[Show abstract][Hide abstract] ABSTRACT: Helicobacter cinaedi is the most common enterohepatic Helicobacter species that causes bacteremia in humans, but its pathogenicity is unclear. Here, we investigated the possible association of H. cinaedi with atherosclerosis in vivo and in vitro. We found that H. cinaedi infection significantly enhanced atherosclerosis in hyperlipidaemic mice. Aortic root lesions in infected mice showed increased accumulation of neutrophils and F4/80(+) foam cells, which was due, at least partly, to bacteria-mediated increased expression of proinflammatory genes. Although infection was asymptomatic, detection of cytolethal distending toxin RNA of H. cinaedi indicated aorta infection. H. cinaedi infection altered expression of cholesterol receptors and transporters in cultured macrophages and caused foam cell formation. Also, infection induced differentiation of THP-1 monocytes. These data provide the first evidence of a pathogenic role of H. cinaedi in atherosclerosis in experimental models, thereby justifying additional investigations of the possible role of enterohepatic Helicobacter spp. in atherosclerosis and cardiovascular disease.
[Show abstract][Hide abstract] ABSTRACT: Helicobacter cinaedi was first isolated from rectal cultures from homosexual men in 1984. In the 1980s to mid 1990s, the microorganism was mainly isolated from samples from homosexual men or immunocompromised patients; however, during the last two decades, H. cinaedi has been isolated from immunocompromised and from immunocompetent individuals worldwide. In Japan, the isolation of this microorganism was first reported in 2003. Since then, many cases have been reported in hospitals across the country. Despite many reports, the etiological properties and pathogenicity of H. cinaedi remain elusive; however, we are increasingly able to recognize some of the features and the clinical relevance of infection. In particular, a long incubation period is essential for detection in an automatic blood culture system and many of the recent isolates are resistant to both macrolides and quinolones. Furthermore, there is an association between infection and severe or chronic illnesses, such as meningitis or arteriosclerosis, in addition to mild diseases such as fever, abdominal pain, gastroenteritis, proctitis, diarrhea, erysipelas, cellulitis, arthritis, and bacteremia.
In this review, we introduce the current knowledge and our latest findings relating to H. cinaedi.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to establish a broth microdilution method for antimicrobial susceptibility testing of Helicobacter cinaedi and to assess the prevalence and mechanisms of fluoroquinolone resistance in Japanese clinical isolates. A broth microdilution method using modified Levinthal broth was developed and compared with the agar dilution method for testing susceptibility to ampicillin, gentamicin, tetracycline and ciprofloxacin. The minimum inhibitory concentrations obtained by these two methods were almost the same for all the antibiotics tested, demonstrating the broth microdilution method to be a suitable and reliable technique for antimicrobial susceptibility testing. A broth microdilution method for antimicrobial susceptibility test for H. cinaedi was established. This method is expected to help improve treatment.
Microbiology and Immunology 05/2013; 57(5):353-8. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We aimed to establish a broth microdilution method for antimicrobial susceptibility testing of H. cinaedi and to assess the prevalence and mechanisms of fluoroquinolone resistance in Japanese clinical isolates. A broth microdilution method using modified levinthal broth was developed and compared with the agar dilution method for testing susceptibility to ampicillin, gentamicin, tetracycline and ciprofloxacin. The MICs obtained by these two methods were almost the same for all the antibiotics tested demonstrating the broth microdilution method to be a suitable and reliable format for antimicrobial susceptibility testing. We established a broth microdilution method for antimicrobial susceptibility test for H. cinaedi. This method is expected to help improvement of the treatment.
Microbiology and Immunology 03/2013; · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Helicobacter cinaedi is the most frequently reported enterohepatic Helicobacter species isolated from humans. Earlier research suggested that certain patients with H. cinaedi infection may remain undiagnosed or incorrectly diagnosed because of difficulties in detecting the bacteria by conventional culture methods. Here, we report a nested PCR assay that rapidly detects the cytolethal distending toxin (cdt) gene of H. cinaedi with high specificity and sensitivity. Specificity of the assay was validated by using different species of Helicobacter and Campylobacter, as well as known H. cinaedi-positive and -negative samples. The sensitivity of detection for the cdt gene in the assay was 10(2) CFU/ml urine or 10(2) CFU/10(5) infected RAW 264.7 cells. In an H. cinaedi-infected mouse model, cdt gene of H. cinaedi was effectively detected via the assay with urine (6/7), stool (2/3), and blood (2/6) samples. Importantly, it detected H. cinaedi in blood, urine and stool samples of one patient with a suspected H. cinaedi infection and three patients with known infections. The assay was further used clinically to follow-up two H. cinaedi-infected patients after antibiotic treatment. Stool samples of these two patients evaluated by nested PCR after antibiotic therapy showed clearance of bacterial DNA. Finally, analysis of stool specimens of healthy volunteers showed occasional positive reactions (4/30) to H. cinaedi DNA, which suggests intestinal colonization by H. cinaedi in healthy subjects. In conclusion, this nested PCR assay may be useful for the rapid diagnosis, antimicrobial treatment evaluation and epidemiological study of H. cinaedi infection.
Journal of clinical microbiology 09/2012; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Helicobacter cinaedi has been increasingly recognized as an emerging pathogen. Reports of recurrent bacteremia and isolation of H. cinaedi organisms from a patient with myopericarditis led us to postulate that H. cinaedi is associated with chronic inflammatory cardiovascular diseases such as atrial arrhythmias and atherosclerosis. To assess any association of H. cinaedi with atrial arrhythmias, a retrospective case-control study of patients attending Kumamoto University Hospital from 2005 to 2009 was performed. The arrhythmia status of these patients was determined from their electrocardiography and electrophysiological studies. Multiple logistic regression analysis was used to identify independent risk factors. In a comparison of case patients (n= 132) with control subjects (n= 137), H. cinaedi seropositivity was identified as an independent risk factor for atrial arrhythmia (odds ratio, 5.13; 95% confidence interval, 3.0-8.7; P < 0.001). There were no significant differences, however, between these two groups with respect to anti-H. pylori IgG concentrations, anti-Chlamydophila pneumoniae IgG concentrations, and other studied variables. IgG concentrations against H. cinaedi and H. pylori were inversely correlated, which suggests cross-immunity between these two bacteria. Also, to explore any association of H. cinaedi with atherosclerosis, immunohistochemical analysis of atherosclerotic aortic tissues collected post mortem from nine patients was performed. Immunohistochemistry of atherosclerotic aortic tissues from all nine patients detected H. cinaedi antigens inside CD68(+) macrophages. These findings provide the first evidence, to our knowledge, of a possible association of H. cinaedi with atrial arrhythmias and atherosclerosis.
Microbiology and Immunology 02/2012; 56(3):145-54. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 8-Nitro-cGMP (8-nitroguanosine 3',5'-cyclic monophosphate) is a nitrated derivative of cGMP, which can function as a unique electrophilic second messenger involved in regulation of an antioxidant adaptive response in cells. In the present study, we investigated chemical and biochemical regulatory mechanisms involved in 8-nitro-cGMP formation, with particular focus on the roles of ROS (reactive oxygen species). Chemical analyses demonstrated that peroxynitrite-dependent oxidation and myeloperoxidase-dependent oxidation of nitrite in the presence of H2O2 were two major pathways for guanine nucleotide nitration. Among the guanine nucleotides examined, GTP was the most sensitive to peroxynitrite-mediated nitration. Immunocytochemical and tandem mass spectrometric analyses revealed that formation of 8-nitro-cGMP in rat C6 glioma cells stimulated with lipopolysaccharide plus pro-inflammatory cytokines depended on production of both superoxide and H2O2. Using the mitochondria-targeted chemical probe MitoSOX Red, we found that mitochondria-derived superoxide can act as a direct determinant of 8-nitro-cGMP formation. Furthermore, we demonstrated that Nox2 (NADPH oxidase 2)-generated H2O2 regulated mitochondria-derived superoxide production, which suggests the importance of cross-talk between Nox2-dependent H2O2 production and mitochondrial superoxide production. The results of the present study suggest that 8-nitro-cGMP can serve as a unique second messenger that may be implicated in regulating ROS signalling in the presence of NO.
[Show abstract][Hide abstract] ABSTRACT: The biological significance of nitrated guanine derivatives, especially 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), has become evident. Therefore it is important to determine the presence and relative abundance of 8-nitro-cGMP formed in cells and tissues. In the present study, we performed immunocytochemistry with monoclonal antibodies specific for 8-nitroguanine (clone NO2-52) and 8-nitro-cGMP (clone 1G6) in rat C6 glioma cells and rat primary cultured astrocytes. Immunocytochemistry utilizing the anti-8-nitro-cGMP monoclonal antibody (1G6) indicated that immunostaining increased markedly in C6 cells expressing increased amounts of inducible nitric oxide synthase (iNOS) after treatment with lipopolysaccharide (LPS) plus cytokines. Treatment of C6 cells with inhibitors for NOS and soluble guanylate cyclase (sGC) completely nullified the elevated 1G6 immunoreactivity. These results were consistent with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Immunocytochemistry performed using NO2-52 also showed that treatment of cells with inhibitors for NOS and sGC completely nullified the elevated immunoreactivity; this indicated that 8-nitro-cGMP is a major component of 8-nitroguanine derivatives produced in cells. Similar results were obtained in the primary astrocytes stimulated with LPS plus cytokines. Because immunocytochemistry is a conventional, powerful, and fairly straightforward method for determining the presence, localization, and relative abundance of an antigen of interest in cultured cells, anti-8-nitroguanine (NO2-52) and anti-8-nitro-cGMP (1G6) antibodies could be useful tools for analyzing nitrated guanine nucleotides, especially 8-nitro-cGMP, by means of immunocytochemistry.
[Show abstract][Hide abstract] ABSTRACT: A nitrated guanine nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 microm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys(434) of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.
Journal of Biological Chemistry 05/2010; 285(31):23970-84. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO), produced by inducible NO synthase (iNOS) during infection, plays a crucial role in host defense mechanisms. Salmonella typhimurium infection in mice is associated with excessive production of NO from iNOS as a host defense response. An important cytoprotective and antimicrobial function of NO is mediated by induction of heme oxygenase (HO)-1. The signaling mechanism of NO-dependent HO-1 induction has remained unclear, however. We recently discovered a nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), which is formed via guanine nitration with NO and reactive oxygen species. iNOS-dependent 8-nitro-cGMP formation and HO-1 induction were identified in Salmonella-infected mice. Extensive apoptosis observed with iNOS-deficient macrophages infected with Salmonella was remarkably suppressed via HO-1 induced by 8-nitro-cGMP formed in cells. This cytoprotective signaling appears to be mediated by the reaction of 8-nitro-cGMP with protein sulfhydryls to generate a novel post-translational modification named protein S-guanylation. We also found that 8-nitro-cGMP specifically S-guanylates Keap1, a negative regulator of transcription factor Nrf2, which in turn up-regulates transcription of HO-1. Here, we discuss the unique mechanism of NO-mediated host defense that operates via formation of a novel signaling molecule - 8-nitro-cGMP - during microbial infections.
Journal of Clinical Biochemistry and Nutrition 01/2010; 46(1):14-9. · 2.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Signaling mechanisms of NO-mediated host defense are yet to be elucidated. In this study, we report a unique signal pathway for cytoprotection during Salmonella infection that involves heme oxygenase 1 (HO-1) induced by a nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP). Wild-type C57BL/6 mice and C57BL/6 mice lacking inducible NO synthase (iNOS) were infected with Salmonella enterica serovar Typhimurium LT2. HO-1 was markedly up-regulated during the infection, the level being significantly higher in wild-type mice than in iNOS-deficient mice. HO-1 up-regulation was associated with 8-nitro-cGMP formation detected immunohistochemically in Salmonella-infected mouse liver and peritoneal macrophages. 8-Nitro-cGMP either exogenously added or formed endogenously induced HO-1 in cultured macrophages infected with Salmonella. HO-1 inhibition by polyethylene glycol-conjugated zinc-protoporphyrin IX impaired intracellular killing of bacteria in mouse liver and in both RAW 264 cells and peritoneal macrophages. Infection-associated apoptosis was also markedly increased in polyethylene glycol-conjugated zinc-protoporphyrin IX-treated mouse liver cells and cultured macrophages. This effect of HO-1 inhibition was further confirmed by using HO-1 short interfering RNA in peritoneal macrophages. Our results suggest that HO-1 induced by NO-mediated 8-nitro-cGMP formation contributes, via its potent cytoprotective function, to host defense during murine salmonellosis.
