Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii.

BACKGROUND: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2'OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. METHODS: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. CONCLUSION: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.

Characterization of CD4+ T Cell Subsets in Patients with Abdominal Aortic Aneurysms.

BACKGROUND: The mediators produced by CD4+ T lymphocytes are involved in the pathogenesis of aneurysmal lesions in abdominal aortic aneurysm (AAA) patients. The aim of this study was to identify and characterize the CD4+ T cell subsets involved in human AAA. METHODS: The CD4+ T cell subsets in 30 human aneurysmal lesions were determined using flow cytometry (FC) and immunohistochemistry (IHC). The peripheral blood mononuclear cells (PBMCs) from patients with AAA were also analyzed by FC and compared with control subjects. RESULTS: Human aneurysmal lesions contained IFN-γ, IL-12p35, IL-4, IL-23p19, IL-17R, and IL-22 positive cells. PBMCs from AAA patients had higher expression levels of IFN-γ, TNF-α, IL-4, and IL-22 when compared to controls. CONCLUSIONS: Our results show the presence of TH1, TH2, TH17, and TH22 subsets in aneurysmal lesions of AAA patients and suggest that these cells may be mainly activated in situ, where they can induce tissue degradation and contribute to the pathogenesis of AAA.

Paracoccidioidomycosis Associated With a Heterozygous STAT4 Mutation and Impaired IFN-γ Immunity.

Background: Mutations in genes affecting interferon-γ (IFN-γ) immunity have contributed to understand the role of IFN-γ in protection against intracellular pathogens. However, inborn errors in STAT4, which controls interleukin-12 (IL-12) responses, have not yet been reported. Our objective was to determine the genetic defect in a family with a history of paracoccidioidomycosis. Methods: Genetic analysis was performed by whole-exome sequencing and Sanger sequencing. STAT4 phosphorylation (pSTAT4) and translocation to the nucleus, IFN-γ release by patient lymphocytes, and microbicidal activity of patient monocytes/macrophages were assessed. The effect on STAT4 function was evaluated by site-directed mutagenesis using a lymphoblastoid B cell line (B-LCL) and U3A cells. Results: A heterozygous missense mutation, c.1952 A>T (p.E651V) in STAT4 was identified in the index patient and her father. Patient's and father's lymphocytes showed reduced pSTAT4, nuclear translocation, and impaired IFN-γ production. Mutant B-LCL and U3A cells also displayed reduced pSTAT4. Patient's and father's peripheral blood mononuclear cells and macrophages demonstrated impaired fungicidal activity compared with those from healthy controls that improved in the presence of recombinant human IFN-γ, but not rhIL-12. Conclusion: Our data suggest autosomal dominant STAT4 deficiency as a novel inborn error of IL-12-dependent IFN-γ immunity associated with susceptibility to paracoccidioidomycosis.

An Immunomodulatory Peptide Confers Protection in an Experimental Candidemia Murine Model.

Fungal Candida species are commensals present in the mammalian skin and mucous membranes. Candida spp. are capable of breaching the epithelial barrier of immunocompromised patients with neutrophil and cell-mediated immune dysfunctions and can also disseminate to multiple organs through the bloodstream. Here we examined the action of innate defense regulator 1018 (IDR-1018), a 12-amino-acid-residue peptide derived from bovine bactenecin (Bac2A): IDR-1018 showed weak antifungal and antibiofilm activity against a Candida albicans laboratory strain (ATCC 10231) and a clinical isolate (CI) (MICs of 32 and 64 µg · ml-1, respectively), while 8-fold lower concentrations led to dissolution of the fungal cells from preformed biofilms. IDR-1018 at 128 µg · ml-1 was not hemolytic when tested against murine red blood cells and also has not shown a cytotoxic effect on murine monocyte RAW 264.7 and primary murine macrophage cells at the tested concentrations. IDR-1018 modulated the cytokine profile during challenge of murine bone marrow-derived macrophages with heat-killed C. albicans (HKCA) antigens by increasing monocyte chemoattractant protein 1 (MCP-1) and interleukin-10 (IL-10) levels, while suppressing tumor necrosis factor alpha (TNF-α), IL-1ß, IL-6, and IL-12 levels. Mice treated with IDR-1018 at 10 mg · kg-1 of body weight had an increased survival rate in the candidemia model compared with phosphate-buffered saline (PBS)-treated mice, together with a diminished kidney fungal burden. Thus, IDR-1018 was able to protect against murine experimental candidemia and has the potential as an adjunctive therapy.

