Marcatura di molecole biologiche a funzione antigenica per lo studio e la caratterizzazione di protocolli di vaccinoterapia in oncologia medica

Dendritic Cells (DCs) derived from human blood monocytes that have been nurtured in GM-CSF and IL-4, followed by maturation in a monocyte-conditioned medium, are the most potent APCs known. These DCs have many features of primary DCs, including the expression of molecules that enhance antigen capture and selective receptors that guide DCs to and from several sites in the body, where they elicit the T cell mediated immune response. For these features, immature DCs (iDC) loaded with tumor antigen and matured (mDC) with a standard cytokine cocktail, are used for therapeutic vaccination in clinical trials of different cancers. However, the efficacy of DCs in the development of immunocompetence is critically influenced by the type (whole lysate, proteins, peptides, mRNA), the amount and the time of exposure of the tumor antigens used for loading in the presentation phase. The aim of the present study was to create instruments to acquire more information about DC antigen uptake and presentation mechanisms to improve the clinical efficacy of DCbased vaccine. In particular, two different tumor antigen were studied: the monoclonal immunoglobulin (IgG or IgA) produced in Myeloma Multiple, and the whole lysate obtained from melanoma tissues. These proteins were conjugated with fluorescent probe (FITC) to evaluate the kinetic of tumor antigen capturing process and its localization into DCs, by cytofluorimetric and fluorescence microscopy analysis, respectively. iDC pulsed with 100μg of IgG-FITC/106 cells were monitored from 2 to 22 hours after loading. By the cytofluorimetric analysis it was observed that the monoclonal antibody was completely captured after 2 hours from pulsing, and was decreased into mDC in 5 hours after maturation stimulus. To monitor the lysate uptake, iDC were pulsed with 80μg of tumor lysate/106 cells, then were monitored in the 2h to 22 hours interval time after loading. Then, to reveal difference between increasing lysate concentration, iDC were loaded with 20-40-80-100-200-400μg of tumor lysate/106 cells and monitored at 2-4-8-13h from pulsing. By the cytofluorimetric analysis, it was observed that, the 20-40-80-100μg uptake, after 8 hours loading was completed reaching a plateau phase. For 200 and 400μg the mean fluorescence of cells increased until 13h from pulsing. The lysate localization into iDC was evaluated with conventional and confocal fluorescence microscopy analysis. In the 2h to 8h time interval from loading an intensive and diffuse fluorescence was observed within the cytoplasmic compartment. Moreover, after 8h, the lysate fluorescence appeared to be organized in a restricted cloudy-shaded area with a typical polarized aspect. In addition, small fluorescent spots clearly appeared with an increment in the number and fluorescence intensity. The nature of these spot-like formations and cloudy area is now being investigated detecting the colocalization of the fluorescence lysate and specific markers for lysosomes, autophagosomes, endoplasmic reticulum and MHCII positive vesicles.

Studio dei meccanismi molecolari coinvolti nel determinismo sintomatologico di piante infette da virus e virus-satelliti

The aim of our work was to study the molecular mechanisms involved in symptoms appearance of plants inoculated either with a virus or with a virus-satellite complex. In the first case, we tried to set up a reliable method for an early identification of PVYNTN strains present in Italy and causing potato tuber necrosis. This, to prevent their spread in the field and to avoid severe yield losses, especially in seed potato production. We tried to localize the particular genomic region responsible for tuber necrosis. To this purpose, we carried out RT-PCR experiments using various primer combinations, covering PVY genomic regions larger than those previously used by other authors. As the previous researchers, though, we were not able to differentiate all NTN from others PVY strains. This probably because of the frequent virus variability, due to both genomic mutations and possible recombination events among different strains. In the second case, we studied the influence of Y-sat (CaRNA5 satellite) on symptoms of CMV (Cucumber mosaic virus) in Nicotiana benthamiana plants: strong yellowing appearance instead of simple mosaic. Wang et al (2004), inoculating the same infectious complex on tobacco plants transformed with a viral suppressor of plant silencing (HC-PRO), did not experience the occurrence of yellowing anymore and, therefore, hypotesized that changes in symptoms were due to plant post transcriptional gene silencing (PTGS) mechanism. In our case, inoculation of N. benthamiana plants transformed with another PTGS viral suppressor (p19), and other plants defective for RNA polymerase 6 (involved in systemic silencing), still resulted in yellowing appearance. This, to our opinion, suggests that in our system another possible mechanism is involved.

