Regression of Fibrosis and Reversal of Cirrhosis in Rats by Galectin Inhibitors in Thioacetamide-Induced Liver Disease

Galectin-3 protein is critical to the development of liver fibrosis because galectin-3 null mice have attenuated fibrosis after liver injury. Therefore, we examined the ability of novel complex carbohydrate galectin inhibitors to treat toxin-induced fibrosis and cirrhosis. Fibrosis was induced in rats by intraperitoneal injections with thioacetamide (TAA) and groups were treated with vehicle, GR-MD-02 (galactoarabino-rhamnogalaturonan) or GM-CT-01 (galactomannan). In initial experiments, 4 weeks of treatment with GR-MD-02 following completion of 8 weeks of TAA significantly reduced collagen content by almost 50% based on Sirius red staining. Rats were then exposed to more intense and longer TAA treatment, which included either GR-MD-02 or GM-CT-01 during weeks 8 through 11. TAA rats treated with vehicle developed extensive fibrosis and pathological stage 6 Ishak fibrosis, or cirrhosis. Treatment with either GR-MD-02 (90 mg/kg ip) or GM-CT-01 (180 mg/kg ip) given once weekly during weeks 8–11 led to marked reduction in fibrosis with reduction in portal and septal galectin-3 positive macrophages and reduction in portal pressure. Vehicle-treated animals had cirrhosis whereas in the treated animals the fibrosis stage was significantly reduced, with evidence of resolved or resolving cirrhosis and reduced portal inflammation and ballooning. In this model of toxin-induced liver fibrosis, treatment with two galectin protein inhibitors with different chemical compositions significantly reduced fibrosis, reversed cirrhosis, reduced galectin-3 expressing portal and septal macrophages, and reduced portal pressure. These findings suggest a potential role of these drugs in human liver fibrosis and cirrhosis.     

ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation

HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell-cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP-70, a key kinase regulating T-cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP-70, or expressing a kinase-dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP-70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP-70, intracellular Gag localization was impaired. ZAP-70 was required in infected donor cells for efficient cell-to-cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T-cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell-to-cell contacts.     

Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells

Villin is an actin-binding protein present in intestinal and kidney brush borders. Villin has been shown to present in vitro Ca(2+)-dependent bundling and severing F-actin properties. The study of villin knock-out mice allowed us to show that while bundling of F-actin microfilaments is unaffected, this protein is important for the reorganization of the actin cytoskeleton elicited by various signals during both physiological and pathological conditions. Here, we studied the role of villin during infection by Shigella flexneri, the causative agent of bacillary dysentery. This bacterium induces the reorganization of the host actin cytoskeleton to penetrate into epithelial cells and spread from cell to cell. In vivo, we show that unlike newborn vil+/+ mice, which are sensitive to Shigella invasion, resulting in a destructive inflammatory response of the intestinal mucosa following intragastric inoculation, newborn vil-/- mice appear fully resistant to infection. Using primary cultures of intestinal epithelial cells derived from vil+/+ or vil -/- mice, we demonstrate that villin plays an essential role in S. flexneri entry and cell-to-cell dissemination. Villin expression is thus critical for Shigella infection through its ability to remodel the actin cytoskeleton.     

Effect of dietary virgin olive oil on urinary excretion of etheno-DNA adducts

A significant protective effect against cancer and coronary heart disease has been attributed to the Mediterranean diet, in which olive oil is the main source of fat. Dietary antioxidants, as phenolic compounds from virgin olive oil, are candidates for reducing cancer risk by minimizing oxidatively derived DNA damage. Etheno-DNA adducts are formed as a result of oxidative stress and lipid peroxidation. To evaluate whether phenol-rich virgin olive oil influences urinary excretion of the etheno-DNA adducts epsilonAde, epsilondA, and epsilondC as markers of oxidative stress, a randomized, double-blinded, crossover trial with three intervention periods was conducted in 28 healthy men. Each intervention was of 3 weeks' duration and separated by 2-week washout periods. Twenty-five milliliters of similar olive oils, but with differences in their phenolic content (from 2.7 to 366 mg/kg), were supplied to each subject per day. The urinary excretion of the DNA adducts was assayed by LC-MS/MS in samples before and after consumption of high phenolic content olive oil (virgin). The 24-h excretion rate did not differ significantly between baseline and after virgin olive oil consumption: epsilonAde, 105.5 +/- 40.8 vs 116.4 +/- 53.4 pmol epsilonAde/24 h (p = 0.21); epsilondA, 37.9 +/- 24.8 vs 37.6 17 +/- 24.2 pmol epsilondA/24 h (p = 0.93); and epsilondC, 218.7 +/- 157.2 vs 193.5 +/- 64.7 pmol epsilondC/24 h (p = 0.37). Multiple regression analysis showed a significant association between etheno-DNA adduct excretion rate and the dietary intake of linoleic acid (C18:2, omega-6) in healthy men. Consumption of 25 ml per day of phenol-rich virgin olive oil for 3 weeks did not modify to a significant degree the urinary excretion of etheno-DNA adducts in 28 healthy volunteers.     

