Acinetobacter baumannii Lipopolysaccharide Influences Adipokine Expression in 3T3-L1 Adipocytes.

Acinetobacter baumannii is one of the most important nosocomial opportunistic pathogen worldwide. In addition, obesity has been associated with an increased risk of nosocomial infection, suggesting that there may be an association between A. baumannii and white adipose tissue. However, the effects of A. baumannii on adipocytes have not been well studied at the molecular level. Here, we investigated the potential role of A. baumannii-derived lipopolysaccharides (LPS) as signaling molecules that affect adipocyte functionality. We tested the effect of increasing concentrations of A. baumannii-derived LPS (10, 100, or 1000 ng/mL) on the 3T3-L1 adipocyte cell line. Exposure to LPS was found to increase the expression of several adipokines (e.g., MIP-2, MCP-1, TNF-α, IL-6, lipocalin-2, and FABP4) in 3T3-L1 adipocytes and significantly reduced the expression of leptin and adiponectin. The effects of A. baumannii-derived LPS on MIP-2 expression were similar in comparison with that of LPS prepared from Pseudomonas aeruginosa and Escherichia coli in our cell culture-based system. This study suggests that A. baumannii-derived LPS functions as a signaling molecule that impacts the inflammatory function of white adipose tissue on the level of gene expression.

Acinetobacter baumannii escape from neutrophil extracellular traps (NETs).

Acinetobacter baumannii and Pseudomonas aeruginosa are the same aerobic gram-negative bacillus and are usually harmless but cause infectious diseases in compromised hosts. Neutrophils play a critical role in infective protection against the extracellular growth of bacteria. Recently, a new biological defense mechanism called neutrophil extracellular traps (NETs) has been attracting attention. In present study, we investigated the responsiveness of neutrophils to A. baumannii and P. aeruginosa, focusing on NET formation. Neutrophils were co-cultured with A. baumannii or P. aeruginosa, and then DNA, histone and neutrophil elastase were stained, and the formation of NETs was evaluated. Neutrophils stimulated with A. baumannii had spread, but their shapes was maintained, and the nucleus was observed as clearly as that in non-stimulated neutrophils. However, neutrophils stimulated with P. aeruginosa did not maintain their cellular morphology, and the nucleus was disrupted with DNA, histones, and neutrophil elastase released into the extracellular space. These results suggest that A. baumannii does not induce NET formation, in contrast to P. aeruginosa. In addition, we measured expression of myeloperoxidase (MPO), reactive oxygen species (ROS) and superoxide in neutrophils, and we found that these expression in P. aeruginosa-stimulated neutrophils was stronger than that in A. baumannii-stimulated neutrophils. Furthermore, A. baumannii was not killed by neutrophils, in contrast to P. aeruginosa. In this study, we show that the reactivity of neutrophils and their biological defense mechanism are different between A. baumannii and P. aeruginosa, which is important for understanding the pathogenicity of these bacteria.

Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP).

Klebsiella pneumoniae carbapenemases (KPC), which are associated with resistance to carbapenem, have recently spread worldwide and have become a global concern. It is necessary to detect KPC-producing organisms in clinical settings to be able to control the spread of this resistance. We have developed a loop-mediated isothermal amplification (LAMP) method for rapid detection of KPC producers. LAMP primer sets were designed to recognize the homologous regions of blaKPC-2 to blaKPC-17 and could amplify blaKPC rapidly. The specificity and sensitivity of the primers in the LAMP reactions for blaKPC detection were determined. This LAMP assay was able to specifically detect KPC producers at 68 °C, and no cross-reactivity was observed for other types of ß-lactamase (class A, B, C, or D) producers. The detection limit for this assay was found to be 10(0) CFU per tube, in 25 min, which was 10-fold more sensitive than a PCR assay for blaKPC detection. Then, the sensitivity of the LAMP reactions for blaKPC detection in human specimens (sputum samples, urine samples, fecal samples and blood samples) was analyzed; it was observed that the LAMP assay had almost the same sensitivity in these samples as when using purified DNA. The LAMP assay is easy to perform and rapid. It may therefore be routinely applied for detection of KPC producers in the clinical laboratory.

