging activity in chronic inflammation and ischemia-reperfusion injury. Therefore, bilirubin, as an inartificial ROS scavenger, is expected to be used for the development of nanomedicines against more diseases due to the universality of ROS involvement in human pathological conditions.

Caspase-1 involves in bilirubin-induced injury of cultured rat cortical neurons.

BACKGROUND: Bilirubin encephalopathy, the most serious complication of hyperbilirubinemia during the neonatal period, with high mortality and morbidity, often causes irreversible neurological damage. Currently, caspase-1, a member of the cysteinyl aspartate-specific protease caspase family, is regarded as a key mediator of inflammatory processes, attracting widespread attention. The purpose of this study was to investigate whether caspase-1 is involved in bilirubin-induced neuronal injury. METHODS: VX-765, a highly potent and selective inhibitor of caspase-1, was used to investigate the effects of unconjugated bilirubin (UCB) on rat cortical neurons, including cell viability, morphological changes in the cell membrane, and nuclear factor-kappa B (NF-κB) activation. RESULTS: Neurons treated with UCB showed increased caspase-1 activity without the secretion of interleukin (IL)-1ß and IL-18, and caspase-1 was significantly inhibited by pretreatment with VX-765. The cell viability of the VX-765-pretreated neurons was improved, and cell membrane rupture was prevented, as detected by lactate dehydrogenase release and ethidium bromide uptake. Moreover, NF-κB activation by UCB exposure, was attenuated by VX-765 pretreatment. CONCLUSION: Bilirubin-induced neuronal injury involves the activation of caspase-1 and NF-κB, leading to membrane leakage, independently of IL-1ß and IL-18.

Parenteral bilirubin in healthy volunteers: a reintroduction in translational research.

AIMS: Preclinical results suggest therapeutic potential of mild hyperbilirubinemia in T2DM and cardiovascular disease. Translational data are limited, because an appropriate bilirubin formulation for parenteral human use is lacking. Considering its use in both clinical practice and medical research in the past, we explored the feasibility to reintroduce parenteral bilirubin for translational experiments. METHODS: We developed a preparation method in accordance with good manufacturing practice and evaluated the parenteral applicability in healthy volunteers (n = 8). Explorative pharmacokinetic and safety data were compared to the results from a literature study on the former parenteral use of bilirubin. Bilirubin was administered intra-arterially to raise the local plasma concentration in the forearm vascular bed (n = 4) and intravenously to raise the systemic plasma concentration (n = 4). Finally, pharmacokinetic characteristics were studied following a single bolus infusion (n = 3). RESULTS: During parenteral application, no side effects occurred. Adverse events mentioned during the two-week observation period were in general mild and self-limiting. Three more significant adverse events (appendicitis, asymptomatic cardiac arrhythmia and atopic eczema) were judged unrelated by independent physicians. A dose-concentration relationship appeared sufficiently predictable for both intra-arterial and intravenous administration. In line with existing knowledge, bilirubin pharmacokinetics could be described best according to a two-compartment model with a volume of distribution of 9.9 (±2.0) l and a total plasma clearance of 36 (±16) ml per minute. CONCLUSIONS: Supported by previous reports, our data suggest that it is both feasible and safe to perform translational experiments with parenteral albumin bound bilirubin.

Mortality outcomes after busulfan-containing conditioning treatment and haemopoietic cell transplantation in patients with Gilbert's syndrome: a retrospective cohort study.