Actions of acromelic acid on nervous system l-glutamate receptors

Acromelic acid, a naturally occurring kainoid, isolated from the mushroom Clitocybe acromelalga, is a weak displacer of [³H]L-glutamate binding to cockroach (Periplaneta americana) nerve cord membranes. Acromelic acid (1 mM) displaces ～?60% of specifically bound [³H]L-glutamate. When applied by bath perfusion to the cell body membrane of the cockroach fast coxal depressor motor neurone, acromelic acid generated slow, prolonged, dose-dependent depolarizations at concentrations of 0.3 μM and above. Thus acromelic acid is among the most potent of the excitatory amino acids tested to date on insect neurones. © 1994 Wiley-Liss, Inc.     

Cellular Metabolism Is a Major Determinant of HIV-1 Reservoir Seeding in CD4+ T Cells and Offers an Opportunity to Tackle Infection

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Recombinant infectious hematopoietic necrosis viruses induce protection for rainbow trout Oncorhynchus mykiss

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicaemia virus (VHSV) are rhabdoviruses that infect salmonids, producing serious economic losses. Two recombinant IHN viruses were generated by reverse genetics. For one (rIHNV GFP) the IHNV NV gene was replaced with the green fluorescent protein (GFP) gene. In the other (rIHNV-Gvhsv GFP) the G gene was also exchanged for that of VHSV. No mortalities, external signs or histological lesions were observed in experimental infections conducted with the recombinant viruses. Neither the rIHNV GFP nor rIHNV-Gvhsv GFP was detected by RT-PCR in any of the examined tissues from experimentally infected fish. In order to assess their potential as vaccines against the wild type viruses, rainbow trout were vaccinated with the recombinant viruses by intraperitoneal injection and challenged 30 d later with virulent IHNV or VHSV. The GFP viruses provided protection against both wild type viruses. None of the recombinant viruses induced antibody production, and the expression of interferon (IFNalpha4) and interferon induced genes such as Mx protein and ISG-15 was not different to that of controls. The rIHNV-Gvhsv GFP did not inhibit cellular apoptosis as it was observed in an IHNV inoculated fish cell line. These studies suggest that the recombinant rIHNV-Gvhsv GFP is a promising candidate as a live recombinant vaccine and also provides a good model to further study viral pathogenicity and the molecular basis of protection against these viral infections.     

Lysosome motility and distribution: Relevance in health and disease

Lysosomes are dynamic organelles, which can fuse with a variety of targets and undergo constant regeneration. They can move along microtubules in a retrograde and anterograde fashion by using motor proteins, kinesin and dynein, being main players in extracellular secretion, intracellular components degradation and recycling. Moreover, lysosomes interact with other intracellular organelles to regulate their turnover, such as ER, mitochondria and peroxisomes.The correct localization of lysosomes is relevant in several physiological processes, including appropriate antigen presentation, neurotransmission and receptors modulation in neuronal synapsis, whereas hepatic lysosomes and autophagy are master regulators of nutrient homeostasis.Alterations in lysosome function due to mutation of genes encoding lysosomal proteins, soluble hydrolases as well as membrane proteins, lead to lysosomal storage diseases (LSDs). Lysosomes containing undegraded substrates are finally stacked and therefore miss positioned inside the cell, leading to lysosomal dysfunction, which impacts a wide range of cellular functions.