45 research outputs found
Lower Rim Guanidinocalix[4]arenes: Macrocyclic Nonviral Vectors for Cell Transfection
Guanidinium groups were introduced through a spacer at
the lower
rim of calix[4]Âarenes in the cone conformation to give new potential
nonviral vectors for gene delivery. Several structural modifications
were explored, such as the presence or absence of a macrocyclic scaffold,
lipophilicity of the backbone, length of the spacer, and nature of
the charged groups, in order to better understand the factors which
affect the DNA condensation ability and transfection efficiency of
these derivatives. The most interesting compound was a calix[4]Âarene
unsubstituted at the upper rim and having four guanidinium groups
linked at the lower rim through a three carbon atom spacer. This compound,
when formulated with DOPE, showed low toxicity and transfection efficiency
higher than the commercially available lipofectamine LTX in the treatment
of human Rhabdomiosarcoma and Vero cells. Most of the investigated
compounds showed a tendency to self-aggregate in pure water or in
the presence of salts, as evidenced by NMR and AFM studies, and it
was found that the ability to condense DNA plasmids in nanometric
globules is a necessary but not sufficient condition for transfection.
The superiority of macrocyclic vectors over linear Gemini-type analogues
and of guanidinium compared to other ammonium head groups in determining
the biological activity of the vectors was also ascertained
<i>Neospora caninum</i> infection of ASSCs.
<p>Representative phase contrast image (20x) of ASSCs containing <i>Neospora caninum</i> tachyzoites and cell free <i>Neospora caninum</i> tachyzoites at 48 h post infection.</p
Long lasting response of bIL-8-Luc reporter construct.
<p><b>A</b>) Representative images of groups of mice (n = 3 per group) transiently transgenized with bIL-8-Luc or Empty vector DNA and intratracheally instilled with LPS, TNF-α or vehicle (saline solution) at 8, 32, 45 and 60 days post transgenization. Mice were monitored at 3 hours post stimulation by BLI by drawing a region of interest (ROI) over the chest. <b>B</b>) Light intensity quantification of the ROI using the LivingImage software. The experiment was repeated three times and each point represents the mean ± standard deviation of 9 animals. <b>C</b>) Data were expressed as folds of induction over vehicle-treated mice (Saline) and statistical differences were tested by One Way ANOVA followed by Dunnet’s post hoc test for group comparisons. Results are reported as mean ± SD and significance attributed when <i>P</i><0.05 (*) or <i>P</i><0.01(**).</p
ASSC viral infection.
<p>Representative phase contrast images (10x) of uninfected and infected (CpHV-1, BoHV-1 and BVDV) ASSCs 48 h post infection, along with the replication kinetics of the respective viruses expressed as log TCID<sub>50</sub>/ml. The data presented are the means ± standard errors of triplicate measurements (<i>P</i>>0.05 for all time points as measured by Student’s <i>t</i> test).</p
<i>In vitro</i> characterization of bIL-8-Luc reporter construct.
<p><b>A</b>) Schematic diagram (not to scale) of the <i>Bos taurus</i> chromosome 6 with the IL-8 gene locus comprising promoter and exons annotated by numbers. The 2 kb IL-8 promoter sequence, comprising the putative TATA box, the transcriptional start site (+1) and the UTR (red underlined) was cloned into the pGL3 basic vector in front of the open reading frame of the Luciferase reporter. <b>B</b>) RT-PCR products of TLRs from 1 to 9 and their corresponding amplicon size. GAPDH was used as an internal control and the amplification performed in the absence of reverse transcriptase (-RT) as a negative control. <b>C</b>) RT-PCR products of TNF-αRI,II and their corresponding amplicon size. GAPDH was used as an internal control and the amplification performed in the absence of reverse transcriptase (-RT) as a negative control. D) IL-8 promoter activation after treatment of bIL-8-Luc transfected LA-4 cells with LPS or TNF-α, along with the untreated control (Untr.). Each experiment was done in quadruplicate, and each point represents the mean ± standard deviation of three experiments. Data were expressed as folds of induction (2,1 and 2,8 for LPS and TNF-α respectively) over vehicle-treated cells and statistical differences were tested by One Way ANOVA followed by Dunnet’s post hoc test for group comparisons. Results are reported as mean ± SD and significance attributed when <i>P</i><0.05 (*) or <i>P</i><0.01(**).</p
<i>In vivo</i> tumorigenesis assay.
<p>A) Representative in vivo bioluminescence images of nude mice subcutaneously inoculated with 3×10<sup>6</sup> lentiviral transduced ASSCs on one side and 10<sup>6</sup> on the opposite side of their body. Luminescence signal was acquired weekly until it was no longer detectable (B).</p
White blood cells and neutrophils infiltration.
<p>Cellular infiltration into the lung of mice intratracheally instilled with LPS, TNF-α or vehicle. The amount of White blood cells (WBC), Neutrophils (Neut) and Monocytes (Mono) found in BALF was expressed as number of cells per μl at 3 (A) and 24 hours (B) post treatment. The experiment was repeated three times and each point represents the mean ± standard deviation of 9 animals. Data were expressed as folds of induction over vehicle-treated mice (Saline) and statistical differences were tested by One Way ANOVA followed by Dunnet’s post hoc test for group comparisons. Results are reported as mean ± SD and significance attributed when <i>P</i><0.05 (*) or <i>P</i><0.01(**).</p
ASSC biodistribution.
<p>A) Representative in vivo bioluminescence image of ASSCs transduced with different doses (1 and 0.1 transducing units (T.U.)/cell) of a lentiviral vector expressing luciferase. B) Representative <i>in vivo</i> bioluminescence image of FVB mice displaying ASSCs transduced with a lentiviral vector expressing luciferase and localized to the lung 4 h post injection.</p
Cell line characterization.
<p>A) Exponential regression curve by which ASSCs doubling time (DT) and growth rate (GR) was calculated. B) SV40 large and small T antigen expression in ASSCs at different passages (5, 10, 15, 20, 30, 40 and 60) as monitored by Western immunoblotting. A cross reactive protein band (CB) is detected also in the negative control (−), ASSC primary culture. The ladder lane is indicated by Ld. C) Phase contrast and fluorescent images (20x) of ASSCs from the 60<sup>th</sup> passage indicating expression of the stromal marker, vimentin. Counterstained nuclei with 4′, 6-diamidino-2-phenylindole (DAPI) were merged with green fluorescent image (merge). ASSCs from the 3<sup>rd</sup>, 10<sup>th</sup>, and 20<sup>th</sup> passage stained similarly with α-vimentin antibody (not shown).</p
bIL-8-Luc reporter construct response <i>in vivo</i>.
<p><b>A</b>) Representative images of groups of mice (n = 3 per group) transiently transgenized with bIL-8-Luc or Empty vector DNA and intratracheally instilled with LPS, TNF-α or vehicle (saline solution). Mice were monitored at 3, 5 and 24 hours post stimulation by BLI drawing a region of interest (ROI) over the chest. <b>B</b>) Light intensity quantification of the ROI using the LivingImage software. The experiment was repeated three times and each point represents the mean ± standard deviation of 9 animals. <b>C</b>) Data were expressed as folds of induction over vehicle-treated mice (Saline) and statistical differences were tested by One Way ANOVA followed by Dunnet’s post hoc test for group comparisons. Results are reported as mean ± SD and significance attributed when <i>P</i><0.05 (*) or <i>P</i><0.01(**).</p