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Supplementary Figure 1 from A Dietary Tomato Supplement Prevents Prostate Cancer in TRAMP Mice

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Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) represents a valuable alternative for children with nonmalignant disease and ex vivo negative selection of TCR-αβ

PurposeSurvival rates of Childhood Cancer Patients have improved tremendously over the past four decades. However, cancer treatments are associated with an increased risk of developing an anticipated onset of chronic diseases typical of aging. Thus, we aimed to identify molecular/metabolic cellular alterations responsible for early aging in Childhood Cancer Survivors (CCS).Patients and MethodsBiochemical, proteomic and molecular biology analyses were conducted on mononuclear cells (MNCs) isolated from peripheral blood of 196 CCS, comparing the results with those obtained on MNCs of 154 healthy subjects.ResultsData demonstrate that CCS-MNCs show: i) inefficient oxidative phosphorylation associated with low energy status and a metabolic switch to lactate fermentation compared with age-matched normal controls; ii) increment of lipid peroxidation due to an unbalance among the oxidative stress production and the activation of the antioxidant defenses; (iii) significantly lower expression of genes and proteins involved in mitochondrial biogenesis and metabolism regulation, such as CLUH, PGC1-α, and SIRT6 in CCS, not observed in the age-matched healthy or elderly subjects. The application of a mathematical model based on biochemical parameters predicts that CCS have a biological age significantly increased by decades compared to the chronological age. Overall, the results show that the impact of chemo/chemoradiotherapy on mitochondria efficiency in 196 CCS was rather homogeneous, irrespective of cancer type, treatment protocols, and time elapsed from the end of the curative period.ConclusionsOur study identifies some biochemical and molecular alterations possibly contributing to the pathophysiology of anticipated aging and metabolic deficiency described in CCS. These results may be useful in identifying approaches to restore the mitochondrial function, slowing down the aging and the associated pathological conditions in CCS.

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.

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.

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.

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.

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (

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.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.In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A(+) cell numbers were higher than old control non-smokers (p0.05). 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 (p0.01). Angiogenin(+) cell numbers were higher in patients with COPD than both old control groups (p0.05). 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) (p0.01). 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.Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.

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 B 2 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 B 2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B 2 receptor expression in HBAFb. Bradykinin, via bradykinin B 2 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 B 2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B 2 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.

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

// Meriem Hasmim 1, 2, * , Stefania Bruno 3, * , Sandy Azzi 2 , Cindy Gallerne 2 , Julien Giron Michel 2 , Giulia Chiabotto 4 , Vincent Lecoz 2 , Cristina Romei 5 , Grazia Maria Spaggiari 5 , Annalisa Pezzolo 6 , Vito Pistoia 6 , Eric Angevin 1, 7 , Sophie Gad 1, 8 , Sophie Ferlicot 1, 9 , Yosra Messai 1 , Claudine Kieda 10 , Denis Clay 11 , Federica Sabatini 12 , Bernard Escudier 1, 7 , Giovanni Camussi 4 , Pierre Eid 2 , Bruno Azzarone 5 , Salem Chouaib 1 1 INSERM U 1186, Equipe labellisee Ligue Contre le Cancer, Gustave Roussy Campus, Villejuif, France 2 INSERM UMR 1014, Lavoisier Building, Paul Brousse Hospital, Villejuif, France 3 Department of Molecular Biotechnology and Healthy Science, Molecular Biotechnology Center, University of Torino, Turin, Italy 4 Department of Medical Science, University of Torino, Medical School, Torino, Italy 5 DIMES, UNIGE, Genova, Italy 6 Laboratory of Oncology Giannina Gaslini Institute, Genoa, Italy 7 Medical Oncology Department, Gustave Roussy Campus, Villejuif, France 8 Laboratoire de Genetique Oncologique EPHE, Ecole Pratique des Hautes Etudes, Paris, France 9 Universite Paris-Sud, Assistance Publique-Hopitaux de Paris, Service d'Anatomo-Pathologie, Hopital Bicetre, Le Kremlin-Bicetre, France 10 Centre de Biophysique Moleculaire, CNRS UPR 4301, Orleans, France 11 INSERM UMR 972, Paul Brousse Hospital, Villejuif, France 12 Stem Cell and Cell Therapy Laboratory, Istituto G. Gaslini, Genoa, Italy * These authors have contributed equally to this work Correspondence to: Salem Chouaib, e-mail: salem.chouaib@gustaveroussy.fr Bruno Azzarone, e-mail: bazzarone@hotmail.com Keywords: clear cell renal cell carcinoma, cancer stem cells, patient-derived xenografts, CD133, EpCAM Received: July 24, 2015 Accepted: October 08, 2015 Published: November 02, 2015 ABSTRACT 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 CD133 high /CD105-/Ecad low and PDX-2 cells were CD133 low /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-/EpCAM low and RCC-41-PDX-2/CD133 + /EpCAM bright ). 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.

