31 research outputs found

    Development and in Vitro Evaluation of a Microbicide Gel Formulation for a Novel Non-Nucleoside Reverse Transcriptase Inhibitor Belonging to the N-Dihydroalkyloxybenzyloxopyrimidines (N-DABOs) Family

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    17openPreventing HIV transmission by the use of a vaginal microbicide is a topic of considerable interest in the fight against AIDS. Both a potent anti-HIV agent and an efficient formulation are required to develop a successful microbicide. In this regard, molecules able to inhibit the HIV replication before the integration of the viral DNA into the genetic material of the host cells, such as entry inhibitors or reverse transcriptase inhibitors (RTIs), are ideal candidates for prevention purpose. Among RTIs, S- and N-dihydroalkyloxybenzyloxopyrimidines (S-DABOs and N-DABOs) are interesting compounds active at nanomolar concentration against wild type of RT and with a very interesting activity against RT mutations. Herein, novel N-DABOs were synthesized and tested as anti-HIV agents. Furthermore, their mode of binding was studied by molecular modeling. At the same time, a vaginal microbicide gel formulation was developed and tested for one of the most promising candidates.openTintori, Cristina; Brai, Annalaura; DASSO LANG, MARIA CHIARA; Deodato, Davide; Greco, Antonia Michela; Bizzarri, Bruno Mattia; Cascone, Lorena; Casian, Alexandru; Zamperini, Claudio; Dreassi, Elena; Crespan, Emmanuele; Maga, Giovanni; Vanham, Guido; Ceresola, Elisa; Canducci, Filippo; Ariën, Kevin K.; Botta, MaurizioTintori, Cristina; Brai, Annalaura; DASSO LANG, MARIA CHIARA; Deodato, Davide; Greco, Antonia Michela; Bizzarri, Bruno Mattia; Cascone, Lorena; Casian, Alexandru; Zamperini, Claudio; Dreassi, Elena; Crespan, Emmanuele; Maga, Giovanni; Vanham, Guido; Ceresola, Elisa; Canducci, Filippo; Ariën, Kevin K.; Botta, Maurizi

    data_sheet_1_Increased iNKT17 Cell Frequency in the Intestine of Non-Obese Diabetic Mice Correlates With High Bacterioidales and Low Clostridiales Abundance.PDF

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    <p>iNKT cells play different immune function depending on their cytokine-secretion phenotype. iNKT17 cells predominantly secrete IL-17 and have an effector and pathogenic role in the pathogenesis of autoimmune diseases such as type 1 diabetes (T1D). In line with this notion, non-obese diabetic (NOD) mice that spontaneously develop T1D have an increased percentage of iNKT17 cells compared to non-autoimmune strains of mice. The factors that regulate iNKT cell expansion and acquisition of a specific iNKT17 cell phenotype are unclear. Here, we demonstrate that the percentage of iNKT17 cells is increased in the gut more than peripheral lymphoid organs of NOD mice, thus suggesting that the intestinal environment promotes iNKT17 cell differentiation in these mice. Increased intestinal iNKT17 cell differentiation in NOD mice is associated with the presence of pro-inflammatory IL-6-secreting dendritic cells that could contribute to iNKT cell expansion and iNKT17 cell differentiation. In addition, we found that increased iNKT17 cell differentiation in the large intestine of NOD mice is associated with a specific gut microbiota profile. We demonstrated a positive correlation between percentage of intestinal iNKT17 cells and bacterial strain richness (α-diversity) and relative abundance of Bacterioidales strains. On the contrary, the relative abundance of the anti-inflammatory Clostridiales strains negatively correlates with the intestinal iNKT17 cell frequency. Considering that iNKT17 cells play a key pathogenic role in T1D, our data support the notion that modulation of iNKT17 cell differentiation through gut microbiota changes could have a beneficial effect in T1D.</p

    Confocal microscopy on human atherosclerotic carotid sections.

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    <p>The macroscopic aspect of a carotid plaque is reconstructed in A by stereomicroscope and displays a lipidic core and of luminal thrombus. Features are confirmed in B by histology (haematoxylin and eosin). Confocal microscopy in C shows a reconstruction of part of the area close to the lumen (L) in a section stained with Fab7816-FLAG (green), mouse-anti-human Collagen type I (white) and mouse anti-human CD45 (red), revealed by opportune secondary antibodies. In D is an enlargement of the area indicated by § symbol in C with several Fab7816-FLAG+/CD45+ cells in the neointima. indicate a regions magnified. Scale bars indicate the magnification.</p

    Immunofluorescence on human coronary plaqes and Immunohistochemistry on human carotid sections with Fab7816-FLAG.

