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BACKGROUND Although some studies have described the association between serum ferritin levels and specific disorders in child and adolescent psychiatry, few have focused on mental health with low serum ferritin levels in children and adolescents. This study examined the effects of iron administration on psychological state of children and adolescents with reduced serum ferritin concentration. METHODS This prospective study evaluated 19 participants aged 6-15 years with serum ferritin levels

Mast cell-deficient mice are helpful for understanding the roles of mast cells in vivo. To date, a dozen mouse models for mast cell deficiency have been reported. However, mice with a specific depletion of all populations of mast cells have not been reported. We generated knock-in mice, termed Mcpt5/Cma1DTR mice, expressing human diphtheria toxin A (DT) receptor under the endogenous promoter of Mcpt5 (also known as Cma1), which encodes mouse mast cell protease-5. Flow cytometry and histological analysis showed that intraperitoneal injection of DT induced almost complete depletion of mast cells in heterozygote Mcpt5/Cma1DTR/+ mice. The deletion rates of mast cells in peritoneal cavity, mesentery, abdominal skin, ear skin, and glandular stomach were 99.9%, 100%, 98.7%, 97.7%, and 100%, respectively. Passive cutaneous anaphylaxis reaction also revealed mast cell deficiency in ear skin after DT treatment. Other than mast cells, a small percentage of marginal zone B cells in Mcpt5/Cma1DTR/+ mice were killed by DT treatment. In conclusion, the Mcpt5/Cma1DTR/+ mouse model is valuable for achieving conditional depletion of all populations of mast cells without inducing a marked reduction in other cells.

Aim: Tumor-derived small extracellular vesicles (TEVs) are considered for use in inducing tumor antigen-specific immune responses as they contain tumor antigens. The delivery of tumor antigens to the antigen presentation cells (especially dendritic cells [DCs]), and the activation of DCs are the main challenges of TEV therapy. Materials & methods: TEVs were modified with CD40 ligand (CD40L), which can target CD40 expressed on the surface of DCs and can activate them via CD40L-CD40 interactions. Results: It was found that CD40L-TEVs were efficiently taken up by DCs and also activated them. Moreover, tumor antigens were efficiently presented to the T cells by DCs treated with CD40L-TEVs. Conclusion: This study proved that CD40L-modification of TEVs will be helpful for further development of TEV-based tumor vaccination.

Predicting the outbreak of disease is essential when managing shrimp farms. Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus is a serious disease in shrimp. It is essential that shrimp farmers on the east coast of the Mekong Delta detect the disease as early as possible, because the mortality rate can reach 100%. Here, we used machine learning to predict AHPND development based on data collected since 2010 from shrimp farms in Tra Vinh, Ben Tre, Bac Lieu, and Ca Mau provinces. We initially hypothesized that the dependent variable, AHPND, was affected by 31 independent variables, but ultimately used 15 key variables to train the models. Logistic regression, artificial neural network, decision tree, and K-nearest neighbor analyses were performed, and the accuracy of the predictions was evaluated using hold-out and cross-validation tests. Logistic regression, as the most stable algorithm, was thus used to predict AHPND outbreaks in shrimp farms.

Musculocontractural Ehlers-Danlos syndrome (mcEDS) is caused by generalized depletion of dermatan sulfate (DS) due to biallelic pathogenic variants in CHST14 encoding dermatan 4-O-sulfotransferase 1 (D4ST1) (mcEDS-CHST14). Here, we generated mouse models for mcEDS-CHST14 carrying homozygous mutations (1 bp deletion or 6 bp insertion/10 bp deletion) in Chst14 through CRISPR/Cas9 genome engineering to overcome perinatal lethality in conventional Chst14-deleted knockout mice. DS depletion was detected in the skeletal muscle of these genome-edited mutant mice, consistent with loss of D4ST1 activity. The mutant mice showed common pathophysiological features, regardless of the variant, including growth impairment and skin fragility. Notably, we identified myopathy-related phenotypes. Muscle histopathology showed variation in fiber size and spread of the muscle interstitium. Decorin localized diffusely in the spread endomysium and perimysium of skeletal muscle, unlike in wild-type mice. The mutant mice showed lower grip strength and decreased exercise capacity compared to wild type, and morphometric evaluation demonstrated thoracic kyphosis in mutant mice. The established CRISPR/Cas9-engineered Chst14 mutant mice could be a useful model to further our understanding of mcEDS pathophysiology and aid in the development of novel treatment strategies., Summary: CRISPR/Cas9 genome-engineered Chst14−/− mouse models of musculocontractural Ehlers-Danlos syndrome (mcEDS) display similar myopathic features (particularly those caused by the loss of D4ST1) to mcEDS patients and may facilitate further understanding of mcEDS.

Ehlers–Danlos syndromes (EDSs) are heterogeneous group of heritable connective tissue disorders characterized by joint and skin hyperextensibility as well as fragility of various organs. Recently, we described a new type of EDS, musculocontractual EDS (mcEDS-CHST14), caused by pathogenic variants of the carbohydrate sulfotransferase 14 (CHST14) gene mutation. B6;129S5-Chst14tm1Lex/Mmucd (B6;129-Chst14 KO) mice are expected to be an animal model of mcEDS-CHST14. However, >90% of B6;129-Chst14 KO homozygous (B6;129-Chst14−/−) mice show perinatal lethality. Therefore, improvement of the birth rate of Chst14−/− mice is needed to clarify the pathophysiology of mcEDS-CHST14 using this animal model. Some B6;129-Chst14−/− embryos had survived at embryonic day 18.5 in utero, suggesting that problems with delivery and/or childcare may cause perinatal lethality. However, in vitro fertilization and egg transfer did not improve the birth rate of the mice. A recent report showed that backcrossing to C57BL/6 strain induces perinatal death of all Chst14−/− mice, suggesting that genetic background influences the birthrate of these mice. In the present study, we performed backcrossing of B6;129-Chst14 KO mice to a BALB/c strain, an inbred strain that shows lower risks of litter loss than C57BL/6 strain. Upon backcrossing 1 to 12 times, the birth rate of Chst14−/− mice was improved with a birth rate of 6.12–18.64%. These results suggest that the genetic background influences the birth rate of Chst14−/− mice. BALB/c congenic Chst14−/− (BALB.Chst14−/−) mice may facilitate investigation of mcEDS-CHST14. Furthermore, backcrossing to an appropriate strain may contribute to optimizing animal experiments.

Morphological deformities of the jaw and vertebrae arise as hindrances to seed production of Japanese eel Anguilla japonica. To obtain basic information related to development in these areas, we analyzed eel larva skeletal system formation by staining with alizarin red S. Results demonstrated that dentary and maxillary ossification at the jaw begins by 10 days after hatching, about 8 mm total length. Subsequent progression of ossification is slow until metamorphosis. For instance, ossification of the angular and quadrate, which are connected to the dentary, begins remarkably later than ossification of the dentary. Ossification of the head develops quickly during metamorphosis and produces a primitive skull in glass eels. Ossification of the vertebral centrum progresses rapidly from the posterior end to the anterior-most during metamorphosis. Our results demonstrate that the process of ossification at the head is shared among some species of Elopomorpha, but that ossification of vertebral centra differed. Furthermore, our results imply consistency of the period between ossification and the occurrence of some morphological deformities around the jaw and vertebrae.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication and are thereby expected to be promising carriers for drug delivery. Understanding the factors that affect sEV pharmacokinetics is crucial for its application as a drug delivery carrier. In this study, the role of sEV surface glycans was investigated by evaluating the effects of enzymatic deglycosylation treatment on sEV pharmacokinetics. First, control glycoprotein fetuin was used to optimize the glycosidase treatment conditions. B16-BL6-derived sEVs labeled with fusion proteins comprising Gag protein and Gaussia luciferase (gLuc) (Gag-gLuc) were then treated with glycosidases, Peptide-N-Glycosidase F or O-glycosidase, which cleaves N- and O-glycans, respectively. Glycosidase-treated sEVs showed physicochemical characteristics comparable to those of the untreated sEVs. However, removal of N-glycans from B16-BL6 sEVs enhanced cellular uptake by the peritoneal macrophages, while the removal of O-glycans had minimal impact, as evaluated by flow cytometry. To determine the effect of surface glycans on the sEV pharmacokinetics, Gag-gLuc labeled B16-BL6 sEVs treated with or without glycosidases were then intravenously administered to mice. Glycosidase-treated sEVs showed almost identical clearance from the blood circulation as that of the untreated sEVs. These results suggest minimal impact of surface glycans on sEV pharmacokinetics, despites its effect on cellular uptake.

Adriamycin (ADR)-induced nephropathy is frequently utilized in rodent models of podocytopathy. However, the application of this model in mice is limited to a few strains, such as BALB/c mice. The most commonly used mouse strain, C57BL/6 (B6), is resistant to ADR-induced nephropathy, as are all mouse strains with a B6 genetic background. Reportedly, the R2140C variant of the Prkdc gene is the cause of susceptibility to ADR-induced nephropathy in mice. To verify this hypothesis, we produced Prkdc mutant B6 mice, termed B6-PrkdcR2140C, that possess the R2140C mutation. After administration of ADR, B6-PrkdcR2140C mice exhibited massive proteinuria and glomerular and renal tubular injuries. In addition, there was no significant difference in the severity between B6-PrkdcR2140C and BALB/c. These findings demonstrated that B6-PrkdcR2140C show ADR-induced nephropathy susceptibility at a similar level to BALB/c, and that the PRKDC R2140C variant causes susceptibility to ADR-induced nephropathy. In future studies, ADR-induced nephropathy may become applicable to various kinds of genetically modified mice with a B6 background by mating with B6-PrkdcR2140C.

We aimed to compare the efficacy of the VIO soft coagulation system (VSCS) for the treatment of air leaks by sealing with fibrin glue, and also assess the histological alterations that occur after soft coagulation. A mouse pulmonary air leak model was designed. The pulmonary fistula was subsequently coagulated with the VSCS or sealed with fibrin glue with polyglycolic acid (PGA) sheets. The burst pressure at air leak recurrence was measured in each group, and the results were compared. We also evaluated the histological alterations in the mouse pulmonary air leak model after soft coagulation with the VSCS. The burst pressure in the soft coagulation group (80 W/Effect 5) (median 42.8; range 35.4–53.8 cmH2O) was similar to that in the fibrin glue group (median 41.5; range 34.6–43.9 cmH2O) (p = 0.21). Histological examinations revealed that the visceral pleura remained torn, the structure of the pulmonary alveolus was maintained, and the coagulated fistula was covered with a fibrin membrane in the soft coagulation group. The pressure resistance following soft coagulation was equivalent to that after sealing using fibrin glue with PGA sheets. The air leaks were likely controlled by covering the fistula with a fibrin membrane after soft coagulation with the VSCS.

Diabetic animal models have made significant contributions to understanding the etiology of diabetes and to the development of new medications. Our research group recently developed a novel diabetic mouse strain, the insulin hyposecretion (ihs)mouse. The strain involves neither obesity nor insulitis but exhibits notable pancreatic β-cell dysfunction, distinguishing it from other well-characterized animal models. In ihs mice, severe impairment of insulin secretion from pancreas has been elicited by glucose or potassium chloride stimulation. To clarify the genetic basis of impaired insulin secretion, beginning with identifying the causative gene, genetic linkage analysis was performed using [(C57BL/6 × ihs) F1 × ihs] backcross progeny. Genetic linkage analysis and quantitative trait loci analysis for blood glucose after oral glucose loading indicated that a recessively acting locus responsible for impaired glucose tolerance was mapped to a 14.9-Mb region of chromosome 18 between D18Mit233 and D18Mit235 (the ihs locus). To confirm the gene responsible for the ihs locus, a congenic strain harboring the ihs locus on the C57BL/6 genetic background was developed. Phenotypic analysis of B6.ihs-(D18Mit233-D18Mit235) mice showed significant glucose tolerance impairment and markedly lower plasma insulin levels during an oral glucose tolerance test. Whole-genome sequencing and Sanger sequencing analyses on the ihs genome detected two ihs-specific variants changing amino acids within the ihs locus; both variants in Slc25a46 and Tcerg1 were predicted to disrupt the protein function. Based on information regarding gene functions involving diabetes mellitus and insulin secretion, reverse-transcription quantitative polymerase chain reaction analysis revealed that the relative abundance of Reep2 and Sil1 transcripts from ihs islets was significantly decreased whereas that of Syt4 transcripts were significantly increased compared with those of control C57BL/6 mice. Thus, Slc25a46, Tcerg1, Syt4, Reep2 and Sil1 are potential candidate genes for the ihs locus. This will be the focus of future studies in both mice and humans.