Prove di caratterizzazione meccanica del legame di adesione fra FRP ed acciaio

Il progresso nella tecnica di recupero e di rinforzo nelle strutture metalliche con i polimeri fibro-rinforzati FRP (fibre reinforced polymers). 1.1 Introduzione nelle problematiche ricorrenti delle strutture metalliche. Le strutture moderne di una certa importanza, come i grattacieli o i ponti, hanno tempi e costi di costruzione molto elevati ed è allora di importanza fondamentale la loro durabilità, cioè la lunga vita utile e i bassi costi di manutenzione; manutenzione intesa anche come modo di restare a livelli prestazionali predefiniti. La definizione delle prestazioni comprende la capacità portante, la durabilità, la funzionalità e l’aspetto estetico. Se il livello prestazionale diventa troppo basso, diventa allora necessario intervenire per ripristinare le caratteristiche iniziali della struttura. Strutture con una lunga vita utile, come per la maggior parte delle strutture civili ed edilizie, dovranno soddisfare esigenze nuove o modificate: i mezzi di trasporto ad esempio sono diventati più pesanti e più diffusi, la velocità dei veicoli al giorno d'oggi è aumentata e ciò comporta anche maggiori carichi di tipo dinamico.

Studio di biomateriali usati come scaffold per Tissue Engineering e loro caratterizzazione con tecniche spettroscopiche vibrazionali e di analisi termica

This research investigated someone of the main problems connected to the application of Tissue Engineering in the prosthetic field, in particular about the characterization of the scaffolding materials and biomimetic strategies adopted in order to promote the implant integration. The spectroscopic and thermal analysis techniques were usefully applied to characterize the chemico-physical properties of the materials such as – crystallinity; – relative composition in case of composite materials; – Structure and conformation of polymeric and peptidic chains; – mechanism and degradation rate; – Intramolecular and intermolecular interactions (hydrogen bonds, aliphatic interactions). This kind of information are of great importance in the comprehension of the interactions that scaffold undergoes when it is in contact with biological tissues; this information are fundamental to predict biodegradation mechanisms and to understand how chemico-physical properties change during the degradation process. In order to fully characterize biomaterials, this findings must be integrated by information relative to mechanical aspects and in vitro and in vivo behavior thanks to collaborations with biomedical engineers and biologists. This study was focussed on three different systems that correspond to three different strategies adopted in Tissue Engineering: biomimetic replica of fibrous 3-D structure of extracellular matrix (PCL-PLLA), incorporation of an apatitic phase similar to bone inorganic phase to promote biomineralization (PCL-HA), surface modification with synthetic oligopeptides that elicit the interaction with osteoblasts. The characterization of the PCL-PLLA composite underlined that the degradation started along PLLA fibres, which are more hydrophylic, and they serve as a guide for tissue regeneration. Moreover it was found that some cellular lines are more active in the colonization of the scaffold. In the PCL-HA composite, the weight ratio between the polymeric and the inorganic phase plays an essential role both in the degradation process and in the biomineralization of the material. The study of self-assembling peptides allowed to clarify the influence of primary structure on intermolecular and intermolecular interactions, that lead to the formation of the secondary structure and it was possible to find a new class of oligopeptides useful to functionalize materials surface. Among the analytical techniques used in this study, Raman vibrational spectroscopy played a major role, being non-destructive and non-invasive, two properties that make it suitable to degradation studies and to morphological characterization. Also micro-IR spectroscopy was useful in the comprehension of peptide structure on oxidized titanium: up to date this study was one of the first to employ this relatively new technique in the biomedical field.

