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

Dissemination of <i>S.</i> Typhi during systemic infection.

<p>Typhoid is usually contracted by ingestion of food or water contaminated by fecal or urinary carriers excreting <i>S.</i> Typhi. The incubation period is usually 7 to 14 d. In the small intestine the bacteria adhere to the mucosa and then invade the epithelial cells. The Peyer's patches, which are aggregrated lymphoid nodules of the terminal ileum, play an important role in the transport to the underlying lymphoid tissue. Specialized epithelial cells such as M cells overlying these Peyer's patches are probably the site of internalization of <i>S.</i> Typhi. Once the bacteria have penetrated the mucosal barrier, the invading organism translocates to the intestinal lymphoid follicles and the draining mesenteric lymph nodes, and some pass on to the reticuloendothelial cells of the liver and spleen. During the bacteremic phase, the bacteria are widely disseminated throughout the body. Secondary infection can occur with liver, spleen, bone-marrow, gallbladder, and Peyer's patches as the most preferred sites. The gallbladder is the main reservoir during a chronic infection with <i>S.</i> Typhi and invasion occurs either directly from the blood or by retrograde spread from the bile. Of interest, the ability of <i>Salmonella</i> to form biofilms on gallstones is likely to be a critical factor in establishment of chronic carriage and shedding of <i>S.</i> Typhi <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002933#ppat.1002933-Crawford1" target="_blank">[88]</a>. The bacteria that are excreted in the bile can then reinvade the intestinal wall by the mechanism previously described or are excreted by feces. Typical clinical symptoms are fever, malaise, and abdominal discomfort. Clinical features such as a tender abdomen, hepatomegaly, splenomegaly, and a relative bradycardia are common. Rose spots, the classical skin lesions associated with typhoid fever, are relatively uncommon and occur in 5%–30% of cases. The most severe manifestations of typhoid leading to sepsis and death are either necrosis of the Peyer's patches resulting in gut perforation and peritonitis or a toxic encephalopathy associated with myocarditis and haemodynamic shock <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002933#ppat.1002933-Parry1" target="_blank">[8]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002933#ppat.1002933-Everest1" target="_blank">[89]</a>.</p

<i>Salmonella</i> and its first encounter with the host.

<p>(a) The intracellular life of <i>Salmonella</i>. Invasion of phagocytic and non-phagocytic cells. <i>Salmonella</i> is a facultative intracellular pathogen that can be found in a variety of phagocytic and non-phagocytic cells, in which it is able to survive and replicate. To establish this intracellular niche, the T3SS1 and -2 play a predominant role; key virulence factors are involved in accessing and utilizing these cells <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002933#ppat.1002933-Ibarra1" target="_blank">[36]</a>. After ingestion, intestinal colonization follows and <i>Salmonella</i> enters enterocytes and dendritic cells in the intestinal epithelium <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002933#ppat.1002933-Ibarra1" target="_blank">[36]</a>. Subsequently, <i>Salmonella</i> that reach the submucosa can be internalized by resident macrophages via different mechanisms: by phagocytosis, active invasion using the T3SS1 or T3SS1-independent invasion using fimbriae or other adhesins on the bacterial surface. (1) <i>Salmonella</i>-containing-vacuole. Following internalization <i>Salmonella</i> remains within a modified phagosome known as the <i>Salmonella</i> containing vacuole (SCV) and injects a limited number of effector proteins, such as SipA, SipC, SopB/SigD, SodC-1, SopE2, and SptP into the cytoplasm. These effectors cause rearrangements of the actin cytoskeleton and SCV morphology among other changes. (2) Replication within the SCV. <i>Salmonella</i> survives and replicates within the SCV, where it is able to avoid host antimicrobial effector mechanisms. The T3SS2 is required for systemic virulence in the mouse and survival within macrophages. (3) Transport of <i>Salmonella</i> to distant sites. After penetration of the M cells, the invading microorganisms translocate to the intestinal lymphoid follicles and the draining mesenteric lymph nodes, and some pass on to the reticuloendothelial cells of the liver and spleen. <i>Salmonella</i> organisms are able to survive and multiply within the mononuclear phagocytic cells of the lymphoid follicles, liver, and spleen <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002933#ppat.1002933-Ibarra1" target="_blank">[36]</a>. (b) Host–pathogen interaction in typhoid and non-typhoid <i>Salmonella</i>. Simplified scheme of the first encounter between <i>Salmonella</i> spp. and the immune system. Specified cells such as neutrophils, macrophages, dendritic, phagocytic, and epithelial cells recognize specific pathogen associated molecular patterns (PAMPs) and danger-associated-molecular patterns (DAMPs), thereby eliciting an immune response. PAMPs such as LPS, Flagella, and bacterial DNA can trigger TRL4, TRL5, and TRL9, respectively. TLR-induced activation of NF-κB is essential for the production of pro-IL-1β, pro-IL-18, which can be negatively regulated by IRAK-M <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002933#ppat.1002933-Kobayashi1" target="_blank">[90]</a>. The NLRs are situated in the cytosol and can also recognize PAMPs. However, NLRP3 is triggered by a different, yet unknown, mechanism, although DAMPs are thought to play a crucial role. TLR, toll-like receptors; LPS, lipopolysaccharide; NF-κB, regulated nuclear factor kappa-light-chain-enhancer of activated B cells; IRAK-M, IL-1R-assiociated kinase-M; IL, Interleukin; ASC, apoptotic speck protein containing a caspase recruitment domain; NLR, NOD-like receptors (including NLRP3 and NLRC4); MyD88, myeloid differentiation primary response gene <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002933#ppat.1002933-Crawford1" target="_blank">[88]</a>.</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

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

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

Extracellular levels of granzyme A and B in patients with typhoid fever and healthy controls, and correlation with IFN- γ levels.

<p>Plasma levels of granzyme A (A) and B (B) measured in healthy controls (n = 38) compared to admission samples of hospitalized typhoid fever patients (n = 28). Medians are shown. Significance determined via Mann-Whitney <i>U</i> tests. ***P<0.001. Granzyme A is correlated to granzyme B (C). Levels of granzyme A (D) and granzyme B (E) are correlated to interferon (IFN)-γ in patients. Correlation coefficient reported is for Spearman's Rho. Gzm: granzyme.</p

Extracellular levels of granzyme A and B on admission and during discharge in patients.

<p>Typhoid fever patients (n = 15) who were discharged from hospital had lower levels of granzymes at follow-up when patients were clinically improved (granzyme A; A), although this did only reach statistical significance for granzyme B (B). Medians are shown. Significance determined via Mann-Whitney <i>U</i> tests. *P<0.05. Gzm: granzyme.</p