37 results on '"Asperti C"'
Search Results
2. The role of skin tests with polyethylene glycol and polysorbate 80 in the vaccination campaign for COVID-19: results from an Italian multicenter survey
- Author
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Montera, M.C., primary, Giordano, A., additional, Asperti, C., additional, Aruanno, A., additional, Barzaghi, C.E., additional, Bignardi, D., additional, and et, al., additional
- Published
- 2023
- Full Text
- View/download PDF
3. The role of skin tests with polyethylene glycol and polysorbate 80 in the vaccination campaign for COVID-19: results from an Italian multicenter survey
- Author
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Montera, M C, Giordano, A, Asperti, C, Aruanno, Arianna, Barzaghi, C E, Bignardi, D, Borrelli, P, Bommarito, L, Busa, Mattia Fernando, Calafiore, P, Carusi, V, Cinquini, M, Cortellini, G, Cocchi, Riccardo, D'Auria, F, De Caro, F, Demonte, A, Di Leo, E, Di Lizia, M, Di Rienzo, A, Fumagalli, Francesca, Kihlgren, P, Lodi Rizzini, F, Macchia, D, Manzotti, G, Marra, A M, Mileto, P, Mietta, S, Montagni, M, Nettis, E, Nucera, Eleonora, Peveri, S, Pivetta, D, Pirisi, M, Ramirez, G A, Rivolta, F, Rizzi, Angela, Savoia, Andrea Alessio, Pedicini, A, Scarpa, A, Zisa, G, Yacoub, M-R, Aruanno, A, Busa, M, Cocchi, R, Fumagalli, F, Nucera, E (ORCID:0000-0002-0565-7680), Rizzi, A (ORCID:0000-0002-6795-746X), Savoia, A, Montera, M C, Giordano, A, Asperti, C, Aruanno, Arianna, Barzaghi, C E, Bignardi, D, Borrelli, P, Bommarito, L, Busa, Mattia Fernando, Calafiore, P, Carusi, V, Cinquini, M, Cortellini, G, Cocchi, Riccardo, D'Auria, F, De Caro, F, Demonte, A, Di Leo, E, Di Lizia, M, Di Rienzo, A, Fumagalli, Francesca, Kihlgren, P, Lodi Rizzini, F, Macchia, D, Manzotti, G, Marra, A M, Mileto, P, Mietta, S, Montagni, M, Nettis, E, Nucera, Eleonora, Peveri, S, Pivetta, D, Pirisi, M, Ramirez, G A, Rivolta, F, Rizzi, Angela, Savoia, Andrea Alessio, Pedicini, A, Scarpa, A, Zisa, G, Yacoub, M-R, Aruanno, A, Busa, M, Cocchi, R, Fumagalli, F, Nucera, E (ORCID:0000-0002-0565-7680), Rizzi, A (ORCID:0000-0002-6795-746X), and Savoia, A
- Abstract
Background. International guidelines suggested skin tests with Polyethylene-glycol (PEG) and polysorbate 80 (PS-80), to investigate a possible hypersensitivity to these excipients either to identify subjects at risk of developing allergic reactions to Covid-19 vaccines, or in patients with suspected IgE mediated hypersensitivity reactions (HR) to the Covid-19 vaccine. The main purpose of this study was to investigate the prevalence of PEG and PS sensitization in patients with a clinical history of HR to drugs containing PEG/PS and in patients with a suspected Covid-19 vaccine immediate HR. Methods. This was a multicenter retrospective study conducted by allergists belonging to 20 Italian medical centers. Skin testing was performed in 531 patients with either a clinical history of suspected hypersensitivity reaction (HR) to drugs containing PEG and/or PS-80 (group 1:362 patient) or a suspected HR to Covid-19 vaccines (group 2: 169 patient), as suggested by the AAIITO/SIAAIC guidelines for the "management of patients at risk of allergic reactions to Covid-19 vaccines" [1]. Results. 10/362 (0.02%) had positive skin test to one or both excipients in group 1, 12/169 (7.1%) in group 2 (p less than 0.01). In group 2 HRs to Covid-19 vaccines were immediate in 10/12 of cases and anaphylaxis occurred in 4/12 of patients. Conclusions. The positivity of skin test with PEG and or PS before vaccination is extremely rare and mostly replaceable by an accurate clinical history. Sensitization to PEG and PS has to be investigated in patients with a previous immediate HR to a Covid-19 vaccine, in particular in patients with anaphylaxis.
- Published
- 2023
4. AB0448 ADVERSE PREGNANCY OUTCOMES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS: THE ROLE OF THERAPY.
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Cilona, M. B., primary, Canti, V., additional, Dalpiaz, I., additional, De Lorenzo, R., additional, Inguscio, G., additional, Asperti, C., additional, Rosa, S., additional, Castiglioni, M. T., additional, and Rovere-Querini, P., additional
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- 2022
- Full Text
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5. Liprin-α1 regulates breast cancer cell invasion by affecting cell motility, invadopodia and extracellular matrix degradation
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Astro, V, Asperti, C, Cangi, G, Doglioni, C, and de Curtis, I
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- 2011
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6. The tumor vessel targeting agent NGR-TNF controls the different stages of the tumorigenic process in transgenic mice by distinct mechanisms
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Porcellini S., Asperti C., Valentinis B., Tiziano E., Mangia P., Rizzardi G. P, C. Traversari, BORDIGNON , CLAUDIO, Porcellini, S., Asperti, C., Valentinis, B., Tiziano, E., Mangia, P., Bordignon, Claudio, Rizzardi G., P, and C., Traversari
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immune system ,angiogenesi ,vascular targeting - Abstract
NGR-TNF is a vascular targeting agent in advanced clinical development, coupling tumor necrosis factor-a (TNF) with the CNGRCG peptide, which targets a CD13 isoform specifically expressed by angiogenic vessels. Antitumor efficacy of NGR-TNF has been described in different transplantation tumor models. Nevertheless, the mechanism underlying its activity is not fully understood. In the wild type and in the immunodeficient (RAG¡/¡) RIP1-Tag2 models of multistage pancreatic carcinogenesis, we demonstrate that CD13 is highly expressed on endothelial cells of hyperplastic and angiogenic islets, whereas its expression is down regulated in tumors where it partially colocalize with pericytes. In vivo CNGRCG peptides coupled to fluorescent nanoparticles (quantum dots) bind to CD13 and colocalize with anti-CD31, in pancreatic islets. At early stage, low doses of NGR-murine (m)TNF have a direct cytotoxic effect inducing endothelial cell apoptosis, reducing vessel density and eventually inhibiting the development of angiogenic islets. At a later stage, NGRmTNF is able to reduce tumor growth inducing vascular normalization, exclusively when treatment is carried out in the immunocompetent mice. Interestingly, NGR-mTNF-treated tumors from these mice are characterized by CD8C T cell infiltration. At molecular level, overexpression of genes involved in vessels normalization was detected only in NGRmTNF- treated tumors from immunocompetent mice. These findings identified a new mechanism of action of NGR-mTNF, providing support for the development of new therapeutic strategies combining chemotherapy or active/adoptive immunotherapies to low dose NGR-TNF treatment.
