Unravelling the role of sphingolipids in cystic fibrosis lung disease.

Abstract Cystic fibrosis (CF), one of the most common lethal hereditary diseases of white European populations, is caused by loss-of-function mutations in the CF Transmembrane conductance Regulator (CFTR) gene. One of the main causes of mortality is the onset of CF lung disease, which is characterized by chronic infection and inflammation resulting in the progressive remodelling, irreversible damage and fibrosis of the airways. An increasing number of studies indicate that sphingolipids are crucial players in pulmonary manifestations of CF, even if their direct involvement in CF lung disease is still unclear. In this review, we give an overview of the role of sphingolipids in CF pulmonary disease, focusing on the relationship between glycosphingolipids and lung inflammation, which represents the main hallmark of this disease

Integrin αvβ5 is a primary receptor for adenovirus in CAR-negative cells

<p>Abstract</p> <p>Background</p> <p>Viruses bind to specific cellular receptors in order to infect their hosts. The specific receptors a virus uses are important factors in determining host range, cellular tropism, and pathogenesis. For adenovirus, the existing model of entry requires two receptor interactions. First, the viral fiber protein binds Coxsackie and Adenovirus Receptor (CAR), its primary cellular receptor, which docks the virus to the cell surface. Next, viral penton base engages cellular integrins, coreceptors thought to be required exclusively for internalization and not contributing to binding. However, a number of studies reporting data which conflicts with this simple model have been published. These observations have led us to question the proposed two-step model for adenovirus infection.</p> <p>Results</p> <p>In this study we report that cells which express little to no CAR can be efficiently transduced by adenovirus. Using competition experiments between whole virus and soluble viral fiber protein or integrin blocking peptides, we show virus binding is not dependent on fiber binding to cells but rather on penton base binding cellular integrins. Further, we find that binding to low CAR expressing cells is inhibited specifically by a blocking antibody to integrin αvβ5, demonstrating that in these cells integrin αvβ5 and not CAR is required for adenovirus attachment. The binding mediated by integrin αvβ5 is extremely high affinity, in the picomolar range.</p> <p>Conclusions</p> <p>Our data further challenges the model of adenovirus infection in which binding to primary receptor CAR is required in order for subsequent interactions between adenovirus and integrins to initiate viral entry. In low CAR cells, binding occurs through integrin αvβ5, a receptor previously thought to be used exclusively in internalization. We show for the first time that integrin αvβ5 can be used as an alternate binding receptor.</p

Role of CFTR expressed by neutrophils in modulating acute lung inflammation and injury in mice

Objective and designCystic fibrosis transmembrane conductance regulator (CFTR) regulates infection and inflammation. In this study, we investigated whether a lack of functional CFTR in neutrophils would promote lipopolysaccharide (LPS)-induced lung inflammation and injury.Materials and methodsCFTR-inhibited or F508del-CFTR-mutated neutrophils were stimulated with LPS and cultured to evaluate production of cytokines and NF-κB activation. Wild-type mice were reconstituted with F508del neutrophils or bone marrow and then intratracheally challenged with LPS to observe lung inflammatory response.ResultsPharmacologic inhibition and genetic mutation of CFTR in neutrophils activated NF-κB and facilitated macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) production. Wild-type mice reconstituted with F508del neutrophils and bone marrow had more severe lung inflammation and injury after LPS challenge compared to wild-type mice receiving wild-type neutrophils or bone marrow reconstitution.ConclusionsLack of functional CFTR in neutrophils can promote LPS-induced acute lung inflammation and injury

Role of Cellular Heparan Sulfate Proteoglycans in Infection of Human Adenovirus Serotype 3 and 35

Species B human adenoviruses (Ads) are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs). We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection

Mechanisms of the noxious inflammatory cycle in cystic fibrosis

Multiple evidences indicate that inflammation is an event occurring prior to infection in patients with cystic fibrosis. The self-perpetuating inflammatory cycle may play a pathogenic part in this disease. The role of the NF-κB pathway in enhanced production of inflammatory mediators is well documented. The pathophysiologic mechanisms through which the intrinsic inflammatory response develops remain unclear. The unfolded mutated protein cystic fibrosis transmembrane conductance regulator (CFTRΔF508), accounting for this pathology, is retained in the endoplasmic reticulum (ER), induces a stress, and modifies calcium homeostasis. Furthermore, CFTR is implicated in the transport of glutathione, the major antioxidant element in cells. CFTR mutations can alter redox homeostasis and induce an oxidative stress. The disturbance of the redox balance may evoke NF-κB activation and, in addition, promote apoptosis. In this review, we examine the hypotheses of the integrated pathogenic processes leading to the intrinsic inflammatory response in cystic fibrosis

Anatomical Differences Determine Distribution of Adenovirus after Convection-Enhanced Delivery to the Rat Brain

Background: Convection-enhanced delivery (CED) of adenoviruses offers the potential of widespread virus distribution in the brain. In CED, the volume of distribution (Vd) should be related to the volume of infusion (Vi) and not to dose, but when using adenoviruses contrasting results have been reported. As the characteristics of the infused tissue can affect convective delivery, this study was performed to determine the effects of the gray and white matter on CED of adenoviruses and similar sized super paramagnetic iron oxide nanoparticles (SPIO). Methodology/Principal Findings: We convected AdGFP, an adenovirus vector expressing Green Fluorescent Protein, a virus sized SPIO or trypan blue in the gray and white matter of the striatum and external capsule of Wistar rats and towards orthotopic infiltrative brain tumors. The resulting Vds were compared to Vi and transgene expression to SPIO distribution. Results show that in the striatum Vd is not determined by the Vi but by the infused virus dose, suggesting diffusion, active transport or receptor saturation rather than convection. Distribution of virus and SPIO in the white matter is partly volume dependent, which is probably caused by preferential fluid pathways from the external capsule to the surrounding gray matter, as demonstrated by co-infusing trypan blue. Distant tumors were reached using the white matter tracts but tumor penetration was limited. Conclusions/Significance: CED of adenoviruses in the rat brain and towards infiltrative tumors is feasible when regional anatomical differences are taken into account while SPIO infusion could be considered to validate proper catheter positioning and predict adenoviral distribution

Chloride conductance in membrane vesicles from human placenta using a fluorescent probe. Implications for cystic fibrosis

Previous evidence suggests that the molecular defect in cystic fibrosis (CF) could reside in an altered chloride conductance of epithelial tissues. Since the brush border of the syncytiotrophoblast of the chorionic villi of human placenta is an abundant source of epithelial membranes and it is unaltered by secondary pathology or treatment we chose to characterize its chloride conductance and to compare it in normal and CF membranes. Chloride transport was studied in microvillar vesicles (MVV) by the quencing of the fluorescent probe 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Chloride conductance at 23\ub0C: (a) increased by 39% under a membrane potential change of 70 mV; (b) was inhibited by diphenylamine 2-carboxylate (K(i) = 150 \u3bcM); (c) displayed an activation energy of 3.5 kcal \ub7 mol-1. The comparison of the chloride conductance for an inwardly directed gradient of 150 mM Cl- at 23\ub0C (membrane potential set at 0 mV) between CF and control membranes was not significantly different. These findings demonstrate the presence of a chloride conductive pathway in microvillar vesicles from human placenta and preliminary results exclude major differences in the conductance of CF derived material in the absence of neurohormonal stimuli

Molecular basis of cystic fibrosis: from bench to bedside

Cystic fibrosis (CF), is an autosomal recessive disease affecting different organs. The lung disease, characterized by recurrent and chronic bacterial infection and inflammation since infancy, is the main cause of morbidity and precocious mortality of these individuals. The innovative therapies directed to repair the defective CF gene should account for the presence of more than 200 disease-causing mutations of the CF transmembrane conductance regulator (CFTR) gene. The review will recall the different experimental approaches in discovering CFTR protein targeted molecules, such as the high throughput screening on chemical libraries to discover correctors and potentiators of CFTR protein, dual-acting compounds, read-through molecules, splicing defects repairing tools, CFTR "amplifiers"

Heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions

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 \u3bcg/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. (C) 2000 Academic Press

Heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions

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 \u3bcg/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. (C) 2000 Academic Press