Costruzione di uno scaffold vascolarizzato per la chirurgia ortopedica

Specific aims The aim is to improve the treatment of the bone losses at the metacarpal bones level (both diaphysis and epiphysis) combining microsurgery, tissue engineering and biomaterials, so to minimize the donor side morbidity and optimize healing and outcomes. Methods Pre-operative controlateral X-ray or 3-D CT to allow custom-made HA scaffolds. Cement as temporary spacer in acute lesion and monitoring of infective risks. Treatment of the bone loss recurring to pre-fabricated or custom-made HA scaffolds, adding platelet gel or growth factor OP1. Stable synthesis. Control group with auto/omografts. Outcome indices: % of bone-union; finger TAM, Kapandji, DASH score; NMR and Scintigraphy at 180 days for revascularisation and bio-substitution of the scaffold. Preliminary results The authors just treated 6 patients, 4 males and 2 females, with an average age of 38.5 yrs, affected by segmental bone losses at the hand and wrist, recurring to pre-fabricated not vascularised scaffolds. In all cases the synthesis was performed with angular stability plates and a stable synthesis achieved. All patients have been controlled at a mean follow-up of 10.5 months (from 2 to 16 ). In all case but one the bone-scaffold osteo-integration was achieved at an average of 38 days at the hand, and 46 days at the wrist. The outcome studies, according to the DASH score, finger TAM, and Kapandji, were good and excellent in 5 cases, poor in one.

Sul rinforzo di elementi in C.A.: confronto tra varie normative

Il documento pre-normativo italiano sul rinforzo di strutture in c.a. mediante l’uso di materiale fibrorinforzato. 1.1 INTRODUZIONE La situazione unica dell’Italia per quanto riguarda la conservazione delle costruzioni esistenti, è il risultato della combinazione di due aspetti, come primo, il medio-alto rischio sismico di una gran parte di territorio, come testimoniato dalla zonizzazione sismica recente, e come secondo aspetto, l'estrema complessità di un ambiente edilizio che non ha confronto nel mondo. Le tipologie della costruzione in Italia si distinguono a quelle stimate come patrimonio storico, che in alcuni casi risalgono a circa 2000 anni fa, a quelle che sono state costruite in ultimi cinque secoli, durante e dopo il Rinascimento, che sono considerate come patrimonio culturale ed architettonico dell' Italia (e del mondo!), infine a quelle fatte in tempi recenti, considerevolmente durante e dopo il boom economico del l960 ed ora visti come antiquate. Le due prime categorie in gran parte sono composte dalle edilizie di muratura, mentre agli ultimi principalmente appartengono le costruzioni di cemento armato.

Variazione dei markers di attivata coagulazione indotta dalla terapia infusionale con Infliximab in pazienti affetti da malattie infiammatorie croniche intestinali

INTRODUCTION: A relationship between inflammatory response and coagulation is suggested by many observations. In particular, pro-inflammatory cytokines, such as TNFalpha, promote the activation of coagulation and reduce the production of anticoagulant molecules. It is known that inflammatory bowel diseases show a prothrombotic state and a condition of hypercoagulability. Aim of our study was to evaluate whether anti-TNFalpha therapy induces changes in the levels of coagulation activation markers in IBD patients. MATERIALS AND METHODS: We analyzed 48 plasma samples obtained before and 1 hour after 24 infliximab infusions (5 mg/kg) in 9 IBD patients (5 men and 4 women; mean age: 47.6+17.6 years; 4 Crohn's disease, 4 Ulcerative Colitis,1 Indeterminate Colitis). F1+2 and D-dymer levels were measured in each sample using ELISA methods.The data were statistically analyzed by means of Wilcoxon matched paired test. RESULTS: Median F1+2 levels were markdely reduced 1 hour after anti-TNFα infusion (median pre-infusion levels were 247.0 pmol/L and median post-infusion levels were 185.3 pmol/L) (p<0.002). Median D-dymer levels were also significantly reduced, from 485.2 ng/mL to 427.6 ng/mL (p< 0.001). These modifications were more evident in patients naive for infliximab therapy (p<0.02 for F1+2 and p<0.02 for D-dymer) and in Crohn's disease compared with Ulcerative Colitis patients (p=0.01 for F1+2 and p<0.007 for D-dymer).CONCLUSIONS: Infusion of infliximab significantly reduces the activation of coagulation cascade in IBD patients. This effect is early enough to suggest a direct effect of infliximab on the coagulation cascade and a possible new anti-inflammatory mechanism of action of this molecule.