Vermamoeba vermiformis
represents one of the most common free-living amoebae identified in worldwide environmental surveys. We analyzed 56 water samples with varying characteristics, including ...temperature and the particular settings in which humans may be exposed to water, plus one corneal scraping from a keratitis patient, with the following aims: (i) to investigate the presence of
V. vermiformis
; (ii) to identify the isolate subtypes; (iii) to place the Italian isolates in the broader picture of the genetic diversity within
V. vermiformis.
Twenty-two isolates were identified upon culturing and sequencing of > 600 bp in the 18S ribosomal RNA (rRNA) gene sequence, bringing to 27 the number of sequences recovered from Italian sources. By adding deposited sequences, we assembled a dataset of 74 isolates. Three of our isolates were characterized by allelic code 7-5-1-1, never reported before, and two showed 100% identity with an uncultured eukaryote and carried the 719T>C variant. We show that the variable segments E5, E3, F, and G convey most of the information on diversity, enabling the clustering of the isolates in a replicable fashion. The presence of different strains in natural thermal waters and in distribution systems indicated heterogeneity of the amoebic populations. Also, ours and the only other sequence from human infection were mapped in different clades. Overall, we enlarged the repertoire of single nucleotide and indel variants and the list of allelic codes, proceeding one step further in the description of the diversity within the genus.
The interest in the development of nanoscale plasmonic technologies has dramatically increased in recent years. The photonic properties of plasmonic nanopatterns can be controlled and tuned via their ...size, shape, or the arrangement of their constituents. In this work, we propose a 2D hybrid metallic polymeric nanostructure based on the octupolar framework with enhanced sensing property. We analyze its plasmonic features both numerically and experimentally, demonstrating the higher values of their relevant figures of merit: we estimated a surface-enhanced Raman spectroscopy (SERS) enhancement factor of 9 × 107 and a SPR bulk sensitivity of 430 nm/RIU. In addition, our nanostructure exhibits a dual resonance in the visible and near-infrared region, enabling our system toward multispectral plasmonic analysis. Finally, we illustrate our design engineering strategy as enabled by electron beam lithography by the outstanding performance of a SERS-based biosensor that targets the Shiga toxin 2a, a clinically relevant bacterial toxin. To the best of our knowledge, this is the first time that a SERS fingerprint of this toxin has been evidenced.
Acetylcholine (ACh), synthesized by Choline Acetyl‐Transferase (ChAT), exerts its physiological effects via mAChRM3 in epithelial cells. We hypothesized that cigarette smoke affects ChAT, ACh, and ...mAChRM3 expression in the airways from COPD patients promoting airway disease. ChAT, ACh, and mAChRM3 were assessed: “ex vivo” in the epithelium from central and distal airways of COPD patients, Healthy Smoker (S) and Healthy Subjects (C), and “in vitro” in bronchial epithelial cells stimulated with cigarette smoke extract (CSE). In central airways, mAChRM3, ChAT, and ACh immunoreactivity was significantly higher in the epithelium from S and COPD than in C subjects. mAChRM3, ChAT, and ACh score of immunoreactivity was high in the metaplastia area of COPD patients. mAChRM3/ChAT and ACh/ChAT co‐localization of immunoreactivity was observed in the bronchial epithelium from COPD. In vitro, CSE stimulation significantly increased mAChRM3, ChAT, and ACh expression and mAChRM3/ChAT and ACh/ChAT co‐localization in 16HBE and NHBE, and increased 16HBE proliferation. Cigarette smoke modifies the levels of mAChMR3, ChAT expression, and ACh production in bronchial epithelial cells from COPD patients. Non‐neuronal components of cholinergic system may have a role in the mechanism of bronchial epithelial cell proliferation, promoting alteration of normal tissue, and of related pulmonary functions.
Cigarette smoke is involved in the alteration of non‐neuronal cholinergic system components expression, contributing to the airway remodeling processes, and to the progressive decline in lung function in COPD patients.
