Anti-tumor necrosis factor α treatment promotes apoptosis and prevents liver regeneration in a transgenic mouse model of chronic hepatitis C.

UNLABELLED: Tumor necrosis factor α (TNFα) has been implicated in a variety of inflammatory diseases, and anti-TNFα has been shown to improve therapy when added to standard of care in chronic hepatitis C virus (HCV) infection. In addition, patients with chronic HCV have increased serum levels of TNFα and the macrophage-attracting chemokine (C-C motif) ligand 2 (CCL2). A mouse model of chronic HCV with hepatic nonstructural (NS) 3/4A protein expression mimics the human infection through a reduced response to double-stranded RNA and cleavage of the T cell protein tyrosine phosphatase. The mice also display a resistance to TNFα in vivo. We therefore analyzed the relationship between NS3/4A and TNFα. Wild-type and NS3/4A-transgenic (Tg) mice were treated with TNFα/D-galactosamine (D-galN), acting through the TNF receptor 1 on hepatocytes and macrophages, or lipopolysaccharide (LPS)/D-galN, acting through Toll-like receptor 4 on sinusoidal endothelial cells, macrophages, and dendritic cells. Mice were analyzed for hepatic signaling, liver damage, TNFα, and CCL2. Similar to HCV-infected humans, NS3/4A-Tg mice displayed elevated basal levels of TNFα and CCL2. Treatment of NS3/4A-Tg mice with TNFα/D-galN or LPS/D-galN led to increased hepatic nuclear factor kappa B (NFκB) activation, increased TNFα and CCL2 levels, decreased apoptosis, and increased hepatocyte regeneration. Importantly, blocking NFκB activation (bortezomib) or administering anti-TNFα (infliximab) 4 hours after LPS/D-galN injection reversed the resistance of NS3/4A-Tg mice to TNFα-induced liver injury. CONCLUSION: Resistance to TNFα seen in NS3/4A-Tg mice is explained by a hepatoprotective effect of NFκB and TNFα. Hence, anti-TNFα agents block these effects and are antiviral by promoting hepatocyte apoptosis and preventing hepatocyte regeneration.

Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells.

Giardia lamblia, an important cause of diarrheal disease, resides in the small intestinal lumen in close apposition to epithelial cells. Since the disease mechanisms underlying giardiasis are poorly understood, elucidating the specific interactions of the parasite with the host epithelium is likely to provide clues to understanding the pathogenesis. Here we tested the hypothesis that contact of Giardia lamblia with intestinal epithelial cells might lead to release of specific proteins. Using established co-culture models, intestinal ligated loops and a proteomics approach, we identified three G. lamblia proteins (arginine deiminase, ornithine carbamoyl transferase and enolase), previously recognized as immunodominant antigens during acute giardiasis. Release was stimulated by cell-cell interactions, since only small amounts of arginine deiminase and enolase were detected in the medium after culturing of G. lamblia alone. The secreted G. lamblia proteins were localized to the cytoplasm and the inside of the plasma membrane of trophozoites. Furthermore, in vitro studies with recombinant arginine deiminase showed that the secreted Giardia proteins can disable host innate immune factors such as nitric oxide production. These results indicate that contact of Giardia with epithelial cells triggers metabolic enzyme release, which might facilitate effective colonization of the human small intestine.

Annexin-like alpha giardins: a new cytoskeletal gene family in Giardia lamblia.

Through a genome survey and phylogenetic analysis, we have identified and sequenced 14 new coding regions for alpha-giardins in Giardia lamblia. These proteins are related to annexins and comprise a multi-gene family with 21 members. Many alpha giardins are highly expressed proteins that are very immunogenic during acute giardiasis in humans. However, little is known about the function of these proteins. By using PCR with different combinations of gene-specific primers, we demonstrated that several of the genes localised to the same chromosomal fragment. These data point towards a molecular evolution through gene duplication and subsequent functional divergence. Semi-quantitative reverse transcriptase-PCR analysis of the Giardia life cycle revealed large differences in mRNA expression levels of the alpha giardins. Epitope tagging of the alpha-giardins localised them to different cytoskeletal components, such as the flagella and the adhesive disc, but also to the plasma membrane. These localisation experiments suggest alpha-giardins play a role in cell motility, attachment and membrane stability.

