Clinical outcomes of COVID-19 caused by the Alpha variant compared with one by wild type in Kobe, Japan. A multi-center nested case-control study.

OBJECTIVES: The emergence of the Alpha variant of novel coronavirus 2019 (SARS-CoV-2) is a concerning issue but their clinical implications have not been investigated fully. METHODS: We conducted a nested case-control study to compare severity and mortality caused by the Alpha variant (B.1.1.7) with the one caused by the wild type as a control from December 2020 to March 2021, using whole-genome sequencing. 28-day mortality and other clinically important outcomes were evaluated. RESULTS: Infections caused by the Alpha variant were associated with an increase in the use of oxygen (43.4% vs 26.3%. p = 0.017), high flow nasal cannula (21.2% vs 4.0%, p = 0.0007), mechanical ventilation (16.2% vs 6.1%, p = 0.049), ICU care (30.3% vs 14.1%, p = 0.01) and the length of hospital stay (17 vs 10 days, p = 0.031). More patients with the Alpha variant received medications such as dexamethasone. However, the duration of each modality did not differ between the 2 groups. Likewise, there was no difference in 28-day mortality between the 2 groups (12% vs 8%, p = 0.48), even after multiple sensitivity analyses, including propensity score analysis. CONCLUSION: The Alpha variant was associated with a severe form of COVID-19, compared with the non-Alpha wild type, but might not be associated with higher mortality.

Analysis of Predictive Factors for Diarrhea after the Administration of Naldemedine.

Naldemedine (NAL), a peripherally acting µ-opioid receptor antagonist, is effective for opioid-induced constipation (OIC). However, diarrhea is the most common adverse event. We investigated the incidence of NAL-induced diarrhea in patients who started NAL at Nagasaki University Hospital between June 2017 and March 2019. Predictors of NAL-induced diarrhea were analyzed using a multivariate logistic regression model. Two hundred and forty-two patients were included in the present study, and NAL-induced diarrhea was observed in 17.8% (43 patients). The results of multiple logistic regression analyses identified the administration of opioid analgesics for 8 d or longer before the initiation of NAL (odds ratio (OR): 2.20, 95% confidence interval (95% CI): 1.04-4.64, p = 0.039), the combination of a laxative (OR: 2.22, 95%CI: 1.03-4.81, p = 0.042), and the combination of CYP3A4 inhibitors (strong/moderate) (OR: 2.80, 95%CI: 1.02-7.67, p = 0.045) as risk factors. Therefore, the development of diarrhea needs to be considered in patients with these risk factors. Furthermore, diarrhea may be controlled by the initiation of NAL within 7 d of opioid analgesics and, where possible, the discontinuation of or change in the combination of moderate or strong CYP3A4 inhibitors.

Clinical and Molecular Characteristics of Klebsiella pneumoniae Isolates Causing Bloodstream Infections in Japan: Occurrence of Hypervirulent Infections in Health Care.

Although hypervirulent Klebsiella pneumoniae (hvKp) has been associated with severe community-acquired infections that occur among relatively healthy individuals, information about hvKp infections in health care settings remains limited. Here, we systematically analyzed the clinical and molecular characteristics of K. pneumoniae isolates causing bloodstream infections in a cross-sectional study. Clinical characteristics of K. pneumoniae bloodstream infections from hospitals across Japan were analyzed by a review of the medical records. Whole-genome sequencing of the causative isolates was performed. Bacterial species were confirmed and hvKp were identified using whole-genome sequencing data. Clinical characteristics of hvKp infections were compared with those of non-hvKp infections by bivariate analyses. Of 140 cases of K. pneumoniae bloodstream infections, 26 cases (18.6%) were caused by various clones of hvKp defined by the carriage of cardinal virulence genes. Molecular identification revealed that 24 (17.1%) and 14 (10%) cases were caused by Klebsiella variicola and Klebsiella quasipneumoniae, respectively. Patients with hvKp infections had higher proportions of diabetes mellitus (risk ratio [RR], 1.75; 95% confidence interval [CI], 1.05 to 2.94), and their infections had significantly higher propensity to involve pneumonia (RR, 5.85; 95% CI, 1.39 to 24.6), liver abscess (RR, 5.85; 95% CI, 1.39 to 24.6), and disseminated infections (RR, 6.58; 95% CI, 1.16 to 37.4) than infections by other isolates. More than one-half of hvKp infections were health care associated or hospital acquired, and a probable event of health care-associated transmission of hvKp was documented. hvKp isolates, which are significantly associated with severe and disseminated infections, are frequently involved in health care-associated and hospital-acquired infections in Japan.

[Efforts toward Venous Access of Anti-Cancer Drug by Nurses].

Our hospital has an established outpatient chemotherapy room, and medical doctors have accessed veins for infusion so far. We trialed venous access by nurses for the purpose of managing safe and proper cancer chemotherapy, reducing the work of doctors, and reducing patient waiting time. A questionnaire was conducted in June 2013, and nurses secured routes at 19 facilities(58%)of the 33 national university hospitals. In November of the same year, the working group was established, and from September 2016 to March 2017, lectures, practical skills, a paper test, and a practical test were conducted; successful applicants were approved as in-hospital certified nurses. From April 2017, we started intravenous injection of anti-cancer drugs by nurses in outpatient chemotherapy rooms and always waiting for doctor in chemotherapy room. There have been many favorable reports of reduced pain and less route failure from patients, and issues, such as extravasation and dyspnea, have not occurred yet. The doctors who were interrupting their work by 29 minutes(20 minutes to and from the patient for a 9-minute procedure)could concentrate on their own tasks. However, patient waiting time increased from 14 minutes to 21 minutes because the amount of work for nurses increased. In the future, reducing the burden on nurses, for example, by increasing the number of nurses, is warranted.

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.

Evaluation of Antiemetic Therapy for Hepatic Transcatheter Arterial Infusion Chemotherapy with Cisplatin.

Antiemetic prophylaxis with aprepitant, a 5-hydroxytryptamine3 (5-HT3) receptor antagonist and dexamethasone is recommended for patients receiving intravenous cisplatin chemotherapy. Whether the same antiemetic regime is superior for hepatic transcatheter arterial infusion chemotherapy with cisplatin (CDDP-TAI) is unknown. We conducted a retrospective study of antiemetic prophylaxis protection against chemotherapy-induced nausea and vomiting (CINV) in CDDP-TAI at Nagasaki University Hospital. The rate of complete response (CR) to antiemetics in the acute (<24 h) and delayed phases (24-120 h) was measured. Twenty-four patients were treated with a 5-HT3 receptor antagonist (granisetron or azasetron) and dexamethasone on the day of chemotherapy (day 1 only). There was a significant difference between the CR rates in the acute and delayed phases, 91.6, and 69.7%, respectively. Combination of a 5-HT3 antagonist and dexamethasone on day 1 is effective against acute CINV, but not delayed CINV during CDDP-TAI. These results may help guide the management of nausea and vomiting during CDDP-TAI to achieve better tolerance and compliance for fewer interventions and increased favorable therapeutic outcomes.

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.

Pentosan polysulfate treatment ameliorates motor function with increased serum soluble vascular cell adhesion molecule-1 in HTLV-1-associated neurologic disease.

The main therapeutic strategy against human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) characterized by lower extremity motor dysfunction is immunomodulatory treatment, with drugs such as corticosteroid hormone and interferon-α, at present. However, there are many issues in long-term treatment with these drugs, such as insufficient effects and various side effects. We now urgently need to develop other therapeutic strategies. The heparinoid, pentosan polysulfate sodium (PPS), has been safely used in Europe for the past 50 years as a thrombosis prophylaxis and for the treatment of phlebitis. We conducted a clinical trial to test the effect of subcutaneous administration of PPS in 12 patients with HAM/TSP in an open-labeled design. There was a marked improvement in lower extremity motor function, based on reduced spasticity, such as a reduced time required for walking 10 m and descending a flight of stairs. There were no significant changes in HTLV-I proviral copy numbers in peripheral blood contrary to the inhibitory effect of PPS in vitro for intercellular spread of HTLV-I. However, serum soluble vascular cell adhesion molecule (sVCAM)-1 was significantly increased without significant changes of serum level of chemokines (CXCL10 and CCL2). There was a positive correlation between increased sVCAM-1and reduced time required for walking 10 m. PPS might induce neurological improvement by inhibition of chronic inflammation in the spinal cord, through blocking the adhesion cascade by increasing serum sVCAM-1, in addition to rheological improvement of the microcirculation. PPS has the potential to be a new therapeutic tool for HAM/TSP.

Hepatic gene delivery system electrostatically assembled with glycyrrhizin.

In this study, a novel liver-targeted gene delivery vector was developed by electrostatically coating the cationic complex of pDNA and polyethylenimine (PEI) with glycyrrhizin (GL). The ternary complex, pDNA/PEI/GL, had approximately 100 nm stable particles with a negative charge surface. pDNA/PEI/GL showed high gene expression comparable to that of the complex of pDNA and PEI (pDNA/PEI) in human hepatoma cell line HepG2 without cytotoxicity and agglutination. After intravenous injection of pDNA/PEI/GL into mice, the highest gene expression was observed in the liver. pDNA/PEI/GL showed significantly higher gene expression in parenchymal cells than in nonparenchymal cells. On the basis of these results, we evaluated the pharmacological activity of the ternary complex including the pDNA encoding insulin (pCMV-Ins). The pCMV-Ins/PEI/GL decreased blood glucose concentrations 24 h after its intravenous administration to mice. The ternary complex of pDNA, PEI, and GL may be a promising liver-targeted gene vector.

Novel siRNA delivery system using a ternary polymer complex with strong silencing effect and no cytotoxicity.

We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and γ-polyglutamic acid (γ-PGA), which showed silencing effect and no cytotoxicity. The binary complexes of siRNA with PEI were approximately 73-102 nm in particle size and 45-52 mV in ζ-potential. The silencing effect of siRNA/PEI complexes increased with an increase of PEI, and siRNA/PEI complexes with a charge ratio greater than 16 showed significant luciferase knockdown in a mouse colon carcinoma cell line regularly expressing luciferase (Colon26/Luc cells). However, strong cytotoxicity and blood agglutination were observed in the siRNA/Lipofectamine complex and siRNA/PEI16 complex. Recharging cationic complexes with an anionic compound was reported to be a promising method for overcoming these toxicities. We therefore prepared ternary complexes of siRNA with PEI (charge ratio 16) by the addition of γ-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of γ-PGA content, which decreased the ζ-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including γ-PGA with an anionic surface charge. The high incorporation of ternary complexes into Colon26/Luc cells was confirmed with fluorescence microcopy. Having achieved knockdown of an exogenously transfected gene, the ability of the complex to mediate knockdown of an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was assessed in B16-F10 cells. The ternary complex (siRNA/PEI16/γ-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system.

Ternary complex of plasmid DNA electrostatically assembled with polyamidoamine dendrimer and chondroitin sulfate for effective and secure gene delivery.