BACKGROUND: Gilbert's syndrome is a common inherited disorder of bilirubin metabolism, characterised by mild, unconjugated hyperbilirubinaemia. However, the effect of Gilbert's syndrome on the disposition of some drugs can lead to unexpected toxicity. We tested the hypothesis that patients undergoing myeloablative conditioning and haemopoietic cell transplantation would have different mortality outcomes depending on whether or not they had laboratory evidence of Gilbert's syndrome. METHODS: In this retrospective cohort study, we used clinical and laboratory data of patients who had haemopoietic cell transplantation from Jan 1, 1991, to Dec 31, 2011. Patients were included if they had received high-dose conditioning regimens of cyclophosphamide plus total body irradiation (CY/TBI), busulfan plus cyclophosphamide (BU/CY), busulfan plus melphalan plus thioTEPA (BUMELTT), or melphalan before transplant. Patients were excluded if their original consent forms to report transplant outcomes were not signed, if consent was withdrawn, or if they were a prisoner. Patients with Gilbert's syndrome were defined as having laboratory values before the start of conditioning therapy for unconjugated serum bilirubin concentrations of at least 17·1 µmol/L (≥1 mg/dL), normal conjugated serum bilirubin, and no evidence of hepatitis, cholestasis, or haemolysis. We assessed the association of Gilbert's syndrome with overall mortality and non-relapse mortality using adjusted Cox regression models at day 200 after transplantation. FINDINGS: Our study cohort was 3379 patients-1855 (55%) allograft and 1524 (45%) autograft recipients. 211 (6%) patients had Gilbert's syndrome and 3168 (94%) did not have this condition. Most patients were adults (median age 45·8 years [IQR 33·2-55·5]) with haematological malignancies. For overall mortality 664 (20%) patients had died by day 200 after transplant (47 [22%] of 211 who had Gilbert's syndrome vs 617 [19%] of 3168 who did not have Gilbert's syndrome), and for non-relapse mortality 499 (92%) patients had died before relapse was recorded (38 [18%] who had Gilbert's syndrome vs 461 [15%] who did not have Gilbert's syndrome). The effect of Gilbert's syndrome on the risk of overall mortality and non-relapse mortality by transplant day 200 varied between the conditioning regimens and donor groups. In patients conditioned with a myeloablative regimen that contained busulfan (n=1131), those with Gilbert's syndrome (n=60) were at a significantly increased risk of death and non-relapse mortality by day 200 compared with those without Gilbert's syndrome (n=1071; hazard ratio [HR] 2·30, 95% CI 1·47-3·61, p=0·00030; and 2·77, 1·71-4·49, p<0·0001). In patients who received CY/TBI or melphalan conditioning regimens, those with Gilbert's syndrome had similar outcomes to those without Gilbert's syndrome (overall mortality at day 200 HR 0·90, 95% CI 0·60-1·34, p=0·60; non-relapse mortality at day 200: 0·90, 0·56-1·45, p=0·65). Analyses of causes of death and busulfan disposition provided no mechanistic explanation for the differences in mortality. INTERPRETATION: Overall mortality and non-relapse mortality at day 200 after transplant were significantly worse in patients with Gilbert's syndrome who received busulfan-containing myeloablative conditioning regimens, compared with non-Gilbert's syndrome patients. Patients with Gilbert's syndrome should receive busulfan-containing myeloablative conditioning regimens with caution. FUNDING: US National Institutes of Health.

The role of gamma-aminobutyric acid/glycinergic synaptic transmission in mediating bilirubin-induced hyperexcitation in developing auditory neurons.

Hyperbilirubinemia is a common clinical phenomenon observed in human newborns. A high level of bilirubin can result in severe jaundice and bilirubin encephalopathy. However, the cellular mechanisms underlying bilirubin excitotoxicity are unclear. Our previous studies showed the action of gamma-aminobutyric acid (GABA)/glycine switches from excitatory to inhibitory during development in the ventral cochlear nucleus (VCN), one of the most sensitive auditory nuclei to bilirubin toxicity. In the present study, we investigated the roles of GABAA/glycine receptors in the induction of bilirubin hyperexcitation in early developing neurons. Using the patch clamp technique, GABAA/glycine receptor-mediated spontaneous inhibitory synaptic currents (sIPSCs) were recorded from bushy and stellate cells in acute brainstem slices from young mice (postnatal day 2-6). Bilirubin significantly increased the frequency of sIPSCs, and this effect was prevented by pretreatments of slices with either fast or slow Ca(2+) chelators BAPTA-AM and EGTA-AM suggesting that bilirubin can increase the release of GABA/glycine via Ca(2+)-dependent mechanisms. Using cell-attached recording configuration, we found that antagonists of GABAA and glycine receptors strongly attenuated spontaneous spiking firings in P2-6 neurons but produced opposite effect in P15-19 neurons. Furthermore, these antagonists reversed bilirubin-evoked hyperexcitability in P2-6 neurons, indicating that excitatory action of GABA/glycinergic transmission specifically contribute to bilirubin-induced hyperexcitability in the early stage of development. Our results suggest that bilirubin-induced enhancement of presynaptic release GABA/Glycine via Ca(2+)-dependent mechanisms may play a critical role in mediating neuronal hyperexcitation associated with jaundice, implicating potential new strategies for predicting, preventing, and treating bilirubin neurotoxicity.

