49 results on '"Bezzerri, Valentino"'
Search Results
2. Growth Charts for Shwachman-Diamond Syndrome at Ages 0 to 18 Years.
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Pegoraro A, Bezzerri V, Tridello G, Brignole C, Lucca F, Pintani E, Danesino C, Cesaro S, Fioredda F, and Cipolli M
- Abstract
Shwachman-Diamond syndrome (SDS) is one of the most common inherited bone marrow failure syndromes. SDS is characterized by hypocellular bone marrow, with a severe impairment of the myeloid lineage, resulting in neutropenia, thrombocytopenia, and, more rarely, anemia. Almost 15% of patients with SDS develop myelodysplastic syndrome or acute myeloid leukemia as early as childhood or young adulthood. Exocrine pancreatic insufficiency is another common feature of SDS. Almost all patients with SDS show failure to thrive, which is associated with skeletal abnormalities due to defective ossification. Considering these observations, it remains unfeasible to use the common growth charts already available for the general population. To address this issue, we report how we drew up growth charts of patients with SDS aged 0 to 18 years. We analyzed height, weight, and body max index (BMI) in 121 Italian patients with SDS. Results indicated that the 50th and 3rd percentiles of weight and height of the pediatric general population correspond to the 97th and 50th percentiles of patients with SDS aged 0-18 years, respectively. In addition, the percentage increment in weight of subjects aged 14-18 years was higher in patients with SDS than in the general population. SDS-specific growth charts, such as those described here, afford a new tool, which is potentially useful for both clinical and research purposes in SDS.
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- 2024
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3. Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.
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Cipolli M, Boni C, Penzo M, Villa I, Bolamperti S, Baldisseri E, Frattini A, Porta G, Api M, Selicato N, Roccia P, Pollutri D, Marinelli Busilacchi E, Poloni A, Caporelli N, D'Amico G, Pegoraro A, Cesaro S, Oyarbide U, Vella A, Lippi G, Corey SJ, Valli R, Polini A, and Bezzerri V
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- Humans, Shwachman-Diamond Syndrome, Tumor Suppressor Protein p53 genetics, Codon, Nonsense, Myelopoiesis, Neutrophils metabolism, Chemotaxis, Ribosomes metabolism, Lipomatosis genetics, Bone Marrow Diseases genetics, Bone Marrow Diseases therapy, Exocrine Pancreatic Insufficiency genetics
- Abstract
Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)
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- 2024
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4. Shwachman-Diamond syndromes: clinical, genetic, and biochemical insights from the rare variants.
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Kawashima N, Oyarbide U, Cipolli M, Bezzerri V, and Corey SJ
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- Humans, Shwachman-Diamond Syndrome, Mutation, Signal Recognition Particle genetics, Lipomatosis genetics, Lipomatosis metabolism, Lipomatosis pathology, Bone Marrow Diseases genetics, Bone Marrow Diseases pathology, Exocrine Pancreatic Insufficiency genetics, Exocrine Pancreatic Insufficiency metabolism, Exocrine Pancreatic Insufficiency pathology
- Abstract
Shwachman-Diamond syndrome is a rare inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. In 10-30% of cases, transformation to a myeloid neoplasm occurs. Approximately 90% of patients have biallelic pathogenic variants in the SBDS gene located on human chromosome 7q11. Over the past several years, pathogenic variants in three other genes have been identified to cause similar phenotypes; these are DNAJC21, EFL1, and SRP54. Clinical manifestations involve multiple organ systems and those classically associated with the Shwachman-Diamond syndrome (bone, blood, and pancreas). Neurocognitive, dermatologic, and retinal changes may also be found. There are specific gene-phenotype differences. To date, SBDS, DNAJC21, and SRP54 variants have been associated with myeloid neoplasia. Common to SBDS, EFL1, DNAJC21, and SRP54 is their involvement in ribosome biogenesis or early protein synthesis. These four genes constitute a common biochemical pathway conserved from yeast to humans that involve early stages of protein synthesis and demonstrate the importance of this synthetic pathway in myelopoiesis.
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- 2023
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5. The Molecular and Genetic Mechanisms of Inherited Bone Marrow Failure Syndromes: The Role of Inflammatory Cytokines in Their Pathogenesis.
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Kawashima N, Bezzerri V, and Corey SJ
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- Humans, Congenital Bone Marrow Failure Syndromes genetics, Shwachman-Diamond Syndrome genetics, Interferon-alpha, Intracellular Signaling Peptides and Proteins, Cytokines genetics, Dyskeratosis Congenita
- Abstract
Inherited bone marrow failure syndromes (IBMFSs) include Fanconi anemia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, dyskeratosis congenita, severe congenital neutropenia, and other rare entities such as GATA2 deficiency and SAMD9/9L mutations. The IBMFS monogenic disorders were first recognized by their phenotype. Exome sequencing has validated their classification, with clusters of gene mutations affecting DNA damage response (Fanconi anemia), ribosome structure (Diamond-Blackfan anemia), ribosome assembly (Shwachman-Diamond syndrome), or telomere maintenance/stability (dyskeratosis congenita). The pathogenetic mechanisms of IBMFSs remain to be characterized fully, but an overarching hypothesis states that different stresses elicit TP53-dependent growth arrest and apoptosis of hematopoietic stem, progenitor, and precursor cells. Here, we review the IBMFSs and propose a role for pro-inflammatory cytokines, such as TGF-β, IL-1β, and IFN-α, in mediating the cytopenias. We suggest a pathogenic role for cytokines in the transformation to myeloid neoplasia and hypothesize a role for anti-inflammatory therapies.
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- 2023
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6. Counteracting the Common Shwachman-Diamond Syndrome-Causing SBDS c.258+2T>C Mutation by RNA Therapeutics and Base/Prime Editing.
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Peretto L, Tonetto E, Maestri I, Bezzerri V, Valli R, Cipolli M, Pinotti M, and Balestra D
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- Humans, DNA genetics, Mutation, RNA Splice Sites, Alternative Splicing genetics, Gene Editing, Shwachman-Diamond Syndrome genetics, Shwachman-Diamond Syndrome therapy
- Abstract
Shwachman-Diamond syndrome (SDS) represents one of the most common inherited bone marrow failure syndromes and is mainly caused by SBDS gene mutations. Only supportive treatments are available, with hematopoietic cell transplantation required when marrow failure occurs. Among all causative mutations, the SBDS c.258+2T>C variant at the 5' splice site (ss) of exon 2 is one of the most frequent. Here, we investigated the molecular mechanisms underlying aberrant SBDS splicing and showed that SBDS exon 2 is dense in splicing regulatory elements and cryptic splice sites, complicating proper 5'ss selection. Studies ex vivo and in vitro demonstrated that the mutation alters splicing, but it is also compatible with tiny amounts of correct transcripts, which would explain the survival of SDS patients. Moreover, for the first time for SDS, we explored a panel of correction approaches at the RNA and DNA levels and provided experimental evidence that the mutation effect can be partially counteracted by engineered U1snRNA, trans-splicing, and base/prime editors, ultimately leading to correctly spliced transcripts (from barely detectable to 2.5-5.5%). Among them, we propose DNA editors that, by stably reverting the mutation and potentially conferring positive selection to bone-marrow cells, could lead to the development of an innovative SDS therapy.
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- 2023
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7. SARS-CoV-2 viral entry and replication is impaired in Cystic Fibrosis airways due to ACE2 downregulation.
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Bezzerri V, Gentili V, Api M, Finotti A, Papi C, Tamanini A, Boni C, Baldisseri E, Olioso D, Duca M, Tedesco E, Leo S, Borgatti M, Volpi S, Pinton P, Cabrini G, Gambari R, Blasi F, Lippi G, Rimessi A, Rizzo R, and Cipolli M
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- Humans, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Down-Regulation, Receptors, Virus genetics, Receptors, Virus metabolism, Spike Glycoprotein, Coronavirus metabolism, Virus Replication, Angiotensin-Converting Enzyme 2 genetics, Angiotensin-Converting Enzyme 2 metabolism, COVID-19, Cystic Fibrosis genetics, SARS-CoV-2 physiology, Virus Internalization
- Abstract
As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis could be considered a comorbidity for coronavirus disease 2019. Instead, current clinical evidence seems to be heading in the opposite direction. To clarify whether host factors expressed by the Cystic Fibrosis epithelia may influence coronavirus disease 2019 progression, here we describe the expression of SARS-CoV-2 receptors in primary airway epithelial cells. We show that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral entry and replication in Cystic Fibrosis cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin 6 in healthy donor-derived primary airway epithelial cells, but a very weak response in primary Cystic Fibrosis cells. Collectively, these data support that Cystic Fibrosis condition may be at least partially protecting from SARS-CoV-2 infection., (© 2023. The Author(s).)
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- 2023
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8. Immunogenicity and Safety of the BNT162b2 COVID-19 Vaccine in Patients with Cystic Fibrosis with or without Lung Transplantation.
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Lucca F, Bezzerri V, Danese E, Olioso D, Peserico D, Boni C, Cucchetto G, Montagnana M, Tridello G, Meneghelli I, Ros M, Lippi G, and Cipolli M
- Subjects
- Humans, Antibodies, Viral, BNT162 Vaccine, Immunoglobulin A, Immunoglobulin G, SARS-CoV-2, COVID-19 prevention & control, COVID-19 Vaccines adverse effects, Cystic Fibrosis complications, Lung Transplantation adverse effects
- Abstract
Cystic fibrosis (CF) is characterized by a progressive decline in lung function, which may be further impaired by viral infections. CF is therefore considered a comorbidity of coronavirus disease 2019 (COVID-19), and SARS-CoV-2 vaccine prioritization has been proposed for patients with (pw)CF. Poor outcomes have been reported in lung transplant recipients (LTR) after SARS-CoV-2 infections. LTR have also displayed poor immunization against SARS-CoV-2 after mRNA-based BNT162b2 vaccination, especially in those undergoing immunosuppressive treatment, mostly those receiving mycophenolate mofetil (MMF) therapy. We aimed to determine here the immunogenicity and safety of the BNT162b2 vaccine in our cohort of 260 pwCF, including 18 LTR. Serum levels of neutralizing anti-SARS-CoV-2 IgG and IgA antibodies were quantified after the administration of two doses. PwCF displayed a vaccine-induced IgG and IgA antiviral response comparable with that seen in the general population. We also observed that the immunogenicity of the BNT162b2 vaccine was significantly impaired in the LTR subcohort, especially in patients undergoing MMF therapy. The BNT162b2 vaccine also caused minor adverse events as in the general population, mostly after administration of the second dose. Overall, our results justify the use of the BNT162b2 vaccine in pwCF and highlight the importance of a longitudinal assessment of the anti-SARS-CoV-2 IgG and IgA neutralizing antibody response to COVID-19 vaccination.
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- 2023
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9. Novel Translational Read-through-Inducing Drugs as a Therapeutic Option for Shwachman-Diamond Syndrome.
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Bezzerri V, Lentini L, Api M, Busilacchi EM, Cavalieri V, Pomilio A, Diomede F, Pegoraro A, Cesaro S, Poloni A, Pace A, Trubiani O, Lippi G, Pibiri I, and Cipolli M
- Abstract
Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS protein expression in bone marrow stem cells isolated from SDS patients carrying the nonsense mutation K62X. In this study, the authors evaluated the effect of a panel of ataluren analogues in restoring SBDS protein resynthesis and function both in hematological and non-hematological SDS cells. Besides confirming that ataluren can efficiently induce SBDS protein re-expression in SDS cells, the authors found that another analogue, namely NV848, can restore full-length SBDS protein synthesis as well, showing very low toxicity in zebrafish. Furthermore, NV848 can improve myeloid differentiation in bone marrow hematopoietic progenitors, enhancing neutrophil maturation and reducing the number of dysplastic granulocytes in vitro. Therefore, these findings broaden the possibilities of developing novel therapeutic options in terms of nonsense mutation suppression for SDS. Eventually, this study may act as a proof of concept for the development of similar approaches for other IBMFS caused by nonsense mutations.
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- 2022
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10. Enhanced p53 Levels Are Involved in the Reduced Mineralization Capacity of Osteoblasts Derived from Shwachman-Diamond Syndrome Subjects.