Reduced systemic levels of IL-10 are associated with the severity of obstructive sleep apnea and insulin resistance in morbidly obese humans.

Obstructive sleep apnea (OSA) has been related to elevation of inflammatory cytokines and development of insulin resistance in morbidly obese (MO) subjects. However, it is still unclear whether the systemic concentration of anti-inflammatory mediators is also affected in MO subjects directly related to the severity of OSA and level of insulin resistance. Normal weight and MO subjects were subjected to overnight polysomnography in order to establish the severity of OSA, according to the apnea-hypopnea index (AHI). Blood samples were obtained for estimation of total cholesterol and triglycerides, insulin, glucose, insulin resistance, tumor necrosis factor alpha (TNF-α), interleukin 12 (IL12), and interleukin 10 (IL-10). Serum levels of IL-10 were significantly lower in MO subjects with OSA than in MO and control individuals without OSA. Besides being inversely associated with serum TNF-α and IL-12, decreased IL-10 levels were significantly related to increased AHI, hyperinsulinemia, and insulin resistance. Serum IL-10 is significantly reduced in morbidly obese subjects with severe OSA while also showing a clear relationship with a state of hyperinsulinemia and insulin resistance probably regardless of obesity in the present sample. It may be of potential clinical interest to identify the stimulatory mechanisms of IL-10 in obese individuals with OSA.

Differential expression of interleukin-12 p35 and p40 subunits in response to Aeromonas hydrophila and Aquareovirus infection in grass carp, Ctenopharyngodon idella.

The grass carp (Ctenopharyngodon idella) aquaculture industry in Asia is prone to bacterial and viral hemorrhagic diseases. Effective adjuvants for vaccine formulation are the need of the hour for control of these diseases and long-term sustainability of grass carp farming. In this study, the involvement of interleukin-12 (IL-12) from grass carp (gcIL­12) in anti-bacterial and anti-viral immune responses was demonstrated via expression profiles of gcIL-12 subunits in immune tissues of the fish, following infection by Aeromonas hydrophila and Aquareovirus. Additionally, cDNA of the gcIL-12 subunits, p35 and p40 was cloned and characterized. We found that most of the structurally and functionally important features of vertebrate orthologues were conserved in gcIL-12 subunits, p35 and p40, with some features specific to grass carp. High levels of gcIL-12 p35 expression in the brain and gills suggest that IL-12 plays an important role in neural and immune systems. High expression levels in the heart, blood, and immune-related tissues suggest an important role in circulation and the immune system as well. Infections by both, A. hydrophila and Aquareovirus stimulated the mRNA expression of gcIL-12 subunits, p35 and p40 in most immune tissues. Significant upregulation or downregulation of gcIL-12 subunits, p35 and p40 by bacterial and viral infection confirms their potential role in anti-bacterial and anti-viral immune responses in fish.

Previous stress exposure enhances both anxiety-like behaviour and p35 levels in the basolateral amygdala complex: modulation by midazolam.

Stress exposure induces long lasting neurobiological changes in selected brain areas, which could be associated with the emergence of negative emotional responses. In the present study, previously restrained animals exhibited excessive anxiety one day later in the elevated plus maze. We explore whether stress exposure affects the expression levels of cyclin-dependent kinase 5 (Cdk5) and of its activator protein p35, in diverse amygdaloid nuclei. Stress exposure enhanced p35 levels selectively in the basolateral amygdala (BLA). This up-regulation might be functionally associated with the occurrence of exaggerated anxiety since such emotional response was selectively reversed by an intra-BLA infusion of olomoucine, a Cdk5 inhibitor, 15 min prior to the restraint session. Moreover, pre-treatment with midazolam, a benzodiazepine ligand, not only prevented the excessive anxiety but also attenuated the p35 increase in the BLA of stressed rats. In conclusion, we suggest a pivotal role of the Cdk5/p35 complex, specifically in BLA in the excessive anxiety induced by a previous stressful experience.