Gene expression analysis of TREM1 and GRK2 in polymorphonuclear leukocytes as the surrogate biomarkers of acute bacterial infections.

OBJECTIVE: In the acute stage of infectious diseases such as pneumonia and sepsis, sequelae hypercytokinemia and cytokine storm are often observed simultaneously. During bacterial infections, activated polymorphonuclear leukocytes (PMNs) cause inflammation and organ dysfunction in severely ill patients. Gene expression of the triggering receptor on myeloid cells (TREM)-1 and G-coupled-protein receptor kinase (GRK)-2 in PMNs isolated from patients was analysed to identify genes correlated with the severity of pathophysiological conditions. METHODS: mRNA levels of TREM1 and GRK2 in the PMNs from 26 patients (13 with pneumonia, 5 with severe sepsis, and 8 with septic shock) were analysed by using quantitative real-time PCR. The synthesised soluble form (s)TREM-1 was incubated with normal PMNs to investigate its biological functions in vitro. RESULTS: Copies of TREM1 transcript were 0.7- to 2.1-fold higher in patients with pneumonia compared to those of normal subjects; the average fold-change was 1.1-fold. The mRNA levels of patients suffering from severe sepsis and septic shock were 0.34- and 0.33-fold lower compared to those of healthy subjects, respectively. TREM1 mRNA levels in 5 of 26 patients in convalescent stages recovered to normal levels. The mRNA levels of GRK2 in the PMNs of patients were also downregulated. The synthesised sTREM-1 upregulated the mRNA levels of TREM1 in normal PMNs. CONCLUSIONS: TREM1 mRNA levels were inversely correlated with the severity of pathophysiological conditions in acute bacterial infections. The gene expression levels of TREM1 in PMNs isolated from patients with bacterial infections may be used as a surrogate biomarker for determining the severity.

Immunobiological effects of lipopolysaccharide derived from Helicobacter pylori and influence of a proton pump inhibitor lansoprazole on human polymorphonuclear leukocytes.

Helicobacter pylori colonizes the human gastric mucosa of more than half of the human population and has a unique lipopolysaccharide (LPS) structure. LPS is the most dominant and suitable pathogen-associated molecular pattern that is detected via pattern recognition receptors. Although the priming effect of H. pylori LPS on reactive oxygen species (ROS) production of PMNs is lower than that of Escherichia coli O111:B4 LPS, LPS released from H. pylori associated with antibiotics eradication therapy may activate PMNs and increase ROS production. In addition, we describe the effects of H. pylori and E. coli O111:B4 LPSs on gene expression and the anti-inflammatory effect of lansoprazole (LPZ) in human polymorphonuclear leukocytes. LPS isolated from H. pylori and E. coli O111:B4 alters toll-like receptor 2 (TLR) and TLR4 expressions similarly. However, LPS from E. coli O111:B4 and H. pylori caused a 1.8-fold and 1.5-fold increase, respectively, in CD14 expression. All LPS subtypes upregulated TNFα and IL6 expression in a concentration-dependent manner. Although E. coli O111:B4 LPS upregulated IL8R mRNA levels, H. pylori LPS did not (≦ 100 ng/mL). Gene expression levels of ITGAM demonstrated no significant change on using both LPSs. These different effects on the gene expression in PMNs may depend on variations in LPS structural modifications related to the acquired immunomodulatory properties of H. pylori LPS. Proton pump inhibitors, i.e., LPZ, are used in combination with antibiotics for the eradication therapy of H. pylori. LPZ and its acid-activated sulphenamide form AG-2000 suppress ROS production of PMNs in a dose-dependent manner. These results suggest that LPZ combination with antibiotics for H. pylori eradication reduces gastric inflammation by suppressing ROS release from PMNs.

Effects of erythromycin and rifampicin on immunomodulatory gene expression and cellular function in human polymorphonuclear leukocytes.

BACKGROUND: We investigated the effects of two antibiotics, erythromycin and rifampicin, on the immunomodulatory gene expression and cellular function of human polymorphonuclear leukocytes (PMNs). METHODS: We used real-time quantitative PCR to examine the expression of immunomodulatory genes. The production of reactive oxygen species (ROS) was determined by fluorescence-activated cell sorting. PMN chemotaxis was analyzed using a KK chemotaxis chamber. RESULTS: Stimulation of PMNs with lipopolysaccharide (LPS) resulted in increases in the mRNA levels of immunomodulatory genes. Rifampicin significantly inhibited the overexpression of TLR2, TLR4, CD14 and IL8Rs. However, erythromycin suppressed only the upregulation of TLR2 and TNFA. Neither antibiotic had an effect on the production of ROS. Rifampicin significantly inhibited PMN chemotaxis, but erythromycin had no effect. CONCLUSIONS: Erythromycin and rifampicin may play anti-inflammatory roles by affecting the expression levels of immunomodulatory genes or the chemotaxis of PMNs.

Priming effects of tumor necrosis factor-α on production of reactive oxygen species during Toxoplasma gondii stimulation and receptor gene expression in differentiated HL-60 cells.

Neutrophils are among the principal effector cells that protect against infectious agents, in part by producing reactive oxygen species (ROS) via the actions of tumor necrosis factor-α (TNF-α). In this study, we investigated whether HL-60 cells that had been differentiated into neutrophil-like cells by all-trans retinoic acid could be primed with TNF-α similar to human neutrophils. Our results showed that when differentiated HL-60 (dHL-60) cells were primed with TNF-α for 10 min, ROS production induced by zymosan A or phorbol myristate acetate (PMA) was enhanced in a TNF-α-dose-dependent manner. In addition, when dHL-60 cells were stimulated with live tachyzoites of Toxoplasma gondii after TNF-α priming, ROS production was also enhanced. Thus, dHL-60, similar to neutrophils, produced ROS after PMA, zymosan A, or T. gondii stimulation. Furthermore, we examined gene expression in dHL-60 cells after TNF-α treatment. The pro-inflammatory cytokine IL-6 was up-regulated more than 1.6-fold by 0.1 ng/mL TNF-α. Endogenous TNF-α was down-regulated by priming. IL-8 receptors genes were not affected by priming with 0.1 ng/mL or 1 ng/mL TNF-α. Complement receptor (CR) 1 and CR3 gene expression was not affected by TNF-α priming for 10 min. However, when the priming period was extended to 1 h, CR1 and CR3 genes were up-regulated 1.3 and 1.4-fold, respectively. Expression of the cell-surface CR3 (CD11b) was not significantly affected by TNF-α for 15 min but was slightly enhanced after priming for 2 h. These results suggest that dHL-60 cells may be used as a substitute for neutrophils when evaluating the effects of cytokines or immunomodulator agents.

Analysis of membrane antigens on neutrophils from patients with sepsis.

The aim of the present study was to assess changes of cell membrane antigens on neutrophils in septic patients. Expression levels of neutrophil membrane antigens were measured employing a FACS calibur flow cytometer with several fluorescence-labeled monoclonal antibodies. Expression levels of the CD14 antigen were higher in patients with sepsis than in healthy individuals. In particular, the expression levels of CD14 increased in patients complicated by septic shock. Expression levels of TLR-4 were higher in patients with sepsis or septic shock than in healthy individuals. Expression levels of CD11b and CD16 were lower in patients with sepsis or septic shock than in healthy individuals and were even lower in those complicated by septic shock. Expression levels of neutrophil membrane antigens in patients with sepsis markedly changed in the acute phase. However, these levels tended to return to those of healthy individuals in the convalescing phase. Analyses of the surface antigens on neutrophils strongly involved in biological defense or tissue injury are informative for understanding the pathology of sepsis and for conducting therapy targeting neutrophils in the future.

Tigecycline Suppresses the Virulence Factors of Multidrug-Resistant Acinetobacter baumannii Allowing Human Neutrophils to Act.

Purpose: To determine the ability of human neutrophils to kill multidrug-resistant Acinetobacter baumannii (MDRAB) in the presence of tigecycline (TGC). Methods: Clinical isolates of MDRAB were cultured with human neutrophils and H2O2 in the presence of TGC. The numbers of viable bacteria, catalase activity, gene expression at the K locus of the MDRAB, reactive oxygen species (ROS) production, and granule exocytosis in human neutrophils were determined. Results: There was a time-dependent increase in the numbers of MDRAB after co-culturing with human neutrophils, whereas there was a significant decrease in the MDRAB numbers when co-cultured with both, human neutrophils and TGC for 6 h. The presence or absence of TGC did not affect total ROS production or the expression of CD11b, CD15, and CD63 on human neutrophils occurred when co-cultured with MDRAB. TGC significantly suppressed catalase activity and gene expression at the K locus of MDRAB, and significantly reduced the thickness of the capsule. Additionally, the bacterial viability of TGC-treated MDRAB cultured with H2O2 was lower than that without H2O2 after 6 h of culture. Conclusion: TGC significantly suppressed the expression of catalase and the capsule in MDRAB without adverse effects on neutrophil function, allowing human neutrophils to kill MDRAB. TGC is an effective antibiotic for treating MDRAB infections.

Effects of colistin and tigecycline on multidrug-resistant Acinetobacter baumannii biofilms: advantages and disadvantages of their combination.

We investigated the antimicrobial effects of colistin (CST) and tigecycline (TGC), either alone or in combination, on biofilm-dispersed and biofilm-embedded multidrug-resistant Acinetobacter baumannii (MDRAB) strains R1 and R2. The bacterial growth of biofilm-dispersed MDRAB was inhibited by CST or TGC. However, the inhibitory effects were attenuated by a combination of CST and low concentrations of TGC. The bactericidal effects of CST, but not TGC, were observed on biofilm-dispersed MDRAB. Notably, the bactericidal effects increased with a combination of CST and high concentrations of TGC, whereas they were attenuated with the combination of CST and low concentrations of TGC. Although biofilm formation by MDRAB decreased with increasing concentrations of CST or TGC, there was no complete disruption of the biofilms. Additionally, the biofilms increased with a combination of 1-2 µg/mL CST and TGC at 2 µg/mL and 2-4 µg/mL for strains R1 and R2, respectively. Biofilm-embedded MDRAB was eradicated with CST, but not TGC. Notably, the eradication effects increased with a combination of CST and high concentrations of TGC, whereas attenuation happened with the combination of CST and low concentrations of TGC. These results provide information on the combined effects of CST and TGC in the treatment of biofilm-associated MDRAB infection.

Immunomodulatory gene expression analysis in LPS-stimulated human polymorphonuclear leukocytes treated with antibiotics commonly used for multidrug-resistant strains.

Conventional antibiotics used for the treatment of severe infections such as sepsis and septic shock confer immunomodulatory benefits. However, the growing problem of multidrug resistant infections has led to an increase in the administration of non-conventional last-resort antibiotics, including quinolones, aminoglycosides, and polypeptides, and the effects of these drugs on immunomodulatory gene expression in activated human polymorphonuclear leukocytes (PMNs) have not been reported. In this study, lipopolysaccharide-stimulated PMNs were incubated with piperacillin, rifampicin, fosfomycin (FOM), levofloxacin (LVFX), minocycline (MINO), colistin, tigecycline, or amikacin, and the mRNA expression levels of pattern recognition receptors (TLR2, TLR4, and CD14), inflammatory cytokines (TNFα and IL6), and chemokine receptors (IL8Rs and ITGAM) in these cells were quantitated using real-time qPCR. Many of the tested antibiotics altered the expression of the investigated cytokines. Notably, FOM, LVFX, and MINO significantly downregulated the expression of IL6, which is associated with pro- and anti-inflammatory defense mechanisms. Treatment of FOM and LVFX reduced IL-6 production as well as observed for IL6 gene expression. These findings indicated transcription and translation cooperation under the used experimental conditions. Therefore, our findings suggest that administration of these antibiotics suppresses the host anti-inflammatory response.

Acinetobacter baumannii LOS Regulate the Expression of Inflammatory Cytokine Genes and Proteins in Human Mast Cells.

Herein, we investigated the effect of bacterial lipooligosaccharides (LOS), from Acinetobacter baumannii, on the expression of pro-inflammatory genes that play an essential role in bacterial clearance. LAD2 human mast cells were stimulated with LOS derived from two strains of A. baumannii-ATCC 19606 and MDRA T14. LOS exposure induced the expression of genes for pro-inflammatory mediators, including TNF-α, IL-8, LTC4S, CCL4, and TLR4. The mRNA expression levels of a majority of the pro-inflammatory genes, except TLR4, in A. baumannii-LOS stimulated mast cells were increased. Moreover, co-culture of neutrophils with the supernatant obtained from LOS (ATCC 19606 and MDRA T14)-induced LAD2 cells increased the transmigration of neutrophils, which plays a critical role in the early protection against bacterial infections. The results of the present study suggest that LOS could be involved in the pathogenicity of A. baumannii by inducing inflammatory responses via mast cells and that IL-8 is involved in recruiting neutrophils in response to bacterial invasion.

Histopathological Analysis of Acinetobacter baumannii Lung Infection in a Mouse Model.

Acinetobacter baumannii is the main causative pathogen of nosocomial infections that causes severe infections in the lungs. In this study, we analyzed the histopathological characteristics of lung infection with two strains of A. baumannii (ATCC 19606 and the clinical isolate TK1090) and Pseudomonas aeruginosa PAO-1 in C3H/HeN mice to evaluate the virulence of A. baumannii. Survival was evaluated over 14 days. At 1, 2, 5, or 14 days postinfection, mice of C3H/HeN were sacrificed, and histopathological analysis of lung specimens was also performed. Histopathological changes and accumulation of neutrophils and macrophages in the lungs after infection with A. baumannii and P. aeruginosa were analyzed. Following intratracheal inoculation, the lethality of ATCC 19606- and TK1090-infected mice was lower than that of PAO-1-infected mice. However, when mice were inoculated with a sub-lethal dose of A. baumannii, the lung bacterial burden remained in the mice until 14 days post-infection. Additionally, histopathological analysis revealed that macrophages infiltrated the lung foci of ATCC 19606-, TK1090-, and PAO-1-infected mice. Although neutrophils infiltrated the lung foci of ATCC 19606- and TK1090-infected mice, they poorly infiltrated the lung foci of PAO-1-infected mice. Accumulation of these cells in the lung foci of ATCC 19606- and TK1090-infected mice, but not PAO-1-infected mice, was observed for 14 days post-infection. These results suggest that A. baumannii is not completely eliminated despite the infiltration of immune cells in the lungs and that inflammation lasts for prolonged periods in the lungs. Further studies are required to understand the mechanism of A. baumannii infection, and novel drugs and vaccines should be developed to prevent A. baumannii infection.

Prevalence and Relatedness of mcr-1-Mediated Colistin-Resistant Escherichia coli Isolated From Livestock and Farmers in Japan.

