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Massimo Conese
Ruolo
Professore Associato
Organizzazione
Università degli Studi di Foggia
Dipartimento
Dipartimento di Scienze Mediche e Chirurgiche
Area Scientifica
Area 06 - Scienze mediche
Settore Scientifico Disciplinare
MED/04 - Patologia Generale
Settore ERC 1° livello
LS - Life sciences
Settore ERC 2° livello
LS7 Diagnostic Tools, Therapies and Public Health: Aetiology, diagnosis and treatment of disease, public health, epidemiology, pharmacology, clinical medicine, regenerative medicine, medical ethics
Settore ERC 3° livello
LS7_6 Gene therapy, cell therapy, regenerative medicine
High-mobility group box 1 (HMGB1) is a nonhistone protein secreted by airway epithelial cells in hyperinflammatory diseases such as asthma. In order to down-regulate HMGB1 expression in airway epithelial cells, siRNA directed against HMGB1 was delivered through nanocomplexes based on a cationic copolymer of poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) by using H441 cells. Two copolymers were used in these experiments bearing respectively spermine side chains (PHEA-Spm) and both spermine and PEG2000 chains (PHEA-PEG-Spm). PHEA-Spm and PHEA-PEG-Spm derivatives complexed dsDNA oligonucleotides with a w/w ratio of 1 and higher as shown by a gel retardation assay. PHEA-Spm and PHEA-PEG-Spm siRNA polyplexes were sized 350–650 nm and 100–400 nm respectively and ranged from negativity/neutrality (at 0.5 ratio) to positivity (at 5 ratio) as ζ potential. Polyplexes formed either at a ratio of 0.5 (partially complexing) or at the ratio of 5 (fully complexing) were tested in subsequent experiments. Epifluorescence revealed that nanocomplexes favored siRNA entry into H441 cells in comparison with naked siRNA. As determined by flow cytometry and a trypan blue assay, PHEA-Spm and PHEA-PEG-Spm allowed siRNA uptake in 42–47% and 30% of cells respectively, however only with PHEA-Spm at w/w ratio of 5 these percentages were significantly higher than those obtained with naked siRNA (20%). Naked siRNA or complexed scrambled siRNA did not exert any effect on HMGB1mRNA levels, whereas PHEA-Spm/siRNA at the w/w ratio of 5 down-regulated HMGB1 mRNA up to 58% of control levels (untransfected cells). PEGylated PHEA-Spm/siRNA nanocomplexes were able to down-regulate HMGB1 mRNA levels up to 61% of control cells. MTT assay revealed excellent biocompatibility of copolymer/siRNA polyplexes with cells. In conclusion, we have found optimal conditions for down-regulation of HMGB1 by siRNA delivery mediated by polyaminoacidic polymers in airway epithelial cells in the absence of cytotoxicity. Functional and in-vivo studies are warranted.
BACKGROUND: It is not known whether antibiotic therapy for lung disease in cystic fibrosis (CF) has an influence on circulating polymorphonuclear neutrophil (PMN) function and apoptosis. PATIENTS AND METHODS: Blood PMNs were obtained from 14 CF patients before and after antibiotic treatment for an acute exacerbation, and from 10 healthy controls. PMNs were evaluated for production of reactive oxygen species (ROS) by spectrophotometry, of cytokines in the conditioned medium by ELISA, and apoptotic response by cytofluorimetry. RESULTS: ROS and interleukin (IL)-8 were produced at higher levels by CF PMNs pre-therapy than control PMNs under basal conditions. IL-8 levels further increased after therapy. Early apoptotic response was higher in CF PMNs pre-therapy than in control PMNs, and this pattern did not change after antibiotic treatment. CONCLUSIONS: Circulating PMNs are primed in CF acute patients. Further studies are needed to consider PMN-produced IL-8 as a biomarker to evaluate response to antibiotic therapy in CF patients.
Nelle malattie respiratorie croniche, come l'asma allergico e la broncopneumopatia cronica ostruttiva (BPCO), lo stress ossidativo porta ad una infiammazione incontrollata con iperproduzione di muco, il quale impedisce un delivery ottimale di farmaci alle cellule epiteliali respiratorie. Per superare tale barriera, il progetto propone di veicolare sostanze antiossidanti naturali mediante due livelli di complessità: uno relativo alla formulazione di tali sostanze in nanoparticelle lipidiche o polimeriche mucopenetranti; un altro concernente la concentrazione di tali sistemi nanoparticolati sulle cellule epiteliali respiratorie grazie alla loro aerosolizzazione e magnetofezione (drug targeting fisico). Proponiamo di usare antiossidanti naturali presenti nel pomodoro e nell'uva, prodotti di ampia produzione pugliese.I risultati potenziali attesi includono: 1) la comprensione delle potenzialità farmacologiche di antiossidanti naturali per malattie croniche respiratorie; 2) la messa a punto di nuove tecnologie farmaceutiche indirizzate al delivery di farmaci in ambito polmonare; 3) l'utilizzo di un metodo non invasivo di drug delivery nella salute pubblica, con particolare riguardo a malattie debilitanti e socialmente impegnative.Rispetto all'ambito di riferimento, il delivery di antiossidanti porterà sicuramente un beneficio ai soggetti affetti da tali malattie, che attualmente rappresentano nel mondo occidentale la quarta causa di morte.
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