Airway epithelium is emerging as a regulator of innate immune responses to a variety of insults including cigarette smoke. The main goal of this study was to explore the effects of cigarette smoke ...extracts (CSE) on Toll-like receptor (TLR) expression and activation in a human bronchial epithelial cell line (16-HBE). The CSE increased the expression of TLR4 and the lipopolysaccharide (LPS) binding, the nuclear factor-κB (NF-κB) activation, the release of interleukin-8 (IL-8) and the chemotactic activity toward neutrophils. It did not induce TLR2 expression or extracellular signal-regulated signal kinase 1/2 (ERK1/2) activation. The LPS increased the expression of TLR4 and induced both NF-κB and ERK1/2 activation. The combined exposure of 16-HBE to CSE and LPS was associated with ERK activation rather than NF-κB activation and with a further increase of IL-8 release and of chemotactic activity toward neutrophils. Furthermore, CSE decreased the constitutive interferon-inducible protein-10 (IP-10) release and counteracted the effect of LPS in inducing both the IP-10 release and the chemotactic activity toward lymphocytes. In conclusion, cigarette smoke, by altering the expression and the activation of TLR4 via the preferential release of IL-8, may contribute to the accumulation of neutrophils within the airways of smokers.
Background Lipoxins are biologically active eicosanoids with anti-inflammatory properties. Lipoxin A4 (LXA4 ) signaling blocks asthmatic responses in human and experimental model systems. There is ...evidence that patients with respiratory diseases, including severe asthma (SA), display defective generation of lipoxin signals despite glucocorticoid therapy. Objective We investigated airway levels of formyl peptide receptor 2–lipoxin receptor (FPR2/ALXR), LXA4 , and its counterregulatory compound, leukotriene B4 (LTB4 ), in patients with childhood asthma. We addressed the potential interplay of the LXA4 -FPR2/ALXR axis and glucocorticoids in the resolution of inflammation. Methods We examined LXA4 and LTB4 concentrations in induced sputum supernatants from children with intermittent asthma (IA), children with SA, and healthy control (HC) children. In addition, we investigated FPR2/ALXR expression in induced sputum cells obtained from the study groups. Finally, we evaluated in vitro the molecular interaction between LXA4 and glucocorticoid receptor–based mechanisms. Results We found that children with SA have decreased LXA4 concentrations in induced sputum supernatants in comparison with children with IA. In contrast to decreases in LXA4 concentrations, LTB4 concentrations were increased in children with asthma independent of severity. LXA4 concentrations negatively correlated with LTB4 concentrations and with exacerbation numbers in children with SA. FPR2/ALXR expression was reduced in induced sputum cells of children with SA compared with that seen in HC subjects and children with IA. Finally, we describe in vitro the existence of crosstalk between LXA4 and glucocorticoid receptor at the cytosolic level mediated by G protein–coupled FPR2/ALXR in peripheral blood granulocytes isolated from HC subjects, children with IA, and children with SA. Conclusion Our findings provide evidence for defective LXA4 generation and FPR2/ALXR expression that, associated with increased LTB4 , might be involved in a reduction in the ability of inhaled corticosteroids to impair control of airway inflammation in children with SA.
