A ribosomal RNA fragment with 2',3'-cyclic phosphate and GTP-binding activity acts as RIG-I ligand.

The RNA helicase RIG-I plays a key role in sensing pathogen-derived RNA. Double-stranded RNA structures bearing 5'-tri- or diphosphates are commonly referred to as activating RIG-I ligands. However, endogenous RNA fragments generated during viral infection via RNase L also activate RIG-I. Of note, RNase-digested RNA fragments bear a 5'-hydroxyl group and a 2',3'-cyclic phosphate. How endogenous RNA fragments activate RIG-I despite the lack of 5'-phosphorylation has not been elucidated. Here we describe an endogenous RIG-I ligand (eRL) that is derived from the internal transcribed spacer 2 region (ITS2) of the 45S ribosomal RNA after partial RNase A digestion in vitro, RNase A protein transfection or RNase L activation. The immunostimulatory property of the eRL is dependent on 2',3'-cyclic phosphate and its sequence is characterized by a G-quadruplex containing sequence motif mediating guanosine-5'-triphosphate (GTP) binding. In summary, RNase generated self-RNA fragments with 2',3'-cyclic phosphate function as nucleotide-5'-triphosphate binding aptamers activating RIG-I.

Identification of an Optimal TLR8 Ligand by Alternating the Position of 2'-O-Ribose Methylation.

Recognition of RNA by receptors of the innate immune system is regulated by various posttranslational modifications. Different single 2'-O-ribose (2'-O-) methylations have been shown to convert TLR7/TLR8 ligands into specific TLR8 ligands, so we investigated whether the position of 2'-O-methylation is crucial for its function. To this end, we designed different 2'-O-methylated RNA oligoribonucleotides (ORN), investigating their immune activity in various cell systems and analyzing degradation under RNase T2 treatment. We found that the 18S rRNA-derived TLR7/8 ligand, RNA63, was differentially digested as a result of 2'-O-methylation, leading to variations in TLR8 and TLR7 inhibition. The suitability of certain 2'-O-methylated RNA63 derivatives as TLR8 agonists was further demonstrated by the fact that other RNA sequences were only weak TLR8 agonists. We were thus able to identify specific 2'-O-methylated RNA derivatives as optimal TLR8 ligands.

A Comprehensive UHPLC Ion Mobility Quadrupole Time-of-Flight Method for Profiling and Quantification of Eicosanoids, Other Oxylipins, and Fatty Acids.

Analysis of oxylipins by liquid chromatography mass spectrometry (LC/MS) is challenging because of the small mass range occupied by this diverse lipid class, the presence of numerous structural isomers, and their low abundance in biological samples. Although highly sensitive LC/MS/MS methods are commonly used, further separation is achievable by using drift tube ion mobility coupled with high-resolution mass spectrometry (DTIM-MS). Herein, we present a combined analytical and computational method for the identification of oxylipins and fatty acids. We use a reversed-phase LC/DTIM-MS workflow able to profile and quantify (based on chromatographic peak area) the oxylipin and fatty acid content of biological samples while simultaneously acquiring full scan and product ion spectra. The information regarding accurate mass, collision-cross-section values in nitrogen (DTCCSN2), and retention times of the species found are compared to an internal library of lipid standards as well as the LIPID MAPS Structure Database by using specifically developed processing tools. Features detected within the DTCCSN2 and m/ z ranges of the analyzed standards are flagged as oxylipin-like species, which can be further characterized using drift-time alignment of product and precursor ions distinctive of DTIM-MS. This not only helps identification by reducing the number of annotations from LIPID MAPS but also guides discovery studies of potentially novel species. Testing the methodology on Salmonella enterica serovar Typhimurium-infected murine bone-marrow-derived macrophages and thrombin activated human platelets yields results in agreement with literature. This workflow has also annotated features as potentially novel oxylipins, confirming its ability in providing further insights into lipid analysis of biological samples.

Investigating the control of Listeria monocytogenes on a ready-to-eat ham product using natural antimicrobial ingredients and postlethality interventions.

Ready-to-eat (RTE) meat and poultry products manufactured with natural or organic methods are at greater risk for Listeria monocytogenes growth, if contaminated, than their conventional counterparts due to the required absence of preservatives and antimicrobials. Thus, the objective of this study was to investigate the use of commercially available natural antimicrobials and postlethality interventions in the control of L. monocytogenes growth and recovery on a RTE ham product. Antimicrobials evaluated were cranberry powder (90MX), vinegar (DV), and vinegar/lemon juice concentrate (LV1X). Postlethality interventions studied were high hydrostatic pressure at 400 (HHP400) or 600 (HHP600) MPa, lauric arginate (LAE), octanoic acid (OA), and postpackaging thermal treatment (PPTT). Parameters evaluated through 98 days of storage at 4±1°C were residual nitrite concentrations, pH, a(w), and viable L. monocytogenes on modified Oxford (MOX) media. On day 1, OA, 90MX, DV, and LV1X yielded lower residual nitrite concentrations than the control, whereas HHP400, HHP600, and LAE did not. LAE, HHP400, and OA reduced L. monocytogenes population compared to the control after 1 day of storage by 2.38, 2.21, and 1.73 log10 colony-forming units per gram, respectively. PPTT did not achieve a significant reduction in L. monocytogenes populations. L. monocytogenes recovered and grew in all postlethality intervention treatments except HHP600. 90MX did not inhibit the growth of L. monocytogenes, while DV and LV1X did. Results of this study demonstrate the bactericidal properties of HHP, OA, and LAE and the bacteriostatic potential of natural antimicrobial ingredients such as DV and LV1X against L. monocytogenes.

A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae reveals novel insights into factors that stabilize chromatin structure on newly replicated DNA.

The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.

Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood.

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.

Platelet Receptor Glycoprotein VI-Dimer Is Overexpressed in Patients with Atrial Fibrillation at High Risk of Ischemic Stroke.

Introduction Atrial fibrillation (AF) increases the risk of ischemic stroke (IS). We hypothesized that the functional form of platelet receptor glycoprotein (GP) VI, GPVI-dimer, which binds to collagen and fibrin causing platelet activation, is overexpressed in patients with AF who have not had a stroke. Methods A total of 75 inpatients with AF were recruited. None were admitted with or had previously had thrombotic events, including IS or myocardial infarction. Platelet surface expression of total GPVI, GPVI-dimer, and the platelet activation marker P-selectin were quantitated by whole blood flow cytometry. Serum biomarkers were collected in AF patients. Results were compared against patients contemporaneously admitted to hospital with similar age and vascular risk-factor profiles without AF (noAF, n = 30). Results Patients with AF have similar total GPVI surface expression ( p = 0.58) and P-selectin exposure ( p = 0.73) on their platelets compared with noAF patients but demonstrate significantly higher GPVI-dimer expression ( p = 0.02 ). Patients with paroxysmal AF express similar GPVI-dimer levels compared with permanent AF and GPVI-dimer levels were not different between anticoagulated groups. Serum N-terminal pro b-type natriuretic peptide ( p < 0.0001 ) and high sensitivity C-reactive protein ( p < 0.0001 ) were significantly correlated with GPVI-dimer expression in AF platelets. AF was the only vascular risk factor that was independently associated with higher GPVI-dimer expression in the whole population ( p = 0.02 ) . Conclusion GPVI inhibition is being explored in clinical trials as a novel target for IS treatment. As GPVI-dimer is elevated in AF patients' platelets, the exploration of targeted GPVI-dimer inhibition for stroke prevention in patients at high risk of IS due to AF is supported.

Role of heat shock protein 47 in platelet glycoprotein VI dimerization and signaling.

Background: Heat shock protein 47 (HSP47) is an intracellular chaperone protein with an indispensable role in collagen biosynthesis in collagen-secreting cells. This chaperone has also been shown to be released and present on the surface of platelets. The inhibition of HSP47 in human platelets or its ablation in mouse platelets reduces platelet function in response to collagen and the glycoprotein (GP) VI collagen receptor agonist CRP-XL. Objectives: In this study, we sought, through experiments, to explore cellular distribution, trafficking, and influence on GPVI interactions to understand how HSP47 modulates collagen receptor signaling. Methods: HSP47-deficient mouse platelets and SMIH- treated human platelets were used to study the role of HSP47 in collagen mediated responses and signaling. Results: Using subcellular fractionation analysis and immunofluorescence microscopy, HSP47 was found to be localized to the platelet-dense tubular system. Following platelet stimulation, HSP47 mobilization to the cell surface was shown to be dependent on actin polymerization, a feature common to other dense tubular system resident platelet proteins that are released to the cell surface during activation. In this location, HSP47 was found to contribute to platelet adhesion to collagen or CRP-XL but not to GFOGER peptide (an integrin α2ß1-binding sequence within collagens), indicating selective effects of HSP47 on GPVI function. Dimerization of GPVI on the platelet surface increases its affinity for collagen. GPVI dimerization was reduced following HSP47 inhibition, as was collagen and CRP-XL-mediated signaling. Conclusion: The present study identifies a role for cell surface-localized HSP47 in modulating platelet responses to collagen through dimerization of GPVI, thereby enhancing platelet signaling and activation.

Effects of high-pressure processing and enzymatic dephosphorylation on phosvitin properties.

BACKGROUND: Egg phosvitin could be a good source of functional peptides. Enzymatic dephosphorylation and high-pressure processing combined with thermal treatment applied before proteolysis could produce phosvitin hydrolysates with different properties compared to its native form. RESULTS: Phosvitin structure was maintained overall during high-pressure treatment of 600 MPa applied at an initial temperature of 65 °C regardless of the pH and duration of treatment, confirming the high structural stability of this phosphoprotein. Treatment of phosvitin with phosphatase increased the degree of dephosphorylation from 24% to 63%, after 2 and 18 h, respectively. Moderate dephosphorylation of phosvitin prior to proteolytic digestion improved its hydrolysis, allowing formation of peptides with a molecular weight lower than 17,000 kDa as determined by size exclusion chromatography. Angiotensin-converting enzyme (ACE) inhibition and antioxidant activity of dephosphorylated and protease-treated phosvitin was increased by 52% and 39%, respectively, as compared to protease-digested native phosvitin. CONCLUSION: Enzymatic dephosphorylation before proteolysis mimicking in vivo gut conditions improved ACE inhibition and antioxidant activity of phosvitin hydrolysates.

Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI-dimer-An interaction that may modulate fibrin crosslinking.

Background: In the fibrin-forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel-like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ-chains and α-chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. Objectives: We determined if GPVI interacts with FXIII and how this might modulate clot formation. Methods: We measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI-dimer (GPVI-Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin-activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ-chain crosslinking were analyzed by immunoblotting. Results: GPVI-dimer, but not GPVI-monomer, bound to FXIII. GPVI-dimer selectively bound to the FXIII A-subunit, but not to the B-subunit, an interaction that was decreased or abrogated by the GPVI-dimer-specific antibody mFab-F. The GPVI-dimer-FXIII interaction decreased the extent of γ-chain crosslinking, indicating a role in the regulation of clot formation. Conclusions: This is the first report of the specific interaction between GPVI-dimer and the A-subunit of FXIII, as determined in an in vitro system with defined components. GPVI-dimer-FXIII binding was inhibitory toward FXIII-catalyzed crosslinking of fibrin γ-chains in fibrin clots. This raises the possibility that GPVI-dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability.

Many Ways to Communicate-Crosstalk between the HBV-Infected Cell and Its Environment.

Chronic infection with the hepatitis B virus (HBV) affects an estimated 257 million people worldwide and can lead to liver diseases such as cirrhosis and liver cancer. Viral replication is generally considered not to be cytopathic, and although some HBV proteins may have direct carcinogenic effects, the majority of HBV infection-related disease is related to chronic inflammation resulting from disrupted antiviral responses and aberrant innate immune reactions. Like all cells, healthy and HBV-infected cells communicate with each other, as well as with other cell types, such as innate and adaptive immune cells. They do so by both interacting directly and by secreting factors into their environment. Such factors may be small molecules, such as metabolites, single viral proteins or host proteins, but can also be more complex, such as virions, protein complexes, and extracellular vesicles. The latter are small, membrane-enclosed vesicles that are exchanged between cells, and have recently gained a lot of attention for their potential to mediate complex communication and their potential for therapeutic repurposing. Here, we review how HBV infection affects the communication between HBV-infected cells and cells in their environment. We discuss the impact of these interactions on viral persistence in chronic infection, as well as their relation to HBV infection-related pathology.

Platelet surface receptor glycoprotein VI-dimer is overexpressed in stroke: The Glycoprotein VI in Stroke (GYPSIE) study results.

OBJECTIVES: Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. METHODS: 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). RESULTS: The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r2 = 0.88, P<0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001). CONCLUSIONS: Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.

Microchip electrospray: improvements in spray and signal stability during gradient elution by an inverted postcolumn makeup flow.