The Journal of Immunology 04/2009; 182(6):3746-56. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) produced by inducible NO synthase (iNOS) during infection plays a crucial role in host defense mechanisms, via its antimicrobial and cytoprotective activities. Infection of Salmonella typhimurium in mice induces excessive production of NO, as a host defense response. We found much greater bacterial growth and apoptotic changes in iNOS-deficient (iNOS-/-) mice than in wild-type mice. However, the mechanism of NO-mediated cytoprotection during Salmonella infection remained unclear. An important signaling mechanism induced by NO is heme oxygenase (HO)-1, a significant cytoprotective molecule produced by oxidative stress. Thus, we sought to clarify NO-dependent cytoprotective and antimicrobial host defense, with a particular focus on the signaling mechanism of HO-1 induction. We recently discovered a nitrated cyclic nucleotide, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), which is formed via NO possibly with reactive oxygen species. We observed strong immunoreactivity for 8-nitro-cGMP in Salmonella-infected wild-type mouse liver and peritoneal macrophages in culture but not in iNOS-/- mouse liver and macrophages. Moreover, a higher apoptosis was observed in iNOS-/- macrophages compared with wild-type macrophages after Salmonella infection, but the difference was nullified when iNOS-/- cells were treated with 8-nitro-cGMP. Finally, authentic 8-nitro-cGMP induced HO-1 in cultured macrophages infected with Salmonella. The signaling function of 8-nitro-cGMP appears to be mediated by its unique reaction with the sulfhydryl group of cysteine, thus forming a proteinS-cGMP adduct, which is an important mechanism of post-translational modification of proteins called protein S-guanylation. More importantly, we found 8-nitro-cGMP-dependent S-guanylation of Keap1, a regulatory protein of transcription factor Nrf2, which regulates the transcription of HO-1. In this review, we focus on a unique mechanism of NO-mediated host defense via formation of a novel signaling molecule, 8-nitro-cGMP in microbial infections.
Nihon Hansenbyo Gakkai zasshi = Japanese journal of leprosy: official organ of the Japanese Leprosy Association 03/2009; 78(1):41-7.
[Show abstract][Hide abstract] ABSTRACT: 8-Nitroguanosine is a nitratively modified nucleoside that is formed endogeneously under inflammatory conditions dependent on nitric oxide production, particularly associated with cancer risks. Here, we investigated the mutagenic potential of 8-nitroguanosine in mammalian cells. Treatment with 8-nitroguanosine (10-1000 microM) for 1h significantly increased (by 6-8 times) the mutation frequency of the xanthine-guanine phosphoribosyltransferase (gpt) gene in AS52 cells without cytotoxic effects. 8-Nitroguanosine treatment induced a G-to-T transversion in gpt gene at position 86. It also significantly increased levels of abasic sites in DNA. These observations suggest that formation of 8-nitroguanosine may contribute to the pathogenesis of inflammation-associated carcinogenesis.
Cancer Letters 05/2008; 262(2):239-47. · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Helicobacter cinaedi infection is now recognized as an increasingly important emerging disease. Its pathogenesis and epidemiological features are not fully understood, however. Here, we investigated the antigenic protein of H. cinaedi and the immunological response to it in H. cinaedi-infected patients. We constructed a genomic library of H. cinaedi from an H. cinaedi clinical isolate, and various H. cinaedi recombinant proteins were expressed. We identified the 30-kDa protein, encoded in an 822-bp H. cinaedi genome, as a major antigen, which was specifically recognized by serum from an H. cinaedi-immunized rabbit and H. cinaedi-infected patients. The gene encoding this 30-kDa antigen had high sequence similarity with genes encoding putative membrane proteins of bacteria. To evaluate whether the 30-kDa protein can be applied in serological testing for H. cinaedi infections, the recombinant protein was expressed in Escherichia coli as a His-tagged fusion protein and purified by Ni(2+) affinity chromatography. Western blot analysis revealed strong immunoreactivity of the 31-kDa fusion protein with serum antibody from patients infected with H. cinaedi, but such an immunoreaction was absent or was very weak with uninfected control serum. An enzyme-linked immunosorbent assay using this H. cinaedi major antigen showed significantly high antibody titers for H. cinaedi-infected subjects compared with those of various control groups. We therefore conclude that the 30-kDa putative membrane protein is a major antigen of H. cinaedi and is useful for immunological and serological testing for clinical diagnosis and for further epidemiological study of H. cinaedi infection in humans.
[Show abstract][Hide abstract] ABSTRACT: Salmonella species normally infect hosts via the oral-fecal route. We previously reported that NO had potent host defense functions in murine salmonellosis, not only via a direct antibacterial effect but also because it was cytoprotective for infected host cells. Here, we used an oral route to infect iNOS-deficient mice infected with S. enterica serovar Typhimurium to further investigate the cytoprotective role of NO in preventing damage caused by Salmonella organisms in PP. Oral bacterial challenge (2 x 10(5) CFU, or >100 LD(50)) produced a more severe infection and greater lethality in iNOS-deficient mice than in iNOS-competent mice. We used specific antibodies to S. enterica Typhimurium, neutrophils, iNOS, nitrotyrosine, and dendritic cells (CD11c-positive) in histochemical and immunohistochemical studies to examine infected PP tissues. S. enterica Typhimurium colonization in PP from iNOS-deficient mice was significantly higher than that in wild-type mice. Histochemical assays showed extensive cellular damage in PP. We then examined PP tissues for apoptosis by means of in situ TUNEL analysis and by measuring caspase-3 specific activity in tissue homogenates. Increased numbers of TUNEL-positive cells and severe granulomatous inflammation with increased infiltration of neutrophils and macrophages were observed during infection in iNOS-deficient mice compared with wild-type mice. iNOS-deficient mice had increased numbers of dendritic cells and significantly higher caspase-3-specific activity in PP. These data confirm that NO exerts its protective function not only through direct antibacterial action, but also by preventing apoptosis and thereby contributing to antimicrobial defense during salmonellosis.
Microbiology and Immunology 01/2008; 52(4):197-208. · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.
Nature Chemical Biology 12/2007; 3(11):727-35. · 12.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: At various times after orthopedic operations (more than a few weeks, with an average of 29.9 days), 11 patients had a sudden onset of high temperature (average 38.9 degrees C) and local cellulitis at different sites on the operated sides. The wounds had completely healed, without complicated infections, when the cellulitis occurred. The clinical picture of cellulitis in all patients was atypical: diffuse salmon-pink skin color, local heat, swelling, spontaneous pain, and tenderness but no eruptions. No patient had any underlying immunocompromising conditions or had been given immunosuppressive agents. Gram-negative spiral bacteria were isolated from blood cultures and were identified as Helicobacter cinaedi on the basis of 16S rRNA gene sequencing and DNA-DNA hybridization using standard strains. By means of phylogenetic analysis, we divided these clinical isolates into two clones. The H. cinaedi strain isolated via fecal cultures from two patients without intestinal symptoms was the same clone as the blood isolate. All isolates were quite susceptible to various antibiotics, and clinical and inflammatory symptoms of bacteremia and cellulitis improved after treatment with penicillins and cephalosporins. A relatively high incidence of recurrence of the same disease was observed, however. Almost all patients responded immunologically to the infection, as evidenced by the production of serum antibody against H. cinaedi. We thus suggest that H. cinaedi should not be regarded as simply an opportunistic pathogen but that it may be a pathogen in immunocompetent hosts and may cause infections together with bacteremia and cellulitis.
Journal of Clinical Microbiology 02/2007; 45(1):31-8. · 4.07 Impact Factor