Rapid assay for detecting gyrA and parC mutations associated with fluoroquinolone resistance in Enterobacteriaceae.

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.

Recurrent Helicobacter cinaedi cellulitis and bacteremia in a patient with systemic lupus erythematosus.

A 31-year-old woman who had developed systemic lupus erythematosus at 17 years of age was admitted to the hospital for suspected cellulitis in the lower extremities. A blood culture performed upon admission to the hospital detected Helicobacter cinaedi (H. cinaedi), which was also isolated in blood and fecal cultures obtained on the 42nd hospital day. Bacterial translocation of H. cinaedi present in the intestines may have led to the development of recurrent bacteremia and cellulitis. In cases such as this, appropriate antibiotics therapy might be needed for more than one month. Moreover, H. cinaedi, a cause of emerging infections, requires a long period of time to grow; therefore it is important to extend the culture duration when the presence of this bacterium is suspected.

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.

Apoptotic signaling in endothelial cells with neutrophil activation.

As is the case for tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), degranulated substances (DS) released from polymorphonuclear leukocytes (PMN) and H(2)O(2) cause endothelial cell apoptosis through the phosphorylation of members of the mitogen-activated protein kinase (MAPK) family. Stimulation of human umbilical vein endothelial cells (HUVEC) with IL-1ß or TNF-α/cycloheximide (CHX) was found to enhance the phosphorylation of p38 and Jun-N-terminal kinase (JNK) in a time-dependent fashion, but did not affect the time-dependent phosphorylation of extracellular signal-regulated kinase. In addition, IL-1ß and TNF-α/CHX induced the phosphorylation of activating transcription factor-2 (ATF-2), but not c-Jun. Moreover, the p38 in HUVEC was phosphorylated by DS released from PMN and also by H(2)O(2), but not by •O(2) (-) induced by myeloperoxidase (MPO) of PMN. On the other hand, caspase 8 in HUVEC was activated by DS, but not by H(2)O(2) and/or •O(2) (-). In addition, caspases 3 and 7 were cleaved by the treatment of DS and turned into active forms. DS was concentrated, analyzed by electrophoresis, and revealed to contain precursor and subunits of MPO (90, 60, and 14 kDa) and another peptide with a molecular weight of about 28 kDa. Because SB203580 that was an inhibitor of p38 MAPK did not repress phosphorylation of ATF-2 in HUVEC, it was suggested that JNK was more important than p38 in a series of signaling courses. These results suggest the possibility that not only TNF-α/CHX and IL-1ß but also DS released from PMN and the cell-permeable reactive oxygen species H(2)O(2) induce blood vessel injury through endothelial apoptosis.

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.

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.

Effects of grepafloxacin on the function of human polymorphonuclear leukocytes and the phosphorylation of p38 mitogen-activated protein kinase.

BACKGROUND: Some new quinolones (NQs) modulate polymorphonuclear leukocyte (PMN) functions. We investigated these effects on PMN functions at concentrations <10 microg/ml. METHODS: Chemotactic activity and the production of reactive oxygen species (ROS) were measured using a 48-well chemotaxis chamber and by luminol-dependent chemiluminescence (CL) activity, respectively. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was measured by Western blot using specific antibodies to its phosphorylation sites. RESULTS: Grepafloxacin (GPFX) at concentrations >5 microg/ml increased the chemotactic activity and ROS production of PMNs after stimulation with N-formylmethionyl-leucyl-phenylalanine (fMLP), whereas prulifloxacin (PUFX) showed no effect. In contrast to PUFX, GPFX at concentrations >1 microg/ml stimulated the phosphorylation of p38 MAPK. CONCLUSIONS: GPFX enhanced the chemotactic activity and ROS production of PMNs after stimulation with fMLP at concentrations <10 microg/ml. These effects of GPFX on PMNs could be in part due to the enhancement of p38 MAPK phosphorylation.

Role of F4/80Mac-1 adherent non-parenchymal liver cells in concanavalin A-induced hepatic injury in mice.

AIM: Non-parenchymal liver cells (NPLC) play an important role in the regulation of immune responses and the inflammatory process. In this study, we hypothesized that F4/80(+)Mac-1(high+) cells were involved in the regulative feedback-modulated regulation of inflammatory responses during concanavalin A (Con A)-induced hepatitis. METHODS: Hepatitis was induced in BALB/c mice by the intravenous injection of Con A. Liver injury was assessed using serum aminotransferase and pathology. The function of NPLC was assessed by FACS analysis. Accessory cell function of adherent Con A NPLC was performed with an ovalbumin specific T-helper 1 (Th1) clone proliferation assay. The culture supernatant nitric oxide (NO) content was quantified by the Griess reaction. Inducible NO synthase (iNOS) expression was demonstrated by immunohistochemistry and Western blot analysis. RESULTS: The number of hepatic F4/80(+)Mac-1(high+)cells increased in a time-dependent manner after Con A administration, which consequently suppressed Th1 cell proliferation by a mechanism likely to involve NO. The iNOS expression of NPLC was elevated at 24 h post-Con A injection. In nude mice, F4/80(+)Mac-1(high+)cells did not increase in the Con A-treated liver; the NPLC did not suppress Th1 clone proliferation. CONCLUSION: These findings suggest that the in vivo activation of F4/80(+)Mac-1(high+)cells by Con A administration suppresses Th1 cell proliferation by increasing NO, and subsequently reducing liver injury.

Up-regulation of adhesion molecule expression in glomerular endothelial cells by anti-myeloperoxidase antibody.

BACKGROUND: Anti-neutrophil cytoplasmic antibody directed against myeloperoxidase (MPO-ANCA) has been implicated in pauci-immune crescentic glomerulonephritis. It stimulates primed neutrophils to adhere to glomerular endothelial cells (GECs), thereby releasing reactive oxygen and other toxic substances and ultimately damaging the GECs. Though, a pathogenic role for MPO-ANCA is not fully understood, we hypothesized that MPO-ANCA modulates GEC functions by the increases in expression of adhesion molecules. METHODS: A polyclonal rabbit anti-recombinant mouse MPO antibody (anti-rmMPO IgG) was evaluated in mouse GEC (mGEC) for its effect on adhesion molecule expression. The primary culture of mGEC was incubated with anti-rmMPO IgG or isotype control and the expression of intercellular adhesion molecules-1 (ICAM-1) was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis and ICAM-1 cell ELISA. RESULTS: The real-time RT-PCR analysis showed that a treatment with 100 microg/ml anti-rmMPO IgG increased the expression of mRNAs for ICAM-1, vascular cell adhesion molecule-1 and E-selectin by approximately 12.5, 7.5 and 10.5-fold, respectively. ICAM-1 cell ELISA also substantiated increased expression of ICAM-1. This enhancement of ICAM-1 expression was mediated by the antigen specificity of anti-rmMPO IgG. In addition, there were several proteins in mGEC specifically immunoprecipitated with anti-rmMPO IgG. CONCLUSIONS: These results showed that anti-MPO antibody activates not only neutrophils, but also GEC, indicating that anti-rmMPO IgG-induced direct activation of GEC contributes to neutrophil adhesion to GEC, thereby increasing glomerular neutrophil infiltration in initiation and progression of pauci-immune glomerulonephritis.

Opsonic activity assessment of human intravenous immunoglobulin preparations against drug-resistant bacteria.

We have used the ability of opsonized bacteria to stimulate luminol-enhanced chemiluminescence (CL) of human polymorphonuclear leukocytes (PMN) to examine the opsonic capabilities of commercially available human intravenous immunoglobulin (i.v.Ig) preparations. The method was tested against 14 strains of drug-resistant gram-positive bacteria (including methicillin-resistant Staphylococcus aureus, hetero-vancomycin-resistant S. aureus, vancomycin-resistant enterococci, penicillin-resistant Streptococcus pneumoniae), and 23 strains of gram-negative bacteria (including extended-spectrum beta-lactamase-producing bacteria, metallo-beta-lactamase-producing bacteria, beta-lactamase-negative ampicillin-resistant Haemophilus influenzae). An Fc-intact i.v.Ig preparation treated with polyethylene glycol (PEG) was evaluated for opsonization effectiveness against these bacteria in vitro. The opsonization of these organisms was enhanced by an Fc-intact i.v.Ig, and the opsonic activity was dose dependent. A pepsin-treated i.v.Ig preparation exhibited poor opsonic activity for all bacteria tested. These results suggest that Fc-intact i.v.Ig, which augments opsonic activity against various drug-resistant bacteria, will be a useful addition to the treatment of severe bacterial infections in immunocompromised patients with impaired serum opsonic capacity.

Production of anti-Candida antibodies in mice with gut colonization of Candida albicans.

BACKGROUND: Production of antibodies that are specific for allergens is an important pathological process in inflammatory allergic diseases. These contain the antibodies against antigens of Candida albicans, one of the normal microbial flora in an intestinal tract. We studied the effects of the prednisolone administration on the production of anti-Candida antibodies in the gastrointestinally C. albicans-colonized mice. METHODS AND MATERIALS: BALB/c mice, treated with antibacterial antibiotics to decontaminate indigenous intestinal bacterial flora, were inoculated intragastrically with C. albicans. The mice, in which C. albicans grows intestinally, were administered prednisolone to induce temporary immunosuppression. The Candida growth in their intestinal tract and their antibody response to Candida were examined. RESULTS: Antibiotic treatment allowed establishment of C. albicans gastrointestinal colonization, but did not cause subsequent systemic dissemination of C. albicans in all the animals. When these animals received an additional treatment with prednisolone, they showed a significantly higher population of C. albicans in their feces than those of animals treated with antibiotics alone, and the organisms were recovered even from their kidney. This systemic dissemination by C. albicans appeared to be temporal, because all the mice survived without any symptoms for more than 2 months. Examination of the serum titers of total immunoglobulin (Ig)E antibodies and specific IgE and IgG antibodies against Candida antigens demonstrated that titers of total IgE increased, partially by day 14 and clearly at day 27, in prednisolone-treated Candida-colonized mice. Without prednisolone treatment, an increment of the serum titer was scarcely observed. By day 27, corresponding to the increase of total IgE, the anti-Candida IgE and IgG titer increased in mice of the prednisolone-treated group. CONCLUSION: Administration of prednisolone to Candida-colonized mice can induce production of the IgG, IgE antibodies against Candida antigens, perhaps through temporal systemic dissemination of Candida from the intestinal tract.

[Transferability of vanA gene from vancomycin-resistant Enterococcus faecalis in the digestive tract of specific pathogen-free mice].

We evaluated the transferability of vanA gene from vancomycin-resistant Enterococcus faecalis (VREF) to vancomycin-sensitive E. faecalis (VSEF) in vitro and in vivo. In vitro conjugal transfer experiment by filter mating, the vanA gene of VREF was transferable at the high frequency to VSEF and a mutant strain which cured vanA gene of VREF. In vivo studies in the digestive tract of specific pathogen-free mice pretreated with oral antibiotics, transconjugants were also detected from the feces of a mouse at the lower frequency. However, the colonization of transconjugants was transient. The vanA gene in the donor and the transconjugant strain was confirmed by using a polymerase chain reaction method. These results suggest that VSEF colonizing in the human digestive tract might be developed to VREF by transferring of the vanA gene.