Dynamic changes in mobile phase composition during high-performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray modes. We investigate the impact of the eluent composition on spray stability and MS response by infusion and injection experiments with a small tetrapeptide in water-acetonitrile mixtures. The employed HPLC/electrospray (ESI)-MS configuration uses a microchip equipped with an enrichment column, a separation column, and a makeup flow (MUF) channel. One nano pump is connected to the separation column, while a second one delivers solvent of exactly inverted composition to the MUF channel. Both solvent streams are united behind the separation column, before the ESI tip, such that the resulting electrosprayed solution always has identical composition during a gradient elution. Analyte peak parameters without and with MUF compensation are determined and discussed with respect to the electrospray mode and eluent composition. The postcolumn MUF significantly improves spray and signal stability over the entire solvent gradient, without compromising the performance of the HPLC separation column. It can also be conveniently implemented on microchip platforms.

Fast, accurate, and convenient analysis of bed densities for columns packed with fine reversed-phase particles.

We determine the interparticle porosities of commercially available, analytical, reversed-phase HPLC columns by Donnan exclusion of a small, unretained, co-ionic tracer (nitrate ions). The columns contained packings of C(18)-modified, endcapped, silica particles, which differed in their nominal particle diameters (1.8-5 µm) and construction (fully porous or core-shell). Experiments were carried out by monitoring the elution volumes of nitrate samples in a mobile phase of acetonitrile/water 80:20 v/v at increasing concentrations of Tris-HCl buffer (pH 8.1) from 0.01 to 60 mM. At low buffer concentrations, nitrate ions are completely electrostatically excluded from the intraparticle mesopore space, which is reflected by a plateau region in the elution curves. The elution volume in the plateau region equals the interparticle void volume. Clearly defined plateau regions were observed for all columns, even those densely packed with core-shell and sub-2 µm particles, enabling the accurate determination of interparticle porosities to three decimal places in a fast and convenient way.

Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites.

RNAi is widely applied to inhibit expression of specific genes, but it is limited by variable efficiency and specificity of empirically designed siRNA or shRNA constructs. This complicates studies targeting individual genes and significantly impairs large-scale screens using genome-wide knockdown libraries. Here, we show that ectopic expression of the RISC slicer Argonaute-2 (Ago2, eIF2C2) dramatically enhances RNAi specifically for mRNA targets with perfectly matched binding sites. This effect depends on its endonuclease activity and is uncoupled from its regulation of microRNA expression. To model the application of Ago2 coexpression with shRNA knockdown, we targeted the EGF receptor (EGFR) in lung cancer cells exhibiting oncogene addiction to EGFR. Whereas multiple empirically designed shRNA constructs exhibited highly divergent efficiencies in mediating EGFR knockdown and cell killing, coexpression of Ago2 resulted in uniform and highly specific target gene suppression and apoptosis in EGFR-dependent cells. Codelivery of Ago2 with shRNA constructs or siRNA duplexes thus provides a strategy to enhance the efficacy and the specificity of RNAi in experimental and potentially therapeutic settings.

The Endogenous RIG-I Ligand Is Generated in Influenza A-Virus Infected Cells.

As a result of a viral infection, viral genomes are not only recognized by RIG-I, but also lead to the activation of RNase L, which cleaves cellular RNA to generate the endogenous RIG-I ligand (eRL). The eRL was previously identified as a specific sequence derived from the internal transcribed spacer region 2, which bears a 2'3' cyclic phosphate instead of the common 5' triphosphate. By now, the generation of the eRL and its immunostimulatory effect were shown both in vitro and in reporter systems. In this work, we aimed to elucidate whether the eRL is also generated in Influenza A (IAV) and vesicular stomatitis virus (VSV) infected cells. RNA was extracted from virus-infected cells and used for immunostimulations as well as specific PCR-strategies to detect eRL cleavage. We show that the eRL is generated in IAV infected HEK293 cells, but we could not detect specific eRL fragments in VSV infected cells. Further, RIG-I mediated IFN-response depends not only on viral genomes but also on the eRL, as immunostimulatory properties remain present under 5'triphosphate degrading conditions. In summary, we prove the IAV infection induced eRL generation in HEK293 cells, amplifying the innate immune response.

Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen.

OBJECTIVE: The platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI-dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. METHODS AND RESULTS: ELISA using biotinyl-fibrinogen immobilized on streptavidin-coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near-plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI-Fc2 or Revacept) or monomer (GPVI-His: single chain of Revacept GPVI domain, with His tag). Clot-bound proteins were analyzed by SDS-PAGE/immunoblotting. GPVI-dimer bound to noncrosslinked fibrin clots with classical one-site binding kinetics, with µM-level KD , and to crosslinked clots with higher affinity. Anti-GPVI-dimer (mFab-F) inhibited the binding. However, GPVI-His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot-bound platelet-derived proteins were integrin αIIbß3, present at high levels, and GPVI. CONCLUSIONS: We conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KD to those obtained for its binding to fibrinogen D-fragment and D-dimer, suggesting that fibrin(ogen)'s GPVI-binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot.

