Mechanism of reactive oxygen species generation and oxidative DNA damage induced by acrylohydroxamic acid, a putative metabolite of acrylamide.

Acrylamide is formed during the heating of food and is also found in cigarette smoke. It is classified by the International Agency for Research on Cancer as a probable human carcinogen (Group 2A). Glycidamide, an epoxide metabolite of acrylamide, is implicated in the mechanism of acrylamide carcinogenicity. Acrylamide causes oxidative DNA damage in target organs. We sought to clarify the mechanism of acrylamide-induced oxidative DNA damage by investigating site-specific DNA damage and reactive oxygen species (ROS) generation by a putative metabolite of acrylamide, acrylohydroxamic acid (AA). Our results, using 32P-5'-end-labeled DNA fragments, indicated that, although AA alone did not damage DNA, AA treated with amidase induced DNA damage in the presence of Cu(II). DNA cleavage occurred preferentially at T and C, and particularly at T in 5'-TG-3' sequences, and the DNA cleavage pattern was similar to that of hydroxylamine. The DNA damage was inhibited by methional, catalase, and Cu(I)-chelator bathocuproine, suggesting that H2O2 and Cu(I) are involved in the mechanism of DNA damage induced by AA treated with amidase. In addition, amidase-treated AA increased 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in calf thymus DNA, an indicator of oxidative DNA damage, in a dose-dependent manner. In conclusion, hydroxylamine, possibly produced from AA treated with amidase, was autoxidized via the Cu(II)/Cu(I) redox cycle and H2O2 generation, suggesting that oxidative DNA damage induced by ROS plays an important role in acrylamide-related carcinogenesis.

Combination of syringaresinol-di-O-ß-D-glucoside and chlorogenic acid shows behavioral pharmacological anxiolytic activity and activation of hippocampal BDNF-TrkB signaling.

Mental stress, such as anxiety and conflict, causes physiological changes such as dysregulation of autonomic nervous activity, depression, and gastric ulcers. It also induces glucocorticoid production and changes in hippocampal brain-derived neurotrophic factor (BDNF) levels. We previously reported that Acanthopanax senticosus HARMS (ASH) exhibited anxiolytic activity. Thus, we attempted to identify the anxiolytic constituents of ASH and investigated its influence on hippocampal BDNF protein expression in male Sprague Dawley rats administered chlorogenic acid (CHA), ( +)-syringaresinol-di-O-ß-D-glucoside (SYG), or a mixture of both (Mix) for 1 week using the open field test (OFT) and improved elevated beam walking (IEBW) test. As with ASH and the benzodiazepine anxiolytic cloxazolam (CLO), Mix treatment significantly increased locomotor activity in the OFT. CHA and Mix increased the time spent in the open arm in the IEBW test. SYG and Mix treatment inhibited the significant increase in normalized low-frequency power, indicative of sympathetic nervous activity, and significant decrease in normalized high-frequency power, indicative of parasympathetic nervous activity, as observed in the IEBW test. SYG and Mix treatment significantly increased hippocampal BDNF protein expression. The combination of CHA and SYG possibly induces anxiolytic behavior and modulates autonomic regulation, activates hippocampal BDNF signaling as with ASH.

Reliability and validity of on-road driving tests in vulnerable adults: a systematic review.

The on-road driving test is considered a 'gold standard' evaluation; however, its validity and reliability have not been sufficiently reviewed. This systematic review aimed to map out and synthesize literature regarding on-road driving tests using the Consensus-based Standards for the Selection of Health Measurement Instruments checklist. Cochrane Library, PubMed, CINAHL, and Web of Science databases were searched from initiation through February 2018. All articles addressing reliability or validity of on-road driving tests involving adult rehabilitation patients were included. The search output identified 513 studies and 36 articles, which were included in the review. The Washington University Road Test/Rhode Island Road Test, performance analysis of driving ability, test ride for investigating practical fitness-to-drive, and K-score demonstrated high reliability and validity in regard to the Consensus-based Standards for the Selection of Health Measurement Instruments checklist. The Washington University Road Test/Rhode Island Road Test and test ride for investigating practical fitness-to-drive were analyzed based on Classical Test Theory techniques, and performance analysis of driving ability and K-score were analyzed based on Item Response Theory techniques. The frequency of studies were Washington University Road Test/Rhode Island Road Test (n=9), Test Ride for Investigating Practical fitness-to-drive (n=8), performance analysis of driving ability (n=4), and K-score (n=1). From the viewpoint of accuracy and generalization, the Washington University Road Test/Rhode Island Road Test, test ride for investigating practical fitness-to-drive, and performance analysis of driving ability were identified as highly qualified concerning on-road driving tests. However, the ability to assess real-world driving depends on various environmental conditions.

Identification of the minimal region of peptide derived from ADP-ribosylation factor1 (ARF1) that inhibits IgE-mediated mast cell activation.

Mast cells play a pivotal role in allergic reactions and inflammations. Aggregation of the high affinity IgE receptor (FcεRI) eventually leads to the release of granule components such as histamine, as well as the de novo synthesis of inflammatory cytokines and lipid mediators. These substances are involved in the development of allergy and inflammation. Therefore, efficient inhibitors of mast cell activation would be therapeutically beneficial. We previously demonstrated that the synthetic peptide derived from the NH2-terminal region (2-17: GNIFANLFKGLFGKKE) of a small GTPase ARF1 (ADP-ribosylation factor1) inhibited FcεRI-induced mast cell degranulation. However, detailed structure-activity relationship study of NH2-terminal portion of ARF1 peptide has not been done. In addition, it is still unclear whether the NH2-terminal peptide of ARF1 suppresses FcεRI-induced production of cytokines and lipid mediators such as leukotriene C4 (LTC4) from mast cells. Here we show that amino acid residues K10-K16 are necessary for ARF1 peptide to efficiently inhibit FcεRI-induced activation of bone marrow-derived mast cells (BMMCs), indicated by decreased mast cell degranulation, cytokine secretion and leukotriene release. Furthermore, we show that ARF1 peptide inhibits IgE-mediated passive cutaneous anaphylaxis reaction. Our results suggest that the peptide derived from ARF1 could be developed into a novel anti-allergic agent for therapeutic intervention in allergy and mast cell-related pathologies.

Development of an activity-based probe for amyloid ß-hydrolyzing antibodies.

We report developing an activity-based probe containing an amyloid ß peptide (Aß) 17-27 and an electrophilic phosphonate diester at the C-terminus. A probe containing an electrophilic moiety is able to react with the nucleophiles on an antibody or an antibody with proteinase activity. The probe reacted with an Aß specific monoclonal antibody and formed a covalent complex. The covalent binding also occurred specifically when the probe reacted with serum containing anti-Aß antibodies. These results suggest that the probe would serve as a powerful tool to isolate Aß specific antibodies that are capable of Aß hydrolysis activity.

6-(4-Amino-2-butyl-imidazoquinolyl)-norleucine: Toll-like receptor 7 and 8 agonist amino acid for self-adjuvanting peptide vaccine.

Generally, small peptides by themselves are weak to induce antibody responses. Toll-like receptor (TLR) ligands are attractive candidates of vaccine adjuvants to improve their antigenicity. The covalent conjugation of TLR ligands with antigens to produce self-adjuvanting peptide vaccine is a promising approach. Based on the structure of TLR7/8 ligands, a series of synthetic amino acids 6-imidazoquinolyl-norleucines were synthesized, wherein an imidazoquinoline structure as the TLR7/8 agonistic pharmacophores was constructed on the ε-NH2 group of Lys. Of them, 6-(4-amino-2-butyl-imidazoquinolyl)-norleucine showed the most potent TLR7 and TLR8 agonistic activities with EC50 values of 8.55 and 106 µM, respectively. Subsequently, mice were immunized with the influenza A virus M2e antigen mixed with or covalently conjugated to the TLR7/8 agonist amino acid, which led to induction of M2e specific antibody productions in the absence of other adjuvant. We successfully developed a novel efficient tool for self-adjuvanting peptide vaccines targeting TLR7/8.

Relationship between Functional Visual Acuity and Useful Field of View in Elderly Drivers.