Tumor-cell-derived exosomes contain endogenous tumor antigens and can be used as a potential cancer vaccine without requiring identification of the tumor-specific antigen. To elicit an effective antitumor effect, efficient tumor antigen presentation by MHC class I molecules on dendritic cells (DC) is desirable. Because DC endocytose exosomes, an endosomal escape mechanism is required for efficient MHC class I presentation of exosomal tumor antigens. In the present study, efficient cytosolic delivery of exosomal tumor antigens was performed using genetically engineered tumor-cell-derived exosomes and pH-sensitive fusogenic GALA peptide. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a streptavidin (SAV; a protein that binds to biotin with high affinity)-lactadherin (LA; an exosome-tropic protein) fusion protein to obtain SAV-LA-modified exosomes (SAV-exo). SAV-exo was mixed with biotinylated GALA to obtain GALA-modified exosomes (GALA-exo). Fluorescent microscopic observation using fluorescent-labeled GALA showed that the exosomes were modified with GALA. GALA-exo exerted a membrane-lytic activity under acidic conditions and efficiently delivered exosomal cargos to the cytosol. Moreover, DC treated with GALA-exo showed enhanced tumor antigen presentation capacity by MHC class I molecules. Thus, genetically engineered GALA-exo are effective in controlling the intracellular traffic of tumor-cell-derived exosomes and for enhancing tumor antigen presentation capacity.

Exosomes are small membrane vesicles containing lipids, proteins, and nucleic acids. Recently, researchers have uncovered that exosomes are involved in various biological events, such as tumor growth, metastasis, and the immune response, by delivering their cargos to exosome-receiving cells. Moreover, exosomes are expected to be used in therapeutic treatments, such as tissue regeneration therapy and antitumor immunotherapy, because exosomes are effective delivery vehicles for proteins, nucleic acids, and other bioactive compounds. To elucidate the biological functions of exosomes, and for the development of exosome-based therapeutics, the pharmacokinetics of exosomes is important. In this review, we aim to summarize current knowledge about the pharmacokinetics and biodistribution of exosomes. The pharmacokinetics of exogenously administered exosomes is discussed based on the tissue distribution, types of cells taking up exosomes, and key molecules in the pharmacokinetics of exosomes. In addition, recent progress in the methods to control the pharmacokinetics of exosomes is reviewed.

Cytosine-phosphate-guanine (CpG) DNA is known to increase the potency of vaccines. Here, in vitro and in vivo stimulation of toll-like receptor 9 by CpG DNA incorporated into polypod-like DNA nanostructures was evaluated by measuring the levels of tumor necrosis factor alpha released from macrophage-like RAW 264.7 cells and plasma interleukin (IL)-12p40 in vivo following intravenous injection into mice. Phosphodiester CpG1668 was selected as the CpG DNA, and tripodna and hexapodna, which were CpG1668-containing tripod and hexapod-like DNA nanostructures, respectively, were designed. CpG-tripodna and CpG-hexapodna induced tumor necrosis factor alpha release from RAW 264.7 cells about 10- and ∼30-fold higher than single-stranded CpG1668 (CpG-SS). Moreover, in all cases examined, plasma IL-12p40 concentrations increased after intravenous injection into mice, with peak levels depending on the samples and the doses. The area under the plasma concentration-time curves indicated that the CpG-hexapodna was approximately 20-fold more efficient in inducing IL-12p40 production than CpG-SS. The efficiency of CpG-tripodna and CpG-hexapodna to increase the potency of CpG-SS in vivo was comparable to that observed in cultured RAW 264.7 cells. These results provide experimental evidence that in vitro studies can be used to estimate the in vivo immunostimulatory activity of CpG DNA incorporated into DNA nanostructures.

Leaf cuticular membranes (CMs) of Sonneratia alba are considered to play an important role in environmental tolerance, and chemical and mechanical properties of their CMs are crucial factors which provide protective barrier and mechanical supports. Leaf CMs were enzymatically isolated from both adaxial and abaxial sides of young matured leaves (L3), and their chemical and mechanical properties were compared. Chemical compositions of both CMs were similar reflecting their morphological similarity. The adaxial CM was consisted of 23.4% wax, 64.2% cutin, 7.4% cutan and 6.1% polysaccharides with thickness of 9 µm, and the abaxial CM had the values of 23.9%, 63.2%, 9.4%, 4.2% and 8 µm, respectively. Major monomers in both cutins were 9,10-epoxy-18-hydroxyoctadecanoic acid and 9(10),16-dihydroxyhexadecanoic acid. Mechanical properties of both CMs were also similar reflecting their morphological and chemical compositional similarities.

We previously demonstrated that the immunostimulatory activity of CpG DNA is increased by the formation of polypod-like structures. The present study was designed to elucidate the mechanism underlying this increase. Tripodna (three pods) and hexapodna (six pods) were prepared. The cellular uptake of Alexa Fluor 488-labeled DNA samples was examined in several cell lines by measuring the MFI of cells. TNF-α release from RAW264.7 cells was measured after addition of polypodna containing CpG motifs. Dissociation of double stranded DNA was evaluated using FRET. Tripodna and hexapodna were efficiently taken up by macrophage-like RAW264.7 cells and dendritic DC2.4 cells, but not by fibroblast or endothelial cell lines. The uptake by RAW264.7 cells was highest for hexapodna, followed by tripodna, dsDNA, and ssDNA. The release of TNF-α from RAW264.7 cells was also highest for hexapodna. The ratio of TNF-α release to cellular uptake was highest for ssDNA, and lowest for dsDNA. Tripodna and hexapodna were more easily dissociated into single strands after cellular uptake than was dsDNA. The efficient cellular uptake and prompt dissociation into single strands can be directly related to the high immunostimulatory activity of polypod-like structured DNAs containing CpG motifs.

Accelerated formation of fibrin clots in a tumor microenvironment can be used for targeted delivery of interferon gamma (IFNγ) to tumor cells. Here, we selected cysteine-arginine-glutamic acid-lysine-alanine (CREKA) as the fibrin clot-binding peptide and designed 2 types of fusion proteins for tumor targeting. The CREKA peptide was fused to IFNγ's C-terminus, with or without a matrix metalloproteinase-2 (MMP2)-cleavable linker (IFNγ-mmp-CREKA or IFNγ-CREKA, respectively). The former was designed to release IFNγ from IFNγ-mmp-CREKA bound to fibrin clots, to ensure IFNγ's function in the tumor milieu. IFNγ-activated sequence-dependent reporter gene expression in B16-BL6 cells revealed that the biological activities of IFNγ-CREKA and IFNγ were comparable, whereas that of IFNγ-mmp-CREKA was approximately 60% that of IFNγ. Plasma concentrations of IFNγ-CREKA and IFNγ-mmp-CREKA remained at effective levels for at least 4 weeks after gene transfer into mice. After gene transfer to tumor-bearing mice, intratumoral concentration of IFNγ in pCpG-IFNγ-mmp-CREKA group was tended to be higher than those of the other groups. Inhibition of colon-26 tumor growth was significantly more with gene transfer of IFNγ-mmp-CREKA than with IFNγ or IFNγ-CREKA. These results indicate that targeted delivery of IFNγ to fibrin clots through IFNγ-mmp-CREKA fusion can enhance the therapeutic efficacy of IFNγ in cancer gene therapy.