Background: Pain and vasomotor symptoms are common side effects in breast cancer patients. Traditional Chinese medicine (TCM) may be an option of care for these side effects in a multidisciplinary pathway. Aim: The purpose of this study was to evaluate if TCM is safe and effective in hot flashes and pain of women affected by breast cancer. Materials and Methods: In the Centre Fior di Prugna of the Local Unit Tuscany centre from January 2012 to August 2016 128 questionnaires were analyzed. These were administered to 115 women with breast cancer (operated and/or undergoing chemotherapy, and/or radiotherapy and/or hormonal treatment who had hot flashes or pain or both the symptoms). All the women received acupuncture according to a pre-specified protocol including plum blossom needle, somatic acupuncture and auriculotherapy. The effectiveness in hot flashes was evaluated by the questionnaires Hot Flash Score (HFS) and that in the pain at rest and during movement by the questionnaires with the verbal scale Present Pain Intensity (PPI) and Numeric Pain Intensity Scale (NRS) Results: No relevant side effects were registered. All the outcome indices have significantly improved. The average number of hot flashes in 24 hours changed from 14.2 before treatment to 7.52 at the end (p value

Introduction: Pain in musculoskeletal disorders is a common problem that involves high social and individual costs. Systematic reviews and randomized controlled trials show that a number of treatments usually applied in this field are not effective. On the other hand, there is clinical evidence of the effectiveness of physical exercise, acupuncture and auriculotherapy. Side effects of current medication limit their use. Aim: To evaluate the effectiveness of auriculotherapy on the pain of patients suffering from chronic muscoloskeletal disorders (non-oncological pain). Methods: From February 2010 to April 2017, in the Centre of Traditional Chinese Medicine Fior di Prugna - Local Health Unit Tuscany centre, 1,180 questionnaires were analyzed. After the first visit with anamnesis and objective examination, patients underwent 6/8 sessions of auriculotherapy, once a week, according to a pre-established protocol that included the search for painful points in the ear through baresthesic diagnosis and adding general points (functional and psychic points). Points in the ear were treated unilaterally by needles; afterwards permanent Vaccaria seeds or press needles were applied and left in situ for a week. The effectiveness on pain at rest and during movement was evaluated by verbal scale Present Pain Intensity (PPI) and Numeric Pain Intensity Scale (NRS) self-administered before and at the end of treatment. Results: Outcome indices have significantly improved. Based on the PPI questionnaire, auriculotherapy reduced pain both at rest and during movement. Patients with strong and atrocious pain decreased from 231 to 45 and 604 to 163, respectively at rest and during movement. The median value of the NRS decreased from 5 to 2 at rest and from 7 to 4 during movement. Conclusions: Auriculotherapy can be effective to reduce pain in musculoskeletal disorders, also in combination with usual care. Randomized controlled trials are needed to verify the efficacy of this protocol of treatment.

