Vera Cherepanov

Remote ischemic preconditioning decreases adhesion and selectively modifies functional responses of human neutrophils.

OBJECTIVES Preconditioning of cells or organs by transient sublethal ischemia-reperfusion (IR), termed ischemic preconditioning (IPC), protects the cell or organ from a subsequent prolonged ischemic insult. The mechanisms of this effect remain to be fully elucidated. We have recently reported that IPC of a forearm results in alterations in gene expression profiles of circulating polymorphonuclear leukocytes. The goal of the current study was to determine if the observed changes in gene expression lead to functional changes in neutrophils. METHODS We examined the effect of repetitive transient human forearm ischemia (three cycles of 5 min ischemia, followed by 5 min of reperfusion) on the function of circulating neutrophils. Neutrophil functions were examined before, after 1 d, and after 10 d of daily transient forearm ischemia. To modulate IR-induced inflammation the neutrophils were stimulated with N-formyl-methionyl-leucyl phenylalanine (FMLP) and lipopolysaccharide (LPS). RESULTS Neutrophil adhesion was significantly decreased on day 1 and remained low on day 10 (P = 0.0149) without significant change in CD11b expression. Phagocytosis was significantly suppressed on day 10 compared with day 0 (P < 0.0001). Extracellular cytokine levels were low in the absence of an exogenous stimulus but stimulation with LPS induced significant changes on day 10. We observed a trend in reduction of apoptosis on day 1 and day 10 that did not reach statistical significance (P < 0.08). CONCLUSION This study indicates that repetitive IPC of the forearm results in substantial alterations in neutrophil function, including reduced adhesion, exocytosis, phagocytosis, and modified cytokine secretion.

Control of vesicle fusion by a tyrosine phosphatase.

The tyrosine phosphatase PTP-MEG2 is targeted by its amino-terminal Sec14p homology domain to the membrane of secretory vesicles. There it regulates vesicle size by promoting homotypic vesicle fusion by a mechanism that requires its catalytic activity. Here, we identify N-ethylmaleimide-sensitive factor (NSF), a key regulator of vesicle fusion, as a substrate for PTP-MEG2. PTP-MEG2 reduced the phosphotyrosine content of NSF and co-localized with NSF and syntaxin 6 in intact cells. Furthermore, endogenous PTP-MEG2 co-immunoprecipitated with endogenous NSF. Phosphorylation of NSF at Tyr 83, as well as an acidic substitution at the same site, increased its ATPase activity and prevented αSNAP binding. Conversely, expression of a Y83F mutant of NSF caused spontaneous fusion events. Our results suggest that the molecular mechanism by which PTP-MEG2 promotes secretory vesicle fusion involves the local release of NSF from a tyrosine-phosphorylated, inactive state. This represents a novel mechanism for localized regulation of NSF and the first demonstrated role for a protein tyrosine phosphatase in the regulated secretory pathway.

Immune function in patients with Shwachman–Diamond syndrome

Shwachman–Diamond syndrome (SDS) is an inherited multisystem disorder characterized by exocrine pancreatic dysfunction and varying degrees of cytopenia. In addition, various immunological abnormalities have been noted. To clarify the issue of immunological competence or incompetence in SDS, we prospectively studied immune function in 11 patients with SDS. Seven suffered from recurrent bacterial infections and six from recurrent viral infections. Varying degrees of impairment were readily identified. All patients had neutropenia; total lymphocyte counts, however, were normal in all except one patient. Nine patients had B‐cell defects comprising one or more of the following abnormalities: low IgG or IgG subclasses, low percentage of circulating B lymphocytes, decreased in vitro B‐lymphocyte proliferation and a lack of specific antibody production. Seven out of nine patients studied had at least one T‐cell abnormality comprising a low percentage of total circulating T lymphocytes or CD3+/CD4+ cell subpopulations or decreased in vitro T‐lymphocyte proliferation. Five out of six patients studied had decreased percentages of circulating natural killer cells. Moreover, neutrophil chemotaxis was significantly low in all the patients studied. These data point to a major immunodeficiency component in SDS that places patients at heightened risk of infections, even if neutrophil numbers are protective. This finding broadens the definition of the syndrome substantially: it suggests that the SDS marrow defect occurs at the level of an early haematological–lymphocytic stem cell or that a combined marrow and thymic stromal defect accounts for the aberrant function of haematopoietic and lymphopoietic lineages.

Plasma interferon‐gamma‐inducible protein‐10 (IP‐10) levels during acute hepatitis C virus infection

Systemic levels of interferon‐gamma‐inducible protein‐10 (IP‐10) are predictive of treatment‐induced clearance in chronic hepatitis C virus (HCV). In the present study, factors associated with plasma IP‐10 levels at the time of acute HCV detection and the association between IP‐10 levels and spontaneous clearance were assessed in three cohorts of acute HCV infection. Among 299 individuals, 245 (181 male, 47 human immunodeficiency virus‐positive [HIV+]) were HCV RNA+ at acute HCV detection. In adjusted analysis, factors independently associated with IP‐10 levels ≥150 pg/mL (median level) included HCV RNA levels >6 log IU/mL, HIV coinfection and non‐Aboriginal ethnicity. Among 245 HCV RNA+ at acute HCV detection, 214 were untreated (n = 137) or had persistent infection (infection duration ≥26 weeks) at treatment initiation (n = 77). Spontaneous clearance occurred in 14% (29 of 214). Individuals without spontaneous clearance had significantly higher mean plasma IP‐10 levels at the time of acute HCV detection than those with clearance (248 ± 32 versus 142 ± 22 pg/mL, P = 0.008). The proportion of individuals with spontaneous clearance was 0% (0 of 22, P = 0.048) and 16% (27 of 165) and in those with and without plasma IP‐10 levels ≥380 pg/mL. In adjusted analyses, favorable IL28B genotype was associated with spontaneous clearance, while higher HCV RNA level was independently associated with lower odds of spontaneous clearance. Conclusion: High IP‐10 levels at acute HCV detection were associated with failure to spontaneously clear HCV. Patients with acute HCV and high baseline IP‐10 levels, particularly >380 pg/mL, should be considered for early therapeutic intervention, and those with low levels should defer therapy for potential spontaneous clearance. (HEPATOLOGY 2013;)

S-adenosyl methionine improves early viral responses and interferon-stimulated gene induction in hepatitis C nonresponders

BACKGROUND & AIMS Less than half of patients infected with hepatitis C virus (HCV) achieve sustained viral clearance after pegylated interferon (peginterferon) and ribavirin therapy. S-adenosyl methionine (SAMe) improves interferon signaling in cell culture. We assessed the effect of SAMe on the kinetics of the early antiviral response and interferon signaling in nonresponders to previous antiviral therapy and investigated the mechanisms involved. METHODS Nonresponders with HCV genotype 1 were given peginterferon alfa-2a and ribavirin for 2 weeks (course A, baseline/control). After 1 month, patients received SAMe (1600 mg daily) for 2 weeks and then peginterferon and ribavirin for 48 weeks (course B; completed by 21 of 24 patients). Viral kinetics and interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells (PBMCs) were compared between courses. RESULTS The decrease in HCV RNA from 0 to 48 hours (phase 1) was similar with and without SAMe. However, the second phase slope of viral decline was improved with SAMe (course A, 0.11 ± 0.04 log(10) IU/mL/wk; course B, 0.27 ± 0.06; P = .009); 11 patients (53%) achieved an early virological response, and 10 (48%) had undetectable HCV RNA by week 24. Induction of ISGs in PBMCs was significantly greater during course B. In cultured cells, SAMe increased induction of ISGs and the antiviral effects of interferon by increasing STAT1 methylation, possibly affecting STAT1-DNA binding. CONCLUSIONS The addition of SAMe to peginterferon and ribavirin improves the early viral kinetics and increases ISG induction in nonresponders to previous therapy. SAMe might be a useful adjunct to peginterferon-based therapies in chronic HCV infection.

Human neutrophil peptides and phagocytic deficiency in bronchiectatic lungs.