Dendritic cell-derived small extracellular vesicles (DC-sEVs) are proposed as a novel candidate for tumor antigen-based cancer immunotherapy. In order to improve the DC-sEV-induced antitumor immunity, production of DC-sEVs capable of inducing potent antigen-specific humoral and cellular immune responses is necessary. Here, we collected sEVs from DCs and added ovalbumin (OVA), which was used as model antigen, as well as LPS and IFN-γ, to prepare DC-sEVs with high immune activity. After confirming that the collected sEVs, named activated-DCOVA-sEVs, contained OVA and possessed immunologically relevant components (MHC class I molecule displaying antigen epitopes and co-stimulatory molecules, as well as sEV marker proteins), we found that activated-DCOVA-sEV stimulated macrophages and DCs through Toll-like receptor 4 signaling and boosted innate immunity in the tumor microenvironment. Moreover, activated-DCOVA-sEVs induced potent antigen-specific humoral and cellular immune responses both in vitro and in vivo. Finally, immunization with activated-DCOVA-sEVs exhibited stronger in vivo antitumor effects in tumor-bearing mice induced by inoculation with OVA-expressing tumor cells.

Tensin2 (TNS2) is a focal adhesion-localized protein possessing N-terminal tandem protein tyrosine phosphatase (PTPase) and C2 domains, and C-terminal tandem Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains. Genetic deletion of Tns2 in a susceptible murine strain leads to podocyte alterations after birth. To clarify the domain contributions to podocyte maintenance, we generated two Tns2-mutant mice with the genetic background of the susceptible FVB/NJ strain, Tns2∆C and Tns2CS mice, carrying a SH2-PTB domain deletion and a PTPase domain inactivation, respectively. The Tns2∆C mice developed massive albuminuria, severe glomerular injury and podocyte alterations similarly to those in Tns2-deficient mice. In contrast, the Tns2CS mice showed no obvious phenotypic abnormalities. These results indicate that the TNS2 SH2-PTB domain, but not its PTPase activity, plays a role in podocyte maintenance. Furthermore, in a podocyte cell line, the truncated TNS2 mutant lacking the SH2-PTB domain lost the ability to localize to focal adhesion. Taken together, these data suggest that TNS2 recruitment to focal adhesion is required to maintain postnatal podocytes on a susceptible genetic background.

Fenoxasulfone is a novel rice herbicide that was discovered and developed by Kumiai Chemical Industry Co., Ltd. It displays excellent herbicidal activity against Echinochloa spp. and other annual weeds at 150-200 g a.i./ha with long residual activity and has a favorable toxicological, ecotoxicological, and environmental profile. Fenoxasulfone's mode of action was investigated, and it has been shown to inhibit the biosynthesis of very-long-chain fatty acids in plants. Fenoxasulfone was registered in Japan in 2014, and various products containing fenoxasulfone have been launched. With its high efficacy and long residual activity, we believe that fenoxasulfone will contribute to efficient food production in the future.

Immunostimulatory DNA hydrogel (sDNA hydrogel) containing unmethylated cytosine-phosphate-guanine (CpG) sequences has been demonstrated to be a useful antigen delivery system, which can effectively induce an antigen-specific immune response through stimulation of the innate immune system. However, relatively rapid release of antigens from the sDNA hydrogel limits its potential. To enhance the potency of the sDNA hydrogel via improvement of its sustained release property, we selected chitosan, a biocompatible cationic polymer which electrostatically interacts with DNA, and mixed it with the sDNA hydrogel. Compared to unmixed sDNA hydrogel, sDNA hydrogel mixed with chitosan (Chitosan-sDNA hydrogel) was more stable, tougher, had more bound water, released a model antigen ovalbumin (OVA) more slowly in vitro, and provided longer retention of OVA at the injection site after intradermal injection into mice. Intradermal immunization of mice with the OVA-loaded Chitosan-sDNA hydrogel resulted in the induction of a higher level of OVA-specific IgG in serum compared with OVA-loaded sDNA hydrogel with no chitosan. These results indicate that the Chitosan-sDNA hydrogel is an improved sustained release formulation for efficient induction of antigen-specific immune responses.

For cancer immunotherapy via tumor antigen vaccination in combination with an adjuvant, major challenges include the identification of a particular tumor antigen and efficient delivery of the antigen as well as adjuvant to antigen-presenting cells. In this study, we proposed an efficient exosome-based tumor antigens-adjuvant co-delivery system using genetically engineered tumor cell-derived exosomes containing endogenous tumor antigens and immunostimulatory CpG DNA. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a fusion streptavidin (SAV; a protein that binds to biotin with high affinity)-lactadherin (LA; an exosome-tropic protein) protein, yielding genetically engineered SAV-LA-expressing exosomes (SAV-exo). SAV-exo were combined with biotinylated CpG DNA to prepare CpG DNA-modified exosomes (CpG-SAV-exo). Fluorescent microscopic observation revealed the successful modification of exosomes with CpG DNA by SAV-biotin interaction. CpG-SAV-exo showed efficient and simultaneous delivery of exosomes with CpG DNA to murine dendritic DC2.4 cells in culture. Treatment with CpG-SAV-exo effectively activated DC2.4 cells and enhanced tumor antigen presentation capacity. Immunization with CpG-SAV-exo exhibited stronger in vivo antitumor effects in B16BL6 tumor-bearing mice than simple co-administration of exosomes and CpG DNA. Thus, genetically engineered CpG-SAV-exo is an effective exosome-based tumor antigens-adjuvant co-delivery system that will be useful for cancer immunotherapy.

Gene therapy techniques aiming to induce the long-term interferon-β (IFN-β) expression are desirable for inhibiting cancer growth. However, there has been no success in this regard because IFN-β significantly inhibits transgene expression. This study used the IFN-inducible Mx promoter to promote IFN-β expression. The pMx-IFN-β plasmid was constructed to achieve long-term IFN-β expression. In cultured cells transfected with the Mx promoter-driven reporter protein plasmid, IFN-β induced concentration-dependent expression of the reporter protein. After the hydrodynamic injection of pMx-IFN-β into mice, the serum concentration of IFN-β was maintained at ≥100 pg/mL for1 month. IFN-β expression was significantly suppressed by the co-injection of small interfering RNA targeting the interferon-α/β receptor (IFNAR), suggesting that IFN-β binding to IFNAR increased IFN-β expression. Moreover, the hydrodynamic injection of pMx-IFN-β significantly suppressed the growth of colon26 tumors in mice. In contrast, a conventional promoter-driven plasmid was less effective than pMx-IFN-β in all the experiments. Taken together, these results indicate that the interferon-inducible Mx promoter-driven expression system effectively achieves long-term expression of IFN-β and represents a potential tool for cancer gene therapy.

Background Tensin2 is a focal adhesion-localized multidomain protein expressed in various tissues, and its dysfunction leads to alterations in podocytes. However, these podocyte-related manifestations are dependent on murine strain. Tensin2 dysfunction results in susceptible strains developing podocyte foot process effacement and massive albuminuria, whereas podocytes in resistant strains remain almost intact. In our previous studies, quantitative trait loci analysis and congenic analysis using resistant C57BL/6J and susceptible ICGN mice identified a modifier locus associated with podocyte injury caused by tensin2 dysfunction on chromosome 2. However, the effect of this modifier locus on chromosome 2 is insufficient to explain the resistance of C57BL/6J mice to tensin2 dysfunction, indicating the existence of other modifier genes. Results Whereas previous studies focused on the severity of chronic kidney disease, the present study focused on podocyte injury. We performed a genome-wide linkage analysis of backcrosses between two tensin2-deficient mouse strains, B6.ICGN-Tns2nph and FVB.ICGN-Tns2nph, and detected a novel major modifier locus on chromosome 10. The combined effect of the C57BL/6J alleles of the two loci on chromosomes 2 and 10 reduced the urinary albumin excretion caused by tensin2 dysfunction to a level comparable to that of C57BL/6J mice. Conclusions These data indicate that the resistance to podocyte injury caused by tensin2 dysfunction is mainly produced by the effects of the modifier genes on the two loci. The identification of these modifier genes is expected to help elucidate the mechanism underlying podocyte injury. Electronic supplementary material The online version of this article (10.1186/s12863-018-0611-1) contains supplementary material, which is available to authorized users.

Exosomes, which are expected to be delivery systems for biomolecules such as nucleic acids, are collected by several methods. However, the effect of exosome isolation methods on the characteristics of exosomes as drug carriers, such as recovery efficiency after sterile filtration and pharmacokinetics, has not been investigated despite the importance of these characteristics for the development of exosome-based delivery systems. In the present study, exosomes collected from murine melanoma B16-BL6 cells by several methods were compared with respect to dispersibility, recovery rate after filtering, and clearance from the blood circulation in mice. The exosomes were collected by three ultracentrifugation-based methods: simple ultracentrifugation/pelleting (pelleting method), ultracentrifugation with an iodixanol cushion (cushion method), and ultracentrifugation on an iodixanol density gradient (gradient method). The isolation methods had little effect on the particle number of exosomes. In contrast, transmission electron microscopy observation and size distribution measurement using tunable resistive pulse sensing indicated that the exosomes of the gradient method were more dispersed than the others. The exosomes were labeled with Gaussia luciferase and intravenously injected into mice. Clearance of injected exosomes from the blood circulation did not significantly change with isolation methods. When the exosomes were filtered using a 0.2-μm filter, the recovery rate was 82% for the exosomes of the gradient method, whereas it was less than 50% for the others. These results indicate that the exosome isolation method markedly affects the dispersibility and filtration efficiency of the exosomes.

Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines. From the Clinical Editor: Toll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.

We previously have shown that multicellular spheroids containing insulin-secreting cells are an effective therapy for diabetic mice. Here we attempted to increase insulin secretion by incorporating other cell types into spheroids. Multicellular spheroids of mouse MIN6 pancreatic β cells were formed in microwells alone and with aortic vascular endothelial MAEC cells or embryo fibroblast NIH3T3 cells. mRNA expression of insulin genes and insulin secretion of MIN6 cells in each spheroid were measured by real-time PCR and an insulin ELIZA kit. Moreover, collagen IV expression in each spheroid was analyzed by western blot. In all cases, uniformly sized (about 300 μm) multicellular spheroids were obtained. MAEC or NIH3T3 cell incorporation into MIN6 spheroids significantly increased mRNA expression of insulin genes and insulin secretion. In addition, collagen IV expression, which was reported to enhance insulin secretion from pancreatic β cells, also increased in their spheroids. The formation of mixed multicellular spheroids containing collagen IV-expressing cells can improve the insulin secretion from insulin-secreting MIN6 cells, and mixed multicellular spheroids can be a potent therapeutic option for patients with type I diabetes mellitus.

Previous studies indicate that immunostimulatory DNA-based injectable hydrogels harboring unmethylated cytosine-phosphate-guanine (CpG) dinucleotides meet the requirements of an effective antigen delivery system, including safety, biodegradability, ease of administration, and stimulation of the innate immune system. However, rapid release of the model antigen ovalbumin (OVA) from the hydrogel limits its potential. Here, the aim is to achieve sustained OVA release from a DNA hydrogel through cationization of the antigen. Ethylenediamine (ED)-conjugated cationized OVA (ED-OVA), but not OVA, forms a complex with hexapod-like structured DNA, a component of the DNA hydrogel. The release of ED-OVA from the hydrogel is significantly slower than that of OVA. ED-OVA mixed with CpG DNA hydrogel efficiently binds to mouse dendritic DC2.4 cells and results in high antigen presentation. Intratumoral injections of ED-OVA/CpG DNA hydrogel significantly delays tumor growth of OVA-expressing EG7-OVA cells in mice. Then, a cationic OVA peptide antigen (R8-L2-pepI) consisting of an OVA MHC class I epitope, octaarginine, and a linker is designed. Intratumoral injections of R8-L2-pepI/CpG DNA hydrogel eradicate tumors in five out of six mice. Thus, it is concluded that a vaccine consisting of immunostimulatory CpG DNA hydrogel and cationized antigens can be effective for cancer immunotherapy., Article first published online: 13 AUG 2015

Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be investigated. Bisulfite sequencing and reactivation by hydrodynamic injection of isotonic solution were employed to investigate methylation statues of CpG in transiently expressing plasmid, pCMV-Luc, in mouse liver after hydrodynamic delivery. The cytosines of CpGs in the promoter region of pCMV-Luc were methylated in mouse liver, but the methylation was much later than the decline in the expression. The expression from pre-methylated pCMV-Luc was insensitive to reactivation. Neither an inhibitor of DNA methylation nor an inhibitor of histone deacetylation had significant effects on transgene expression after hydrodynamic injection of pCMV-Luc. Partial hepatectomy, which reduces the transgene expression from the non-integrated vector into the genome, significantly reduced the transgene expression of human interferon γ from a long-term expressing plasmid pCpG-Huγ, suggesting that the CpG-reduced plasmid was not significantly integrated into the genomic DNA. These results indicate that the CpG-reduced plasmids achieve prolonged transgene expression without integration into the host genome, although the methylation status of CpG sequences in plasmids will not be associated with the prolonged expression.

