30. 針刺麻酔に関する文献的ならびに基礎的研究(第519回千葉医学会例会 第8回佐藤外科例会)

<p>Cytokine response in lung homogenates, BALF and plasma of WT and <i>Trem-2</i>^<i>-/-</i> mice during experimental melioidosis.</p

NOD2 deficiency reduces the capacity of alveolar macrophages and neutrophils to internalize <i>S</i>. <i>pneumoniae in vitro</i>.

<p>Growth arrested, FITC labeled <i>S</i>. <i>pneumoniae</i> D39 were incubated with alveolar macrophages (A) or CFSE-labeled <i>S</i>. <i>pneumoniae</i> D39 with peripheral blood neutrophils (B) from wild-type (Wt) and <i>Nod2</i>^{<i>−/−</i>} mice at 4°C (n = 3–4 per mouse strain) or 37°C (n = 6–8 per mouse strain) for 1 hour after which phagocytosis was quantified. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation; *<i>P</i><0.05, ***<i>P</i><0.001 versus Wt cells.</p

Cytokine and chemokine concentrations in lung homogenates of wild-type and <i>Nod2</i>^<i>-/-</i>mice during pneumococcal pneumonia caused by serotype 2 <i>S</i>. <i>pneumoniae</i> (D39).

<p>Proinflammatory cytokine (TNF-α, IL-1β, IL-6) and chemokine (KC, MIP-2 and CCL2) levels in lung homogenates at 6, 24 and 48 hours after intranasal <i>S</i>. <i>pneumoniae</i> D39 infection in wild-type (Wt) and <i>Nod2</i>^<i>-/-</i> mice. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (8 mice per group at each time point); * <i>P</i><0.05, ** <i>P</i><0.01.</p

NOD2 deficiency results in defective pulmonary clearance of non-encapsulated mutant <i>S</i>. <i>pneumoniae</i> D39Δ<i>cps</i>.

<p>Wild-type (Wt) and <i>Nod2</i>^{<i>−/−</i>} mice were intranasally infected with 10⁸ CFU of <i>S</i>. <i>pneumoniae</i> D39Δ<i>cps</i> and sacrificed 6 or 24 hours later. Bacterial loads were determined in lung homogenates. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (8 mice per group at each time point); * <i>P</i><0.05.</p

Cytokine and chemokine concentrations in lung homogenates of wild-type (Wt) and <i>Nod2</i>^<i>-/-</i> mice during pneumococcal pneumonia caused by an unencapsulated mutant strain serotype 2 <i>S</i>. <i>pneumoniae</i> D39Δ<i>cps</i>.

<p>Proinflammatory cytokine (TNF-α, IL-1β, IL-6) and chemokine (KC, MIP-2 and CCL2) levels in lung homogenates at 6 and 24 hours after intranasal <i>S</i>. <i>pneumoniae</i> D39Δ<i>cps</i> infection in wild-type (Wt) and <i>Nod2</i>^<i>-/-</i> mice. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (8 mice per group at each time point); * <i>P</i><0.05.</p

NOD2 deficiency does not influence lung pathology and neutrophil recruitment during pneumonia caused by an unencapsulated mutant <i>S</i>. <i>pneumoniae</i> D39Δ<i>cps</i>.

<p>Wild-type (Wt) and <i>Nod2</i>^{<i>−/−</i>} mice were intranasally infected with 10⁸ CFU of <i>S</i>. <i>pneumoniae</i> D39Δ<i>cps</i> and sacrificed 6 or 24 hours later. Representative hematoxylin and eosin (HE) stainings of lung tissue of Wt (A) and <i>Nod2</i>^<i>-/-</i> mice (B) 24 hours after inoculation with <i>S</i>. <i>pneumoniae</i> (original magnification ×200). Quantification of pulmonary Ly-6G positivity (F) and MPO levels in whole lung homogenates (G) 6 or 24 hours after intranasal infection with <i>S</i>. <i>pneumoniae</i> D39Δ<i>cps</i> of wild-type (Wt) and <i>Nod2</i>^{<i>−/−</i>} mice. Representative neutrophil stainings (brown) of Wt (D) and <i>Nod2</i>^{<i>−/−</i>} mice (E) 24 hours after induction of pneumococcal pneumonia are shown (original magnification ×200). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (8 mice per group at each time point). Differences between groups were not significant.</p

NOD2 deficiency does not influence lung pathology and neutrophil recruitment during pneumonia caused by serotype 2 <i>S</i>. <i>pneumoniae</i> (D39).

<p>Wild-type (Wt) and <i>Nod2</i>^{<i>−/−</i>} mice were intranasally infected with 10⁷ CFU of <i>S</i>. <i>pneumoniae</i> D39 and sacrificed 6, 24 or 48 hours later. Representative hematoxylin and eosin (HE) stainings of lung tissue of Wt (A) and <i>Nod2</i>^<i>-/-</i> mice (B) 24 hours after inoculation with <i>S</i>. <i>pneumoniae</i> D39 (original magnification ×200). Quantification of pulmonary Ly-6G positivity (F) and MPO levels in whole lung homogenates (G) 6, 24 or 48 hours after intranasal infection with <i>S</i>. <i>pneumoniae</i> D39 of wild-type (Wt) and <i>Nod2</i>^{<i>−/−</i>} mice. Representative neutrophil stainings (brown) of Wt (D) and <i>Nod2</i>^{<i>−/−</i>} mice (E) 24 hours after induction of pneumococcal pneumonia are shown (original magnification ×200). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (8 mice per group at each time point). Differences between groups were not significant.</p

Effect of TREM-1 deficiency on bacterial clearance, pulmonary neutrophil influx and organ damage during experimental melioidosis.

<p>WT (closed circles/black bars) and <i>Trem-1/3</i>^<i>-/-</i> mice (open circles/ white bars) were intranasally infected with 5 x 102 CFU of <i>B</i>. <i>pseudomallei</i> and sacrificed 24 and 72 h post-infection, followed by determination of bacterial loads in lung homogenate <b>(</b><i>A</i><b>),</b> BALF <b>(</b><i>B</i><b>),</b> blood <b>(</b><i>C</i><b>)</b> and liver <b>(</b><i>D</i><b>).</b> Neutrophil influx as determined by % Ly6G positive surface of lung slides was calculated for WT and <i>Trem-1/3</i>^<i>-/-</i> mice <b>(</b><i>E</i><b>).</b> Lung <b>(</b><i>F</i><b>)</b> and spleen <b>(</b><i>G</i><b>)</b> pathology was scored as described in the Methods section. Aspartate transaminase (AST; <i>H</i><b>),</b> alanine transaminase (ALT; <i>I</i>), lactate dehydrogenase (LDH; <i>J</i><b>)</b> and blood urea nitrogen (BUN; <i>K</i>) were measured as a marker for end organ damage. Data are expressed as mean ± SEM. n = 7–8 mice per group. *<i>P</i> < 0.05; **<i>P</i>< 0.01 (Mann-Whitney <i>U</i> test).</p

Reduced neutrophil influx in lungs of <i>Trem-2</i> ^<i>-/-</i> mice, without affecting lung pathology.

<p>Lung pathology was determined in wild-type (WT; black bars) and <i>Trem-2</i>^<i>-/-</i> mice (white bars) infected with 5 x 102 CFU <i>B</i>. <i>pseudomallei</i> at 72h post-infection as described in the Methods section (<i>A</i>). Representative lung slides of WT (<i>B</i>) and <i>Trem-2</i>^<i>-/-</i> mice (<i>C</i>) (original magnification 10x). Neutrophil influx was defined by Ly6G positivity (expressed as % of total lung surface; <i>D</i>). Representative photographs of Ly6G-immunostaining for granulocytes on lung slides of WT (<i>E</i>) and <i>Trem-2</i>^<i>-/-</i> mice (<i>F</i>) (original magnification 10x). Data are expressed as mean ± SEM, n = 5–6 mice per group per time point. * <i>P</i> < 0.05. (Mann-Whitney <i>U</i> test).</p

Survival of <i>Trem-2</i>^<i>-/-</i> mice, but not of <i>Trem-1/3</i>^<i>-/-</i> mice, is enhanced in experimental melioidosis.

<p>Survival was observed for every 4-6h, up to a maximum of 14 days after intranasal inoculation with 5 x 102 CFU <i>B</i>. <i>pseudomallei</i> in wild-type (WT; closed circles) and <i>Trem-1/3</i>^<i>-/-</i> mice (open circles; <i>A</i>). Similarly, survival of WT (closed circles) and <i>Trem-2</i>^<i>-/-</i> mice (open circles) was determined (<i>B</i>) (n = 20 per group). The <i>P</i> value indicates significance of the difference in survival between <i>Trem-2</i>^<i>-/-</i> and WT mice (Kaplan-Meier analysis, followed by a log-rank test). ns = not significant. In addition, WT (closed circles) and <i>Trem-2</i>^<i>-/-</i> mice (open circles) were infected with 5 x 102 colony forming units (CFU) of <i>B</i>. <i>pseudomallei</i> intranasally (n = 5–6 mice per group) and sacrificed 72 h post-infection, in order to determine bacterial loads in lung homogenates (<i>C</i>), broncho-alveolar lavage fluid (BALF) (<i>D</i>), whole blood (<i>E</i>), liver (<i>F</i>) and spleen (<i>G</i>). Data are expressed as mean ± SEM, n = 5-6/group. ** <i>P</i>< 0.01. BC+ denotes positive blood cultures (Mann-Whitney <i>U</i> test).</p