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

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

No effect of TREM-1 deficiency on the cellular responsiveness and phagocytosis or intracellular killing of <i>B</i>. <i>pseudomallei</i>.

<p>Whole blood (<i>A</i>), bone marrow derived macrophages (BMDM; <i>B</i>) and alveolar macrophages (AM; <i>C</i><b>)</b> of WT and <i>Trem-1/3</i>^<i>-/-</i> mice were stimulated with medium, <i>E</i>.<i>coli</i> LPS(100 ng/ml) or heat-inactivated wild type <i>B</i>. <i>pseudomallei</i> (107 CFU/ml at a MOI of 50). TNF-α levels were measured in the supernatant obtained after 20 h of stimulation. BMDM (<i>D</i>) and AM (<i>E</i>) of WT and <i>Trem-1/3</i>^<i>-/-</i> mice were incubated at 37°C with FITC labeled heat-inactivated <i>B</i>. <i>pseudomallei</i> after which time-dependent phagocytosis was determined. Data are expressed as mean ± SEM and are representative of two or three independent experiments. n = 4 or 8 (for the whole blood assay) per group. *<i>P</i>< 0.05 (Mann-Whitney <i>U</i> test).</p

Increased TREM-1 and TREM-2 expression in experimental melioidosis.

<p>TREM-1 and TREM-2 mRNA expression was determined in wild type (WT) mice prior to infection or at 24 or 72h post-infection with 5 x 102 CFU <i>B</i>.<i>pseudomallei</i> intranasally. TREM-1 mRNA expression in lung (<i>A</i>) and liver (<i>B</i>) was determined. Likewise, TREM-2 mRNA expression was measured in lung (<i>C</i>) and liver (<i>D</i>) tissue. Data are presented as fold induction compared to the mRNA expression in uninfected mice (all RNA data are normalized to GAPDH). Data are mean ± SEM, n = 4–5 mice/group. * <i>P</i>< 0.05, ** <i>P</i> < 0.01, compared to gene-expression at t = 0h (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

TLR4^-/- and TLR2x4^-/-, but not TLR2^-/- mice, display diminished cytokine responses to <i>B</i>.<i>pseudomallei</i> LPS-induced pulmonary inflammation in mice.

<p>mTNF-α (A), interleukin (IL)-6 (B) and KC (C) levels were determined in bronchoalveolair fluid (BALF) of wild-type (WT), TLR2^-/-, TLR4^-/- and TLR2x4^-/- mice 6h post-intranasal administration of 10 ug LPS of <i>B</i>.<i>pseudomallei</i>. Following a Kruskal-Wallis test, Mann-Whitney U-tests were performed. Data are presented as means ± SEM. N = 7 or 9 per group and experiments were performed in duplicate. *** <i>P</i>< 0.001 vs. WT.</p

Successful extraction and purification of <i>B</i>.<i>pseudomallei-</i>LPS.

<p>LPS from <i>B</i>.<i>pseudomallei</i> 1026b was purified by a modified hot phenol-water extraction method including proteinase K treatment. 10 μl of LPS (0.5 mg/ml) was fractionated by SDS-PAGE electrophoresis followed by silver staining (A) which showed the characteristic ladder pattern of LPS banding of Gram-negative bacteria, without any indications of protein contamination (detection limit of 0.5–5 ng) and Coomassie blue staining (B) which demonstrated no protein contamination as well (detection limit 50 ng). Contamination of the extracted LPS was also assessed by the addition of LPS-binding Polymyxin B (PMB) (C). For this purpose, the murine alveolar macrophage cell line MH-S was stimulated with RPMI 1640 medium [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145397#pone.0145397.ref056" target="_blank">56</a>], LPS of <i>E</i>. <i>coli</i> 0111:B4 or <i>B</i>.<i>pseudomallei</i> 1026b and incubated with increasing concentrations of PMB for 6h, followed by TNF-α measurement in the supernatant. Results are representative for three independent experiments. (M = ladder, B.ps = <i>B</i>.<i>pseudomallei</i>-LPS)</p

LPS of <i>B</i>.<i>pseudomallei</i> signals solely via murine TLR4.

<p>Murine whole blood (A) and peritoneal macrophages (B) harvested from wild-type (WT), TLR2^-/- and TLR4^-/- mice were stimulated for 24h with RPMI 1640 + 10% FCS medium, LPS of <i>E</i>. <i>coli</i> 0111:B4 (100 ng/ml), LPS of <i>B</i>.<i>pseudomallei</i> (100 ng/ml), heat-killed <i>B</i>.<i>pseudomallei</i> 1026b (100 ng/ml), or its O-antigen lacking mutant SRM117 (both 10⁷ CFU/ml) before measurement of murine TNF-α (n = 4). Following a Kruskal-Wallis test, Mann-Whitney U-tests were performed. Data are means ± SEM. Results are representative of two or three independent experiments. *<i>P</i>< 0.05 vs. WT.</p

Profound changes in fecal microbiota composition during melioidosis.

<p>Fecal pellets were sampled from eight mice before (t = 0) they were infected intranasally with 150 CFU of <i>B</i>. <i>pseudomallei</i> and 72 hours after (t = 72). Microbial composition was analysed by IS-pro, using the number of nucleotides between the genes for ribosomal subunit 16 and 23 in the DNA (interspacer region) of the bacterium as a unique classification characteristic. (A) Clustering analysis, by unweighted pair group method with arithmetic mean (UPGMA) on cosine distances, shows the similarity of samples; individual mice are indicated by a number. Colors represent the most important bacterial phyla (purple, Actinobacteria; red, Bacteroidetes; blue, Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV); yellow, Proteobacteria). Length of the interspacer regions in basepairs is indicated on the y-axis; lines indicate the presence of PCR products. Color intensity increases with the presence of PCR product. (B) Diversity of microbial communities before and 72 hours after induction of melioidosis, expressed as Shannon index (green: total bacteria; red: Bacteroidetes; Blue: Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV); yellow: Proteobacteria). Data are presented as box- and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. ** p<0.01 pre- versus post-infection.</p