Acetylcholine may play a role in cell activation and airway inflammation. We evaluated the levels of both mRNA and protein of muscarinic M
1, M
2, M
3 receptors in human bronchial epithelial cell ...line (16HBE). 16HBE cells were also stimulated with acetylcholine and extracellular signal-regulated kinase1/2 (ERK1/2) and NFkB pathway activation as well as the IL-8 release was assessed in the presence or absence of the inhibitor of Protein-kinase (PKC) (GF109203X), of the inhibitor of mitogenic activated protein-kinase kinase (MAPKK) (PDO9805), of the inhibitor of kinaseB-α phosphorilation (pIkBα) (BAY11-7082), and of muscarinic receptor antagonists tiotropium bromide, 4-Diphenylacetoxy-
N-methylpiperidine methiodide (4-DAMP), telenzepine, gallamine. Additionally, we tested the IL-8-mediated neutrophil chemotactic activity of 16HBE supernatants stimulated with acetylcholine in the presence or absence of tiotropium. 16HBE cells expressed both protein and mRNA for muscarinic M
3, M
2 and M
1 receptors with levels of muscarinic M
3 receptor
>
muscarinic M
1 receptor
>
muscarinic M
2 receptor. Acetylcholine (10 μM) significantly stimulated ERK1/2 and NFkB activation as well as IL-8 release in 16HBE cells when compared to basal values. Furthermore, while the use of tiotropium, 4-DAMP, GF109203X, PDO98059, BAY11-7082 completely abolished these events, the use of telenzepine and gallamine were only partially able to downregulate these effects. Additionally, acetylcholine-mediated IL-8 release from 16HBE cells significantly increased chemotaxis toward neutrophils and this effect was blocked by tiotropium. In conclusion, acetylcholine activates the release of IL-8 from 16HBE involving PKC, ERK1/2 and NFkB pathways via muscarinic receptors, suggesting that it is likely to contribute to IL-8 related neutrophilic inflammatory disorders in the airway. Thus, muscarinic antagonists may contribute to control inflammatory processes in airway diseases.
Abstract Background The main limiting factor to major hepatic resections is the amount of the future liver remnant (FLR). Associating Liver Partition with Portal Vein Ligation for Staged Hepatectomy ...(ALPPS) is a procedure which induces a rapid hypertrophy of the FLR in patients with non-resectable liver tumours. Methods ALPPS is a surgical technique of in-situ splitting of the liver along the main portal scissura or the right side of the falciform ligament, in association with portal vein ligation in order to induce a rapid hypertrophy of the left FLR. Results The median FLR volume increase was 18.7% within one week after the first step and 38.6% after the second step. At the first step the median operating time was 300 min, blood transfusions were not required in any case, median blood loss was 150 cc. At the second step median operating time was 180 min, median blood loss was 50 cc, none of the patients required intra-operative blood. All patients are alive at a median follow up of 9 months. Conclusions This novel strategy seems to be feasible even in the context of a cirrhotic liver, and demonstrates the capacity to reach a sufficient FLR within a shorter interval of time.
•The liver was enriched of Vγ9Vδ2 T-cells expressing an effector/activated phenotype, independently from HCV infection.•An enrichment of PD-1+Vγ9Vδ2 T-cells was observed both in the peripheral blood ...and in the liver of HCVpos patients.•HCV induced a dysregulation in IFN-γ production by intrahepatic Vγ9Vδ2 T-cells by mantaining their anti-HCV activity.
Hepatitis C virus (HCV) persistence results from inefficiencies of both innate and adaptive immune responses to eradicate the infection. A functional impairment of circulating Vγ9Vδ2 T-cells was described but few data are available on Vγ9Vδ2 T-cells in the liver that, however, represents the battlefield in the HCV/host interaction.
Aim of this work was to compare circulating and intrahepatic Vγ9Vδ2 T-cells in chronic HCV-infected patients (HCVpos) and in HCV-negative (HCVneg) subjects. Phenotypic and functional analysis was performed by flow cytometry. Anti-HCV activity was analyzed by using an in vitro autologous liver culture system.
Independently from HCV infection, the liver was enriched of Vγ9Vδ2 T-cells expressing an effector/activated phenotype. In contrast, an enrichment of PD-1 expressing Vγ9Vδ2 T-cells was observed both in the peripheral blood and in the liver of HCVpos patients, probably due to a persistent antigenic stimulation. Moreover, a lower frequency of IFN-γ producing Vγ9Vδ2 T-cells was observed in the liver of HCVpos patients, suggesting a functional impairment in the cytokine production in HCVpos liver. Despite this hypo-responsiveness, intrahepatic Vγ9Vδ2 T-cells are able to exert an anti-HCV activity after specific stimulation.
Altogether, our data show that HCV infection induced a dysregulation of intrahepatic Vγ9Vδ2 T cells that maintain their anti-HCV activity after specific stimulation. A study aimed to evaluate the mechanisms of the antiviral activity may be useful to identify new pathways able to improve Vγ9Vδ2 T-cells intrahepatic function during HCV infection.
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