Ionic Liquid-Based Transcutaneous Peptide Antitumor Vaccine: Therapeutic Effect in a Mouse Tumor Model.

Traditional vaccinations need to be injected with needles, and since some people have a strong aversion to needles, a needle-free alternative delivery system is important. In this study, we employed ionic liquids (ILs) for transcutaneous delivery of cancer antigen-derived peptides to obtain anticancer therapeutic effects in a needle-free manner. ILs successfully increased the in vitro skin permeability of a peptide from Wilms tumor 1 (WT1), one of the more promising cancer antigens, plus or minus an adjuvant, resiquimod (R848), a toll-like receptor 7 agonist. In vivo studies demonstrated that concomitant transcutaneous delivery of WT1 peptide and R848 by ILs induced WT1-specific cytotoxic T lymphocyte (CTL) in mice, resulting in tumor growth inhibition in Lewis lung carcinoma-bearing mice. Interestingly, administrating R848 in ILs before WT1 peptides in ILs increased tumor growth inhibition effects compared to co-administration of both. We found that the prior application of R848 increased the infiltration of leukocytes in the skin and that subsequent delivery of WT1 peptides was more likely to induce WT1-specific CTL. Furthermore, sequential immunization with IL-based formulations was applicable to different types of peptides and cancer models without induction of skin irritation. IL-based transcutaneous delivery of cancer antigen-derived peptides and adjuvants, either alone or together, could be a novel approach to needle-free cancer therapeutic vaccines.

Mutagenesis, breeding, and characterization of sake yeast strains with low production of dimethyl trisulfide precursor.

Dimethyl trisulfide (DMTS) is one of the main components responsible for hineka, the aroma associated with deteriorated Japanese sake during storage. The molecule 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) has been previously identified as a major precursor compound of DMTS. Furthermore, it had been suggested that the yeast methionine salvage pathway is involved in the production of DMTS-P1. In sake brewing tests, DMTS-P1 and the DMTS producing potential (DMTS-pp; DMTS amount of sake after accelerated storage) were significantly reduced in mde1 or mri1 strain, which lack genes of the methionine salvage pathway. Industrial use of the gene-disrupting strains may not be accepted in the Japanese food industry. In order to obtain mde1 or mri1 mutants, we established a method to screen 5'-methylthioadenosine (MTA) non-utilizing strains using minimum culture medium containing methionine or MTA by ethyl methanesulfonate (EMS) mutagenesis with methionine-auxotrophic sake yeast haploid. As expected, mde1 and mri1 mutants were identified among the obtained mutants by an established screening method. The obtained strains had poor fermentation ability in sake brewing tests, so back-crossing was performed on the mutants to obtain mde1 or mri1 homozygous mutants. These strains had improved brewing characteristics, and DMTS-P1 and the DMTS-pp of the produced sake were significantly lower than those of the parent strains. These strains are expected to contribute to improving the maintenance of sake quality during storage.

Effective concentration of intravenous immunoglobulin for neutralizing Panton-Valentine leukocidin in human blood.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infects healthy individuals, although the precise cause remains unclear. CA-MRSA produces Panton-Valentine leukocidin (PVL), which often causes severe invasive infection; however, antitoxin drugs against PVL are limited. Intravenous immunoglobulin (IVIg) possesses antitoxin activity, but unfortunately, the optimal dose is unknown. Here, we measured the PVL neutralizing antibody titer in the plasma of Japanese individuals and sera of American donors. Next, we compared the cytotoxic effects of PVL on neutrophils in phosphate buffered saline (PBS) or whole blood to determine the effect of the neutralizing antibody. Finally, we evaluated the effective concentration of IVIg required to neutralize PVL in PBS and whole blood. We observed that the titer of PVL neutralizing antibody in healthy individuals polarized as high and low/none group. Additionally, the PVL neutralizing antibody titer considerably affected the concentration at which IVIg elicited its effect. This suggests that PVL-producing CA-MRSA might be involved in determining the severity of infection in healthy individuals without neutralizing antibody against PVL. The neutralizing effect of IVIg was observed in both PBS and whole blood. However, the optimal concentration of IVIg required for neutralizing PVL varied between PBS and whole blood. In addition, since the PVL-neutralizing activity of IVIg also largely depends on blood composition, such as neutralizing antibody concentration, the optimal dosage of IVIg as an antitoxin drug should be decided in a timely manner after considering the patient's medical background.

