Intraperitoneal injection of lipopolysaccharide (LPS; 100 microg) in mice resulted in the disappearance of almost all proteose peptone-induced polymorphonuclear neutrophils (PMNs) with high-level fluorescence for the cell surface marker Gr-1 (Gr-1(high)) at 15 min postinjection, followed by doubling of their proportion at 30 min postinjection. High staining levels of 3'-acetyl-2'-carboxyl-6',7'-(dihyropyran-2'-one)-5 or 6-carboxyfluorescein diacethoxylmethyl ester-labeled PMNs injected into the peritoneal cavity were detected in mesenteric lymph nodes 15 min postinjection of LPS. Therefore, the time of decrease of Gr-1(high) PMNs coincided with that of the increase in cell accumulation in mesenteric lymph nodes. Since milk fat globule-EGF factor 8 (MFG-E8), which is secreted by macrophages, bound many PMNs exhibiting Gr-1(high) and Gr-1(medium) at 30 min postinjection of LPS, the staining level of annexin V on those cells was very low because its binding site is the same as the receptor for MFG-E8. At 60 min postinjection of LPS, the proportion of Gr-1(high) PMNs decreased, and almost all Gr-1(medium) PMNs tended to shift to the right compared with those at 30 min postinjection. The geomeans of Toll-like receptor 4 (TLR4) expression on PMNs at 15, 30, and 60 min postinjection of LPS were 63, 66, and 24%, respectively, compared with that on normal PMNs, indicating that the expression of TLR4 decreases in response to exposure to LPS. Our results suggest that LPS induced PMN death and that many PMNs expressing Gr-1(high) undergo apoptosis 180 min postinjection of LPS.
Intraperitoneal injection of lipopolysaccharide (LPS; 100 g) in mice resulted in the disappearance of almost all proteose peptone-induced polymorphonuclear neutrophils (PMNs) with high-level fluorescence for the cell surface marker Gr-1 (Gr-1 high ) at 15 min postinjection, followed by doubling of their proportion at 30 min postinjection. High staining levels of 3-acetyl-2-carboxyl-6,7-(dihyropyran-2-one)-5 or 6-carboxyfluorescein diacethoxylmethyl ester-labeled PMNs injected into the peritoneal cavity were detected in mesenteric lymph nodes 15 min postinjection of LPS. Therefore, the time of decrease of Gr-1 high PMNs coincided with that of the increase in cell accumulation in mesenteric lymph nodes. Since milk fat globule-EGF factor 8 (MFG-E8), which is secreted by macrophages, bound many PMNs exhibiting Gr-1 high and Gr-1 medium at 30 min postinjection of LPS, the staining level of annexin V on those cells was very low because its binding site is the same as the receptor for MFG-E8. At 60 min postinjection of LPS, the proportion of Gr-1 high PMNs decreased, and almost all Gr-1 medium PMNs tended to shift to the right compared with those at 30 min postinjection. The geomeans of Toll-like receptor 4 (TLR4) expression on PMNs at 15, 30, and 60 min postinjection of LPS were 63, 66, and 24%, respectively, compared with that on normal PMNs, indicating that the expression of TLR4 decreases in response to exposure to LPS. Our results suggest that LPS induced PMN death and that many PMNs expressing Gr-1 high undergo apoptosis 180 min postinjection of LPS. The toxic effects of lipopolysaccharide (LPS) are mediated by endogenous cytokines and other mediators released by LPSactivated host cells, such as macrophages. Recent studies suggest that the administration of LPS initiates the production and release of various cytokines, such as tumor necrosis factor alpha (TNF-), interleukin 1 (IL-1), IL-6, and gamma inter
The present study was designed to compare in vitro antibacterial activities of linezolid and vancomycin against vancomycin-susceptible Enterococcus faecalis (VSEF) and vancomycin-resistant enterococci (VRE) isolated in Japan with those of quinupristin–dalfopristin, teicoplanin and minocycline, and the in vitro short time bactericidal activity and the in vivo activities of linezolid and vancomycin against vancomycin-susceptible and -resistant E. faecalis. The MIC90s of linezolid, quinupristin–dalfopristin, vancomycin, teicoplanin and minocycline for VSEF and VRE were both 2 mg/L, both 2 mg/L, 2 and >128 mg/L, 0.25 and >128 mg/L, and both 32 mg/L, respectively. The efficacy of linezolid for mice with bacteraemia caused by VSEF was similar to that of vancomycin, but the elimination ratio of viable organisms from the blood of mice treated with vancomycin was significantly higher than in linezolid-treated and untreated mice at 2 h post-administration, and those of the two groups at 4 and 6 h were significantly higher than in untreated mice. Moreover, linezolid was highly active in mice with bacteraemia caused by vancomycin-resistant E. faecalisbecause this drug had potent in vitro activity against the organisms. Our results indicate that linezolid is suitable for the treatment of VRE and VSEF bacteraemia, and vancomycin is suitable for VSEF bacteraemia.
Thymus- and activation-regulated chemokine (TARC, also known as CCL17) is used as a biomarker for atopic dermatitis. The methods currently used for its measurement are complex, time-consuming, and require large machinery, warranting the need for a method that is simple, has a quick turnaround time, and requires less complex machinery. We evaluated the analytical performance of a novel latex turbidimetric immunoassay method, "Nanopia TARC", on 174 residual serum samples from patients with skin or allergic diseases. This evaluation included the assessment of the limit of blank/detection/quantification (LOB/D/Q), precision, accuracy, linearity, interference, and commutability between Nanopia TARC and "HISCL TARC", based on the chemiluminescent enzyme immunoassay (CLEIA) method. The LOB/D/Q values were 13, 57, and 141 pg/mL, respectively. The coefficient of variation of the repeatability was 0.9-3.8%, and that of the intermediate precision was 2.1-5.4%. The total error of the accuracy was 1.9-13.4%. The linearity was 141 and 19,804 pg/mL for TARC. The correlation coefficient between Nanopia TARC and HISCL TARC determined using the Passing-Bablok regression analysis was 0.999. Furthermore, the concordance of diagnostic criteria with AD was 92%. Nanopia TARC was confirmed to have the same analytical performance for TARC measurement as the existing CLEIA method.
Bacteraemia caused by vancomycin-resistant enterococci (VRE) is an important clinical problem because there are only a few potent antimicrobial agents against such bacteria. Therefore, understanding the pathogenic mechanisms of VRE bacteraemia is important for prophylaxis. This study shows that treatment of mice with cyclophosphamide and a combination of metronidazole, kanamycin and vancomycin reduced normal intestinal flora and induced systemic VRE bacteraemia. Translocation of VRE and the normal intestinal flora to the mesenteric lymph nodes, liver, spleen and blood, and mortality rate were dependent on treatment with cyclophosphamide and each of the three antimicrobial drugs. Among the different strains studied, C57BL/6 mice were the most susceptible to VRE. The virulence of vancomycin-resistant Enterococcus faecalis was greater than that of vancomycin-resistant Ent. faecium. On the day after inoculation of VRE, Escherichia coli was also detected in many VRE-positive specimens including blood, liver and the mesenteric lymph nodes. Moreover, both VRE and E. coli were detected simultaneously in almost all blood samples obtained from dead and dying mice, and VRE organisms outnumbered E. coli in those samples by 100:1 or more. These results indicate that changes in normal intestinal flora by administration of antimicrobial drugs and severity of neutropenia induced by cyclophosphamide are important factors that contribute to the development of systemic VRE bacteraemia. E. coli may be intimately associated with the establishment of VRE translocation.
We describe a novel technology for detecting nucleic acids: Probe Alteration Link Self-Assembly Reactions (PALSAR). PALSAR comprises DNA self-assembly of pairs of short DNA probes formed by alternate hybridization of three complementary regions in a pair of honeycomb probes (HCPs). Self-assembly occurs at designated salt concentrations and reaction temperatures and requires no enzymes. We prepared pairs of HCPs to detect mRNAs encoded by the GAPDH gene β-actin (BA) gene, CD3D gene, CD4 gene, major vault protein (MV) gene and the signalling lymphocytic activation molecule-associated protein (SAP) gene, and succeeded in quantitatively detecting these mRNAs. PALSAR could detect mRNA directly without synthesizing cDNA. Moreover, multiple mRNAs could be detected simultaneously in a single reaction tube and there was a good correlation between the results obtained PALSAR and those by real-time PCR.