A novel newborn rat kernicterus model created by injecting a bilirubin solution into the cisterna magna.

BACKGROUND: Kernicterus still occurs around the world; however, the mechanism of bilirubin neurotoxicity remains unclear, and effective treatment strategies are lacking. To solve these problems, several kernicterus (or acute bilirubin encephalopathy) animal models have been established, but these models are difficult and expensive. Therefore, the present study was performed to establish a novel kernicterus model that is simple and affordable by injecting unconjugated bilirubin solution into the cisterna magna (CM) of ordinary newborn Sprague-Dawley (SD) rats. METHODS: On postnatal day 5, SD rat pups were randomly divided into bilirubin and control groups. Then, either bilirubin solution or ddH2O (pH = 8.5) was injected into the CM at 10 µg/g (bodyweight). For model characterization, neurobehavioral outcomes were observed, mortality was calculated, and bodyweight was recorded after bilirubin injection and weaning. Apoptosis in the hippocampus was detected by H&E staining, TUNEL, flow cytometry and Western blotting. When the rats were 28 days old, learning and memory ability were evaluated using the Morris water maze test. RESULTS: The bilirubin-treated rats showed apparently abnormal neurological manifestations, such as clenched fists, opisthotonos and torsion spasms. Bodyweight gain in the bilirubin-treated rats was significantly lower than that in the controls (P<0.001). The early and late mortality of the bilirubin-treated rats were both dramatically higher than those of the controls (P = 0.004 and 0.017, respectively). Apoptosis and necrosis in the hippocampal nerve cells in the bilirubin-treated rats were observed. The bilirubin-treated rats performed worse than the controls on the Morris water maze test. CONCLUSION: By injecting bilirubin into the CM, we successfully created a new kernicterus model using ordinary SD rats; the model mimics both the acute clinical manifestations and the chronic sequelae. In particular, CM injection is easy to perform; thus, more stable models for follow-up study are available.

Developmental onset of bilirubin-induced neurotoxicity involves Toll-like receptor 2-dependent signaling in humanized UDP-glucuronosyltransferase1 mice.

Biological and signaling events that connect developmentally induced hyperbilirubinemia to bilirubin-induced neurological dysfunction (BIND) and CNS toxicity in humans are poorly understood. In mammals, UDP-glucuronosyltransferase 1A1 (UGT1A1) is the sole enzyme responsible for bilirubin glucuronidation, a rate-limiting step necessary for bilirubin metabolism and clearance. Humanized mice that express the entire UGT1 locus (hUGT1) and the UGT1A1 gene, develop neonatal hyperbilirubinemia, with 8-10% of hUGT1 mice succumbing to CNS damage, a phenotype that is presented by uncontrollable seizures. We demonstrate that neuroinflammation and reactive gliosis are prominent features of bilirubin brain toxicity, and a disturbed redox status resulting from activation of NADPH oxidase is an important contributing mechanism found in BIND. Using knock-out mice and primary brain cells, we connect a key pattern recognition receptor, Toll-like receptor 2 (TLR2), to hyperbilirubinemia-induced signaling. We illustrate a requirement for TLR2 signaling in regulating gliosis, proinflammatory mediators, and oxidative stress when neonatal mice encounter severe hyperbilirubinemia. TLR2-mediated gliosis strongly correlates with pronounced neuroinflammation in the CNS with up-regulation of TNFα, IL-1ß, and IL-6, creating a pro-inflammatory CNS environment. Gene expression and immunohistochemistry staining show that hUGT1/Tlr2(-/-) mice fail to activate glial cells, proinflammatory cytokines, and stress response genes. In addition, bilirubin-induced apoptosis was significantly enhanced by blocking TLR2 signaling indicating its anti-apoptotic property. Consequently, a higher neonatal death rate (57.1%) in hUGT1/Tlr2(-/-) mice was observed when compared with hUGT1 mice (8.7%). These results suggest that TLR2 signaling and microglia neuroinflammation are linked to a repair and/or protection mode against BIND.

