NADPH Oxidase Limits Collaborative Pattern-Recognition Receptor Signaling to Regulate Neutrophil Cytokine Production in Response to Fungal Pathogen-Associated Molecular Patterns.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by genetic defects in leukocyte NADPH oxidase, which has both microbicidal and immunomodulatory roles. Hence, CGD is characterized by recurrent bacterial and fungal infections as well as aberrant inflammation. Fungal cell walls induce neutrophilic inflammation in CGD; yet, underlying mechanisms are incompletely understood.

Systemic levels of anti-PAD4 autoantibodies correlate with airway obstruction in cystic fibrosis.

Cystic fibrosis (CF) airway disease is characterized by the long-term presence of neutrophil granulocytes. Formation of neutrophil extracellular traps (NETs) and/or autoantibodies directed against extracellular components of NETs are possible contributors to neutrophil-mediated lung damage in CF. The goal of this study was to measure their levels in CF adults compared to healthy controls and subjects with rheumatologic diseases known to develop NET-related autoantibodies and pathologies, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

Haploinsufficiency of NADPH Oxidase Subunit Neutrophil Cytosolic Factor 2 Is Sufficient to Accelerate Full-Blown Lupus in NZM 2328 Mice.

Objective: We have previously established that the gene for neutrophil cytosolic factor 2 (NCF-2) predisposes to lupus, and we have identified lupus patients with point mutations that are predicted to cause reduced NADPH oxidase activity. We undertook this study to investigate the relationship between reduced leukocyte NADPH oxidase activity and immune dysregulation associated with systemic lupus erythematosus (SLE).

Methods: We generated NCF-2-null mice, in which NADPH oxidase activity is absent, on the nonautoimmune C57BL/6 (B6) mouse background and on the NZM 2328 mouse background, a polygenic model in which mice spontaneously develop lupus.

Swimming Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by Flagellated Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections often characterized by robust neutrophilic infiltration. Neutrophils provide the first line of defense against P. aeruginosa.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro.

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs).

Release of cystic fibrosis airway inflammatory markers from Pseudomonas aeruginosa-stimulated human neutrophils involves NADPH oxidase-dependent extracellular DNA trap formation.

Cystic fibrosis (CF) airways are characterized by bacterial infections, excess mucus production, and robust neutrophil recruitment. The main CF airway pathogen is Pseudomonas aeruginosa. Neutrophils are not capable of clearing the infection.

NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.

Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function.

Pseudogout-associated inflammatory calcium pyrophosphate dihydrate microcrystals induce formation of neutrophil extracellular traps.

Pseudogout is an autoinflammatory condition triggered by calcium pyrophosphate dehydrate (CPPD) crystal deposition in the joints. The innate immune system is irritated by and responds to the presence of the crystals with an inflammatory response. The synovial fluid contains activated inflammatory macrophages and neutrophil granulocytes.

Cross-protection by co-immunization with influenza hemagglutinin DNA and inactivated virus vaccine using coated microneedles.

The need for annual revaccination against influenza is a burden on the healthcare system, leads to low vaccination rates and makes timely vaccination difficult against pandemic strains, such as during the 2009 H1N1 influenza pandemic. In an effort toward achieving a broadly protective vaccine that provides cross-protection against multiple strains of influenza, this study developed a microneedle patch to co-immunize with A/PR8 influenza hemagglutinin DNA and A/PR8 inactivated virus vaccine. We hypothesize that this dual component vaccination strategy administered to the skin using microneedles will provide cross-protection against other strains of influenza.

Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase.

Beyond intracellular killing, a novel neutrophil-based antimicrobial mechanism has been recently discovered: entrapment and killing by neutrophil extracellular traps (NETs). NETs consist of extruded nuclear DNA webs decorated with granule proteins. Although NET formation is an important innate immune mechanism, uncontrolled NET release damages host tissues and has been linked to several diseases including cystic fibrosis (CF).

Stability of whole inactivated influenza virus vaccine during coating onto metal microneedles.

Immunization using a microneedle patch coated with vaccine offers the promise of simplified vaccination logistics and increased vaccine immunogenicity. This study examined the stability of influenza vaccine during the microneedle coating process, with a focus on the role of coating formulation excipients. Thick, uniform coatings were obtained using coating formulations containing a viscosity enhancer and surfactant, but these formulations retained little functional vaccine hemagglutinin (HA) activity after coating.

Protective effect of Korean red ginseng extract on the infections by H1N1 and H3N2 influenza viruses in mice.

Ginseng has been used in humans for thousands of years and is known to have multiple biological and immunomodulatory effects. In this study, we investigated whether Korean red ginseng extract would have preventive and antiviral effects on influenza virus infection. Oral administration to mice of red ginseng extract prior to infection significantly increased survival after infection with the 2009 pandemic H1N1 virus.

Protective effect of ginseng polysaccharides on influenza viral infection.

Ginseng polysaccharide has been known to have multiple immunomodulatory effects. In this study, we investigated whether Panax ginseng polysaccharide (GP) would have a preventive effect on influenza infection. Administration of mice with GP prior to infection was found to confer a survival benefit against infection with H1N1 (A/PR/8/34) and H3N2 (A/Philippines/82) influenza viruses.

Stability of influenza vaccine coated onto microneedles.

A microneedle patch coated with vaccine simplifies vaccination by using a patch-based delivery method and targets vaccination to the skin for superior immunogenicity compared to intramuscular injection. Previous studies of microneedles have demonstrated effective vaccination using freshly prepared microneedles, but the issue of long-term vaccine stability has received only limited attention. Here, we studied the long-term stability of microneedles coated with whole inactivated influenza vaccine guided by the hypothesis that crystallization and phase separation of the microneedle coating matrix damages influenza vaccine coated onto microneedles.

MyD88 plays an essential role in inducing B cells capable of differentiating into antibody-secreting cells after vaccination.

We investigated the roles of MyD88, an innate adaptor signaling molecule, in inducing protective humoral immunity after vaccination with influenza virus-like particles (VLPs). MyD88 knockout C57BL/6 mice (MyD88(-/-) mice) vaccinated with influenza VLPs showed significant defects in inducing IgG2a/c isotype antibodies and in generating splenic recall memory B cell responses and antibody-secreting plasma cells in the bone marrow. The protective efficacy of influenza VLP vaccination was lower in MyD88(-/-) mice than in the wild-type mice.

Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells.

In this study, we evaluated the role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the tumor necrosis factor-alpha (TNF-alpha) induced cyclooxygenase-2 (COX-2) expression using A549 lung adenocarcinoma cells. TNF-alpha induced the expression of COX-2 in A549 cells, but did not induce BEAS-2B expression. The expression of COX-2 in A549 cells was TNF-alpha dose-dependent (5~100 ng/ml).

Microneedle delivery of H5N1 influenza virus-like particles to the skin induces long-lasting B- and T-cell responses in mice.

A simple method suitable for self-administration of vaccine would improve mass immunization, particularly during a pandemic outbreak. Influenza virus-like particles (VLPs) have been suggested as promising vaccine candidates against potentially pandemic influenza viruses, as they confer long-lasting immunity but are not infectious. We investigated the immunogenicity and protective efficacy of influenza H5 VLPs containing the hemagglutinin (HA) of A/Vietnam/1203/04 (H5N1) virus delivered into the skin of mice using metal microneedle patches and also studied the response of Langerhans cells in a human skin model.

Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like particles.

We generated influenza virus-like particles (VLPs) containing the wild type (WT) H5 hemagglutinin (HA) from A/Viet Nam/1203/04 virus or a mutant H5 HA with a deletion of the multibasic cleavage motif. VLPs containing mutant H5 HA were found to be as immunogenic as VLPs containing WT HA. A single intramuscular vaccination with either type of H5 VLPs provided complete protection against lethal challenge.

Intradermal vaccination with influenza virus-like particles by using microneedles induces protection superior to that with intramuscular immunization.

Influenza virus-like particles (VLPs) are a promising cell culture-based vaccine, and the skin is considered an attractive immunization site. In this study, we examined the immunogenicity and protective efficacy of influenza VLPs (H1N1 A/PR/8/34) after skin vaccination using vaccine dried on solid microneedle arrays. Coating of microneedles with influenza VLPs using an unstabilized formulation was found to decrease hemagglutinin (HA) activity, whereas inclusion of trehalose disaccharide preserved the HA activity of influenza VLP vaccines after microneedles were coated.

Influenza immunization with trehalose-stabilized virus-like particle vaccine using microneedles.

Morbidity and mortality due to seasonal and pandemic influenza could be reduced by simpler vaccination methods that enable improved vaccination coverage. In this study, solid metal microneedles coated with influenza virus-like particle (VLP) vaccine were inserted into skin for intradermal immunization. Microneedles were applied to the skin by hand and designed for simple administration with little or no training.

Enhanced memory responses to seasonal H1N1 influenza vaccination of the skin with the use of vaccine-coated microneedles.

Background: Morbidity and mortality due to influenza could be reduced by improved vaccination.

Methods: To develop a novel skin delivery method that is simple and allows for easy self-administration, we prepared microneedle patches with stabilized influenza vaccine and investigated their protective immune responses.

Results: Mice vaccinated with a single microneedle dose of trehalose-stabilized influenza vaccine developed strong antibody responses that were long-lived.

Stabilization of influenza vaccine enhances protection by microneedle delivery in the mouse skin.

Background: Simple and effective vaccine administration is particularly important for annually recommended influenza vaccination. We hypothesized that vaccine delivery to the skin using a patch containing vaccine-coated microneedles could be an attractive approach to improve influenza vaccination compliance and efficacy.

Methodology/principal Findings: Solid microneedle arrays coated with inactivated influenza vaccine were prepared for simple vaccine delivery to the skin.

Improved influenza vaccination in the skin using vaccine coated microneedles.

Easy and effective vaccination methods could reduce mortality rates and morbidity due to vaccine-preventable influenza infections. In this study, we examined the use of microneedle patches to increase patient coverage through possible self-administration and enhance vaccine immunogenicity by targeted delivery to skin. We carried out a detailed study of protective immune responses after a single influenza vaccination to the skin of mice with a novel microneedle patch designed to facilitate simple and reliable vaccine delivery.

Induction of long-term protective immune responses by influenza H5N1 virus-like particles.

Background: Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic.

Methodology/principal Findings: Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing.

Kinetics of immune responses to influenza virus-like particles and dose-dependence of protection with a single vaccination.

The format of influenza virus-like particles (VLPs) as a nonreplicating particulate vaccine candidate is a promising alternative to conventional egg-based vaccines. In this study, we have investigated the detailed kinetics of immune responses and protective efficacy after a single intranasal immunization with different doses of VLPs alone or in the presence of an Escherichia coli mutant heat-labile enterotoxin [mLT(R192G)] or cholera toxin subunit B as adjuvants. Analysis of immune responses showed differential kinetics in a VLP antigen dose-dependent manner and dynamic changes in the ratios of antibody immunoglobulin G isotypes over the time course.