Combined Dopamine and Grape Seed Extract-Loaded Solid Lipid Nanoparticles: Nasal Mucosa Permeation, and Uptake by Olfactory Ensheathing Cells and Neuronal SH-SY5Y Cells.

We have already formulated solid lipid nanoparticles (SLNs) in which the combination of the neurotransmitter dopamine (DA) and the antioxidant grape-seed-derived proanthocyanidins (grape seed extract, GSE) was supposed to be favorable for Parkinson's disease (PD) treatment. In fact, GSE supply would reduce the PD-related oxidative stress in a synergic effect with DA. Herein, two different methods of DA/GSE loading were studied, namely, coadministration in the aqueous phase of DA and GSE, and the other approach consisting of a physical adsorption of GSE onto preformed DA containing SLNs. Mean diameter of DA coencapsulating GSE SLNs was 187 ± 4 nm vs. 287 ± 15 nm of GSE adsorbing DA-SLNs. TEM microphotographs evidenced low-contrast spheroidal particles, irrespective of the SLN type. Moreover, Franz diffusion cell experiments confirmed the permeation of DA from both SLNs through the porcine nasal mucosa. Furthermore, fluorescent SLNs also underwent cell-uptake studies by using flow cytometry in olfactory ensheathing cells and neuronal SH-SY5Y cells, evidencing higher uptake when GSE was coencapsulated rather than adsorbed onto the particles.

Treatment of Cystic Fibrosis Patients Homozygous for F508del with Lumacaftor-Ivacaftor (Orkambi®) Restores Defective CFTR Channel Function in Circulating Mononuclear Cells.

The treatment of cystic fibrosis (CF) patients homozygous for the F508del mutation with Orkambi®, a combination of a corrector (lumacaftor) and a potentiator (ivacaftor) of the mutated CFTR protein, resulted in some amelioration of the respiratory function. However, a great variability in the clinical response was also observed. The aim of this study was to evaluate the response to Orkambi® in a small cohort of F508del/F508del patients (n = 14) in terms of clinical and laboratory parameters, including ex vivo CFTR activity in mononuclear cells (MNCs), during a 12-month treatment. Patients responded with an increase in percent predicted forced expiratory volume in 1 s (FEV1%) and body mass index (BMI) as well as with a decrease in white blood cell (WBC) total counts and serum C-reactive protein (CRP) levels, although not significantly. Sweat chloride and CFTR-dependent chloride efflux were found to decrease and increase, respectively, as compared with pre-therapy values. CFTR and BMI showed a statistically significant correlation during Orkambi® treatment. Clustering analysis showed that CFTR, BMI, sweat chloride, FEV1%, and WBC were strongly associated. These data support the notion that CFTR-dependent chloride efflux in MNCs should be investigated as a sensitive outcome measure of Orkambi® treatment in CF patients.

G-CSF and GM-CSF Modify Neutrophil Functions at Concentrations found in Cystic Fibrosis.

The role of colony stimulating factors (CSFs) in cystic fibrosis (CF) circulating neutrophils has not been thoroughly evaluated, considering that the neutrophil burden of lung inflammation in these subjects is very high. The aim of this study was to assess granulocyte-CSF (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) levels in CF patients in various clinical conditions and how these cytokines impact on activation and priming of neutrophils. G-CSF and GM-CSF levels were measured in sputum and serum samples of stable CF patients (n = 21) and in CF patients with acute exacerbation before and after a course of antibiotic therapy (n = 19). CSFs were tested on non CF neutrophils to investigate their effects on reactive oxygen species (ROS) production, degranulation (CD66b, elastase, lactoferrin, MMP-9), and chemotaxis. At very low concentrations found in CF patients (0.005-0.1 ng/ml), both cytokines inhibited ROS production, while higher concentrations (1-5 ng/ml) exerted a stimulatory effect. While either CSF induced elastase and MMP-9 secretion, lactoferrin levels were increased only by G-CSF. Chemotaxis was inhibited by GM-CSF, but was increased by G-CSF. However, when present together at low concentrations, CSFs increased basal and fMLP-stimulated ROS production and chemotaxis. These results suggest the CSF levels that circulating neutrophils face before extravasating into the lungs of CF patients may enhance their function contributing to the airway damage.

Insulin-like growth factor-6 (IGFBP-6) stimulates neutrophil oxidative burst, degranulation and chemotaxis.

The aim of this study was to understand whether insulin-like growth factor-binding protein-6 (IGFBP-6) has functional effects on neutrophils, in particular when they cross epithelium during inflammation. We found that IGFBP-6 increased ROS production (cytofluorimetry), degranulation of primary and tertiary granules (ELISA) and transmigration through the epithelial monolayer. No priming by IGFBP-6 on neutrophils stimulated by either PMA or fMLP was observed. IGFBP-6 is an agonist of neutrophils' functions, most likely when these cells have been already activated by other stimuli.

CFTR-dependent chloride efflux in cystic fibrosis mononuclear cells is increased by ivacaftor therapy.

AIM: The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiator ivacaftor (Kalydeco®) improves clinical outcome in G551D cystic fibrosis (CF) patients. Here, we have investigated whether ivacaftor has a clinical impact on non-G551D gating mutations and function of circulating leukocytes as well. METHODS: Seven patients were treated with ivacaftor and evaluated at baseline, and at 1-3 and 6 months. Besides clinical and systemic inflammatory parameters, circulating mononuclear cells (MNC) were evaluated for CFTR-dependent chloride efflux by spectrofluorimetry, neutrophils for oxidative burst by cytofluorimetry and HVCN1 mRNA expression by real time PCR. RESULTS: Ivacaftor determined a significant decrease in sweat chloride concentrations at all time points during treatment. Body mass index (BMI), FEV1 , and FVC showed an increasing trend. While C-reactive protein decreased significantly at 2 months, the opposite behavior was noticed for circulating monocytes. CFTR activity in MNC was found to increase significantly at 3 and 6 months. Neutrophil oxidative burst peaked at 2 months and then decreased to baseline. HVCN1 mRNA expression was significantly higher than baseline at 1-3 months and decreased after 6 months of treatment. The chloride efflux in MNC correlated positively with both FEV1 and FVC. On the other hand, sweat chloride correlated positively with CRP and WBC, and negatively with both respiratory function tests. A cluster analysis confirmed that sweat chloride, FEV1 , FVC, BMI, and MNC chloride efflux behaved as a single entity over time. DISCUSSION: In patients with non-G551D mutations, ivacaftor improved both chloride transport in sweat ducts and chloride efflux in MNC, that is, functions directly imputed to CFTR.

Evaluation of genome-wide expression profiles of blood and sputum neutrophils in cystic fibrosis patients before and after antibiotic therapy.

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to "healthy" condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and ß-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.

Increase in interleukin-8 production from circulating neutrophils upon antibiotic therapy in cystic fibrosis patients.

BACKGROUND: It is not known whether antibiotic therapy for lung disease in cystic fibrosis (CF) has an influence on circulating polymorphonuclear neutrophil (PMN) function and apoptosis. PATIENTS AND METHODS: Blood PMNs were obtained from 14 CF patients before and after antibiotic treatment for an acute exacerbation, and from 10 healthy controls. PMNs were evaluated for production of reactive oxygen species (ROS) by spectrophotometry, of cytokines in the conditioned medium by ELISA, and apoptotic response by cytofluorimetry. RESULTS: ROS and interleukin (IL)-8 were produced at higher levels by CF PMNs pre-therapy than control PMNs under basal conditions. IL-8 levels further increased after therapy. Early apoptotic response was higher in CF PMNs pre-therapy than in control PMNs, and this pattern did not change after antibiotic treatment. CONCLUSIONS: Circulating PMNs are primed in CF acute patients. Further studies are needed to consider PMN-produced IL-8 as a biomarker to evaluate response to antibiotic therapy in CF patients.

Streptococcus pneumoniae modulates the respiratory burst response in human neutrophils.

