A Prospective Study of Hematologic Complications and Long-Term Survival of Italian Patients Affected by Shwachman-Diamond Syndrome.

OBJECTIVE: To describe the hematologic outcome and long-term survival of patients enrolled in the Shwachman-Diamond syndrome Italian Registry. STUDY DESIGN: A retrospective and prospective study of patients recorded in the Shwachman-Diamond syndrome Italian Registry. RESULTS: The study population included 121 patients, 69 males and 52 females, diagnosed between 1999 and 2018. All patients had the clinical diagnosis confirmed by mutational analysis on the SBDS gene. During the study period, the incidence of SDS was 1 in 153 000 births. The median age of patients with SDS at diagnosis was 1.3 years (range, 0-35.6 years). At the first hematologic assessment, severe neutropenia was present in 25.8%, thrombocytopenia in 25.5%, and anemia in 4.6% of patients. A normal karyotype was found in 40 of 79 patients, assessed whereas the most frequent cytogenetic abnormalities were isochromosome 7 and interstitial deletion of the long arm of chromosome 20. The cumulative incidence of severe neutropenia, thrombocytopenia, and anemia at 30 years of age were 59.9%, 66.8%, and 20.2%, respectively. The 20-year cumulative incidence of myelodysplastic syndrome/leukemia and of bone marrow failure/severe cytopenia was 9.8% and 9.9%, respectively. Fifteen of 121 patients (12.4%) underwent allogeneic stem cell transplantation. Fifteen patients (12.4%) died; the probability of overall survival at 10 and 20 years was 95.7% and 87.4%, respectively. CONCLUSIONS: Despite an improvement in survival, hematologic complications still cause death in patients with SDS. Further studies are needed to optimize type and modality of hematopoietic stem cell transplantation and to assess the long-term outcome in nontransplanted patients.

Normative growth charts for Shwachman-Diamond syndrome from Italian cohort of 0-8 years old.

OBJECTIVES: Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder. Its predominant manifestations include exocrine pancreatic insufficiency, bone marrow failure and skeletal abnormalities. Patients frequently present failure to thrive and susceptibility to short stature. Average birth weight is at the 25th percentile; by the first birthday, >50% of patients drop below the third percentile for height and weight.The study aims at estimating the growth charts for patients affected by SDS in order to give a reference tool helpful for medical care and growth surveillance through the first 8 years of patient's life. SETTING AND PARTICIPANTS: This retrospective observational study includes 106 patients (64 M) with available information from birth to 8 years, selected among the 122 patients included in the Italian National Registry of SDS and born between 1975 and 2016. Gender, birth date and auxological parameters at repeated assessment times were collected. The General Additive Model for Location Scale and Shape method was applied to build the growth charts. A set of different distributions was used, and the more appropriate were selected in accordance with the smallest Akaike information criterion. RESULTS: A total of 408 measurements was collected and analysed. The median number of observations per patient amounted to 3, range 1-11. In accordance with the methods described, specific SDS growth charts were built for weight, height and body mass index (BMI), separately for boys and girls.The 50th and 3rd percentiles of weight and height of the healthy population (WHO standard references) respectively correspond to the 97th and 50th percentiles of the SDS population (SDS specific growth charts), while the difference is less evident for the BMI. CONCLUSIONS: Specific SDS growth charts obtained through our analysis enable a more appropriate classification of patients based on auxological parameters, representing a useful reference tool for evaluating their growth during childhood.

Peripheral blood immunophenotyping in a large cohort of patients with Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes, characterized by neutropenia, occasional thrombocytopenia, and anemia. Bone marrow evaluation reveals an increased number of monocytes and mature B cells along with decreased granulocytes. However, little is known about the subpopulations of peripheral blood cells, and few previous publications have been based on a small number of patients. Here, we report a comprehensive immunophenotypic analysis from a cohort of 37 SDS patients who display impairment mostly in the myeloid compartment with a deficiency also in the number of B cells and CD4/CD8 double-negative T cells.

ß-Sitosterol Reduces the Expression of Chemotactic Cytokine Genes in Cystic Fibrosis Bronchial Epithelial Cells.

Extracts from Nigella arvensis L. seeds, which are widely used as anti-inflammatory remedies in traditional medicine of Northern Africa, were able to inhibit the expression of the pro-inflammatory neutrophil chemokine Interleukin (IL)-8 in Cystic Fibrosis (CF) bronchial epithelial IB3-1 cells exposed to the Gram-negative bacterium Pseudomonas aeruginosa. The chemical composition of the extracts led to the identification of three major components, ß-sitosterol, stigmasterol, and campesterol, which are the most abundant phytosterols, cholesterol-like molecules, usually found in plants. ß-sitosterol (BSS) was the only compound that significantly reproduced the inhibition of the P. aeruginosa-dependent expression of IL-8 at nanomolar concentrations. BSS was tested in CF airway epithelial CuFi-1 cells infected with P. aeruginosa. BSS (100 nM), showed a significant and consistent inhibitory activity on expression of the P. aeruginosa-stimulated expression chemokines IL-8, GRO-α GRO-ß, which play a pivotal role in the recruitment of neutrophils in CF inflamed lungs. Preliminary mechanistic analysis showed that BSS partially inhibits the P. aeruginosa-dependent activation of Protein Kinase C isoform alpha, which is known to be involved in the transmembrane signaling activating IL-8 gene expression in bronchial epithelial cells. These data indicate BSS as a promising molecule to control excessive lung inflammation in CF patients.

Diffuse alterations in grey and white matter associated with cognitive impairment in Shwachman-Diamond syndrome: evidence from a multimodal approach.

Shwachman-Diamond syndrome is a rare recessive genetic disease caused by mutations in SBDS gene, at chromosome 7q11. Phenotypically, the syndrome is characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal dysplasia and variable cognitive impairments. Structural brain abnormalities (smaller head circumference and decreased brain volume) have also been reported. No correlation studies between brain abnormalities and neuropsychological features have yet been performed. In this study we investigate neuroanatomical findings, neurofunctional pathways and cognitive functioning of Shwachman-Diamond syndrome subjects compared with healthy controls. To be eligible for inclusion, participants were required to have known SBDS mutations on both alleles, no history of cranial trauma or any standard contraindication to magnetic resonance imaging. Appropriate tests were used to assess cognitive functions. The static images were acquired on a 3 × 0 T magnetic resonance scanner and blood oxygen level-dependent functional magnetic resonance imaging data were collected both during the execution of the Stroop task and at rest. Diffusion tensor imaging was used to assess brain white matter. The Tract-based Spatial Statistics package and probabilistic tractography were used to characterize white matter pathways. Nine participants (5 males), half of all the subjects aged 9-19 years included in the Italian Shwachman-Diamond Syndrome Registry, were evaluated and compared with nine healthy subjects, matched for sex and age. The patients performed less well than norms and controls on cognitive tasks (p = 0.0002). Overall, cortical thickness was greater in the patients, both in the left (+10%) and in the right (+15%) hemisphere, significantly differently increased in the temporal (left and right, p = 0.04), and right parietal (p = 0.03) lobes and in Brodmann area 44 (p = 0.04) of the right frontal lobe. The greatest increases were observed in the left limbic-anterior cingulate cortex (≥43%, p < 0.0004). Only in Broca's area in the left hemisphere did the patients show a thinner cortical thickness than that of controls (p = 0.01). Diffusion tensor imaging showed large, significant difference increases in both fractional anisotropy (+37%, p < 0.0001) and mean diffusivity (+35%, p < 0.005); the Tract-based Spatial Statistics analysis identified six abnormal clusters of white matter fibres in the fronto-callosal, right fronto-external capsulae, left fronto-parietal, right pontine, temporo-mesial and left anterior-medial-temporal regions. Brain areas activated during the Stroop task and those active during the resting state, are different, fewer and smaller in patients and correlate with worse performance (p = 0.002). Cognitive impairment in Shwachman-Diamond syndrome subjects is associated with diffuse brain anomalies in the grey matter (verbal skills with BA44 and BA20 in the right hemisphere; perceptual skills with BA5, 37, 20, 21, 42 in the left hemisphere) and white matter connectivity (verbal skills with alterations in the fronto-occipital fasciculus and with the inferior-longitudinal fasciculus; perceptual skills with the arcuate fasciculus, limbic and ponto-cerebellar fasciculus; memory skills with the arcuate fasciculus; executive functions with the anterior cingulated and arcuate fasciculus).

Effects of decoy molecules targeting NF-kappaB transcription factors in Cystic fibrosis IB3-1 cells: recruitment of NF-kappaB to the IL-8 gene promoter and transcription of the IL-8 gene.

One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3-1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3-1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.

Further characterization of Shwachman-Diamond syndrome: psychological functioning and quality of life in adult and young patients.

To assess psychosocial functioning and quality of life in a representative group of adult and young patients with Shwachman-Diamond syndrome (SDS), all patients 3 years old and over included in the Italian SDS Registry were investigated using an ad-hoc questionnaire for information about demography, education, socialization, rehabilitation therapy, and standardized questionnaires [SF-36, Child Behavior Check-List (CBCL)] for quality of life and behavior. Results were compared with those of a Cystic Fibrosis (CF) patient group, matched for age and sex. Eighty-one percent of patients answered. All but one adult patient lived with their parents, 24% had independent income, and 57% had a driver's license. Different levels (from mild to severe) of cognitive impairment were reported by 76% of the adults and by 65% of the young patients. These data are significantly lower than those of the CF group. Both groups present low scores in the emotional and mental health evaluations at SF-36, but SDS patients reported significantly more limitations in physical functioning (PF) and more body pain (BP) experiences. As reported by parents at CBCL, young SDS patients show more "social problems" (in the clinical area 31% SDS vs. 6% CF), "attention deficits disorder" (29% SDS vs. 0%CF), and "somatic complaints" (24% SDS vs. 12% CF). Psychosocial functioning is impaired in the majority of SDS patients, significantly more than in patients affected by CF.

Modulators of sphingolipid metabolism reduce lung inflammation.

The investigation of novel targets for the treatment of cystic fibrosis (CF) lung inflammation is a major priority, considering that no effective therapy is available for this purpose. Consistent with the evidence that the sphingolipid (SL) ceramide regulates airway inflammation and infection in mice and patients with CF, SLs were identified as targets for treating pulmonary disorders, including CF. Because miglustat, an inhibitor of the synthesis of glycosphingolipids, reduces the Pseudomonas aeruginosa-dependent transcription of the IL-8 gene in bronchial cells, we examined the effects of miglustat and amitriptyline, another drug affecting ceramide metabolism, on the expression of 92 genes implicated in host immune defense. Infection with the P. aeruginosa strain PAO1 up-modulated the expression of 14 (27%) genes in IB3-1 cells and 15 (29%) genes in CF primary respiratory epithelia grown at an air-liquid interface, including chemokines (IL-8, growth-regulated Gro-α/ß/γ proteins, and granulocyte chemotactic peptide-2 [GCP-2]), proinflammatory cytokines (IL-1α/ß, IL-6, and TNF-α), and the intercellular adhesion molecule-1, nuclear factor kB1, toll like receptor 2, and human defensin B4 genes, confirming that bronchial epithelium is an important source of inflammatory mediators. Both miglustat and amitriptyline reduced the immune response, an effect that paralleled a decrease in the P. aeruginosa-induced accumulation of ceramide. Miglustat (100 mg/kg), given to C57BL/6 mice once daily for a period of 3 consecutive days before lipopolysaccharide (LPS) challenge, strongly reduced the number of neutrophils recruited in the airways and the expression of the keratinocyte-derived chemokine in lung extracts. Collectively, these results indicate that targeting the metabolism of SLs can down-modulate the recruitment of neutrophils into the lung.

Phospholipase C-ß3 is a key modulator of IL-8 expression in cystic fibrosis bronchial epithelial cells.