Extracellular vesicles (EVs) are small membrane vesicles secreted from cells and have great potential as drug delivery carriers. Surface proteins on EV membranes might play roles in pharmacokinetics. One method which can be used to study the role of surface membrane of EV is to modify the inner space of EV. In the present study, we constructed a plasmid DNA expressing a fusion protein of Gag protein derived from Moloney murine leukemia virus (Gag) and Gaussia luciferase (gLuc) (Gag-gLuc) to modify the inner space of EVs. EVs were collected from B16BL6 melanoma cells, transfected with the plasmid, and isolated by a differential ultracentrifugation method. Gag-gLuc EVs were negatively charged globular vesicles with a diameter of approximately 100 nm. gLuc labeling of the Gag-gLuc EVs was stable in serum. gLuc activity of Gag-gLuc EVs was minimally decreased by proteinase K (ProK) treatment, indicating that gLuc was modified in the inner space of EV. Then, to evaluate the effect of the surface proteins of EVs on their pharmacokinetics, Gag-gLuc EVs treated with ProK were intravenously administered to mice. Volume of distribution (Vd) was significantly smaller for treated EVs than untreated EVs. Moreover, integrin α

We previously succeeded in the visualization of tissue distribution of B16BL6 cells-derived exosomes by labeling with Gaussia luciferase (gLuc)-LA, a fusion protein of gLuc (a reporter protein) and lactadherin (LA; an exosome-tropic protein). However, total amount of B16BL6-derived exosomes delivered to each organ could not be evaluated because of the reduction of luminescent signal from gLuc-LA. The aim of the present study was to quantitatively evaluate the tissue distribution of B16BL6-derived exosomes. To this end, we labeled B16BL6-derived exosomes with iodine-125 ((125) I) based on streptavidin (SAV)-biotin system. A plasmid vector encoding fusion protein, SAV-LA, was constructed, and B16BL6 cells were transfected with the plasmid to obtain SAV-LA-coupled exosomes. SAV-LA-coupled exosomes were incubated with (3-(125) I-iodobenzoyl) norbiotinamide ((125) I-IBB) to obtain (125) I-labeled B16BL6 exosomes. After intravenous injection of (125) I-labeled B16BL6 exosomes into mice, radioactivity quickly disappeared from the blood circulation. At 4 h, 28%, 1.6%, and 7% of the injected radioactivity/organ was detected in the liver, spleen, and lung, respectively. These results indicate that (125) I-labeling of exosomes using SAV-biotin system is a useful method to quantitatively evaluate the amount of exogenously administered exosomes delivered to each organ and that the liver is the major organ in the clearance of exogenously administered B16BL6-derived exosomes.

Computational fluid dynamics (CFD) analysis was conducted for a biplane-type otter board model to validate the use of CFD analysis in designing otter boards. In the base mode (aspect ratio 3.0, camber ratio 15%, gap-chord ratio 0.91, and stagger angle 30°), CFD analysis results show a maximum lift coefficient value of 1.65 for angle of attack 17.5°, which is in agreement with results of the flume tank experiment (CLmax = 1.64 when α = 20°) under the same conditions. In addition, the streamline and separation zones around the otter board were visualized and compared to the results from past tank experiment studies. CFD analysis results showed patterns similar to the experimental results, and the details of flow with a trailing vortex were confirmed. We concluded that CFD analysis is useful in the designing and development of otter boards.

Treatment of hepatitis C virus (HCV) infection has greatly improved in the last 5 years because of the identification of direct-acting antivirals (DAAs). However, concerns exist regarding the emergence of drug resistance-associated substitutions (RASs). In this study, we evaluated the in vivo antiviral effect of three classes of interferons (IFNs), namely, types I, II, and III IFNs, on DAA-resistant HCVs. IFN-α2, IFN-γ, and IFN-λ1 were selected as typical types I, II, and III IFNs, respectively. Human hepatocyte-transplanted chimeric mice were infected with NS3-D168, NS5A-L31-, and NS5A-Y93-mutated HCVs, and the antiviral effect of IFN-α2, IFN-γ, and IFN-λ1 on these HCV RASs was examined. Chimeric mice infected with NS3- and NS5A-mutated HCVs were hydrodynamically injected with IFN-expressing plasmids to evaluate the antiviral effect of IFNs. Serum concentrations of IFNs were maintained for at least 42 days. We found that serum HCV level significantly decreased and serum and hepatic HCV levels reached below detection limit in 5/5 and 3/5 chimeric mice injected with IFN-γ- and IFN-λ1-expressing plasmids, respectively. The antiviral effect of IFN-α2 on DAA-resistant HCVs was weaker than that of IFN-γ and IFN-λ1. Serum ALT levels showed a small and transient increase in mice injected with the IFN-γ-expressing plasmid but not in mice injected with the IFN-λ1-expressing plasmid. However, no apparent histological damage was observed in the liver sections of mice injected with the IFN-γ-expressing plasmid. These results indicate that IFN-γ and IFN-λ1 are an attractive therapeutic option for treating infection caused by NS3- and NS5A-mutated HCV.

Our previous study indicates that cationization of an antigen is effective for sustained release of both immunostimulatory DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG DNA, and antigen from a DNA hydrogel. Another approach to sustained antigen release would increase the applicability and versatility of the system. In this study, a hydrophobic interaction-based sustained release system of ovalbumin (OVA), a model antigen, from immunostimulatory CpG DNA hydrogel is developed by the use of cholesterol-modified DNA and urea-denatured OVA (udOVA). Cholesterol-modified DNA forms a hydrogel, Dgel(chol), and induces IL-6 mRNA expression in mouse skin after intradermal injection, as DNA without cholesterol does. Cholesterol-modified DNA associated with OVA and denaturation of OVA using urea increases the interaction. The release of udOVA from Dgel(chol) is significantly slower than that from DNA hydrogel with no cholesterol, Dgel. Moreover, intratumoral injections of udOVA/Dgel(chol) significantly inhibit the growth of EG7-OVA tumors in mice. These results indicate that sustained release of antigen from Dgel can be achieved by the combination of urea denaturation and cholesterol modification, and retardation of antigen release is effective to induce antigen-specific cancer immunity.

Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepared multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 µm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates containing the microwells resulted in the formation of HepG2 spheroids with average diameters of 195, 320, 493, and 548 µm. The cell number per spheroid positively correlated with its diameter, and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when preparing HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids.

A recent study demonstrated that heat stress induces mitochondrial biogenesis in C2C12 myotubes, thereby implying that heat stress may be an effective treatment to enhance endurance training-induced mitochondrial adaptations in skeletal muscle. However, whether heat stress actually induces mitochondrial adaptations in skeletal muscle in vivo is unclear. In the present study, we report the novel findings that 1) whole body heat stress produced by exposure of ICR mice to a hot environment (40°C, 30 min/day, 5 days/wk, 3 wk) induced mitochondrial adaptations such as increased mitochondrial enzyme activity (citrate synthase and 3-hydroxyacyl CoA dehydrogenase) and respiratory chain protein content (complexes I–V) in skeletal muscle in vivo and 2) postexercise whole body heat stress additively enhanced endurance training-induced mitochondrial adaptations (treadmill running, 25 m/min, 30 min/day, 5 days/wk, 3 wk). Moreover, to determine the candidate mechanisms underlying mitochondrial adaptations, we investigated the acute effects of postexercise whole body heat stress on the phosphorylation status of cellular signaling cascades that subsequently induce mitochondrial gene transcription. We found that whole body heat stress boosted the endurance exercise-induced phosphorylation of p38 MAPK, increased the phosphorylation status of p70S6K, a biomarker of mammalian target of rapamycin complex 1 activity, and unexpectedly dephosphorylated AMP-activated protein kinase and its downstream target acetyl-CoA carboxylase in skeletal muscle. Our present observations suggest that heat stress can act as an effective postexercise treatment. Heat stress treatment appeared to be clinically beneficial for people who have difficulty participating in sufficient exercise training, such as the elderly, injured athletes, and patients.

Nucleic acids, DNA and RNA, not only allow transfer and replication of densely coded genetic information, but also act as danger signals triggering innate immune response. Recent progress in the design and construction of nano-sized structures using DNA has opened a new field of nanotechnology. The unique properties of nano-sized DNA constructs can be exploited to develop programmable materials for efficient delivery of bioactive compounds. In this review, recent advances in DNA nanotechnology and its applications as delivery systems are summarized. In particular, we focus on the delivery of DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotide, or CpG motif, to immune cells expressing Toll-like receptor 9. Recent studies have shown that precisely designed DNA constructs, such as multi-branched DNA, polyhedral DNA, and DNA origami, can be used to enhance the biological activity of CpG DNA.

Interferon γ (IFN-γ), an anticancer agent, is a strong inducer of indoleamine 2, 3-dioxygenase 1 (IDO1), which is a tryptophan-metabolizing enzyme involved in the induction of tumor immune tolerance. In this study, we investigated the IDO1 expression in organs after IFN-γ gene transfer to mice. IFN-γ gene transfer greatly increased the mRNA expression of IDO1 in many tissues with the highest in the liver. This upregulation was associated with reduced L-tryptophan levels and increased L-kynurenine levels in serum, indicating that IFN-γ gene transfer increased the IDO activity. Then, Lewis lung carcinoma (LLC) tumor-bearing wild-type and IDO1-knockout (IDO1 KO) mice were used to investigate the effects of IDO1 on the antitumor activity of IFN-γ. IFN-γ gene transfer significantly retarded the tumor growth in both strains without any significant difference in tumor size between the two groups. By contrast, the IDO1 activity was increased only in the wild-type mice by IFN-γ gene transfer, suggesting that cells other than LLC cells, such as tumor stromal cells, are the major contributors of IDO1 expression in LLC tumor. Taken together, these results imply that IFN-γ gene transfer mediated IDO1 upregulation in cells other than LLC cells has hardly any effect on the antitumor activity of IFN-γ.

Investigation of mouse macrophage-like RAW264.7 cells showed that the immunostimulatory activity of CpG DNA is increased by formation of polypod-like structured DNA (polypodna), an assembly consisting of three or more oligodeoxynucleotides. To apply CpG polypodna to immunotherapy, its activity was examined in murine dendritic DC2.4 cells, splenic macrophages, and bone marrow-derived dendritic cells (BMDCs). In all cell types, increasing the pod number increased the cellular uptake of DNA and cytokine release. No significant release of cytokines was observed in macrophages lacking Toll-like receptor 9. Similar results were obtained after intradermal injection of polypodna. The polypodna preparations produced significantly higher amounts of interferon α in human peripheral blood mononuclear cells (PBMCs) compared with single-stranded DNA. The conditioned medium of hexapodna-treated human PBMCs effectively inhibited the activity of a hepatitis C virus subgenomic replicon reporter system. These results indicate that polypodna preparations are useful as an immunostimulator. From the Clinical Editor This study demonstrates the utility of polypoid-like structured DNA (polypodna) preparations as potent immunostimulators in a murine model.

Background Induction of the immune response to transgene products is a serious concern in gene therapy, and is generally known to be influenced by the transgene expression profile, as well as the types of cells that express the transgene. However, the exact nature of the association between the transgene expression profile and immune induction following gene transfer is unclear. Methods In the present study, plasmids, pCpG-fLuc or pCMV-fLuc, used for driving long- or short-term expression of firefly luciferase, respectively, were injected into mice by hydrodynamic injections along with a reporter plasmid expressing Gaussia luciferase (pROSA-gLuc) to evaluate the transgene expression profile in the liver. Single pROSA-gLuc administration resulted in stable gLuc activity in serum for more than 1 year; thus, gLuc activity was used for monitoring immune responses to the liver cells expressing both gLuc and fLuc after co-injection. Results A significant reduction in gLuc activity was observed 2 weeks after co-injection of pROSA-gLuc with pCpG-fLuc, whereas stable gLuc activity was observed when pROSA-gLuc was co-injected with pCMV-fLuc. A high level of fLuc-specific immunoglobulin G was detectable in pCpG-fLuc-injected mice; furthermore, histological analysis of the liver sections of these mice indicated CD8+ cell infiltration, implying that the transgene-expressing hepatocytes were removed by the infiltrating cells. Conclusions Our results demonstrate that sustained transgene expression in hepatocytes triggers antigen-specific immune responses, although short-term expression of the same transgene product elicits little, if any, immune response. Copyright © 2014 John Wiley & Sons, Ltd.

Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, liver failure, and hepatocellular carcinoma. Although the combination therapy employing pegylated interferon (IFN)-α and ribavirin is effective, this treatment is effective in only approximately 50% patients with genotype 1 HCV infection. IFN-γ is a potent anti-HCV agent that exhibits its antiviral action through a receptor distinct from that for IFN-α. Therefore, IFN-γ application might provide an alternative approach to IFN-α-based therapies. However, recombinant IFN-γ protein exhibits a poor pharmacokinetic property, that is, a very short half-life. It is our hypothesis that sustained IFN-γ serum concentrations produced by gene transfer could effectively eliminate HCV in vivo. We examined the in vivo antiviral activity in human hepatocyte chimeric mice infected with genotype 1b HCV at high HCV RNA titers (10(5)-10(7) copies/ml). The human IFN-γ-expressing plasmid vector pCpG-huIFNγ exhibited prolonged transgene expression in mice compared with the plasmid vector pCMV-huIFNγ. Moreover, the gene transfer of pCpG-huIFNγ eliminated HCV from the liver of the chimeric mice for a sustained period. On the contrary, administration of pCMV-huIFNγ could not eliminate HCV. In conclusion, we found that a single pCpG-huIFNγ injection resulted in long-term elimination of HCV RNA in chimeric mice, providing, for the first time, direct evidence that chronic infection with high titer HCV in vivo can be treated by sustained IFN-γ treatment.