Colistin is used to treat infectious diseases in humans and livestock; it has also been used as a feed additive for livestock for approximately 50 years. Since the mcr-1 plasmid-mediated colistin resistance gene was discovered in China in 2015, it has been detected worldwide, mainly in livestock. In this study, we investigated the prevalence and characteristics of mcr-mediated colistin-resistant Escherichia coli in livestock and farmers in Japan. We collected fecal samples from 295 healthy livestock (202 cattle and 93 swine) and 62 healthy farmers from 72 livestock farms (58 cattle farms and 14 swine farms) between 2013 and 2015. Twenty-eight mcr-1-harboring E. coli strains were isolated from 25 livestock (six cattle and 19 swine) and three farmers (two cattle farmers and one swine farmer). The prevalence rates of mcr-1-harboring E. coli in livestock and farmers were 8.47 and 4.84%, respectively. Of the 28 strains, the resistance genes of three were transferable via the mcr-1-coding plasmids to E. coli J53 at low frequencies (10-7-10-8). Six strains coharbored mcr-1 with CTX-M ß-lactamases (CTX-M-14, CTX-M-27, or CTX-M-156). Of the isolates obtained from livestock and farmers in four farms (farms C, I, N, and P), nine strains had the same genotypical characteristics (sequence types and pulsed-field gel electrophoresis band patterns), plasmid characteristics (incompatibility group and plasmid transferability), and minimum inhibitory concentrations. Thus, the findings suggested that clonal strains could spread among livestock and farmers within farms. To our knowledge, this is the first study to detect clonal relatedness of mcr-1-mediated colistin-resistant E. coli in livestock and farmers. It is suggested that farmers are at a higher risk of acquiring mcr-1-harboring strains, calling for our attention based on the One Health concept.

Suppression of phosphorylation of extracellular-signal-regulated kinase and p38 mitogen-activated protein kinase in polymorphonuclear leukocytes by the proton pump inhibitor lansoprazole.

Lansoprazole (LPZ) is a proton pump inhibitor that suppresses gastric secretion and exerts anti-inflammatory effects on immune cells. Recently, LPZ has been used for the treatment of peptic ulcer and gastritis, which can be caused by Helicobacter pylori, due to its potent acid-suppressive effects. We focused the aim to the anti-inflammatory effects on the over-activation of neutrophils, and investigated the effects of LPZ on the signal transduction of the mitogen-activated protein kinase (MAPK) family. LPZ slightly phosphorylated p38 MAPK of neutrophils at a concentration of 10 microg/ml , but did not phosphorylate extracellular-signal regulated kinase (ERK) 1/2. Pretreatment of neutrophils with (1-5 microg/ml ) LPZ strongly attenuated the phorbol-12-myristate-13-acetate-stimulated phosphorylation of ERK1/2, and LPZ slightly suppressed the lipopolysaccharide (LPS)- and N-formylmethionylleucylphenylalanine-stimulated phosphorylation of p38. ERK1/2 produces the mitochondrial anti-apoptotic proteins, and the signaling pathway from LPS and N-formylmethionylleucylphenylalanine to p38 is the main pathway for reactive oxygen species production. The mechanism of anti-inflammatory effect of LPZ on hyper-activated neutrophils is suggested to be the suppression of signal transduction of ERK1/2 and p38 MAPK.

Analysis of Immune Responses in Acinetobacter baumannii-Infected Klotho Knockout Mice: A Mouse Model of Acinetobacter baumannii Infection in Aged Hosts.