PURPOSE: To investigate the relationship between the functional visual acuity (FVA) and useful field of view (UFOV) in elderly drivers and assess the usefulness of the FVA test to screen driving aptitude in elderly drivers. METHODS: This study included 45 elderly drivers (31 men, 14 women; mean age, 68.1 years) and 30 younger drivers (26 men, 4 women; mean age, 34.2 years) who drive regularly. All participants underwent measurement of the binocular corrected distant visual acuity (CDVA), binocular corrected distant FVA (CDFVA), and Visual Field with Inhibitory Tasks Elderly Version (VFIT-EV) to measure UFOV. The tear function and cognitive status also were evaluated. RESULTS: The CDVA, the CDFVA, cognitive status, and the correct response rate (CAR) of the VFIT-EV were significantly worse in the elderly group than in the control group (P = 0.000 for all parameters). The cognitive status was correlated significantly with the CDVA (r = -0.301, P = 0.009), CDFVA (r = -0.402, P = 0.000), and the CAR of the VFIT-EV (r = 0.348, P = 0.002) in all subjects. The results of the tear function tests were not correlated with the CDVA, CDFVA, or VFIT-EV in any subjects. Stepwise regression analysis for all subjects in the elderly and control groups showed that the CDFVA predicted the CAR most significantly among the clinical factors evaluated. CONCLUSION: The FVA test is a promising method to screen the driving aptitude, including both visual and cognitive functions, in a short time.

Vaccination with lipid core peptides fails to induce epitope-specific T cell responses but confers non-specific protective immunity in a malaria model.

Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP) vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4(+) and/or CD8(+) T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8(+) T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8(+) T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge.

Structure-activity relationship for the development of a self-adjuvanting mucosally active lipopeptide vaccine against Streptococcus pyogenes.

Infection with group A streptococcus (GAS) can result in a number of diseases, some of which are potentially life-threatening. The oral-nasal mucosa is a primary site of GAS infection, and a mucosally active vaccine candidate could form the basis of an antidisease and transmission-blocking GAS vaccine. In the present study, a peptide from the GAS M protein (J14) representing a B cell epitope was incorporated alongside a universal T cell helper epitope and a Toll-like receptor 2 targeting lipid moiety to form lipopeptide constructs. Through structure activity studies, we identified a vaccine candidate that induces J14-specific mucosal and systemic antibody responses when administered intranasally without additional adjuvants. The systemic antibodies elicited were capable of inhibiting the growth of GAS. In addition, J14-specific mucosal antibodies corresponded with reduced throat colonization after respiratory GAS challenge. These preclinical experiments show that this lipopeptide could form the basis of an optimal needle-free mucosal GAS vaccine.

Overview and outlook of Toll-like receptor ligand-antigen conjugate vaccines.

The discovery of Toll-like receptors (TLRs) facilitated our understanding of the innate and adaptive immune systems, and has raised the potential to develop novel methods of vaccine and immunotherapy. For effective vaccination, antigens and adjuvants must be administered simultaneously via the same route. Many studies have demonstrated that TLR ligands covalently coupled to the antigens have several benefits over nonconjugated antigens. This review introduces the applications of TLR ligands as vaccine adjuvants, focusing on the development of vaccines composed of antigen and TLR ligand in single molecules (TLR ligand-antigen conjugates) using Pam3/2Cys, lipid A analogues, recombinant flagellin, imidazoquinoline analogues and unmethylated CpG motifs to activate immune systems through TLR2, TLR4, TLR5, TLR7/8 and TLR9, respectively.

[Problems impeding the introduction of care services under the long-term care insurance system].

