Tsuneyuki Ubagai

Influences of aflatoxin B1 on reactive oxygen species generation and chemotaxis of human polymorphonuclear leukocytes

The effects of aflatoxin (AF), a hepatotoxic agent and the strongest carcinogen in nature, on polymorphonuclear leukocyte (PMN) chemotaxis and chemiluminescence (CL) were studied. Luminol-dependent CL activity, which reflects the production of reactive oxygen species (ROS) from PMNs, was up-regulated to approximately 150% when PMNs were treated with 0.05 ng/ml of AFB1 upon stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) or zymosan. On the other hand, PMN CL activity was down-regulated to approximately 25% of the control when PMNs were treated with 50 ng/ml of AFB1 upon stimulation with zymosan. The effect of AFB1 on PMN chemotaxis was also investigated using the Boyden chamber method. The chemotactic ability of PMNs when interleukin (IL)-8 was used as a chemoattractant was inhibited in a dose-dependent manner at AFB1 concentrations of >10 ng/ml. In reverse transcriptase (RT)-PCR analysis, gene expression levels of CXC chemokine receptor (CXCR)1/2, whose ligands are IL-8, granulocyte chemotactic protein (GCP)-2, neutrophil attractant-activation protein (NAP)-2, and epithelial neutrophil-activating protein (ENA)-78, which regulate PMN chemotaxis, were also down-regulated in a dose-dependent manner by 50 ng/ml AFB1. mRNA expression levels of CXCR1/2 were down-regulated to approximately 85% of that in the controls when PMNs were treated with 100 ng/ml AFB1. These results suggest that a high concentration of AFB1 reduces the chemotactic ability of PMNs via the CXCR1/2 cascade indirectly.

Gr-1high Polymorphonuclear Leukocytes and NK Cells Act via IL-15 to Clear Intracellular Haemophilus influenzae in Experimental Murine Peritonitis and Pneumonia

Polymorphonuclear leukocytes (PMNs) can be divided into Gr-1high and Gr-1low subpopulations, but the differences in the functions of these cells in the host are unknown. This study investigated the roles of these two cell populations in the clearance of an intracellular pathogen (Haemophilus influenzae) causing murine peritonitis and pneumonia. Microarray analysis and quantitative real-time PCR analysis of proteose peptone-elicited peritoneal murine PMNs showed that IL-15 mRNA levels were significantly higher in Gr-1high PMNs than in Gr-1low PMNs. In addition, IL-15 was produced only by Gr-1-positive PMNs, especially Gr-1high PMNs. IL-15 was required for efficient clearance of experimental murine H. influenzae pneumonia, as 4 days postinfection lungs from IL-15 knockout mice contained 50- to 100-fold more bacteria than did wild-type mouse lungs. Gr-1 PMN-depleted C57BL/6 mice were more susceptible to H. influenzae pneumonia than were Gr-1 PMN replete C57BL/6 mice or C57BL/6 nude mice, demonstrating that Gr-1 PMNs are important in the clearance of intracellular bacteria. IL-15-activated NK cells killed H. influenzae in PMNs. Flow cytometry confirmed the expression of CD69 on the cell membrane of IL-15-activated NK cells. Our results show that Gr-1high PMNs produce more IL-15 than Gr-1low PMNs, and that IL-15-activated NK cells protect against early infection by H. influenzae.

