Development of the Complement C5 Assay by LC-MS/MS in Monkey Serum and Comparison with Enzyme-Linked Immunosorbent Assay.

Complement C5 (C5) is the key component for the complement activation pathway, which is important for innate immunity, and inhibition of C5 is considered to be effective in antibody-mediated rejection in organ transplantation. Thus determination of C5 levels in systemic circulation is a simple way to understand efficacy of drugs that aim to inhibit C5 production. We have developed a simple liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay for C5 in cynomolgus monkey serum. C5 in monkey serum was subjected to tryptic digestion, and two signature peptides, DSSVPNTGTAR and LQGTLPVEAR, were assayed by LC-MS/MS with electrospray ionization in the positive ion mode. Assay reproducibility in serum samples was evaluated, and the assay was applied to the C5 assay in monkey serum after administration of C5 siRNA encapsulated in lipid nanoparticles to monkeys. The time profiles of C5 after administration of C5 siRNA were comparable between the two signature peptides by LC-MS/MS and were also similar to those by an enzyme-linked immunosorbent assay using an assay kit. These findings suggest that the established LC-MS/MS assay of C5 is reliable to determine C5 levels in monkey serum.

Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells.

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

Identification of potent siRNA targeting complement C5 and its robust activity in pre-clinical models of myasthenia gravis and collagen-induced arthritis.

Complement component 5 (C5), an important molecule in the complement cascade, blockade by antibodies shows clinical efficacy in treating complement-mediated disorders. However, insufficient blockading induced by single-nucleotide polymorphisms in the C5 protein or frequent development of "breakthrough" intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria treated with eculizumab have been reported. Herein, we developed a lipid nanoparticle (LNP)-formulated siRNA targeting C5 that was efficiently delivered to the liver and silenced C5 expression. We identified a potent C5-siRNA with an in vitro IC50 of 420 pM and in vivo ED50 of 0.017 mg/kg following a single administration. Single or repeated administrations of the LNP-formulated C5-siRNA allowed robust and durable suppression of liver C5 expression in mice. Complement C5 silencing ameliorated C5b-dependent anti-acetylcholine receptor antibody-induced myasthenia gravis and C5a-dependent collagen-induced arthritis symptoms. Similarly, in nonhuman primates, a single administration of C5-siRNA/LNP-induced dose-dependent plasma C5 suppression and concomitantly inhibited serum complement activity; complement activity recovered to the pre-treatment levels at 65 days post administration, thus indicating that the complement activity can be controlled for a specific period. Our findings provide the foundation for further developing C5-siRNA delivered via LNPs as a potential therapeutic for complement-mediated diseases.

First description of Lachnoanaerobaculum orale as a possible cause of human bacteremia.

Lachnoanaerobaculum spp. is an obligate anaerobic, gram-positive, spore-forming, rod-shaped bacillus. Here, we report the first known case of bacteremia due to L. orale, which was detected in a patient with acute lymphoblastic leukemia. A 69-year-old man developed neutropenic fever with severe stomatitis during chemotherapy for leukemia. The bacteria strain isolated from blood culture was successfully identified as L. orale via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Susceptibility testing revealed that the strains showed low minimum inhibitory concentrations (MICs) of beta-lactams, clindamycin, and metronidazole, but higher MICs of fluoroquinolones. The present case study indicates that Lachnoanaerobaculum can be a cause of human infection, including bloodstream infection.

Clinical Surveillance of Candidemia at Our Hospital.

Treatment of Candidemia has become increasingly complicated as more and more non-albicans Candida species are being isolated in recent years.We launched an investigation of the species, the MIC value, and the state of administration of antifungal drugs for all the cases with Candida spp. confirmed by blood cultures for the 7-year period from 2012 to 2018 at our hospital. In total, 192 cases were found and 206 strains of Candida species were isolated. Overall, 49.5% of the 206 isolated strains were Candida albicans (102 strains), followed by Candida glabrata (40 strains, 19.4%), and Candida parapsilosis (38 strains, 18.4%). The most frequently used antifungal drug for the initial dose was MCFG (120 cases, 59.2%), while the most frequently switched antifungal agent was L-AMB. Cases with an inappropriate end-of-treatment time represented 58.7% of all the cases.We investigated the Candidemia situation at our hospital for a period of seven years. We believe that it is important for medical institutions to gather detailed data on candidemia at their own hospitals. Likewise, the hospital's Infection Control Team/Antimicrobial Stewardship Team should inform the physicians-in-charge about the appropriate diagnosis and treatment based on the data obtained.

Role of Anti-Fractalkine Antibody in Suppression of Joint Destruction by Inhibiting Migration of Osteoclast Precursors to the Synovium in Experimental Arthritis.