The purpose of this study was to develop a ternary complex of plasmid DNA (pDNA) electrostatically assembled with polyamidoamine (PAMAM) dendrimer and chondroitin sulfate (CS) for effective and secure gene delivery. PAMAM dendrimers are new cationic polymers that are expected to be used as gene delivery vectors. However, cationic non-viral gene vectors showed cytotoxicity by binding to negative cellular membranes. We therefore prepared a ternary complex by adding CS, an anionic polymer, and examined its usefulness. The pDNA/PAMAM dendrimer complex (PAMAM dendriplex) and the PAMAM dendriplex coated by CS (CS complex) showed nanoparticles with positive ζ-potential and negative ζ-potential, respectively. The CS complex had no cytotoxicity against B16-F10 cells and no agglutination activity, although severe cytotoxicity and high agglutination were observed in the PAMAM dendriplex. As a result of an in vitro gene expression study of B16-F10 cells, not only the PAMAM dendriplex but also the CS complex showed high transfection efficiency. The transfection efficiency of the CS complex was significantly inhibited by clathrin-mediated endocytosis inhibitor (chlorpromazine), caveolae-mediated endocytosis inhibitor (genistein), and hypothermia. Tail-vein injection of the CS complex into mice led to significantly higher gene expression in the spleen than the PAMAM dendriplex. Thus, the ternary complex of pDNA electrostatically assembled with PAMAM denriplex and CS showed safe high gene expression in the spleen. This vector is expected to be useful for useful gene delivery.

Ocular gene delivery systems using ternary complexes of plasmid DNA, polyethylenimine, and anionic polymers.

In this experiment, we developed anionic ternary complexes for effective and safe ocular gene delivery. Ternary complexes were constructed by coating plasmid DNA (pDNA)/polyethylenimine (PEI) complex with anionic polymers such as γ-polyglutamic acid (γ-PGA) and chondroitin sulfate (CS). The cationic pDNA/PEI complex showed high gene expression on the human retinal pigment epithelial cell line, ARPE-19 cells. The pDNA/PEI complexes, however, also showed high cytotoxicity on the cells and aggregated strongly in the vitreous body. On the other hand, the anionic ternary complexes showed high gene expression on ARPE-19 cells without such cytotoxicity and aggregation. After intravitreous administration of the complexes, the anionic ternary complexes showed high gene expression in the retina. These results strongly indicate that anionic ternary complexes are suitable for effective and safe ocular gene therapy.

Safe and effective delivery of small interfering RNA with polymer- and liposomes-based complexes.

We developed binary and ternary complexes based on polymers and liposomes for safe and effective delivery of small interfering RNA (siRNA). Anti-luciferase siRNA was used as a model of nucleic acid medicine. The binary complexes of siRNA were prepared with cationic polymers and cationic liposomes such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-arginine (PLA), trimethyl[2,3-(dioleoxy)-propyl]ammonium chloride (DOTMA), and cholesteryl 3ß-N-(dimetylaminnoethyl)carbamate hydrochloride (DC-Chol). The ternary complexes were constructed by the addition of γ-polyglutamic acid (γ-PGA) to the binary complexes. The complexes were approximately 54-153 nm in particle size. The binary complexes showed a cationic surface charge although an anionic surface charge was observed in the ternary complexes. The polymer-based complexes did not show a silencing effect in the mouse colon carcinoma cell line expressing luciferase regularly (Colon26/Luc cells). The binary complexes based on liposomes and their ternary complexes coated by γ-PGA showed a significant silencing effect. The binary complexes showed significant cytotoxicity although the ternary complexes coated by γ-PGA did not show significant cytotoxicity. The ternary complexes coated by γ-PGA suppressed luciferase activity in the tumor after their direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, we have newly identified safe and efficient ternary complexes of siRNA for clinical use.

Ternary complex of plasmid DNA with protamine and γ-polyglutamic acid for biocompatible gene delivery system.

