Effect of tetracycline on the cell viability and the expression of MMPs in human keratinocytes treated with <i>L</i>. <i>laeta</i> venom.

<p>HaCaT cell cultures were incubated with 100 μg/mL of <i>L</i>. <i>laeta</i> venom (V) or control medium (C) in the presence or absence of tetracycline (T). After 72 hours of treatment, cell viability was analyzed by MTT assay <b>(A)</b> and cell supernatants were analyzed by zymography <b>(B),</b> western blotting <b>(C)</b> and Luminex assay (tetracycline: 150 μg/mL) <b>(D).</b> Results are representative of three independent experiments and are expressed as the mean of triplicates ± standard deviation. Significant differences (*) <i>P</i><0.05; (**) <i>P</i><0.01 and (***) <i>P</i><0.001 from control or (^#) <i>P</i><0.05; (^##) <i>P</i><0.01 and (^###) <i>P</i><0.001 from venom treated cells.</p

Histological analysis of the dermonecrotic lesion induced by <i>L</i>. <i>laeta</i> venom after tetracycline treatment.

<p>Rabbits were injected with 3 μg/mL of <i>L</i>. <i>laeta</i> venom and treated, twice a day during 48 hours, with tetracycline containing cream. Control sites were injected with an equal volume of PBS. Panels correspond to the panoramic view of skin sections from rabbits injected with PBS <b>(A),</b> <i>L</i>. <i>laeta</i> venom <b>(B),</b> <i>L</i>. <i>laeta</i> venom and treated with lanolin cream vehicle <b>(C),</b> <i>L</i>. <i>laeta</i> venom and treated with lanolin cream containing tetracycline <b>(D)</b>. Arrows indicate areas of leukocyte infiltration. Panels 1–2 show details of collagenous area of the dermis of the same sections. Black bars at the top of each panel indicate 100 μm.</p

<i>L</i>. <i>laeta</i> venom and Class 1 SMase D induce the expression of matrix metaloproteinases in human keratinocytes.

<p><i>Zymography analysis</i>: HaCaT cell culture supernatants collected after 72 hours of the treatment with <i>L</i>. <i>laeta</i> venom <b>(A)</b> or 48 hours with the SMase D Class I (SMase I) <b>(B)</b> were run on gelatin containing 10% SDS-PAGE gels under non-reducing conditions. Control supernatants were harvested after 48 and 72 hours from cells incubated with medium plus saline. <b>(C)</b> <i>Western blot analysis</i>: HaCaT cell culture supernatants, collected after 24, 48 72 hours of the treatment with medium plus saline (C), venom or SMases D from Class 1 (SMase I from <i>L</i>. <i>laeta</i>) were run on 12.5% SDS-PAGE gels, blotted and developed using MoAbs against human MMP7. Figures are representative of three independent experiments.</p

Effect of <i>L</i>. <i>laeta</i> venom and Class 1 SMase D on human keratinocytes cell viability.

<p>HaCaT cell cultures (2x10⁴ cells) were incubated with increasing concentrations (25 to 200 μg/ml) of <i>Loxosceles</i> venom <b>(A)</b> or SMase I <b>(B)</b>. After 24, 48 and 72 hours of treatment, cell viability was analyzed by MTT assay. Results are representative of three independent experiments and expressed as the mean of triplicates ± standard deviation. Significant differences (***) <i>P</i><0.001 from control.</p

Class 1 SMase D (SMase I from <i>L</i>. <i>laeta</i>) but not SMase Class 2 induced expression of MMP7 in human keratinocytes.

<p>HaCaT cell culture supernatants, collected after 48 hours of the treatment with medium plus saline (Control) or 100 μg/mL of SMases D from Class 1 (SMase I from <i>L</i>. <i>laeta</i>) or Class 2 (P1 and P2 from <i>L</i>. <i>intermedia</i>), were subjected to zymography analysis <b>(A)</b>, Western blotting <b>(B)</b> using MoAbs against human MMP7, and Luminex assay <b>(C)</b> for human MMP2, MMP9 and MMP7. Results are representative of three independent experiments and are expressed as the mean of triplicates ± standard deviation. Significant differences (*) <i>P</i><0.05; (**) <i>P</i><0.01 and (***) <i>P</i><0.001 from control or (^#) <i>P</i><0.05; (^##) <i>P</i><0.01 and (^###) <i>P</i><0.001 from SMases D treated cells.</p

<i>Loxosceles</i> and <i>Sicarius ornatus</i> venom SMases D incorporate into human erythrocytes and cause loss of glycophorin C expression.

