Three-dimensional (3D) Printed Model to Plan the Endoscopic Treatment of Upper Airway Stenosis

Background: Endoscopic management of tracheal stenosis may be challenging, especially in the case of complex stenosis placed near the vocal folds, and needing stent placement. Herein, we evaluated the utility of the three-dimensional (3D) airway model for procedural planning in a consecutive series of patients with complex airway stenosis and scheduled for endoscopic treatment. Methods: This strategy was applied to 7 consecutive patients with tracheal stenosis unfit for surgery. The model was printed in a rubber-like material, and almost 7 hours were needed to create it. All patients presented respiratory failure with a mean value of 3.4±0.4 Medical Research Council (MRC) dyspnea scale, 47±3.9 forced expiratory volume in 1 second (FEV1%), and an impairment in the 6-minute walking test (6MWT) (mean value, 175±53 m). The mean length of the stenosis was 19±3.4 mm; 3 of the 7 (43%) patients presented a subglottic stenosis. In 4/7 (57%) patients the stenosis was >5 mm, but its treatment required the placement of a stent because of the presence of tracheal cartilage injury. Results: The mean operation time was 22.7±6.6 minutes. No complications were observed during and after the procedure. A significant increase of MRC (3.4±0.4 vs. 1.6±0.5; P=0.003), of FEV1% (47±3.9 vs. 77±9.7; P=0.001), and of 6MWT (175±53 vs. 423±101; P=0.0002) was observed after the procedure (mean follow-up, 11.1±8.8 mo). Conclusion: Our 3D airway model in the management of airway stenosis is useful for procedural planning, rehearsal, and education. The fidelity level of the 3D model remains the main concern for its wider use in patient care. Thus, our impressions should be confirmed by future prospective studies

Impact of pe_pgrs33 gene polymorphisms on mycobacterium tuberculosis infection and pathogenesis

PE_PGRS33 is a surface-exposed protein of Mycobacterium tuberculosis (Mtb) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the pe_pgrs33 gene of Mtb clinical isolates and evaluate their impact on protein functions. We sequenced pe_pgrs33 in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the Mtb complex (MTBC). Overall, an association between pe_pgrs33 alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on pe_pgrs33 against deleterious SNPs. Among a total of 19 pe_pgrs33 alleles identified in this study, 5 were cloned and used to complement the pe_pgrs33 knock-out mutant strain of Mtb H37Rv (Mtb\uce\u9433) to assess the functional impact of the respective polymorphisms in in vitro infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of Mtb H37Rv, impairing the cell entry capacity of Mtb, but neither its intracellular replication rate nor its immunomodulatory properties. In vivo studies performed in the murine model of tuberculosis (TB) demonstrated that the Mtb\uce\u9433 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, Mtb\uce\u9433 showed an enhanced virulence during the chronic steps of infection compared to Mtb H37Rv. Similarly, the complementation of Mtb\uce\u9433 with a frameshift allele also resulted in a Mtb strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of Mtb

Evaluation of PE_PGRS33 as a potential surface target for humoral responses against Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) PE_PGRS33 is a surface-exposed protein that was shown to interact with Toll-like receptor 2 on host macrophages to induce inflammatory signals and promote entry in macrophages. In this study, we investigated PE_PGRS33 as a potential target of a humoral response aimed at hampering key processes in tuberculosis pathogenesis. PE_PGRS33 protein was successfully expressed and purified under native condition in Escherichia coli. The purified protein retained its native functional and biological properties, showing the ability to elicit proinflammatory signals in murine and human macrophages. Interestingly, a polyclonal antiserum raised against native PE_PGRS33 showed no cross-reactions with other mycobacterial proteins. The anti-PE_PGRS33 serum was also able to inhibit Mtb entry into macrophages, but it did not reduce entry of the Mtb\u394pe_pgrs33 strain. Addition of native recombinant PE_PGRS33 to the Mtb\u394pe_pgrs33 strain during infection restored the Mtb wild-type entry phenotype in macrophage. Moreover, the anti-PE_PGRS33 serum was able to neutralize the proinflammatory activity of PE_PGRS33 in vitro. Furthermore, mice immunized with native recombinant PE_PGRS33, but not with a DNA vaccine expressing PE_PGRS33, were able to restrict M. smegmatis in vivo. These results highlight the potential use of PE_PGRS33 as a target of a neutralizing humoral response against tuberculosis