The purpose of the present study was to investigate the usefulness of the ternary complex with protamine and γ-polyglutamic acid (γ-PGA), which are biodegradable materials for foods and medical products, as a safe gene delivery vector. We formed cationic binary complexes (plasmid DNA (pDNA)/protamine complexes) with high transfection efficiency. The binary complex showed slight toxicity probably related to its total cationic charge. Then, we formed ternary complexes (pDNA/protamine/γ-PGA complexes) by addition of anionic polymer, γ-PGA, and they showed no cytotoxicity. The transfection efficiency of the pDNA/protamine/γ-PGA complexes was as high as that of the pDNA/protamine complexes, although their zeta potentials were different. Inhibition study of the gene expressions in B16-F10 cells suggested that pDNA/protamine complexes were taken up by caveolae-mediated endocytosis and macropinocytosis. On the other hand, pDNA/protamine/γ-PGA complexes were taken up by clathrin-mediated endocytosis and macropinocytosis. Thus, we succeeded in developing the ternary complex as a safe gene delivery vector with biocompatible materials.

Secure splenic delivery of plasmid DNA and its application to DNA vaccine.

In this experiment, we developed a novel safe and effective gene delivery vector coated with γ-polyglutamic acid (γ-PGA-coated complexes). The γ-PGA-coated complex was composed of chiseled spherical nano-particles with anionic charges. The plasmid DNA/polyethyleneimine complex (non-coated complex) showed high transgene efficiency in the spleen and lung after intravenous administration in mice, with high liver toxicity and lethality. On the other hand, γ-PGA-coated complex selectively showed high transgene efficiency in the spleen without such toxicity. Furthermore, the γ-PGA-coated complex highly accumulated and showed high gene expression in the marginal zone of the spleen. Those results strongly indicated that γ-PGA-coated complex was suitable as a DNA vaccine vector. We therefore applied γ-PGA-coated complex to melanoma DNA vaccine, pUb-M. The γ-PGA-coated complex containing pUb-M significantly inhibited the growth and metastasis of a melanoma cell line, B16-F10 cells. In conclusion, we developed a splenic gene vector, γ-PGA-coated complex, as a novel technology for clinical vaccination.

Development of effective cancer vaccine using targeting system of antigen protein to APCs.

PURPOSE: To develop a novel cancer vaccine using the targeting system of antigen protein to antigen-presenting cells (APCs) for efficient and safe cancer therapy. METHODS: The novel delivery system was constructed with antigen protein, benzalkonium chloride (BK), and γ-polyglutamic acid (γ-PGA), using ovalbumin (OVA) as a model antigen protein and evaluating its immune induction effects and utilities for cancer vaccine. RESULTS: BK and γ-PGA enabled encapsulation of OVA and formed stable anionic particles at nanoscale, OVA/BK/γ-PGA complex. Complex was taken up by dendritic cell line DC2.4 cells efficiently. We subcutaneously administered the complex to mice and examined induction of IgGs. The complex induced not only Th2-type immunoglobulins but also Th1-type immunoglobulins. OVA/BK/γ-PGA complex inhibited tumor growth of E.G7 cells expressing OVA regularly; administered OVA/BK/γ-PGA complex completely rejected tumor cells. CONCLUSION: The novel vaccine could be platform technology for a cancer vaccine.

Nanoparticles electrostatically coated with folic acid for effective gene therapy.

We developed a novel vector, electrostatically coated poly(ethylenimine) (PEI)/pDNA complexes with folic acid (FA). Without covalent binding, the FA molecules could coat the PEI/pDNA complexes, and stable anionic nanoparticles were formed at a charge ratio greater than 60. The addition of FA markedly decreased the cytotoxicity of the cationic PEI/pDNA complexes to the melanoma cell line, B16-F10 cells, which regularly expressed FA-specific receptor (FR). Furthermore, the anionic FA60/PEI/pDNA complexes showed high transgene efficiency via the FR-mediated pathway in B16-F10 cells. The FA60/PEI/pDNA complexes did not show agglutination with erythrocytes. After the intravenous injection of FA60/PEI/pDNA complexes into mice, a higher transgene efficiency than PEI/pDNA complexes was observed in the liver, kidney, spleen, and lung with FR. The gene expressions of FA60/PEI/pDNA complexes were significantly inhibited by preadministration of FA. Thus, the FA60/PEI/pDNA complexes were useful for effective gene therapy.