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Frattini A, Bolamperti S, Valli R, Cipolli M, Pinto RM, Bergami E, Frau MR, Cesaro S, Signo M, Bezzerri V, Porta G, Khan AW, Rubinacci A, and Villa I
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- Cells, Cultured, Female, Humans, Male, Osteoblasts pathology, Proteins genetics, Proteins metabolism, Shwachman-Diamond Syndrome genetics, Shwachman-Diamond Syndrome pathology, Tumor Suppressor Protein p53 genetics, Calcification, Physiologic, Osteoblasts metabolism, Shwachman-Diamond Syndrome metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, caused by loss-of-function mutations in the SBDS gene, a factor involved in ribosome biogenesis. By analyzing osteoblasts from SDS patients (SDS-OBs), we show that SDS-OBs displayed reduced SBDS gene expression and reduced/undetectable SBDS protein compared to osteoblasts from healthy subjects (H-OBs). SDS-OBs cultured in an osteogenic medium displayed a lower mineralization capacity compared to H-OBs. Whole transcriptome analysis showed significant differences in the gene expression of SDS-OBs vs. H-OBs, particularly in the ossification pathway. SDS-OBs expressed lower levels of the main genes responsible for osteoblastogenesis. Of all downregulated genes, Western blot analyses confirmed lower levels of alkaline phosphatase and collagen type I in SDS-OBs than in H-OBs. Interestingly, SDS-OBs showed higher protein levels of p53, an inhibitor of osteogenesis, compared to H-OBs. Silencing of Tp53 was associated with higher collagen type I and alkaline phosphatase protein levels and an increase in SDS-OB mineralization capacity. In conclusion, our results show that the reduced capacity of SDS-OBs to mineralize is mediated, at least in part, by the high levels of p53 and highlight an important role of SBDS in osteoblast functions.
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- 2021
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11. Does cystic fibrosis constitute an advantage in COVID-19 infection?
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Bezzerri V, Lucca F, Volpi S, and Cipolli M
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- Adult, COVID-19, COVID-19 Testing, Clinical Laboratory Techniques methods, Cohort Studies, Coronavirus Infections diagnosis, Coronavirus Infections prevention & control, Cystic Fibrosis diagnosis, Female, Humans, Incidence, Italy epidemiology, Male, Middle Aged, Pandemics prevention & control, Pneumonia, Viral prevention & control, Pulmonary Disease, Chronic Obstructive diagnosis, Real-Time Polymerase Chain Reaction methods, Reference Values, Retrospective Studies, Risk Assessment, Coronavirus Infections epidemiology, Cystic Fibrosis epidemiology, Pandemics statistics & numerical data, Pneumonia, Viral epidemiology, Pulmonary Disease, Chronic Obstructive epidemiology
- Abstract
The Veneto region is one of the most affected Italian regions by COVID-19. Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), may constitute a risk factor in COVID-19. Moreover, respiratory viruses were generally associated with severe pulmonary impairment in cystic fibrosis (CF). We would have therefore expected numerous cases of severe COVID-19 among the CF population. Surprisingly, we found that CF patients were significantly protected against infection by SARS-CoV-2. We discussed this aspect formulating some reasonable theories.
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- 2020
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12. Nonsense Suppression Therapy: New Hypothesis for the Treatment of Inherited Bone Marrow Failure Syndromes.
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Bezzerri V, Api M, Allegri M, Fabrizzi B, Corey SJ, and Cipolli M
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- Aminoglycosides pharmacology, Congenital Bone Marrow Failure Syndromes genetics, Humans, Oxadiazoles pharmacology, Aminoglycosides therapeutic use, Codon, Nonsense drug effects, Congenital Bone Marrow Failure Syndromes therapy, Nonsense Mediated mRNA Decay drug effects, Oxadiazoles therapeutic use
- Abstract
Inherited bone marrow failure syndromes (IBMFS) are a group of cancer-prone genetic diseases characterized by hypocellular bone marrow with impairment in one or more hematopoietic lineages. The pathogenesis of IBMFS involves mutations in several genes which encode for proteins involved in DNA repair, telomere biology and ribosome biogenesis. The classical IBMFS include Shwachman-Diamond syndrome (SDS), Diamond-Blackfan anemia (DBA), Fanconi anemia (FA), dyskeratosis congenita (DC), and severe congenital neutropenia (SCN). IBMFS are associated with high risk of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumors. Unfortunately, no specific pharmacological therapies have been highly effective for IBMFS. Hematopoietic stem cell transplantation provides a cure for aplastic or myeloid neoplastic complications. However, it does not affect the risk of solid tumors. Since approximately 28% of FA, 24% of SCN, 21% of DBA, 20% of SDS, and 17% of DC patients harbor nonsense mutations in the respective IBMFS-related genes, we discuss the use of the nonsense suppression therapy in these diseases. We recently described the beneficial effect of ataluren, a nonsense suppressor drug, in SDS bone marrow hematopoietic cells ex vivo. A similar approach could be therefore designed for treating other IBMFS. In this review we explain in detail the new generation of nonsense suppressor molecules and their mechanistic roles. Furthermore, we will discuss strengths and limitations of these molecules which are emerging from preclinical and clinical studies. Finally we discuss the state-of-the-art of preclinical and clinical therapeutic studies carried out for IBMFS.
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- 2020
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13. mTOR and STAT3 Pathway Hyper-Activation is Associated with Elevated Interleukin-6 Levels in Patients with Shwachman-Diamond Syndrome: Further Evidence of Lymphoid Lineage Impairment.
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Vella A, D'Aversa E, Api M, Breveglieri G, Allegri M, Giacomazzi A, Marinelli Busilacchi E, Fabrizzi B, Cestari T, Sorio C, Bedini G, D'Amico G, Bronte V, Poloni A, Benedetti A, Bovo C, Corey SJ, Borgatti M, Cipolli M, and Bezzerri V
- Abstract
Shwachman-Diamond syndrome (SDS) is a rare inherited bone marrow failure syndrome, resulting in neutropenia and a risk of myeloid neoplasia. A mutation in a ribosome maturation factor accounts for almost all of the cases. Lymphoid involvement in SDS has not been well characterized. We recently reported that lymphocyte subpopulations are reduced in SDS patients. We have also shown that the mTOR-STAT3 pathway is hyper-activated in SDS myeloid cell populations. Here we show that mTOR-STAT3 signaling is markedly upregulated in the lymphoid compartment of SDS patients. Furthermore, our data reveal elevated IL-6 levels in cellular supernatants obtained from lymphoblasts, bone marrow mononuclear and mesenchymal stromal cells, and plasma samples obtained from a cohort of 10 patients. Of note, everolimus-mediated inhibition of mTOR signaling is associated with basal state of phosphorylated STAT3. Finally, inhibition of mTOR-STAT3 pathway activation leads to normalization of IL-6 expression in SDS cells. Altogether, our data strengthen the hypothesis that SDS affects both lymphoid and myeloid blood compartment and suggest everolimus as a potential therapeutic agent to reduce excessive mTOR-STAT3 activation in SDS., Competing Interests: The authors declare no conflict of interest.
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- 2020
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14. Peripheral blood immunophenotyping in a large cohort of patients with Shwachman-Diamond syndrome.
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Bezzerri V, Vella A, Gennaro GD, Ortolani R, Nicolis E, Cesaro S, Fabrizzi B, Bronte V, Corey SJ, and Cipolli M
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- Adolescent, Adult, Bone Marrow Diseases pathology, Case-Control Studies, Child, Child, Preschool, Cohort Studies, Exocrine Pancreatic Insufficiency pathology, Female, Follow-Up Studies, Humans, Infant, Lipomatosis pathology, Male, Prognosis, Shwachman-Diamond Syndrome, Young Adult, B-Lymphocytes immunology, Bone Marrow Diseases blood, Bone Marrow Diseases immunology, Exocrine Pancreatic Insufficiency blood, Exocrine Pancreatic Insufficiency immunology, Immunophenotyping methods, Leukocytes, Mononuclear immunology, Lipomatosis blood, Lipomatosis immunology
- Abstract
Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes, characterized by neutropenia, occasional thrombocytopenia, and anemia. Bone marrow evaluation reveals an increased number of monocytes and mature B cells along with decreased granulocytes. However, little is known about the subpopulations of peripheral blood cells, and few previous publications have been based on a small number of patients. Here, we report a comprehensive immunophenotypic analysis from a cohort of 37 SDS patients who display impairment mostly in the myeloid compartment with a deficiency also in the number of B cells and CD4/CD8 double-negative T cells., (© 2019 Wiley Periodicals, Inc.)
- Published
- 2019
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15. Shwachman-Diamond Syndrome: Molecular Mechanisms and Current Perspectives.
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Bezzerri V and Cipolli M
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- Animals, Bone Marrow Diseases diagnosis, Bone Marrow Diseases therapy, Disease Models, Animal, Exocrine Pancreatic Insufficiency diagnosis, Exocrine Pancreatic Insufficiency therapy, Hematopoiesis genetics, Humans, Lipomatosis diagnosis, Lipomatosis therapy, Mutation genetics, Shwachman-Diamond Syndrome, Bone Marrow Diseases genetics, Exocrine Pancreatic Insufficiency genetics, Lipomatosis genetics
- Abstract
Shwachman-Diamond syndrome (SDS) is a rare inherited disease mainly caused by mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene. However, it has recently been reported that other genes, including DnaJ heat shock protein family (Hsp40) member C21 (DNAJC21), elongation factor-like 1 (EFL1) and signal recognition particle 54 (SRP54) are also associated with an SDS-like phenotype. Interestingly, SBDS, DNAJC21, EFL1 and SRP54 are involved in ribosome biogenesis: SBDS, through direct interaction with EFL1, promotes the release of the eukaryotic initiation factor 6 (eIF6) during ribosome maturation, DNAJC21 stabilizes the 80S ribosome, and SRP54 facilitates protein trafficking. These findings strengthen the postulate that SDS is a ribosomopathy. SDS is a multiple-organ disease mainly characterized by bone marrow failure, bone malformations, pancreatic insufficiency and cognitive disorders. Almost 15-20% of patients with SDS present myelodysplastic syndrome with a high risk of acute myeloid leukemia (AML) transformation. Unfortunately, besides bone marrow transplantation, no gene-based therapy for SDS has yet been developed. This review aims to recapitulate the recent findings on the molecular mechanisms of SDS underlying bone marrow failure, hematopoiesis and AML development and to draw a realistic picture of current perspectives.
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- 2019
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16. Is cellular senescence involved in cystic fibrosis?
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Bezzerri V, Piacenza F, Caporelli N, Malavolta M, Provinciali M, and Cipolli M
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- Cystic Fibrosis physiopathology, Cystic Fibrosis Transmembrane Conductance Regulator, Humans, Cellular Senescence, Cystic Fibrosis pathology
- Abstract
Pulmonary disease is the main cause of the morbidity and mortality of patients affected by cystic fibrosis (CF). The lung pathology is dominated by excessive recruitment of neutrophils followed by an exaggerated inflammatory process that has also been reported to occur in the absence of apparent pathogenic infections. Airway surface dehydration and mucus accumulation are the driving forces of this process. The continuous release of reactive oxygen species and proteases by neutrophils contributes to tissue damage, which eventually leads to respiratory insufficiency. CF has been considered a paediatric problem for several decades. Nevertheless, during the last 40 years, therapeutic options for CF have been greatly improved, turning CF into a chronic disease and extending the life expectancy of patients. Unfortunately, chronic inflammatory processes, which are characterized by a substantial release of cytokines and chemokines, along with ROS and proteases, can accelerate cellular senescence, leading to further complications in adulthood. The alterations and mechanisms downstream of CFTR functional defects that can stimulate cellular senescence remain unclear. However, while there are correlative data suggesting that cellular senescence may be implicated in CF, a causal or consequential relationship between cellular senescence and CF is still far from being established. Senescence can be both beneficial and detrimental. Senescence may suppress bacterial infections and cooperate with tissue repair. Additionally, it may act as an effective anticancer mechanism. However, it may also promote a pro-inflammatory environment, thereby damaging tissues and leading to chronic age-related diseases. In this review, we present the most current knowledge on cellular senescence and contextualize its possible involvement in CF.
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- 2019
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17. PLCB3 Loss of Function Reduces Pseudomonas aeruginosa-Dependent IL-8 Release in Cystic Fibrosis.
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Rimessi A, Bezzerri V, Salvatori F, Tamanini A, Nigro F, Dechecchi MC, Santangelo A, Prandini P, Munari S, Provezza L, Garreau de Loubresse N, Muller J, Ribeiro CMP, Lippi G, Gambari R, Pinton P, and Cabrini G
- Subjects
- Bronchi pathology, Calcium Signaling, Cell Line, Computer Simulation, Humans, Mucus metabolism, Mutation genetics, Phospholipase C beta chemistry, Phospholipase C beta genetics, Phospholipase C beta metabolism, Serine metabolism, Structure-Activity Relationship, Cystic Fibrosis metabolism, Cystic Fibrosis pathology, Interleukin-8 metabolism, Phospholipase C beta deficiency, Pseudomonas aeruginosa physiology
- Abstract
The lungs of patients with cystic fibrosis (CF) are characterized by an exaggerated inflammation driven by secretion of IL-8 from bronchial epithelial cells and worsened by Pseudomonas aeruginosa infection. To identify novel antiinflammatory molecular targets, we previously performed a genetic study of 135 genes of the immune response, which identified the c.2534C>T (p.S845L) variant of phospholipase C-β3 (PLCB3) as being significantly associated with mild progression of pulmonary disease. Silencing PLCB3 revealed that it potentiates the Toll-like receptor's inflammatory signaling cascade originating from CF bronchial epithelial cells. In the present study, we investigated the role of the PLCB3-S845L variant together with two synthetic mutants paradigmatic of impaired catalytic activity or lacking functional activation in CF bronchial epithelial cells. In experiments in which cells were exposed to P. aeruginosa, the supernatant of mucopurulent material from the airways of patients with CF or different agonists revealed that PLCB3-S845L has defects of 1) agonist-induced Ca
2+ release from endoplasmic reticulum and rise of Ca2+ concentration, 2) activation of conventional protein kinase C isoform β, and 3) induction of IL-8 release. These results, besides identifying S845L as a loss-of-function variant, strengthen the importance of targeting PLCB3 to mitigate the CF inflammatory response in bronchial epithelial cells without blunting the immune response.- Published
- 2018
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18. Ataluren-driven restoration of Shwachman-Bodian-Diamond syndrome protein function in Shwachman-Diamond syndrome bone marrow cells.
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Bezzerri V, Bardelli D, Morini J, Vella A, Cesaro S, Sorio C, Biondi A, Danesino C, Farruggia P, Assael BM, D'amico G, and Cipolli M
- Subjects
- Apoptosis drug effects, Bone Marrow Diseases pathology, Cells, Cultured, Codon, Nonsense drug effects, Colony-Forming Units Assay, Exocrine Pancreatic Insufficiency pathology, Gene Expression Regulation drug effects, Humans, Lipomatosis pathology, Monocytes cytology, Monocytes drug effects, Phosphorylation drug effects, Protein Processing, Post-Translational drug effects, Shwachman-Diamond Syndrome, TOR Serine-Threonine Kinases metabolism, Bone Marrow Cells metabolism, Bone Marrow Diseases metabolism, Exocrine Pancreatic Insufficiency metabolism, Lipomatosis metabolism, Oxadiazoles pharmacology, Proteins genetics
- Abstract
Shwachman-Diamond syndrome (SDS) is a rare inherited recessive disease mainly caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which encodes for the homonymous protein SBDS, whose function still remains to be fully established. SDS affects several organs causing bone marrow failure, exocrine pancreatic insufficiency, skeletal malformations, and cognitive disorders. About 15% of SDS patients develop myelodysplastic syndrome (MDS) and are at higher risk of developing acute myeloid leukemia (AML). Deficiency in SBDS expression has been associated with increased apoptosis and lack of myeloid differentiation in bone marrow hematopoietic progenitors. Importantly, most SDS patients carry nonsense mutations in SBDS. Since ataluren is a well-characterized small molecule inhibitor that can suppress nonsense mutations, here, we have assessed the efficacy of this drug in restoring SBDS expression in hematopoietic cells obtained from a cohort of SDS patients. Remarkably, we show that ataluren treatment readily restores SBDS protein expression in different cell types, particularly bone marrow stem cells. Furthermore, ataluren promotes myeloid differentiation in hematopoietic progenitors, reduces apoptotic rate in primary PBMCs, and brings mammalian target of rapamycin phosphorylation levels back to normal in both lymphoblasts and bone marrow mesenchymal stromal cells (BM-MSCs). Since a specific therapy against SDS is currently lacking, these results provide the rationale for ataluren repurposing clinical trials., (© 2018 Wiley Periodicals, Inc.)
- Published
- 2018
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19. An antisense peptide nucleic acid against Pseudomonas aeruginosa inhibiting bacterial-induced inflammatory responses in the cystic fibrosis IB3-1 cellular model system.
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Montagner G, Bezzerri V, Cabrini G, Fabbri E, Borgatti M, Lampronti I, Finotti A, Nielsen PE, and Gambari R
- Subjects
- Apoptosis genetics, Cell Line, Cell Proliferation genetics, Humans, Inflammation complications, Inflammation genetics, Inflammation microbiology, Interleukin-8 genetics, Pseudomonas aeruginosa growth & development, Up-Regulation genetics, Cystic Fibrosis pathology, Oligonucleotides, Antisense genetics, Peptide Nucleic Acids genetics, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa physiology
- Abstract
Discovery of novel antimicrobial agents against Pseudomonas aeruginosa able to inhibit bacterial growth as well as the resulting inflammatory response is a key goal in cystic fibrosis research. We report in this paper that a peptide nucleic acid (PNA3969) targeting the translation initiation region of the essential acpP gene of P. aeruginosa, and previously shown to inhibit bacterial growth, concomitantly also strongly inhibits induced up-regulation of the pro-inflammatory markers IL-8, IL-6, G-CSF, IFN-γ, IP-10, MCP-1 and TNF-α in IB3-1 cystic fibrosis cells infected by P. aeruginosa PAO1. Remarkably, no effect on PAO1 induction of VEGF, GM-CSF and IL-17 was observed. Analogous experiments using a two base mis-match control PNA did not show such inhibition. Furthermore, no significant effects of the PNAs were seen on cell growth, apoptosis or secretome profile in uninfected IB3-1 cells (with the exception of a PNA-mediated up-regulation of PDGF, IL-17 and GM-CSF). Thus, we conclude that in cell culture an antimicrobial PNA against P. aeruginosa can inhibit the expression of pro-inflammatory cytokines otherwise induced by the infection. In particular, the effects of PNA-3969 on IL-8 gene expression are significant considering the key role of this protein in the cystic fibrosis inflammatory process exacerbated by P. aeruginosa infection., (Copyright © 2017. Published by Elsevier B.V.)
- Published
- 2017
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20. β-Sitosterol Reduces the Expression of Chemotactic Cytokine Genes in Cystic Fibrosis Bronchial Epithelial Cells.
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Lampronti I, Dechecchi MC, Rimessi A, Bezzerri V, Nicolis E, Guerrini A, Tacchini M, Tamanini A, Munari S, D'Aversa E, Santangelo A, Lippi G, Sacchetti G, Pinton P, Gambari R, Agostini M, and Cabrini G
- Abstract
Extracts from Nigella arvensis L. seeds, which are widely used as anti-inflammatory remedies in traditional medicine of Northern Africa, were able to inhibit the expression of the pro-inflammatory neutrophil chemokine Interleukin (IL)-8 in Cystic Fibrosis (CF) bronchial epithelial IB3-1 cells exposed to the Gram-negative bacterium Pseudomonas aeruginosa . The chemical composition of the extracts led to the identification of three major components, β-sitosterol, stigmasterol, and campesterol, which are the most abundant phytosterols, cholesterol-like molecules, usually found in plants. β-sitosterol (BSS) was the only compound that significantly reproduced the inhibition of the P. aeruginosa -dependent expression of IL-8 at nanomolar concentrations. BSS was tested in CF airway epithelial CuFi-1 cells infected with P. aeruginosa . BSS (100 nM), showed a significant and consistent inhibitory activity on expression of the P. aeruginosa -stimulated expression chemokines IL-8, GRO-α GRO-β, which play a pivotal role in the recruitment of neutrophils in CF inflamed lungs. Preliminary mechanistic analysis showed that BSS partially inhibits the P. aeruginosa -dependent activation of Protein Kinase C isoform alpha, which is known to be involved in the transmembrane signaling activating IL-8 gene expression in bronchial epithelial cells. These data indicate BSS as a promising molecule to control excessive lung inflammation in CF patients.
- Published
- 2017
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21. Differential Effects of Angelicin Analogues on NF- κ B Activity and IL-8 Gene Expression in Cystic Fibrosis IB3-1 Cells.
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Lampronti I, Manzione MG, Sacchetti G, Ferrari D, Spisani S, Bezzerri V, Finotti A, Borgatti M, Dechecchi MC, Miolo G, Marzaro G, Cabrini G, Gambari R, and Chilin A
- Subjects
- Cell Line, Humans, Interleukin-8 genetics, Molecular Structure, NF-kappa B genetics, Cystic Fibrosis metabolism, Furocoumarins chemistry, Furocoumarins pharmacology, Interleukin-8 metabolism, NF-kappa B metabolism
- Abstract
The angelicin analogue 4,6,4'-trimethylangelicin (TMA) was recently reported as a strong inhibitor of nuclear factor- κ B (NF- κ B) activity and of the expression of the interleukin-8 (IL-8) gene in bronchial epithelial cells in which the inflammatory response has been challenged with P. aeruginosa , the most common bacterium found in the airways of patients affected by cystic fibrosis (CF). These findings encouraged us to analyze new synthetic analogues of TMA in order to evaluate their biological activities on human bronchial epithelial CF IB3-1 cells and to find more potent anti-NF- κ B agents exhibiting only minor antiproliferative effects. Analogues able to inhibit NF- κ B/DNA interaction at lower concentration than TMA were found and selected to investigate their biological activity on IB3-1 cells induced with TNF- α . In this biological system, NF- κ B-mediated IL-8 gene expression was investigated. Some analogues showed similar activity to the lead compound TMA. Other analogues displayed higher activities; in particular, the most interesting compounds showing relevant anti-inflammatory effects were found to cause 56-83% reduction of IL-8 mRNA expression at low concentrations (1-10 μ M), without changes in cell proliferation pattern, demonstrating their potential interest for a possible development of anti-inflammatory therapy of cystic fibrosis.
- Published
- 2017
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22. Transient Receptor Potential Ankyrin 1 Channels Modulate Inflammatory Response in Respiratory Cells from Patients with Cystic Fibrosis.
- Author
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Prandini P, De Logu F, Fusi C, Provezza L, Nassini R, Montagner G, Materazzi S, Munari S, Gilioli E, Bezzerri V, Finotti A, Lampronti I, Tamanini A, Dechecchi MC, Lippi G, Ribeiro CM, Rimessi A, Pinton P, Gambari R, Geppetti P, and Cabrini G
- Subjects
- A549 Cells, Adult, Bronchi pathology, Cystic Fibrosis genetics, Cystic Fibrosis pathology, Cytokines genetics, Epithelial Cells metabolism, Epithelial Cells pathology, Female, Gene Silencing, Humans, Interleukin-8 genetics, Interleukin-8 metabolism, Male, Middle Aged, Models, Biological, Nerve Tissue Proteins antagonists & inhibitors, Pneumonia genetics, Pseudomonas aeruginosa physiology, RNA, Messenger genetics, RNA, Messenger metabolism, TRPA1 Cation Channel, Tissue Donors, Transcription, Genetic, Transient Receptor Potential Channels antagonists & inhibitors, Young Adult, Calcium Channels metabolism, Cystic Fibrosis complications, Lung pathology, Nerve Tissue Proteins metabolism, Pneumonia complications, Pneumonia pathology, Transient Receptor Potential Channels metabolism
- Abstract
Pseudomonas aeruginosa colonization, prominent inflammation with massive expression of the neutrophil chemokine IL-8, and luminal infiltrates of neutrophils are hallmarks of chronic lung disease in patients with cystic fibrosis (CF). The nociceptive transient receptor potential ankyrin (TRPA) 1 calcium channels have been recently found to be involved in nonneurogenic inflammation. Here, we investigate the role of TRPA1 in CF respiratory inflammatory models in vitro. Expression of TRPA1 was evaluated in CF lung tissue sections and cells by immunohistochemistry and immunofluorescence. Epithelial cell lines (A549, IB3-1, CuFi-1, CFBE41o
- ) and primary cells from patients with CF were used to: (1) check TRPA1 function modulation, by Fura-2 calcium imaging; (2) down-modulate TRPA1 function and expression, by pharmacological inhibitors (HC-030031 and A-967079) and small interfering RNA silencing; and (3) assess the effect of TRPA1 down-modulation on expression and release of cytokines upon exposure to proinflammatory challenges, by quantitative RT-PCR and 27-protein Bioplex assay. TRPA1 channels are expressed in the CF pseudostratified columnar epithelium facing the bronchial lumina exposed to bacteria, where IL-8 is coexpressed. Inhibition of TRPA1 expression results in a relevant reduction of release of several cytokines, including IL-8 and the proinflammatory cytokines IL-1β and TNF-α, in CF primary bronchial epithelial cells exposed to P. aeruginosa and to the supernatant of mucopurulent material derived from the chronically infected airways of patients with CF. In conclusion, TRPA1 channels are involved in regulating the extent of airway inflammation driven by CF bronchial epithelial cells.- Published
- 2016
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23. New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes.
