Giulio Cabrini

Heparan Sulfate Glycosaminoglycans Are Receptors Sufficient To Mediate the Initial Binding of Adenovirus Types 2 and 5

ABSTRACT Cell infection by adenovirus serotypes 2 and 5 (Ad2/5) initiates with the attachment of Ad fiber to the coxsackievirus and Ad receptor (CAR) followed by αv integrin-mediated entry. We recently demonstrated that heparan sulfate glycosaminoglycans (HS GAGs) expressed on cell surfaces are involved in the binding and infection of Ad2/5 (M. C. Dechecchi, A. Tamanini, A. Bonizzato, and G. Cabrini, Virology 268:382–390, 2000). The role of HS GAGs was investigated using extracellular soluble domain 1 of CAR (sCAR-D1) and heparin as soluble receptor analogues of CAR and HS GAGs in A549 and recombinant CHO cell lines with differential levels of expression of the two receptors and cultured to various densities. Complete inhibition of binding and infection was obtained by preincubating Ad2/5 with both heparin (10 μg/ml) and sCAR-D1 (200 μg/ml) in A549 cells. Partial inhibition was observed when heparin and sCAR-D1 were preincubated separately with Ad. The level of heparin-sensitive [3H]Ad2/5 binding doubled in sparse A549 cells (50 to 70,000 cells/cm2) with respect to that of cells grown to confluence (200 to 300,000 cells/cm2), in parallel with increased expression of HS GAGs. [3H]Ad2 bound to sparse CAR-negative CHO cells expressing HS GAGs (CHO K1). No [3H]Ad2 binding was observed in CHO K1 cells upon competitive inhibition with heparin and in HS GAG-defective CHO A745, D677, and E606 clones. HS-sensitive Ad2 infection was obtained in CAR-negative sparse CHO K1 cells but not in CHO A745 cells, which were permissive to infection only upon transfection with CAR. These results demonstrate that HS GAGs are sufficient to mediate the initial binding of Ad2/5.

A His-155 to Tyr Polymorphism Confers Gain-of-Function to the Human P2X7 Receptor of Human Leukemic Lymphocytes

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.

IL-4 Is a Potent Modulator of Ion Transport in the Human Bronchial Epithelium In Vitro

Recent data show that proinflammatory stimuli may modify significantly ion transport in the airway epithelium and therefore the properties of the airway surface fluid. We have studied the effect of IL-4, a cytokine involved in the pathogenesis of asthma, on transepithelial ion transport in the human bronchial epithelium in vitro. Incubation of polarized bronchial epithelial cells with IL-4 for 6–48 h causes a marked inhibition of the amiloride-sensitive Na+ channel as measured in short circuit current experiments. On the other hand, IL-4 evokes a 2-fold increase in the current activated by a cAMP analog, which reflects the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, IL-4 enhances the response to apical UTP, an agonist that activates Ca2+-dependent Cl− channels. These effects are mimicked by IL-13 and blocked by an antagonist of IL-4Rα. RT-PCR experiments show that IL-4 elicits a 7-fold decrease in the level of the γ amiloride-sensitive Na+ channel mRNA, one of the subunits of the amiloride-sensitive Na+ channel, and an increase in CFTR mRNA. Our data suggest that IL-4 may favor the hydration of the airway surface by decreasing Na+ absorption and increasing Cl− secretion. This could be required to fluidify the mucus, which is hypersecreted during inflammatory conditions. On the other hand, the modifications of ion transport could also affect the ion composition of airway surface fluid.

Oxidative stress and antioxidant therapy in cystic fibrosis.

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.

Interaction of Adenovirus Type 5 Fiber with the Coxsackievirus and Adenovirus Receptor Activates Inflammatory Response in Human Respiratory Cells

ABSTRACT The innate immune response to adenovirus (Ad)-derived gene transfer vectors has been shown to initiate immediately after interaction of Ad with respiratory epithelial cells, through the induction of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and JNK mitogen-activated protein kinase (MAPK), nuclear factor κB (NF-κB), and different proinflammatory genes. Ad serotypes 2 or 5 (Ad2/5) enter respiratory epithelia after initial binding of fiber with the coxsackievirus-adenovirus receptor (CAR) or, alternatively, with cell surface heparan sulfate glycosaminoglycans. Ad2/5 internalization is triggered by binding of penton base to cellular RGD-binding integrins. Here we investigated the role of the Ad5 surface domain proteins constituting the vector capsid, namely, the fiber, the penton base, and the hexon, on the transmembrane signals leading to the transcription of the different proinflammatory genes in the human respiratory A549 cell line. Interaction of Ad fiber with CAR activates both ERK1/2 and JNK MAPK and the nuclear translocation of NF-κB, whereas no activation was observed after exposing A549 cells to penton base and hexon proteins. Moreover, interaction of Ad fiber with CAR, but not heparan sulfate proteoglycans, promotes transcription of the chemokines interleukin-8, GRO-α, GRO-γ, RANTES, and interferon-inducible protein 10. These results identify the binding of Ad5 fiber with the cellular CAR as a key proinflammatory activation event in epithelial respiratory cells that is independent of the transcription of Ad5 genes.

Analysis of the complete coding region of the CFTR gene in a cohort of CF patients from North-Eastern Italy: identification of 90% of the mutations

A complete coding-region analysis on 225 cystic fibrosis (CF) chromosomes from a cohort that includes all the affected subjects born in two North-Eastern Italian regions over eight years was performed. In a previous study, we identified mutations on 166/225 (73.8%) CF chromosomes after screening for 62 mutations. To characterise the remaining 59 CF chromosomes, we carried out automated direct DNA sequencing (exons 9 and 13), RNA single-strand conformation polymorphism (exons 1–8 and 10–12) and denaturing gradient gel electrophoresis (exons 14a–24) of the 27 exons and flanking regions of the CF transmembrane conductance regulator gene. We identified 22 mutations, four of which are novel, viz. 711+5G→A, R709X, 3132delTG and 2790-2An→G, and we characterised 90.2% (203/225) of the CF chromosomes. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters, assessed at the age of 12 years, is presented. We confirm some previously reported genotype-phenotype correlations and we report a new nonsense mutation (R709X) associated with a pancreatic sufficient phenotype.

Mitochondrial Ca2+-dependent NLRP3 activation exacerbates the Pseudomonas aeruginosa-driven inflammatory response in cystic fibrosis

The common pathological manifestation of cystic fibrosis (CF) is associated with an excessive lung inflammatory response characterized by interleukin-1β 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. aeruginosa-dependent mitochondrial perturbation, in which flagellin is the inducer and mitochondrial Ca(2+) uniporter (MCU) is a signal-integrating organelle member for NLRP3 activation and IL-1β and IL-18 processing. Our work elucidates the regulation of the NLRP3 inflammasome by mitochondrial Ca(2+) in the P. aeruginosa-dependent inflammatory response and deepens our understanding of the significance of mitochondria in the Ca(2+)-dependent control of inflammation.

Genetic history of cystic fibrosis mutations in italy. I. Regional distribution

Earlier analysis of the Italian population showed patterns of genetic differentiation that were interpreted as being the result of population settlements going back to pre‐Roman times. DNA disease mutations may be a powerful tool in further testing this hypothesis since the analysis of diseased individuals can detect variants too rare to be resolved in normal individuals. We present data on the relative frequencies of 60 cystic fibrosis (CF) mutations in Italy and the geographical distribution of the 12 most frequent CF mutations screened in 3492 CF chromosomes originating in 13 Italian regions. The 12 most frequent mutations characterize about 73% of the Italian CF chromosomes. The most common mutation, ΔF508, has an average frequency of 51%, followed by N1303K and G542X, both with average frequencies around 5%. Multivariate analyses show that the relative frequencies of CF mutations are heterogeneous among Italian regions, and that this heterogeneity is weakly correlated with the geographical pattern of non‐DNA ‘classical’ genetic markers. The northern regions are well differentiated from the central‐southern regions and within the former group the western and eastern regions are remarkably distinct. Moreover, Sardinia shows the presence of mutation T338I, which seems absent in any other European CF chromosome. The north‐western regions of Italy, characterized by the mutation 1717‐1G→A, were under Celtic influence, while the north‐east regions, characterized by the mutations R1162X, 2183AA→G and 711 + 5G→A, were under the influence of the Venetic culture.

Pyrogallol, an active compound from the medicinal plant Emblica officinalis, regulates expression of pro-inflammatory genes in bronchial epithelial cells

The most relevant cause of morbidity and mortality in cystic fibrosis (CF) patients is the lung pathology characterized by chronic infection and inflammation sustained mainly by Pseudomonas aeruginosa (P. aeruginosa). Innovative pharmacological approaches to control the excessive inflammatory process in the lung of CF patients are thought to be beneficial to reduce the extensive airway tissue damage. Medicinal plants from the so-called traditional Asian medicine are attracting a growing interest because of their potential efficacy and safety. Due to the presence of different active compounds in each plant extract, understanding the effect of each component is important to pursue selective and reproducible applications. Extracts from Emblica officinalis (EO) were tested in IB3-1 CF bronchial epithelial cells exposed to the P. aeruginosa laboratory strain PAO1. EO strongly inhibited the PAO1-dependent expression of the neutrophil chemokines IL-8, GRO-alpha, GRO-gamma, of the adhesion molecule ICAM-1 and of the pro-inflammatory cytokine IL-6. Pyrogallol, one of the compounds extracted from EO, inhibited the P. aeruginosa-dependent expression of these pro-inflammatory genes similarly to the whole EO extract, whereas a second compound purified from EO, namely 5-hydroxy-isoquinoline, had no effect. These results identify Pyrogallol as an active compound responsible for the anti-inflammatory effect of EO and suggest to extend the investigation in pre-clinical studies in airway animal models in vivo, to test the efficacy and safety of this molecule in CF chronic lung inflammatory disease.

Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07

This study compares the effect of three chemically unrelated cystic fibrosis transmembrane conductance regulator (CFTR) activators on epithelial cell monolayers expressing the G551D‐CFTR mutant. We measured Cl− transport as the amplitude of short‐circuit current in response to the membrane permeable cAMP analogue 8‐(4‐chlorophenylthio)adenosine‐3′‐5′‐cyclic monophosphate (CPT‐cAMP) alone or in combination with a CFTR opener. The correction of G551D‐CFTR defect was quantified by comparison with maximal activity elicited in cells expressing wild type CFTR. To this end we used Fisher rat thyroid (FRT) cells transfected with wild type or G551D CFTR, and primary cultures of human nasal epithelial cells. In both types of epithelia, cAMP caused activation of Cl− transport that was inhibited by glibenclamide and not by 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonic acid. After normalising for CFTR expression, the response of FRT‐G551D epithelia was 1% that of wild type monolayers. Addition of genistein (10–200 μM), but not of 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX, 1–100 μM) or of the benzo[c]quinolizinium MPB‐07 (10–200 μM) to FRT‐G551D epithelia pre‐treated with cAMP, stimulated a sustained current that at maximal genistein concentration corresponded to 30% of the response of wild type epithelia. The genistein dose‐response curve was bell‐shaped due to inhibitory activity at the highest concentrations. The dose‐dependence in G551D cells was shifted with respect to wild type CFTR so that higher genistein concentrations were required to observe activation and inhibition, respectively. On human nasal epithelia the correction of G551D‐CFTR defective conductance obtained with genistein was 20% that of wild type. The impressive effect of genistein suggests that it might correct the Cl− transport defect on G551D patients.