A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

Background: Intestinal allograft rejection resembles Crohn's disease clinically and pathologically. An understanding of its mechanism could impact this life-saving procedure, as well as provide insight into the pathophysiology of inflammatory bowel disease. The NOD2 protein has been implicated as a key player in intestinal immune health, as a consequence of the discovery of three polymorphisms linked with Crohn's disease. An investigation was carried out to determine whether epithelial immune function and graft survival were influenced by NOD2 mutations in an intestinal transplant population. Methods: The NOD2 genotypes of 34 transplants performed consecutively over the past 3 years were determined. The NOD2 genotypes were related to clinical outcomes and the expression of certain intestinal antimicrobial peptides (AMPs) believed to protect the epithelium. Results: An unexpectedly high percentage of recipients, 35%, possessed NOD2 polymorphisms, while 8.6% of donors had comparable mutations. The likelihood of allograft failure was about 100-fold higher in recipients with mutant NOD2 alleles compared with recipients with wild-type NOD2 loci. Rejection in NOD2 mutant recipients was characterised by decreased expression of certain Paneth cell and enterocyte AMPs, prior to the onset of epithelial injury and inflammation.

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The role of retinoic acid (RA) in liver fibrogenesis was previously studied in cultured hepatic stellate cells (HSCs). RA suppresses the expression of alpha2(I) collagen by means of the activities of specific nuclear receptors RARalpha, RXRbeta and their coregulators. In this study, the effects of RA in fibrogenesis were examined in carbon tetrachloride (CCl4) induced liver fibrosis in mice. Mice were treated with CCl4 or RA and CCl4, along side control groups, for 12weeks. RA reduced the amount of histologically detectable fibrosis produced by CCl4. This was accompanied by a attenuation of the CCl4 induced increase in alpha2(I) collagen mRNA and a lower (2-fold versus 3-fold) increase in liver hydroxyproline. Furthermore, RA reduced the levels of 3-nitrotyrosine (3-NT) protein adducts and thiobarbituric acid (TBA) reactive substance (TBARS) in the liver, which are formed as results of oxidative stress induced by CCl4 treatment. These in vivo findings support our previous in vitro studies in cultured HSC of the inhibitory effect of RA on type I collagen expression. The data also provide evidence that RA reduces CCl4 induced oxidative stress in liver, suggesting that the anti-fibrotic role of RA is not limited to the inhibition of type I collagen expression.

This study shows that concentrations of AC, which occur in vivo after the ingestion of alcoholic beverages, result in the formation of ROS in rat hepatic stellate cells. The increases in ROS are known to activate stellate cells promoting fibrogenesis.

Hepatic fibrosis is due to the increased synthesis and deposition of type I collagen. Acetaldehyde activates type I collagen promoters. Nuclear factor B (NF-B) was previously shown to inhibit expression of murine ␣ 1 (I) and human ␣ 2 (I) collagen promoters. The present study identifies binding of NF-B, present in nuclear extracts of stellate cells, to a region between ؊553 and ؊537 of the murine ␣ 2 (I) collagen promoter. The NF-B (p65) expression vector inhibited promoter activity. Mutation of the promoter at the NF-B-binding site increased basal promoter activity and abrogated the activating and inhibitory effects of transforming growth factor ␤ and tumor necrosis factor ␣, respectively, on promoter activity. Acetaldehyde increased I B-␣ kinase activity and phosphorylated I B-␣, NF-B nuclear protein, and its binding to the promoter. However, the activating effect of acetaldehyde was not affected by the mutation of the promoter. In conclusion, although acetaldehyde increases the binding of NF-B to the murine ␣ 2 (I) collagen promoter, this binding does not mediate the activating effect of acetaldehyde on promoter activity. The effects of acetaldehyde in increasing the translocation of NF-B to the nucleus with increased DNA binding activity may be important in mediating the effects of acetaldehyde on other genes.

Deficiencies in NAD(P)H oxidase and in iNOS separately reduce, but do not eliminate carbon tetrachloride-induced liver fibrosis. Likely causes for this inhibitory effects are decreases in the production of ROS in NAD(P)H deficiency and of peroxinitrite radicals in iNOS deficiency.

TNFalpha and acetaldehyde independently activate NF-kappaB by rapid enhancement of IkappaB-alpha kinase activity and degradation of IkB-alpha protein. Increased TNFalpha is the principal mechanism for the elevation of NF-kappaB in severe alcoholic hepatitis. The elevation of NF-kappaB due to TNFalpha enhance liver injury, but inhibit fibrogenesis. In contrast, the effect of acetaldehyde in activating NF-kappaB is associated with increases in both liver injury and fibrogenesis, indicating that the effects of acetaldehyde on fibrogenesis are mediated by cytokines and by trans-acting factors other than NF-kappaB.