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Bezzerri V, Vella A, Calcaterra E, Finotti A, Gasparello J, Gambari R, Assael BM, Cipolli M, and Sorio C
- Abstract
Shwachman-Diamond syndrome (SDS) is an inherited disease caused by mutations of a gene encoding for SBDS protein. So far little is known about SBDS exact function. SDS patients present several hematological disorders, including neutropenia and myelodysplastic syndrome (MDS), with increased risk of leukemic evolution. So far, the molecular mechanisms that underlie neutropenia, MDS and AML in SDS patients have been poorly investigated. STAT3 is a key regulator of several cellular processes including survival, differentiation and malignant transformation. Moreover, STAT3 has been reported to regulate neutrophil granulogenesis and to induce several kinds of leukemia and lymphoma. STAT3 activation is known to be regulated by mTOR, which in turn plays an important role in cellular growth and tumorigenesis. Here we show for the first time, to the best of our knowledge, that both EBV-immortalized B cells and primary leukocytes obtained from SDS patients present a constitutive hyper-activation of mTOR and STAT3 pathways. Interestingly, loss of SBDS expression is associated with this process. Importantly, rapamycin, a well-known mTOR inhibitor, is able to reduce STAT3 phosphorylation to basal levels in our experimental model. A novel therapeutic hypothesis targeting mTOR/STAT3 should represent a significant step forward into the SDS clinical practice.
- Published
- 2016
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24. miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma.
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Khalil S, Fabbri E, Santangelo A, Bezzerri V, Cantù C, Di Gennaro G, Finotti A, Ghimenton C, Eccher A, Dechecchi M, Scarpa A, Hirshman B, Chen C, Ferracin M, Negrini M, Gambari R, and Cabrini G
- Subjects
- Brain Neoplasms metabolism, DNA Modification Methylases genetics, DNA Repair Enzymes genetics, Gene Expression Profiling methods, Glioblastoma metabolism, Humans, Oligonucleotide Array Sequence Analysis methods, Tumor Suppressor Proteins genetics, Brain Neoplasms genetics, DNA Modification Methylases biosynthesis, DNA Repair Enzymes biosynthesis, Gene Expression Regulation, Neoplastic genetics, Glioblastoma genetics, MicroRNAs genetics, Tumor Suppressor Proteins biosynthesis
- Abstract
The levels of expression of O6-methylguanine-DNA methyltransferase (MGMT) are relevant in predicting the response to the alkylating chemotherapy in patients affected by glioblastoma. MGMT promoter methylation and the published MGMT regulating microRNAs (miRNAs) do not completely explain the expression pattern of MGMT in clinical glioblastoma specimens. Here we used a genome-wide microarray-based approach to identify MGMT regulating miRNAs. Our screen unveiled three novel MGMT regulating miRNAs, miR-127-3p, miR-409-3p, and miR-124-3p, in addition to the previously identified miR-181d-5p. Transfection of these three novel miRNAs into the T98G glioblastoma cell line suppressed MGMT mRNA and protein expression. However, their MGMT- suppressive effects are 30-50% relative that seen with miR-181d-5p transfection. In silico analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) revealed that miR-181d-5p is the only miRNA that consistently exhibited inverse correlation with MGMT mRNA expression. However, statistical models incorporating both miR-181d-5p and miR-409-3p expression better predict MGMT expression relative to models involving either miRNA alone. Our results confirmed miR-181d-5p as the key MGMT-regulating miRNA. Other MGMT regulating miRNAs, including the miR-409-3p identified in this report, modify the effect of miR-181d-5p on MGMT expression. MGMT expression is, thus, regulated by cooperative interaction between key MGMT-regulating miRNAs., Competing Interests: None for all authors.
- Published
- 2016
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25. Regulation of IL-8 gene expression in gliomas by microRNA miR-93.
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Fabbri E, Brognara E, Montagner G, Ghimenton C, Eccher A, Cantù C, Khalil S, Bezzerri V, Provezza L, Bianchi N, Finotti A, Borgatti M, Moretto G, Chilosi M, Cabrini G, and Gambari R
- Subjects
- Base Sequence, Binding Sites, Brain Neoplasms metabolism, Brain Neoplasms pathology, Cell Line, Tumor, Cluster Analysis, Gene Expression, Gene Expression Profiling, Glioma metabolism, Glioma pathology, Humans, In Situ Hybridization, Interleukin-8 chemistry, Interleukin-8 metabolism, MicroRNAs chemistry, Models, Biological, Neoplasm Grading, Nucleic Acid Conformation, RNA Interference, RNA, Messenger chemistry, Transfection, Vascular Endothelial Growth Factor A genetics, Vascular Endothelial Growth Factor A metabolism, Brain Neoplasms genetics, Gene Expression Regulation, Neoplastic, Glioma genetics, Interleukin-8 genetics, MicroRNAs genetics, RNA, Messenger genetics
- Abstract
Background: Different strategies have been proposed to target neoangiogenesis in gliomas, besides those targeting Vascular Endothelial Growth Factor (VEGF). The chemokine Interleukin-8 (IL-8) has been shown to possess both tumorigenic and proangiogenic properties. Although different pathways of induction of IL-8 gene expression have been already elucidated, few data are available on its post-transcriptional regulation in gliomas., Methods: Here we investigated the role of the microRNA miR-93 on the expression levels of IL-8 and other pro-inflammatory genes by RT-qPCR and Bio-Plex analysis. We used different disease model systems, including clinical samples from glioma patients and two glioma cell lines, U251 and T98G., Results: IL-8 and VEGF transcripts are highly expressed in low and high grade gliomas in respect to reference healthy brain; miR-93 expression is also increased and inversely correlated with transcription of IL-8 and VEGF genes. Computational analysis showed the presence of miR-93 consensus sequences in the 3'UTR region of both VEGF and IL-8 mRNAs, predicting possible interaction with miR-93 and suggesting a potential regulatory role of this microRNA. In vitro transfection with pre-miR-93 and antagomiR-93 inversely modulated VEGF and IL-8 gene expression and protein release when the glioma cell line U251 was considered. Similar data were obtained on IL-8 gene regulation in the other glioma cell line analyzed, T98G. The effect of pre-miR-93 and antagomiR-93 in U251 cells has been extended to the secretion of a panel of cytokines, chemokines and growth factors, which consolidated the concept of a role of miR-93 in IL-8 and VEGF gene expression and evidenced a potential regulatory role also for MCP-1 and PDGF (also involved in angiogenesis)., Conclusion: In conclusion, our results suggest an increasing role of miR-93 in regulating the level of expression of several genes involved in the angiogenesis of gliomas.
- Published
- 2015
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26. Mitochondrial Ca2+-dependent NLRP3 activation exacerbates the Pseudomonas aeruginosa-driven inflammatory response in cystic fibrosis.
- Author
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Rimessi A, Bezzerri V, Patergnani S, Marchi S, Cabrini G, and Pinton P
- Subjects
- Bronchi metabolism, Bronchi microbiology, Bronchi pathology, Calcium Channels genetics, Calcium Channels metabolism, Calcium Signaling, Carrier Proteins agonists, Carrier Proteins metabolism, Cells, Cultured, Cystic Fibrosis genetics, Cystic Fibrosis microbiology, Cystic Fibrosis pathology, Cystic Fibrosis Transmembrane Conductance Regulator, Epithelial Cells microbiology, Epithelial Cells pathology, Flagellin genetics, Flagellin metabolism, Gene Expression Regulation, Host-Pathogen Interactions, Humans, Inflammation, Interleukin-18 biosynthesis, Interleukin-18 metabolism, Interleukin-1beta biosynthesis, Interleukin-1beta metabolism, Mitochondria microbiology, Mitochondria pathology, NLR Family, Pyrin Domain-Containing 3 Protein, Pseudomonas aeruginosa pathogenicity, Calcium metabolism, Carrier Proteins genetics, Cystic Fibrosis metabolism, Epithelial Cells metabolism, Mitochondria metabolism, Pseudomonas aeruginosa physiology
- Abstract
The common pathological manifestation of cystic fibrosis (CF) is associated with an excessive lung inflammatory response characterized by interleukin-1β accumulation. CF airway epithelial cells show an exacerbated pro-inflammatory response to Pseudomonas aeruginosa; however, it is unclear whether this heightened inflammatory response is intrinsic to cells lacking CF transmembrane conductance regulator (CFTR). Here we demonstrate that the degree and quality of the inflammatory response in CF are supported by P. aeruginosa-dependent mitochondrial perturbation, in which flagellin is the inducer and mitochondrial Ca(2+) uniporter (MCU) is a signal-integrating organelle member for NLRP3 activation and IL-1β and IL-18 processing. Our work elucidates the regulation of the NLRP3 inflammasome by mitochondrial Ca(2+) in the P. aeruginosa-dependent inflammatory response and deepens our understanding of the significance of mitochondria in the Ca(2+)-dependent control of inflammation.
- Published
- 2015
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27. Pseudomonas aeruginosa reduces the expression of CFTR via post-translational modification of NHERF1.
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Rubino R, Bezzerri V, Favia M, Facchini M, Tebon M, Singh AK, Riederer B, Seidler U, Iannucci A, Bragonzi A, Cabrini G, Reshkin SJ, and Tamanini A
- Subjects
- Animals, Bronchi cytology, Bronchi metabolism, Bronchi microbiology, Cell Line, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Humans, Lung cytology, Lung metabolism, Lung microbiology, Mice, Mutation, Phosphoproteins genetics, Phosphorylation, Pseudomonas aeruginosa, Respiratory Mucosa microbiology, Sodium-Hydrogen Exchangers genetics, Ubiquitination, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Phosphoproteins metabolism, Protein Processing, Post-Translational, Pseudomonas Infections metabolism, Respiratory Mucosa metabolism, Sodium-Hydrogen Exchangers metabolism
- Abstract
Pseudomonas aeruginosa infections of the airway cells decrease apical expression of both wild-type (wt) and F508del CFTR through the inhibition of apical endocytic recycling. CFTR endocytic recycling is known to be regulated by its interaction with PDZ domain containing proteins. Recent work has shown that the PDZ domain scaffolding protein NHERF1 finely regulates both wt and F508delCFTR membrane recycling. Here, we investigated the effect of P. aeruginosa infection on NHERF1 post-translational modifications and how this affects CFTR expression in bronchial epithelial cells and in murine lung. Both in vitro in bronchial cells, and in vivo in mice, infection reduced CFTR expression and increased NHERF1 molecular weight through its hyper-phosphorylation and ubquitination as a consequence of both bacterial pilin- and flagellin-mediated host-cell interaction. The ability of P. aeruginosa to down-regulate mature CFTR expression was reduced both in vivo in NHERF1 knockout mice and in vitro after silencing NHERF1 expression or mutations blocking its phosphorylation at serines 279 and 301. These studies provide the first evidence that NHERF1 phosphorylation may negatively regulate its action and, therefore, the assembly and function of multiprotein NHERF1 complexes in response to infection. The identification of molecular mechanisms responsible for these effects could identify novel targets to block potential P. aeruginosa interference with the efficacy of potentiator and/or corrector compounds.
- Published
- 2014
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28. Antibacterial and anti-inflammatory activity of a temporin B peptide analogue on an in vitro model of cystic fibrosis.