Intravenous immunoglobulin preparation attenuates neurological signs in rat experimental autoimmune neuritis with the suppression of macrophage inflammatory protein -1α expression.

To clarify the mechanism of action of an intravenous immunoglobulin (IVIG) preparation in chronic inflammatory demyelinating polyneuropathy, the effects of IVIG were investigated using an experimental autoimmune neuropathy model in the rat. IVIG significantly suppressed the progression of neurologic signs and sciatic nerve conduction velocity with the inhibition of inflammatory cell infiltration, mainly of macrophages, to the peripheral nerves. A significant suppressive effect on the expression of macrophage inflammatory protein 1-α (MIP-1α) was simultaneously observed in the nerves. These results suggest that IVIG is effective for inflammatory demyelinating polyneuropathy by inhibiting the chemotactic factor of macrophages.

Tag-assisted liquid-phase peptide synthesis using hydrophobic benzyl alcohols as supports.

A soluble tag-assisted liquid-phase peptide synthesis was successfully established based on simple hydrophobic benzyl alcohols, which can be easily prepared from naturally abundant materials. Excellent precipitation yields can be obtained at each step, combining the best properties of solid-phase and liquid-phase techniques. This approach can also be applied efficiently to fragment couplings, allowing chemical synthesis of several bioactive peptides.

Evaluation of the in vivo radiosensitizing activity of etanidazole using tumor-bearing chick embryo.

Chick embryos have been used as alternative experimental animals in various research fields, including virology, immunology, toxicology, oncology, and embryology. Until now, there have been no in vivo models using chick embryo to evaluate radiosensitizing activity. Here, the in vivo radiosensitizing activity of etanidazole, a well-known hypoxic cell radiosensitizer, was evaluated using tumor-bearing chick embryo. On the basis of tumor growth, drug administration and X-ray irradiation were performed on day 15 chick embryo, with the endpoint being day 18 chick embryo. In day 15 chick embryo, an X-ray irradiation dose of equal or less than 10 Gy did not cause significant tumor growth suppression. Intravenous administration of equal or less than 1.0 mg of etanidazole did not cause tumor growth suppression. Neither doses of equal or less than 8 Gy of irradiation nor 1.0 mg of etanidazole caused fatality of the chick embryo. On the basis of these results, we evaluated the radiosensitizing effect of a combination treatment with 8 Gy of irradiation and 1.0 mg of etanidazole. As noted above, 1.0 mg of etanidazole alone and 8 Gy of irradiation alone did not show tumor growth suppression. In contrast, a combination treatment with 8 Gy of irradiation and 1.0 mg of etanidazole showed 35% of significant tumor growth suppression. Thus, we succeeded in evaluating the in vivo radiosensitizing activity of etanidazole using tumor-bearing chick embryo. These results suggest that the use of tumor-bearing chick embryo may be part of a promising system for evaluating radiosensitizing activity.

Complement-mediated bacteriolysis after binding of specific antibodies to drug-resistant Pseudomonas aeruginosa: morphological changes observed by using a field emission scanning electron microscope.

A bactericidal mechanism mediated by human serum was investigated by a field emission scanning electron microscope and a strain of drug-resistant Pseudomonas aeruginosa. When the bacteria were treated with meropenem, a carbapenem antibiotic, spheroplasts and bulges (spheroidization) appeared after 1-3 h. When 40% serum was added to the bacteria, the bacteria agglutinated within 2 min and then lysed after 5-30 min. Immunoelectron micrographic analyses showed dispositions of complement component C9 molecules on the cell surface of lysed bacteria by the serum treatment that might suggest formation of a membrane attack complex. Immunoglobulin G (IgG) depletion from the serum diminished the lytic activity and adding human intravenous immunoglobulin (IVIG) restored it, suggesting that lysis was induced by specific IgG binding to the bacteria. IVIG may help patients with less IgG against bacteria to overcome severe infection.

