169,798 research outputs found
Il team riabilitativo. La formazione degli operatori: il fisiatra
No full textThe goal of Rehabilitation medicine is to prevent disability and to provide therapy and compensation dor disability and its consequences, and intervene in order to facilitate social integration. Multidisciplinary approach is a key factor in Rehabilitation Medicin
Caratterizzazione di due anticorpi monoclonali umani cross-neutralizzanti diretti contro la glicoproteina E2 del virus dell'epatite C
No full textNell’80% dei casi di infezione, il virus dell’epatite C (HCV) supera le difese dell’ospite e
stabilisce un’infezione persistente, che espone il paziente al rischio di sviluppare cirrosi e
epatocarcinoma. La terapia basata su ribavirina e interferone è costosa, poco efficace e gravata da
effetti collaterali. Nonostante la grande variabilità genetica del virus, concentrata soprattutto a
livello delle glicoproteine E1E2 dell’envelope, sono state descritte alcune regioni di E2 conservate
tra i diversi genotipi, suggerendo che la scarsa variabilità di tali regioni è necessaria per mantenere
alcune funzioni cruciali della glicoproteina nel ciclo virale. Pertanto l’identificazione e la
caratterizzazione di anticorpi diretti contro queste porzioni conservate di E2 potrebbe fornire un
contributo per lo sviluppo di una valida immunoterapia passiva e per la realizzazione di un vaccino
efficace.
In questa tesi sono stati caratterizzati due frammenti anticorpali monoclonali umani (Fab e20 e
Fab e137) diretti contro la glicoproteina E2. e20 ed e137 sono stati clonati dal repertorio linfocitario
di una paziente infetta in modo cronico da HCV genotipo 1b. Per selezionare dei cloni
potenzialmente cross-reattivi, la library anticorpale ottenuta dalla paziente è stata screenata contro
una glicoproteina E2 ricombinante derivata da un sottotipo diverso: 1a. Tramite studi di binding,
e20 ed e137 sono risultati in grado di legare E2 di tutti i genotipi (tranne il genotipo 5 per il Fab
e137). Esperimenti condotti con peptidi lineari, saggi di competizione con anticorpi diretti contro
epitopi noti e la generazione di mutanti sito-specifici di E2 hanno dimostrato che e20 ed e137 sono
diretti contro un epitopo conformazionale. In particolar modo, l’epitopo del Fab e20 coinvolge i
residui aminoacidici: W437, F442, W529, G530 e D535; mentre quello del Fab e137 coinvolge:
T416, W420, W437, L438, L441, F442, W529, G530 e D535.
I residui W529, G530, D535 risultano essere conservati fra i vari genotipi e coinvolti nel legame
di E2 al CD81. Saggi di competizione fra il Fab e20 o il Fab e137 e CD81 confermano che i Fab
sono diretti contro una regione di E2 importante per il legame del virus al recettore cellulare. Si è
osservato che i Fab riconoscono dei residui contenuti della porzione della glicoproteina E2 che va
dall’aminoacido 436 al 447, regione descritta in letteratura come quella riconosciuta dagli anticorpi
non neutralizzati in grado di interferire con l’attività dei cloni neutralizzanti. I nostri dati
sembrerebbero contrastare questa affermazione, infatti i Fab e20 ed e137 non solo sono in grado di
riconoscere alcuni residui contenuti in questa regione, ma possiedono anche un’attività
neutralizzante rilevante. Inoltre il legame del Fab e137 viene abrogato anche da due mutazioni a
livello dell’epitopo riconosciuto dall’anticorpo murino AP33, descritto in letteratura come il clone
con un ampia cross-reattività ed un’elevata attività neutralizzante.
I Fab e20 ed e137 possiedono una potente attività cross-neutralizzante. Nel saggio di
neutralizzazione con pseudovirus, e20 ed e137 neutralizzano i genotipi 1a, 2a e 4 (IC50 intorno ai 7
μg/mL) e meno efficientemente i genotipi 1b e 2b. L’attività neutralizzante è stata confermata in un
saggio di neutralizzazione basato sull’uso di un particolare isolato virale in grado di replicare in
vitro (IC50 intorno ai 2 μg/mL). Grazie agli studi di cinetica di neutralizzazione è emersa la capacità
dei Fab e20 ed e137 di inibire l’infettività virale agendo dopo il binding del virus alle cellule
bersaglio; in particolare interferiscono con gli eventi immediatamente successivi al legame tra virus
e recettori a bassa affinità. Analizzando questi dati, possiamo concludere che abbiamo identificato e
caratterizzato due frammenti anticorpali con un ampia cross-reattività ed una potente attività crossneutralizzante,
in quanto sono in grado di legare dei residui aminoacidici sulla glicoproteina E2 che
svolgono un ruolo chiave nel ciclo virale e che risultano essere conservati tra i vari genotipi. Per cui
questi Fab risultano essere fondamentali per lo sviluppo di una immunoterapia passiva efficace e
per lo studio dell’interazione virus-ospite.In approximately 80% of the cases of infection, hepatitis C virus (HCV) overcomes the host
immune response, leading to a chronic infection associated with an increased risk of severe liver
diseases. Current therapy (ribavirin and interferon) is expensive, can have adverse side effects and
is ineffective for approximately 50% of patients. Despite the genetic variability of the virus at the
level of E1E2 envelope glycoproteins, some regions conserved among different E2 genotypes have
been described, suggesting that no variability at this level is required to maintain critical functions
of the glycoprotein in the life cycle of the virus. Therefore, the identification and characterization of
antibodies directed against these regions can theoretically provide a valuable contribution to the
creation of an effective vaccine and to the development of passive immunotherapy.
In this thesis, two human monoclonal antibody fragments (Fab e20 and Fab e137) directed
against the HCV E2 glycoprotein, have been characterized. The antibodies were cloned from
lymphocytes of a patient chronically infected with HCV genotype 1b. In order to select crossreactive
clones, the library obtained from the patient was screened against a recombinant
glycoprotein E2 derived from a different subtype: 1a. In binding experiment, e20 and e137 are
capable of binding E2 of all genotypes (e137 is not able to bind genotype 5).
Several studies with linear peptides representing different portions of E2, competition assays
with antibodies directed against known epitopes and the generation of site-specific mutants of E2
showed that e20 and e137 are directed against a conformational epitope. In particular, the epitope of
Fab e20 includes the amino acids: W437, F442, W529, G530 e D535; whereas the epitope of Fab
e137 includes the amino acids: T416, W420, W437, L438, L441, F442, W529, G530 e D535.The
residues W529, G530, D535 are conserved between different genotypes and involved in the binding
of E2 to CD81. Additional tests of competition, between e20 or e137 and CD81, confirmed that the
Fabs are directed against a region of E2 important for the binding of virus to cellular receptor. Fab
e20 and e137 are able to recognize amino acids within the region 436-447, that is described to bind
unneutralizing antibodies, that interfere with the neutralizing antibodies. Furthermore, the data
highlight that the epitope of e137 includes two conserved residues (aa 416 and 420) that were
described to be within the epitope recognized by mouse monoclonal antibody AP33.
The Fab e20 and Fab e137 have a potent cross-neutralizing activity. In the neutralization assay
with pseudovirus e20 and e137 neutralize genotypes 1a, 2a and 4 (IC50 approximately 7 μg/mL) and
less efficiently genotypes 1b and 2b. The neutralizing activity was also confirmed in a different
neutralization assay based on a particular virus isolate that can replicate in vitro (IC50 approximately
2 μg/mL). Finally, the kinetic of neutralization study has shown the ability of the Fab e20 and Fab
e137 to inhibit viral infectivity by acting after viral binding to target cells, in particular they are
capable of interfering with the events immediately after the binding between virus and low affinity
receptor.The data highlight that we identified and characterized two different broadly cross-reacting
and neutralizing human monoclonal antibody fragments direct against highly conserved residues of
E2 glycoprotein, that are crucial for CD81 binding and HCVpp infectivity.
Overall, the availability of cross-reactive monoclonal antibodies with strong neutralizing activity
(i) allows a better understanding of the virus-host interplay, (ii) provides new opportunities to
develop antigens potentially able to elicit a broadly neutralizing immune response, and (iii) may
assist in the development of an effective passive immunotherapy for HCV infection
Hepatitis C virus (HCV) infection may elicit neutralizing antibodies targeting epitopes conserved in all viral genotypes
No full textAnti-hepatitis C virus (HCV) cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20) were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535) are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp) incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc) JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection
Pulmonary metastasectomy : an overview
No full textMetastasectomy is the most frequent surgical resection undertaken by thoracic surgeons, being the lung the second common site of metastases. The present oncological criteria for pulmonary metastasectomy are: (I) the primary cancer need to be controlled or controllable; (II) no extrathoracic metastasis-that is not controlled or controllable-exists; (III) all of the tumor must be resectable, with adequate pulmonary reserve; (IV) there are no alternative medical treatment options with lower morbidity. General favourable prognostic features in patients with pulmonary metastases are: (I) one or few metastases; (II) long disease free interval; (III) normal CEA levels in colorectal cancers. Negative predictive features in patients candidate to pulmonary metastasectomies are: (I) active primary cancer; (II) extrathoracic metastases; (III) inability to obtain surgical radicality; (IV) mediastinal lymphatic spread. The lack of controlled trials and studies limited by short follow-up and small cohorts did not allow to overcome some skepticism; moreover, the heterogeneity of these patients in terms of demographic, biologic and histologic characteristics represents a clear limit even in the largest series. On the basis of present knowledge, without results coming from on-going randomized trials, radical resection, histology, and disease free interval seem to be independent prognostic factors identifying a cohort of patients maximally benefitting from lung metastasectomy
VATS bilaterale con sospensione sternale per timectomia radicale in paziente miastenica con neoformazione intraventricolare sinistra
No full textNei pazienti miastenici la chirurgia mininvasiva offre importanti benefici in termini di morbidità postoperatoria. Presentiamo un caso di un approccio VATS bilaterale con sospensione sternale per timectomia radicale in una paziente affetta da miastenia gravis e con una massa intraventricolare sinistra
Have lunar cycles and zodiac signs an impact on the mortality and morbidity of the lung cancer surgery?
No full textGoing Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Molecular characterization of the human neutralizing response against hepatitis C virus and its role in the prediction of the infection outcome
No full textThe hepatitis C virus (HCV) adopts several escape mechanisms and is able to evade the host immune response in the majority of
patients. During primary infection, HCV is not cleared in 80% of cases
resulting in chronic infection. The current treatment for HCV
infection is mainly represented by the administration of a combined
therapy (IFN-α, ribavirin) and by the use of new anti-viral drugs
(protease inhibitors). Unfortunately, only 50% of the infected patients
respond completely to these therapies. It has been demonstrated how a
neutralizing antibody response is correlated with lower HCV titer in
acute infection. Moreover, it is also demonstrated how a rapid induction
of neutralizing antibodies can be correlated with the viral clearance.
Under these purposes, results clear how neutralizing antibodies can be
important for the HCV infection control. In addition, they can represent
good candidates for passive immunotherapy. They also can be applied
both in diagnosis, as useful tools for the evaluation of the presence
of cross-neutralizing antibodies in patients sera, and in research studies
to better understand the virus–host interplay, an aspect that can be
crucial in predicting the infection clinical outcome. In this study, we
characterized the synergistic neutralization of HCV by two broadly
neutralizing human monoclonal antibodies directed against HCV/E2
glycoprotein, named e20 and e137
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