Reliability of causality assessment for drug, herbal and dietary supplement hepatotoxicity in the Drug‐Induced Liver Injury Network (DILIN)

Background & AimsBecause of the lack of objective tests to diagnose drug‐induced liver injury (DILI), causality assessment is a matter of debate. Expert opinion is often used in research and industry, but its test–retest reliability is unknown. To determine the test–retest reliability of the expert opinion process used by the Drug‐Induced Liver Injury Network (DILIN).MethodsThree DILIN hepatologists adjudicate suspected hepatotoxicity cases to one of five categories representing levels of likelihood of DILI. Adjudication is based on retrospective assessment of gathered case data that include prospective follow‐up information. One hundred randomly selected DILIN cases were re‐assessed using the same processes for initial assessment but by three different reviewers in 92% of cases.ResultsThe median time between assessments was 938 days (range 140–2352). Thirty‐one cases involved >1 agent. Weighted kappa statistics for overall case and individual agent category agreement were 0.60 (95% CI: 0.50–0.71) and 0.60 (0.52–0.68) respectively. Overall case adjudications were within one category of each other 93% of the time, while 5% differed by two categories and 2% differed by three categories. Fourteen per cent crossed the 50% threshold of likelihood owing to competing diagnoses or atypical timing between drug exposure and injury.ConclusionsThe DILIN expert opinion causality assessment method has moderate interobserver reliability but very good agreement within one category. A small but important proportion of cases could not be reliably diagnosed as ≥50% likely to be DILI.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111130/1/liv12540.pd

ELEVATED PHENYLACETIC ACID LEVELS DO NOT CORRELATE WITH ADVERSE EVENTS IN PATIENTS WITH UREA CYCLE DISORDERS OR HEPATIC ENCEPHALOPATHY AND CAN BE PREDICTED BASED ON THE PLASMA PAA TO PAGN RATIO

Background Phenylacetic acid (PAA) is the active moiety in sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB, HPN-100), both are approved for treatment of urea cycle disorders (UCDs) - rare genetic disorders characterized by hyperammonemia. PAA is conjugated with glutamine in the liver to form phenylacetyleglutamine (PAGN), which is excreted in urine. PAA plasma levels ≥500 μg/dL have been reported to be associated with reversible neurological adverse events (AEs) in cancer patients receiving PAA intravenously. Therefore, we have investigated the relationship between PAA levels and neurological AEs in patients treated with these PAA pro-drugs as well as approaches to identifying patients most likely to experience high PAA levels. Methods The relationship between nervous system AEs, PAA levels and the ratio of plasma PAA to PAGN were examined in 4683 blood samples taken serially from: [1] healthy adults [2], UCD patients ≥2 months of age, and [3] patients with cirrhosis and hepatic encephalopathy (HE). The plasma ratio of PAA to PAGN was analyzed with respect to its utility in identifying patients at risk of high PAA values. Results Only 0.2% (11) of 4683 samples exceeded 500 ug/ml. There was no relationship between neurological AEs and PAA levels in UCD or HE patients, but transient AEs including headache and nausea that correlated with PAA levels were observed in healthy adults. Irrespective of population, a curvilinear relationship was observed between PAA levels and the plasma PAA:PAGN ratio, and a ratio > 2.5 (both in μg/mL) in a random blood draw identified patients at risk for PAA levels > 500 μg/ml. Conclusions The presence of a relationship between PAA levels and reversible AEs in healthy adults but not in UCD or HE patients may reflect intrinsic differences among the populations and/or metabolic adaptation with continued dosing. The plasma PAA:PAGN ratio is a functional measure of the rate of PAA metabolism and represents a useful dosing biomarker

The Chlamydia psittaci Genome: A Comparative Analysis of Intracellular Pathogens

Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.A comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.This study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions

A Plant Kavalactone Desmethoxyyangonin PreventsInflammation and Fulminant Hepatitis in Mice

Alpinia pricei Hayata is a Formosan plant which has been popularly used as nutraceutical or folk medicine for inflammation and various disorders. An active compound of the plant rhizomes, desmethoxyyangonin (DMY), was identified in this study for its novel effect against endotoxin lipopolysaccharide (LPS)-stimulated inflammation in murine macrophages and LPS/D-galactosamine (LPS/D-GalN)-induced fulminant hepatitis in mice. DMY was observed to significantly inhibit proliferation and activation of T cells ex vivo and the activity of several pro-inflammatory mediators in vitro. DMY also protected LPS/D-GalN−induced acute hepatic damages in mice through inhibiting aminotransferases activities and infiltrations of inflammatory macrophages, neutrophils and pathogenic T cells into the liver tissues. In addition, pretreatment with DMY significantly improved the survival rate of LPS/D-GalN−treated mice to 90% (9/10), compared to LPS/D-GalN−treated group (40%, 4/10). UPLC/MS platform-based comparative metabolomics approach was used to explore the serum metabolic profile in fulminant hepatic failure (FHF) mice with or without the DMY pretreatment. The results showed that LPS/D-GalN−induced hepatic damage is likely through perturbing amino acid metabolism, which leads to decreased pyruvate formation via catalysis of aminotransferases, and DMY treatment can prevent to a certain degree of these alterations in metabolic network in mouse caused by LPS/D-GalN. Mechanistic investigation demonstrated that DMY protects LPS or LPS/D-GalN−induced damages in cell or liver tissues mainly through de-regulating IKK/NFκB and Jak2/STAT3 signaling pathways. This report provides evidence-based knowledge to support the rationale for the use of A. pricei root extract in anti-inflammation and also its new function as hepatoprotetive agent against fulminant hepatitis

Intracellular Bacteria Encode Inhibitory SNARE-Like Proteins

Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival

Inhibition of Endothelin-1-Mediated Contraction of Hepatic Stellate Cells by FXR Ligand

Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen and the enhanced contractile response to vasoactive mediators such as endothelin-1 (ET-1). The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidneys, adrenals, and intestine. FXR is also expressed in HSCs and activation of FXR in HSCs is associated with significant decreases in collagen production. However, little is known about the roles of FXR in the regulation of contraction of HSCs. We report in this study that treatment of quiescent HSCs with GW4064, a synthetic FXR agonist, significantly inhibited the HSC transdifferentiation, which was associated with an inhibition of the upregulation of ET-1 expression. These GW4064-treated cells also showed reduced contractile response to ET-1 in comparison to HSCs without GW4064 treatment. We have further shown that GW4064 treatment inhibited the ET-1-mediated contraction in fully activated HSCs. To elucidate the potential mechanism we showed that GW4064 inhibited ET-1-mediated activation of Rho/ROCK pathway in activated HSCs. Our studies unveiled a new mechanism that might contribute to the anti-cirrhotic effects of FXR ligands

Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

BACKGROUND: Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function. METHODS: We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions. RESULTS: We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp), C. caviae (GPIC), and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi. CONCLUSION: Caveolin-2 associates with the chlamydial inclusion independently of caveolin-1. The function of caveolin-2, either in the uninfected cell or in the chlamydial developmental cycle, remains to be elucidated. Nevertheless, this second caveolin protein can now be added to the small number of host proteins that are associated with the inclusions of this obligate intracellular pathogen