Complement-mediated bactericidal effect of antibodies in human intravenous preparation against multi-drug resistant pseudomonas aeruginosa.

After incubation of four of ten strains of multi-drug resistant Pseudomonas aeruginosa (MDRP) with the healthy human serum for one hour, the bacteria lysed and the viable cell count decreased with the disappearance of the C3 complement. Over 99% of IgG was deleted from the serum by passing the serum through a Protein G Sepharose column. In such IgG-deleted serum, the bactericidal activity on P. aeruginosa was cancelled. The bactericidal activity recovered by adding human intravenous immunoglobulin preparation (IVIG) at the concentration of 2-4 mg/mL to the serum. Moreover, a strong bactericidal activity was shown in the case of some strains that the antibody titers of IVIG were high. It was thought that specific antibodies to some of P aeruginosa are contained in IVIG and activated the classical pathway of complement system; as a result, the bacterial lyses had been caused. Sera from healthy persons contain antibodies to P aeruginosa as well as complement components. Since the complement-mediated bactericidal activity disappeared by removing IgG in this study. These results suggest that the IVIG therapy in bacterial infections is expected to recover the immune function so as complement-mediated bactericidal activity as well as opsonic activity against P aeruginosa, especially in the patient with neutropenia.

Design, synthesis, and radiosensitizing activities of sugar-hybrid hypoxic cell radiosensitizers.

We have designed sugar-hybrid TX-1877 derivatives conjugated with sugar moieties including beta-glucose (beta-Glc), beta-galactose (beta-Gal), alpha-mannose (alpha-Man) and N-acetyl-beta-galactosamine (beta-GalNAc). Compound 1 (TX-1877) was glycosylated with appropriate peracetylated sugars using BF(3)-OEt(2) to give acetylated sugar-hybrids, 5 (TX-2244), 6 (TX-2245), 7 (TX-2246), and 10 (TX-2243). Removal of the acetyl groups afforded the sugar-hybrids having free hydroxyl groups, 11 (TX-2141), 12 (TX-2218), 13 (TX-2217) and 14 (TX-2068). We evaluated their radiosensitizing activities by an in vitro radiosensitization assay. All free hydroxyl hybrids have lower enhancement ratio (ER) values (ER1.43) and lower n-octanol/water partition coefficient (P(oct)) values (P(oct)<1.00x10(-2)) than does 1 (TX-1877, ER=1.75, P(oct): 5.60x10(-2)). All acetylated hybrids have similar P(oct) values (3.55x10(-2)-1.05x10(-1)) to 1 (TX-1877) and have improved ER values (ER>or=1.47) compared to the hybrids having free hydroxyl groups. Among these, 5 (TX-2244) is the most active radiosensitizer (ER=2.30). We found a good correlation (r=0.866) between the magnitude of P(oct) (logP(oct)) and the ER value of 5 (TX-2244), 6 (TX-2245), 7 (TX-2246), 10 (TX-2243) and 1 (TX-1877), suggesting that increasing the hydrophobicity is reflected in increased in vitro radiosensitizing activity. In the present study, we have succeeded in producing sugar-hybrid hypoxic cell radiosensitizers that have an increased radiosensitizing activity that does not depend on increased hydrophobicity.

A new murine model to define the critical pathologic and therapeutic mediators of polymyositis.

OBJECTIVE: To establish a new murine model of polymyositis (PM) for the understanding of its pathologic mechanisms and the development of new treatment strategies. METHODS: C protein-induced myositis (CIM) was induced by a single immunization of recombinant human skeletal C protein in C57BL/6 mice, as well as in CD4-depleted, CD8-depleted, and mutant mice as controls. Some mice were treated with high-dose intravenous immunoglobulin (IVIG) after disease induction. Muscle tissues were examined histologically. RESULTS: In mice with CIM, inflammation was confined to the skeletal muscles. Histologic examination revealed a common pathologic feature of CIM and PM, involving abundant infiltration of CD8 and perforin-expressing cells in the endomysial site of the injured muscle. Suppression of myositis was achieved by depletion of both CD4 and CD8 T cells. Despite the development of serum anti-C protein antibodies in wild-type mice, severe myositis was induced in mice deficient in B cells. Induction of myositis was suppressed in interleukin-1alpha/beta (IL-1alpha/beta)-null mutant mice, but not in tumor necrosis factor alpha (TNFalpha)-null mutant mice. Use of IVIG, a treatment with proven efficacy in PM, suppressed CIM in the subgroup of treated mice. CONCLUSION: CIM mimics PM pathologically and clinically. Infiltration of CD8 T cells is the most likely mechanism of muscle injury, and IL-1, but not B cells or TNFalpha, is crucial in the development of CIM. IVIG has therapeutic effects in CIM, suggesting that the effects of IVIG are not mediated by suppression of antibody-mediated tissue injury. This murine model provides a useful tool for understanding the pathologic mechanisms of PM and for developing new treatment strategies.

[Neutralizing activity of human immunoglobulin preparation against toxic shock syndrome toxin-1].

Human intravenous immunoglobulin (IVIG) preparation containing a variety of antibodies is widely used against severe infectious diseases. Although IVIG is supposed to promote phagocytosis of opsonized bacteria and neutralize several bacterial toxins, it is unclear what antibodies are responsible for the effect in clinical use. In this study, we found that IVIG showed neutralizing activity against toxic shock syndrome toxin-1 (TSST-1), produced by methicillin-resistant Staphylococcus aureus (MRSA). Whereas intravenous inoculation with culture medium of MRSA 1945 strain into ICR mice causes immediate death, all of the mice survived in case of previous administration of IVIG. Such effect might be attributed to neutralization of TSST-1. Murine splenocytes incubated with TSST-1 (1.0 ng/mL) for 48 hours produced IFN-gamma. By addition of IVIG at 100 micrograms/mL into culture medium, production of IFN-gamma was completely inhibited. From IVIG, anti-TSST-1 antibody was purified by affinity chromatography as one of the effective antibodies. MRSA 1945 mixed with dextran-based microcarrier beads and injected subcutaneously into mice resulted in the formation of abscess and excretion of TSST-1 in serum for 14 days. IVIG and anti-TSST-1 antibody neutralized TSST-1 in blood 24 hours after infection, although bacterial count was kept constant. Experimental rabbit TSS model was established by synergism with lipopolysaccharide (LPS) of Escherichia coli. Anti-TSST-1 antibody protected NZW rabbits from lethal challenge with TSST-1 (1 microgram/kg, i.v.) 4 hours before LPS in a dose-dependent manner (10 micrograms/kg, i.v.), whereas all of the NZW rabbits died in the control group. Thus, IVIG may be a useful tool in the prevention and perhaps therapy of staphylococcal infections and TSS.

Synergistic Antibacterial Activity of a New Quinolone, T-3762, and Human Gamma Globulin Against Intracellular Salmonella.

The bactericidal activity of antibacterial agents against intracellular bacteria using a Salmonella-lined macrophage model (TtT/M-87), established from mouse pituitary tumor-associated macrophages, was investigated in this study. Because a virulent strain of Salmonella enteritidis 116-54 is able to proliferate in phagocytized macrophages, the bactericidal activity against intracellular bacteria can be determined using this model. We found that a new quinolone, T-3762, has strong bactericidal activity against virulent S. enteritidis in macrophages. The bactericidal activity of T-3762 was augmented by the addition of human γ-globulin. The combination of T-3762 and human γ-globulin showed significantly higher bactericidal activity than that of ofloxacin and human γ-globulin. These results suggest that T-3762 can penetrate mammalian cells in an active form and that human γ-globulin may be able to enhance the bactericidal activity of macrophages by opsonization of bacteria with antibody in the γ-globulin fraction.