Utility of Blood Culture in Patients with Community-Acquired Pneumonia: A Propensity Score-Matched Analysis Based on a Japanese National Health Insurance Database.

Community-acquired pneumonia (CAP) is an acute pulmonary parenchymal infection acquired outside the hospital. The utility of blood cultures in inpatients with CAP to reduce mortality and length of hospital stay is controversial. This study aimed to determine the utility of blood cultures on the first day of hospitalization for CAP inpatients and its influence on mortality, length of hospital stay, and antibiotics use. We conducted a fact-finding survey on the implementation of blood culture in inpatients with CAP in Japan. A propensity score (PS)-matched analysis based on the National Database of Health Insurance Claims and Specific Health Check-ups of Japan database was conducted. Overall, 163173 patients were included in the analysis, and PS matching extracted 68104 pairs. The results of the comparison between the PS-matched blood culture group and PS-matched control group were as follows: mortality and length of hospital stay were significantly lower in the PS-matched blood culture group than in the control group. The adjusted odds ratio (OR) (95% confidence interval (CI)) for in-hospital mortality with blood culture test was 0.73 (0.68-0.79). Moreover, for days of antibiotic usage, number of antibiotics used were significantly higher in the PS-matched blood culture group than that in the control group. Our findings indicated that performing a blood culture on the first day of hospitalization for inpatients with CAP was associated with reduced mortality. To our knowledge, this is the largest epidemiological study to assess the utility of blood culture in Japanese inpatients with CAP. This testing method shows potential for application in clinical practice.

Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues.

The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and γ-polyglutamic acid (γ-PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination.

[Influence of Storage Conditions after One-dose Packaging on Stability of Magnesium Oxide Tablets].

The intestinal motor function declines in the elderly. Many patients have constipation and ingest magnesium oxide for a prolonged period. One-dose packaging of drugs is adopted for many elderly patients to help them to avoid forgetting to take an increase in the number of oral drugs to be taken and improve convenience. Several studies on differences in stability among magnesium oxide preparations including generic drugs have been reported, but no study on the influence of the storage conditions after one-dose packaging on the properties of magnesium oxide tablets has been reported. Three types of magnesium oxide tablet were one-dose-packaged and stored for 12 weeks with and without a desiccant. The difference in hardness and weight significantly increased in all tablets stored without a desiccant. The disintegration time significantly extended in the tablets stored without a desiccant from 2 weeks after the initiation of storage and that of the B tablet markedly extended. However, all tablets passed the disintegration criteria of the disintegration test. The pH-area under the concentration-time curve significantly decreased in the tablets stored without a desiccant. It was suggested that when the one-dose-packaged magnesium oxide preparation is stored, it should be placed in a bag containing a desiccant.

Methotrexate-Coated Complexes of Plasmid DNA and Polyethylenimine for Gene Delivery.

Folate receptors are overexpressed on the surface cancer cells. We successfully constructed a new gene delivery vector of methotrexate (MTX)-coated plasmid DNA-polyethylenimine (pDNA-PEI) complexes (PEI complexes) by electrostatic binding. The stable anionic nanoparticle was optimized at MTX charge ratios of 120 or more. pDNA-PEI-MTX complexes (MTX complexes) demonstrated gene expression efficiency as high as cationic pDNA-PEI complexes in the mouse melanoma cell line, B16-F10. The MTX complexes were taken up by the cell-specific uptake mechanisms via the folate receptor. MTX-coated complexes are useful as endocytosis ligands. The MTX120 complexes exhibited no blood aggregation. The transgene efficiency of MTX120 complexes in the liver and spleen after their intravenous administration was higher than that of PEI complexes. Therefore, MTX complexes are expected as a new gene vector in the future.

Splenic Delivery System of pDNA through Complexes Electrostatically Constructed with Protamine and Chondroitin Sulfate.

We developed and optimized a novel gene delivery vector constructed electrostatically with an anionic biological component and a cationic biological component. Cationic binary complexes of plasmid DNA (pDNA) with novo-protamine sulfate as a medical product (PRT complexes) demonstrated high gene expression with minimal cytotoxicity, likely related with its total cationic charge. Subsequently, anionic compounds were added to the PRT complexes to form ternary complexes with neutral or anionic charges. Among the anionic compounds examined, chondroitin sulfate sodium (CS) as a medical product encapsulated the PRT complexes to produce stable ternary complexes (CS complexes) at charge ratios of ≥4 with pDNA. CS complexes exhibited high gene expression without cytotoxicity in mouse melanoma cell line, B16-F10 cells, in vitro. An inhibition study with endocytosis inhibitors suggested that PRT complexes were mainly taken up by caveolae-mediated endocytosis, and CS complexes were mainly taken up by clathrin-mediated endocytosis in B16-F10 cells. We found that CS complexes including pDNA encoding Oplophorus gracilirostris luciferase induced selective gene expression in the spleen after intravenous administration into ddY male mice. Thus, we successfully constructed useful gene vectors with biological components as medical products.

Gene delivery system of pDNA using the blood glycoprotein fetuin.

Fetuin is a biocompatible plasma protein and strongly enhances phagocytosis of bacteria, DNA and apoptotic cells by peripheral blood cells such as monocytes, macrophages and dendritic cells. We developed a novel gene delivery system: ternary complexes constructed with pDNA, polyethylenimine (PEI) and fetuin. Without covalent binding, fetuin was able to coat pDNA-PEI complexes, and stable anionic nanoparticles formed at a weight ratio greater than 30. Optimised pDNA-PEI-fetuin complexes significantly decreased the cytotoxicity of pDNA-PEI complexes in the melanoma cell line B16F10. Furthermore, the pDNA-PEI-fetuin complexes had higher transgene efficiency compared to that of commercial lipofectin previously reported in B16F10 cells despite an anionic surface. The pDNA-PEI-fetuin complexes did not agglutinate with erythrocytes. The pDNA-PEI-fetuin complexes had high gene expression in the spleen after intravenous administration in mice. Thus, the pDNA-PEI-fetuin complexes were a useful in vivo gene delivery system with tropism for the spleen.

DNA Microcapsule for Photo-Triggered Drug Release Systems.

In this study we constructed spherical photo-responsive microcapsules composed of three photo-switchable DNA strands. These strands first formed a three-way junction (TWJ) motif that further self-assembled to form microspheres through hybridization of the sticky-end regions of each branch. To serve as the photo-switch, multiple unmodified azobenzene (Azo) or 2,6-dimethyl-4-(methylthio)azobenzene (SDM-Azo) were introduced into the sticky-end regions via a d-threoninol linker. The DNA capsule structure deformed upon trans-to-cis isomerization of Azo or SDM-Azo induced by specific light irradiation. In addition, photo-triggered release of encapsulated small molecules from the DNA microcapsule was successfully achieved. Moreover, we demonstrated that photo-triggered release of doxorubicin caused cytotoxicity to cultured cells. This biocompatible photo-responsive microcapsule has potential application as a photo-controlled drug-release system.

Biocompatible complex coated with glycosaminoglycan for gene delivery.

The purpose of this study was to develop a ternary complex of plasmid DNA (pDNA) electrostatically assembled with dendrigraft poly-l-lysine (DGL) and biodegradable glycosaminoglycan for effective and secure gene delivery. High gene expression of pDNA/DGL complex was confirmed with slight cytotoxicity and erythrocyte agglutination. Anionic ternary complexes of 55.4-223.8 nm were formed by the addition of a glycosaminoglycan such as chondroitin sulfate A (CS-A), chondroitin sulfate B (CS-B), chondroitin sulfate C (CS-C) or hyaluronic acid (HA). Using the cell line B16-F10, most of the ternary complexes showed only weak gene expression and little cytotoxicity, although the pDNA/DGL/CS-A complexes maintained a certain level of gene expression. In particular, the pDNA/DGL/CS-A8 complexes showed significantly higher gene expression than pDNA/DGL complexes in the presence of fetal bovine serum. Gene expression from the pDNA/DGL/CS-A8 complex was inhibited by a high concentration of CS-A and endocytosis inhibitors. After intravenous administration of the pDNA/DGL/CS-A8 complex and the pDNA/DGL complex into ddY mice, high gene expression was observed in the reticuloendothelial systems, the pDNA/DGL/CS-A complex is expected to be useful for gene therapy.

Application of biodegradable dendrigraft poly-l-lysine to a small interfering RNA delivery system.

Dendrigraft poly-l-lysine (DGL), including its central core, consists entirely of lysine, hence it is completely biodegradable. We applied DGL in a small interfering RNA (siRNA) delivery system. Binary complexes with siRNA and DGL had particle sizes of 23-73 nm and ζ-potentials of 34-42 mV. The siRNA-DGL complexes showed significant silencing effects in a mouse colon carcinoma cell line expressing luciferase (Colon26/Luc cells). The siRNA-DGL complexes induced slight cytotoxicity and hematological toxicity at a high charge ratio of DGL to siRNA, probably because of their cationic charges. Therefore, we recharged the siRNA-DGL complexes with γ-polyglutamic acid (γ-PGA), a biodegradable anionic compound, which was reported to reduce the cytotoxicity of cationic complexes. The ternary complexes showed particle sizes of 35-47 nm at a charge ratio of greater than 14 to siRNA with negative charges. Strong silencing effects of the ternary complexes were observed in Colon26/Luc cells without cytotoxicity or hematological toxicity. The cellular uptake and degradation of the binary and ternary complexes were confirmed by fluorescence microscopy. The ternary complexes suppressed luciferase activity in the tumor after direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, a potentially important siRNA delivery system was constructed using biodegradable DGL.

Splenic gene delivery system using self-assembling nano-complex with phosphatidylserine analog.

The recognition of phosphatidylserine on the erythrocyte membrane mediates erythrophagocytosis by resident spleen macrophages. The application of phosphatidylserine to a gene vector may be a novel approach for splenic drug delivery. Therefore, we chose 1,2-dioleoyl-sn-glycero-3-phospho-L-serin (DOPS) as an analogue of phosphatidylserine for splenic gene delivery of plasmid DNA (pDNA). In the present study, we successfully prepared a stable pDNA ternary complex using DOPS and polyethyleneimine (PEI) and evaluated its efficacy and safety. The pDNA/PEI complex had a positive charge and showed high transgene efficacy, although it caused cytotoxicity and agglutination. The addition of DOPS changed the ζ-potential of the pDNA/PEI complex to negative. It is known that anionic complexes are not taken up well by cells. Surprisingly, however, the pDNA/PEI/DOPS complex showed relatively high transgene efficacy in vitro. Fluorescence microscope observation revealed that the pDNA/PEI/DOPS complex internalized the cells while maintaining the complex formation. The injection of the pDNA/PEI complex killed most mice within 24 h at high doses, although all mice in the pDNA/PEI/DOPS complex group survived. The ternary complex with DOPS showed markedly better safety compared with the pDNA/PEI complex. The pDNA/PEI/DOPS complex showed high gene expression selectively in the spleen after intravenous injection into mice. Thus the ternary complex with DOPS can be used to deliver pDNA to the spleen, in which immune cells are abundant. It appears to have an excellent safety level, although further study to determine the mechanism of action is necessary.