Functions of neutrophils, major participant in host defense mechanisms, are known to be regulated by various types of immunomodulators. Capacity of immunomodulators which are reported to show antitumor effect in vivo to induce neutorophil adherence response in vitro was investigated. Several bacterial immunomodulators (OK-432, Corynebacterium parvum, B.C.G.) and components of bacteria cell walls (lipopolysaccharide (LPS), lipid A, lipoteicoic acid, N-cell wall skelton (N-CWS), muramyl dipeptide (MDP)) and fungal polysaccharides (lentinan, zymosan A, etc.) were tested. Neutrophils prepared from peripheral blood of healthy men were incubated with each immunomodulator at 37°C for 60 min in 96 well plastic plates, then neutrophils adherent to substratum were stained by crystal violet and their optical density at 570 nm was measured as a parameter of neutrophil adherence. Although purified polysaccharides mainly prepared from fungi did not induce the adherent response, not only bacterial bodies and their components but also tumor necrosis factor-α (TNF-α) clearly induced it. On the base of these results, functional classification and typing of immunomodulators by different activities in neutrophil adherence was discussed.
Protonophoric uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP) decreases the proton motive force (ΔP) of the mitochondrial inner membrane and results in inhibition of oxidative phosphorylation. In this study, a CCCP-resistant clone was isolated from a random gene trap insertional mutant library of Chinese hamster ovary (CHO)-K1 cells which was constructed by infecting a retrovirus vector, ROSAβgeo. Although we expected the isolation of the mutants defective in nuclear genes responsible for mitochondrial functions, the disrupted gene of the isolated mutant that we named R1 cells was identified as one of the alleles for ribosomal protein 5 of large subunit (RPL5). The R1 cells express as much as 80% RPL5 protein compared with the parental CHO-K1 cells, possibly due to enhanced transcription from a remaining wild-type RPL5 allele in R1 cells. Furthermore, the protein amount is not decreased by CCCP in R1 cells, in contrast to its clear reduction by CCCP in parental cells. Since mutations of RPL5 and other ribosomal proteins are responsible for the ribosomopathies and cancer, the present mutant may be a useful cellular model of such human diseases from a viewpoint of energy metabolism as well as a tool for the study of ribosome biogenesis and extra-ribosomal function of the RPL5 protein.
The antiviral activities of a nucleoside analog antiviral drug (ribavirin) and a non-nucleoside drug (mycophenolate mofetil) against human parainfluenza virus type 2 (hPIV-2) were investigated, and the restoration of the inhibition by guanosine and S-(4-nitrobenzyl)-6-thioinosine (NBTI: equilibrative nucleoside transporter 1 inhibitor) were also investigated. Ribavirin (RBV) and mycophenolate mofetil (MMF) inhibited cell fusion induced by hPIV-2. Both RBV and MMF considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV and MMF as determined by polymerase chain reaction (PCR) and real time PCR. mRNA syntheses were also reduced. An indirect immunofluorescence study showed that RBV and MMF largely inhibited viral protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP), it was found that virus entry into the cells and multinucleated giant cell formation were almost completely blocked by RBV and MMF. RBV and MMF did not disrupt actin microfilaments or microtubules. Both guanosine and NBTI completely or partially reversed the inhibition by RBV and MMF in the viral replication, syntheses of genome RNA, mRNA and protein, and multinucleated giant cell formation. NBTI caused a little damage in actin microfilaments, but had no effect on microtubules. Both RBV and MMF inhibited the replication of hPIV-2, mainly by inhibiting viral genome RNA, mRNA and protein syntheses. The inhibition was almost completely recovered by guanosine. These results indicate that the major mechanism of the inhibition is the depletion of intracellular GTP pools.