Developmental changes in the adhesive disk during Giardia differentiation.

Giardia lamblia is a protozoan parasite infecting the upper mammalian small intestine. Infection relies upon the ability of the parasite to attach to the intestine via a unique cytoskeletal organelle, the ventral disk. We determined the composition and structure of the disk throughout the life cycle of the parasite and identified a new disk protein, SALP-1. SALP-1 is an immunodominant protein related to striated fiber-assemblin (SFA). The disk is disassembled during encystation and stored as four fragments in the immobile cyst. Serial Analysis of Gene Expression (SAGE) showed that the mRNA levels of the disk proteins decreased in encystation but two-dimensional protein gels showed that the protein levels were more constant. The parasite emerges without a functional disk but the four disk fragments are quickly reassembled into two new disks on the dividing, early excysting form. Thus, disk proteins are stored within the cyst, ready to be used in the rapid steps of excystation.

Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity.

Alpha-1 giardin is an immunodominant protein in the intestinal protozoan parasite Giardia lamblia. The Triage((R)) parasite panel, used to detect copro-antigens in stool from giardiasis patients, reacts with an epitope between amino acids 160 and 200 in alpha-1 giardin. This region of the protein is also highly immunogenic during human infections. Alpha-1 giardin is related to annexins and like many other annexins it was shown to be plasma membrane associated. Immunoelectron and immunofluorescence microscopy revealed that some alpha-1 giardin are displayed on the surface of recently excysted cells. Recombinant alpha-1 giardin displayed a Ca(2+)-dependent binding to glycosaminoglycans (GAGs), in particular heparan sulphate, a common GAG in the intestinal tract. Recombinant alpha-1 giardin bound to thin sections of human small intestine, a binding which could be inhibited by adding increasing concentrations of sulphated sugars. A surface associated trypsin activated Giardia lectin (taglin) has been suggested to be important for G. lamblia attachment. In this study we show that a monoclonal antibody that inhibits taglin recognises alpha-1 and alpha-2 giardin. Thus, alpha-1 giardin is a highly immunoreactive GAG-binding protein, which may play a key role in the parasite-host interaction. Our results further show a conserved function of annexins from lower to higher eukaryotes.

Identification of immunoreactive proteins during acute human giardiasis.

The protozoan Giardia lamblia is a major cause of parasite-induced diarrhea in humans. Humoral immunity has been shown to be important for clearance of the infection, but only a few antigens have been identified. In this study, we focused on the immunoreactivity of nonvariant antigens. Serum samples from 93 patients with acute giardiasis who were infected during a waterborne outbreak in a nonendemic country were screened on 1-dimensional Western blots. Representative serum samples that reacted strongly with proteins of different molecular weights were further analyzed on 2-dimensional Western blots. Sixteen immunoreactive proteins were identified using mass spectrometry analysis, among them variable surface proteins, alpha-giardins, arginine deiminase, ornithine carbamoyl transferase, and fructose-1,6-bisphosphate aldolase. Several of the identified proteins were immunoreactive in recombinant form, and they may be important in the development of new diagnostic tools and vaccines.

Giardia lamblia: identification and characterization of Rab and GDI proteins in a genome survey of the ER to Golgi endomembrane system.

To investigate the complexity of the endomembrane transport system in the early diverging eukaryote, Giardia lamblia, we characterized homologues of the GTP-binding proteins, Rab1 and Rab2, involved in regulating vesicular trafficking between the endoplasmic reticulum and Golgi in higher eukaryotes, and GDI, which plays a key role in the cycling of Rab proteins. G. lamblia Rab1, 2.1, and GDI sequences largely resemble yeast and mammalian homologues, are transcribed as 0.66-, 0.62-, and 1.4-kb messages, respectively, and are expressed during growth and encystation. Western analyses detected an abundant Rab/GDI complex at approximately 80 kDa, and free GDI (60 kDa) in both trophozoites and encysting cells. Immunoelectron microscopy with antibody to Rab1 localized Rab with ER, encystation secretory vesicles, and lysosome-like peripheral vesicles. GDI associated with these structures, and with small vesicles found throughout the cytoplasm, consistent with GDI's key role in Rab cycling between organelles within the cell.