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

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

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

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

Antibiotic microbiota disruption does not affect neutrophil influx.

<p>Lungs were obtained at the indicated time points after intranasal inoculation with 500 CFU <i>B</i>. <i>pseudomallei</i>. Paraffin-embedded lung tissue sections were stained with haematoxylin/eosin to score different parameters for pathology (A-B, 2x magnification, representative images). The combined score, given by a blinded pathologist, was not different between the two groups (C). Sections from the same samples were stained for Ly-6GC as a neutrophil marker (representative microphotographs, 2x magnification) (D-E). The percentage Ly-6GC-positive surface of the total lung surface was calculated using ImageJ (F). The number of cells per mL BALF was counted using a Coulter counter (G). Myeloperoxidase was quantified in lung homogenates as a measure for neutrophil degranulation (H). Bone marrow and blood was obtained from naïve control and antibiotic pre-treated mice and using FACS analysis the percentage of Ly6GC+, CD11b+ cells within the viable CD45+ population was determined (I). Data are presented as box- and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. White bars represent control mice, grey bars antibiotic treated mice. No statistically significant differences were found.</p

Limited effect of antibiotic induced gut microbiota disruption on survival and organ damage.

<p>Survival (A) and clinical observation score (B) of control (white dots) and antibiotic treated mice (grey dots) after intranasal inoculation with 150 CFU <i>B</i>. <i>pseudomallei</i> (n = 20 mice per group, depicted is the mean). No statistically significant differences were detected. Aspartate aminotranspherase (AST, C), alanine aminotranspherase (ALT, D), urea (E) and lactate dehydrogenase (LDH, F) were measured in plasma after inoculation with 500 CFU <i>B</i>. <i>pseudomallei</i> as markers for liver-, renal- and general damage. Data are presented as box- and whisker plots showing the smallest observation, lower quartile, median, upper quartile and largest observation. White bars represent control mice, grey bars antibiotic treated mice. N = 5–6 samples per group. ND = not detectable.</p

Cytokine- and chemokine levels in lung homogenate and plasma.

<p>Cytokine- and chemokine levels in lung homogenate and plasma.</p