Aflatoxin B1 modulates the insulin-like growth factor-2 dependent signaling axis

Although aflatoxin B(1) (AFB(1)) is known as a mycotoxin that induces hepatocellular carcinoma (HCC), its effects on HCC cells have not been sufficiently investigated. The HCC cell lines HepG2, Huh-6, Huh-7, and PLC were cultured (5 x 10(5)cells/ml) and various concentrations of AFB(1) were added. The expression levels of the alpha-fetoprotein (AFP), insulin-like growth factor-2 (IGF-2), and insulin-like growth factor-1 receptor (IGF-1R) genes in each sample were determined by real-time PCR, with the following results: (1) The level of AFP expression in HepG2 increased at 5-50 ng/ml of AFB(1) in a dose-dependent manner. The AFP expression level in Huh-6 increased at 0.01-5 ng/ml of AFB(1) in a dose-dependent manner and decreased to half controls level at 50 ng/ml of AFB(1). The AFP expression level in Huh-7 decreased to one-third the original level at 0.5-50 ng/ml of AFB(1). The AFP expression level in PLC decreased at 0-0.5 ng/ml of AFB(1) in a dose-dependent manner, and decreased to one-third at concentrations of AFB(1) between 0.5 and 50 ng/ml. (2) The IGF-2 and IGF-1R expression levels in Huh-6 increased more than 10-fold at 0.5-5 ng/ml of AFB(1), but decreased to half at 50 ng/ml of AFB(1). The IGF-2 and IGF-1R expression levels in other cell lines increased in a dose-dependent manner. AFB(1) induced translations of IGF-2 and IGF-1R and cell proliferation: When 50 ng/ml AFB(1) was administrated, cell numbers were 2.0-, 1.7-, and 1.5-fold higher than those of controls after 3 days of culture in HepG2, Huh-7, and PLC, respectively. Particularly, in Huh-6, it increased 2.5-fold higher than those of controls following 5 ng/ml AFB(1) administration. The ratio of fold-change phospho-IGF-1R in all cell lines that were treated with AFB(1), increased 1.1-1.5-fold. These results indicate that AFB(1) may enhance HCC cell proliferation through an IGF-2-dependent signal axis, although it remains to be investigated whether those effects are associated with human hepatocarcinogenesis resulting from AFB(1) exposure.

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 β-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.

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.

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.

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.

A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin‐8

Hospital‐acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii‐activated neutrophils showed increased IL‐8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL‐8. A. baumannii appears to use neutrophils for transport by activating these cells via IL‐8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL‐8 release for bacterial portage. This mechanism might be a new treatment target.

Gene expression analysis in human polymorphonuclear leukocytes stimulated by LPSs from nosocomial opportunistic pathogens

Innate immunity coordinates LPS detection via TLR4 on polymorphonuclear leukocytes (PMNs) to elicit responses to many Gram-negative bacteria. In this study, we describe the effects of five subtypes of LPS [isolated from Escherichia coli B4, Pseudomonas aeruginosa PAO1, multidrug-resistant P. aeruginosa (MDRP), Acinetobacter baumannii and multidrug-resistant A. baumannii (MDRA)] on gene expression in PMNs. LPS isolated from B4, PAO1, and A. baumannii did not significantly alter TLR2 expression. However, LPS from MDRP and MDRA caused a 0.6-fold decrease and 2.7-fold increase, respectively, in TLR2 expression. Similarly, TLR4 expression was not significantly altered by LPS isolated from B4, PAO1 and A. baumannii but was down-regulated by LPS isolated from MDRP and MDRA by 0.1- and 0.6-fold, respectively. All LPS subtypes, excluding PAO1, down-regulated CD14 expression in PMNs. However, all five LPS subtypes up-regulated TNFA, IL1B, IL6, IL10 and TREM1 expression in a concentration-dependent manner, with the most substantial responses observed following exposure to LPS from MDRP and MDRA. These different effects on the gene expression in PMNs may depend on variation in LPS structural modifications related to acquired drug resistance, such as acylation and/or glycosylation.

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 µg/ml) were added to the PMNs (1 × 106 cells/ml), which were stimulated with LPS (100 ng/ml) and incubated at 37°C for 1 or 3 h. When LPS-stimulated PMNs were treated with LPZ at ≥5.0 µg/ml for 1 h, mRNA expression levels of CXCR1/2 and TNFα were suppressed in a dose-dependent manner. The gene expression level of CD14 was also downregulated by LPZ at ≥0.1 µg/ml, with expression suppressed to 50% by 10 µg/ml LPZ. However, LPZ at 0.01–5.0 µg/ml had no significant effect on the expression of TLR-4 or CD11b/CD18 mRNA. LPZ at 10 µg/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 ≥0.01 µg/ml LPZ, in a dose-dependent manner. LPZ may suppress the biological functions of PMNs, such as chemotaxis and inflammatory chemokine production.