<p>Erythrocytes were treated with venoms from <i>Sicarius ornatus</i> or <i>Loxosceles</i> or with VBS²⁺ buffer (control) and analyzed for the expression GPC by flow cytometry [A]. The ability of the toxins to insert into the erythrocyte surface was analyzed using a monospecific polyclonal rabbit serum against <i>Loxosceles intermedia</i> SMases D [B]. Results are representative for three different experiments and expressed as median of fluorescence of duplicates ± SD. The variation among experiments was around 10%. (*) Significant differences (<i>P</i><0.05) from control; (#) Significant differences from <i>L. laeta</i> venom (<i>P</i><0.05); (•) Significant difference between female and male <i>Sicarius</i> venoms (<i>P</i><0.05).</p

Molecular model of Lox-FB.

<p>Construction of molecular model based on structure of human factor B (PDB 2ok5.1), using the tool SWISS-Model Workspace available on <a href="http://swissmodel.expasy.org/workspace/" target="_blank">http://swissmodel.expasy.org/workspace/</a>. The further analyses were performed using the software SwissPDBViewer.</p

Multiple alignment of the vWFA and serine protease domains from Lox-FB with sequences of FB/C2 proteins from other organisms.

<p>The alignment was performed using MUSCLE algorithm available in MEGA software. Amino acids that are highlighted in bold indicate identical regions. The amino acids residues that are functionally important at Factor D or C1s cleavage site; the metal ion dependent binding site (MIDAS) and the protease active sites are indicated by dark arrows; the three amino acids residues (T⁴³¹, A⁴³³, S⁶¹⁶) that are important on stabilization of catalytic triad are indicated by sign #; the conserved cysteines residues are indicated by asterisks and the two extra cysteines present in Lox-FB, Hd-FB3 and Rd-FB are highlighted in grey. <i>Loxosceles laeta</i> (Lox-FB), <i>Hasarius adansoni</i> (Hd-FB1; HD-FB2; Hd-FB3), <i>Tachypleus tridentatus</i> (Tt-FB1;Tt-FB-2), <i>Ruditapes decussatus</i> (Rd-FB), <i>Nematostella vectensis</i> (Nv-FB), <i>Branchiostoma belcheri</i> (Bb-FB), <i>Strongylocentrotus purpuratus</i> (Sp-FB), <i>Apostichopus japonicus</i> (Aj-FB), <i>Homo sapiens</i> (Hu-C2;Hu-FB).</p

Hemolysis dependent of complement system.

<p>Human Erythrocytes, pre-treated with VBS²⁺ or with <i>Sicarius ornatus</i> or <i>Loxosceles</i> spp. venoms, were incubated with autologous normal human serum. After incubation for 1 h at 37°C, unlysed cells were spun down; the absorbance of the supernatants was measured at 414 nm and expressed as percentage of lysis. Results are representative for three different experiments and expressed as mean ± SD of duplicates. The variation among experiments was around 10%. (*) Significant differences (<i>P</i><0.05) from control; (#) Significant differences from <i>Loxosceles</i> spp. venoms (<i>P</i><0.05); (•) Significant difference between female and male <i>Sicarius ornatus</i> venoms (<i>P</i><0.05).</p

Protein content of <i>Sicarius ornatus</i> and <i>Loxosceles</i> venoms.

<p>The protein content of <i>Loxosceles laeta</i> females (n = 68), female (n = 5) and male (n = 3) adult <i>Sicarius ornatus</i> spiders venoms samples were determined using the BCA colorimetric method. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002394#s3" target="_blank">Results</a> are expressed as mean ± SD. (*) Significant difference (<i>P</i><0.05) from <i>L. laeta</i> venom.</p