Interleukin-33 and thymic stromal lymphopoietin, but not interleukin-25, are crucial for development of airway eosinophilia induced by chitin.

Exposure to various antigens derived from house dust mites (HDM) is considered to be a risk factor for development of certain allergic diseases such as atopic asthma, atopic dermatitis, rhinitis and conjunctivitis. Chitin is an insoluble polysaccharide (ß-(1-4)-poly-N-acetyl-D-glucosamine) and a major component in the outer shell of HDMs. Mice exposed to chitin develop asthma-like airway eosinophilia. On the other hand, several lines of evidence show that the effects of chitin on immune responses are highly dependent on the size of chitin particles. In the present study, we show that chitin induced production of IL-33 and TSLP by alveolar and bronchial epithelial cells, respectively, in mice. IL-25, IL-33 and TSLP were reported to be important for group 2 innate lymphoid cell (ILC2)-, but not Th2 cell-, dependent airway eosinophilia in a certain model using chitin beads. Here, we show that-in our murine models-epithelial cell-derived IL-33 and TSLP, but not IL-25, were crucial for activation of resident lung Th2 cells as well as group 2 innate lymphoid cells (ILC2s) to produce IL-5, resulting in development of chitin-induced airway eosinophilia. Our findings provide further insight into the underlying mechanisms of development of HDM-mediated allergic disorders.

Chitin promotes antigen-specific Th2 cell-mediated murine asthma through induction of IL-33-mediated IL-1ß production by DCs.

Chitin, which is a major component of house dust mites (HDM), fungi, crustaceans, etc., can activate immune cells, suggesting that it contributes to development of allergic disorders such as asthma. Although the pathophysiological sensitization route of asthmatic patients to allergens is considered via the respiratory tract, the roles of intranasally-administered chitin in development of asthma remain unclear. After ovalbumin (OVA) challenge, development of airway inflammation was profoundly exacerbated in mice sensitized with OVA in the presence of chitin. The exacerbation was dependent on IL-33, but not IL-25, thymic stromal lymphopoietin or IL-17A. Chitin enhanced IL-33-dependent IL-1ß production by dendritic cells (DCs). Furthermore, chitin- and IL-33-stimulated DC-derived IL-1ß promoted OVA-specific Th2 cell activation, resulting in aggravation of OVA-induced airway inflammation. These findings indicate the adjuvant activity of chitin via a new mechanism and provide important clues for development of therapeutics for allergic disorders caused by HDM, fungi and crustaceans.

Establishment of an Endogenous Clostridium difficile Rat Infection Model and Evaluation of the Effects of Clostridium butyricum MIYAIRI 588 Probiotic Strain.

Clostridium difficile is well known as an agent responsible for pseudomembranous colitis and antibiotic-associated diarrhea. The hamster model utilizing an oral route for infection of C. difficile has been considered to be the standard model for analysis of C. difficile infection (CDI) but this model exhibits differences to human CDI, most notably as most hamsters die without exhibiting diarrhea. Therefore, we attempted to develop a new non-lethal and diarrheal rat CDI model caused by endogenous C. difficile using metronidazole (MNZ) and egg white. In addition, the effects of probiotic strain Clostridium butyricum MIYAIRI 588 (CBM) on CDI were examined using this model. Syrian Golden hamsters received clindamycin phosphate orally at 30 mg/kg on 5 days before challenge with either C. difficile VPI10463 (hypertoxigenic strain) or KY34 (low toxigenic clinical isolate). Mortality and the presence of diarrhea were observed twice a day for the duration of the experiment. Wistar rats received 10% egg white dissolved in drinking water for 1 week ad libitum following intramuscular administration of 200 mg/kg MNZ twice a day for 3 days. Diarrhea score was determined for each day and fecal water content, biotin concentration, and cytotoxin titer in feces were examined. More than 70% of hamsters orally infected with C. difficile died without exhibiting diarrhea regardless of toxigenicity of strain. The rats receiving egg white after MNZ administration developed diarrhea due to overgrowth of endogenous C. difficile. This CDI model is non-lethal and diarrheal, and some rats in this model were spontaneously cured. The incidence of diarrhea was significantly decreased in C. butyricum treated rats. These results indicate that the CDI model using egg white and MNZ has potentially better similarity to human CDI, and implies that treatment with C. butyricum may reduce the risk of CDI.

Role of IL-17A and IL-10 in the antigen induced inflammation model by Mycoplasma pneumoniae.

BACKGROUND: Mycoplasma pneumoniae is one of the causative organisms of community-acquired pneumonia which is found commonly in younger patients. Extrapulmonary complications similar to autoimmune disease are caused by M. pneumoniae following the initial infection. The mechanism and pathology of onset is not clear, but it is considered that excessive host immunoreactions play a part in the onset of mycoplasmal pneumonia and its extrapulmonary complications. In this study, we investigated the participation of the immune response, excluding the participation of Th1 and Th2 which has previously been investigated. RESULTS: In this study, the host immune response of an antigen induced inflammation model using SPF mice repeatedly sensitized with M. pneumoniae antigens was analyzed. The specificity of M. pneumoniae antigens in the Th17 response of murine lymphocytes in vitro was also examined. Frequent and concentrated sensitization induced exacerbation of lung inflammation immunologically and pathologically, and evoked intrapulmonary IL-17A and IL-10 production. M. pneumoniae antigen stimulation induced proliferation of mouse lymphocytes and caused production of IL-17A and IL-10. In addition, it was shown that IL-17A and IL-10 production was increased in the presence of IL-6 and TGF-ß1. CONCLUSIONS: It was shown that M. pneumoniae antigens induced potent immunoreaction and enhanced the Th17 cell response both in vivo and in vitro, and that both Treg and IL-10 are involved in the suppression of IL-17A production. This raises the possibility that breakdown of the immune balance may be part of the process leading to subsequent development of extrapulmonary mycoplasmal pneumonia.

Molecular and microbiological characterization of Clostridium difficile isolates from single, relapse, and reinfection cases.

In this study, we investigated the correlation between the microbiological characteristics of Clostridium difficile clinical isolates and the recurrence of C. difficile-associated disease (CDAD). Twenty C. difficile isolates recovered from 20 single infection cases and 53 isolates from 20 recurrent cases were analyzed by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping, and the cytotoxicity, antimicrobial susceptibility, and sporulation/germination rates of the isolates were examined. Recurrent cases were divided into relapse or reinfection cases by the results of C. difficile DNA typing. Among the 20 recurrent cases, 16 cases (80%) were identified to be relapse cases caused by the initial strain and the remaining 4 cases (20%) were identified to be reinfection cases caused by different strains. All 73 isolates were susceptible to both vancomycin and metronidazole, but resistance against clindamycin, ceftriaxone, erythromycin, and ciprofloxacin was found in 87.7%, 93.2%, 87.7%, and 100% of the isolates, respectively. No correlations between DNA typing group, cytotoxicity, and sporulation rate of isolates and infection status, i.e., single, relapse, or reinfection, were observed. However, the isolates recovered from relapse cases showed a significantly higher germination rate when incubated in medium lacking the germination stimulant sodium taurocholate. These results indicate that the germination ability of C. difficile may be a potential risk factor for the recurrence of CDAD.

Mycoplasma pneumoniae induces interleukin-8 production via the epidermal growth factor receptor pathway.

In mycoplasmal pneumonia, the bronchi are histopathologically filled with polymorphonuclear leukocytes. The EGFR pathway is involved in IL-8 production. We investigated the contribution of the EGFR pathway to IL-8 production by bronchial epithelial cells (A549) stimulated with Mp-Ag. The IL-8 production by A549 cells stimulated with Mp-Ag was decreased by the addition of an EGFR kinase inhibitor or transfection with small interfering RNA against EGFR. The levels of epiregulin mRNA in A549 cells were increased by stimulation with Mp-Ag. In conclusion, the EFGR pathway participates in IL-8 production by bronchial epithelial cells stimulated with Mp-Ag.

Cimetidine enhances antigen-specific IgE and Th2 cytokine production.

BACKGROUND: Treatment with anti-ulcer drugs has been shown to enhance IgE production against food antigens. However, little is known about the immunological effects of cimetidine, a histamine receptor type 2 (H2R) antagonist that is widely used as an anti-ulcer drug, in allergy. Therefore, the present study investigated the role of cimetidine in Th2 immune responses in mice. METHODS: BALB/c mice were immunized intraperitoneally with ovalbumin (OVA) with and without cimetidine. The levels of cytokines in supernatants of spleen cells cultured in the presence of OVA for 4 days and the levels of total and OVA-specific IgG(1), IgG(2a) and/or IgE in sera from these mice were determined by ELISA. RESULTS: Administration of cimetidine to OVA-sensitized BALB/c mice promoted Th2 cytokine secretion by OVA-stimulated spleen cells in vitro and increased serum levels of OVA-specific IgE, IgG(1) and IgG(2a). CONCLUSIONS: These results indicate that cimetidine can enhance Th2 responses, suggesting that cimetidine may contribute to IgE production in allergies.

Synthesis and hybridization properties of 2'-O-methylated oligoribonucleotides incorporating 2'-O-naphthyluridines.

2'-O-(1-Naphthyl)uridine and 2'-O-(2-naphthyl)uridine were synthesized by a microwave-mediated reaction of 2,2'-anhydrouridine with naphthols. Using the 3'-phosphoramidite building blocks, these 2'-O-aryluridine derivatives were incorporated into 2'-O-methylated oligoribonucleotides. Incorporation of five 2'-O-(2-naphthyl)uridines into a 2'-O-methylated RNA sense strand significantly increased the thermostability of the duplex with a 2'-O-methylated RNA antisense strand. Circular dichroism spectroscopy and molecular dynamic simulation of the duplexes formed between the modified RNAs and 2'-O-methyl RNAs suggested that there are π-π interactions between two neighboring naphthyl groups in a sequence of the five consecutively modified nucleosides.

Identification of a mechanism for lung inflammation caused by Mycoplasma pneumoniae using a novel mouse model.

Human Mycoplasma pneumoniae (MP) pneumonia is characterized by alveolar infiltration with neutrophils and lymphocytes and lymphocyte/plasma cell infiltrates in the peri-bronchovascular area (PBVA). No mouse model has been able to mimic the pathological features seen in human MP pneumonia, such as plasma cell-rich lymphocytic infiltration in PBVA. To figure out the mechanism for inflammation by MP infection using a novel mouse model that mimics human MP pneumonia, mice were pre-immunized intraperitoneally with Th2 stimulating adjuvant, alum, alone or MP extracts with an alum, followed by intratracheal challenge with MP extracts. The toll-like receptor-2, which is the major receptor for mycoplasma cell wall lipoproteins, was strongly up-regulated in alveolar macrophages in a latter group after the pre-immunization but prior to the intratracheal challenge. Those findings demonstrated that acceleration of innate immunity by antecedent antigenic stimulation can be an important positive-feedback mechanism in lung inflammation during MP pneumonia.

Antimicrobial and immunomodulatory effect of clarithromycin on macrolide-resistant Mycoplasma pneumoniae.

Macrolide antibiotics are frequently administered to treat mycoplasmal pneumonia. However, macrolide-resistant Mycoplasma pneumoniae has recently been isolated from clinical specimens in Japan. Clarithromycin (CAM) is a 14-membered-ring macrolide that has host immunomodulatory activity. Here, we established a gnotobiotic mouse model that was monoassociated with macrolide-resistant M. pneumoniae, and pathologically and microbiologically analysed the effects of antibiotics against mycoplasmal pneumonia. We also examined the immunomodulatory activities of macrolide antibiotics in human lung carcinoma A549 cells in vitro and in a specific-pathogen-free (SPF) mouse model of pneumonia induced by M. pneumoniae antigen in vivo. CAM anti-mycoplasma antibiotics decreased the number of macrolide-sensitive and -resistant M. pneumoniae in the lungs of gnotobiotic mice. Thus, in SPF mice, CAM modulated pulmonary inflammation induced by M. pneumoniae antigens.

Microwave-assisted synthesis of 2'-O-aryluridine derivatives.

A new method for the synthesis of 2'-O-aryluridines was developed via the microwave-mediated reaction of 2,2'-anhydrouridine with aromatic alcohols. Aminophenol and aminonaphthol derivatives underwent selective 2'-O-arylation with 2,2'-anhydrouridine to produce 2'-O-(aminoaryl)uridine derivatives. These reactions proved to proceed without the need for any bases or solvents, but better results were obtained by use of N,N-dimethylacetamide (DMA) as the solvent in some cases.

[Clostridium difficile TOX A/B II TEST evaluation].

We compared the performance of two commercial toxin detection kits, C. difficile toxin A/B (C. difficile TOX A/B II test; TOX A/B II) and C. difficile toxin A (Uniquick), for (i) detection using highly purified toxin A solution; (ii) cross-reactivity using culture supernatants of toxin A-positive and B-positive C. difficile, toxin A-negative and B-positive C. difficile, and toxin A-negative and B-negative C. difficile strains and other bacteria; and (iii) sensitivity and specificity using clinical specimens. Results indicated that TOX A/B II detected toxin A at concentrations of 0.35 ng/mL and Uniquick at concentrations of 0.7 ng/mL. Uniquick performance was specific for detecting toxin A alone, while TOX A/B II detected toxin A/B specifically. Kit performance was then evaluated using 99 fecal specimens--43 specimens from patients with toxin B-positive C. difficile and 56 from those without. Sensitivity of TOX A/B II vs Uniquick was 95.3% vs 76.7%, specificity 98.2% vs 98.2%, positive predictive 97.6% vs 97.1%, and negative predictive value 96.5% vs 84.6%. Findings thus indicate that TOX A/B II is a more suitable diagnostic aid for CDAD than Uniquick because it correlates well with toxin B-positive C. difficile culture results. Stool culture for C. difficile is also required, however.

Synthesis and properties of nucleoside derivatives acylated by chemically stable 2-(trimethylsilyl)benzoyl group.

We report the synthesis and properties of nucleoside derivatives acylated by 2-(trimethylsilyl)benzoyl (TMSBz) that proved to be extremely stable under basic conditions when introduced into the 5'-hydroxyl group of thymidine, the 4-amino group of deoxycytidine and the 2'-hydroxyl group of uridine. In particular, 2'-O-TMSBz-uridine could be isolated and was more stable in pyridine, while it isomerized in CH(2)Cl(2) in the presence of Et(3)N to yield a mixture of the 2'-O- and 3'-O-acylated species.

Immunological analysis and pathological examination of gnotobiotic mice monoassociated with Mycoplasma pneumoniae.

Although mycoplasmal pneumonia has been generally considered to be a disease with good prognosis, a pathogenic host immune response has been associated with its occurrence. In the present study, the pathogenic significance of the immune response was examined using germ-free mice either infected intranasally with Mycoplasma pneumoniae or inoculated with M. pneumoniae antigens (soluble antigen and partially purified antigen). In gnotobiotic mice monoassociated with M. pneumoniae, 10(4) c.f.u. M. pneumoniae per lung were isolated 2-28 days after infection. Inflammatory changes with infiltration of lymphocytes were histopathologically detected in the perivascular area at 2 and 7 days after infection. In the mice intranasally inoculated with soluble antigen or partially purified antigens (F6 and F10 antigens), infiltration of neutrophils and lymphocytes was histopathologically detected at 2 days after inoculation. Severe pneumonia with tissue destruction was observed in the mice inoculated with F6 antigen. A gamma interferon (IFN-gamma) dominant response in endogenous cytokine expression was observed in all the treated mice. These results indicate that inflammatory changes in the lung tissue were prolonged in gnotobiotic mice monoassociated with M. pneumoniae compared with mice inoculated with M. pneumoniae antigen. In addition, it was shown that IFN-gamma plays an important role in the pathogenesis of pneumonia in mice either infected with M. pneumoniae or inoculated with its antigen. In particular, the F6 antigen has been considered to be an important virulence factor in terms of induction of tissue injury causing infiltration of lymphocytes and neutrophils in the lung, suggesting a close interaction between the immune response and the occurrence of M. pneumoniae pneumonia.

Synthesis and properties of oligonucleotides having a chemically stable 2-(trimethylsilyl)benzoyl group.

Acyl groups such as benzoyl and phenoxyacetyl have been used as a fundamental tool for protection of reactive hydroxyl and amino groups in chemical synthesis of DNA and RNA oligomers. It is well known that such protecting groups can be easily cleaved under basic conditions. Here we report 2-(trimethylsilyl)benzoyl (TMSBz), i.e., a chemically stabilized benzoyl group that is substituted with a trimethylsilyl group at its 2-position. To the best of our knowledge, the TMSBz group is the most resistant to basic conditions as an acyl group in nucleic acid chemistry.

Efficient synthesis of functionalized oligodeoxyribonucleotides with base-labile groups using a new silyl linker.

In this study, we developed new 3'-terminal deoxyribonucleoside-loading reagents 1 with a new silyl-type linker. These reagents could increase the efficiency of introduction of 3'-terminal deoxyribonucleoside components into polymer supports to a level of 17-29micromol/g. The efficiency was higher than that of previous T-loading reagents because reagents 1 contain a 4-aminobutyryl residue as a spacer. Moreover, we could synthesize not only unmodified DNA oligomers but also a base-labile modified DNA oligomer using resins 9a-d in the activated phosphite method without base protection.

Chlamydia pneumoniae growth inhibition in cells by the steroid receptor antagonist RU486 (mifepristone).

Since steroids are powerful anti-inflammatory agents and increase susceptibility to a variety of infections, including Chlamydia (Chlamydophila) pneumoniae respiratory tract infections, the effect of the steroid receptor antagonist RU486 (mifepristone) on C. pneumoniae growth in epithelial HEp-2 cells was examined. Treatment of HEp-2 cells with RU486 significantly inhibited the growth of C. pneumoniae in a dose-dependent manner. Electron microscopic studies also revealed that the treatment of infected cells with RU486 resulted in a marked destruction of infecting organisms. The addition of the host cell protein synthesis inhibitor cycloheximide to the infected cells did not alter the inhibition of C. pneumoniae growth by RU486. Pretreatment of C. pneumoniae organisms with RU486 before addition to culture also did not result in any modulation of bacterial growth in the cells. However, the binding of RU486 to C. pneumoniae organisms in cells at 24 h after infection was demonstrated by immune electron microscopy with anti-RU486 antibody. Incubation of cells with anti-RU486 antibody completely diminished the inhibition of C. pneumoniae growth by RU486. These results indicate that RU486 may directly bind to the bacteria within cells and cause the destruction of C. pneumoniae. This novel mode of regulation of C. pneumoniae growth in cells by RU486 might provide a new approach to understanding complicated aspects of C. pneumoniae infection.

'Protected DNA Probes' capable of strong hybridization without removal of base protecting groups.

We propose a new strategy called the 'Protected DNA Probes (PDP) method' in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac(4)C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac(6)az(8)c(7)A). It was found that PDP containing ac(4)C and ac(6)az(8)c(7)A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.

Synthesis and properties of DNA oligomers containing 2'-deoxynucleoside N-oxide derivatives.

Cytosine and adenine N-oxide derivatives have long been known as products resulting from the oxidative damage of DNA by peroxides such as hydrogen peroxide. Although the synthesis and properties of 2'-deoxynucleoside N-oxide derivatives have been well described, little has been reported about the chemical and biochemical behavior of initially formed DNA oligomers containing these N-oxide bases. In this study, we established a convenient method for the solid-phase synthesis of oligodeoxynucleotides incorporating 2'-deoxycytidine N-oxide (dC O) or 2'-deoxyadenosine N-oxide (dA O) by using the postsynthetic oxidation of N-protected DNA oligomers except for the target dC or dA site with m-CPBA in MeOH in a highly selective manner. In this strategy, the benzoyl, phthaloyl, and (4-isopropylphenoxy)acetyl groups proved to serve as base protecting groups to avoid oxidation of adenine, cytosine, and guanine, respectively, at the unmodified sites.

Synthesis and properties of oligodeoxynucleotides containing 5-carboxy-2'-deoxycytidines.

5-Carboxy-2'-deoxycytidine (dC(COO-)) was synthesized as an anion-carrier to seek a new possibility of modified oligodeoxynucleotides capable of stabilization of duplexes and triplexes. The base pairing properties of this compound were evaluated by use of ab initio calculations. These calculations suggest that the Hoogsteen-type base pair of dC(COO-)-G is less stable than that of the canonical C+-G pair and the Watson-Crick-type base pair of dC(COO-)-G is slightly more stable than the natural G-C base pair. The modified cytosine base showed a basicity similar to that of cytosine (pKa 4.2). It turned out that oligodeoxynucleotides 13mer and 14mer incorporating dC(COO-) could form duplexes with the complementary DNA oligomer, which were more stable than the unmodified duplex. In contrast, it formed a relatively unstable triplex with the target ds DNA.