Effects of unconjugated bilirubin on chromosomal damage in individuals with Gilbert's syndrome measured with the micronucleus cytome assay.

Circulating unconjugated bilirubin (UCB) has been reported to protect against lung and colorectal cancer. The present study aimed to explore, for the first time, whether mildly elevated circulating UCB, as found in Gilbert`s syndrome (GS), is associated with changes of DNA damage. A random 76 individuals, matched for age and gender, were recruited from the general population and allocated into the GS group (UCB ≥ 17.1 µM; n = 38) or control group (UCB <17.1 µM; n = 38). Chromosomal and cytological changes were determined in lymphocytes and buccal cells using the cytokinesis-block micronucleus cytome assay (CBMN) and buccal micronucleus cytome assay (BMcyt). No significant differences were found between GS subjects and the control group in the CBMN and BMcyt determined endpoints. Subsequently, when age dependency of effects were analysed, lower formation of buccal micronucleated cells (by 73.3%) and buccal nuclear buds (by 70.9%) in the GS subgroup ≥ 30 years were found, compared to the GS subgroup <30 years. These findings suggest DNA protection in epithelial tissue of older individuals with GS.

Osteoporosis en la cirrosis hepática / Osteoporosis in liver cirrhosis

La osteoporosis es una complicación frecuente en la enfermedad hepática crónica, especialmente en las etapas finales de la enfermedad. El problema es más crítico en los pacientes trasplantados, cuando la pérdida ósea se acelera durante el período inmediatamente después de la cirugía. El principal mecanismo implicado en el desarrollo de osteoporosis en los hepatópatas es el déficit de la formación ósea, por el efecto nocivo de sustancias como la bilirrubina y los ácidos biliares, o bien por el efecto tóxico del alcohol o el hierro sobre los osteoblastos.Para la prevención y el tratamiento de la osteoporosis es recomendable una buena nutrición y la administración de suplementos de calcio y vitamina D. No hay pautas concretas en su tratamiento farmacológico, pero se ha demostrado que los bisfosfonatos son eficaces para aumentar la masa ósea en pacientes con colestasis crónica, con un buen perfil de seguridad (AU)

Osteoporosis is a common complication of chronic liver disease, especially in the final stages. This entity is more critical in liver transplant recipients, when bone loss accelerates during the immediate postoperative period. The main mechanism involved in the development of osteoporosis in liver disease is deficient bone formation due to the harmful effects of substances such as bilirubin and bile acids or the toxic effect of alcohol or iron on osteoblasts.To prevent and treat osteoporosis, good nutrition and calcium and vitamin D supplementation are required. There are no specific recommendations on drug treatment but bisphosphonates are effective in increasing bone mass in patients with chronic cholestasis and have a good safety profile (AU)

Bilirubin-induced neurological damage.

The incidence of the neurological damage of due to severe neonatal jaundice is increasing mainly due to early discharge from the hospital. However the molecular mechanisms at the basis of the neurological damage are still largely unknown. We here summarize what is known and what is not yet known on how bilirubin may cause cellular damage to selective regions of the brain. This may hopefully help to develop diagnostic and therapeutic strategies for a more effective handling of this invalidating condition.

Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation.

Microglia constitute the brain's immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target.