The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for high morbidity and mortality worldwide. A major feature of pneumococcal pneumonia is an abundant neutrophil infiltration. In this work we observed that the R6 nonencapsulated S. pneumoniae strain induced a higher oxidative burst in neutrophils compared with its capsulated progenitor D39, by triggering neutrophil NADPH oxidase to produce more reactive oxygen intermediates (ROI) and by interfering with the neutrophil kinase signalling pathway. In addition, we evaluated the possibility that the capsule, lacking in R6 but present in D39, could modulate the S. pneumoniae-induced neutrophil respiratory burst. In this respect, three knock-out isogenic mutants (D39ΔCPS2E, D39ΔCPS-R6 and R6ΔCPS-R6) that were unable to synthesize the capsule, were tested for their capability of inducing the release of neutrophil-ROIs. The results indicate that the mutants behaved similarly to their wild-type parental strains in enhancing respiratory burst activity, suggesting that the capsule itself is not directly involved in modulating the neutrophil oxidative burst induced by S. pneumoniae, but that other genetic differences between D39 and R6 present elsewhere in the genome could be responsible for these mechanisms.

Streptococcus pneumoniae induces mast cell degranulation.

Streptococcus pneumoniae colonizes the nasopharynx of healthy human carriers, but occasionally can spread in the body causing severe diseases. The mucosa of the respiratory tract is enriched in mast cells, key players of the innate immune response. Here, we report on the interaction of various strains of S. pneumoniae with the mast cell line RBL-2H3. Live, but not heat-killed, bacteria were found to induce mast cell degranulation in a dose- and time-dependent manner, only partially controlled by cytosolic calcium, with no production of TNF-alpha and IL-6. Non-encapsulated pneumococcal strains exhibited different potencies in triggering mast cells. We propose here that the induction of mast cell degranulation by pneumococcal factors not accompanied by the production of pro-inflammatory cytokines may be a specific strategy elaborated by this bacterium to promote its own spreading from the respiratory mucosa into the environment.

The neutrophil-activating protein of Helicobacter pylori.

Infection of the stomach mucosa by the gastric pathogen Helicobacter pylori is accompanied by a large infiltration of neutrophils and monocytes which are believed to contribute substantially to H. pylori-induced gastritis. A protein was identified (HP-NAP for neutrophil-activating protein from H. pylori) that was capable of increasing the adhesion of neutrophils to endothelial cells. We have demonstrated that HP-NAP is a dodecamer composed of identical 17-kDa subunits that induces the production of reactive oxygen radicals (ROIs) by neutrophils via a cascade of intracellular activation events. HP-NAP has also been shown to be chemotactic for neutrophils and monocytes, and a majority of H. pylori-infected patients have been found to produce antibodies specific for HP-NAP making it a strong vaccine candidate. More recently it has been shown that HP-NAP can stimulate tissue factor and plasminogen activator inhibitor-2 production by human monocytes. While structurally similar to the Escherichia coli DNA-binding protein Dps, HP-NAP has characteristics that are more similar to bacterioferritins being capable of binding up to 500 atoms of iron in vitro. Further study, however, has revealed that synthesis of HP-NAP in H. pylori is not altered by the addition or subtraction of metal ions from its growth medium suggesting that the primary role of the protein in vivo is not as a metal-binding protein. A number of other reports have proposed that HP-NAP acts as an adhesin being capable of binding several different compounds in vitro. Sequence analysis of the genomes of several other bacteria reveal that many possess Dps/HP-NAP-like proteins. The preliminary characterisation of some of these proteins will be discussed.

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a potent stimulant of mast cells.

Infection by Helicobacter pylori causes an acute inflammatory response followed by a chronic infection of the human gastric mucosa. A neutrophil-activating protein (HP-NAP) has been identified in H.pylori, and its role in infection and immune response is currently under investigation. Here, we show that HP-NAP induces beta-hexosaminidase release and interleukin-6 production in peritoneal mast cells, two actions which are completely inhibited by pertussis toxin. We also show that in polarized epithelial cell monolayers HP-NAP translocates from the apical to the basolateral domain, where mast cells are located. These findings characterize HP-NAP as an inflammatory factor of H.pylori that is effective from the beginning of the inflammatory cascade.