Human Neutrophils Couple Nitric Oxide Production and Extracellular Traps Formation in Allergic Asthma.

RATIONALE: Nitric oxide (NO) is elevated in the airways and serum of allergic asthmatic patients, suggesting an important role in asthma. NO production has been widely attributed to the canonical inducible nitric oxide synthase (iNOS). Much effort has been made to inhibit this enzyme with two outcomes: no asthma improvement; and partial NO reduction, suggesting the involvement of an iNOS-independent source. OBJECTIVES: Neutrophils produce NO under inflammatory conditions and their role in asthma has been overlooked. The present study analyzes their possible role as source of NO. METHODS: Our hypothesis was tested in 99 allergic patients with intermittent bronchial asthma and 26 healthy donors. NO production by blood and sputum neutrophils in response to allergens, anti-IgE, and anti-IgE receptors Abs was assessed by Griess, flow cytometry and confocal microscopy. Extracellular traps (ETs) formation, as a possible consequence of NO production, was quantified by western blot and confocal microscopy, and reactive oxygen species by luminol-enhanced chemiluminescence. RESULTS: Among blood and sputum granulocytes from allergic asthmatic patients, only neutrophils, produce NO by an IgE-dependent mechanism. This production is independent of NOS, but dependent on a reaction between L-arginine and reactive oxygen species from NOX2. NO and ETosis are induced in parallel, and NO amplifies ETs formation, which is a key mediator in asthma. CONCLUSIONS: Our findings reveal a novel role of neutrophils as the unique allergen/IgE-dependent NO source in allergic asthma enhancing ETs formation. These results suggest that NO produced by neutrophils needs further consideration in the treatment of allergic asthma.

Au@16-pH-16/miR-21 mimic nanosystem: An efficient treatment for obesity through browning and thermogenesis induction.

Despite the abundance of registered clinical trials worldwide, the availability of effective drugs for obesity treatment is limited due to their associated side effects. Thus, there is growing interest in therapies that stimulate energy expenditure in white adipose tissue. Recently, we demonstrated that the delivery of a miR-21 mimic using JetPEI effectively inhibits weight gain in an obese mouse model by promoting metabolism, browning, and thermogenesis, suggesting the potential of miR-21 mimic as a treatment for obesity. Despite these promising results, the implementation of more advanced delivery system techniques for miR-21 mimic would greatly enhance the advancement of safe and efficient treatment approaches for individuals with obesity in the future. Our objective is to explore whether a new delivery system based on gold nanoparticles and Gemini surfactants (Au@16-ph-16) can replicate the favorable effects of the miR-21 mimic on weight gain, browning, and thermogenesis. We found that dosages as low as 0.2 µg miR-21 mimic /animal significantly inhibited weight gain and induced browning and thermogenic parameters. This was evidenced by the upregulation of specific genes and proteins associated with these processes, as well as the biogenesis of beige adipocytes and mitochondria. Significant increases in miR-21 levels were observed in adipose tissue but not in other tissue types. Our data indicates that Au@16-ph-16 could serve as an effective delivery system for miRNA mimics, suggesting its potential suitability for the development of future clinical treatments against obesity.

Associated Factors of Pneumonia in Individuals with Chronic Obstructive Pulmonary Disease (COPD) Apart from the Use of Inhaled Corticosteroids.

Inhaled corticosteroids (ICSs) are widely used in chronic obstructive pulmonary disease (COPD) and in combination with long-acting ß2 agonists (LABAs) to reduce exacerbations and improve patient lung function and quality of life. However, ICSs have been associated with an increased risk of pneumonia in individuals with COPD, although the magnitude of this risk remains unclear. Therefore, it is difficult to make informed clinical decisions that balance the benefits and adverse effects of ICSs in people with COPD. There may be other causes of pneumonia in patients with COPD, and these causes are not always considered in studies on the risks of using ICSs in COPD. We consider it very useful to clarify these aspects in assessing the influence of ICSs on the incidence of pneumonia and their role in the treatment of COPD. This issue has important implications for current practice and the evaluation and management of COPD, since COPD patients may benefit from specific ICS-based treatment strategies. Many of the potential causes of pneumonia in patients with COPD can act synergistically, so they can be included in more than one section.

Regulation and directed inhibition of ECP production by human neutrophils.