Hepatitis-D Virus Infection Is Not Impaired by Innate Immunity but Increases Cytotoxic T-Cell Activity.

Approximately 70 million humans worldwide are affected by chronic hepatitis D, which rapidly leads to liver cirrhosis and hepatocellular carcinoma due to chronic inflammation. The triggers and consequences of this chronic inflammation, induced by co-infection with the hepatitis D virus (HDV) and the hepatitis B virus (HBV), are poorly understood. Using CRISPR technology, we characterized the recognition of HDV mono- and co-infection by intracellular innate immunity and determined its influence on the viral life cycle and effector T-cell responses using different HBV and HDV permissive hepatoma cell lines. We showed that HDV infection is detected by MDA5 and -after a lag phase -induces a profound type I interferon response in the infected cells. The type I interferon response, however, was not able to suppress HDV replication or spread, thus providing a persistent trigger. Using engineered T-cells directed against the envelope proteins commonly used by HBV and HDV, we found that HDV immune recognition enhanced T-cell cytotoxicity. Interestingly, the T-cell effector function was enhanced independently of antigen presentation. These findings help to explain immune mediated tissue damage in chronic hepatitis D patients and indicate that combining innate triggers with T-cell activating therapies might allow for a curative approach.

Are integrin alpha(2)beta(1), glycoprotein Ib and vWf levels correlated with their contributions to platelet adhesion on collagen under high-shear flow?

Platelets in flowing blood at high-shear stress are recruited to exposed subendothelial collagen of injured vessels by GPIb-von Willebrand factor (vWf) and integrin alpha(2)beta(1) (alpha(2)beta(1))-collagen interactions. Platelet adhesion to type I collagen depends mainly on the alpha(2)beta(1)-collagen interaction and that to type III collagen depends on the GPIb-vWf interaction due to vWf's weak affinity for type I collagen. Contributions of these two interactions would differ depending on expressions of alpha(2)beta(1), vWf, or GPIb. We quantitated platelet adhesion to low- and high-density collagen under high-shear flow conditions in the presence of anti-alpha(2)beta(1) (Gi9) and anti-GPIb (NNKY5-5) antibodies to determine if their inhibitory effects were correlated with the amounts of alpha(2)beta(1), GPIb and vWf. Gi9 inhibition of adhesion to type I collagen was decreased in platelets with more integrin alpha(2)beta(1). Gi9 and NNKY5-5 are more inhibitory against adhesion to low-density type III and I, respectively. Higher alpha(2)beta(1) expression decreases adhesion to low-density type III and increases Gi9 inhibition of adhesion to high-density type III, suggesting crosstalk between the alpha(2)beta(1)-collagen and GPIb-vWf interactions in adhesion to type III. Integrin alpha(2)beta(1)-collagen and GPIb-vWf interactions both contribute to platelet adhesion to collagen under high-shear flow. In adhesion under high-shear stress, the two interactions would compensate for each other, when there is a deficiency in one or the other. The alpha(2)beta(1)-collagen interaction was also suggested to have an inhibitory effect on platelet adhesion to type III collagen, through a yet undefined mechanism.

Innate immune recognition and modulation in hepatitis D virus infection.

Hepatitis D virus (HDV) is a global health threat with more than 15 million humans affected. Current treatment options are largely unsatisfactory leaving chronically infected humans at high risk to develop liver cirrhosis and hepatocellular carcinoma. HDV is the only human satellite virus known. It encodes only two proteins, and requires Hepatitis B virus (HBV) envelope protein expression for productive virion release and spread of the infection. How HDV could evolve and why HBV was selected as a helper virus remains unknown. Since the discovery of Na+-taurocholate co-transporting polypeptide as the essential uptake receptor for HBV and HDV, we are beginning to understand the interactions of HDV and the immune system. While HBV is mostly regarded a stealth virus, that escapes innate immune recognition, HBV-HDV coinfection is characterized by a strong innate immune response. Cytoplasmic RNA sensor melanoma differentiation antigen 5 has been reported to recognize HDV RNA replication and activate innate immunity. Innate immunity, however, seems not to impair HDV replication while it inhibits HBV. In this review, we describe what is known up-to-date about the interplay between HBV as a helper and HDV's immune evasion strategy and identify where additional research is required.