Development of Potent Inhibitors of the Mycobacterium tuberculosis Virulence Factor Zmp1 and Evaluation of Their Effect on Mycobacterial Survival inside Macrophages

The enzyme Zmp1 is a zinc-containing peptidase that plays a critical role in the pathogenicity of Mycobacterium tuberculosis. Herein we describe the identification of a small set of Zmp1 inhibitors based on a novel 8-hydroxyquinoline-2-hydroxamate scaffold. Among the synthesized compounds, N-(benzyloxy)-8-hydroxyquinoline-2-carboxamide (1 c) was found to be the most potent Zmp1 inhibitor known to date, and its binding mode was analyzed both by kinetics studies and molecular modeling, identifying critical interactions of 1 c with the zinc ion and residues in the active site. The effect of 1 c on intracellular Mycobacterium survival was assayed in J774 murine macrophages infected with M. tuberculosis H37Rv or M. bovis BCG and human monocyte-derived macrophages infected with M. tuberculosis H37Rv. Cytotoxicity and genotoxicity were also assessed. Overall, inhibitor 1 c displays interesting in vitro antitubercular properties worthy of further investigation

Polar localization of PE_PGRS30^GFP spatial distribution in <i>M. smegmatis</i>.

<p>A) Confocal images of <i>M. smegmatis</i> expressing PE_PGRS30^GFP and its functional GFP-tagged chimeras obtained with a 63× objective. In the inbox, a 100× image obtained overlapping green channel and transmission image is shown. B) Sample line profile obtained quantifying the fluorescence along mycobacterial cell. C) Ratio between the GFP emission intensity at bacterium pole (considered as the value 200 nm far from the bacterium border) and GFP emission intensity in the cytoplasm (measured at the 50% of the bacterium length). Twenty R values were analyzed for each <i>M. smegmatis</i> strain under study. Two-tailed Student's <i>t</i>-test was used to analyze R ratio (<b>*</b><i>p</i><0.05, <b>**</b><i>p<</i>0.01).</p

Differential polar localization between PE_PGRS30 and PE_PGRS33 spatial distribution in <i>Mtb</i>.

<p>A) Schematic representation of the PE_PGRS33-derived chimeras expressed in <i>Mtb</i> H37Rv. B) Confocal images of <i>Mtb</i> H37Rv expressing PE_PGRS33^GFP and ₃₃PE^GFP using a 63× objective. In the inbox, a 100× image obtained overlapping green channel and transmission image is shown. Sample line profile and ratio between the GFP emission intensity at the bacterium pole and the GFP emission intensity in the cytoplasm between PE_PGRS30^GFP and PE_PGRS33^GFP (C) and between ₃₀PE^GFP and ₃₃PE^GFP (D). Twenty R values were analyzed for each <i>Mtb</i> strain under study. Two-tailed Student's <i>t</i>-test was used to analyze R ratio (<b>*</b><i>p</i><0.05, <b>**</b><i>p<</i>0.01).</p

Principio di responsabilità e ricerca pedagogica

Il volume raccoglie gli scritti di molti studiosi, italiani e stranieri, in onore di Umberto Margiotta: testimone attivo e protagonista dell'innovazione scientifica pedagogica nell'ultimo mezzo secolo. Come curatori, abbiamo operato per trovare, nella complessità del suo percorso, alcune direttrici che più di altre hanno contrassegnato l'evoluzione delle sue indagini scientifiche. La prima parte si concentra sull'approccio fenomenologico-critico della pedagogia, approccio incardinato nelle scienze umane e nelle scienze sociali, approdato in Margiotta all'analisi delle ontologie pedagogiche e alla formazione capacitante dei talenti, concepite in termini di razionalità incrementale. La seconda parte si occupa di quei dispositivi che hanno reso esplicita l’agentività di Margiotta, che non può fermarsi alla dimensione speculativa, deve originare percorsi di ricerca caratterizzati da evidenze, percorsi dove prevale la visione pragmatica del mondo

Impact of protein domains on PE_PGRS30 polar localization in Mycobacteria

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism