Serum markers of hepatocyte death and apoptosis are non invasive biomarkers of severe fibrosis in patients with alcoholic liver disease

Background

Quantification of hepatotocyte death is useful to evaluate the progression of alcoholic liver diseases. Our aims were to quantify and correlate the circulating levels of Cytokeratin 18 (CK18) and its caspases-generated fragment to disease severity in heavy alcoholics.

Methodology/Principal Findings

CK18 and CK18-fragment were evaluated in the serum of 143 heavy alcoholics. Serum levels of markers of hepatocyte death (CK18), apoptosis (CK18 fragment) and necrosis (CK18 -CK18 fragment) increased in patients with severe fibrosis compared to patients with mild fibrosis. These markers strongly correlated with Mallory-Denk bodies, hepatocyte ballooning, fibrosis and with hepatic TNFα and TGFβ assessed in the liver of 24 patients. Elevated levels of serum hepatocyte death and apoptotic markers were independent risk factors in predicting severe fibrosis in a model combining alkaline phosphatase, bilirubin, prothrombin index, hyaluronate, hepatocyte death and apoptotic markers. The level of markers of hepatocyte death and apoptosis had an area under the receiving operator curve that predicted severe fibrosis of 0.84 and 0.76, respectively.

Conclusion/Significance

Death of hepatocytes can be easily evaluated with serum markers and correlated with severe fibrosis in heavy alcohol drinkers. These biomarkers could be useful to rapidly evaluate liver injuries and the efficacy of therapies.     

Osteopontin deficiency aggravates hepatic injury induced by ischemia–reperfusion in mice

Osteopontin (OPN) is a multifunctional protein involved in hepatic steatosis, inflammation, fibrosis and cancer progression. However, its role in hepatic injury induced by ischemia–reperfusion (I–R) has not yet been investigated. We show here that hepatic warm ischemia for 45 min followed by reperfusion for 4 h induced the upregulation of the hepatic and systemic level of OPN in mice. Plasma aspartate aminotransferase and alanine aminotransferase levels were strongly increased in Opn^−/− mice compared with wild-type (Wt) mice after I–R, and histological analysis of the liver revealed a significantly higher incidence of necrosis of hepatocytes. In addition, the expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNFα), interleukin 6 (IL6) and interferon-γ were strongly upregulated in Opn^−/− mice versus Wt mice after I–R. One explanation for these responses could be the vulnerability of the OPN-deficient hepatocyte. Indeed, the downregulation of OPN in primary and AML12 hepatocytes decreased cell viability in the basal state and sensitized AML12 hepatocytes to cell death induced by oxygen–glucose deprivation and TNFα. Further, the downregulation of OPN in AML12 hepatocytes caused a strong decrease in the expression of anti-apoptotic Bcl2 and in the ATP level. The hepatic expression of Bcl2 also decreased in Opn^−/− mice versus Wt mice livers after I–R. Another explanation could be the regulation of the macrophage activity by OPN. In RAW macrophages, the downregulation of OPN enhanced iNOS expression in the basal state and sensitized macrophages to inflammatory signals, as evaluated by the upregulation of iNOS, TNFα and IL6 in response to lipopolysaccharide. In conclusion, OPN partially protects from hepatic injury and inflammation induced in this experimental model of liver I–R. This could be due to its ability to partially prevent death of hepatocytes and to limit the production of toxic iNOS-derived NO by macrophages.     

Homology modeling, simulation and molecular docking studies of catechol-2, 3-Dioxygenase from Burkholderia cepacia: Involved in degradation of Petroleum hydrocarbons

Catechol 2, 3-dioxygenase is present in several types of bacteria and undergoes degradation of environmental pollutants through an important key biochemical pathways. Specifically, this enzyme cleaves aromatic rings of several environmental pollutants such as toluene, xylene, naphthalene and biphenyl derivatives. Hence, the importance of Catechol 2, 3-dioxygenase and its role in the degradation of environmental pollutants made us to predict the three-dimensional structure of Catechol 2, 3-dioxygenase from Burkholderia cepacia. The 10ns molecular dynamics simulation was carried out to check the stability of the modeled Catechol 2, 3- dioxygenase. The results show that the model was energetically stable, and it attains their equilibrium within 2000 ps of production MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3-dioxygenase reveals that the benzene, O-xylene, Toluene, Fluorene, Naphthalene, Carbazol, Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl makes strong hydrogen bond and Van der waals interaction with the active site residues of H150, L152, W198, H206, H220, H252, I254, T255, Y261, E271, L276 and F309. Free energy of binding and estimated inhibition constant of these compounds demonstrates that they are energetically stable in their binding cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except Pyrene all the substrates made close contact with Fe atom by the distance ranges from 1.67 to 2.43 Å. In addition to that, the above mentioned substrate except pyrene all other made π-π stacking interaction with H252 by the distance ranges from 3.40 to 3.90 Å. All these docking results reveal that, except Chrysene all other substrate has good free energy of binding to hold enough in the active site and makes strong VdW interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of catalyzing the above-mentioned substrate.     

IGF-1 modulates gene expression of proteins involved in inflammation,cytoskeleton, and liver architecture

Even though the liver synthesizes most of circulating IGF-1, it lacks its receptor under physiological conditions. However, according to previous studies, a damaged liver expresses the receptor. For this reason, herein, we examine hepatic histology and expression of genes encoding proteins of the cytoskeleton, extracellular matrix, and cell-cell molecules and inflammation-related proteins. A partial IGF-1 deficiency murine model was used to investigate IGF-1’s effects on liver by comparing wild-type controls, heterozygous igf1^+/?, and heterozygous mice treated with IGF-1 for 10 days. Histology, microarray for mRNA gene expression, RT-qPCR, and lipid peroxidation were assessed. Microarray analyses revealed significant underexpression of igf1 in heterozygous mice compared to control mice, restoring normal liver expression after treatment, which then normalized its circulating levels. IGF-1 receptor mRNA was overexpressed in Hz mice liver, while treated mice displayed a similar expression to that of the controls. Heterozygous mice showed overexpression of several genes encoding proteins related to inflammatory and acute-phase proteins and underexpression or overexpression of genes which coded for extracellular matrix, cytoskeleton, and cell junction components. Histology revealed an altered hepatic architecture. In addition, liver oxidative damage was found increased in the heterozygous group. The mere IGF-1 partial deficiency is associated with relevant alterations of the hepatic architecture and expression of genes involved in cytoskeleton, hepatocyte polarity, cell junctions, and extracellular matrix proteins. Moreover, it induces hepatic expression of the IGF-1 receptor and elevated acute-phase and inflammation mediators, which all resulted in liver oxidative damage.     

An analysis of Bat Hawk Macheiramphus alcinus diet in the Melaky Region of lowland western Madagascar

We present information on the prey taken by the Bat Hawk Macheiramphus alcinus in two different areas of lowland western central Madagascar. These are the first dietary data from Madagascar for this widespread Old World species. The recovered remains were almost exclusively of bats and birds, with a few examples of reptiles and insects. In total, 178 pellets were analysed. On the basis of minimum number of individuals and biomass, bats accounted for 58.3% and 30.3%, respectively, and birds 36.1% and 69.7%, respectively. Amongst the nine species of bats recovered from the pellets, four were represented by multiple individuals, particularly taxa belonging to the families Molossidae and Vespertilionidae that fly in open areas, and for the 11 species of identified birds, all were represented by a single individual. These patterns are interpreted as a specialisation of feeding on bats during a narrow window of time at dusk, as they leave day roost sites, and then using birds in a more general manner to fill in nutritional needs.     

Données cytochimiques et ultrastructurales sur les tubes sécréteurs des glandes de la glu chez Peripatus acacioi Marcus et Marcus (Onychophore)

Résumé Dans les glandes de la glu, formations propres aux Onychophores, les tubes sécréteurs comprennent un épithélium glandulaire entouré d'une enveloppe conjonctive et musculaire. L'enveloppe conjonctive, délimitée par des membranes basales de mucopolysaccharides neutres et de mucoprotéines, contient deux sortes de fibrilles: des fibrilles collagènes et un type particulier de fibrilles élastiques. Les fibres musculaires, éparses, d'orientation circulaire, montrent une organisation beaucoup plus rudimentaire que dans les muscles somatiques.L'épithélium glandulaire est formé de cellules prismatiques toutes semblables, à gros noyau polyploïde en position basale, avec nucléole très volumineux. L'ensemble du cytoplasme de ces cellules est chargé de ribonucléines sous forme d'ergastoplasme granulifère à citernes concentriques et de nombreux ribosomes libres. Les structures lamellaires concentriques de l'ergastoplasme, qui se différencient du côté basal, près des noyaux, s'accroissent dans la région médiane des cellules et se désorganisent à l'apex, en libérant des grandes quantités de ribosomes. La sécrétion de ces cellules est formée essentiellement de protéines qui s'accumulent dans la région apicale sans passer par l'appareil de Golgi, lequel est d'ailleurs peu développé. Ces protéines ne sont donc pas concentrées en granules de sécrétion, mais déchargées dans la cavité glandulaire, directement à l'état diffus, par l'intermédiaire d'épanchements cytoplasmiques de la face apicale cellulaire dans lesquels passent également de nombreux ribosomes libres. Polyploïdie, grand volume nucléolaire, hyperdéveloppement de l'ergastoplasme granulifère, abondance des ribosomes libres, non-intervention de l'appareil de Golgi et absence de concentration de la sécrétion en granules sont mis en relation avec la synthèse rapide et continue des protéines qui forment le principal constituant de la glu.

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Cytochemical and ultrastructural data on the secretory tubes of the slime glands in Peripatus acacioi Marcus and Marcus (Onychophora)

Summary In the peculiar slime glands of the Onychophora, the secretory tubes consist of a glandular epithelium surrounded by a layer of connective and muscular tissue. The connective layer, bounded by basal membranes of neutral mucopolysaccharides and mucoproteins, contains two kinds of fibrils: collagenous fibrils and an especial type of elastic fibrils. The muscle fibers, which are scattered and circularly oriented, are more simply organized than those of the somatic muscles.The glandular epithelium is constituted by prismatic cells of the same type, with a large polyploid basally situated nucleus, containing a very large nucleolus. The cytoplasm of these cells is filled with ribonucleins in form of ergastoplasm and in numerous free ribosomes. Concentric lamellar structures of ergastoplasm, which originate basally close to the nucleus, are well developed in the central region of the cell and become disorganized at the apex, where they liberate great quantities of ribosomes. The secretion of these cells consists essentially of proteins, which accumulate in the apical region without passing through the poorly developed Golgi apparatus. These proteins, therefore, are not concentrated in secretory granules, rather they are released in a diffuse form, into the glandular cavity. They are discharged in cytoplasmic expansions detached from the apical region, together with a large quantity of free ribosomes. Polyploidy, large nucleolus, well developed granular ergastoplasm, abundance of free ribosomes, non-participation of the Golgi apparatus and lack of concentration in secretory granules are correlated with rapid and continuous synthesis of proteins which are the principal component of the slime.

Avec la collaboration technique de Mlle Eliana Parisi et l'aide de la Fundação de Amparo à Pesquisa do Estado de São Paulo.