The therapeutic effect of TNFR1-selective antagonistic mutant TNF-alpha in murine hepatitis models

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in a wide variety of inflammatory pathologies, such as hepatitis, via the TNF receptor-1 (TNFR1). To develop TNFR1-targeted anti-inflammatory drugs, we have already succeeded in creating a TNFR1-selective antagonistic mutant TNF-alpha (R1antTNF) and shown that R1antTNF efficiently inhibits TNF-alpha/TNFR1-mediated biological activity in vitro. In this study, we examined the therapeutic effect of R1antTNF in acute hepatitis using two independent experimental models, induced by carbon tetrachloride (CCl(4)) or concanavalin A (ConA). In a CCl(4)-induced model, treatment with R1antTNF significantly inhibited elevation in the serum level of ALT (alanine aminotransferase), a marker for liver damage. In a ConA-induced T-cell-mediated hepatitis model, R1antTNF also inhibited the production of serum immune activated markers such as IL-2 and IL-6. These R1antTNF-mediated therapeutic effects were as good as or better than those obtained using conventional anti-TNF-alpha antibody therapy. Our results suggest that R1antTNF may be a clinically useful TNF-alpha antagonist in hepatitis.     

Transcriptomic characterization of gall tissue of Japanese elm tree (Ulmus davidiana var. japonica) induced by the aphid Tetraneura nigriabdominalis

Insect galls are abnormal plant tissues induced by parasitic insect(s) for use as their habitat. In previous work, we suggested that gall tissues induced by the aphid Tetraneura nigriabdominalis on Japanese elm trees are less responsive than leaf tissues to jasmonic acid (JA), which is involved in the production of volatile organic compounds as a typical defensive reaction of plants against attack by insect pests. A comprehensive analysis of gene expression by RNA sequencing indicated that the number of JA responsive genes was markedly lower in gall tissues than in leaf tissues. This suggests that gall tissues are mostly defective in JA signaling, although JA signaling is not entirely compromised in gall tissue. Gene ontology analysis sheds light on some stress-related unigenes with higher expression levels in gall tissues, suggesting that host plants sense aphids as a biotic stress but are defective in the JA-mediated defense response in gall tissues.     

PEGylated adenovirus vectors containing RGD peptides on the tip of PEG show high transduction efficiency and antibody evasion ability

BACKGROUND: PEGylation of adenovirus vectors (Ads) is an attractive strategy in gene therapy. Although many types of PEGylated Ad (PEG-Ads), which exhibit antibody evasion activity and long plasma half-life, have been developed, their entry into cells has been prevented by steric hindrance by polyethylene glycol (PEG) chains. Likewise, sufficient gene expression for medical treatment could not be achieved. METHODS: A set of PEG-Ads, which have different PEG modification rates, was constructed, and gene expression was evaluated using A549 cells. A novel PEGylated Ad (RGD-PEG-Ad), which contained RGD (Arg-Gly-Asp) peptides on the tip of PEG, was developed. We evaluated gene expression both in Coxsackie-adenovirus receptor (CAR)-positive as well as -negative cells, and in vivo gene expression was also determined. Furthermore, the antibody evasion ability and the specificity of infection exhibited by this RGD-PEG-Ad were also evaluated. RESULTS: Whereas PEG-Ads decreased gene expression in CAR-positive cells, RGD-PEG-Ad enhanced gene expression notably, to a level about 200-fold higher than that of PEG-Ads. Moreover, gene expression of RGD-PEG-Ad was almost equal to that of Ad-RGD, which contains an RGD-motif in the fiber and exhibits among the highest gene expression of CAR-positive and -negative cells. Furthermore, although Ad-RGD gene expression decreased remarkably in the presence of anti-Ad antiserum, RGD-PEG-Ad maintained its activity against antibodies. In vivo experiments also demonstrated that the modification of Ads with RGD-PEG induced efficient gene expression. CONCLUSIONS: In the present study, we demonstrated that a new strategy, which combined integrin-targeting the RGD peptide on the tip of PEG and modified the Ad using this material, could enhance gene expression in both CAR-positive and -negative cells. At the same time, this novel PEGylated Ad maintained strong protective activity against antibodies. This strategy could also be easily modified for developing other vectors using other targeting molecules.     

Adaptive change and conservatism in host specificity in two local populations of the thistle-feeding ladybird beetle Epilachna niponica

We investigated geographic differences in the host specificity of Epilachna niponica Lewis (Coleoptera: Coccinellidae). The Yuwaku population feeds mainly on Cirsium matsumurae Nakai (Asteraceae) and secondarily on Cirsrium kagamontanum Nakai. The Asiu population, located 150 km away from the Yuwaku, feeds exclusively on C. ashiuense Yokoyama et T. Shimizu. Under laboratory conditions, we examined the differences between the two populations in adult feeding acceptance, adult feeding preference, and larval performance, using several closely related thistle species and varieties, including their native hosts. In the Asiu population, adult beetles clearly avoided the host of the Yuwaku population, C. kagamontanum, and no larvae were able to complete their development, whereas in the Yuwaku population, adults accepted and even preferred it to some other thistle species, and about 10% of first instar larvae became adults. This indicates that the Yuwaku population evolved its feeding preference and physiological adaptation to C. kagamontanum through a utilization of this low‐ranked host under natural conditions. Apart from C. kagamontanum, the two populations showed a similar host susceptibility pattern, indicating that this ladybird beetle has a conserved hierarchy in feeding preference and growth performance. We also observed adult leaf choice behavior when given different thistle species, and found that difference in biting rate after palpation determined the leaf areas consumed, implying that factors on the leaf surface played an important role in the choice.     

Titanium dioxide induces different levels of IL-1β production dependent on its particle characteristics through caspase-1 activation mediated by reactive oxygen species and cathepsin B

Although titanium dioxide (TiO₂) is widely used, its inhalation can induce inflammatory diseases accompanied by interleukin-1β (IL-1β) production. The particle characteristics of TiO₂ are important factors in its biological effects. It is urgently necessary to investigate the relationship between the particle characteristics and biological responses for the development of safe forms of TiO₂. Here, we systematically compared the production of IL-1β in response to various forms of TiO₂ by macrophage-like human THP-1 cells using various sizes (nano to micro), crystal structures (anatase or rutile), and shapes (spherical or spicular) of TiO₂. The production of IL-1β depended dramatically on the characteristics of the TiO₂. Notably, smaller anatase and larger rutile particles provoked higher IL-1β production. In addition, IL-1β production depended on active cathepsin B and reactive oxygen species production independent of the characteristics of TiO₂. Our results provide basic information for the creation of safe and effective novel forms of TiO₂.     

Comparison of a Stool Antigen Test and Serology for the Diagnosis of Helicobacter pylori Infection in Mass Survey

Background: Serum antibody to Helicobacter pylori is tested in mass screening for gastric cancer along with the level of serum pepsinogens (PG) I and II. Recently, stool antigen tests have been developed as a new non-invasive test. We examined H. pylori infection by both serology and stool antigen test in a mass survey and compared the results to estimate applicability of stool antigen test for mass survey.
Methods: A total of 994 healthy adults who received mass survey in April 2005 were tested. There were 379 men and 615 women, and the mean age was 57.7 years old. Stool samples were used to measure a H. pylori- specific antigen by enzyme immunoassay. Serum samples were tested for the prevalence of IgG antibody to H. pylori , and the level of PGs I and II was also measured to determine the presence of atrophic gastritis.
Results: Infection of H. pylori was defined as positive 61.4% and 56.4% by serology and stool antigen test, respectively. The concordance of both tests was not affected by gender and age of the subjects but difference was seen in subjects with atrophic gastritis. In particular, positivity of stool antigen test (81.8%) was significantly lower than that of serology (88.7%, p  < .05) in 303 subjects with severe atrophic gastritis.
Conclusions: Stool antigen test, which detects present but not previous infection of H. pylori , would be applicable to diagnose H. pylori infection in mass survey. Usefulness of stool antigen tests for the screening of gastric cancer should be examined.     

Regulation of Organ Formation by Cytokinin and Auxin in Lilium Bulbscales Grown In Vitro

Regulation of organ formation by cytokinin and auxin was investigatedin vitro using Lilium auratum Lindl. (wild species habituatedin Japan) and Lilium speciosum Thunb. cv. "Uchida". The interactionof -naphthylacetic acid (NAA) and kinetin on bulbscale or rootdifferentiation was examined. NAA and kinetin showed mainlyindividual but also some synergistic effects. The effects ofbenzyladenine (BA) and kinetin were compared and the resultindicated that BA has a stronger physiological effect on organformation than kinetin and that their effects on Lilium auratumand Lilium speciosum were BA or kinetin-specific. The actionof kinetin on Lilium was affected by sucrose concentration andthe strength of the Murashige and Skoog medium (MS medium),and thus their high concentrations inhibited the kinetin-inducedbulbscale differentiation. Furthermore, a high sucrose levelnegated the kinetin inhibition of root formation, while highMS medium strength in itself inhibited root formation. Morphologicalobservation of bulbscale differentiation induced under a highkinetin level revealed that the new-formed structures are homologousto normally grown bulbs in soil in spite of their particularfeatures. (Received August 3, 1981; Accepted November 2, 1981)     

The Relationship of the Feces Protein Particles to Rice Protein Bodies

Feces protein particles (FPP), spherical or oval in shape, 1.0~3.5 μ in diameter, which were observed in the fresh feces of the Japanese, in an average density of 5 × 10⁹/g, resembled the rice protein bodies (RPB) in electron-microscopical fine structure. The FPP decreased in the feces of the man who had been given a rice-free diet consisting of steamed sweet potato; when boiled rice had been fed, the FPP increased, and reached the level of the original density within 5 days. The FPP were concluded to be derived from an indigestible fraction of the RPB which were supplied to the Japanese with the daily intake of rice.     

A single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with Meth-A fibrosarcoma

Cytokine-encoding viral vectors are considered to be promising in cancer gene immunotherapy. Interleukin 12 (IL-12) has been used widely for anti-tumor treatment, but the administration route and tumor characteristics strongly influence therapeutic efficiency. Meth-A fibrosarcoma has been demonstrated to be insensitive to IL-12 treatment via systemic administration. In the present study, we developed an IL-12-encoding fiber-mutant adenoviral vector (AdRGD-IL-12) that showed enhanced gene transfection efficiency in Meth-A tumor cells, and the production of IL-12 p70 in the culture supernatant from transfected cells was confirmed by ELISA. In therapeutic experiments, a single low-dose (2 x 10(7) plaque-forming units) intratumoral injection of AdRGD-IL-12 elicited pronounced anti-tumor activity and notably prolonged the survival of Meth-A fibrosarcoma-bearing mice. Immunohistochemical staining revealed that the IL-12 vector induced the accumulation of T cells in tumor tissue. Furthermore, intratumoral administration of the vector induced an anti-metastasis effect as well as long-term specific immunity against syngeneic tumor challenge.     

Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen.