Publications by authors named "Federica Sabatini"

39 Publications

Immunotherapeutic Strategies for Neuroblastoma: Present, Past and Future.

Vaccines (Basel) 2021 Jan 13;9(1). Epub 2021 Jan 13.

Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Istituto Giannina Gaslini (Istituto di Ricerca e Cura a Carattere Scientifico-IRCCS), Via G. Gaslini 5, 16147 Genova, Italy.

Neuroblastoma is the most common extracranial pediatric solid tumor with a heterogeneous clinical course, ranging from spontaneous regression to metastatic disease and death, irrespective of intensive chemotherapeutic regimen. On the basis of several parameters, children affected by neuroblastoma are stratified into low, intermediate and high risk. At present, more than 50% of high-risk patients with metastatic spread display an overall poor long-term outcome also complicated by devastating long-term morbidities. Thus, novel and more effective therapies are desperately needed to improve lifespan of high-risk patients. In this regard, adoptive cell therapy holds great promise and several clinical trials are ongoing, demonstrating safety and tolerability, with no toxicities. Starting from the immunological and clinical features of neuroblastoma, we here discuss the immunotherapeutic approaches currently adopted for high-risk patients and different innovative therapeutic strategies currently under investigation. The latter are based on the infusion of natural killer (NK) cells, as support of consolidation therapy in addition to standard treatments, or chimeric antigen receptor (CAR) T cells directed against neuroblastoma associated antigens (e.g., disialoganglioside GD2). Finally, future perspectives of adoptive cell therapies represented by γδ T lymphocyes and CAR NK cells are envisaged.
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http://dx.doi.org/10.3390/vaccines9010043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828327PMC
January 2021

Iron overload alters the energy metabolism in patients with myelodysplastic syndromes: results from the multicenter FISM BIOFER study.

Sci Rep 2020 06 8;10(1):9156. Epub 2020 Jun 8.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Myelodysplastic syndromes (MDS) are hematological malignancies characterized by ineffective hematopoiesis and increased apoptosis in the bone marrow, which cause peripheral cytopenia. Mitochondria are key regulators of apoptosis and a site of iron accumulation that favors reactive oxygen species (ROS) production with detrimental effects on cell survival. Although the energy metabolism could represent an attractive therapeutic target, it was poorly investigated in MDS. The purpose of the study was to analyze how the presence of myelodysplastic hematopoiesis, iron overload and chelation impact on mitochondrial metabolism. We compared energy balance, OxPhos activity and efficiency, lactic dehydrogenase activity and lipid peroxidation in mononuclear cells (MNCs), isolated from 38 MDS patients and 79 healthy controls. Our data show that ATP/AMP ratio is reduced during aging and even more in MDS due to a decreased OxPhos activity associated with an increment of lipid peroxidation. Moreover, the lactate fermentation enhancement was observed in MDS and elderly subjects, probably as an attempt to restore the energy balance. The biochemical alterations of MNCs from MDS patients have been partially restored by the in vitro iron chelation, while only slight effects were observed in the age-matched control samples. By contrast, the addition of iron chelators on MNCs from young healthy subjects determined a decrement in the OxPhos efficiency and an increment of lactate fermentation and lipid peroxidation. In summary, MDS-MNCs display an altered energy metabolism associated with increased oxidative stress, due to iron accumulation. This condition could be partially restored by iron chelation.
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http://dx.doi.org/10.1038/s41598-020-66162-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280296PMC
June 2020

Transplantation Induces Profound Changes in the Transcriptional Asset of Hematopoietic Stem Cells: Identification of Specific Signatures Using Machine Learning Techniques.

J Clin Med 2020 Jun 1;9(6). Epub 2020 Jun 1.

Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.

During the phase of proliferation needed for hematopoietic reconstitution following transplantation, hematopoietic stem/progenitor cells (HSPC) must express genes involved in stem cell self-renewal. We investigated the expression of genes relevant for self-renewal and expansion of HSPC (operationally defined as CD34+ cells) in steady state and after transplantation. Specifically, we evaluated the expression of ninety-one genes that were analyzed by real-time PCR in CD34+ cells isolated from (i) 12 samples from umbilical cord blood (UCB); (ii) 15 samples from bone marrow healthy donors; (iii) 13 samples from bone marrow after umbilical cord blood transplant (UCBT); and (iv) 29 samples from patients after transplantation with adult hematopoietic cells. The results show that transplanted CD34+ cells from adult cells acquire an asset very different from transplanted CD34+ cells from cord blood. Multivariate machine learning analysis (MMLA) showed that four specific gene signatures can be obtained by comparing the four types of CD34+ cells. In several, but not all cases, transplanted HSPC from UCB overexpress reprogramming genes. However, these remarkable changes do not alter the commitment to hematopoietic lineage. Overall, these results reveal undisclosed aspects of transplantation biology.
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http://dx.doi.org/10.3390/jcm9061670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355574PMC
June 2020

Discrete Changes in Glucose Metabolism Define Aging.

Sci Rep 2019 07 17;9(1):10347. Epub 2019 Jul 17.

UOC Laboratorio Cellule Staminali post-natali e Terapie Cellulari, IRCCS Istituto Giannina Gaslini, Genoa, Italy.

Aging is a physiological process in which multifactorial processes determine a progressive decline. Several alterations contribute to the aging process, including telomere shortening, oxidative stress, deregulated autophagy and epigenetic modifications. In some cases, these alterations are so linked with the aging process that it is possible predict the age of a person on the basis of the modification of one specific pathway, as proposed by Horwath and his aging clock based on DNA methylation. Because the energy metabolism changes are involved in the aging process, in this work, we propose a new aging clock based on the modifications of glucose catabolism. The biochemical analyses were performed on mononuclear cells isolated from peripheral blood, obtained from a healthy population with an age between 5 and 106 years. In particular, we have evaluated the oxidative phosphorylation function and efficiency, the ATP/AMP ratio, the lactate dehydrogenase activity and the malondialdehyde content. Further, based on these biochemical markers, we developed a machine learning-based mathematical model able to predict the age of an individual with a mean absolute error of approximately 9.7 years. This mathematical model represents a new non-invasive tool to evaluate and define the age of individuals and could be used to evaluate the effects of drugs or other treatments on the early aging or the rejuvenation.
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http://dx.doi.org/10.1038/s41598-019-46749-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637183PMC
July 2019

Altered lipid metabolism could drive the bone marrow failure in fanconi anaemia.

Br J Haematol 2019 02 12;184(4):693-696. Epub 2018 Mar 12.

Haematology Unit, Istituto Giannina Gaslini, Genova, Italy.

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http://dx.doi.org/10.1111/bjh.15171DOI Listing
February 2019

Patient Safety in Complementary Medicine through the Application of Clinical Risk Management in the Public Health System.

Medicines (Basel) 2017 Dec 16;4(4). Epub 2017 Dec 16.

Regional Centre for Clinical Risk Management and Patient Safety, 50100 Florence, (T.B.).

To develop a systematic approach to detect and prevent clinical risks in complementary medicine (CM) and increase patient safety through the analysis of activities in homeopathy and acupuncture centres in the Tuscan region using a significant event audit (SEA) and failure modes and effects analysis (FMEA). SEA is the selected tool for studying adverse events (AE) and detecting the best solutions to prevent future incidents in our Regional Healthcare Service (RHS). This requires the active participation of all the actors and external experts to validate the analysis. FMEA is a proactive risk assessment tool involving the selection of the clinical process, the input of a multidisciplinary group of experts, description of the process, identification of the failure modes (FMs) for each step, estimates of the frequency, severity, and detectability of FMs, calculation of the risk priority number (RPN), and prioritized improvement actions to prevent FMs. In homeopathy, the greatest risk depends on the decision to switch from allopathic to homeopathic therapy. In acupuncture, major problems can arise, mainly from delayed treatment and from the modalities of needle insertion. The combination of SEA and FMEA can reveal potential risks for patients and suggest actions for safer and more reliable services in CM.
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http://dx.doi.org/10.3390/medicines4040093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750617PMC
December 2017

Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation.

Cell Mol Life Sci 2018 03 3;75(5):889-903. Epub 2017 Oct 3.

Stem Cell Laboratory and Cell Therapy Center, IRCCS Istituto Giannina Gaslini, 16147, Genoa, Italy.

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.
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http://dx.doi.org/10.1007/s00018-017-2665-zDOI Listing
March 2018

Exosomes from human mesenchymal stem cells conduct aerobic metabolism in term and preterm newborn infants.

FASEB J 2016 Apr 11;30(4):1416-24. Epub 2015 Dec 11.

*Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy.

Exosomes are secreted nanovesicles that are able to transfer RNA and proteins to target cells. The emerging role of mesenchymal stem cell (MSC) exosomes as promoters of aerobic ATP synthesis restoration in damaged cells, prompted us to assess whether they contain an extramitochondrial aerobic respiration capacity. Exosomes were isolated from culture medium of human MSCs from umbilical cord of ≥37-wk-old newborns or between 28- to 30-wk-old newborns (i.e.,term or preterm infants). Characterization of samples was conducted by cytofluorometry. Oxidative phosphorylation capacity was assessed by Western blot analysis, oximetry, and luminometric and fluorometric analyses. MSC exosomes express functional respiratory complexes I, IV, and V, consuming oxygen. ATP synthesis was only detectable in exosomes from term newborns, suggestive of a specific mechanism that is not completed at an early gestational age. Activities are outward facing and comparable to those detected in mitochondria isolated from term MSCs. MSC exosomes display an unsuspected aerobic respiratory ability independent of whole mitochondria. This may be relevant for their ability to rescue cell bioenergetics. The differential oxidative metabolism of pretermvs.term exosomes sheds new light on the preterm newborn's clinical vulnerability. A reduced ability to repair damaged tissue and an increased capability to cope with anoxic environment for preterm infants can be envisaged.-Panfoli, I., Ravera, S., Podestà, M., Cossu, C., Santucci, L., Bartolucci, M., Bruschi, M., Calzia, D., Sabatini, F., Bruschettini, M., Ramenghi, L. A., Romantsik, O., Marimpietri, D., Pistoia, V., Ghiggeri, G., Frassoni, F., Candiano, G. Exosomes from human mesenchymal stem cells conduct aerobic metabolism in term and preterm newborn infants.
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http://dx.doi.org/10.1096/fj.15-279679DOI Listing
April 2016

Isolation and characterization of renal cancer stem cells from patient-derived xenografts.

Oncotarget 2016 Mar;7(13):15507-24

INSERM U 1186, Equipe labellisée Ligue Contre le Cancer, Gustave Roussy Campus, Villejuif, France.

As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133high/CD105-/Ecadlow and PDX-2 cells were CD133low/CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAMlow and RCC-41-PDX-2/CD133+/EpCAMbright). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133- over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133+ over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132+ and RCC-41-PDX-2/CD133+ displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132+ tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution.Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.
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http://dx.doi.org/10.18632/oncotarget.6266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941257PMC
March 2016

Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples.

PLoS One 2015 29;10(9):e0138680. Epub 2015 Sep 29.

Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, IRCCS Istituto Giannina Gaslini, Genoa, Italy.

Background: Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups.

Methods And Results: Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15-4.8) vs 0.3% (0.032-2.23) p = 0.0001 and in neonates <32 weeks of gestational age (GA) compared to those ≥32 wks GA: 0.95% (range 0.18-4.8) and 0.36% (0.15-3.2) respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term) and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP) did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL) resulted higher compared to term (p = 0.004) and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017). The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168).

Conclusions: We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs) from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144)The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138680PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4587939PMC
June 2016

Expression of vascular remodelling markers in relation to bradykinin receptors in asthma and COPD.

Thorax 2013 Sep 5;68(9):803-11. Epub 2013 Jun 5.

Division of Respiratory Disease, Department of Clinical and Biological Sciences, A.O.U. San Luigi Hospital, University of Torino, Orbassano, Torino, Italy.

Background: Vascular remodelling plays a central role in asthma and chronic obstructive pulmonary disease (COPD). Bradykinin (BK) is a vasoactive proinflammatory peptide mediating acute responses in asthma. We investigated the role of angiogenic factors in relation to BK receptors in asthma and COPD.

Methods: Bronchial biopsies from 33 patients with COPD, 24 old (≥50 years) patients with (≥50 years) asthma, 18 old control smokers, 11 old control non-smokers, 15 young (≤40yrs) patients with (≤40 years) asthma and 10 young control non-smokers were immunostained for CD31, vascular endothelial growth factor-A (VEGF-A), angiogenin and BK receptors (B2R and B1R). Fibroblast and endothelial co-localisation of relevant molecules were performed by immunofluorescence. BK-induced VEGF-A and angiogenin release was studied (ELISA) in bronchial fibroblasts from subjects with asthma and COPD.

Results: In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A(+) cell numbers were higher than old control non-smokers (p<0.05). Angiogenin(+), B2R(+) and B1R(+) cell numbers in old patients with asthma were higher than in old control non-smokers, control smokers and patients with COPD (p<0.01). Angiogenin(+) cell numbers were higher in patients with COPD than both old control groups (p<0.05). In all patients with asthma the number of B2R(+) cells was positively related to the numbers of B1R(+) (rs=0.43), angiogenin(+) (rs=0.42) and CD31 cells (rs=0.46) (p<0.01). Angiogenin(+) cell numbers were negatively related to forced expiratory volume in 1 s (rs=-0.415, p=0.008). Double immunofluorescence revealed that CD31 cells of capillary vessels coexpressed B2R and that fibroblasts coexpressed B2R, VEGF-A and angiogenin. BK (10(-6)M) induced significant angiogenin release in fibroblasts from asthma and to a lesser extent in COPD.

Conclusions: Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.
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http://dx.doi.org/10.1136/thoraxjnl-2012-202741DOI Listing
September 2013

Bradykinin-induced asthmatic fibroblast/myofibroblast activities via bradykinin B2 receptor and different MAPK pathways.

Eur J Pharmacol 2013 Jun 12;710(1-3):100-9. Epub 2013 Apr 12.

Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy.

Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B2 receptor expression by Western blot analysis; cell proliferation by [(3)H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B2 receptor expression in HBAFb. Bradykinin, via bradykinin B2 receptor, significantly increased fibroblast proliferation at lower concentration (10(-11)M) and α-SMA expression/polymerization at higher concentration (10(-6)M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.
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http://dx.doi.org/10.1016/j.ejphar.2013.03.048DOI Listing
June 2013

Pharmacological modulation of the bradykinin-induced differentiation of human lung fibroblasts: effects of budesonide and formoterol.

J Asthma 2012 Dec 22;49(10):1004-11. Epub 2012 Oct 22.

Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy.

Objective: Bradykinin (BK) induces differentiation of lung fibroblasts into myofibroblasts, which play an important role in extracellular matrix remodeling in the airways of asthmatic patients. It is unclear whether this process is affected by antiasthma therapies. Here, we evaluated whether a glucocorticoid, budesonide (BUD), and a long-acting β2-agonist, formoterol (FM), either alone or in combination, modified BK-induced lung fibroblast differentiation, and affected the BK-activated intracellular signaling pathways.

Methods: Human fetal lung fibroblasts were incubated with BUD (0.001-0.1 μM) and/or FM (0.0001-0.1 μM) before exposure to BK (0.1 or 1 μM). Fibroblast differentiation into α-smooth-muscle-actin-positive (α-SMA⁺) myofibroblasts, BK2 receptor (B2R) expression, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation (p-ERK1/2), intracellular Ca²⁺ concentration ([Ca²⁺]i), and p65 nuclear factor kappa B translocation were evaluated.

Results: BUD (0.1 μM) and FM (0.1 μM), either alone or in combination, completely inhibited BK-induced α-SMA protein expression and decreased the numbers of α-SMA⁺ fibroblasts, with a clear reduction in α-SMA stress fibers organization. BUD also completely inhibited the increase of B2R, whereas FM with or without BUD had no effect. BK-induced increases of [Ca²⁺]i and p-ERK1/2 were significantly reduced to similar levels by BUD and FM, either alone or in combination, whereas p65 translocation was completely inhibited by all treatments.

Conclusion: Both BUD and FM, either alone or in combination, effectively inhibited the BK-induced differentiation of fibroblasts into α-SMA⁺ myofibroblasts and the intracellular signaling pathways involved in fibroblast activation. These results suggest that BUD and FM combination therapy has potential to inhibit fibroblast-dependent matrix remodeling in the airways of asthmatic patients.
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http://dx.doi.org/10.3109/02770903.2012.729633DOI Listing
December 2012

Bradykinin- and lipopolysaccharide-induced bradykinin B2 receptor expression, interleukin 8 release and "nitrosative stress" in bronchial epithelial cells BEAS-2B: role for neutrophils.

Eur J Pharmacol 2012 Nov 23;694(1-3):30-8. Epub 2012 Aug 23.

Department of Clinical and Biological Sciences, Division of Respiratory Disease, University of Torino, Torino, Italy.

Bradykinin-induced interleukin (IL)-8 release should potentially activate neutrophils releasing myeloperoxidase (MPO) and subsequently generating "nitrosative stress". We studied bradykinin-induced expression of bradykinin B(2) receptor and bradykinin- and lipopolysaccharide (LPS)-induced IL-8 release, MPO (marker of neutrophil activation) and 3-nitrotyrosine (3-NT; marker of "nitrosative stress") production in human bronchial epithelial cells BEAS-2B alone or in co-culture with human neutrophils. We evaluated B(2) receptor protein expression in BEAS-2B cells by immunostainings and Western blot analysis, and measured respectively bradykinin- or LPS-induced IL-8 release in BEAS-2B cells and bradykinin- and/or LPS-induced MPO and 3-NT production in BEAS-2B cells co-cultured with human neutrophils by ELISA. In addition, we evaluated bradykinin- and/or LPS-induced 3-NT formation in BEAS-2B cells co-cultured with human neutrophils by immunocytochemistry. Bradykinin up-regulates B(2) receptor expression (P<0.05) and stimulate IL-8 release (P<0.001) in BEAS-2B cells. Either the selective bradykinin B(2) receptor antagonist HOE 140 or the selective bradykinin B(1) receptor antagonist Lys-(des-Arg(9), Leu(8))-bradykinin alone halved IL-8 release and the combination of both drugs suppressed this effect. In BEAS-2B cells co-cultured with human neutrophils bradykinin increased MPO release and 3-NT production compared to BEAS-2B cells with human neutrophils (P<0.001), and the addition of LPS in BEAS-2B cells with human neutrophils and bradykinin induced a further dramatically increase of MPO release and 3-NT formation (P<0.001). Bradykinin and LPS provoked "nitrosative stress", potentially mediated by IL-8, in bronchial epithelium co-cultured with neutrophils suggesting a role for bradykinin in the amplification of chronic airway inflammation via production of "nitrosative stress".
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http://dx.doi.org/10.1016/j.ejphar.2012.07.051DOI Listing
November 2012

Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway.

Lab Invest 2012 Aug;92(8):1140-8

Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy.

Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-a, IL-4 and IFN-g to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as G(T) values; (d) epidermal growth factor receptor (EGFR)-dependent mitogenactivated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-a (47±3%), IL-4 (43±1%) and INF-g (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. G(T) values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm²)1000 in control cultures and in those exposed to TNF-a, IFN-g and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 μM) or a MAP kinase inhibitor (U0126, 25 μM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.
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http://dx.doi.org/10.1038/labinvest.2012.67DOI Listing
August 2012

A mixture of bacterial mechanical lysates is more efficient than single strain lysate and of bacterial-derived soluble products for the induction of an activating phenotype in human dendritic cells.

Immunol Lett 2011 Jul 29;138(1):86-91. Epub 2011 Mar 29.

Laboratorio di Immunologia, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.

Dendritic cells (DCs), following an optimal maturation, are able to drive an efficient immune-response. For this, both co-stimulatory molecules (CD80 and CD86), activation molecules (CD83) and peptide presenting molecules (HLA) are over-expressed. The in vitro treatment of immature DC with fragments of bacterial strains, obtained by using a mechanical lysis as well as with bacterial-derived molecules (such as lipopolysaccharide and protido-glycan), induced the maturation of DCs and the secretion of a panel of cytokines and chemokines. Of note, ex vivo treated circulating DCs and plasmacytoid DCs were also activated by these bacterial bodies. However, while the particulate fraction of single bacterial strains or soluble bacterial-derived molecules induced a sub-optimal maturation (as evaluated by the expression of an activating phenotype on DCs and the amount of cytokine secretion), the addition of the mixture of the particulate fractions of the different bacterial strains was able to mediate an optimal maturation. These results were also confirmed by using the secretion of both cytokines and chemokines as markers of DC activation. All these findings suggest that the particulate fraction of bacterial lysate mixtures, because of their ability to interact with different surface structures, might be exploited not only as an immunogen, but also as an adjuvant treatment to boost an immune-response to poorly "antigenic" proteins, such as cancer antigens or allergens.
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http://dx.doi.org/10.1016/j.imlet.2011.03.006DOI Listing
July 2011

High frequency of development of B cell lymphoproliferation and diffuse large B cell lymphoma in Dbl knock-in mice.

J Mol Med (Berl) 2011 May 11;89(5):493-504. Epub 2011 Jan 11.

Laboratory of Molecular Biology, G. Gaslini Institute, Largo Gaslini 5, Genoa, Italy.

Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.
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http://dx.doi.org/10.1007/s00109-010-0712-4DOI Listing
May 2011

A dietary tomato supplement prevents prostate cancer in TRAMP mice.

Cancer Prev Res (Phila) 2010 Oct 17;3(10):1284-91. Epub 2010 Aug 17.

Department of Oncology and Neurosciences, G. d'Annunzio University, Chieti, Italy.

Transgenic adenocarcinoma of the mouse prostate (TRAMP) is a model for progressive prostate cancer that mirrors the stages of the human form. In this study, the effects of a diet enriched with processed whole tomatoes on survival, tumorigenesis, and progression of prostate cancer, and the antioxidant and inflammatory status of TRAMP mice were investigated. Tomato diet significantly increased overall survival (P < 0.01), delayed progression from prostatic intraepithelial neoplasia to adenocarcinoma, and decreased the incidence of poorly differentiated carcinoma. Biochemical data disclosed an increase in serum antioxidant activity and a reduction of serum inflammation/angiogenesis biomarkers of particular importance in prostate carcinogenesis.
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http://dx.doi.org/10.1158/1940-6207.CAPR-09-0237DOI Listing
October 2010

Bronchial airway epithelial cell damage following exposure to cigarette smoke includes disassembly of tight junction components mediated by the extracellular signal-regulated kinase 1/2 pathway.

Chest 2009 Jun 15;135(6):1502-1512. Epub 2009 May 15.

Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy.

Background: Through a variety of biochemical mechanisms, cigarette smoke (CS) may damage airway epithelium, altering its normal structure and function. Injury to epithelium may include changes in tight junction (TJ) integrity with impairment of epithelial barrier function.

Methods And Results: To study the effect of the exposure to CS condensate (CSC) on TJ integrity, two human bronchial epithelial cell lines (HBECs), BEAS-2B and 16HBE14o-, were used. Exposure of the two HBECs to CSC resulted in a time-dependent and concentration-dependent disassembly of TJs, which were already detectable at 24 h at all the CSC concentrations tested (5%, 10%, and 20%), associated with changes in cell shape, suggesting cell damage. However, a significant inhibition of cell growth and an increase in DNA fragmentation were detected only at the highest CSC concentration tested (20%) at 48 and 72 h, respectively. The involvement of epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase (ERK) 1/2 cascade in CSC-induced damage was shown by the observation that exposure to CSC (5%) induced a marked phosphorylation of ERK1/2, already detectable after 5-min incubation and confirmed by the demonstration that not only ERK1/2 phosphorylation but also CSC-induced TJ disassembly and DNA fragmentation were partially inhibited by a mitogen-activated protein kinase kinase inhibitor (U0126) and completely blocked by a EGFR inhibitor (AG1478).

Conclusion: CSC-induced damage to airway epithelium includes disassembly of TJs, modulated through the EGFR-ERK1/2 signaling pathway.
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http://dx.doi.org/10.1378/chest.08-1780DOI Listing
June 2009

Modulation of human lung fibroblast functions by ciclesonide: evidence for its conversion into the active metabolite desisobutyryl-ciclesonide.

Immunol Lett 2007 Sep 1;112(1):39-46. Epub 2007 Aug 1.

Pulmonary Disease Unit, G. Gaslini Institute, Largo G. Gaslini 5, 16147 Genoa, Italy.

Background: Ciclesonide, an inhaled corticosteroid administered as inactive compound with almost no binding affinity for the glucocorticoid receptor, is clinically effective in asthma being converted by airway epithelial cells into its active metabolite desisobutyryl-(des)-ciclesonide.

Aim: To evaluate whether ciclesonide could directly modulate in vitro bronchial fibroblast functions being converted into des-ciclesonide by these pluripotent cells involved in the regulation of airway inflammation and remodelling.

Methods: Ciclesonide (0.09-9.0 microM) was added to a human adult lung fibroblast cell line (CCL-202), seeded in medium in the presence of the following cytokines and growth factors: (a) basic fibroblast growth factor (bFGF) for cell proliferation, measured by tritiated thymidine ([3H]TdR) incorporation; (b) tumour necrosis factor (TNF)-alpha, to stimulate intercellular adhesion molecule (ICAM)-1 expression and monocyte chemoattractant protein-1 (MCP-1) and eotaxin release, evaluated by flow cytometry and ELISA, respectively; (c) transforming growth factor (TGF)-beta1, for induction of alpha smooth muscle actin (alpha-SMA) protein expression and modification of the organization of alpha-SMA stress fibres, evaluated by Western blot analysis and fluorescence microscopy.

Results: The presence of ciclesonide in cell cultures induced a significant downregulation of: (a) bFGF-induced fibroblast proliferation and TNF-alpha-induced ICAM-1 expression, at the 0.3-9.0 microM concentrations (p<0.05); (b) TNF-alpha-induced MCP-1 release, at all the concentrations tested (p<0.05); (c) TNF-alpha-induced eotaxin release, at the three highest concentrations (0.9-9.0 microM) (p<0.05); (d) TGF-beta1-induced of alpha-SMA protein expression at the 0.3-3.0 microM concentrations, associated with a reduction in the organization of alpha-SMA stress fibres.

Conclusions: These data show at cellular level an effective anti-inflammatory activity of ciclesonide on human lung fibroblasts and support the hypothesis that also these cells, in addition to airway epithelial cells, may be involved in converting the parental compound into its active metabolite in the airways.
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http://dx.doi.org/10.1016/j.imlet.2007.06.010DOI Listing
September 2007

Inhibition of TGF-beta induced lung fibroblast to myofibroblast conversion by phosphodiesterase inhibiting drugs and activators of soluble guanylyl cyclase.

Eur J Pharmacol 2007 Oct 29;572(1):12-22. Epub 2007 Jun 29.

Department of Biochemistry Inflammation, ALTANA Pharma AG, Byk-Gulden-Str.2, 78467 Konstanz, Germany.

Pulmonary fibroblast to myofibroblast conversion is a pathophysiological feature of idiopathic pulmonary fibrosis and COPD. This conversion is induced by transforming growth factor (TGF)-beta derived from epithelial cells as well as activated macrophages that have infiltrated the lung. Preventing this conversion might be a favourable therapeutic approach. Within this study we examined the activity of different members of the phosphodiesterase (PDE) family in primary human lung fibroblasts and various lung fibroblast cell lines both before and after TGF-beta induced differentiation to myofibroblasts as reflected by the expression of alpha-smooth muscle actin. We showed that the predominant PDE activities in lung fibroblasts are attributed to PDE5, PDE1 and to a smaller extent to PDE4. cyclic GMP (cGMP)-hydrolyzing activity declines by about half after differentiation to myofibroblasts in all pulmonary fibroblasts investigated, which is accompanied by a down-regulation of PDE5 protein. Lung fibroblast to myofibroblast differentiation is blocked by treatment with the PDE4 inhibitor piclamilast alone, depending on the TGF-beta concentration applied, and in combination with prostaglandin E(2) (PGE(2)) in a synergistic manner. Despite the high PDE5 activity the PDE5 inhibitor sildenafil by itself as well as in combination with brain natriuretic peptide or the nitric oxide-donor DETA-NONOate shows no inhibiting effects. However, combining sildenafil with the guanylyl cyclase (GC) activator BAY58-2667 and ODQ (which sensitizes GC for activation by BAY58-2667) suppressed TGF-beta induced differentiation. In summary, our data indicate that drugs interfering with the cyclic AMP (cAMP)-as well as with the NO-cGMP-pathway offer the therapeutic opportunity to prevent the differentiation of pulmonary fibroblasts to myofibroblasts in lung fibrosis.
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http://dx.doi.org/10.1016/j.ejphar.2007.06.036DOI Listing
October 2007

The histamine-induced enhanced expression of vascular cell adhesion molecule-1 by nasal polyp-derived fibroblasts is inhibited by levocetirizine.

Am J Rhinol 2006 Sep-Oct;20(5):445-9

Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy.

Background: Histamine, a key chemical mediator in allergic reaction, exhibits an array of pro-inflammatory effects that include the activation of fibroblasts. The aim of this study was to evaluate whether histamine could stimulate nasal polyp-derived fibroblasts to express vascular cell adhesion molecule (VCAM)-1, a surface molecule involved in structural-inflammatory cell interaction and whether levocetirizine could inhibit this induction.

Methods: Primary nasal polyp tissue-derived fibroblasts were stimulated with histamine (10-1000 microM) or interleukin (IL)-4 plus tumor necrosis factor (TNF)-alpha (0.5-5 ng/mL) and VCAM-1 expression was evaluated by flow cytometry analysis. The inhibitory effect of the selective H1-antagonist levocetirizine (0.01-10.0 microM) on VCAM-1 expression was also tested.

Results: Compared with unstimulated cultures, histamine or IL-4 + TNF-alpha, at the highest concentrations tested, significantly increase VCAM-1 expression (p < 0.05). To evaluate the ability of levocetirizine to downregulate VCAM-1 expression, fibroblasts were stimulated with histamine (1000 microM) or IL-4 + TNF-alpha (5 ng/mL), in the presence of the drug (0.01-10.0 microM). The histamine-induced VCAM-1 expression was effectively inhibited by levocetirizine (0.1-10.0 microM) (p < 0.05). No effect of the drug on IL-4 + TNF-alpha-induced VCAM-1 expression was observed.

Conclusions: Histamine upregulates VCAM-1 expression on nasal polyp-derived fibroblasts and this phenomenon, relevant to allergic late-phase inflammation, is effectively inhibited by levocetirizine.
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http://dx.doi.org/10.2500/ajr.2006.20.0796DOI Listing
January 2007

The lung and the gut: common origins, close links.

Paediatr Respir Rev 2006 6;7 Suppl 1:S235-9. Epub 2006 Jun 6.

Pulmonary and Allergy Units, G. Gaslini University Hospital, Genoa, Italy.

Because of the common embryologic origin, alteration of the many factors modulating the development of the alimentary and the respiratory tract often results in structural abnormalities involving the two systems. Indeed, some of the most common embryologic disorders of the airways are frequently associated with anomalies of the gastrointestinal tract. Lung growth occurs as a series of tightly regulated events, depending on a number of factors, including developmental, genetic and environment ones. Abnormalities of any of these factors may causes developmental alterations of the lung leading to a group of disorders termed "bronchopulmonary-foregut malformations". These are usually sporadic, solitary cystic hamartomas, involving conducting airways, arteries, venous drainage, and lung parenchyma, which are now often discovered on routine prenatal sonography. While some lesions may be large and cause serious complications in the foetus or newborn, many will be asymptomatic at birth, raising controversy about management: simple observation or surgery and, if so, at what age? Over the past two decades molecular studies have started to shed light on the complex series of events that control proper formation of the lung, with the hope that a better understanding of the molecular basis of pulmonary maturation will allow the design of new therapeutic strategies.
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http://dx.doi.org/10.1016/j.prrv.2006.04.192DOI Listing
October 2006

The effect of transforming growth factor (TGF)-beta1 and (TGF)-beta2 on nasal polyp fibroblast activities involved upper airway remodeling: modulation by fluticasone propionate.

Immunol Lett 2006 May 30;105(1):61-7. Epub 2006 Jan 30.

Pulmonary Disease Unit, G. Gaslini Institute, Largo G. Gaslini 4, 16148 Genoa, Italy.

Transforming growth factor (TGF)-beta may play a significant role in nasal polyposis pathogenesis, possibly through fibroblast activation. We studied the effects of two TGF-beta isoforms (TGF-beta1 and TGF-beta2) on nasal polyposis fibroblasts by evaluating cell proliferation and differentiation into myofibroblasts. In addition, the inhibitory activity of different concentrations of fluticasone propionate (F.P.) was tested in this in vitro system. Primary nasal polyp tissue-derived fibroblasts were stimulated with different concentrations (1, 10 and 20 ng/ml) of TGF-beta1 and TGF-beta2 for different incubation periods (24, 48 and 72 h) and cell proliferation [3H thymidine ([3H]TdR) incorporation] and alpha-smooth muscle actin (alpha-SMA) expression (immunocytochemistry) was evaluated. The lowest concentration of TGF-beta1 (1 ng/ml) induced a significant increase in [3H]TdR incorporation at 48 and 72 h (p<0.05, each comparison), while in the presence of TGF-beta (10 ng/ml) and TGF-beta2 (1 ng/ml) the enhancement in cell proliferation was significant only after 48 h (p<0.05, each comparison with the unstimulated cells). In contrast, a significant increase in alpha-SMA expression was observed in the presence of the two highest concentration of both TGF-beta isoforms, at 48 and 72 h for TGF-beta1 (p<0.05, each comparison), but only at 72 h for TGF-beta2 (<0.05, each comparison). Finally, at all concentrations tested, F.P. significantly inhibited the TGF-beta1 and TGF-beta2-induced 3HTdR incorporation (p<0.01, each comparison) and the alpha-SMA expression (p<0.05, each comparison). Thus, in vitro different concentrations of TGF-beta1 and TGF-beta2 appear to sequentially stimulate primary nasal polyp tissue-derived fibroblast proliferation and myofibroblast differentiation. These activities are effectively inhibited by F.P.
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http://dx.doi.org/10.1016/j.imlet.2006.01.003DOI Listing
May 2006

Reactive nitrogen species in the respiratory tract.

Eur J Pharmacol 2006 Mar 7;533(1-3):240-52. Epub 2006 Feb 7.

Unit of Pneumology, IRCCS Gaslini Institute, Largo G Gaslini 5, 16147 Genoa, Italy.

Endogenous Nitric Oxide (NO) plays a key role in the physiological regulation of airway functions. In response to various stimuli activated inflammatory cells (e.g., eosinophils and neutrophils) generate oxidants ("oxidative stress") which in conjunction with exaggerated enzymatic release of NO and augmented NO metabolites produce the formation of strong oxidizing reactive nitrogen species, such as peroxynitrite, in various airway diseases including asthma, chronic obstructive pulmonary diseases (COPD), cystic fibrosis and acute respiratory distress syndrome (ARDS). Reactive nitrogen species provoke amplification of inflammatory processes in the airways and lung parenchyma causing DNA damage, inhibition of mitochondrial respiration, protein dysfunction and cell damage ("nitrosative stress"). These effects alter respiratory homeostasis (such as bronchomotor tone and pulmonary surfactant activity) and the long-term persistence of "nitrosative stress" may contribute to the progressive deterioration of pulmonary functions leading to respiratory failure. Recent studies showing that protein nitration can be dynamic and reversible ("denitration mechanisms") open new horizons in the treatment of chronic respiratory diseases affected by the deleterious actions of "nitrosative stress".
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http://dx.doi.org/10.1016/j.ejphar.2005.12.057DOI Listing
March 2006

Activity of budesonide on nasal neutrophilic inflammation and obstruction in children with recurrent upper airway infections. A preliminary investigation.

Int J Pediatr Otorhinolaryngol 2006 Mar 2;70(3):445-52. Epub 2005 Sep 2.

U.O.C. di Pneumologia, I.R.C.C.S. G. Gaslini, Istituto G. Gaslini, Largo G. Gaslini, 5, 16147 Genoa, Italy.

Objective: While it is widely accepted that inhaled glucocorticosteroids represent an effective treatment for allergic rhinitis, little is known on the specific effects of this therapeutic approach in other upper airway disorders of childhood. The aim of the study was to evaluate the improvement of clinical symptoms and changes in local cellular inflammatory reaction induced by budesonide inhalation suspension in children with recurrent nasal infections using budesonide inhalation suspension delivered by Rinowash, a nebulizer designed to treat upper airway structures.

Methods: In a randomized, controlled-open study, 14 children (5.88+/-0.56 years of age) with recurrent upper airway infections and chronic nasal obstruction were enrolled and randomly treated for 7-10 days either with budesonide inhalation suspension (250 microg/bidie) (nine patients) or with saline solution (five patients). Before and after treatment, inflammatory cells in nasal brushing and nasal symptom score were evaluated.

Results: Out of the nine patients treated with budesonide, two were excluded from the analysis because of acute respiratory infections requiring systemic antibiotic treatment. A significant decrease in nasal brushing neutrophil percentage was observed after treatment with budesonide (P=0.016) but not after saline solution treatment (P=1.00). No significant changes in nasal brushing mononuclear cell or eosinophil proportions were observed after treatment with budesonide inhalation suspension or saline solution (P=NS, each comparison). Treatment with budesonide, but not with saline solution, was associated with a significant reduction in nasal obstruction (P=0.016).

Conclusions: These preliminary data indicate that short-term treatment with budesonide inhalation suspension, used for an indication out of label, may significantly reduce local neutrophilic inflammation and nasal obstruction in children with recurrent upper airway infections.
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http://dx.doi.org/10.1016/j.ijporl.2005.07.015DOI Listing
March 2006

IL-8 and airway neutrophilia in children with gastroesophageal reflux and asthma-like symptoms.

Respir Med 2006 Feb 5;100(2):307-15. Epub 2005 Jul 5.

U.O.C. di Pneumologia, Istituto G. Gaslini, Largo G. Gaslini 5, Genova 16147, Italy.

Gastroesophageal reflux (GER) may induce respiratory symptoms (RS) through inhalation of acid gastric contents. To characterize the airway inflammation associated with this condition, 20 children [7.4 (0.9) yr old] with "difficult to treat" RS and a positive 24-h oesophageal pH monitoring (pHm) were studied and bronchoalveolar lavage (BAL) performed. The control group included 10 children [7.3 (1.3) yr], non-atopics, with a respiratory clinical history similar to the cases but no reflux, as demonstrated by a negative 24-h oesophageal pHm. On BAL samples, in addition to inflammatory indexes, the lipid-laden macrophage (LLM) index was determined as index of gastric content inhalation. As compared to controls, GER children had higher neutrophil proportion (P=0.002), higher LLM index (P=0.004) and higher concentrations of interleukin (IL)-8 (P=0.005), myeloperoxidase (MPO) (P=0.001) and elastase (P=0.045) in BAL fluid. In GER children, but not in controls, neutrophil proportion significantly correlated with LLM index (r=0.65, P=0.002), with IL-8 (r=0.62, P=0.003) and MPO levels (r=0.54, P=0.014) but not with elastase concentrations. These results suggest an active pathogenetic role of IL-8 in the recruitment and activation of neutrophils in the airways of children with GER, respiratory symptoms and BAL findings suggestive of gastric content aspiration.
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http://dx.doi.org/10.1016/j.rmed.2005.05.011DOI Listing
February 2006

Human bronchial fibroblasts exhibit a mesenchymal stem cell phenotype and multilineage differentiating potentialities.

Lab Invest 2005 Aug;85(8):962-71

Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy.