The PDE4 inhibitor roflumilast mitigates bleomycin-induced lung fibrotic remodeling in rodents. In the current study it was explored whether roflumilast N-oxide, the active metabolite of roflumilast influences functions of cultured lung fibroblasts. Cells of the human foetal lung fibroblast strain GM06114 were stimulated with TNF-alpha (5 ng ml(-1)) and cell surface ICAM-1 and eotaxin release were assessed. [methyl-(3)H] thymidine incorporation was measured following stimulation with bFGF (10 ng ml(-1)). alpha-Smooth muscle actin (protein), CTGF (mRNA) and fibronectin (mRNA) were determined secondary to TGFbeta1 (1 ng ml(-1)). In the presence of PGE(2) (1 nM), roflumilast N-oxide reduced TNF-alpha-induced ICAM-1 and eotaxin by about 70% and90% with half-maximum inhibition at 0.9 nM and 0.5 nM, respectively. Roflumilast N-oxide also attenuated [methyl-(3)H] thymidine incorporation secondary to bFGF by about 75% with half-maximum inhibition at 0.7 nM when cells were co-incubated with IL-1beta (10 pg ml(-1)). In the presence of this cytokine roflumilast N-oxide (1 microM) diminished TGFbeta1-induced expression of alpha-smooth muscle actin and transcripts of CTGF and fibronectin. In addition, IL-1beta up-regulated PDE4 activity in the lung fibroblasts. Taken together, these findings indicate that roflumilast N-oxide directly targets human lung fibroblasts, which may at least partially explain the efficacy of roflumilast to mitigate a pulmonary fibrotic response in vivo.

Bradykinin (BK) induces fibroblast contraction but the structural changes and intracellular mechanisms involved have not been completely explored. We stimulated HFL-1 fibroblasts with BK to assess: 1) fibroblast contractility; 2) the role of alpha-smooth muscle actin (SMA) in contraction by small interfering RNA (siRNA); 3) alpha-SMA protein expression; 4) alpha-SMA and F-actin structure; 5) intracellular calcium concentration; and 6) phosphorylated myosin light-chain (pMLC) and MLC kinase (MLCK) expression. BK triggered concentration- and time-dependent fibroblast gel contraction in conjunction with alpha-SMA over expression, but not in alpha-SMA-siRNA-treated cells. BK also increased alpha-SMA(+) and F-actin(+) cell number and stress fibre polymerisation (detectable at 5-60 min). These BK-induced changes were associated with an increase in intracellular calcium concentration, which peaked within 15 s, and activation of pMLC, which was detectable at 5-60 min. No MLCK content modification was observed. The different manifestations of the BK-induced fibroblast activation were downregulated at different levels (25-100%) by HOE140, a specific BK B2 receptor (B2R) antagonist and by the Ca(2+) chelator, EGTA. Thus, BK-induced fibroblast contraction, associated with differentiation into alpha-SMA(+) myofibroblasts, is mediated through the activation of the B2R and involves the Ca(2+)/calmodulin pMLC-dependent pathway.

Aim: 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). Methods: 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. Results: 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. Conclusions: The combination of SEA and FMEA can reveal potential risks for patients and suggest actions for safer and more reliable services in CM.

BACKGROUND: Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease. OBJECTIVES: To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I-IV) compared with age-matched control subjects, smokers with normal lung function and never smokers. METHODS: The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB). RESULTS: The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2-15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD. CONCLUSION: The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

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.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.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.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 (p0.05); (b) TNF-alpha-induced MCP-1 release, at all the concentrations tested (p0.05); (c) TNF-alpha-induced eotaxin release, at the three highest concentrations (0.9-9.0 microM) (p0.05); (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.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.

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., Podesta, 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.

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 μM) or interleukin (IL)-4 plus tumor necrosis factor (TNF)-α (0.5–5 ng/mL) and VCAM-1 expression was evaluated by flow cytometry analysis. The inhibitory effect of the selective H1-antagonist levocetirizine (0.01–10.0μM) on VCAM-1 expression was also tested. Results Compared with unstimulated cultures, histamine or IL-4 + TNF-α, 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 μM) or IL-4 + TNF-α (5 ng/mL), in the presence of the drug (0.01–10.0 μM). The histamine-induced VCAM-1 expression was effectively inhibited by levocetirizine (0.1–10.0 μM) (p < 0.05). No effect of the drug on IL-4 + TNF-α-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.

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

Summary 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.

Summary 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.

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 (p0.05, each comparison) and a remarkable chemotactic response to both C5a or rh IL-5 (p0.001, 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 (p0.05). 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 (p0.05, 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.

Although elevated levels of serum immunoglobulin E (IgE) are considered the hallmark of atopic diseases, their clinical value in evaluating subjects with allergic disorders is under debate. To evaluate possible relationships between serum IgE levels and a variety of clinical parameters, 83 mild asthmatic children [10.98-year-old (2.95)], sensitized to house dust mites (HDM) Dermatophagoides pteronyssinus (Dp) or D. farinae (Df), were enrolled. As compared with normal control reference values detected in our laboratory, children with allergic asthma had higher blood eosinophil counts (expressed both as percentage and as absolute number) and higher fractional exhaled nitric oxide (FeNO) levels but similar values in pulmonary function parameters. In the allergic asthmatic population, serum levels of total, Dp-specific or Df-specific IgE correlated positively with eosinophil counts (Rho > or = 0.30, p or = 0.33, p 0.1, each correlation). Finally, significant correlations, although moderate, were found in the allergic asthmatic population between eosinophil counts and FeNO levels (Rho > or = 0.42, p < 0.001, each correlation). Thus, in atopic children sensitized to HDM with mild intermittent asthma, IgE levels in blood appear to reflect systemic (blood eosinophils) and organ-specific (FeNO) markers of allergic inflammation but not pulmonary volumes or the degree of airflow limitation.

Background: In atopic subjects, dysfunctions of the upper and lower airways frequently coexist and allergic rhinitis seems to constitute a risk factor for the occurrence of asthma in predisposed individuals. Aim of the study: To evaluate whether in atopic subjects nasal inflammation could reflect changes in respiratory functions, 11 allergic children, sensitized to house dust mites (HDM), with rhinoconjunctivitis and asthma and 10 nonatopic controls (ctrs) were studied. Methods: All subjects underwent nasal brushing to detect percentages of nasal eosinophils (Eos %) and intercellular adhesion molecule-1 (ICAM-1) expression by nasal epithelial cells. In the same day pulmonary function tests, i.e. forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), forced expiratory flows at 25–75% of the vital capacity (FEF25−75%) and methacholine (MCh) bronchial inhalation challenge were also evaluated. Results: Pulmonary function parameters were not significantly different in allergic children and in ctrs (P > 0.05), while a significant increase in bronchial reactivity to MCh, expressed as Pd20 MCh, was detected in the former population (P

The aim of this study was to compare in atopic and nonatopic asthmatic children correlations between two inflammation parameters, i.e., blood eosinophilia and exhaled nitric oxide (FENO), and pulmonary function values, at baseline and after β2-adrenergic bronchodilators. Ninety-two steroid-naive asthmatic children were evaluated: 26 were skin prick test- and RAST-negative (nonatopic subjects), whereas 66 were atopic, 15 being sensitized only to house dust mites (monosensitized) and 51 to mites and to at least one other class of allergens (polysensitized). Baseline spirometric values (FEV1 and FEF25–75%) were similar in atopic and nonatopic groups (P > 0.1, each comparison). However, when compared to nonatopic subjects, atopic children showed a significantly higher degree of blood eosinophilia (3.0% and 6.7% white blood cell count, respectively; P = 0.0001) and higher FENO levels (6.8 ppb and 16.0 ppb, respectively; P = 0.0001). While a positive correlation between FENO levels and blood eosinophilia was observed in atopic children (r = 0.25, P = 0.041), no correlations between these two inflammation parameters and baseline pulmonary function values were demonstrated in any of the asthmatic groups. Inhalation of a β2-agonist drug induced in the two asthmatic populations similar improvements in FEV1 and FEF25–75% and no changes in FENO levels or blood eosinophilia. However, only in atopic children positive correlations were found between percent variation in FEV1 (Δ%FEV1) and FENO levels (r = 0.35, P = 0.006) or blood eosinophilia (r = 0.26, P = 0.04). Within the atopic group, no differences were found between mono- and polysensitized individuals in all parameters evaluated. Thus only in atopic children did parameters of inflammation correlate with airway obstruction reversibility. Pediatr Pulmonol. 2003; 35:358–363. © 2003 Wiley-Liss, Inc.