Sodium glucose transporter 2 inhibitors (SGLT2is), new antidiabetic agents, were reported to improve not only glycemic parameters but also metabolic and circulatory parameters. Whereas, several adverse events caused by SGLT2is were also reported. We aimed to investigate the changes of glycemic, metabolic, and circulatory parameters as well as safety with low-dose administration of two SGLT2is, canagliflozin and ipragliflozin, and also the difference between the two agents. 25 individuals with type-2 diabetes mellitus (T2DM) were recruited and administered with low-dose SGLT2is, canagliflozin (n = 10, 50 mg/day) and ipragliflozin (n = 15, 25 mg/day). We examined glycemic, metabolic, and circulatory parameters at baseline and 24 weeks after administration. All patients completed the study without complications. Compared with baseline, levels of glycated hemoglobin, fasting plasma glucose, and homeostasis model assessment of β-cell function improved significantly at 24 weeks after administration (p < 0.05). Levels of body weight, low-density lipoproteincholesterol, aspartate transaminase, γ-glutamyl transferase, and urinary excretion of albumin also improved significantly (p < 0.05). Moreover, systolic/diastolic blood pressure and levels of brain natriuretic peptide improved significantly (p < 0.05). The comparison of improvement ratio (values of improvement/values of basement) of each agent revealed that there was a significant difference between low-dose canagliflozin and low-dose ipragliflozin for brain natriuretic peptide (0.4404 vs. 0.0970, p = 0.0275). Hence, low-dose SGLT2is could be useful for patients of T2DM not only for hyperglycemia but also for metabolic and circulatory disorders without eliciting adverse events. In addition, with regard to the efficacy upon cardiovascular function, canagliflozin could be more suitable than ipragliflozin.
Safflower seed is effective against oxidative stress, mediating the activation of the apoptotic signaling pathway in the renal tissues of cisplatin-treated mice. The anticancer activity of safflower in various cancer cell lines has also been reported. The present study was conducted to evaluate the potential synergistic anticancer effects of the co-treatment of safflower seed extracts and cisplatin in RKO cells and in BALB/c mice bearing RKO cell-derived human colorectal tumors. In the cellular system, RKO cells were treated with safflower seed extract in the presence or absence of cisplatin for 48 h and the cytotoxicity was evaluated by using microscopy. In the in vivo system, mice were injected with RKO cells and subsequently orally administered 100 or 200 mg/kg body weight safflower seed extract plus cisplatin-treated or untreated mice for 3 days to examine the inhibitory effect on the tumor. Treatment with safflower seed extract or cisplain to RKO cells resulted in a greater cell death than in with untreated cells. In the RKO cells co-treated with both safflower seed extract and cisplatin, greater cell damage was observed. In addition, mice co-administered safflower seed extract and cisplatin had lower concentrations of serum creatinine, which were indicative of less damage to the kidney, and had a lower solid tumor mass and higher expression of the caspase-3 protein. The results showed that safflower seed extract was highly toxic to RKO cells and inhibited tumor growth in cisplatin-treated mice through renoprotective effects.
Foodborne diseases have become a worldwide problem that threatens public health and welfare. Enteropathogenic Escherichia coli (EPEC) is one of major pathogens of moderate to severe diarrhea. The increased prevalence of EPEC strains that produce extended spectrum β-lactamase (ESBL) has deepened the problem. The fruit of Lonicera caerulea var. emphyllocalyx (LCE) has been used as a traditional food preservative and medicine in northern temperate zones such as Hokkaido Island, Japan. In this study, we investigated the antibacterial effect of LCE fruit extract (LCEE) against EPEC. The antibacterial activities of LCEE were examined by bacterial growth, time-kill curve, soft-agar motility, electron microscopy, and 96 well-microplate biofilm assays. We also investigated the bacterial mRNA expression of biofilm-associated genes (fliC, csgA, and fimA) by quantitative real-time PCR assays. LCEE was found to suppress the growth, time-kill curve, and spread of EPEC. It also reduced the biofilm formation in a dose-dependent manner. Morphological analysis using transmission and scanning electron microscopy revealed that LCEE diminished the function of flagella resulting in reduced motility and biofilm formation. The mRNA expression of all three biofilm associated genes was downregulated under LCEE treatment. Extracts of the fruit of LCE inhibit the motility and biofilm formation of EPEC as a result of the inhibition of flagella development and function. We propose LCEE as a therapeutic candidate for the effective therapy of EPEC-associated infectious diseases.
Conventional oral preparations generally release incorporated drugs omnidirectionally, including into the lumen, leading to a low bioavailability of drugs that are unstable in the gastrointestinal tract. Here, we designed Janus microspheres for efficient mucosal drug delivery as single-sided-release microspheres with the oriented attachment to mucus and evaluated their attachment to and orientation on a Caco-2 (human Caucasian colon adenocarcinoma cell line) monolayer. The microspheres comprised a mucus-oriented hemisphere of an ammonioalkyl methacrylate copolymer and a protective hemisphere of a hard fat. Fluorescein isothiocyanate-dextran with an average molecular weight of 3,000-5,000 Da (FD4) was used as a model hydrophilic drug. A water-in-oil emulsion-type solvent evaporation method was employed for fabrication of the Janus microspheres. The yield of Janus microspheres was found to be dependent on the polymer-to-hard fat ratio, with a maximum yield of over 90% being obtained at a ratio of 1:2, whereas lower and higher ratios resulted in monolithic or star-shaped microspheres. FD4 was specifically localized in the polymeric hemisphere. A cell culture study revealed that the Janus microspheres attached to a Caco-2 monolayer via their polymeric hemispheres with the hard fat hemisphere providing a protective sealing. This may lead to the development of an effective enteral drug delivery system for biomedicines, such as polypeptides and nucleic acids.
Crohn's disease (CD) development is thought to involve genetic factors related to immune response as well as environmental factors, such as intestinal bacteria and diet, though no clear cause has yet been identified. In our previous study, we found that the concentrations of linoleic acid, stearic acid, and metabolites in erythrocytes differed between CD patients and healthy subjects. These factors related to lipid metabolism are controlled by Δ6 desaturase (fatty acid desaturase 2, FADS2) and elongase 6 (ELOVL6), respectively. In the present study, we analyzed the gene sequences of FADS2 and ELOVL6 in 52 Japanese CD patients, and then compared mutation frequencies with findings in healthy individuals. Nineteen FADS2 mutations and 33 ELOVL6 mutations were found. Furthermore, a new variant in the promoter region was shown in both genes, though no mutation in the coding region was found in either. For the FADS2 intron, the allele frequency of rs227784 (0.3365; 95% CI = 0.0337-0.01460) was higher than that in healthy subjects (0.0190). Furthermore, allele rs227784 had a greater association with CD (odds ratio = 4.4; 95% CI = 2.1-9.3). As compared with healthy Japanese healthy individuals, no mutations were found with a largely deviated allele frequency in the present CD group. However, the number of patients examined was small, thus the reliability of our results is limited. The present findings regarding genetic effects on CD onset and lipid metabolism may be weak.
Characterization of microbial communities in the skin in healthy individuals and diseased patients holds valuable information for understanding pathogenesis of skin diseases and as a source for developing novel therapies. Notably, resources regarding skin microbiome are limited in developing countries where skin disorders from infectious diseases are extremely common. A simple method for sample collection and processing for skin microbiome studies in such countries is crucial. The aim of this study is to confirm the feasibility of collecting skin microbiota from individuals in Yaoundé, a capital city of Cameroon, and subsequent extraction of bacterial DNA in a resource limited setting. Skin swabs from several individuals in Yaoundé were successfully obtained, and sufficient amount of bacterial 16S ribosomal RNA-coding DNA was collected, which was confirmed by quantitative PCR. The median copy number of 16S ribosomal RNA gene varied across participants and collection sites, with significantly more copies in samples collected from the forehead compared to the left and right forearm, or back. This study demonstrated that collecting surface skin microbes using our swabbing method is feasible in a developing country. We further showed that even with limited resources, we could collect sufficient amount of skin microbiota from the inhabitants in Yaoundé where no studies of skin microbiome were reported, which can be passed to further metagenomic analysis such as next generation sequencing.
In order to deepen the health system reform and improve the mechanism for the formation of drug prices, in January 2019, the General Office of the State Council of the People's Republic of China issued the "National centralized drug purchasing and using pilot program", selected 11 cities in mainland China to carry out "4+7" city drug volume based purchasing pilot work. This paper introduces the specific implementation plan, organizational structure and drug selection process of China's "4+7" city drug volume-based purchasing pilot work, and expounds the initial effects, existing problems and policy development after the implementation of the policy. After the implementation of the policy, the prices of 25 selected drugs were significantly lower, compared with the minimum purchase price of the same drugs in 11 pilot cities in 2017, the average drop was 52%. After the pilot scope was extended to the nation, compared with the minimum purchase price of the Union in 2018, the proposed price of the 25 drugs have an average price drop of 59%, compared with the selected price of the "4+7" pilot cities, the average price drop was 25%, and the price of drugs dropped further. By the end of August 2019, the implementation progress of 25 selected drugs in the "4+7" city drug volume-based purchasing was better than expected, the burden of patients’ drug expenses was reduced, and the pilot work was beginning to bear fruit. The long-term influence and effect of the "4+7" city drug volume-based purchasing and policy implementation after the expansion needs to be further observed.