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- 2015
7. Erratum: Liprin-α1 regulates breast cancer cell invasion by affecting cell motility, invadopodia and extracellular matrix degradation
- Author
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Astro, V, Asperti, C, Cangi, M G, Doglioni, C, and de Curtis, I
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- 2011
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8. EFFICACY AND SAFETY OF COVID-19-SPECIFIC TREATMENTS IN SLE: A MULTICENTRE CASECONTROL STUDY.
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Ramirez, G. A., Gerosa, M., Arroyo-Sánchez, D., Asperti, C., Argolini, L. M., Gallina, G., Bellocchi, C., Cornalba, M., Scotti, I., Suardi, I., Beretta, L., Moroni, L., Bozzolo, E., Caporali, R., and Dagna, L.
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- 2023
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9. Liprin-alpha1 affects the distribution of low-affinity beta1 integrins and stabilizes their permanence at the cell surface
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ASPERTI C, PETTINATO E, DE CURTIS , IVANMATTEO, Asperti, C, Pettinato, E, and DE CURTIS, Ivanmatteo
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Focal adhesions ,Liprin ,Internalization - Abstract
Integrins mediate the interaction between cells and extracellular matrix by assembling adhesive structures that need to be dynamically modulated to allow cell motility. We have recently identified liprin-alpha1 as an essential regulator of integrin dynamics required for efficient cell motility. Here we investigated the effects of liprin-alpha1 expression on beta1 integrin receptors. We found that increased levels of liprin-alpha1 affected the localization of inactive, low-affinity integrins, while increasing the average size of beta1 integrin-positive focal adhesions. Although a direct interaction between beta1 integrins and liprin-alpha1 could not be revealed biochemically, a striking colocalization between redistributed inactive beta1 integrins and liprin-alpha1 was observed. The tight association of overexpressed and endogenous liprin-alpha1 to the cytoplasmic side of the ventral plasma membrane suggested a possible role of liprin in stabilizing integrin receptors at the cell surface. In support of this hypothesis, we demonstrated an inhibitory effect of liprin overexpression on antibody-induced beta1 integrin internalization. On the other hand, depletion of endogenous liprin-alpha by small interfering RNA increased the rate of integrin internalization. Overall, these results support the hypothesis that liprin-alpha1 exerts its action on focal adhesion turnover by influencing the localization and stability of integrin receptors at the cell surface.
- Published
- 2010
10. Liprin-alpha1 promotes cell spreading on the extracellular matrix by affecting the distribution of activated integrins
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ASPERTI C, ASTRO V, TOTARO A, PARIS S, DE CURTIS , IVANMATTEO, Asperti, C, Astro, V, Totaro, A, Paris, S, and DE CURTIS, Ivanmatteo
- Published
- 2009
11. Liprin-α1 regulates breast cancer cell invasion by affecting cell motility, invadopodia and extracellular matrix degradation
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Astro, V, primary, Asperti, C, additional, Cangi, G, additional, Doglioni, C, additional, and de Curtis, I, additional
- Published
- 2010
- Full Text
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12. C. CIOCIOLA, L’impazienza di più lunghi conversari. Dal carteggio di Carlo Dionisotti con Gianfranco Contini, in Il mondo e la storia. Studi in onore di Claudia Villa, a cura di F. LO MONACO e L.C. ROSSI, Firenze, SISMEL-Edizioni del Galluzzo, 2014, pp. 179-185
- Author
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CIOCIOLA, CLAUDIO, G. ALBANESE, S. ASPERTI, C. BOLOGNA, V. VON BUREN, S. CARRAI, M. CENTANNI, P. CHIESA, C. CIOCIOLA, M. FERRARI, F. LO MONACO, M. PASSALACQUA, M. REEVE, M. REFOLIOSI, L.C. ROISSI, F. STELLA, F. LO MONACO, L.C. ROSSI, and Ciociola, Claudio
- Subjects
FILOLOGIA ITALIANA, FILOLOGIA ROMANZA, STORIA DELLA FILOLOGIA, GIANFRANCO CONTINI, CARLO DIONISOTTI - Abstract
Presentazione e edizione di quattro lettere del carteggio tra Carlo Dionisotti e Gianfranco Contini, del quale l'autore sta preparando l'edizione integrale.
- Published
- 2014
13. Liprin-α1 regulates breast cancer cell invasion by affecting cell motility, invadopodia and extracellular matrix degradation
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V, Astro, C, Asperti, M G, Cangi, G, Cangi, C, Doglioni, I, de Curtis, Astro, V., Asperti, C., Cangi, G., Doglioni, Claudio, and de Curtis, I.
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Cancer Research ,Invadopodium ,Cell ,Motility ,Extracellular matrix ,Cell Movement ,Cell Line, Tumor ,Genetics ,medicine ,Humans ,Molecular Biology ,invadopodia ,Adaptor Proteins, Signal Transducing ,biology ,Cell migration ,invasion ,Cell biology ,Extracellular Matrix ,medicine.anatomical_structure ,liprin ,Invadopodia ,Immunology ,biology.protein ,Female ,Extracellular Matrix Degradation ,Cortactin - Abstract
Migration of cells and degradation of the extracellular matrix (ECM) are required for efficient tumor cell invasion, but the underlying molecular mechanisms are only partially known. The PPFIA1 gene for liprin-alpha 1 is frequently amplified in human breast cancers. We recently demonstrated that liprin-alpha 1 is an important regulator of cell edge dynamics during motility. We show, herein, that the liprin-alpha 1 protein is highly expressed in human breast tumors. Functional analysis shows that liprin-alpha 1 is specifically required for the migration and invasion of highly invasive human breast cancer MDA-MB-231 cells. We used time-lapse analysis to demonstrate defects in the motility of liprin-alpha 1-depleted cells that include a striking instability of the lamellipodia. Liprin-alpha 1 levels altered by either RNA interference or overexpression affected also cell spreading and the number of invadopodia per cell, but not the density of invadopodia per unit of surface area. On the other hand, silencing of liprin-alpha 1 inhibited the degradation of the ECM, whereas its overexpression enhanced degradation, resulting in significant negative or positive effects, respectively, on the area of degradation per invadopodium. Transfection of fluorescent-labeled cortactin revealed that depletion of liprin-alpha 1 also affected the assembly and disassembly of invadopodia, with decrease of their lifetime. Our results strongly support a novel important role of liprin-alpha 1 in the regulation of human tumor cell invasion. Oncogene (2011) 30, 1841-1849; doi:10.1038/onc.2010.562; published online 13 December 2010 OI DE CURTIS, Ivanmatteo/0000-0002-7651-1515 Z8 0 ZR 0 ZS 0 ZB 12 Migration of cells and degradation of the extracellular matrix (ECM) are required for efficient tumor cell invasion, but the underlying molecular mechanisms are only partially known. The PPFIA1 gene for liprin-α1 is frequently amplified in human breast cancers. We recently demonstrated that liprin-α1 is an important regulator of cell edge dynamics during motility. We show, herein, that the liprin-α1 protein is highly expressed in human breast tumors. Functional analysis shows that liprin-α1 is specifically required for the migration and invasion of highly invasive human breast cancer MDA-MB-231 cells. We used time-lapse analysis to demonstrate defects in the motility of liprin-α1-depleted cells that include a striking instability of the lamellipodia. Liprin-α1 levels altered by either RNA interference or overexpression affected also cell spreading and the number of invadopodia per cell, but not the density of invadopodia per unit of surface area. On the other hand, silencing of liprin-α1 inhibited the degradation of the ECM, whereas its overexpression enhanced degradation, resulting in significant negative or positive effects, respectively, on the area of degradation per invadopodium. Transfection of fluorescent-labeled cortactin revealed that depletion of liprin-α1 also affected the assembly and disassembly of invadopodia, with decrease of their lifetime. Our results strongly support a novel important role of liprin-α1 in the regulation of human tumor cell invasion.
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- 2010
14. Identification of an Intramolecular Interaction Important for the Regulation of GIT1 Functions
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Ivan de Curtis, Antonio Totaro, Claudia Asperti, Simona Paris, Totaro, A, Paris, S, Asperti, C, and DE CURTIS, Ivanmatteo
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Protein domain ,Cell Cycle Proteins ,Plasma protein binding ,Biology ,Cell Line ,Chlorocebus aethiops ,Cell Adhesion ,Animals ,Humans ,Cell adhesion ,Cell Cycle Protein ,Molecular Biology ,Paxillin ,Adaptor Proteins, Signal Transducing ,COS cells ,C-terminus ,Cell Biology ,Articles ,Subcellular localization ,Cell biology ,Fibronectins ,COS Cells ,Mutation ,biology.protein ,Protein Binding - Abstract
G-protein coupled receptor kinase-interacting protein (GIT) proteins include an N-terminal Arf GTPase-activating protein domain, and a C terminus that binds proteins regulating adhesion and motility. Given their ability to form large molecular assemblies, the GIT1 protein must be tightly regulated. However, the mechanisms regulating GIT1 functions are poorly characterized. We found that carboxy-terminal–truncated fragments of GIT1 bind their partners with higher efficiency compared with the full-length GIT1. We have explored the hypothesis that GIT1 is regulated by an intramolecular mechanism, and we identified two distinct intramolecular interactions between the N and C terminus of GIT1. The release of these interactions increases binding of GIT1 to paxillin and liprin-α, and it correlates with effects on cell spreading. Analysis of cells plated on fibronectin has shown that different deletion mutants of GIT1 either enhance or inhibit spreading, depending on their subcellular localization. Moreover, although the association between βPIX and GIT1 is insufficient to activate GIT1 binding to paxillin, binding of a PAK1 fragment including the βPIX-binding domain enhances paxillin binding to βPIX/GIT1, indicating that p21-activated kinase can activate the binding of paxillin to GIT1 by a kinase-independent mechanism. The release of the identified intramolecular interaction seems to be an important mechanism for the regulation of GIT1 functions.
- Published
- 2007
15. Biochemical and Functional Characterization of the Interaction between Liprin-α1 and GIT1: Implications for the Regulation of Cell Motility
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Ivan de Curtis, Angela Bachi, Emanuela Pettinato, Veronica Astro, Claudia Asperti, Simona Paris, Asperti, C, Astro, V, Pettinato, E, Paris, S, Bachi, A, and DE CURTIS, Ivanmatteo
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Scaffold protein ,Integrins ,Cell ,lcsh:Medicine ,Motility ,Cell Cycle Proteins ,Plasma protein binding ,Biology ,Focal adhesion ,cell spreading ,Cell Movement ,Molecular Cell Biology ,Chlorocebus aethiops ,Cell Adhesion ,medicine ,Signaling in Cellular Processes ,Animals ,Guanine Nucleotide Exchange Factors ,Humans ,focal adhesion ,lcsh:Science ,Cell Cycle Protein ,Cell Shape ,Paxillin ,Adaptor Proteins, Signal Transducing ,Multidisciplinary ,lcsh:R ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,COS Cells ,biology.protein ,lcsh:Q ,Lamellipodium ,signaling ,Cell Movement Signaling ,Rho Guanine Nucleotide Exchange Factors ,Research Article ,Signal Transduction ,HeLa Cells ,Protein Binding ,Subcellular Fractions - Abstract
We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.
- Published
- 2011
16. Impact of asthma on severe food-induced allergic reactions: a systematic review and meta-analysis.
- Author
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Cilona MB, Ramirez GA, Asperti C, Ferlito A, Benanti G, Nannipieri S, Abdul Hadi RM, Capellini C, Dagna L, Cottini M, and Yacoub MR
- Abstract
Summary: Background. Food allergy can range from mild to severe, life-threatening reactions with various symptoms and organ involvement. The impact of asthma on severe food-induced allergic reactions is not completely understood. In the hypothesis that asthma increases the risk of severe food-induced allergic reactions, the aim of this study is to compare the incidence of severe food-induced allergic reactions in patients with history of asthma compared with patients without history of asthma. Methods. We performed a systematic research on electronic databases, including PubMed, Scopus, and Web of Science. Observational studies, studies reporting medical characteristics of patients diagnosed with food allergy, and studies reporting medical history of patients with allergic reactions were included. The primary outcome was the incidence of severe food-induced allergic reactions in patients with history of asthma compared with patients without history of asthma. The protocol of this review was registered in PROSPERO (CRD42023448293). Results. Eight studies with a total of 90,367 patients met the inclusion criteria and were included, with a total population of 28,166 of patients with food allergy. The incidence of severe food-induced allergic reactions in patients with history of asthma compared with patients without history of asthma was increased (OR = 1.28; 95% CI 1.03-1.59; p = 0.03; I2 = 59%). Conclusions. Individuals with both food allergy and asthma are at high risk of severe, potentially fatal allergic reactions. Healthcare professionals should prioritize prevention and management strategies for these subjects.
- Published
- 2024
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17. The role of skin tests with polyethylene glycol and polysorbate 80 in the vaccination campaign for COVID-19: results from an Italian multicenter survey.
- Author
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Montera MC, Giordano A, Asperti C, Aruanno A, Barzaghi CE, Bignardi D, Borrelli P, Bommarito L, Busa M, Calafiore P, Carusi V, Cinquini M, Cortellini G, Cocchi R, D'Auria F, De Caro F, Demonte A, Di Leo E, Di Lizia M, Di Rienzo A, Fumagalli F, Kihlgren P, Lodi Rizzini F, Macchia D, Manzotti G, Marra AM, Mileto P, Mietta S, Montagni M, Nettis E, Nucera E, Peveri S, Pivetta D, Pirisi M, Ramirez GA, Rivolta F, Rizzi A, Savoia A, Pedicini A, Scarpa A, Zambito M, Zisa G, and Yacoub MR
- Subjects
- Humans, Polysorbates adverse effects, Polyethylene Glycols adverse effects, COVID-19 Vaccines adverse effects, Excipients adverse effects, Retrospective Studies, Immunization Programs, Skin Tests, Italy epidemiology, COVID-19 epidemiology, COVID-19 prevention & control, Anaphylaxis diagnosis, Anaphylaxis epidemiology, Hypersensitivity, Immediate
- Abstract
Summary: Background. International guidelines suggested skin tests with Polyethylene-glycol (PEG) and polysorbate 80 (PS-80), to investigate a possible hypersensitivity to these excipients either to identify subjects at risk of developing allergic reactions to Covid-19 vaccines, or in patients with suspected IgE mediated hypersensitivity reactions (HR) to the Covid-19 vaccine. The main purpose of this study was to investigate the prevalence of PEG and PS sensitization in patients with a clinical history of HR to drugs containing PEG/PS and in patients with a suspected Covid-19 vaccine immediate HR. Methods. This was a multicenter retrospective study conducted by allergists belonging to 20 Italian medical centers. Skin testing was performed in 531 patients with either a clinical history of suspected hypersensitivity reaction (HR) to drugs containing PEG and/or PS-80 (group 1:362 patient) or a suspected HR to Covid-19 vaccines (group 2: 169 patient), as suggested by the AAIITO/SIAAIC guidelines for the "management of patients at risk of allergic reactions to Covid-19 vaccines" [1]. Results. 10/362 (0.02%) had positive skin test to one or both excipients in group 1, 12/169 (7.1%) in group 2 (p less than 0.01). In group 2 HRs to Covid-19 vaccines were immediate in 10/12 of cases and anaphylaxis occurred in 4/12 of patients. Conclusions. The positivity of skin test with PEG and or PS before vaccination is extremely rare and mostly replaceable by an accurate clinical history. Sensitization to PEG and PS has to be investigated in patients with a previous immediate HR to a Covid-19 vaccine, in particular in patients with anaphylaxis.
- Published
- 2024
- Full Text
- View/download PDF
18. Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4 + T-cells for the treatment of Hyper IgM1.
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Canarutto D, Asperti C, Vavassori V, Porcellini S, Rovelli E, Paulis M, Ferrari S, Varesi A, Fiumara M, Jacob A, Sergi Sergi L, Visigalli I, Ferrua F, González-Granado LI, Lougaris V, Finocchi A, Villa A, Radrizzani M, and Naldini L
- Subjects
- Humans, Genome, T-Lymphocytes, CD4-Positive T-Lymphocytes, CRISPR-Cas Systems, Gene Editing methods
- Abstract
Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4
+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)- Published
- 2023
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19. IgE response to Aed al 13 and Aed al 14 recombinant allergens from Aedes albopictus saliva in humans.
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Arnoldi I, Villa M, Mancini G, Varotto-Boccazzi I, Yacoub MR, Asperti C, Mascheri A, Casiraghi S, Epis S, Bandi C, Dagna L, Forneris F, and Gabrieli P
- Abstract
Background: Mosquito bite is normally associated with mild allergic responses, but severe localized or systemic reactions are also possible. Reliable tools for the diagnosis of mosquito allergy are still unavailable. Here, we investigated the IgE response to 3 potential salivary allergens identified in the saliva of the tiger mosquito Aedes albopictus ., Methods: Serum from 55 adult individuals (28 controls and 27 allergic people), were analysed using an in-house Enzyme Linked ImmunoSorbent Assay (ELISA) against the Salivary Gland Extract (SGE) and the recombinant proteins alb D7l2 (Aed al 2), alb Antigen5-3 (Aed al 13) and alb LIPS-2 (Aed al 14)., Results: Fifteen of the 27 (56%) individuals having hypersensitive reactions to mosquito bites had IgE serum levels recognizing SGE. Negative sera did not show detectable levels of IgE targeting the SGE from the most common sympatric mosquito Culex pipiens. Among the positive individuals, 2 subjects displayed IgE targeting Aed al 2 (13%), while IgE recognizing Aed al 13 and Aed al 14 were detected in ten (67%) and seven (47%) individuals, respectively. Two sera from non-hypersensitive subjects had detectable levels of IgE targeting Aed al 13, suggesting possible cross-reaction with the homologue salivary proteins of multiple mosquito species or, more generally, of hematophagous insects., Conclusions: Our results indicate that Aed al 13 and Aed al 14 hold the potential to be developed as tools for the diagnosis of allergy to Ae. albopictus bites. Such tools would facilitate epidemiological studies on tiger mosquito allergy in humans and might foster the development of further protein-based assays to investigate cross-species allergies., Competing Interests: The authors report no competing interests., (© 2023 The Author(s).)
- Published
- 2023
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20. Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells.
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Vavassori V, Ferrari S, Beretta S, Asperti C, Albano L, Annoni A, Gaddoni C, Varesi A, Soldi M, Cuomo A, Bonaldi T, Radrizzani M, Merelli I, and Naldini L
- Subjects
- Humans, Hematopoietic Stem Cells metabolism, RNA metabolism, CRISPR-Cas Systems, Gene Editing methods, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism
- Abstract
Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses the delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo via electroporation, and when aiming for homology-driven correction of a DNA template, often provided by viral vectors together with a nuclease editor. Although HSPCs activate a robust p53-dependent DNA damage response upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multiomics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism, and inducing an inflammatory response. Nuclease RNA delivery using lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding a higher number of edited cells compared with using electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared with electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for the treatment of human diseases., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
- Published
- 2023
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- View/download PDF
21. Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo .
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Asperti C, Canarutto D, Porcellini S, Sanvito F, Cecere F, Vavassori V, Ferrari S, Rovelli E, Albano L, Jacob A, Sergi Sergi L, Montaldo E, Ferrua F, González-Granado LI, Lougaris V, Badolato R, Finocchi A, Villa A, Radrizzani M, and Naldini L
- Abstract
Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand ( CD40LG ) gene with a median survival of 25 years, potentially treatable with in situ CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of CD40LG in edited healthy donor and patient cells recapitulating the physiological CD40LG regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile., Competing Interests: L.N., C.A., M.R., V.V., A.V., S.F., S.P., D.C., and A.J. are inventors of patent applications owned by Ospedale San Raffaele S.r.l. and Fondazione Telethon ETS, including one patent application on CD40LG gene editing. L.N. is founder, quota holder, and consultant of GeneSpire S.r.l., (© 2023 The Authors.)
- Published
- 2023
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22. Efficacy and Safety of Anti-SARS-CoV-2 Antiviral Agents and Monoclonal Antibodies in Patients with SLE: A Case-Control Study.
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Ramirez GA, Gerosa M, Bellocchi C, Arroyo-Sánchez D, Asperti C, Argolini LM, Gallina G, Cornalba M, Scotti I, Suardi I, Moroni L, Beretta L, Bozzolo EP, Caporali R, and Dagna L
- Subjects
- Humans, Antibodies, Monoclonal adverse effects, Antiviral Agents adverse effects, Case-Control Studies, Retrospective Studies, SARS-CoV-2, COVID-19 complications, Lupus Erythematosus, Systemic drug therapy
- Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related disease (COVID-19) has spread pandemically with high rates of morbidity and mortality. COVID-19 has also posed unprecedented challenges in terms of rapid development of pharmacological countermeasures to prevent or contrast SARS-CoV-2 pathogenicity. Anti-SARS-CoV-2 antiviral agents and monoclonal antibodies have been specifically designed to attenuate COVID-19 morbidity and prevent mortality in vulnerable subjects, such as patients with immune-mediated diseases, but evidence for the safe and effective use of these drugs in this latter population group is scarce. Therefore, we designed a retrospective, multicentre, observational, case-control study to analyse the impact of these treatments in COVID-19 patients with systemic lupus erythematosus (SLE), a paradigmatic, multi-organ autoimmune disease. We identified 21 subjects treated with antivirals and/or monoclonal antibodies who were matched with 42 untreated patients by age, sex, SLE extension and duration. Treated patients had higher baseline SLE disease activity index 2000 scores [SLEDAI-2K median (interquartile range) = 4 (1-5) vs. 0 (0-2); p = 0.009], higher prednisone doses [5 (0-10) mg vs. 0 (0-3) mg; p = 0.002], and more severe COVID-19 symptoms by a five-point World Health Organisation-endorsed analogue scale [1 (0-1) vs. 0 (0-1); p < 0.010] compared to untreated patients. There was no difference between groups in terms of COVID-19 outcomes and sequelae, nor in terms of post-COVID-19 SLE exacerbations. Three subjects reported mild adverse events (two with monoclonal antibodies, one with nirmatrelvir/ritonavir). These data suggest that anti-SARS-CoV-2 antivirals and monoclonal antibodies might be safely and effectively used in patients with SLE, especially with active disease and more severe COVID-19 symptoms at presentation.
- Published
- 2023
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23. Hypersensitivity reactions to anti-SARS-CoV-2 vaccines: Basophil reactivity to excipients.
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Pignatti P, Ramirez GA, Russo M, Marraccini P, Nannipieri S, Asperti C, Torre FD, Tiri A, Gatti BM, Gurrado A, Meriggi A, Benanti G, Cilona MB, Pigatto P, Burastero SE, Dagna L, and Yacoub MR
- Subjects
- Humans, Basophils, Excipients adverse effects, COVID-19 Vaccines adverse effects, SARS-CoV-2, COVID-19 prevention & control, Hypersensitivity, Hypersensitivity, Delayed
- Abstract
Basophil activation test (BAT) can tackle multiple mechanisms underlying acute and delayed hypersensitivity to drugs and vaccines and might complement conventional allergy diagnostics but its role in anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine-related hypersensitivity is ill-defined. Therefore, 89 patients with possible hypersensitivity (56 % with delayed mucocutaneous manifestations) to anti-SARS-CoV-2 vaccines were tested with BAT for Macrogol 3350, DMG-PEG 2000, PEG 20000, polysorbate-80 and trometamol and compared to 156 subjects undergoing pre-vaccine BAT. A positive BAT was associated with delayed reaction onset (p = 0.010) and resolution (p = 0.011). BAT was more frequently positive to DMG-PEG 2000 than to other excipients in both groups (p < 0.001). DMG-PEG 2000 reactivity was less frequent in vaccine-naïve (6 %) than vaccinated subjects (35 %, p < 0.001) and associated with mRNA-1273 vaccination. DMG-PEG 2000 BAT might therefore have a diagnostic role in subjects with delayed hypersensitivity reactions. Natural immunity might be a key player in basophil activation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)
- Published
- 2023
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24. Heart Rate Variability in Subjects with Severe Allergic Background Undergoing COVID-19 Vaccination.
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Cilona MB, D'Amico F, Asperti C, Ramirez GA, Turi S, Benanti G, Bohane SM, Nannipieri S, Labanca R, Gervasini M, Russetti F, Viapiana N, Lezzi M, Landoni G, Dagna L, and Yacoub MR
- Abstract
Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccination is the world's most important strategy for stopping the pandemic. Vaccination challenges the body's immune response and can be complicated by hypersensitivity reactions. The autonomic nervous system can modulate the inflammatory immune response, therefore constituting a potential marker to characterize individuals at high risk of hypersensitivity reactions. Autonomic nervous system functionality was assessed through measurement of the heart rate variability (HRV) in subjects with a history of severe allergic reactions and 12 control subjects. HRV parameters included the mean electrocardiograph RR interval and the standard deviation of all normal R-R intervals (SDNN). All measurements were performed immediately before the anti-SARS-CoV-2 vaccination. The median RR variability was lower in the study than in the control group: 687 ms (645-759) vs. 821 ms (759-902); p = 0.02. The SDNN was lower in the study group than in the control group: 32 ms (23-36) vs. 50 ms (43-55); p < 0.01. No correlation was found between age and the SDNN. Autonomic nervous system activity is unbalanced in people with a severe allergy background.
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- 2023
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25. Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS): Focus on the Pathophysiological and Diagnostic Role of Viruses.
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Ramirez GA, Ripa M, Burastero S, Benanti G, Bagnasco D, Nannipieri S, Monardo R, Ponta G, Asperti C, Cilona MB, Castagna A, Dagna L, and Yacoub MR
- Abstract
Drug reaction with eosinophilia and systemic symptoms (DRESS) is a heterogeneous, multiorgan and potentially life-threatening drug-hypersensitivity reaction (DHR) that occurs several days or weeks after drug initiation or discontinuation. DHRs constitute an emerging issue for public health, due to population aging, growing multi-organ morbidity, and subsequent enhanced drug prescriptions. DRESS has more consistently been associated with anticonvulsants, allopurinol and antibiotics, such as sulphonamides and vancomycin, although new drugs are increasingly reported as culprit agents. Reactivation of latent infectious agents such as viruses (especially Herpesviridae) plays a key role in prompting and sustaining aberrant T-cell and eosinophil responses to drugs and pathogens, ultimately causing organ damage. However, the boundaries of the impact of viral agents in the pathophysiology of DRESS are still ill-defined. Along with growing awareness of the multifaceted aspects of immune perturbation caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the ongoing SARS-CoV-2-related disease (COVID-19) pandemic, novel interest has been sparked towards DRESS and the potential interactions among antiviral and anti-drug inflammatory responses. In this review, we summarised the most recent evidence on pathophysiological mechanisms, diagnostic approaches, and clinical management of DRESS with the aim of increasing awareness on this syndrome and possibly suggesting clues for future research in this field.
- Published
- 2023
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26. Interactions between Severe Allergy and Anxiety in Anti-SARS-CoV-2 Vaccinees.
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Asperti C, Benanti G, Ramirez GA, Russo M, Vai B, Bramé B, Viapiana N, Nannipieri S, Cilona MB, Mazzetti M, Zuffada S, Di Mattei VE, Benedetti F, Dagna L, and Yacoub MR
- Abstract
Severe drug allergy affects patient hesitancy to new treatments, posing unprecedented challenges to anti-SARS-CoV-2 vaccination campaigns. We aimed to analyze the psychological profile of vaccinees with a history of severe allergy in comparison to subjects with a milder allergy history. Patients attending a dedicated vaccination setting were administered an anonymized questionnaire including clinical data and the State-Trait Anxiety Inventory (STAI) scale (score range 20−80). Patients were also asked whether being in a protected setting affected their attitude toward vaccination. Data are expressed as median (interquartile range). We enrolled 116 patients (78% women), of whom 79% had a history of drug anaphylaxis. The median state anxiety score was 36.5 (30−47.2), while the trait anxiety score was 37 (32−48). State anxiety was higher in those with severe than mild allergy [39 (32−50) vs. 30 (25−37); p < 0.001], with the highest score found in a patient with previous drug anaphylaxis (42.5 [32−51.7]). More than 50% of patients reported that being in a protected setting had lowered their anxiety. Severe allergy is associated with a higher burden of situational anxiety in the setting of vaccination without affecting patient constitutional (trait) levels of anxiety. Vaccination in dedicated facilities might overcome issues related to hesitancy and improve patients’ quality of life.
- Published
- 2022
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27. Basal Serum Diamine Oxidase Levels as a Biomarker of Histamine Intolerance: A Retrospective Cohort Study.
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Cucca V, Ramirez GA, Pignatti P, Asperti C, Russo M, Della-Torre E, Breda D, Burastero SE, Dagna L, and Yacoub MR
- Subjects
- Biomarkers, Headache, Histamine adverse effects, Humans, Retrospective Studies, Amine Oxidase (Copper-Containing) metabolism
- Abstract
Background: Histamine Intolerance (HIT) is a multifaceted pseudoallergic disorder possibly due to defective histamine metabolism. Diamine oxidase (DAO) contributes to histamine degradation and can be measured in the serum. The role of DAO measurement in the diagnostic work-up of HIT still remains unclear, and conflicting results have been reported in the literature. Therefore, we aimed to evaluate the possible clinical usefulness and consistency of DAO value ranges as provided by the assay manufacturer and verify whether they could predict the response to treatment. Methods: We retrospectively analyzed 192 outpatients with HIT symptoms and measured serum DAO values at baseline. Patients were prescribed either with low-histamine diet and/or enzymatic supplementation according to symptom severity and re-evaluated six to eight months later. Patients were stratified into three groups according to DAO levels: <3 U/mL, 3−10 U/mL, and >10 U/mL. HIT severity was assessed on a scale of 1 to 5 before and after treatment. Results: A total of 146 patients completed the study. Gastrointestinal and cutaneous symptoms, often associated with headache, were more frequent in subjects with DAO < 10 U/mL. Symptom severity and DAO ranges were correlated. Patients with intermediate DAO levels (3−10 U/mL) showed a more complex clinical phenotype but also a more significant improvement in symptom severity (score reduction 50%, interquartile range (IQR) = 33−60%) when compared to patients with low DAO (40%, IQR = 20−60%; p = 0.045) or high DAO (33%, IQR = 0−50%; p < 0.001). Complex clinical phenotypes were also more frequent in patients with intermediate DAO levels. Conclusions: HIT is characterized by typical symptoms and low levels of DAO activity. Symptom severity was associated with the degree of DAO deficiency. Patients with DAO values between 3 and 10 U/mL show the best response to treatment (low-histamine diet and/or DAO supplementation). DAO value could arguably be considered as a predictor of clinical response to treatment. Prospective studies are needed to confirm these data.
- Published
- 2022
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28. Efficacy of a rational algorithm to assess allergy risk in patients receiving the BNT162b2 vaccine.
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Yacoub MR, Cucca V, Asperti C, Ramirez GA, Della-Torre E, Moro M, Zandalasini C, Di Napoli D, Ambrosio A, Signorelli C, Colombo S, Beretta L, Ciceri F, Zangrillo A, and Dagna L
- Subjects
- Algorithms, BNT162 Vaccine, COVID-19 Vaccines, Humans, SARS-CoV-2, Anaphylaxis chemically induced, Anaphylaxis diagnosis, Anaphylaxis epidemiology, COVID-19, Vaccines adverse effects
- Abstract
Among 6146 hospital employees, 118 subjects with severe allergic background were identified through a screening questionnaire and stratified into 3 groups (Low-risk (LR), Intermediate (IR) and High-risk (HR) group), based on their allergic anamnesis. Data reports on hypersensitivity reactions (HypR) have been collected in both allergic and non-allergic subjects. Seventeen patients (14%) in the allergic population had a HypR after the first, the second or both doses. Skin manifestations were the most frequent ones. Allergic events were more frequent in HR (35%) than IR (10%; p = 0.005) or LR (0%; p = 0.074) subjects. No patient had anaphylaxis. All patients completed the vaccination schedule. 13 HypR occurred in patients without severe allergic background (13/6028, 0,2%) including one (1/6148, 0.016% of total population) WAO grade-4 anaphylaxis. Our data suggest that BNT162b2 mRNA Covid-19 vaccine is relatively safe also in patients with severe allergic background; however, some precautions are required for high-risk patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
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29. Challenges to Vaccination against SARS-CoV-2 in Patients with Immune-Mediated Diseases.
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Ramirez GA, Asperti C, Cucca V, and Yacoub MR
- Abstract
Aberrant deployment of the immune response is a hallmark pathogenic feature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related disease (COVID-19), possibly accounting for high morbidity and mortality, especially in patients with comorbidities, including immune-mediated disorders. Immunisation with SARS-COV-2 vaccines successfully instructs the immune system to limit viral spread into tissues, mitigate COVID-19 manifestations and prevent its most detrimental inflammatory complications in the general population. Patients with immune-mediated diseases have been excluded from vaccine registration trials, foreclosing the acquisition of specific efficacy and safety data. In this review, we aimed to summarise and critically discuss evidence from real-world studies addressing this issue to provide a comprehensive view of the impact of vaccination practices in patients with allergy, autoimmunity or immunodeficiency. We analysed clinical and laboratory data from 34 studies involving more than 13,000 subjects with various immune disorders who were vaccinated with mRNA- DNA- or inactivated viral particle-based vaccines. These data globally support the safe and effective use of SARS-CoV-2 vaccines in patients with immune-mediated diseases, although patient-tailored strategies to determine vaccination timing, vaccine choice and background therapy management are warranted to optimise vaccination outcomes. More data are needed regarding patients with primary immunodeficiencies.
- Published
- 2021
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30. Characterization and Functional Analysis of CD44v6.CAR T Cells Endowed with a New Low-Affinity Nerve Growth Factor Receptor-Based Spacer.
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Stornaiuolo A, Valentinis B, Sirini C, Scavullo C, Asperti C, Zhou D, Martinez De La Torre Y, Corna S, Casucci M, Porcellini S, and Traversari C
- Subjects
- Cell Line, Tumor, Humans, Immunotherapy, Adoptive, Receptor, Nerve Growth Factor, Receptors, Antigen, T-Cell genetics, Receptors, Nerve Growth Factor, T-Lymphocytes, Xenograft Model Antitumor Assays, Receptors, Chimeric Antigen genetics
- Abstract
Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the single-chain variable fragment (scFv) for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low-affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical-grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in terms of killing capability, and in vivo antitumor activity against hematological and solid tumors. Clinical Trial Registration numbers: clinicaltrial.gov NCT04097301; ClinicalTrials.gov, NCT00423124.
- Published
- 2021
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31. CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice.
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Porcellini S, Asperti C, Corna S, Cicoria E, Valtolina V, Stornaiuolo A, Valentinis B, Bordignon C, and Traversari C
- Subjects
- Adenocarcinoma immunology, Animals, Antigens, CD19, Cell Line, Tumor, Cell Proliferation, Female, Genes, Transgenic, Suicide, Humans, Hyaluronan Receptors genetics, Immunotherapy, Adoptive, Lung pathology, Mice, Mice, Transgenic, Molecular Targeted Therapy, Ovary metabolism, Ovary pathology, Receptors, Antigen, T-Cell genetics, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen immunology, T-Lymphocytes immunology, Adenocarcinoma therapy, Receptors, Antigen, T-Cell immunology, Receptors, Chimeric Antigen therapeutic use
- Abstract
The main challenge of adoptive therapy with Chimeric Antigen Receptor modified T cells (CAR T) is the application to the field of solid tumors, where the identification of a proper antigen has emerged as one of the major drawbacks to CAR T cell treatment success. CD44 is a glycoprotein involved in cell-cell and cell-matrix interactions. The isoform containing the variant domain 6 of CD44 gene (CD44v6) has been implicated in tumorigenesis, tumor cell invasion and metastasis and represents an attractive target for CAR T cell therapies. Targeting CD44v6 antigen has been shown to control tumor growth in acute myeloid leukemia and multiple myeloma mouse models. While CAR T approach for the treatment of B cell malignancies has shown great success, response rates among patients with solid cancer are less favorable. The purpose of our study was to test the efficacy of CD44v6.CAR T cells, produced in compliance with Good Manufacturing Practice (GMP), in adenocarcinoma tumor models. We generated a bicistronic retroviral vector containing the CD44v6 CAR and the HSV-TK Mut2 suicide gene to enhance the safety of the proposed CAR T cell therapy. CD44v6 transduced CAR T cells were homogeneously positive for ΔLNGFR selection marker, were enriched in T central memory (T
CM ) and T memory stem cells (TSCM ) and displayed a highly activated phenotype. In vitro assays revealed antigen-specific activation and cytotoxicity of human CD44v6.CAR T cells against CD44v6 expressing tumor cell lines. When infused in immunodeficient tumor bearing mice, human CD44v6.CAR T cells were able to reach, infiltrate and proliferate at tumor sites, finally resulting in tumor growth control. Next, we checked if cells produced in compliance with GMP grade standards retained the same antitumor activity of those produced with research grade materials and protocols. Noteworthy, no differences in the potency of the CAR T obtained with the two manufacturing processes were observed. In conclusion, our preclinical results suggest that CD44v6.CAR T based adoptive therapy could be a promising strategy in solid cancer treatment., (Copyright © 2020 Porcellini, Asperti, Corna, Cicoria, Valtolina, Stornaiuolo, Valentinis, Bordignon and Traversari.)- Published
- 2020
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32. Mechanism of Action of the Tumor Vessel Targeting Agent NGR-hTNF: Role of Both NGR Peptide and hTNF in Cell Binding and Signaling.
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Valentinis B, Porcellini S, Asperti C, Cota M, Zhou D, Di Matteo P, Garau G, Zucchelli C, Avanzi NR, Rizzardi GP, Degano M, Musco G, and Traversari C
- Subjects
- Angiogenesis Inhibitors chemistry, Cell Line, Tumor, Crystallography, X-Ray, Human Umbilical Vein Endothelial Cells, Humans, Models, Molecular, Oligopeptides chemistry, Recombinant Fusion Proteins chemistry, Signal Transduction drug effects, Tumor Necrosis Factor-alpha chemistry, Angiogenesis Inhibitors pharmacology, Neoplasms blood supply, Oligopeptides pharmacology, Recombinant Fusion Proteins pharmacology, Tumor Necrosis Factor-alpha pharmacology
- Abstract
NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.
- Published
- 2019
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33. Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein.
- Author
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Zucchelli E, Pema M, Stornaiuolo A, Piovan C, Scavullo C, Giuliani E, Bossi S, Corna S, Asperti C, Bordignon C, Rizzardi GP, and Bovolenta C
- Abstract
Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34
+ cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.- Published
- 2017
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34. The tumor vessel targeting agent NGR-TNF controls the different stages of the tumorigenic process in transgenic mice by distinct mechanisms.
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Porcellini S, Asperti C, Valentinis B, Tiziano E, Mangia P, Bordignon C, Rizzardi GP, and Traversari C
- Abstract
NGR-TNF is a vascular targeting agent in advanced clinical development, coupling tumor necrosis factor-α (TNF) with the CNGRCG peptide, which targets a CD13 isoform specifically expressed by angiogenic vessels. Antitumor efficacy of NGR-TNF has been described in different transplantation tumor models. Nevertheless, the mechanism underlying its activity is not fully understood. In the wild type and in the immunodeficient (RAG
-/- ) RIP1-Tag2 models of multistage pancreatic carcinogenesis, we demonstrate that CD13 is highly expressed on endothelial cells of hyperplastic and angiogenic islets, whereas its expression is down regulated in tumors where it partially colocalize with pericytes. In vivo CNGRCG peptides coupled to fluorescent nanoparticles (quantum dots) bind to CD13 and colocalize with anti-CD31, in pancreatic islets. At early stage, low doses of NGR-murine (m)TNF have a direct cytotoxic effect inducing endothelial cell apoptosis, reducing vessel density and eventually inhibiting the development of angiogenic islets. At a later stage, NGR-mTNF is able to reduce tumor growth inducing vascular normalization, exclusively when treatment is carried out in the immunocompetent mice. Interestingly, NGR-mTNF-treated tumors from these mice are characterized by CD8+ T cell infiltration. At molecular level, overexpression of genes involved in vessels normalization was detected only in NGR-mTNF-treated tumors from immunocompetent mice. These findings identified a new mechanism of action of NGR-mTNF, providing support for the development of new therapeutic strategies combining chemotherapy or active/adoptive immunotherapies to low dose NGR-TNF treatment.- Published
- 2015
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35. Molecular dynamics reveal that isoDGR-containing cyclopeptides are true αvβ3 antagonists unable to promote integrin allostery and activation.
- Author
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Ghitti M, Spitaleri A, Valentinis B, Mari S, Asperti C, Traversari C, Rizzardi GP, and Musco G
- Subjects
- Allosteric Regulation, Integrin alphaVbeta3 metabolism, Models, Molecular, Oligopeptides chemistry, Peptides, Cyclic chemistry, Integrin alphaVbeta3 antagonists & inhibitors, Molecular Dynamics Simulation, Oligopeptides pharmacology, Peptides, Cyclic pharmacology
- Abstract
Ain't got that swing(-out): The cyclopeptide isoDGR is emerging as a new αvβ3 integrin binding motif. Agreement between the results of computational and biochemical studies reveals that isoDGR-containing cyclopeptides are true αvβ3 integrin antagonists that block αvβ3 in its inactive conformation (see scheme). isoDGR-based ligands may give αvβ3 antagonists without paradoxical effects., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
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36. Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.
- Author
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Asperti C, Astro V, Pettinato E, Paris S, Bachi A, and de Curtis I
- Subjects
- Animals, COS Cells, Cell Shape, Chlorocebus aethiops, Guanine Nucleotide Exchange Factors metabolism, HeLa Cells, Humans, Paxillin metabolism, Protein Binding, Protein Transport, Rho Guanine Nucleotide Exchange Factors, Subcellular Fractions, Adaptor Proteins, Signal Transducing metabolism, Cell Cycle Proteins metabolism, Cell Movement
- Abstract
We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.
- Published
- 2011
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37. Identification of an intramolecular interaction important for the regulation of GIT1 functions.
- Author
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Totaro A, Paris S, Asperti C, and de Curtis I
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Animals, COS Cells, Cell Adhesion, Cell Cycle Proteins genetics, Cell Line, Chlorocebus aethiops, Fibronectins metabolism, Humans, Mutation genetics, Paxillin metabolism, Protein Binding, Adaptor Proteins, Signal Transducing metabolism, Cell Cycle Proteins metabolism
- Abstract
G-protein coupled receptor kinase-interacting protein (GIT) proteins include an N-terminal Arf GTPase-activating protein domain, and a C terminus that binds proteins regulating adhesion and motility. Given their ability to form large molecular assemblies, the GIT1 protein must be tightly regulated. However, the mechanisms regulating GIT1 functions are poorly characterized. We found that carboxy-terminal-truncated fragments of GIT1 bind their partners with higher efficiency compared with the full-length GIT1. We have explored the hypothesis that GIT1 is regulated by an intramolecular mechanism, and we identified two distinct intramolecular interactions between the N and C terminus of GIT1. The release of these interactions increases binding of GIT1 to paxillin and liprin-alpha, and it correlates with effects on cell spreading. Analysis of cells plated on fibronectin has shown that different deletion mutants of GIT1 either enhance or inhibit spreading, depending on their subcellular localization. Moreover, although the association between betaPIX and GIT1 is insufficient to activate GIT1 binding to paxillin, binding of a PAK1 fragment including the betaPIX-binding domain enhances paxillin binding to betaPIX/GIT1, indicating that p21-activated kinase can activate the binding of paxillin to GIT1 by a kinase-independent mechanism. The release of the identified intramolecular interaction seems to be an important mechanism for the regulation of GIT1 functions.
- Published
- 2007
- Full Text
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