Acinetobacter baumannii is an important opportunistic pathogen that primarily afflicts elderly people. To clarify the pathogenicity of A. baumannii in the elderly, we investigated immune responses to A. baumannii ATCC 19606 infection in klotho knockout (KO) mice, the mouse model of aging. Following intravenous inoculation, the mice seldom displayed severe symptoms. However, the survival rate was 56% at 7 days post-infection. Bacteria were detected in the lungs of klotho KO mice but not klotho wildtype (WT) mice at 7 days post-infection. Neutrophils, eosinophils, interstitial macrophages, and monocyte/dendritic cell subset in the lungs of klotho KO mice were transiently induced after infection with A. baumannii. The number of alveolar macrophages in klotho KO mice was lower than that in klotho WT mice, except for 1 day post-infection. CD11b expression on neutrophils and alveolar macrophages in the lungs of klotho KO mice was seldom upregulated by the infection. These results suggested that immune functions eliminating bacteria in the lungs of klotho KO mice were insufficient. CD11blow conventional DC cells hardly increased in klotho KO mice infected with A. baumannii. Additionally, the production of interleukin (IL)-10 in the sera of klotho KO mice was significantly higher than that in klotho WT mice, whereas that production of interferon-gamma was not detected in the sera of klotho KO mice. These results suggested that acquired immune responses were hardly induced in klotho KO mice. IL-1ß, CXCL1, CXCL2, and CCL2 expression was significantly higher in the lungs of klotho KO mice infected with A. baumannii than in those of klotho WT mice at 1 day post-infection. These results suggested that pulmonary inflammation was elicited in klotho KO mice during early infection. The expression levels of proinflammatory cytokines significantly correlated with TLR9 expression in the lungs of klotho KO mice. The collective results demonstrate an A. baumannii infection state in aged hosts and suggest that pulmonary inflammation and bacterial burden should be noted in aged hosts even in the absence of severe symptoms of A. baumannii infection.

Lipopolysaccharide-Deficient Acinetobacter baumannii Due to Colistin Resistance Is Killed by Neutrophil-Produced Lysozyme.

Acinetobacter baumannii causes nosocomial infections due to its multidrug resistance and high environmental adaptability. Colistin is a polypeptide antibacterial agent that targets lipopolysaccharide (LPS) and is currently used to control serious multidrug-resistant Gram-negative bacterial infections, including those caused by A. baumannii. However, A. baumannii may acquire colistin resistance by losing their LPS. In mouse models, LPS-deficient A. baumannii have attenuated virulence. Nevertheless, the mechanism through which the pathogen is cleared by host immune cells is unknown. Here, we established colistin-resistant A. baumannii strains and analyzed possible mechanisms through which they are cleared by neutrophils. Colistin-resistant, LPS-deficient strains harbor mutations or insertion sequence (IS) in lpx genes, and introduction of intact lpx genes restored LPS deficiency. Analysis of interactions between these strains and neutrophils revealed that compared with wild type, LPS-deficient A. baumannii only weakly stimulated neutrophils, with consequent reduced levels of reactive oxygen species (ROS) and inflammatory cytokine production. Nonetheless, neutrophils preferentially killed LPS-deficient A. baumannii compared to wild-type strains. Moreover, LPS-deficient A. baumannii strains presented with increased sensitivities to antibacterial lysozyme and lactoferrin. We revealed that neutrophil-secreted lysozyme was the antimicrobial factor during clearance of LPS-deficient A. baumannii strains. These findings may inform the development of targeted therapeutics aimed to treat multidrug-resistant infections in immunocompromised patients who are unable to mount an appropriate cell-mediated immune response.

Downregulation of immunomodulator gene expression in LPS-stimulated human polymorphonuclear leukocytes by the proton pump inhibitor lansoprazole.

Lansoprazole (LPZ) has anti-inflammatory activity and repairs cells damaged by phagocytic cells. In the present study, we evaluated the effects of LPZ on gene expression, especially that of immunomodulator genes, in human polymorphonuclear leukocytes (PMNs) activated by lipopolysaccharide (LPS). Several concentrations of LPZ (final concentrations, 0-10 microg/ml) were added to the PMNs (1 x 10(6) cells/ml), which were stimulated with LPS (100 ng/ml) and incubated at 37 degrees C for 1 or 3 h. When LPS-stimulated PMNs were treated with LPZ at >or=5.0 microg/ml for 1 h, mRNA expression levels of CXCR1/2 and TNFalpha were suppressed in a dose-dependent manner. The gene expression level of CD14 was also downregulated by LPZ at >or=0.1 microg/ml, with expression suppressed to 50% by 10 microg/ml LPZ. However, LPZ at 0.01-5.0 microg/ml had no significant effect on the expression of TLR-4 or CD11b/CD18 mRNA. LPZ at 10 microg/ml downregulated the levels of these mRNAs to 80% and 50%, respectively. On the other hand, when the reaction period was extended to 3 h with the same conditions, all mRNA expression levels were downregulated by >or=0.01 microg/ml LPZ, in a dose-dependent manner. LPZ may suppress the biological functions of PMNs, such as chemotaxis and inflammatory chemokine production.

SND1, a component of RNA-induced silencing complex, is up-regulated in human colon cancers and implicated in early stage colon carcinogenesis.

Colon cancers have been shown to develop after accumulation of multiple genetic and epigenetic alterations with changes in global gene expression profiles, contributing to the establishment of widely diverse phenotypes. Transcriptional and posttranscriptional regulation of gene expression by small RNA species, such as the small interfering RNA and microRNA and the RNA-induced silencing complex (RISC), is currently drawing major interest with regard to cancer development. SND1, also called Tudor-SN and p100 and recently reported to be a component of RISC, is among the list of highly expressed genes in human colon cancers. In the present study, we showed remarkable up-regulation of SND1 mRNA in human colon cancer tissues, even in early-stage lesions, and also in colon cancer cell lines. When mouse Snd1 was stably overexpressed in IEC6 rat intestinal epithelial cells, contact inhibition was lost and cell growth was promoted, even after the cells became confluent. Intriguingly, IEC6 cells with high levels of Snd1 also showed an altered distribution of E-cadherin from the cell membrane to the cytoplasm, suggesting loss of cellular polarity. Furthermore, the adenomatous polyposis coli (Apc) protein was coincidentally down-regulated, with no significant changes in the Apc mRNA level. Immunohistochemical analysis using chemically induced colonic lesions developed in rats revealed overexpression of Snd1 not only in colon cancers but also in aberrant crypt foci, putative precancerous lesions of the colon. Up-regulation of SND1 may thus occur at a very early stage in colon carcinogenesis and contribute to the posttranscriptional regulation of key players in colon cancer development, including APC and beta-catenin.

Multidrug-resistant Acinetobacter baumannii resists reactive oxygen species and survives in macrophages.

We investigated the intracellular survival of multidrug-resistant Acinetobacter baumannii (MDRAB) clinical isolates in macrophages, after phagocytosis, to determine their virulence characteristics. After ATCC 19606 and 5 clinical isolates of MDRAB were phagocytosed by mouse and human macrophages, the bacterial count of MDRAB strains, R4 and R5, increased in the mouse macrophages, 24 hours after phagocytosis. Bacterial count of the strains, R1 and R2, was almost equal 4 and 24 hours after phagocytosis. Intracellular reactive oxygen species was detected in the macrophages after phagocytosis of these bacteria. Further, the strains R1, R2, R4, and R5 showed higher catalase activity than ATCC 19606. Additionally, strains R1, R4, and R5 grew more efficiently than ATCC 19606 in the presence of H2O2, whereas growth of strains R2 and R3 was marginally more than that of ATCC 19606 in the presence of H2O2. The MDRAB clinical isolates altered the expression of TNF-α, IL-1ß, IL-6, and MIP-2 mRNA induced in J774A.1 cells, 24 hours after phagocytosis. These results provide insights into the renewed virulence characteristics of MDRAB clinical isolates. Finally, tigecycline killed MDRAB phagocytosed by the macrophages more effectively than colistin, although colistin and tigecycline are both considered effective antibiotics for the treatment of MDRAB.