No abstract

Gene therapy vectors derived from subgroup C adenoviruses of the serotype 5 (Ad5) and 2 (Ad2) resulted in inefficient infection of well differentiated respiratory cells, both in vitro and in vivo. The level of expression and localization of the primary receptor for Ad5 and Ad2, termed CAR, do not completely explain why the infection efficiency varies greatly in different experimental conditions. The possibility that additional receptors like proteoglycans are involved in the infection of Ad5 and Ad2 was investigated, because several pathogenic microorganisms use heparan sulfate-glycosaminoglycans (HS-GAGs) as coreceptors for multistep attachment to target cells. The HS-GAG analog heparin decreased Ad5- and Ad2-mediated infection and binding starting from the concentration of 0.1 microgram/ml, up to a maximum of 50%. A similar reduction in Ad5 binding and infection was obtained by treatment of cells with heparin lyases I, II, and III but not with chondroitin ABC lyase. The effect of heparin on Ad5 binding has not been observed in surface GAG-defective Raji cells and after treating A549 cells with heparin lyases I, II,and III. The binding of Ad5 was completely abolished when both CAR was blocked with RmcB antibody and HS-GAGs were competitively inhibited by heparin. Parallel experiments demonstrate that HS-GAGs are irrelevant to binding and infection of the subgroup B adenovirus type 3. Collectively, these results demonstrate for the first time that HS-GAGs expressed on the cell surface are involved in the binding of Ad5 and Ad2 to host cells.

Cystic fibrosis is a lethal autosomal recessive condition caused by a defect of the transmembrane conductance regulator gene that has a key role in cell homeostasis. A dysfunctional cystic fibrosis transmembrane conductance regulator impairs the efflux of cell anions such as chloride and bicarbonate, and also that of other solutes such as reduced glutathione. This defect produces an increased viscosity of secretions together with other metabolic defects of epithelia that ultimately promote the obstruction and fibrosis of organs. Recurrent pulmonary infections and respiratory dysfunction are main clinical consequences of these pathogenetic events, followed by pancreatic and liver insufficiency, diabetes, protein-energy malnutrition, etc. This complex comorbidity is associated with the extensive injury of different biomolecular targets by reactive oxygen species, which is the biochemical hallmark of oxidative stress. These biological lesions are particularly pronounced in the lung, in which the extent of oxidative markers parallels that of inflammatory markers between chronic events and acute exacerbations along the progression of the disease. Herein, an abnormal flux of reactive oxygen species is present by the sustained activation of neutrophils and other cystic fibrosis-derived defects in the homeostatic processes of pulmonary epithelia and lining fluids. A sub-optimal antioxidant protection is believed to represent a main contributor to oxidative stress and to the poor control of immuno-inflammatory pathways in these patients. Observed defects include an impaired reduced glutathione metabolism and lowered intake and absorption of fat-soluble antioxidants (vitamin E, carotenoids, coenzyme Q-10, some polyunsaturated fatty acids, etc.) and oligoelements (such as Se, Cu and Zn) that are involved in reactive oxygen species detoxification by means of enzymatic defenses. Oral supplements and aerosolized formulations of thiols have been used in the antioxidant therapy of this inherited disease with the main aim of reducing the extent of oxidative lesions and the rate of lung deterioration. Despite positive effects on laboratory end points, poor evidence was obtained on the side of clinical outcome so far. These aspects examined in this critical review of the literature clearly suggest that further and more rigorous trials are needed together with new generations of pharmacological tools to a more effective antioxidant and anti-inflammatory therapy of cystic fibrosis patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

The P2X7R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X7R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X7R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X7R function was monitored by measuring ATP-induced Ca2+ influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X7 allelic variant. Ca2+ influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca2+ flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X7R confirmed an increased ATP-dependent activation of the P2X7 489T mutant with respect to the wild type receptor, as assessed by both by [Ca2+]i influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X7R.

The common pathological manifestation of cystic fibrosis (CF) is associated with an excessive lung inflammatory response characterized by interleukin-1b accumulation. CF airway epithelial cells show an exacerbated pro-inflammatory response to Pseudomonas aeruginosa; however, it is unclear whether this heightened inflammatory response is intrinsic to cells lacking CF transmembrane conductance regulator (CFTR). Here we demonstrate that the degree and quality of the inflammatory response in CF are supported by P. aeruginosadependent mitochondrial perturbation, in which flagellin is the inducer and mitochondrial Ca 2 þ uniporter (MCU) is a signal-integrating organelle member for NLRP3 activation and IL-1b and IL-18 processing. Our work elucidates the regulation of the NLRP3 inflammasome by mitochondrial Ca 2 þ in the P. aeruginosa-dependent inflammatory response and deepens our understanding of the significance of mitochondria in the Ca 2 þ -dependent control of inflammation. hronic airway infection by Pseudomonas aeruginosa is a common pathological manifestation in cystic fibrosis (CF) patients, and the role of calcium ions (Ca 2 þ ) in this process seems pivotal in controlling the P. aeruginosa-dependent innate immune response 1 . This manifestation is associated with an excessive inflammatory response characterized by the accumulation of large amounts of cytokines, including interleukin-1b (IL-1b) 2 .Previous studies support the role of IL-1b in the pathogenesis of CF inflammatory lung disease. The levels of IL-1b are increased in bronchoalveolar lavage fluid of CF patients 2,3 , and polymorphisms in the IL-1b gene have been associated with varying degrees of disease severity 4 .The maturation of IL-1b and IL-18 are directed by the caspase-1 (casp-1)-activating multiprotein platform, the inflammasome. Four distinct inflammasomes that contain a nucleotidebinding and oligomerization domain (NOD)-like receptor have been identified: NLRP1, IPAF, NLRP3 and AIM2 (ref. 5). NODlike receptors (NLRs) are intracellular pattern-recognition receptors that recognize pathogen-associated molecular patterns and activate the pro-inflammatory response through specific pathways 6 . It is generally accepted that the release of IL-1b and IL-18 require two distinct signals; however, the nature of these signals during inflammation is not completely defined. Studies indicate that the 'first signal' can be triggered by various pathogen-associated molecular patterns following Toll-like receptor (TLR) activation, which induces the synthesis of proIL-1b and proIL-18. The 'second signal' is provided by activation of the inflammasome and casp-1, leading to IL-1b and IL-18 processing.P. aeruginosa reportedly activates IPAF, leading to IL-1b processing 7 . IPAF interacts directly with procaspase-1, which contains the N-terminal caspase recruitment domain 8 . However, maximum casp-1 activation in response to IPAF agonists requires the involvement of apoptosis-associated specklike protein (ASC) 9,10 , a bipartite, caspase recruitment domainand py...