Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi-or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells. INTRODUCTIONHaemophilus influenzae is a Gram-negative human pathogen that colonizes asymptomatically the nasopharynx of healthy individuals. Disease occurs when the bacterium reaches privileged anatomical sites under various predisposing conditions, including, among others, age, viral infections or a constant exposure to pollutants, leading to otitis media, sinusitis, meningitis, septicaemia or respiratory infections (Foxwell et al., 1998;Rao et al., 1999). Nontypable H. influenzae (NTHi) is the most frequently isolated bacterial pathogen in the lungs of patients suffering from chronic obstructive pulmonary disease (COPD), and furthermore, NTHi accounts for the majority of bacterial COPD exacerbation episodes (Sethi et al., 2002;Sethi & Murphy, 2001.One of the NTHi virulence factors that has been most extensively analysed is its lipooligosaccharide (LOS). It consists of lipid A, an inner core consisting of a Abbreviations: COPD, chronic obstructive pulmonary disease; EEA1, early endosome antigen 1; FISH, fluorescent in situ hybridization; LOS, lipooligosaccharide; MbCD, methyl-b-cyclodextrin; NHBE cells, normal human bronchial epithelial cells; NTHi, nontypable Haemophilus influenzae; NTHi-CV, NTHi-containing vacuole; PCho, phosphocholine; PFA, paraformaldehyde; PI3K, phosphatidylinositol 3-kinase; SCV, Salmonella-containing vacuole; TEM, transmission electron microscopy. phosphorylated 2-keto-3-deoxyoctulosonic acid (KDO) linked to three heptoses and an outer core consisting of a heteropolymer of glucose and galactose (Schweda et al., 2007). Similar to other bacterial pathogens adapted to mucosal surfaces,...

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Spinning cone column (SCC) distillation has been shown to be a commercially suitable technique for dealcoholized wine (DW) manufacturing, but there are not enough studies about its influence on the DW quality. So, the effect of this technique on the antioxidant activity (% of remaining 1,1-diphenyl-2-picrylhydrazyl radical) and the phenolic compound composition of red, rose, and white DW, obtained at pilot plant scale, has been analyzed. Nineteen raw wines (RWs) from different grape varieties and five different Spanish viticultural regions have been studied before and after dealcoholization. The total phenolic content, flavonols, tartaric esters, and anthocyanins, was determined by spectrophotometry, while the content of phenolic compounds such as stilbenes (trans- and cis-resveratrol), flavonols (rutin, quercetin, and myricetin), flavan-3-ols [(+)-catechin and (-)-epicatechin], anthocyanins (malvidin 3-glucoside), and non-flavonoids (gallic, caffeic, and p-coumaric acids) was determined by high-performance liquid chromatography (HPLC). The resveratrol contents in red wines were between 1.81 and 34.01 mg/L in RWs and between 2.12 and 39.57 mg/L in DWs, Merlot being the grape producing the RWs and DWs with higher resveratrol content. In general, the percent of remaining DPPH(*) was similar or slightly higher (until 5 units of % of remaining DPPH(*)) in DWs versus RWs. This small difference may be due to removal of SO2 (that is an antioxidant) from RWs during distillation. DWs and RWs show similar contents of the studied phenolic compounds, with a tendency, in some cases, to exhibit increases after dealcoholization, caused by the concentration effect via removal of the ethanol. From this work, we can deduce that SCC distillation is a dealcoholization technique minimally destructive with the wine phenolic compounds.

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