Background: Neutrophils are involved in the pathophysiology of allergic asthma, where the Eosinophil Cationic Protein (ECP) is a critical inflammatory mediator. Although ECP production is attributed to eosinophils, we reported that ECP is also present in neutrophils from allergic patients where, in contrast to eosinophils, it is produced in an IgE-dependent manner. Given the key role of ECP in asthma, we investigated the molecular mechanisms involved in ECP production as well as the effects induced by agonists and widely used clinical approaches. We also analyzed the correlation between ECP production and lung function. Methods: Neutrophils from allergic asthmatic patients were challenged with allergens, alone or in combination with cytokines, in the presence of cell-signaling inhibitors and clinical drugs. We analyzed ECP levels by ELISA and confocal microscopy. Lung function was assessed by spirometry. Results: IgE-mediated ECP release is dependent on phosphoinositide 3-kinase, the extracellular signal-regulated kinase (ERK1/2) and the production of reactive oxygen species by NADPH-oxidase. Calcineurin phosphatase and the transcription factor NFAT are also involved. ECP release is enhanced by the cytokines interleukin (IL)-5 and granulocyte macrophage-colony stimulating factor, and inhibited by interferon-γ, IL-10, clinical drugs (formoterol, tiotropium and budesonide) and allergen-specific IT. We also found an inverse correlation between asthma severity and ECP levels. Conclusions: Our results suggest the molecular pathways involved in ECP production and potential therapeutic targets. We also provide a new method to evaluate disease severity in asthmatic patients based on the quantification of in vitro ECP production by peripheral neutrophils.

Targeted inhibition of allergen-induced histamine production by neutrophils.

Histamine is a critical inflammatory mediator in allergic diseases. We showed in a previous work that neutrophils from allergic patients produce histamine in response to allergens to which the patients were sensitized. Here, we investigate the molecular mechanisms involved in this process using peripheral blood neutrophils. We challenged these cells in vitro with allergens and analyzed histamine release in the culture supernatants. We also explored the effect of common therapeutic drugs that ameliorate allergic symptoms, as well as allergen-specific immunotherapy. Additionally, we examined the expression of histidine decarboxylase and diamine oxidase, critical enzymes in the metabolism of histamine, under allergen challenge. We show that allergen-induced histamine release is dependent on the activation of the phosphoinositide 3-kinase, mitogen-activated protein kinase p38, and extracellular signal-regulated kinase 1/2 signaling pathways. We also found a contribution of the phosphatase calcineurin to lesser extent. Anti-histamines, glucocorticoids, anti-M3-muscarinic receptor antagonists, and mainly ß2 -receptor agonists abolished the allergen-dependent histamine release. Interestingly, allergen-specific immunotherapy canceled the histamine release through the downregulation of histidine decarboxylase expression. Our observations describe novel molecular mechanisms involved in the allergen-dependent histamine release by human neutrophils and provide new targets to inhibit histamine production.

The renin angiotensin system (RAS) mediates bifunctional growth regulation in melanoma and is a novel target for therapeutic intervention.

Despite emergence of new systemic therapies, metastatic melanoma remains a challenging and often fatal form of skin cancer. The renin-angiotensin system (RAS) is a major physiological regulatory pathway controlling salt-water equilibrium, intravascular volume and blood pressure. Biological effects of the RAS are mediated by the vasoactive hormone angiotensin II (AngII) via two receptor subtypes, AT1R (encoded by AGTR1) and AT2R (encoded by AGTR2). We report decreasing expression and increasing CpG island methylation of AGTR1 in metastatic versus primary melanoma and detection in serum of methylated genomic DNA from the AGTR1 CpG island in metastatic melanoma implying that AGTR1 encodes a tumour suppressor function in melanoma. Consistent with this hypothesis, antagonism of AT1R using losartan or shRNA-mediated knockdown in melanoma cell lines expressing AGTR1 resulted in acquisition of the ability to proliferate in serum-free conditions. Conversely, ectopic expression of AGTR1 in cell lines lacking endogenous expression inhibits proliferation irrespective of the presence of AngII implying a ligand-independent suppressor function for AT1R. Treatment of melanoma cell lines expressing endogenous AT2R with either AngII or the AT2R-selective agonist Y6AII induces proliferation in serum-free conditions whereas the AT2R-specific antagonists PD123319 and EMA401 inhibit melanoma growth and angiogenesis and potentiate inhibitors of BRAF and MEK in cells with BRAF V600 mutations. Our results demonstrate that the RAS has both oncogenic and tumour suppressor functions in melanoma. Pharmacological inhibition of AT2R may provide therapeutic opportunities in melanomas expressing this receptor and AGTR1 CpG island methylation in serum may serve as a novel biomarker of metastatic melanoma.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

Myocardial Ischemic Subject's Thymus Fat: A Novel Source of Multipotent Stromal Cells.

OBJECTIVE: Adipose Tissue Stromal Cells (ASCs) have important clinical applications in the regenerative medicine, cell replacement and gene therapies. Subcutaneous Adipose Tissue (SAT) is the most common source of these cells. The adult human thymus degenerates into adipose tissue (TAT). However, it has never been studied before as a source of stem cells. MATERIAL AND METHODS: We performed a comparative characterization of TAT-ASCs and SAT-ASCs from myocardial ischemic subjects (n = 32) according to the age of the subjects. RESULTS: TAT-ASCs and SAT-ASCs showed similar features regarding their adherence, morphology and in their capacity to form CFU-F. Moreover, they have the capacity to differentiate into osteocyte and adipocyte lineages; and they present a surface marker profile corresponding with stem cells derived from AT; CD73+CD90+CD105+CD14-CD19-CD45-HLA-DR. Interestingly, and in opposition to SAT-ASCs, TAT-ASCs have CD14+CD34+CD133+CD45- cells. Moreover, TAT-ASCs from elderly subjects showed higher adipogenic and osteogenic capacities compared to middle aged subjects, indicating that, rather than impairing; aging seems to increase adipogenic and osteogenic capacities of TAT-ASCs. CONCLUSIONS: This study describes the human TAT as a source of mesenchymal stem cells, which may have an enormous potential for regenerative medicine.

Allergens Induce the Release of Lactoferrin by Neutrophils from Asthmatic Patients.

BACKGROUND: Despite the evidence that Lactoferrin (Lf) is involved in allergic asthma processes, it is unknown whether neutrophils can be one of the main cellular sources of this key inflammatory mediator directly in response of an IgE mediated stimulus. The present study was undertaken to analyze this question. METHODS: Neutrophils from healthy subjects (n = 34) and neutrophils from allergic asthmatic patients (n = 102) were challenged in vitro with specific allergens to which the patients were sensitized, PAF, or agonist mAbs against IgE-receptors, and the levels of Lf were measured in the culture supernatant. The levels of serum IgE together with the severity of symptoms were also analyzed. RESULTS: Lf was released into the culture supernatant of neutrophils from allergic asthmatic patients in response to allergens and PAF. This response was highly allergen-specific, and did not happen in neutrophils from healthy donors. Allergen effect was mimicked by Abs against FcεRI and galectin-3 but not by FcεRII. The levels of released Lf correlated well with the levels of serum specific IgE and severity of asthma symptoms. These observations represent a novel view of neutrophils as an important source of Lf in allergic asthma. Importantly, the levels of released Lf by neutrophils could therefore be used to evaluate disease severity in allergic asthmatic patients.

Oskar is targeted for degradation by the sequential action of Par-1, GSK-3, and the SCF⁻Slimb ubiquitin ligase.

Translation of oskar messenger RNA (mRNA) is activated at the posterior of the Drosophila oocyte, producing Long Oskar, which anchors the RNA, and Short Oskar, which nucleates the pole plasm, containing the posterior and germline determinants. Here, we show that Oskar is phosphorylated by Par-1 and GSK-3/Shaggy to create a phosphodegron that recruits the SCF(-Slimb) ubiquitin ligase, which targets Short Oskar for degradation. Phosphorylation site mutations cause Oskar overaccumulation, leading to an increase in pole cell number and embryonic patterning defects. Furthermore, the nonphosphorylatable mutant produces bicaudal embryos when oskar mRNA is mislocalized. Thus, the Par-1/GSK-3/Slimb pathway plays important roles in limiting the amount of pole plasm posteriorly and in degrading any mislocalized Oskar that results from leaky translational repression. These results reveal that Par-1 controls the timing of pole plasm assembly by promoting the localization of oskar mRNA but inhibiting the accumulation of Short Oskar protein.