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    <p>a) Representative section stained by Movat’s pentchrome (left panel) showed the morphology of a portion of the coronary plaque tissue (plaque ID-A) displaying slightly damaged media rich in smooth muscle cells. Confocal microscopy (right panels) showed the presence of CD45<sup>+</sup> cells (red) labelled by Fab7816-FLAG revealed by MAb M2 anti-FLAG-FITC (green) in the coronary plaque tissue region indicated by symbol (#). DAPI stained the nuclei (blue). b) Immunoperoxidase on carotid plaque samples demonstrated the presence of several cells reacting with Fab7816-FLAG in an area close to the lumen (left panels) as revealed by MAb M2 anti-FLAG-HRP developed with DAB (brown). The signal is absent in a serial section where the Fab7816-FLAG is omitted (ctrl-, right panels). Magnified images in the bottom panels evidence the spindle shape of Fab7816-FLAG+ cells and their localization in between a clusters of altered cells, possibly foam cell. Haematoxylin (blue) stains nuclei. Scale bars indicate the magnification.</p

    Cross reactivity of commercial available monoclonal antibodies with TAGLN and OMPs.

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    <p>A) WB of OmpK36 with five commercial monoclonal mouse anti-human TAGLN antibodies (used at 1 or 5 <b>µ</b>g/ml). Three commercial mouse monoclonal antibodies were used as negative controls. Anti-6xHIS antibody (Roche) was used as positive control B) ELISA with all representative and five commercial monoclonal mouse anti-human TAGLN antibodies on purified human TAGLN or C) bacterial OMPs. Reactivity against bovine serum albumin (BSA) used as blocking antigen, is also shown.</p

    Cross reactivity of representative Fabs from all patients with TAGLN and OMPs.

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    <p>A) WB of purified human TAGLN (400 ng) with all representative Fabs (10<b> µ</b>g/mL). Unrelated human e8Fab-FLAGwas used as negative control. Anti-MYC-tag (C-terminal tag) and commercial anti-TAGLN were used as positive controls. While the commercial anti-TAGLN recognize selectively only one form of TAGLN, cloned human Fabs recognized both forms. B) WB of OmpK36 (500 ng) with all representative Fabs (10 ng/mL). C) ELISA with all representative Fabs on bacterial OMPs. Reactivity against bovine serum albumin (BSA) used as blocking antigen, is also shown.</p

    Biopanning selection with combinatorial IgG/k library ID-A.

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    <p>Library ID-A was selected by immunoaffinity on atherosclerotic plaque lysate. Results of screening ELISA assays of 30 clones after four rounds of biopanning selection is shown. Sequence analysis of the positively selected clones (O.D.450 nm >0.25 above background) is shown next to the ELISA screening.</p

    Western Blotting of bacterial lysates and on bacterial OMPs with Fab 7816.

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    <p>A) Western blotting of bacterial lysates with Fab7816. 1<b> µ</b>g of each bacterial lysate was loaded in each lane. Fab 7816 clearly reacted with <i>Proteus mirabilis</i> and <i>Klebsiella pneumoniae</i> lysates. B) Western blotting on induced/non induced BL21(DE3) bacterial cells transformed with pET28b vector expressing OmpK36. Two pET vectors were constructed: the NcoI/XhoI wild type OmpK36 vector or the HIS-tagged Ompk36 BamHI/XhoI vector. In both cases Fab 7816 recognized only IPTG induced bacteria.</p

    Confocal microscopy on human atherosclerotic carotid sections.

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    <p>Multiple staining of two carotid plaques are displayed to characterize the Fab7816-FLAG+ cells type. First plaque A-F panels, 2<sup>nd</sup> plaque G-I to N. The reconstruction of a section from 1<sup>st</sup> plaque stained with haematoxylin and eosin (HE) obtained by multiple images grabbing tool of Lucia-G software is in A; asterisk indicates the lumen in correspondence of Fab7816-FLAG+ cells, on the shoulder of the atheromasic lesion demonstrated in B by confocal lmicroscopy. Confocal microscopy images in B,C and G,H demonstrated by Fab7816-FLAG (green), goat-anti-human TAGLN (white), mouse-anti-human CD68 (red) the presence of triple positive cells in the intima, closely to the lumen. Single or double staining are shown in D-F and I-N. Squares and arrows indicated the enlarged areas. DAPI stains the nuclei (blue). Scale bars indicate the magnification.</p
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