In addition to be involved in airway remodelling observed in asthmatic patients, lung fibroblasts may directly contribute to pulmonary inflammation through the release of mediators and through the expression of surface molecules involved in cell-cell interaction. The aim of the study was to evaluate whether two cytokines involved in asthma pathogenesis, IL-4 and TNF-alpha, could modulate the expression of adhesion molecules (VCAM-1 and ICAM-1) and the secretion of chemokines (eotaxin and MCP-1) related to eosinophil recruitment and activation. The constitutive expression of VCAM-1 by unstimulated fibroblasts was over 2-fold lower than that of ICAM-1 (P 0.05 each comparison). In contrast, a significant increase in MCP-1 and eotaxin release was observed in the presence of TNF-alpha (P 0.05). These data show that two 'proinflammatory' cytokines, such as IL-4 and TNF-alpha, may have different and complementary effects on functions involved in the cross-talking between fibroblasts and eosinophils.

In atopic individuals, exposure to allergens is followed by recruitment of blood eosinophils in the target tissue. We investigated whether allergen inhalation challenge could result in depletion of blood eosinophils overexpressing adhesion molecules involved in eosinophil migration.Blood eosinophils were isolated from seven atopic asthmatic patients and seven control subjects and the "at baseline" expression of lymphocyte function-associated antigen-1 (LFA-1), macrophage antigen-1 (Mac-1) and very late antigen-4 (VLA-4) was assessed by monoclonal antibody staining and flow cytometry analysis. Asthmatic patients underwent allergen challenge and the expression of LFA-1, Mac-1 and VLA-4 by blood eosinophils was again evaluated 3 h and 24 h after allergen challenge.As compared to controls, eosinophils from atopics showed at baseline enhanced LFA-1 expression (P=0.0012), but similar Mac-1 or VLA-4 expression (P0.1, each comparison). In atopics, the percentage and absolute number of blood eosinophils were significantly decreased 3 h after allergen challenge (P=0.001 and P=0.022, respectively) but returned to similar values to prechallenge values after an additional 21 h (P0.1). Allergen challenge was also followed by a significant decrease in LFA-1 expression by eosinophils, at 3 h (P=0.002) and at 24 h (P=0.038), while no changes in Mac-1 and VLA-4 were observed. A significant correlation between postchallenge decrease in LFA-1 expression and in blood eosinophilia, both expressed as percentage (r=0.88; P0.01) or absolute number (r=0.87; P0.01) was demonstrated at 3 h (r=0.88; P0.01) but not at 24 h (r=0.64, P0.05 and r=0.11; P0.05, respectively).In allergic asthma, an early recruitment of blood eosinophils overexpressing LFA-1 occurs in the first hours after allergen challenge.

Objective. To evaluate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of local joint inflammation in juvenile idiopathic arthritis (JIA). Methods. Sera from 50 patients affected with JIA and 10 age-matched healthy controls were tested with a commercial ELISA for VEGF. Corresponding synovial fluid (SF) concentrations of VEGF and p75 soluble tumour necrosis factor receptor (sTNFR) were evaluated in 20 active JIA patients. Results. Serum concentrations of VEGF were significantly higher in patients with active polyarticular disease than in patients with active and inactive oligoarticular disease and healthy controls. In JIA patients, serum concentrations of VEGF displayed a significant correlation with a number of clinical and laboratory parameters of disease activity. VEGF concentrations in SF were significantly higher than those detected in corresponding sera. Moreover, a clear correlation was found between corresponding SF and serum VEGF concentrations. In SF, VEGF showed a strong positive correlation with p75 sTNFR. Conclusions. Concentrations of VEGF in SF in patients with JIA are higher than corresponding serum concentrations, suggesting that this pro-angiogenic factor may have a major role in the outgrowth of hyperplastic pannus and tissue damage at the site of tissue inflammations.

Summary Background The mechanisms involved in eosinophil recruitment by cysteinyl-leukotrienes (CysLTs) remain to be defined. Objective We investigated whether CysLTs LTC4, LTD4 and LTE4 could directly stimulate in vitro adhesion molecule expression and cell locomotion of blood eosinophils from atopic asthmatic donors. Methods Mab staining and FACS analysis were used to evaluate Mac-1 and LFA-1 expression on eosinophils before and after CysLTs stimulation. Eosinophil locomotion was tested using a 48-well Boyden microchamber. Results CysLTs, at the concentrations of 1 and 10 nM, were able to significantly up-regulate Mac-1 expression (P 0.05, each comparison). A dose-dependent, eosinophil chemotaxis was also induced by LTC4, LTD4 and LTE4 (0.1–10 nM) (P 0.05, each comparison). Conclusion These data demonstrate that CysLTs are active in vitro in directly up-regulating human eosinophil functions involved in eosinophil recruitment. The down-regulation of Mac-1 expression and eosinophil chemotaxis by the potent and selective CysLT1 receptor antagonist montelukast indicated the specificity of the LTC4-, LTD4- and LTE4-induced response.

Because of its ability to conjugate extensively with fatty acids within lung cells, it has been suggested that budesonide (Bud) may have a prolonged pharmacologic activity, related to retention of the drug in airway tissues. Using human bronchial epithelial cells (HBECs) as target cells, we evaluated whether Bud could have a long-lasting inhibitory effect on ICAM-1 expression and GM-CSF release. HBECs were cultured in Bud (10 microM) or in medium alone (Ctr) for 24 hr, then extensively washed (to remove Bud) and incubated for an additional 6, 12, or 24 hr with IFN-gamma. ICAM-1 expression and GM-CSF release were then measured by flow cytometric analysis. In Ctr HBECs, IFN-gamma induced a time-dependent upregulation of ICAM-1 expression, significant at 6, 12, or 24 hr (p0.05, each comparison), and an increase in GM-CSF release, significant at 24 hr (p0.05). The inhibitory effects of Bud preexposure on IFN-gamma-induced ICAM-1 expression and GM-CSF release were then compared with those of a continuous exposure to the drug during IFN-gamma stimulation. Preexposure to Bud (1 and 10 microM) induced a significant inhibition of IFN-gamma-induced ICAM-1 expression (p0.05, each comparison), but lower than that observed in HBECs continuously exposed at the same Bud concentrations (p0.01, each comparison). In contrast, the inhibition of GM-CSF release was similar in preexposed and in exposed HBECs and statistically significant only at the highest Bud concentration tested (p0.05, each comparison). Thus, Bud is effective in vitro in inducing a downregulation lasting 24 hr of mechanisms involved in leukocyte recruitment.

Gastroesophageal reflux (GER) may be associated with chronic or recurrent asthma-like symptoms secondary to bronchoconstrictor reflexes and/or inhalation of gastric content. The presence of lipid-laden alveolar macrophages has been proposed as an index to establish the degree of gastric aspiration. We evaluated 20 children with "difficult to treat" respiratory symptoms and a clinical history suggestive of GER. All children underwent 24-hr esophageal pH monitoring (pHm) and fiberoptic bronchoscopy with bronchoalveolar lavage (BAL). The amount of lipid per single macrophage was determined by a semiquantitative method, using fluorescence microscopy to detect Nile-Red-stained BAL cells and calculating a lipid-laden macrophage index (LLMI). Eleven children had positive pHm recordings, suggesting the presence of GER (pH-positive patients), and 9 had negative pHm records (pH-negative patients). The pH-positive patients had higher percentages of neutrophils and higher LLMI than the pH-negative children (P 0.05; each correlation). In contrast, a single correlation was found between % BAL neuytrophils and LLMI, both in the pH-positive and in the pH-negative patients (r = 0.72, P = 0.02 and r = 0.71, P = 0.04, respectively). These data demonstrate that a significant proportion of pH-positive patients with respiratory symptoms have BAL abnormalities that suggest airway inflammation and gastric content aspiration. However, the intensity of GER as indicated by pH monitoring does not correspond with BAL data in all patients.

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-alpha, IL-4 and IFN-gamma 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 mitogen-activated 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-alpha (47 +/- 3%), IL-4 (43 +/- 1%) and INF-gamma (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. GT values were, respectively, 1.030 +/- 0.0, 1.300 +/- 0.04, 1.260 +/- 0.020 and 2.220 +/- 0.015 (mS/cm(2)) x 1000 in control cultures and in those exposed to TNF-alpha, IFN-gamma 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 mu M) or a MAP kinase inhibitor (U0126, 25 mu M). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.

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.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.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.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.

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.

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.