[Mechanism of Cryptococcus Meningoencephalitis].

Cryptococcus neoformans and Cryptococcus gattii are fungal pathogens that cause diseases in humans. Cryptococcal species mainly enter the body by inhalation and in most cases are eliminated by host defense mechanisms. Some cases, however, progress to pneumonia and subsequent dissemination of the infection to the central nervous system (CNS), leading to meningoencephalitis. Cryptococcus can cross the blood-brain barrier transcellularly, paracellularly and through infected phagocytes (the Trojan horse mechanism). The reason for the tropism of Cryptococcus to the CNS could be partially explained by the abundance of inositol in the brain, which causes the hyaluronic acid in fungal cells to bind to host CD44 receptors. There are differences in the clinical characteristics of C. neoformans and C. gattii. HIV infection is the most common risk factor for cryptococcosis due to C. neoformans, whereas C. gattii infection with CNS involvement is frequently found in otherwise healthy individuals exposed to plant propagules found in tropical and subtropical regions. As the virulence traits of C. neoformans contributing to CNS disease, high macrophage uptake and laccase activity are associated with the fungal burden and the rate of clearance of the infection from the brain. Recent reports suggested that the C. gattii VGII strain suppresses host immune responses in the lung and causes more lung infections than CNS diseases. Furthermore, the anti-GM-CSF autoantibodies are a risk factor for CNS infection by the C. gattii VGI strain. To understand the mechanism by which Cryptococcus causes CNS disease, it is important to consider the specific characteristics of the species and the molecular types.

Comparative molecular and microbiological diagnosis of 19 infective endocarditis cases in which causative microbes were identified by PCR-based DNA sequencing from the excised heart valves.

Infective endocarditis (IE) is traditionally diagnosed by microbiological analysis of blood cultures, following which therapeutic antibiotics are chosen based on antimicrobial sensitivity tests. However, such conventional techniques do not always lead to an accurate etiological diagnosis. Recently, PCR analysis of the 16S rRNA gene has been employed to identify organisms isolated from excised heart valves. In this study, we analyzed 19 valve samples from patients with confirmed IE, as identified by Duke's criteria. Using broad-range PCR amplification, followed by direct gene sequencing, pathological agents were identified in all samples. Although blood cultures yielded negative results in 4 cases, PCR analysis of valve samples showed positive identification of causative organisms. In 3 cases, there was a difference between blood culture and PCR in identification of pathological agents, which are likely to be misidentified by the conventional method based on the phenotypic database. Postoperative antibiotics were chosen considering the severity of lesions and the results of PCR, Gram staining, and valve cultures. All patients were cured without relapse. The broad-range PCR method was therefore beneficial for the management of IE because it enabled us to identify pathogens directly from the site of infection, even organisms that were difficult to culture or likely to be misidentified by the conventional culture method. Identification of the agents provided precise knowledge of the microbiological spectrum involved in the cases of IE.

Toll-like receptor 9-dependent activation of bone marrow-derived dendritic cells by URA5 DNA from Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised patients. Recently, we reported that Toll-like receptor 9 (TLR9) is involved in host defense against C. neoformans: specifically, it detects the pathogen's DNA. In the present study, we aimed to elucidate the mechanisms underlying TLR9-mediated activation of innate immune responses by using the URA5 gene, which encodes a virulent component of this fungal pathogen. A PCR-amplified 345-bp URA5 gene fragment induced interleukin-12 p40 (IL-12p40) production by bone marrow-derived dendritic cells (BM-DCs) in a TLR9-dependent manner. Similar activity was detected in the 5' 129-bp DNA fragment of URA5 and in a synthesized oligodeoxynucleotide (ODN) with the same sequence. Shorter ODN fragments, which contained GTCGGT or GACGAT but had only 24 or 21 bases, induced IL-12p40 production and CD40 expression by BM-DCs, but this activity vanished when the CG sequence was replaced by GC or when a phosphorothioate modification was introduced. IL-12p40 production caused by active ODN was strikingly enhanced by treatment with DOTAP, a cationic lipid that increases the uptake of DNA by BM-DCs, though DOTAP failed to induce IL-12p40 production by inactive ODN and did not affect the activity of an ODN-containing canonical CpG motif. There was no apparent difference in intracellular trafficking between active and inactive ODNs. Finally, an extremely high dose of inactive ODN suppressed IL-12p40 production by BM-DCs that had been stimulated with active ODN. These results suggest that the C. neoformans URA5 gene activates BM-DCs through a TLR9-mediated signaling pathway, using a mechanism possibly independent of the canonical CpG motif.

Cryptococcus neoformans suppresses the activation of bone marrow-derived dendritic cells stimulated with its own DNA, but not with DNA from other fungi.

DNA from Cryptococcus neoformans activates bone marrow-derived dendritic cells (BM-DCs) in a TLR9-dependent manner. In this study, we examined the effect of the culture supernatants of C. neoformans on the activation of BM-DCs caused by its own DNA. C. neoformans supernatants suppressed IL-12p40, IL-6 production and CD40 expression by BM-DCs stimulated with its own DNA, but not with CpG-ODN and DNA from Candida albicans, Saccharomyces cerevisiae or Escherichia coli. In a confocal microscopic analysis, C. neoformans DNA was colocalized with LAMP-1, a late endosomal marker, and TLR9. The culture supernatants did not show any apparent suppression of these responses. In a luciferase reporter assay, C. neoformans supernatants inhibited NFκB activation caused by its own DNA. These inhibitory activities were attenuated by treatment with heat or trypsin. These results indicate that C. neoformans secrete certain proteinous molecules that suppress the activation of BM-DCs caused by its own DNA.

Activation of pulmonary invariant NKT cells leads to exacerbation of acute lung injury caused by LPS through local production of IFN-γ and TNF-α by Gr-1+ monocytes.

Invariant NK T (iNKT) cells are known to play a critical role in the regulation of inflammatory responses in various clinical settings. In the present study, we assessed the contribution of iNKT cells to the development of acute lung injury (ALI), which was caused by intra-tracheal administration of LPS. Jα18 gene-disrupted mice lacking these cells underwent neutrophilic inflammatory responses in lungs at an equivalent level as control mice. Next, mice were sensitized intra-tracheally with α-galactosylceramide, an activator of iNKT cells, followed by challenge with LPS. In this model, mice showed severe lung injury, and all mice were killed within 72 h after LPS injection. IFN-γ and tumor necrosis factor (TNF)-α were strikingly elevated in the lungs of these mice. Administration of neutralizing mAb against IFN-γ and TNF-α attenuated lung injury in a histopathological analysis and improved their survival rate. Flow cytometric analysis revealed that IFN-γ was expressed in NK cells, iNKT cells and also Gr-1(dull+)Ly-6C(+) monocytes and TNF-α was detected mainly in Gr-1(bright+)Ly-6G(+) neutrophils and Gr-1(dull+)Ly-6C(+) monocytes. Otherwise, in mice treated with LPS alone, IFN-γ was not detected in the lungs and Gr-1(bright+)Ly-6G(+) neutrophil was a main cellular source of TNF-α production. Anti-Gr-1 mAb resulted in the attenuation of ALI and decrease in the level of these cytokines. These results indicated that activation of iNKT cells led to striking exacerbation of ALI caused by LPS and that Gr-1(+) monocytes were recruited in the lungs with expressing IFN-γ and TNF-α and played an important role in the development of these responses.

[A case of infective Aerococcus urinae endocarditis successfully treated by aortic valve replacement].

Aerococcus urinae is a endocarditis rare causative organism with low virulene. We report an A. urinae endocarditis case treated by aortic valve replacement. An 80-year-old woman hospitalized for urinary tract infection and hydronephrosis due to three-week renal calculi. Blood culture on admission isolated Streptococcus acidominimus. During the course, she was transferred to our care for surgical intervention after developing congestive heart failure due to severe aortic regurgitation. Echocardiographic findings indicated infective endocarditis. She underwent aortic valve replacement, and gram staining of the resected valve tissue showed gram-positive cocci, although valve culture was negative. PCR amplification and DNA sequencing using the valve material matched an A. urinae sequence. The woman recovered and was discharged six weeks after antibiotic treatment.

Activation of myeloid dendritic cells by deoxynucleic acids from Cordyceps sinensis via a Toll-like receptor 9-dependent pathway.

The mechanism by which host cells recognize Cordyceps sinensis, a Chinese herbal medicine that is known to exhibit immunomodulating activity, remains poorly understood. In this study, we investigated whether the DNA of this fungus could activate mouse bone marrow-derived dendritic cells (BM-DCs). Upon stimulation with C. sinensis DNA, BM-DCs released IL-12p40 and TNF-alpha and expressed CD40. Cytokine production and CD40 expression were attenuated by chloroquin and bafilomycin A. Activation of BM-DCs by C. sinensis DNA was almost completely abrogated in TLR9KO mice. According to a luciferase reporter assay, C. sinensis DNA activated NF-kappaB in HEK293T cells transfected with the TLR9 gene. Finally, a confocal microscopic analysis showed that C. sinensis DNA was co-localized with CpG-ODN and partly with TLR9 and LAMP-1, a late endosomal marker, in BM-DCs. Our results demonstrated that C. sinensis DNA caused activation of BM-DCs in a TLR9-dependent manner.

Early production of tumor necrosis factor-alpha by Gr-1 cells and its role in the host defense to pneumococcal infection in lungs.

In this study, we elucidated the role of tumor necrosis factor (TNF)-alpha in the host defense to pulmonary infection with Streptococcus pneumoniae and defined the cellular source of this cytokine at an early stage of infection. Administration of anti-TNF-alpha monoclonal antibody (mAb) resulted in the reduced accumulation of neutrophils in bronchoalveolar lavage fluids (BALFs) and severe exacerbation of this infection. In a flow cytometric analysis, the intracellular expression of TNF-alpha was detected in Gr-1(bright+) and Gr-1(dull+) cells during the time intervals postinfection, and F4/80(+) cells expressed intracellular TNF-alpha before Gr-1(dull+) cells appeared. The Gr-1(bright+) and Gr-1(dull+) cells sorted from BALF cells at 24 h were identified as neutrophils and macrophage-like cells, respectively, and the Gr-1(dull+) cells expressing CD11c, partially CD11b and a marginal level of F4/80 secreted TNF-alpha in in vitro cultures. Finally, deletion of Gr-1(+) cells by administration of the specific mAb significantly reduced the concentrations of this cytokine in BALF at 6 and 12 h postinfection, but not the expression of TNF-alpha in F4/80(+) cells. Thus, these results demonstrated that neutrophils, F4/80(+) macrophages and Gr-1(dull+) CD11c(+) macrophage-like cells played an important role in the production of TNF-alpha in lungs at an early stage of infection with S. pneumoniae.

Toll-like receptor 9-dependent activation of myeloid dendritic cells by Deoxynucleic acids from Candida albicans.

The innate immune system of humans recognizes the human pathogenic fungus Candida albicans via sugar polymers present in the cell wall, such as mannan and beta-glucan. Here, we examined whether nucleic acids from C. albicans activate dendritic cells. C. albicans DNA induced interleukin-12p40 (IL-12p40) production and CD40 expression by murine bone marrow-derived myeloid dendritic cells (BM-DCs) in a dose-dependent manner. BM-DCs that lacked Toll-like receptor 4 (TLR4), TLR2, and dectin-1, which are pattern recognition receptors for fungal cell wall components, produced IL-12p40 at levels comparable to the levels produced by BM-DCs from wild-type mice, and DNA from a C. albicans pmr1Delta null mutant, which has a gross defect in mannosylation, retained the ability to activate BM-DCs. This stimulatory effect disappeared completely after DNase treatment. In contrast, RNase treatment increased production of the cytokine. A similar reduction in cytokine production was observed when BM-DCs from TLR9(-/-) and MyD88(-/-) mice were used. In a luciferase reporter assay, NF-kappaB activation was detected in TLR9-expressing HEK293T cells stimulated with C. albicans DNA. Confocal microscopic analysis showed similar localization of C. albicans DNA and CpG-oligodeoxynucleotide (CpG-ODN) in BM-DCs. Treatment of C. albicans DNA with methylase did not affect its ability to induce IL-12p40 synthesis, whereas the same treatment completely eliminated the ability of CpG-ODN to induce IL-12p40 synthesis. Finally, impaired clearance of this fungal pathogen was not found in the kidneys of TLR9(-/-) mice. These results suggested that C. albicans DNA activated BM-DCs through a TLR9-mediated signaling pathway using a mechanism independent of the unmethylated CpG motif.

Toll-like receptor 4-dependent activation of myeloid dendritic cells by leukocidin of Staphylococcus aureus.

Leukocidin (Luk), an exotoxin of Staphylococcus aureus consisting of LukF and LukS, is a hetero-oligomeric pore-forming cytolytic toxin toward human and rabbit polymorphonuclear leukocytes. However, it is uncertain how Luk affects the host immune response. In the present study, we investigated whether Luk has the ability to stimulate mouse bone marrow-derived myeloid dendritic cells (BM-DCs). LukF activated BM-DCs to generate IL-12p40 mRNA, induce intracellular expression and extracellular secretion of this cytokine and express CD40 on their surface, whereas LukS showed a much lower or marginal ability in the activation of BM-DCs than its counterpart component. Similarly, TNF-alpha was secreted by BM-DCs upon stimulation with these components. Combined addition of these components did not lead to a further increase in IL-12p40 secretion. IL-12p40 production caused by LukF was completely abrogated in BM-DCs from TLR4-deficient mice similarly to the response to lipopolysaccharide (LPS). Polymixin B did not affect the LukF-induced IL-12p40 production, although the same treatment completely inhibited the LPS-induced response. Boiling significantly inhibited the response caused by LukF, but not by LPS. Finally, in a luciferase reporter assay, LukF induced the activation of NF-kappaB in HEK293T cells transfected with TLR4, MD2 and CD14, whereas LukS did not show such activity. These results demonstrate that LukF caused the activation of BM-DCs by triggering a TLR4-dependent signaling pathway and suggests that Luk may affect the host inflammatory response as well as show a cytolytic effect on leukocytes.

Human T-cell leukemia virus type I infects human lung epithelial cells and induces gene expression of cytokines, chemokines and cell adhesion molecules.

BACKGROUND: Human T-cell leukemia virus type I (HTLV-I) is associated with pulmonary diseases, characterized by bronchoalveolar lymphocytosis, which correlates with HTLV-I proviral DNA in carriers. HTLV-I Tax seems to be involved in the development of such pulmonary diseases through the local production of inflammatory cytokines and chemokines in T cells. However, little is known about induction of these genes by HTLV-I infection in lung epithelial cells. RESULTS: We tested infection of lung epithelial cells by HTLV-I by coculture studies in which A549 alveolar and NCI-H292 tracheal epithelial cell lines were cocultured with MT-2, an HTLV-I-infected T-cell line. Changes in the expression of several cellular genes were assessed by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay and flow cytometry. Coculture with MT-2 cells resulted in infection of lung epithelial cells as confirmed by detection of proviral DNA, HTLV-I Tax expression and HTLV-I p19 in the latter cells. Infection was associated with induction of mRNA expression of various cytokines, chemokines and cell adhesion molecule. NF-kappaB and AP-1 were also activated in HTLV-I-infected lung epithelial cells. In vivo studies showed Tax protein in lung epithelial cells of mice bearing Tax and patients with HTLV-I-related pulmonary diseases. CONCLUSION: Our results suggest that HTLV-I infects lung epithelial cells, with subsequent production of cytokines, chemokines and cell adhesion molecules through induction of NF-kappaB and AP-1. These changes can contribute to the clinical features of HTLV-I-related pulmonary diseases.

Toll-like receptor 2 (TLR2) and dectin-1 contribute to the production of IL-12p40 by bone marrow-derived dendritic cells infected with Penicillium marneffei.

The present study was designed to elucidate the role of TLR2, TLR4 and dectin-1 in the production of IL-12p40 by bone marrow-derived dendritic cells (BM-DCs) infected with Penicillium marneffei. IL-12p40 production was almost completely abrogated in BM-DCs from TLR2 gene-knockout (KO) and MyD88KO mice, but not from TLR4-defective C3H/HeJ mice compared to those from control mice. Furthermore, BM-DCs from dectin-1KO mice faintly produced IL-12p40 upon stimulation with this fungus. Using a luciferase reporter assay, P. marneffei activated NF-kappaB in HEK293 cells transfected with the TLR2 gene, but not with the dectin-1 gene, and their co-transfection did not lead to further increase in this response. These results indicate that TLR2 and dectin-1 are essential in sensing P. marneffei for the activation of BM-DCs.

Cryptococcus neoformans inhibits nitric oxide synthesis caused by CpG-oligodeoxynucleotide-stimulated macrophages in a fashion independent of capsular polysaccharides.

Cryptococcus neoformans is eradicated by macrophages via production of NO. Unmethylated CpG-ODN protect mice from infection with this fungal pathogen by inducing IFN-gamma. The present study was designed to elucidate the effect of C. neoformans on the synthesis of NO by alveolar macrophages. For this purpose, MH-S, an alveolar macrophage cell line, was stimulated with CpG-ODN in the presence of IFN-gamma. A highly virulent strain of C. neoformans with thick capsule suppressed the production of NO. Capsular polysaccharides were not essential for this suppression, because there was no difference between acapsular mutant (Cap67) and its parent strain. Physical or close interaction of Cap67 with MH-S was necessary, as shown by the loss of such effect when direct contact was interfered by nitrocellulose membrane. Similar effects were observed by disrupted as well as intact Cap67. Whereas the inhibitory effect of intact Cap67 was completely abrogated by heat treatment, disrupted Cap67 did not receive such influence. Finally, disrupted Cap67 did not show any inhibitory effect on the TLR9-mediated activation of NF-kappaB in a luciferase reporter assay with HEK293T cells, although the TLR4-mediated activation was suppressed. These results revealed that C. neoformans suppressed the synthesis of NO by CpG-ODN and IFN-gamma-stimulated macrophages in a fashion independent of capsular polysaccharides, although the precise mechanism remains to be elucidated.

Deoxynucleic acids from Cryptococcus neoformans activate myeloid dendritic cells via a TLR9-dependent pathway.

The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathogen in immunocompromised patients, remains poorly understood. In the present study, we asked whether the DNA of this yeast activates mouse bone marrow-derived myeloid dendritic cells (BM-DCs). BM-DCs released IL-12p40 and expressed CD40 upon stimulation with cryptococcal DNA, and the response was abolished by treatment with DNase, but not with RNase. IL-12p40 production and CD40 expression were attenuated by chloroquine, bafilomycin A, and inhibitory oligodeoxynucleotides (ODN) that suppressed the responses caused by CpG-ODN. Activation of BM-DCs by cryptococcal DNA was almost completely abrogated in TLR9 gene-disrupted (TLR9(-/-)) mice and MyD88(-/-) mice, similar to that by CpG-ODN. In addition, upon stimulation with whole yeast cells of acapsular C. neoformans, TLR9(-/-) BM-DCs produced a lower amount of IL-12p40 than those from wild-type mice, and TLR9(-/-) mice were more susceptible to pulmonary infection with this fungal pathogen than wild-type mice, as shown by increased number of live colonies in lungs. Treatment of cryptococcal DNA with methylase resulted in reduced IL-12p40 synthesis by BM-DCs. Furthermore, using a luciferase reporter assay, cryptococcal DNA activated NF-kappaB in HEK293 cells transfected with the TLR9 gene. Finally, confocal microscopy showed colocalization of fluorescence-labeled cryptococcal DNA with CpG-ODN and the findings merged in part with the distribution of TLR9 in BM-DCs. Our results demonstrate that cryptococcal DNA causes activation of BM-DCs in a TLR9-dependent manner and suggest that the CpG motif-containing DNA may contribute to the development of inflammatory responses after infection with C. neoformans.

Dectin-1 is not required for the host defense to Cryptococcus neoformans.

Dectin-1 is known as a sole receptor for beta-glucan, a major cell wall component of fungal microorganisms. In the current study, we examined the role of this molecule in the host defense to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients. There was no significant difference in the clinical course and cytokine production between dectin-1 gene-deficient and control mice. These results indicate that dectin-1 is not likely essential for the development of host protective responses to C. neoformans.

Adjuvant effect of CpG-oligodeoxynucleotide in anti-fungal chemotherapy against fatal infection with Cryptococcus neoformans in mice.

Cryptococcal meningoencephalitis is a life-threatening infectious disease in immunocompromised patients. Unmethylated CpG-oligodeoxynucleotides (CpG-ODN) protects hosts in a mouse model. In the present study, we tested the adjuvant effect of CpG-ODN in anti-fungal chemotherapy. Administration of either fluconazole (FLCZ) or CpG-ODN was effective in extending survival, accelerating clearance of fungi and preventing disseminated infection. Combination of both agents provided more beneficial effect than either agent alone. Cytokine balance in the infected lungs was biased to Th1-type response by CpGODN, while FLCZ did not further promote. These results suggest that CpG-ODN is a promising adjuvant in chemotherapy against this infection.

Heterozygous deletion of mitotic arrest-deficient protein 1 (MAD1) increases the incidence of tumors in mice.

Mitotic arrest-deficient protein 1 (MAD1) is a component of the mitotic spindle assembly checkpoint. We have created a knockout mouse model to examine the physiologic consequence of reduced MAD1 function. Mad1(+/-) mice were successfully generated, but repeated paired mating of Mad1(+/-) with Mad1(+/-) mice failed to produce a single Mad1(-/-) animal, suggesting that the latter genotype is embryonic lethal. In aging studies conducted for >18 months, Mad1(+/-) mice compared with control wild-type (wt) littermates showed a 2-fold higher incidence of constitutive tumors. Moreover, 42% of Mad1(+/-) (P < 0.03), but 0% of wt, mice developed neoplasia after treatment with vincristine, a microtubule depolymerization agent. Mad1(+/-) mouse embryonic fibroblasts (MEF) were found to be more prone than wt cells to become aneuploid; Mad1(+/-), but not wt, MEFs produced fibrosarcomas when explanted into nude mice. Our results indicate an essential MAD1 function in mouse development and correlate Mad1 haploinsufficiency with increased constitutive tumors.