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate along different pathways including chondrogenic, osteogenic and adipogenic lineages. MSCs with a fibroblast-like morphology have been identified in human fetal lung. However, their frequency and characterization in human adult lung have not been yet evaluated. Therefore, we analyzed the mesenchymal phenotype and differentiation ability of cultured human adult bronchial fibroblast-like cells (Br) in comparison with those of mesenchymal cell progenitors isolated from fetal lung (ICIG7) and adult bone marrow (BM212) tissues. Surface immunophenotyping by flow cytometry revealed a similar expression pattern of antigens characteristic of marrow-derived MSCs, including CD34 (-), CD45 (-), CD90/Thy-1 (+), CD73/SH3, SH4 (+), CD105/SH2 (+) and CD166/ALCAM (+) in Br, ICIG7 and BM212 cells. There was one exception, STRO-1 antigen, which was only weakly expressed in Br cells. Analysis of cytoskeleton and matrix composition by immunostaining showed that lung and marrow-derived cells homogeneously expressed vimentin and nestin proteins in intermediate filaments while they were all devoid of epithelial cytokeratins. Additionally, alpha-smooth muscle actin was also present in microfilaments of a low number of cells. All cell types predominantly produced collagen and fibronectin extracellular matrix as evidenced by staining with the monoclonal antibodies to collagen prolyl 4-hydroxylase and fibronectin isoforms containing the extradomain (ED)-A together with ED-B in ICIG7 cells. Br cells similarly to fetal lung and marrow fibroblasts were able to differentiate along the three adipogenic, osteogenic and chondrogenic mesenchymal pathways when cultured under appropriate inducible conditions. Altogether, these data indicate that MSCs are present in human adult lung. They may be actively involved in lung tissue repair under physiological and pathological circumstances.
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http://dx.doi.org/10.1038/labinvest.3700300DOI Listing
August 2005

Concentration-dependent activity of mometasone furoate and dexamethasone on blood eosinophils isolated from atopic children: modulation of Mac-1 expression and chemotaxis.

Int Immunopharmacol 2004 Dec;4(13):1687-96

Pulmonary Disease Unit, G. Gaslini Institute, Genoa, 16147 Genoa, Italy.

Treatment of asthma with corticosteroids results in downregulation of eosinophilic airway inflammation. We evaluated in vitro the activity of an "inhaled" corticosteroid, mometasone furoate (MF), and of a "systemic" corticosteroid, dexamethasone (DEX), on eosinophil functions, i.e. adhesion molecule expression and cell chemotaxis. Partially purified blood eosinophils were obtained from 18 asthmatic subjects sensitized to house dust mites. The expression of the macrophage antigen (Mac)-1 (CD11b/CD18) was measured by specific monoclonal antibody (mAb) staining and flow cytometry analysis at baseline or after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or with recombinant human (rh) granulocyte macrophage-colony stimulating factor (GM-CSF) plus a mAb anti-human (ah) IgE low affinity receptor [FcepsilonRII or CD23]. Cell chemotaxis toward the complement fragment 5a (C5a) or rh interleukin (IL)-5 was evaluated in Boyden microchambers by light microscopy. Eosinophils showed a significant increase in Mac-1 expression after activation with fMLP or with rh GM-CSF plus ah CD23 mAbs (p<0.05, each comparison) and a remarkable chemotactic response to both C5a or rh IL-5 (p<0.001, each comparison). To test the inhibitory activity of MF and DEX on eosinophil functions, the cells were preincubated for 3 h with four concentrations (0.1, 1, 10 and 100 nM) of each of the two drugs, before being activated by fMLP or by rh GM-CSF plus ah CD23 mAbs or tested with C5a or with rh IL-5. Independently of the stimulus used, both Mac-1 expression and eosinophil migration were effectively downregulated by preincubation with MF or DEX at 1, 10 and 100 nM (p<0.05). The inhibitory activity on cell chemotaxis in response to both C5a or with rh IL-5 was higher for MF than DEX, but only at the highest concentration tested (p<0.05, each comparison). These data demonstrate that concentrations of MF similar to those obtained in vivo are highly effective in inhibiting eosinophil functions involved in airway inflammation.
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http://dx.doi.org/10.1016/j.intimp.2004.07.029DOI Listing
December 2004

The wheezy infant -- immunological and molecular considerations.

Paediatr Respir Rev 2004 ;5 Suppl A:S81-7

Pulmonary Division, G. Gaslini Institute, 16148 Genoa, Italy.

Most of the data on the pathogenesis of asthma is based on information obtained through bronchial biopsies and bronchoalveolar lavage in adults and young adults. Ethical considerations linked to the invasive nature of airway endoscopy have limited the studies on the pathophysiology of asthma in infancy and early childhood. Although there is evidence that an asthma-like inflammation, with increased inflammatory cells and thickening of the lung basement membrane, may be present also at a very early age, clinical and epidemiologic studies suggest that asthma manifestations in preschool children may significantly differ from those observed in older subjects. In western countries, the vast majority of infants and young children has episodic (or intermittent) asthma, and the exacerbations generally defined "wheezing episodes" occur more frequently with a seasonal pattern being usually related to acute viral infections. There is strong epidemiological evidence that approximately 2/3 of all children who wheeze because of viral infections in early life (and are not atopic) have a transient condition that tends to disappear during early school years. All respiratory viruses may be implicated in the wheezing episodes, the principal being respiratory syncytial virus (RSV) and, with a lower frequency, adenovirus and parainfluenza viruses during the first 3 years of life, and rhinoviruses after that age. Infants and preschool children have on average 6-8 "colds" per year, but the illness tends to be limited to the upper respiratory tract alone in a considerable proportion of individuals, without causing symptomatic involvement of the lower respiratory tract. The variety of factors determining the different outcomes are only partially known, but complex interactions between the intrinsic pathogenicity of the virus and host factors, including the socio-economic conditions of the family, are central to define the type of manifestations and the severity of the process.
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http://dx.doi.org/10.1016/s1526-0542(04)90016-4DOI Listing
May 2004