OBJECTIVE: To elucidate the role of the fractalkine (FKN)/CX3 CR1 pathway in joint destruction in rheumatoid arthritis. METHODS: We examined the effect of treatment with anti-mouse FKN (anti-mFKN) monoclonal antibody (mAb) on joint destruction and the migration of osteoclast precursors (OCPs) into the joint, using the collagen-induced arthritis (CIA) model. DBA/1 mice were immunized with bovine type II collagen to induce arthritis, and then treated with anti-mFKN mAb. Disease severity was monitored by arthritis score, and joint destruction was evaluated by soft x-ray and histologic analyses. Plasma levels of joint destruction markers were assessed by enzyme-linked immunosorbent assay. FKN expression on endothelial cells was detected by immunohistochemistry. Bone marrow-derived OCPs were labeled with fluorescein and transferred to mice with CIA, and the migration of the OCPs to the joints was then analyzed. RESULTS: Both prophylactic and therapeutic treatment with anti-mFKN mAb significantly decreased the arthritis and soft x-ray scores. Plasma levels of cartilage oligomeric matrix protein and matrix metalloproteinase 3 decreased after treatment with anti-mFKN mAb. Histologic analysis revealed that anti-mFKN mAb inhibited synovitis, pannus formation, and cartilage destruction, as well as suppressed bone damage, with a marked reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. Anti-mFKN mAb strongly inhibited the migration of bone marrow-derived OCPs into the affected synovium. CONCLUSION: Anti-mFKN mAb notably ameliorates arthritis and joint destruction in the CIA model, as well as inhibits migration of OCPs into the synovium. These results suggest that inhibition of the FKN/CX3 CR1 pathway could be a novel strategy for treatment of both synovitis and joint destruction in rheumatoid arthritis.

Efficient management and maintenance of ultrasonic nebulizers to prevent microbial contamination.

AIM: To seek the cause of Burkholderia cepacia complex (Bcc) infection outbreak and evaluate the efficacy of new methods for nebulizer maintenance. METHODS: We investigated the annual number of Bcc isolates recovered from clinical samples in our hospital between 1999 and 2013. Swab samples were randomly collected for bacterial culture before patient use from 10 each of the two machine types in August 2001; these included 20 samples from each of the following: Drain tubes, operating water chambers, oscillators, and nebulizing chambers. In addition, 10 samples each of nebulizer solutions before and after use were cultured. For environmental investigation, 10 samples were collected from sinks in the nurse stations of the wards where patients positive for Bcc were hospitalized. Numbers of Bcc isolates were compared before and after introduction of new methods for nebulizer maintenance in October 2001. In addition, randomly amplified polymorphic DNA (RAPD) assay was applied to find the genetic divergence of the Bcc isolates obtained from clinical samples and nebulizers. RESULTS: From January 1999 to December 2013, a total of 487 Bcc isolates were obtained from clinical specimens from 181 patients. Notably, 322 (66.1%) Bcc isolates were obtained from clinical specimens from 1999 to 2001, including 244 (115 patients) from sputum and 34 (11 patients) from blood. During this period, 14 isolates were obtained from nebulizer components. Among these, six were derived from nebulizer drain tubes, five from operating water chambers, and one from the oscillator before patient use, and two from nebulizer solutions after patient use. When Bcc was isolated from the nebulizer solution after patient use, Bcc was simultaneously detected in other parts of the nebulizer. Bcc was not isolated from any nebulizer solution before use. RAPD assays revealed similar DNA profiles in isolates obtained from patients and nebulizers. Investigation revealed damaged diaphragms in many nebulizers. The new maintenance methods for nebulizers, including restriction of the usage period, thorough disinfection, and routine check for diaphragm breakage, remarkably reduced Bcc isolation (165 isolates from patients in 12 years and 0 isolate from nebulizers in periodical sampling). In particular, Bcc has been isolated from blood from only one patient since the new methods were introduced. CONCLUSION: Appropriate maintenance of ultrasonic nebulizers is crucial for preventing Bcc contamination of nebulizers and subsequent respiratory tract and blood infections.

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates.

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their components. Therefore, the arrangements in the clusters have been precisely characterized, especially for metal complexes. Contrary to this, characterizations of molecular arrangements in supramolecular clusters composed of organic molecules are limited to a few cases. This is because construction of the supramolecular clusters, especially obtaining a series of the supramolecular clusters, is difficult due to low stability of non-covalent bonds compare to covalent bonds. From this viewpoint, utilization of organic salts is one of the most useful strategies. A series of the supramolecules could be constructed by combinations of a specific organic molecule with various counter ions. Especially, primary ammonium carboxylates are suitable as typical examples of supramolecules because various kinds of carboxylic acids and primary amines are commercially available, and it is easy to change their combinations. Previously, it was demonstrated that primary ammonium triphenylacetates using various kinds of primary amines specifically construct supramolecular clusters, which are composed of four ammoniums and four triphenylacetates assembled by charge-assisted hydrogen bonds, in crystals obtained from non-polar solvents. This study demonstrates an application of the specific construction of the supramolecular clusters as a strategy to conduct systematical symmetric study for clarification of correlations between molecular arrangements in supramolecules and kinds and numbers of their components. In the same way with binary salts composed of triphenylacetates and one kind of primary ammoniums, ternary organic salts composed of triphenylacetates and two kinds of ammoniums construct the supramolecular clusters, affording a series of the supramolecular clusters with various kinds and numbers of the components.

Characterization of supramolecular hidden chirality of hydrogen-bonded networks by advanced graph set analysis.

Supramolecular hidden chirality of hydrogen-bonded (HB) networks of primary ammonium carboxylates was exposed by advanced graph set analysis from a symmetric viewpoint in topology. The ring-type HB (R-HB) networks are topologically regarded as faces, and therefore exhibit prochirality and positional isomerism due to substituents attached on the faces. To describe the symmetric properties of the faces, additional symbols, Re (right-handed or clockwise), Si (left-handed or anticlockwise), and m (mirror), were proposed. According to the symbols, various kinds of faces were classified based on the symmetry. This symmetry consideration of the faces enables us to precisely evaluate supramolecular chirality, especially its handedness, of 0D-cubic, 1D-ladder and 2D-sheet HB networks that are composed of the faces. The 1D-ladder and 2D-sheet HB networks generate chirality by accumulating the chiral faces in 1D and 2D manners, respectively, whereas 0D-cubic HB networks generate chirality based on combinations of eight kinds of faces, similar to the chirality of dice.

Antimicrobial susceptibility and mechanisms of high-level macrolide resistance in clinical isolates of Moraxella nonliquefaciens.

We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan. Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin, ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing ß-lactamase inhibitors showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47 isolates showed high-level MICs to macrolides (MIC ≥128 mg l(-1)) and shared the A2058T mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated strains was significantly higher than that of WT strains (P<0.0001). Thirty-two isolates with high-level macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1 and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA gene. This study represents the first characterization of M. nonliquefaciens.

Dermokine as a novel biomarker for early-stage colorectal cancer.

BACKGROUND: Colorectal cancer is a common disease that is usually detected at an advanced stage, because early-stage cancer is mostly asymptomatic and appropriate serologic biomarkers have not been established. We have previously identified dermokine (DK) as a peptide secreted by keratinocytes and we found that DK-ß/γ was expressed in colorectal tumors. Therefore, we focused on DK-ß/γ as a new candidate diagnostic serum marker for early colorectal cancer. METHODS: DK-ß/γ expression in human colorectal cancer cell lines and tissues was assessed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. We established an experimental enzyme-linked immunosorbent assay (ELISA) to detect DK-ß/γ in the serum of colorectal cancer patients, and we compared the sensitivities of common diagnostic markers, carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, and serum p53 antibody (S-p53). RESULTS: Immunohistochemical staining of colon tumor tissue with anti-DK monoclonal antibody (mAb) revealed that DK-ß/γ was more commonly expressed in the early stages of colorectal cancer (Tis-T1; i.e., cancer in situ, intraepithelial or invasion of lamina propria [Tis]; tumor invades the submucosa [T1]) than in late-stage tumors (T2-T4; i.e., tumor invades the muscularis propria [T2]; tumor invades through the muscularis propria into the subserosa, or into the nonperitonealized pericolic or perirectal tissues [T3]; tumor directly invades other organs or structures and/or perforates visceral peritoneum [T4]). Serum DK-ß/γ levels were determined in 130 patients with colorectal cancer and 25 healthy volunteers. Serum DK-ß/γ was detected in 33.3% of patients with early colorectal cancer (Tis-T1), which was higher than the rates for S-p53 (24.2%), CEA (9.1%), and CA19-9 (0%). The serum DK-ß/γ test was complementary to the other marker tests. Therefore, when the combined four-marker test (DK/CEA/CA19-9/S-p53) was carried out, the diagnostic sensitivity for Tis and T1 tumors reached 60.6%. CONCLUSIONS: Serum DK-ß/γ is the most promising of the existing tumor biomarkers for the diagnosis of early-stage colorectal cancer.

Mouse homologue of skin-specific retroviral-like aspartic protease involved in wrinkle formation.

Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization.