Multiple bottlenecks occur in the subcutaneous routes, but several clones are capable of dissemination.

<p><b>A</b>) Graphs indicate the signature tag proportions found of the bacterial population that resided in the ear of mice infected with the 100% LF⁺ library. Each column is a stacked percentage bar where each clone is represented by a different color. <b>B</b>) Bottlenecks often occur in the draining cLN and are always present by the time the bacteria reach the kidneys. Graphs indicate the signature tag proportions found of the bacterial population that resided in the cLN or kidney of mice infected with the 100% LF⁺ library. Each column is a stacked percentage bar where each clone is represented by a different color.</p

ΔLF clones can be trans complemented when 40% of the library produces LF.

<p><b>A</b>) ΔLF clones are capable of colonization and replication when LF is reduced by 60%. The signature tag proportions found of the bacterial population that resided in the ear of mice infected with the 40% LF⁺ library. Each column is a stacked percentage bar where each clone is represented by a different color. <b>B</b>) ΔLF clones disseminate to the cLN and kidneys when there is a 60% reduction in LF-producing bacteria. The signature tag proportions found of the bacterial population that resided in the cLN and kidney of mice infected with the 40% LF⁺ library. Each column is a stacked percentage bar where each clone is represented by a different color.</p

Decreasing LF producing clones in a signature tag library led to reductions in virulence.

<p><b>A</b>) Decreasing the amount of LF-producing clones in a library reduces the amount of LF that is produced when exotoxin is induced by R-media. All libraries were first normalized by the total protein in the lane. The amount of LF was determined relative to the 100% LF⁺ library. Western blotting was performed two independent times. Columns represent the median and bars represent the range. <b>B</b>) Decreasing the amount of LF-producing clones in a library led to reductions in macrophage cytotoxicity. RAW264.7 cells were plated in 96 well plates and treated with bacterial supernatants from exotoxin inducing R media. LDH release was measured and the %LDH release was calculated relative to the 100% LF⁺ supernatant. Experiments were repeated three independent times with at least 4 replicates per run. Asterisks represent a significant difference (One-way ANOVA with Tukey's multiple comparisons test. ****<i>P-</i>value <0.0001). Error bars represent the 95% confidence interval. <b>C</b>) Kaplan-Meier curve of mice infected with 2×10⁵ CFU spores subcutaneously in the ear pinna. Mice were observed for 7 days. Solid circles represent mice infected with a library where all clones are LF⁺ (n = 22), open circles represent 40% LF⁺ library (n = 14), empty upward triangles represent 10% LF⁺ library (n = 39), empty downward triangles represent 0.3% LF⁺ clones library (n = 8). Asterisk represents a significant difference from the 100% LF⁺ library (Log rank test, *<i>P</i>-value <0.05).</p

Fewer clones disseminate when the library only has 10% of the population produce LF.

<p>There are fewer clones that disseminate when LF-producing bacteria are reduced by 90% in the inoculum. The number of clones that disseminated to the kidneys were enumerated and compared between the libraries. Each symbol represents a mouse. Bars represent the mean and error bars represent the standard deviation. Asterisks represent a statistical difference between the means (Student's T Test, *<i>P</i>-value  = 0.044).</p

Increased survival was due to defects in colonization or dissemination.

<p><b>A</b>) At least 90% of spores in the inoculum germinated 24 hours post infection of ear pinnas. Mice were infected with between 1.5×10⁵–2.0×10⁵ spores of the various libraries and ears were dissected 24 hours post infection. The percentage of germination was measured by comparing the amount of CFU from unheated and heated tissue homogenates. These experiments were done with 3 mice per library and repeated twice. Each symbol represents an individual mouse. Bars represent mean and error bars represent the 95% confidence interval. No statistical difference was found between the populations after performing a one-way ANOVA with Tukey's Multiple Comparison test (<i>P</i>-value >0.05). <b>B</b>) The number of days luminescence was detected at a particular fold difference over background for each signature tag library was not significantly different. Fold difference represents the increase of the infected ear over the non-infected ear. Solid black bars represent mice infected with the 100% LF⁺ (n = 4), grey bars represent 40% LF⁺ library (n = 8), white bars represent 10% LF⁺ library (n = 8), diagonal hashed bars represent 0.3% LF⁺ library (n = 8). Number signs signify that library never had luminescence detected over background at the given fold difference. Asterisks represent a statistical difference from the 0.3% LF⁺ library (Two way ANOVA with Dunnett's Multiple comparison test; **** <i>P</i>-value <0.0001). <b>C</b>) There is no temporal difference in dissemination between the signature tag libraries. Columns indicate the average day luminescence was detected in the draining cervical lymph node and kidney with the 100% LF⁺ library (solid circles, n = 9 for cLN, n = 7 for kidney), 40% LF⁺ library (open circles, n = 9 for cLN, n = 8 for kidney), 10% LF⁺ library (upward facing open triangles, n = 7 for cLN, n = 6 for kidney). Mice were observed at least once per day. Error bars represent the standard deviation. No significant difference when comparing the days for either the cLNs or kidneys (One way ANOVA, <i>P</i>-value>0.05.) <b>D</b>) CFU enumeration (Total bacteria and heat-resistant) in organs infected with differing levels of LF. CFU were enumerated from mice infected with 2×10⁵ CFU spores of either the 100% LF⁺ library (checked boxes, n = 4), 40% LF⁺ signature tag library (gray boxes, n = 6), or 10% LF⁺ signature tag library (white box n = 8). Mice were dissected after luminescence was detected in the kidney, but before mice were moribund. Error bars represent the 10–90 percentiles.</p

ΔLF clones are less capable of survival and dissemination when 10% of the library produces LF.

<p><b>A</b>) BIG23L is the dominant clone in the ear of mice when there is a 90% reduction of LF-producing bacteria in the inoculum. The signature tag proportions found in the bacterial population that resided in the ear of mice infected with the 10% LF⁺ library. Each column is a stacked percentage bar where each clone is represented by a different color. Asterisk represents a significant difference from the initial inoculum (Fisher's Exact test, *** <i>P</i>-value <0.001) <b>B</b>) ΔLF clones are less abundant in the disseminated population when LF is reduced by 90%. The signature tag proportions found of the bacterial population that resided in the cLN and kidneys of mice infected with the 10% LF⁺ library. Each column is a stacked percentage bar where each clone is represented by a different color. Asterisk represents a significant difference from the initial inoculum (Fisher's Exact test, ** <i>P</i>-value <0.01; ****<i>P</i>-value <0.0001).</p

Debridement significantly increases mouse survival from subcutaneous <i>B. anthracis</i> infection.

<p>(A) Black and white photos of a single A/J mouse infected with 1×10⁵ BIG23 spores in the left ear overlaid with false-color representation of photon emission intensity as indicated by the scale on the right in p/s/cm-2/sr. At 12 hours post injection luminescent infected tissue (left photo- before) was removed (debrided) to eliminate all bacterial luminescence (right photo- after). (B to D) BIG23 spores were injected subcutaneously into the left ear of 4–12 week old A/J mice. Control mice either had their left ear unperturbed (control) or non infected tissue on the same ear was removed (cut control). Mice were then monitored for infection and dissemination using in vivo bioluminescent imaging. Mice were monitored for a total of ten days post-infection (p.i.), until luminescence was detected in a major organ, or mouse appeared moribund (indicating imminent death). (B) Mice were inoculated with 1×10⁵ spores of BIG23. Luminescent tissue was debrided at 12 hours p.i. Survival data from a total of seven mice from three independent experiments were analyzed with the log rank test to determine significant differences in survival between debrided and control mice (*, p = 0.0108), and debrided and cut control mice (**, p = 0.0021). There was no statistical difference between control and cut control mice. (C) Mice were inoculated with 1×10⁶ spores of BIG23. Luminescent tissue was debrided at 12 hours p.i. Survival data was analyzed with the log rank test on a total of ten debrided mice and seven mice each for the control groups from three independent experiments to determine significant differences in survival between debrided and control mice (*, p = 0.0109) and debrided and cut control mice (**, p = 0.0026). There was no statistical difference between control and cut control mice. (D) Mice were infected with BIG23 as described above, but debridement of luminescent tissue was performed at 24, 48, or 78 hour p.i. Survival data was analyzed with the log rank test on a total of seven mice from each experimental group, with the exception of the control mice that totaled six, from three independent experiments to determine significant differences from control mice. The survival of mice debrided at 24 hours (*, p = 0.0110) and 48 hours (*, p = 0.0477) p.i. was significantly increased. The survival of mice debrided at 72 hours p.i. was not significantly different than control mice (p = 0.1734).</p

Presence of lethal factor in cervical draining lymph node does not alter cellular activation.

<p>Mice were infected with 1×10⁶ Sterne strain or TKO spores subcutaneously in the left ear. At 24 hours cLN were taken from mice including: Sterne infected mice (solid line non-shaded) along with control non-infected contralateral lymph nodes from Sterne infected mice (solid line shaded), and TKO infected (dashed line non-shaded) and control non-infected contralateral lymph nodes from TKO infected mice (dashed line shaded). cLN were homogenized and cells were stained for flow cytometry. Flow plots were gated for single event live CD45 positive cells. (A) Left- a representative histogram comparing expression of CD69 in draining cLN. Right- The mean fluorescence intensity of lymph nodes draining either Sterne (*, p = 0.0464) or TKO (*, p = 0.0256) infections from seven mice was significantly higher than contralateral control lymph nodes as determined using Student's T test. (B) Left- a representative histogram comparing expression of CD86 in draining cLN. Right- The mean fluorescence intensity of lymph nodes draining either Sterne (*, p = 0.0119) or TKO (**, p = 0.0063) infections from seven mice was significantly higher than contralateral control lymph nodes as determined using Student's T test. (C) A representative comparison of CD80 expression in draining cLN. The mean fluorescence intensity of lymph nodes draining either Sterne or TKO infections from seven mice was not significantly higher than lymph nodes that were not.</p

Lionello Venturi e le polemiche sull’arte astratta in Italia alla metà del XX secolo.

Storia, critica dell'arte e politica nell'Italia del secondo dopoguerra