Respiratory insufficiency is the major cause of morbidity and mortality in patients affected by cystic fibrosis (CF). An excessive neutrophilic inflammation, mainly orchestrated by the release of IL-8 from bronchial epithelial cells and amplified by chronic bacterial infection with Pseudomonas aeruginosa, leads to progressive tissue destruction. The anti-inflammatory drugs presently used in CF patients have several limitations, indicating the need for identifying novel molecular targets. To address this issue, we preliminarily studied the association of 721 single nucleotide polymorphisms from 135 genes potentially involved in signal transduction implicated in neutrophil recruitment in a cohort of F508del homozygous CF patients with either severe or mild progression of lung disease. The top ranking association was found for a nonsynonymous polymorphism of the phospholipase C-ß3 (PLCB3) gene. Studies in bronchial epithelial cells exposed to P. aeruginosa revealed that PLCB3 is implicated in extracellular nucleotide-dependent intracellular calcium signaling, leading to activation of the protein kinase Cα and Cß and of the nuclear transcription factor NF-κB p65. The proinflammatory pathway regulated by PLCB3 acts by potentiating the Toll-like Receptors' signaling cascade and represents an interesting molecular target to attenuate the excessive recruitment of neutrophils without completely abolishing the inflammatory response.

A polymorphism in the 5' UTR of the DEFB1 gene is associated with the lung phenotype in F508del homozygous Italian cystic fibrosis patients.

BACKGROUND: The identification of cystic fibrosis (CF) patients who are at greater risk of lung damage could be clinically valuable. Thus, we attempted to replicate previous findings and verify the possible association between three single nucleotide polymorphisms (SNPs c.-52G>A, c.-44C>G and c.-20G>A) in the 5' untranslated region (5' UTR) of the ß defensin 1 (DEFB1) gene and the CF pulmonary phenotype. METHODS: Genomic DNA from 92 Italian CF patients enrolled in different regional CF centres was extracted from peripheral blood and genotyped for DEFB1 SNPs using TaqMan(®) allele specific probes. In order to avoid genetic confounding causes that can account for CF phenotype variability, all patients were homozygous for the F508del CFTR mutation, and were then classified on the basis of clinical and functional data as mild lung phenotype (Mp, n=50) or severe lung phenotype patients (Sp, n=42). RESULTS: For the c.-20G>A SNP, the frequency of the A allele, as well as the AA genotype, were significantly more frequent in Mp than in Sp patients, and thus this was associated with a protective effect against severe pulmonary disease (OR=0.48 and 0.28, respectively). The effect of the c.-20G>A A allele is consistent with a recessive model, and the protective effect against Sp is exerted only when it is present in homozygosis. For the other two SNPs, no differences were observed as allelic and genotypic frequency in the two subgroups of CF patients. CONCLUSIONS: Our results, although necessary to be confirmed in larger and multiethnic populations, reinforce DEFB1 as a candidate modifier gene of the CF pulmonary phenotype.

Trimethylangelicin reduces IL-8 transcription and potentiates CFTR function.

Chronic inflammatory response in the airway tract of patients affected by cystic fibrosis is characterized by an excessive recruitment of neutrophils to the bronchial lumina, driven by the chemokine interleukin (IL)-8. We previously found that 5-methoxypsoralen reduces Pseudomonas aeruginosa-dependent IL-8 transcription in bronchial epithelial cell lines, with an IC(50) of 10 µM (Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bezzerri V, Bianchi N, Mazzon M, Mancini I, Giri MG, Rizzotti P, Gambari R, Cabrini G. Int Immunopharmacol 9: 1411-1422, 2009). Here, we extended the investigation to analogs of 5-methoxypsoralen, and we found that the most potent effect is obtained with 4,6,4'-trimethylangelicin (TMA), which inhibits P. aeruginosa-dependent IL-8 transcription at nanomolar concentration in IB3-1, CuFi-1, CFBE41o-, and Calu-3 bronchial epithelial cell lines. Analysis of phosphoproteins involved in proinflammatory transmembrane signaling evidenced that TMA reduces the phosphorylation of ribosomal S6 kinase-1 and AKT2/3, which we found indeed involved in P. aeruginosa-dependent activation of IL-8 gene transcription by testing the effect of pharmacological inhibitors. In addition, we found a docking site of TMA into NF-κB by in silico analysis, whereas inhibition of the NF-κB/DNA interactions in vitro by EMSA was observed at high concentrations (10 mM TMA). To further understand whether NF-κB pathway should be considered a target of TMA, chromatin immunoprecipitation was performed, and we observed that TMA (100 nM) preincubated in whole living cells reduced the interaction of NF-κB with the promoter of IL-8 gene. These results suggest that TMA could inhibit IL-8 gene transcription mainly by intervening on driving the recruitment of activated transcription factors on IL-8 gene promoter, as demonstrated here for NF-κB. Although the complete understanding of the mechanism of action of TMA deserves further investigation, an activity of TMA on phosphorylating pathways was already demonstrated by our study. Finally, since psoralens have been shown to potentiate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport, TMA was tested and found to potentiate CFTR-dependent chloride efflux. In conclusion, TMA is a dual-acting compound reducing excessive IL-8 expression and potentiating CFTR function.

Virtual screening against nuclear factor κB (NF-κB) of a focus library: Identification of bioactive furocoumarin derivatives inhibiting NF-κB dependent biological functions involved in cystic fibrosis.

In the present study, a structured-based virtual screening (VS) of differently substituted furocoumarins and analogues has been carried out against nuclear factor kappa B (NF-κB), with the objective of selecting molecules able to inhibit the binding of this transcription factor to the DNA. The focus library was developed starting from chemical structures obtained from the literature, as well as retrieving compounds from available commercial databases. A two dimensional substructure searching method based on four different chemical scaffolds was used for this purpose. Among the 10 highest-scored ligands selected from the docking studies, five commercially available molecules were investigated in biological assays. Four furocoumarin derivatives showed IC(50) values in the range of 40-100 µM in inhibiting NF-κB/DNA interactions studied by electrophoretic mobility shift assay (EMSA). Three compounds significantly inhibited NF-κB dependent biological functions (expression of IL-8) in cellular analysis based on Pseudomonas aeruginosa infection of cystic fibrosis IB3-1 cells. These findings validated the virtual screening approach here presented and reinforce the successful results of our previously computational studies aimed at the identification of molecules targeting NF-κB. The discovered novel compounds could be of relevance to identify more potent inhibitors of NF-κB dependent biological functions beneficial to control lung inflammation occurring in patients affected by cystic fibrosis.

Decoy oligodeoxyribonucleotides and peptide nucleic acids-DNA chimeras targeting nuclear factor kappa-B: inhibition of IL-8 gene expression in cystic fibrosis cells infected with Pseudomonas aeruginosa.

Cystic fibrosis (CF) is characterized by a deep inflammatory process, with production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against IL-8, with the aim of reducing the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. TFD is based on biomolecules mimicking the target sites of transcription factors (TFs) and able to interfere with TF activity when delivered to target cells. Here, we review the inhibitory effects of decoy oligodeoxyribonucleotides (ODNs) on expression of IL-8 gene and secretion of IL-8 by cystic fibrosis cells infected by Pseudomonas aeruginosa. In addition, the effects of decoy molecules based on peptide nucleic acids (PNAs) are discussed. In this respect PNA-DNA-PNA (PDP) chimeras are interesting: (a) unlike PNAs, they can be complexed with liposomes and microspheres; (b) unlike oligodeoxyribonucleotides (ODNs), they are resistant to DNAses, serum and cytoplasmic extracts; (c) unlike PNA/PNA and PNA/DNA hybrids, they are potent decoy molecules. Interestingly, PDP/PDP NF-kappaB decoy chimeras inhibit accumulation of pro-inflammatory mRNAs (including IL-8 mRNA) in P. aeruginosa infected IB3-1, cells reproducing the effects of decoy oligonucleotides. The effects of PDP/PDP chimeras, unlike ODN-based decoys, are observed even in absence of protection with lipofectamine. Since IL-8 is pivotal in pro-inflammatory processes affecting cystic fibrosis, inhibition of its functions might have a clinical relevance.

Virtual screening against p50 NF-kappaB transcription factor for the identification of inhibitors of the NF-kappaB-DNA interaction and expression of NF-kappaB upregulated genes.

Virtual screening against NF-kappaB p50 using docking simulations was applied by starting from a three-dimensional (3D) database containing more than 4.6 million commercially available structures. This database was filtered by specifying a subset of commercially available compounds sharing a (2E,Z)-3-(2-hydroxyphenyl)-2-propenoate substructure and relevant druglike properties. Docking to p50 NF-kappaB was performed with a test set of six known inhibitors of NF-kappaB-DNA interactions. In agreement with docking results, the highest-scored compound displayed a high level of inhibitory activity in electrophoretic mobility shift assay (EMSA) experiments (inhibition of NF-kappaB-DNA interactions) and on biological functions dependent on NF-kappaB activity (inhibition of IL-8 gene expression in cystic fibrosis IB3-1 cells). We found that this in silico screening approach is suitable for the identification of low-molecular-weight compounds that inhibit NF-kappaB-DNA interactions and NF-kappaB-dependent functions. Information deduced from the discovery of the new lead compound and its binding mode could result in further lead optimization resulting in more potent NF-kappaB inhibitors.

Modulation of expression of IL-8 gene in bronchial epithelial cells by 5-methoxypsoralen.

Persistent recruitment of neutrophils in the bronchi of cystic fibrosis patients contributes to airway tissue damage, suggesting the importance of intervening on the expression of the neutrophil chemokine IL-8. Extracts from plants have been investigated to select components able to reduce IL-8 expression in bronchial epithelial cells challenged with Pseudomonas aeruginosa. Extracts and purified components have been added to cells 24 h before pro-inflammatory challenge with P. aeruginosa and IL-8 transcription was quantified in the IB3-1 CF cells in vitro. P. aeruginosa-dependent IL-8 mRNA induction was increased by Argemone mexicana and Vernonia anthelmintica whereas no significant modification of transcription was observed with Aphanamixis polystachya, Lagerstroemia speciosa and Hemidesmus indicus. Finally, inhibition of IL-8 was observed with Polyalthia longifolia (IC50=200 microg/ml) and Aegle marmelos (IC50=20 microg/ml). Compounds from A. marmelos were isolated and identified by GC-MS. No significant effect was observed with butyl-p-tolyl sulphate, whereas the inhibition obtained with 6-methyl-4-chromanone concentration was accompanied by an anti-proliferative effect. On the contrary, 5-methoxypsoralen resulted in IL-8 inhibition at 10 microM concentration, without effects on cell proliferation. In synthesis, 5-methoxypsoralen can be taken into consideration to investigate mechanisms of neutrophil chemotactic signalling and for its potential application in modulating the excessive CF lung inflammation.

Late generation lentiviral vectors: evaluation of inflammatory potential in human airway epithelial cells.

Lentiviruses (LVs) are considered one of the most promising tools for gene transfer, however, their potential to induce pro-inflammatory cytokines on delivery into the respiratory tissue remains to be established. Here we tested a third-generation vesicular stomatitis virus (VSV)-G pseudotyped LV vector in the two respiratory epithelial cell lines A549 and CFT1-C2. We observed that the VSV-G LV vector does not induce (a) activation of the nuclear factor (NF)-kappaB, which intervenes in transcription of pro-inflammatory genes; (b) expression of ICAM-1; and (c) transcription of a panel of cytokines, with the exception of a mild and transient (24h) increase of IFN-gamma mRNA. In contrast, an adenovirus-derived vector strongly activated NF-kappaB and different transcripts such as those of ICAM-1, IL-8, RANTES, IP-10, TNF-alpha, IL-6, IL-1 beta. In conclusion, this third-generation VSV-G pseudotyped LV vector does not elicit major pro-inflammatory signals in human airway epithelial cells and appears to be better suited for gene delivery strategies.