RATIONALE A well-known clinical paradox is that severe bacterial infections persist in the lungs of patients with cystic fibrosis (CF) despite the abundance of polymorphonuclear neutrophils (PMN) and the presence of a high concentration of human neutrophil peptides (HNP), both of which are expected to kill the bacteria but fail to do so. The mechanisms remain unknown. OBJECTIVES This study examined several possible mechanisms to understand this paradox. METHODS PMN were isolated from sputum and blood of subjects with and without CF or non-CF bronchiectasis for phagocytic assays. HNP isolated from patients with CF were used to stimulate healthy PMN followed by phagocytic tests. MEASUREMENTS AND MAIN RESULTS PMN isolated from the sputum of the bronchiectatic patients display defective phagocytosis that correlated with high concentrations of HNP in the lung. When healthy PMN were incubated with HNP, decreased phagocytic capacity was observed in association with depressed surface Fc gamma RIII, actin-filament remodeling, enhanced intracellular Ca(2+), and degranulation. Treatment of PMN with an intracellular Ca(2+) blocker or alpha1-proteinase inhibitor to attenuate the activity of HNP largely prevented the HNP-induced phagocytic deficiency. Intratracheal instillation of HNP in Pallid mice (genetically deficient in alpha1-proteinase inhibitor) resulted in a greater PMN lung infiltration and phagocytic deficiency compared with wild-type mice. CONCLUSIONS HNP or PMN alone exert antimicrobial ability, which was lost as a result of their interaction. These effects of HNP may help explain the clinical paradox seen in patients with inflammatory lung diseases, suggesting HNP as a novel target for clinical therapy.

Lipopolysaccharide and Tumor Necrosis Factor Alpha Inhibit Interferon Signaling in Hepatocytes by Increasing Ubiquitin-Like Protease 18 (USP18) Expression

ABSTRACT Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-α. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-α response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-α/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-α refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-α and LPS) but not others (IL-6 and IL-10) target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential target for intervention in various inflammatory states. IMPORTANCE Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential target for intervention in various inflammatory states.

Plasma interferon-gamma-inducible protein-10 levels are associated with early, but not sustained virological response during treatment of acute or early chronic HCV infection.

Background High plasma levels of interferon-gamma inducible protein-10 (IP-10) have been shown to be associated with impaired treatment response in chronic hepatitis C virus (HCV) infection. Whether IP-10 levels predict treatment in acute HCV infection is unknown. Methods Patients with acute or early chronic HCV infection from the Australian Trial in Acute Hepatitis C (ATAHC) cohort were evaluated. Baseline and on-treatment plasma IP-10 levels were measured by ELISA. IL28B genotype was determined by sequencing. Results Overall, 74 HCV mono-infected and 35 HIV/HCV co-infected patients were treated in ATAHC, of whom 89 were adherent to therapy and were included for analysis. IP-10 levels correlated with HCV RNA levels at baseline (r = 0.48, P<0.001) and during treatment. Baseline IP-10 levels were higher in patients who failed to achieve rapid virological response (RVR). Only one patient with a plasma IP-10 level >600 pg/mL achieved RVR. There was no association with IP-10 levels and early virological response (EVR) or sustained virological response (SVR). Conclusions Baseline IP-10 levels are associated with early viral kinetics but not ultimate treatment outcome in acute HCV infection. Given previous data showing that patients with high baseline IP-10 are unlikely to spontaneously clear acute HCV infection, they should be prioritized for early antiviral therapy.

Inducing Hepatitis C Virus Resistance After Pig Liver Transplantation -”A Proof of Concept of Liver Graft Modification Using Warm Ex Vivo Perfusion”

Normothermic ex vivo liver perfusion (NEVLP) offers the potential to optimize graft function prior to liver transplantation (LT). Hepatitis C virus (HCV) is dependent on the presence of miRNA(microRNA)‐122. Miravirsen, a locked‐nucleic acid oligonucleotide, sequesters miR‐122 and inhibits HCV replication. The aim of this study was to assess the efficacy of delivering miravirsen during NEVLP to inhibit miR‐122 function in a pig LT model. Pig livers were treated with miravirsen during NEVLP or cold storage (CS). Miravirsen absorption, miR‐122 sequestration, and miR‐122 target gene derepression were determined before and after LT. The effect of miravirsen treatment on HCV infection of hepatoma cells was also assessed. NEVLP improved miravirsen uptake versus CS. Significant miR‐122 sequestration and miR‐122 target gene derepression were seen with NEVLP but not with CS. In vitro data confirmed miravirsen suppression of HCV replication after established infection and prevented HCV infection with pretreatment of cells, analogous to the pretreatment of grafts in the transplant setting. In conclusion, miravirsen delivery during NEVLP is a potential strategy to prevent HCV reinfection after LT. This is the first large‐animal study to provide “proof of concept” for using NEVLP to modify and optimize liver grafts for transplantation.

Replication and Oncolytic Activity of an Avian Orthoreovirus in Human Hepatocellular Carcinoma Cells

Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.