Identification of U11snRNA as an endogenous agonist of TLR7-mediated immune pathogenesis.

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.

Actinomyces denticolens as a causative agent of actinomycosis in animals.

The name "Actinomyces suis" was applied to each actinomycete isolate from swine actinomycosis by Grässer in 1962 and Franke in 1973. Nevertheless, this specific species was not included in the "Approved List of Bacterial Name" due to absence of the type cultures. Therefore, "Actinomyces suis" based on the description of Franke 1973 has been considered as "species incertae sedis". We isolated a number of Actinomyces strains from swine. The representative strains of them was designated as Chiba 101 that was closely similar to the description in "Actinomyces suis" reported by Franke in 1973. Interestingly, it was found that the biological characteristics of these strains were also very similar to those of Actinomyces denticolens. Furthermore, the average nucleotide identity (ANI) value between strain Chiba 101 and the type-strain of Actinomyces denticolens (=DSM 20671T) was found to be 99.95%. Sequences of the housekeeping genes and 16S rRNA gene showed 100% homology. These results strongly suggested that "Actinomyces suis" Franke 1973 is the same species as Actinomyces denticolens. Since actinomycosis caused by Actinomyces denticolens have been demonstrated in horses recently, it is necessary to recognize that Actinomyces denticolens is the pathogenic actinomycetes in broader range of animals.

Whole-Genome Sequences of Two Closely Related Bacteria, Actinomyces sp. Strain Chiba101 and Actinomyces denticolens DSM 20671T.

Actinomyces sp. strain Chiba101, isolated from an arthritic leg joint of a pig raised in Japan, is a bacterium closely related to Actinomyces denticolens Here, we deciphered the complete genome sequence of Actinomyces sp. Chiba101 and the high-quality draft genome sequence of A. denticolens DSM 20671T.

Tonsilliphilus suis gen. nov., sp. nov., causing tonsil infections in pigs.

A micro-organism resembling members of the genus Dermatophilus, strain W254(T), which was isolated from the submandibular lymph node of a pig, and an additional 16 strains isolated from swine tonsils, were studied to establish their taxonomic status. Although all 17 strains were isolated anaerobically under an atmosphere of 100 % CO2, all of them were aerotolerant anaerobes. The micro-organisms showed at least five cellular morphologies: (i) a radially protrusive thallus, which proliferated into tuber-like cells; (ii) segmentation in both tubers and thallus followed by multilocule formation, (iii) development of coccoid forms in the locules; (iv) a change from the coccoid forms to zoospores; (v) resting cells, which were able to develop into protrusive thalli again. The micro-organisms were positive for nitrate reduction, but negative for catalase, indole production, hydrolysis of urea and gelatin liquefaction. Milk was not decomposed and none of the strains was haemolytic. A total of 16 compounds, including glucose, were utilized as sole carbon sources and seven compounds, including l-arabinose, were not utilized. Three out of the 17 strains were subjected to further studies. The micro-organisms had meso-diaminopimelic acid in their peptidoglycan and galactose, glucose, madurose and a trace of mannose in their whole-cell sugar patterns. The major phospholipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol.Cellular fatty acids were C15 : 0 (35.7-23.1 %), C16 : 0 (5.9-2.4 %) C17 : 0 (62.9-39.5 %), C17 : 1 (24.4-0 %) and C18 : 0 (3-1.6 %). The predominant menaquinone was MK-8 (H4). The G+C content of the DNA was 69.6-71.8 mol%. Analysis of the 16S rRNA gene sequences showed that the strains clustered with the type strains of members of the family Dermatophilaceae. Based on the polyphasic taxonomic characterization carried out, all 17 strains are considered to belong to a novel species in a new genus, for which the name Tonsilliphilus suis gen. nov., sp. nov. is proposed. The type strain of the type species is W254(T) ( = ATCC 35846(T) = CCM 3774(T) = DSM 21880(T) = JCM 15727(T)).

[Treatment of asbestos-containing waste products to prevent harm to the lungs ].

The amount of industrial wastes with asbestos such as dismantled construction materials has increased. We have reviewed the effect of asbestos-containing products subjected to harmless treatment on the lungs. Usually, the harmless treatment of asbestos is confirmed by the disappearance of fibrous materials and crystal structures by electron microscopy and X-ray diffraction. However, it is very important to perform animal studies and in vitro studies in order to examine the effect of the treated asbestos-containing products on the lungs. From previous treatments of asbestos using acids or high temperature, almost treated materials tended to show decreased toxicity in vitro and in vivo studies. There are some reports of the adverse effects of the treatment. If new harmless treatments of asbestos are developed, it is necessary to perform animal studies and in vitro studies of asbestos-containing products using new harmless treatments.

Safety evaluation of a heavy oil-degrading bacterium, Rhodococcus erythropolis C2.

The safety of an oil-degrading bacterium, C2 strain, was evaluated for utilization in an open system for bioremediation of oil-contaminated environments. The C2 strain was identified as Rhodococcus erythropolis by performing an alignment analysis of the whole 16S rRNA sequence. R. erythropolis was classified as a nonpathogenic (category 1) bacterium. Biological and biochemical properties of the C2 strain also confirmed its nonpathogenicity. The pathogenicity and basic ecotoxicity were studied in laboratory animals and in a variety of test species, respectively. General and inhalation toxicities were not detected; additionally, there was no evidence of skin irritation, mutagenic potential, eye irritation, skin sensitization, ecotoxicity or notable pathogenicity. The comparison of these results with human exposure levels and previously published data indicates that the C2 strain appears to be safe for utilization in bioremediation of polluted environments, requires no special occupational health precautions during the application process, and has a low environmental impact. This study suggests that the C2 strain could be suitable for bioremediation of oil-contaminated environments.

Binding of a highly potent protease-activated receptor-2 (PAR2) activating peptide, [3H]2-furoyl-LIGRL-NH2, to human PAR2.

1 To determine the binding characteristics of a highly potent agonist for protease-activated receptor-2 (PAR2), 2-furoyl-Leu-Ile-Gly-Arg-Leu-amide (2-furoyl-LIGRL-NH(2)), whole-cell binding assays were performed utilising a radioactive ligand, [(3)H]2-furoyl-LIGRL-NH(2). 2 Specific binding of [(3)H]2-furoyl-LIGRL-NH(2) was observed in NCTC2544 cells, dependent upon PAR2 expression, and competitively displaced by the addition of unlabeled PAR2 agonists. Scatchard analysis of specific saturation binding suggested a single binding site, with K(d) of 122+/-26.1 nM and a corresponding B(max) of 180+/-6 f mol in 3.0 x 10(5) cells. 3 The relative binding affinities of a series of modified PAR2 agonist peptides obtained from competition studies paralleled their relative EC(50) values for Ca(2+) mobilisation assays, indicating improved binding affinities by substitution with 2-furoyl at the N-terminus serine. 4 Pretreatment of cells with trypsin reduced specific binding of [(3)H]2-furoyl-LIGRL-NH(2), demonstrating direct competition between the synthetic agonist peptide and the proteolytically revealed tethered ligand for the binding site of the receptor. 5 In HCT-15 cells endogenously expressing PAR2, the binding of [(3)H]2-furoyl-LIGRL-NH(2) was displaced by addition of unlabeled ligands, Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) or 2-furoyl-LIGRL-NH(2). The relative binding affinity of 2-furoyl-LIGRL-NH(2) to SLIGKV-OH was comparable to its relative EC(50) value for Ca(2+) mobilisation assays. 6 The binding assay was successfully performed in monolayers of PAR2 expressing NCTC2544 and human umbilical vein endothelial cells (HUVEC), in 96- and 24-well plate formats, respectively. 7 These studies indicate that [(3)H]2-furoyl-LIGRL-NH(2) binds to human PAR2 at its ligand-binding site. The use of this radioligand will be valuable for characterising chemicals that interact to PAR2.

2-Furoyl-LIGRL-NH2, a potent agonist for proteinase-activated receptor-2, as a gastric mucosal cytoprotective agent in mice.

1. Proteinase-activated receptor-2 (PAR(2)), expressed in capsaicin-sensitive sensory neurons, plays a protective role in gastric mucosa. The present study evaluated gastric mucosal cytoprotective effect of 2-furoyl-LIGRL-NH(2), a novel highly potent PAR(2) agonist, in ddY mice and in wild-type and PAR(2)-knockout mice of C57BL/6 background. 2. Gastric mucosal injury was created by oral administration of HCl/ethanol solution in the mice. The native PAR(2)-activating peptide SLIGRL-NH(2), administered intraperitoneally (i.p.) at 0.3-1 micromol kg(-1) in combination with amastatin, an aminopeptidase inhibitor, but not alone, revealed gastric mucosal protection in ddY mice, which was abolished by ablation of capsaicin-sensitive sensory neurons. 3. I.p. administration of 2-furoyl-LIGRL-NH(2) at 0.1 micromol kg(-1), without combined treatment with amastatin, exhibited gastric mucosal cytoprotective activity in ddY mice, the potency being much greater than SLIGRL-NH(2) in combination with amastatin. This effect was also inhibited by capsaicin pretreatment. 4. Oral administration of 2-furoyl-LIGRL-NH(2) at 0.003-0.03 micromol kg(-1) also protected against gastric mucosal lesion in a capsaicin-reversible manner in ddY mice. 5. I.p. 2-furoyl-LIGRL-NH(2) at 0.1-0.3 micromol kg(-1) caused prompt salivation in anesthetized mice, whereas its oral administration at 0.003-1 micromol kg(-1) was incapable of eliciting salivation. 6. In wild-type, but not PAR(2)-knockout, mice of C57BL/6 background, i.p. administration of 2-furoyl-LIGRL-NH(2) caused gastric mucosal protection. 7. Thus, 2-furoyl-LIGRL-NH(2) is considered a potent and orally available gastric mucosal protective agent. Our data also substantiate a role for PAR(2) in gastric mucosal protection and the selective nature of 2-furoyl-LIGRL-NH(2).