- Author
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Bezzerri V, Avitabile C, Dechecchi MC, Lampronti I, Borgatti M, Montagner G, Cabrini G, Gambari R, and Romanelli A
- Subjects
- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents metabolism, Anti-Inflammatory Agents, Non-Steroidal chemistry, Anti-Inflammatory Agents, Non-Steroidal metabolism, Antimicrobial Cationic Peptides chemistry, Antimicrobial Cationic Peptides metabolism, Bronchi immunology, Bronchi metabolism, Bronchi microbiology, Cell Line, Circular Dichroism, Cystic Fibrosis immunology, Cystic Fibrosis metabolism, Drug Design, Gene Expression Regulation drug effects, Humans, Inflammation Mediators antagonists & inhibitors, Inflammation Mediators metabolism, Lipopolysaccharides antagonists & inhibitors, Lipopolysaccharides chemistry, Lipopolysaccharides metabolism, Microbial Sensitivity Tests, Molecular Conformation, Proteins chemistry, Proteins pharmacology, Pseudomonas Infections immunology, Pseudomonas Infections metabolism, Pseudomonas Infections microbiology, Pseudomonas aeruginosa drug effects, Pseudomonas aeruginosa growth & development, Pseudomonas aeruginosa immunology, Respiratory Mucosa immunology, Respiratory Mucosa metabolism, Respiratory Mucosa microbiology, Spectrometry, Fluorescence, Anti-Bacterial Agents pharmacology, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Antimicrobial Cationic Peptides pharmacology, Bronchi drug effects, Cystic Fibrosis drug therapy, Pseudomonas Infections drug therapy, Respiratory Mucosa drug effects
- Abstract
Natural peptides with antimicrobial properties are deeply investigated as tools to fight bacteria resistant to common antibiotics. Small peptides, as those belonging to the temporin family, are very attractive because their activity can easily be tuned after small modification to their primary sequence. Structure-activity studies previously reported by us allowed the identification of one peptide, analogue of temporin B, TB_KKG6A, showing, unlike temporin B, antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigated the antimicrobial and anti-inflammatory activity of the peptide TB_KKG6A against Pseudomonas aeruginosa. Interestingly, we found that the peptide exhibits antimicrobial activity at low concentrations, being able to downregulate the pro-inflammatory chemokines and cytokines interleukin (IL)-8, IL-1β, IL-6 and tumor necrosis factor-α produced downstream infected human bronchial epithelial cells. Experiments were carried out also with temporin B, which was found to show pro-inflammatory activity. Details on the interaction between TB_KKG6A and the P. aeruginosa LPS were obtained by circular dichroism and fluorescence studies., (Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.)
- Published
- 2014
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29. GBA2-encoded β-glucosidase activity is involved in the inflammatory response to Pseudomonas aeruginosa.
- Author
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Loberto N, Tebon M, Lampronti I, Marchetti N, Aureli M, Bassi R, Giri MG, Bezzerri V, Lovato V, Cantù C, Munari S, Cheng SH, Cavazzini A, Gambari R, Sonnino S, Cabrini G, and Dechecchi MC
- Subjects
- 1-Deoxynojirimycin analogs & derivatives, 1-Deoxynojirimycin pharmacology, Bronchi drug effects, Bronchi enzymology, Bronchi microbiology, Epithelial Cells drug effects, Epithelial Cells enzymology, Epithelial Cells microbiology, Glucosylceramidase, Glycoside Hydrolase Inhibitors pharmacology, Humans, Inflammation microbiology, Pseudomonas Infections microbiology, Cystic Fibrosis enzymology, Inflammation enzymology, Pseudomonas Infections enzymology, Pseudomonas aeruginosa, beta-Glucosidase metabolism
- Abstract
Current anti-inflammatory strategies for the treatment of pulmonary disease in cystic fibrosis (CF) are limited; thus, there is continued interest in identifying additional molecular targets for therapeutic intervention. Given the emerging role of sphingolipids (SLs) in various respiratory disorders, including CF, drugs that selectively target the enzymes associated with SL metabolism are under development. Miglustat, a well-characterized iminosugar-based inhibitor of β-glucosidase 2 (GBA2), has shown promise in CF treatment because it reduces the inflammatory response to infection by P. aeruginosa and restores F508del-CFTR chloride channel activity. This study aimed to probe the molecular basis for the anti-inflammatory activity of miglustat by examining specifically the role of GBA2 following the infection of CF bronchial epithelial cells by P. aeruginosa. We also report the anti-inflammatory activity of another potent inhibitor of GBA2 activity, namely N-(5-adamantane-1-yl-methoxy)pentyl)-deoxynojirimycin (Genz-529648). In CF bronchial cells, inhibition of GBA2 by miglustat or Genz-529648 significantly reduced the induction of IL-8 mRNA levels and protein release following infection by P. aeruginosa. Hence, the present data demonstrate that the anti-inflammatory effects of miglustat and Genz-529648 are likely exerted through inhibition of GBA2.
- Published
- 2014
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30. Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells.
- Author
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Favia M, Mancini MT, Bezzerri V, Guerra L, Laselva O, Abbattiscianni AC, Debellis L, Reshkin SJ, Gambari R, Cabrini G, and Casavola V
- Subjects
- Animals, Cell Line, Cell Membrane genetics, Cell Membrane metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Cystic Fibrosis genetics, Cystic Fibrosis pathology, Endoplasmic Reticulum metabolism, Furocoumarins therapeutic use, HEK293 Cells, Humans, Lung pathology, Protein Transport genetics, Rats, Chlorides metabolism, Cystic Fibrosis drug therapy, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Furocoumarins pharmacology
- Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties., (Copyright © 2014 the American Physiological Society.)
- Published
- 2014
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31. Expression of microRNA-93 and Interleukin-8 during Pseudomonas aeruginosa-mediated induction of proinflammatory responses.
- Author
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Fabbri E, Borgatti M, Montagner G, Bianchi N, Finotti A, Lampronti I, Bezzerri V, Dechecchi MC, Cabrini G, and Gambari R
- Subjects
- 3' Untranslated Regions genetics, Bronchi microbiology, Cell Line, Cystic Fibrosis genetics, Cystic Fibrosis microbiology, Down-Regulation, Epithelial Cells microbiology, Humans, Minichromosome Maintenance Complex Component 7 genetics, NF-kappa B genetics, Protein Processing, Post-Translational, Pseudomonas Infections microbiology, Pseudomonas aeruginosa, RNA, Messenger genetics, Transcription, Genetic, Up-Regulation, Gene Expression Regulation, Inflammation genetics, Inflammation microbiology, Interleukin-8 genetics, MicroRNAs genetics, Pseudomonas Infections genetics
- Abstract
In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3-1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3'-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3'-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3-1, CuFi-1, and NuLi-1 cells by pre-miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB-dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.
- Published
- 2014
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32. Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221.
- Author
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Brognara E, Fabbri E, Bazzoli E, Montagner G, Ghimenton C, Eccher A, Cantù C, Manicardi A, Bianchi N, Finotti A, Breveglieri G, Borgatti M, Corradini R, Bezzerri V, Cabrini G, and Gambari R
- Subjects
- Adult, Analysis of Variance, Annexin A5 metabolism, Apoptosis drug effects, Brain Neoplasms genetics, Cell Line, Tumor, Cell Proliferation drug effects, Cyclin-Dependent Kinase Inhibitor p27 pharmacology, Dose-Response Relationship, Drug, Flow Cytometry, Glioma genetics, Humans, Male, MicroRNAs genetics, Models, Molecular, Time Factors, Brain Neoplasms metabolism, Gene Expression Regulation, Neoplastic drug effects, Glioma metabolism, MicroRNAs metabolism, Peptide Nucleic Acids pharmacology
- Abstract
MicroRNAs are a family of small noncoding RNAs regulating gene expression by sequence-selective mRNA targeting, leading to a translational repression or mRNA degradation. The oncomiR miR-221 is highly expressed in human gliomas, as confirmed in this study in samples of low and high grade gliomas, as well in the cell lines U251, U373 and T98G. In order to alter the biological functions of miR-221, a peptide nucleic acid targeting miR-221 (R8-PNA-a221) was produced, bearing a oligoarginine peptide (R8) to facilitate uptake by glioma cells. The effects of R8-PNA-a221 were analyzed in U251, U373 and T98G glioma cells and found to strongly inhibit miR-221. In addition, the effects of R8-PNA-a221 on p27(Kip1) (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27(Kip1) mRNA content occurs in U251 cells in the presence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27(Kip1) mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27(Kip1) protein expression in the samples treated with R8-PNA-a221 was detected. In addition, R8-PNA-a221 was found able to increase TIMP3 expression (another target of miR-221) in T98G cells. These results suggest that PNAs against oncomiRNA miR-221 might be proposed for experimental treatment of human gliomas.
- Published
- 2014
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33. Effects of decoy molecules targeting NF-kappaB transcription factors in Cystic fibrosis IB3-1 cells: recruitment of NF-kappaB to the IL-8 gene promoter and transcription of the IL-8 gene.
- Author
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Finotti A, Borgatti M, Bezzerri V, Nicolis E, Lampronti I, Dechecchi M, Mancini I, Cabrini G, Saviano M, Avitabile C, Romanelli A, and Gambari R
- Subjects
- Base Sequence, Cell Line, Cystic Fibrosis microbiology, DNA metabolism, Humans, Oligodeoxyribonucleotides genetics, Oligodeoxyribonucleotides metabolism, Peptide Nucleic Acids metabolism, Pseudomonas aeruginosa physiology, Cystic Fibrosis pathology, Interleukin-8 genetics, NF-kappa B metabolism, Oligodeoxyribonucleotides pharmacology, Promoter Regions, Genetic drug effects, Promoter Regions, Genetic genetics, Transcription, Genetic drug effects
- Abstract
One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3-1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3-1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.
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- 2012
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34. Mapping the transcriptional machinery of the IL-8 gene in human bronchial epithelial cells.
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Bezzerri V, Borgatti M, Finotti A, Tamanini A, Gambari R, and Cabrini G
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- Bronchi immunology, Chromatin Immunoprecipitation, Electrophoretic Mobility Shift Assay, Epithelial Cells immunology, HSP27 Heat-Shock Proteins genetics, HSP27 Heat-Shock Proteins immunology, Humans, Interleukin-8 biosynthesis, Phosphoproteins immunology, Pseudomonas Infections immunology, Pseudomonas aeruginosa, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Ribosomal Protein S6 Kinases, 90-kDa genetics, Ribosomal Protein S6 Kinases, 90-kDa immunology, Transcription, Genetic, Transfection, Gene Expression Regulation genetics, Interleukin-8 genetics, Phosphoproteins genetics, Pseudomonas Infections genetics, Respiratory Mucosa immunology, Signal Transduction immunology
- Abstract
IL-8 released from bronchial epithelial cells infected with Pseudomonas aeruginosa plays a crucial role in the chronic lung pathology of patients affected by cystic fibrosis. Novel anti-inflammatory approaches will benefit from a thorough understanding of the regulatory mechanisms involved in the transcription of this chemokine to identify potential pharmacological targets. We addressed this issue by investigating the role of phosphoproteins and transcription factors (TFs) on transcription of IL-8 gene in the human bronchial epithelial IB3-1, CuFi-1, and Calu-3 cells. P. aeruginosa increased the basal phosphorylation of the ERK1/2 pathway components 90-kDa ribosomal S6 kinase (RSK)1/2 and mitogen- and stress-activated kinase-2 and of the p38 MAPK pathway components p38α/δ/γ and heat shock protein 27 (HSP27). The involvement of these kinases in the expression of IL-8 gene was confirmed with pharmacological inhibitors of ERK1/2, RSK, p38, and HSP27 both at transcription and secretion levels. Transfection of TF decoy oligodeoxynucleotides, designed to interfere with the interaction of the TFs NF-κB, NF-IL6, AP-1, CREB, and CHOP with the corresponding consensus sequences identified in the IL-8 promoter, reduced the P. aeruginosa-dependent transcription of IL-8, suggesting their participation in the transcriptional machinery. Stimulation of IB3-1 cells with IL-1β led to a similar pattern of activation, whereas the pattern of phosphoproteins and of TFs modulated by TNF-α differentiated sharply. In conclusion, the results highlight a novel role for RSK1/2 and HSP27 phosphoproteins and of the cooperative role of the TFs NF-κB, NF-IL6, AP-1, CHOP, and CREB in P. aeruginosa-dependent induction of transcription of the IL-8 gene in human bronchial epithelial cells.
- Published
- 2011
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35. Modulators of sphingolipid metabolism reduce lung inflammation.