Type II diabetes is one of the most problematic metabolic disorders and is associated with secondary conditions such as heart disease and eye complications. Interleukin-6 (IL-6), a multifunctional cytokine, could influence conditions of altered glucose metabolism such as insulin resistance in diabetic patients. However, a consensus about the role of IL-6 on glucose metabolism has not been reached. The aim of the present study is to investigate whether the expression of IL-6 affects glucose metabolism in diet-induced obesity (DIO) mice, a model of type II diabetes and obesity, using gene delivery of IL-6. DIO mice received hydrodynamic or intramuscular injection of IL-6-expressing plasmid to investigate the importance of the site of IL-6 expression. DIO mice that received a sustained IL-6 gene transfer showed similar glucose levels to lean mice in a glucose tolerance test. DIO mice exhibited reduced food intake and low body and epididymal fat weights after IL-6 gene transfer. IL-6 gene delivery reduced the mRNA expression of metabolism-related genes in the liver, skeletal muscle, and adipose tissue of DIO mice. The metabolic status of DIO mice receiving intramuscular injections was moderately better than that of DIO mice receiving hydrodynamic injections. The infiltration of inflammatory cells into the sites where the IL-6-expressing plasmid DNA was delivered was observed. Transient expression of IL-6 had limited effects on all parameters examined. These results indicate that the expression of IL-6 has an effect on obesity and the metabolism of glucose and lipid in diabetic mice and that the expression site of IL-6 is not an important factor.

Exosomes are cell-derived extracellular vesicles that function as intercellular delivery carriers. Our previous study demonstrated that macrophages in the liver contributed to the rapid clearance of intravenously administered B16BL6-derived exosomes from the systemic circulation in mice. Phosphatidylserine (PS) may be responsible for this clearance because it is exposed on the surface of exosomes and is recognized by macrophages. In this study, the role of PS exposed on the membranes of exosomes in the uptake of B16BL6-derived exosomes by macrophages was investigated. Negatively charged PS- or phosphatidylglycerol-loaded liposomes suppressed the cellular uptake of PKH67-labeled exosomes by macrophages, whereas phosphatidylcholine-containing liposome did not affect uptake. Subsequently, for the in vivo analysis, exosomes were labeled with Gaussia luciferase, a reporter protein, or (3-125I-iodobenzoyl)norbiotinamide using exosome-tropic fusion proteins comprising the exosome-tropic protein lactadherin. The blood clearance of Gaussia luciferase-labeled exosomes after intravenous injection into mice was significantly delayed by the preinjection of PS- or phosphatidylglycerol-containing liposomes. Moreover, the accumulation of (3-125I-iodobenzoyl)norbiotinamide-labeled exosomes in the liver was decreased by the preinjection of PS-containing liposomes. These results indicate that the negative charge of PS in exosomal membranes is involved in the recognition and clearance of intravenously injected exosomes by macrophages.

Type II interferon (IFNγ) is a representative Th1 cytokine and it possesses a variety of functions, including immune regulation, antiviral and antitumor activity. Because of its multifunctional nature, IFNγ is expected to be applied to the treatment of autoimmune diseases, cancer and viral infection. Although IFNγ has therapeutic potential for such diseases, the clinical use of IFNγ has been limited due to its short in vivo half-life and serious adverse effects. In contrast, gene delivery of IFNγ is an alternative approach to increasing the retention time of IFNγ. To extend transgene expression after plasmid DNA (pDNA) gene transfer, we designed and developed pDNA with varying numbers of CpG motifs. CpG-reduced pDNA resulted in more durable transgene expression than its CpG replete counterpart in mice. Comparison of the effect of promoter/enhancer elements on transgene expression showed that ROSA26 promoter-mediated IFNγ expression was safe because of the lack of an initial surge after hydrodynamic gene transfer. We also designed an IFNγ-mouse serum albumin (MSA) fusion protein, IFNγ-MSA. Gene transfer of this fusion protein resulted in a sustained concentration of IFNγ fusion protein in mouse serum, and inhibited tumor metastasis in mice. These results provide experimental evidence that IFNγ gene therapy can be a useful treatment for a variety of diseases.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-guanine (CpG) dinucleotides can be increased by converting it into branched structures. These structures could be stabilized by ligating the 5′- and 3′-ends to form a closed loop with no terminal ends. To further increase the ability of branched DNA assemblies to induce cytokines, a series of tetrapod-like structured DNA, or tetrapodna, were designed using four 48-base oligodeoxynucleotides (ODNs). All these preparations were designed to have the same sequence except for the nick sites, and all the ODNs of one of the tetrapodna preparations were ligated to obtain circular tetrapodna. The nick site significantly influenced the formation of the structure and melting temperature (Tm), but hardly affected the enzymatic stability of the tetrapodna preparations. Circular tetrapodna exhibited a significantly higher Tm and was more stable in mouse serum than its non-ligated counterparts. The amounts of cytokines released from macrophage-like RAW264.7 cells or dendritic DC2.4 cells after addition of circular tetrapodna were not significantly higher than those after addition of other tetrapodna preparations under conditions when no serum was present. However, when serum was present, circular tetrapodna induced the greatest amount of tumor necrosis factor-α, indicating that circular tetrapodna is effective in inducing cytokines under conditions where DNA-degrading enzymes are present. The cellular association of tetrapodna preparations was almost unaffected by ligation of the terminal ends. These results indicate that circular tetrapodna with no terminal ends is more effective than its non-ligated counterparts in the presence of serum.

Interferon (IFN) gene based therapy has been studied for the treatment of many diseases such as viral infections, cancer and allergic diseases. Non-viral vectors, like plasmid DNA, are promising ways for delivering IFN genes, because of their low immunogenicity and toxicity compared with viral vectors. Potent therapeutic effects of IFN gene transfer will depend on the level and duration of transgene expression after in vivo administration. Therefore, controlling the kinetics of transgene expression of IFNs is a rational approach for improved gene therapy. The design and optimization of plasmid vectors, as well as their route/method of administration, is the key to obtaining high and persistent transgene expression. In this review, we aim to present experimental evidence about the relationships among the properties of plasmid vectors expressing IFNs, the kinetics of transgene expression, and therapeutic effects as well as safety issues.