The effect of <i>p66</i> site-directed integrin binding mutants on vascular transmigration and clearance in <i>Cd1d</i>^<i>-/-</i> mice.

<p><b>A)</b> GFP-expressing <i>B</i>. <i>burgdorferi</i> strains, infectious wild type (GCB847), <i>p66</i>^{<i>D205A</i>,<i>D207A</i>} (GCB3003) and <i>p66</i>^{<i>Δ202–208</i>} (GCB3004) were injected into the tail vein of <i>Cd1d</i>^<i>-/-</i> mice (n = 3/group, 4x10⁸ spirochetes were injected per mouse). After 24 hours, vascular transmigration was scored in the knee joint-proximal tissue in the living mice by intravital microscopy using a spinning disk laser confocal microscope. Blood vessels were stained with PE-conjugated PECAM-1 antibody and green fluorescent spirochetes outside of the vasculature were counted in at least five fields of view (FOV) per mouse. Statistical significance was analyzed using non-parametric Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. <i>P</i>-values for select pairwise comparisons are shown; ns denotes not significant (P-values >0.05). <b>B)</b>. Concentrations of <i>B</i>. <i>burgdorferi</i> in mouse plasma after iv inoculation. <i>Cd1d</i>^<i>-/-</i> mice were injected with <i>B</i>. <i>burgdorferi</i> through the tail vein and blood was withdrawn at 5 and 60 minutes post-inoculation (n = 3/group). Blood cells were allowed to settle overnight as described in Materials and Methods and spirochetes in the plasma were directly counted by dark-field microscopy. The change in spirochete concentration between 5 and 60 minutes was determined for each mouse as the percentage of spirochetes present at 60 minutes relative to the initial 5 minute time point. Statistical significance was analyzed using non-parametric Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. <i>P</i>-values for select pairwise comparisons are shown; ns denotes not significant (P-values >0.05).