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Dechecchi MC, Nicolis E, Mazzi P, Cioffi F, Bezzerri V, Lampronti I, Huang S, Wiszniewski L, Gambari R, Scupoli MT, Berton G, and Cabrini G
- Subjects
- 1-Deoxynojirimycin pharmacology, Animals, Cell Line, Disease Models, Animal, Epithelial Cells immunology, Epithelial Cells metabolism, Epithelial Cells microbiology, Gene Expression Regulation drug effects, Host-Pathogen Interactions drug effects, Humans, Immunity, Innate drug effects, Immunity, Innate genetics, Interleukin-8 genetics, Interleukin-8 metabolism, Lipopolysaccharides, Mice, Mice, Inbred C57BL, Neutrophil Infiltration drug effects, Pneumonia chemically induced, Pneumonia genetics, Pneumonia immunology, Pneumonia metabolism, Pseudomonas aeruginosa pathogenicity, Respiratory Mucosa immunology, Respiratory Mucosa metabolism, Respiratory Mucosa microbiology, 1-Deoxynojirimycin analogs & derivatives, Amitriptyline pharmacology, Anti-Inflammatory Agents pharmacology, Ceramides metabolism, Epithelial Cells drug effects, Inflammation Mediators metabolism, Pneumonia prevention & control, Respiratory Mucosa drug effects
- Abstract
The investigation of novel targets for the treatment of cystic fibrosis (CF) lung inflammation is a major priority, considering that no effective therapy is available for this purpose. Consistent with the evidence that the sphingolipid (SL) ceramide regulates airway inflammation and infection in mice and patients with CF, SLs were identified as targets for treating pulmonary disorders, including CF. Because miglustat, an inhibitor of the synthesis of glycosphingolipids, reduces the Pseudomonas aeruginosa-dependent transcription of the IL-8 gene in bronchial cells, we examined the effects of miglustat and amitriptyline, another drug affecting ceramide metabolism, on the expression of 92 genes implicated in host immune defense. Infection with the P. aeruginosa strain PAO1 up-modulated the expression of 14 (27%) genes in IB3-1 cells and 15 (29%) genes in CF primary respiratory epithelia grown at an air-liquid interface, including chemokines (IL-8, growth-regulated Gro-α/β/γ proteins, and granulocyte chemotactic peptide-2 [GCP-2]), proinflammatory cytokines (IL-1α/β, IL-6, and TNF-α), and the intercellular adhesion molecule-1, nuclear factor kB1, toll like receptor 2, and human defensin B4 genes, confirming that bronchial epithelium is an important source of inflammatory mediators. Both miglustat and amitriptyline reduced the immune response, an effect that paralleled a decrease in the P. aeruginosa-induced accumulation of ceramide. Miglustat (100 mg/kg), given to C57BL/6 mice once daily for a period of 3 consecutive days before lipopolysaccharide (LPS) challenge, strongly reduced the number of neutrophils recruited in the airways and the expression of the keratinocyte-derived chemokine in lung extracts. Collectively, these results indicate that targeting the metabolism of SLs can down-modulate the recruitment of neutrophils into the lung.
- Published
- 2011
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36. Phospholipase C-β3 is a key modulator of IL-8 expression in cystic fibrosis bronchial epithelial cells.
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Bezzerri V, d'Adamo P, Rimessi A, Lanzara C, Crovella S, Nicolis E, Tamanini A, Athanasakis E, Tebon M, Bisoffi G, Drumm ML, Knowles MR, Pinton P, Gasparini P, Berton G, and Cabrini G
- Subjects
- Adenosine Triphosphate pharmacology, Calcium metabolism, Cell Line, Transformed, Cystic Fibrosis metabolism, Cystic Fibrosis pathology, Enzyme Activation, Epithelial Cells microbiology, Gene Expression drug effects, Gene Frequency, Genotype, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Host-Pathogen Interactions, Humans, Interleukin-8 metabolism, Isoenzymes genetics, Isoenzymes metabolism, Lung Diseases genetics, Lung Diseases metabolism, Lung Diseases pathology, Microscopy, Fluorescence, Phospholipase C beta metabolism, Polymorphism, Single Nucleotide, Protein Kinase C genetics, Protein Kinase C metabolism, Protein Kinase C beta, Pseudomonas aeruginosa physiology, RNA Interference, Toll-Like Receptors genetics, Toll-Like Receptors metabolism, Transcription Factor RelA genetics, Transcription Factor RelA metabolism, Cystic Fibrosis genetics, Epithelial Cells metabolism, Interleukin-8 genetics, Phospholipase C beta genetics
- Abstract
Respiratory insufficiency is the major cause of morbidity and mortality in patients affected by cystic fibrosis (CF). An excessive neutrophilic inflammation, mainly orchestrated by the release of IL-8 from bronchial epithelial cells and amplified by chronic bacterial infection with Pseudomonas aeruginosa, leads to progressive tissue destruction. The anti-inflammatory drugs presently used in CF patients have several limitations, indicating the need for identifying novel molecular targets. To address this issue, we preliminarily studied the association of 721 single nucleotide polymorphisms from 135 genes potentially involved in signal transduction implicated in neutrophil recruitment in a cohort of F508del homozygous CF patients with either severe or mild progression of lung disease. The top ranking association was found for a nonsynonymous polymorphism of the phospholipase C-β3 (PLCB3) gene. Studies in bronchial epithelial cells exposed to P. aeruginosa revealed that PLCB3 is implicated in extracellular nucleotide-dependent intracellular calcium signaling, leading to activation of the protein kinase Cα and Cβ and of the nuclear transcription factor NF-κB p65. The proinflammatory pathway regulated by PLCB3 acts by potentiating the Toll-like Receptors' signaling cascade and represents an interesting molecular target to attenuate the excessive recruitment of neutrophils without completely abolishing the inflammatory response.
- Published
- 2011
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37. Trimethylangelicin reduces IL-8 transcription and potentiates CFTR function.
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Tamanini A, Borgatti M, Finotti A, Piccagli L, Bezzerri V, Favia M, Guerra L, Lampronti I, Bianchi N, Dall'Acqua F, Vedaldi D, Salvador A, Fabbri E, Mancini I, Nicolis E, Casavola V, Cabrini G, and Gambari R
- Subjects
- Bronchi cytology, Cell Line, Chlorides metabolism, Dose-Response Relationship, Drug, Epithelial Cells drug effects, Epithelial Cells metabolism, Furocoumarins chemistry, Gene Expression Regulation drug effects, Humans, Interleukin-8 metabolism, NF-kappa B metabolism, Phosphoproteins metabolism, Phosphorylation drug effects, Promoter Regions, Genetic genetics, Protein Binding drug effects, Pseudomonas aeruginosa drug effects, RNA, Messenger genetics, RNA, Messenger metabolism, Trioxsalen chemistry, Trioxsalen pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Furocoumarins pharmacology, Interleukin-8 genetics, Transcription, Genetic drug effects
- Abstract
Chronic inflammatory response in the airway tract of patients affected by cystic fibrosis is characterized by an excessive recruitment of neutrophils to the bronchial lumina, driven by the chemokine interleukin (IL)-8. We previously found that 5-methoxypsoralen reduces Pseudomonas aeruginosa-dependent IL-8 transcription in bronchial epithelial cell lines, with an IC(50) of 10 μM (Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bezzerri V, Bianchi N, Mazzon M, Mancini I, Giri MG, Rizzotti P, Gambari R, Cabrini G. Int Immunopharmacol 9: 1411-1422, 2009). Here, we extended the investigation to analogs of 5-methoxypsoralen, and we found that the most potent effect is obtained with 4,6,4'-trimethylangelicin (TMA), which inhibits P. aeruginosa-dependent IL-8 transcription at nanomolar concentration in IB3-1, CuFi-1, CFBE41o-, and Calu-3 bronchial epithelial cell lines. Analysis of phosphoproteins involved in proinflammatory transmembrane signaling evidenced that TMA reduces the phosphorylation of ribosomal S6 kinase-1 and AKT2/3, which we found indeed involved in P. aeruginosa-dependent activation of IL-8 gene transcription by testing the effect of pharmacological inhibitors. In addition, we found a docking site of TMA into NF-κB by in silico analysis, whereas inhibition of the NF-κB/DNA interactions in vitro by EMSA was observed at high concentrations (10 mM TMA). To further understand whether NF-κB pathway should be considered a target of TMA, chromatin immunoprecipitation was performed, and we observed that TMA (100 nM) preincubated in whole living cells reduced the interaction of NF-κB with the promoter of IL-8 gene. These results suggest that TMA could inhibit IL-8 gene transcription mainly by intervening on driving the recruitment of activated transcription factors on IL-8 gene promoter, as demonstrated here for NF-κB. Although the complete understanding of the mechanism of action of TMA deserves further investigation, an activity of TMA on phosphorylating pathways was already demonstrated by our study. Finally, since psoralens have been shown to potentiate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport, TMA was tested and found to potentiate CFTR-dependent chloride efflux. In conclusion, TMA is a dual-acting compound reducing excessive IL-8 expression and potentiating CFTR function.
- Published
- 2011
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38. A polymorphism in the 5' UTR of the DEFB1 gene is associated with the lung phenotype in F508del homozygous Italian cystic fibrosis patients.
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Crovella S, Segat L, Amato A, Athanasakis E, Bezzerri V, Braggion C, Casciaro R, Castaldo G, Colombo C, Covone AE, De Rose V, Gagliardini R, Lanzara C, Minicucci L, Morgutti M, Nicolis E, Pardo F, Quattrucci S, Raia V, Ravazzolo R, Seia M, Stanzial V, Termini L, Zazzeron L, Cabrini G, and Gasparini P
- Subjects
- Adult, Female, Genotype, Homozygote, Humans, Italy, Male, Phenotype, Polymorphism, Genetic, Young Adult, 5' Untranslated Regions, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Mutation, beta-Defensins genetics
- Abstract
Background: The identification of cystic fibrosis (CF) patients who are at greater risk of lung damage could be clinically valuable. Thus, we attempted to replicate previous findings and verify the possible association between three single nucleotide polymorphisms (SNPs c.-52G>A, c.-44C>G and c.-20G>A) in the 5' untranslated region (5' UTR) of the β defensin 1 (DEFB1) gene and the CF pulmonary phenotype., Methods: Genomic DNA from 92 Italian CF patients enrolled in different regional CF centres was extracted from peripheral blood and genotyped for DEFB1 SNPs using TaqMan(®) allele specific probes. In order to avoid genetic confounding causes that can account for CF phenotype variability, all patients were homozygous for the F508del CFTR mutation, and were then classified on the basis of clinical and functional data as mild lung phenotype (Mp, n=50) or severe lung phenotype patients (Sp, n=42)., Results: For the c.-20G>A SNP, the frequency of the A allele, as well as the AA genotype, were significantly more frequent in Mp than in Sp patients, and thus this was associated with a protective effect against severe pulmonary disease (OR=0.48 and 0.28, respectively). The effect of the c.-20G>A A allele is consistent with a recessive model, and the protective effect against Sp is exerted only when it is present in homozygosis. For the other two SNPs, no differences were observed as allelic and genotypic frequency in the two subgroups of CF patients., Conclusions: Our results, although necessary to be confirmed in larger and multiethnic populations, reinforce DEFB1 as a candidate modifier gene of the CF pulmonary phenotype.
- Published
- 2011
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39. Decoy oligodeoxyribonucleotides and peptide nucleic acids-DNA chimeras targeting nuclear factor kappa-B: inhibition of IL-8 gene expression in cystic fibrosis cells infected with Pseudomonas aeruginosa.
- Author
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Gambari R, Borgatti M, Bezzerri V, Nicolis E, Lampronti I, Dechecchi MC, Mancini I, Tamanini A, and Cabrini G
- Subjects
- Animals, Cell Line, Cystic Fibrosis metabolism, Cystic Fibrosis pathology, Cytokines metabolism, Humans, Inflammation drug therapy, Inflammation metabolism, Inflammation microbiology, Interleukin-8 biosynthesis, Molecular Targeted Therapy, NF-kappa B antagonists & inhibitors, NF-kappa B metabolism, Oligodeoxyribonucleotides genetics, Oligodeoxyribonucleotides pharmacology, Peptide Nucleic Acids genetics, Peptide Nucleic Acids pharmacology, Cystic Fibrosis drug therapy, DNA genetics, Interleukin-8 antagonists & inhibitors, Oligodeoxyribonucleotides therapeutic use, Peptide Nucleic Acids therapeutic use, Pseudomonas aeruginosa
- Abstract
Cystic fibrosis (CF) is characterized by a deep inflammatory process, with production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against IL-8, with the aim of reducing the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. TFD is based on biomolecules mimicking the target sites of transcription factors (TFs) and able to interfere with TF activity when delivered to target cells. Here, we review the inhibitory effects of decoy oligodeoxyribonucleotides (ODNs) on expression of IL-8 gene and secretion of IL-8 by cystic fibrosis cells infected by Pseudomonas aeruginosa. In addition, the effects of decoy molecules based on peptide nucleic acids (PNAs) are discussed. In this respect PNA-DNA-PNA (PDP) chimeras are interesting: (a) unlike PNAs, they can be complexed with liposomes and microspheres; (b) unlike oligodeoxyribonucleotides (ODNs), they are resistant to DNAses, serum and cytoplasmic extracts; (c) unlike PNA/PNA and PNA/DNA hybrids, they are potent decoy molecules. Interestingly, PDP/PDP NF-kappaB decoy chimeras inhibit accumulation of pro-inflammatory mRNAs (including IL-8 mRNA) in P. aeruginosa infected IB3-1, cells reproducing the effects of decoy oligonucleotides. The effects of PDP/PDP chimeras, unlike ODN-based decoys, are observed even in absence of protection with lipofectamine. Since IL-8 is pivotal in pro-inflammatory processes affecting cystic fibrosis, inhibition of its functions might have a clinical relevance., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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40. Virtual screening against p50 NF-kappaB transcription factor for the identification of inhibitors of the NF-kappaB-DNA interaction and expression of NF-kappaB upregulated genes.
- Author
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Piccagli L, Fabbri E, Borgatti M, Bianchi N, Bezzerri V, Mancini I, Nicolis E, Dechecchi CM, Lampronti I, Cabrini G, and Gambari R
- Subjects
- Animals, Cell Line, DNA metabolism, Humans, Interleukin-8 genetics, Mice, Models, Molecular, NF-kappa B p50 Subunit genetics, Protein Binding, Protein Multimerization, RNA, Messenger genetics, Small Molecule Libraries chemistry, Transcription Factor RelB genetics, Gene Expression Regulation drug effects, NF-kappa B p50 Subunit antagonists & inhibitors, NF-kappa B p50 Subunit metabolism, Small Molecule Libraries pharmacology, Transcription Factor RelB antagonists & inhibitors, Transcription Factor RelB metabolism
- Abstract
Virtual screening against NF-kappaB p50 using docking simulations was applied by starting from a three-dimensional (3D) database containing more than 4.6 million commercially available structures. This database was filtered by specifying a subset of commercially available compounds sharing a (2E,Z)-3-(2-hydroxyphenyl)-2-propenoate substructure and relevant druglike properties. Docking to p50 NF-kappaB was performed with a test set of six known inhibitors of NF-kappaB-DNA interactions. In agreement with docking results, the highest-scored compound displayed a high level of inhibitory activity in electrophoretic mobility shift assay (EMSA) experiments (inhibition of NF-kappaB-DNA interactions) and on biological functions dependent on NF-kappaB activity (inhibition of IL-8 gene expression in cystic fibrosis IB3-1 cells). We found that this in silico screening approach is suitable for the identification of low-molecular-weight compounds that inhibit NF-kappaB-DNA interactions and NF-kappaB-dependent functions. Information deduced from the discovery of the new lead compound and its binding mode could result in further lead optimization resulting in more potent NF-kappaB inhibitors.
- Published
- 2009
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41. Modulation of expression of IL-8 gene in bronchial epithelial cells by 5-methoxypsoralen.
- Author
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Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bezzerri V, Bianchi N, Mazzon M, Mancini I, Giri MG, Rizzotti P, Gambari R, and Cabrini G
- Subjects
- 5-Methoxypsoralen, Bronchi pathology, Cell Line, Cell Proliferation, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis immunology, Epithelial Cells immunology, Gene Expression Regulation, Humans, Interleukin-8 genetics, Interleukin-8 immunology, Methoxsalen administration & dosage, Methoxsalen analogs & derivatives, Neutrophils drug effects, Neutrophils pathology, Photosensitizing Agents administration & dosage, Plant Extracts, Pseudomonas aeruginosa pathogenicity, Argemone, Epithelial Cells metabolism, Interleukin-8 metabolism, Pseudomonas aeruginosa immunology, Vernonia
- Abstract
Persistent recruitment of neutrophils in the bronchi of cystic fibrosis patients contributes to airway tissue damage, suggesting the importance of intervening on the expression of the neutrophil chemokine IL-8. Extracts from plants have been investigated to select components able to reduce IL-8 expression in bronchial epithelial cells challenged with Pseudomonas aeruginosa. Extracts and purified components have been added to cells 24 h before pro-inflammatory challenge with P. aeruginosa and IL-8 transcription was quantified in the IB3-1 CF cells in vitro. P. aeruginosa-dependent IL-8 mRNA induction was increased by Argemone mexicana and Vernonia anthelmintica whereas no significant modification of transcription was observed with Aphanamixis polystachya, Lagerstroemia speciosa and Hemidesmus indicus. Finally, inhibition of IL-8 was observed with Polyalthia longifolia (IC50=200 microg/ml) and Aegle marmelos (IC50=20 microg/ml). Compounds from A. marmelos were isolated and identified by GC-MS. No significant effect was observed with butyl-p-tolyl sulphate, whereas the inhibition obtained with 6-methyl-4-chromanone concentration was accompanied by an anti-proliferative effect. On the contrary, 5-methoxypsoralen resulted in IL-8 inhibition at 10 microM concentration, without effects on cell proliferation. In synthesis, 5-methoxypsoralen can be taken into consideration to investigate mechanisms of neutrophil chemotactic signalling and for its potential application in modulating the excessive CF lung inflammation.
- Published
- 2009
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42. Late generation lentiviral vectors: evaluation of inflammatory potential in human airway epithelial cells.
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Copreni E, Nicolis E, Tamanini A, Bezzerri V, Castellani S, Palmieri L, Giri MG, Vella A, Colombatti M, Rizzotti P, Conese M, and Cabrini G
- Subjects
- Adenoviridae genetics, Adenoviridae immunology, Cell Line, Cytokines biosynthesis, Gene Expression Profiling, Humans, Intercellular Adhesion Molecule-1 biosynthesis, Lentivirus genetics, NF-kappa B biosynthesis, Vesiculovirus genetics, Epithelial Cells immunology, Epithelial Cells virology, Genetic Vectors immunology, Lentivirus immunology, Vesiculovirus immunology
- Abstract
Lentiviruses (LVs) are considered one of the most promising tools for gene transfer, however, their potential to induce pro-inflammatory cytokines on delivery into the respiratory tissue remains to be established. Here we tested a third-generation vesicular stomatitis virus (VSV)-G pseudotyped LV vector in the two respiratory epithelial cell lines A549 and CFT1-C2. We observed that the VSV-G LV vector does not induce (a) activation of the nuclear factor (NF)-kappaB, which intervenes in transcription of pro-inflammatory genes; (b) expression of ICAM-1; and (c) transcription of a panel of cytokines, with the exception of a mild and transient (24h) increase of IFN-gamma mRNA. In contrast, an adenovirus-derived vector strongly activated NF-kappaB and different transcripts such as those of ICAM-1, IL-8, RANTES, IP-10, TNF-alpha, IL-6, IL-1 beta. In conclusion, this third-generation VSV-G pseudotyped LV vector does not elicit major pro-inflammatory signals in human airway epithelial cells and appears to be better suited for gene delivery strategies.
- Published
- 2009
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43. Pyrogallol, an active compound from the medicinal plant Emblica officinalis, regulates expression of pro-inflammatory genes in bronchial epithelial cells.
- Author
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Nicolis E, Lampronti I, Dechecchi MC, Borgatti M, Tamanini A, Bianchi N, Bezzerri V, Mancini I, Giri MG, Rizzotti P, Gambari R, and Cabrini G
- Subjects
- Cells, Cultured, Chemokine CXCL1 genetics, Humans, Intercellular Adhesion Molecule-1 genetics, Interleukin-8 genetics, Anti-Inflammatory Agents pharmacology, Bronchi immunology, Gene Expression Regulation drug effects, Phyllanthus emblica chemistry, Pyrogallol pharmacology
- Abstract
The most relevant cause of morbidity and mortality in cystic fibrosis (CF) patients is the lung pathology characterized by chronic infection and inflammation sustained mainly by Pseudomonas aeruginosa (P. aeruginosa). Innovative pharmacological approaches to control the excessive inflammatory process in the lung of CF patients are thought to be beneficial to reduce the extensive airway tissue damage. Medicinal plants from the so-called traditional Asian medicine are attracting a growing interest because of their potential efficacy and safety. Due to the presence of different active compounds in each plant extract, understanding the effect of each component is important to pursue selective and reproducible applications. Extracts from Emblica officinalis (EO) were tested in IB3-1 CF bronchial epithelial cells exposed to the P. aeruginosa laboratory strain PAO1. EO strongly inhibited the PAO1-dependent expression of the neutrophil chemokines IL-8, GRO-alpha, GRO-gamma, of the adhesion molecule ICAM-1 and of the pro-inflammatory cytokine IL-6. Pyrogallol, one of the compounds extracted from EO, inhibited the P. aeruginosa-dependent expression of these pro-inflammatory genes similarly to the whole EO extract, whereas a second compound purified from EO, namely 5-hydroxy-isoquinoline, had no effect. These results identify Pyrogallol as an active compound responsible for the anti-inflammatory effect of EO and suggest to extend the investigation in pre-clinical studies in airway animal models in vivo, to test the efficacy and safety of this molecule in CF chronic lung inflammatory disease.
- Published
- 2008
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44. Anti-inflammatory effect of miglustat in bronchial epithelial cells.
- Author
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Dechecchi MC, Nicolis E, Norez C, Bezzerri V, Borgatti M, Mancini I, Rizzotti P, Ribeiro CM, Gambari R, Becq F, and Cabrini G
- Subjects
- 1-Deoxynojirimycin pharmacology, Bronchi pathology, Cell Culture Techniques, Cell Line, Cystic Fibrosis etiology, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator physiology, Epithelial Cells metabolism, Humans, Inflammation Mediators metabolism, Protein Transport, Respiratory Mucosa metabolism, Respiratory Mucosa pathology, 1-Deoxynojirimycin analogs & derivatives, Bronchi drug effects, Cystic Fibrosis pathology, Enzyme Inhibitors pharmacology, Epithelial Cells drug effects, Respiratory Mucosa drug effects
- Abstract
The role of CFTR deficiency in promoting inflammation remains unclear. Perez et al. [A. Perez, A.C. Issler, C.U. Cotton, T.J. Kelley, A.S. Verkman and P.B. Davis, CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am J Physiol Lung Cell Mol Physiol 2007; 292:L383-L395.] recently demonstrated that the inhibition of function of w/t CFTR produces an inflammatory profile that resembles that observed in CF patients, whereas we found that correction of F508del-CFTR function with MPB-07 down-modulates the inflammatory response to P. aeruginosa in CF bronchial cells [M.C. Dechecchi, E. Nicolis, V. Bezzerri, A. Vella, M. Colombatti, B.M. Assael, et al., MPB-07 reduces the inflammatory response to Pseudomonas aeruginosa in cystic fibrosis bronchial cells. Am J Respir Cell Mol Biol 2007; 36, 615-624.]. Since both evidence support a link between CFTR function and inflammation, we extended our investigation to other F508del-CFTR correctors, such as miglustat (Norez, 2006), an approved drug for Gaucher disease, in comparison with the galactose analogue NB-DGJ. We report here that miglustat but not NB-DGJ restores F508del-CFTR function in CF bronchial epithelial IB3-1 and CuFi-1 cells. Miglustat and NB-DGJ reduce the inflammatory response to P. aeruginosa in both CF and non-CF bronchial cells, indicating that the anti-inflammatory effect is independent of the correction of F508del-CFTR function. Miglustat also inhibits the inflammatory response induced by the supernatant of mucopurulent material obtained from the lower airway tract of cystic fibrosis patients with chronic bacterial colonization (Ribeiro, 2005). Both compounds do not interfere with the adherence of P. aeruginosa to the cells and reduce the expression of IL-8 not only after challenge with P. aeruginosa but also after exposure to TNF alpha or IL-1 beta, suggesting an effect on transduction proteins downstream and in common with different receptors for pathogens. Finally, miglustat has no major effects on overall binding activity of transcription factors NF-kappaBNF-kB and AP-1. Since miglustat is an approved drug, it could be investigated as a novel anti-inflammatory molecule to ameliorate lung inflammation in CF patients.
- Published
- 2008
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45. Docking of molecules identified in bioactive medicinal plants extracts into the p50 NF-kappaB transcription factor: correlation with inhibition of NF-kappaB/DNA interactions and inhibitory effects on IL-8 gene expression.
- Author
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Piccagli L, Fabbri E, Borgatti M, Bezzerri V, Mancini I, Nicolis E, Dechecchi MC, Lampronti I, Cabrini G, and Gambari R
- Subjects
- Aegle chemistry, Animals, Cattle, Cell Line, Cupressus chemistry, Humans, NF-kappa B p50 Subunit antagonists & inhibitors, NF-kappa B p50 Subunit chemistry, Plant Extracts chemistry, Plant Extracts pharmacology, Protein Binding drug effects, Protein Conformation, Pseudomonas drug effects, Pseudomonas metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, DNA metabolism, Gene Expression Regulation drug effects, Interleukin-8 genetics, Models, Molecular, NF-kappa B p50 Subunit metabolism, Plant Extracts metabolism, Plants, Medicinal chemistry
- Abstract
Background: The transcription factor NF-kappaB is a very interesting target molecule for the design on anti-tumor, anti-inflammatory and pro-apoptotic drugs. However, the application of the widely-used molecular docking computational method for the virtual screening of chemical libraries on NF-kappaB is not yet reported in literature. Docking studies on a dataset of 27 molecules from extracts of two different medicinal plants to NF-kappaB-p50 were performed with the purpose of developing a docking protocol fit for the target under study., Results: We enhanced the simple docking procedure by means of a sort of combined target- and ligand-based drug design approach. Advantages of this combination strategy, based on a similarity parameter for the identification of weak binding chemical entities, are illustrated in this work with the discovery of a new lead compound for NF-kappaB. Further biochemical analyses based on EMSA were performed and biological effects were tested on the compound exhibiting the best docking score. All experimental analysis were in fairly good agreement with molecular modeling findings., Conclusion: The results obtained sustain the concept that the docking performance is predictive of a biochemical activity. In this respect, this paper represents the first example of successfully individuation through molecular docking simulations of a promising lead compound for the inhibition of NF-kappaB-p50 biological activity and modulation of the expression of the NF-kB regulated IL8 gene.
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- 2008
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46. Transcription factor oligodeoxynucleotides to NF-kappaB inhibit transcription of IL-8 in bronchial cells.
- Author
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Bezzerri V, Borgatti M, Nicolis E, Lampronti I, Dechecchi MC, Mancini I, Rizzotti P, Gambari R, and Cabrini G
- Subjects
- Bronchi drug effects, Bronchi physiopathology, Cell Nucleus physiology, Cystic Fibrosis genetics, Cystic Fibrosis physiopathology, HIV Long Terminal Repeat, Humans, Pseudomonas aeruginosa physiology, Transcription Factors genetics, Transcription, Genetic drug effects, Interleukin-8 genetics, NF-kappa B genetics, Oligodeoxyribonucleotides pharmacology, Transcription, Genetic genetics
- Abstract
Chronic pulmonary inflammation in patients affected by cystic fibrosis (CF) is characterized by massive bronchial infiltrates of neutrophils, which is sustained by the interaction of pathogens (e.g., Pseudomonas aeruginosa) with surface bronchial cells. To explore new treatment options focused on the reduction of neutrophil chemotaxis, we applied the transcription factor (TF) decoy approach, based on the intracellular delivery of double-stranded oligodeoxynucleotides (ODNs) causing inhibition of the binding of TF-related proteins to the different consensus sequences in the promoter of specific genes. In CF bronchial IB3-1 cells, P. aeruginosa induced transcription of the neutrophil chemokines IL-8 and GRO-gamma, of the adhesion molecule intercellular adhesion molecule (ICAM)-1, and of the cytokines IL-1beta and IL-6. Since consensus sequences for the TF, NF-kappaB, are contained in the promoters of all these genes, IB3-1, CuFi-1, Beas-2B, and CaLu-3 cells were transfected with double-stranded TF "decoy" ODNs mimicking different NF-kappaB consensus sequences. IL-8 NF-kappaB decoy ODN partially inhibited the P. aeruginosa-dependent transcription of IL-8, GRO-gamma, and IL-6, whereas decoy ODNs to both HIV-1 long terminal repeat and Igk produced a strong, 80 to 85% inhibition of transcription of IL-8, without reducing that of GRO-gamma, ICAM-1, IL-1beta, and IL-6. In conclusion, intracellular delivery of "decoy" molecules aimed to compete with the TF, NF-kappaB, is a promising strategy to obtain inhibition of IL-8 gene transcription.
- Published
- 2008
- Full Text
- View/download PDF
47. Induction of IL-6 gene expression in a CF bronchial epithelial cell line by Pseudomonas aeruginosa is dependent on transcription factors belonging to the Sp1 superfamily.
- Author
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Borgatti M, Bezzerri V, Mancini I, Nicolis E, Dechecchi MC, Lampronti I, Rizzotti P, Cabrini G, and Gambari R
- Subjects
- Cell Line, Epithelial Cells immunology, Epithelial Cells microbiology, Humans, Bronchi immunology, Bronchi microbiology, Cystic Fibrosis immunology, Cystic Fibrosis microbiology, Interleukin-6 immunology, Pseudomonas aeruginosa physiology, Sp1 Transcription Factor immunology, Transcription Factors immunology
- Abstract
Cystic fibrosis (CF) is a genetic disease characterized by chronic bacterial lung infection, most commonly sustained by Pseudomonas aeruginosa. Upon infection, elevated concentrations of pro-inflammatory cytokines (i.e. IL-6 and IL1beta) and chemokines (i.e. IL-8 and GROgamma) are found in the bronchoalveolar fluid of CF patients. We report in this paper that: (a) IL-8, IL-6, IL-1beta, ICAM-1, and GRO-gamma genes are upregulated following infection of CF bronchial epithelial IB3-1 cells with P. aeruginosa; (b) Sp1 transcription factor activity is induced following infection of the cystic fibrosis IB3-1 and CuFi-1 cell lines; (c) inhibition of Sp1 activity using transcription factor decoy molecules leads to inhibition of the expression of IL-6 gene. From the theoretical point of view, our results demonstrate that Sp1 transcription factor activity is induced following infection of CF cells with P. aeruginosa, and that this effect is important in the activation of IL-6 gene transcription. From the practical point of view, our data sustain the potential use of decoy molecules targeting the transcription factor Sp1 to control a relevant molecule involved in the inflammatory process associated with the cystic fibrosis airway pathology.
- Published
- 2007
- Full Text
- View/download PDF
48. MPB-07 reduces the inflammatory response to Pseudomonas aeruginosa in cystic fibrosis bronchial cells.
- Author
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Dechecchi MC, Nicolis E, Bezzerri V, Vella A, Colombatti M, Assael BM, Mettey Y, Borgatti M, Mancini I, Gambari R, Becq F, and Cabrini G
- Subjects
- Bronchi microbiology, Chlorides metabolism, Cyclic AMP metabolism, Cystic Fibrosis immunology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Epithelial Cells drug effects, Epithelial Cells immunology, Epithelial Cells microbiology, Epithelial Cells pathology, Gene Expression Regulation drug effects, Genistein pharmacology, Humans, Intercellular Adhesion Molecule-1 genetics, Intercellular Adhesion Molecule-1 metabolism, Interleukin-8 genetics, Interleukin-8 metabolism, NF-kappa B metabolism, Protein Binding drug effects, Pseudomonas Infections, RNA, Messenger genetics, RNA, Messenger metabolism, Transcription Factor AP-1 metabolism, Transcription, Genetic drug effects, Anti-Inflammatory Agents pharmacology, Bronchi immunology, Bronchi pathology, Cystic Fibrosis microbiology, Cystic Fibrosis pathology, Pseudomonas aeruginosa immunology, Quinolizines pharmacology
- Abstract
Chronic lung inflammation in cystic fibrosis (CF) is specifically characterized by predominant endobronchial neutrophil infiltrates, colonization by Pseudomonas aeruginosa, and elevated levels of cytokines and chemokines, first of all IL-8. The extensive inflammatory process in CF lungs is the basis of progressive tissue damage and is largely considered detrimental, making antiinflammatory approaches a relevant therapeutic target. This neutrophil-dominated inflammation seems to be related to an excessive proinflammatory signaling, originating from the same surface epithelial cells expressing the defective CF transmembrane conductance regulator (CFTR) protein, although the underlying mechanisms have not been completely elucidated. To investigate the relationship between defective CFTR and the inflammatory response to P. aeruginosa in CF airway cells, we studied the effect of the DeltaF508 CFTR corrector, benzo(c)quinolizinium (MPB)-07 (Dormer et al., J Cell Science 2001;114:4073-4081). CF bronchial epithelial IB3-1 and CuFi-1 cells overproduced the inflammatory molecules, IL-8 and intercellular adhesion molecule (ICAM)-1, in response to P. aeruginosa, compared with the wild-type, CFTR-expressing bronchial cells, S9, and NuLi-1 cells. In both IB3-1 and CuFi-1 cells, the corrector MPB-07 dramatically reduces the IL-8 and ICAM-1 mRNA expression elicited by P. aeruginosa infection. Correction of CFTR-dependent Cl- efflux was confirmed in MPB-07-treated IB3-1 and CuFi-1 cells. In conclusion, the DeltaF508 CFTR corrector MPB-07 produces an antiinflammatory effect in CF bronchial cells exposed to P. aeruginosa in vitro.
- Published
- 2007
- Full Text
- View/download PDF
49. Interaction of adenovirus type 5 fiber with the coxsackievirus and adenovirus receptor activates inflammatory response in human respiratory cells.
- Author
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Tamanini A, Nicolis E, Bonizzato A, Bezzerri V, Melotti P, Assael BM, and Cabrini G
- Subjects
- Adenoviruses, Human metabolism, Blotting, Western, Capsid Proteins immunology, Cell Line, Cell Nucleus chemistry, Chemokine CCL5 genetics, Chemokine CXCL1, Chemokine CXCL10, Chemokines, CXC genetics, Coxsackie and Adenovirus Receptor-Like Membrane Protein, Cytoplasm chemistry, Gene Expression Regulation, Humans, Interleukin-8 genetics, JNK Mitogen-Activated Protein Kinases metabolism, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, NF-kappa B metabolism, RNA, Messenger analysis, Respiratory Mucosa cytology, Transcription, Genetic, Adenoviruses, Human immunology, Capsid Proteins metabolism, Epithelial Cells virology, Receptors, Virus metabolism, Respiratory Mucosa virology
- Abstract
The innate immune response to adenovirus (Ad)-derived gene transfer vectors has been shown to initiate immediately after interaction of Ad with respiratory epithelial cells, through the induction of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and JNK mitogen-activated protein kinase (MAPK), nuclear factor kappaB (NF-kappaB), and different proinflammatory genes. Ad serotypes 2 or 5 (Ad2/5) enter respiratory epithelia after initial binding of fiber with the coxsackievirus-adenovirus receptor (CAR) or, alternatively, with cell surface heparan sulfate glycosaminoglycans. Ad2/5 internalization is triggered by binding of penton base to cellular RGD-binding integrins. Here we investigated the role of the Ad5 surface domain proteins constituting the vector capsid, namely, the fiber, the penton base, and the hexon, on the transmembrane signals leading to the transcription of the different proinflammatory genes in the human respiratory A549 cell line. Interaction of Ad fiber with CAR activates both ERK1/2 and JNK MAPK and the nuclear translocation of NF-kappaB, whereas no activation was observed after exposing A549 cells to penton base and hexon proteins. Moreover, interaction of Ad fiber with CAR, but not heparan sulfate proteoglycans, promotes transcription of the chemokines interleukin-8, GRO-alpha, GRO-gamma, RANTES, and interferon-inducible protein 10. These results identify the binding of Ad5 fiber with the cellular CAR as a key proinflammatory activation event in epithelial respiratory cells that is independent of the transcription of Ad5 genes.
- Published
- 2006
- Full Text
- View/download PDF
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