OBJECTIVE: To identify problems currently impeding the introduction of care services to seniors who are not yet taking advantage of such services despite the need for some kind of in-home care, and to examine effective solutions by creating a model that clarifies relationships among these problems based on covariance structural analysis. METHODS: An anecdotal self-completion questionnaire was sent by mail to public health nurses who provide consultations to seniors in 657 locations throughout the mainland Japan, Honshu. The cases targeted in this survey were seniors for whom the introduction of care services was perceived to be difficult. Respondents were asked to relate one particularly memorable case encountered since April 2000 in which intervention assistance was provided through home visits. The survey consisted of 43 questions, including demographic information, basic case data, the outcome of intervention assistance in the case cited, and obstacles to introducing nursing services. We analyzed the 311 valid responses received (valid response rate: 47.3%). After performing factor analysis on the problems that were considered to impede the introduction of care services, we examined the relationships among these problems using covariance structural analysis and selected the model that best fit the data. RESULTS: 1) Problems that were considered to impede the introduction of care services were extracted from the results of an item analysis and factor analysis as follows. Factor 1: "Resistance to changing lifestyle." Factor 2: "Relative's lack of understanding or cooperation." Factor 3: "Lack of ability to handle procedures and contracts." Factor 4: "Lack of informal support." Factor 5: "Resistance to undergoing medical exams." 2) We performed a covariance structural analysis using the five factors derived from the factor analysis as the latent variables, and selected the best-fitting model, in which GFI = 0.929, AGFI = 0.901, and CFI = 0.950. The model showed that factors 3, 4, and 5 overlapped with factors 1 and 2 in impeding the introduction of nursing services, thus impeding the introduction of care services. CONCLUSION: The relationships among the problems impeding the introduction of care services were clarified using an anecdotal survey administered to public health nurses. To provide adequate support to these seniors, efforts must be made to examine community-based methods of providing intervention assistance tailored to the needs of individuals, as well as to examine systems of identifying and accommodating seniors who require assistance because they lack the ability to handle bureaucratic procedures themselves and also lack other sources of support.

Immunological evaluation of lipopeptide group A streptococcus (GAS) vaccine: structure-activity relationship.

Streptococcus pyogenes (group A streptococcus, GAS) is a Gram-positive bacterial pathogen responsible for a wide variety of diseases. To date, GAS vaccine development has focused primarily on the M-protein. The M-protein is highly variable at the amino (N)-terminus (determining serotype) but is conserved at the carboxyl (C)-terminus. Previously a 29 amino acid peptide (named J14) from the conserved region of the M-protein was identified as a potential vaccine candidate. J14 was capable of eliciting protective antibodies that recognized many GAS serotypes when co-administered with immuno-stimulants. This minimal epitope however showed no immunogenicity when administered alone. In an attempt overcome this immunological non-responsiveness, we developed a self-adjuvanting vaccine candidate composed of three components: the B-cell epitope (J14), a universal helper T-cell epitope (P25) and a lipid moiety consisting of lipoamino acids (Laas) which target Toll-like receptor 2 (TLR2). Immunological evaluation in B10.BR (H-2k) mice demonstrated that the epitope attachment to the point of lipid moiety, and the length of the Laa alkyl chain have a profound effect on vaccine immunogenicity after intranasal administration. It was demonstrated that a vaccine featuring C-terminal lipid moiety containing alkyl chains of 16 carbons, with P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue was capable of inducing optimal antibody response. These findings have considerable relevance to the development of a broad spectrum J14-based GAS vaccine and in particular provided a rational basis for peptide vaccine design based on this self-adjuvanting lipopeptide technology.

Current status of multiple antigen-presenting peptide vaccine systems: Application of organic and inorganic nanoparticles.

Many studies are currently investigating the development of safe and effective vaccines to prevent various infectious diseases. Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated, killed or inactivated pathogens), carrier proteins and cytotoxic adjuvants. Recently, two main approaches have been used to develop multiple antigen-presenting peptide vaccine systems: (1) the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2) synthetic approaches using size-defined nanomaterials, e.g., self-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms. This review summarizes the recent experimental studies directed to the development of multiple antigen-presenting peptide vaccine systems.

The influence of incorporating lipids or liposaccharides on the particle size of peptide therapeutics.

The characterization of peptide-based drugs is essential to obtain information about their potential suitability. In this study, a therapeutic peptide epitope alone or in combination with a lipid or liposaccharide moiety were assessed to determine their particle sizes by diffusion ordered NMR spectroscopy, dynamic light scattering, and mathematical expressions. These methods were compared and their suitability for different types of peptides is discussed herein. When compared with the mathematical expressions, we found that the NMR method resulted in a particle size that was consistent with the radius of the peptide monomer. The dynamic light scattering method showed that when lipids were conjugated to the peptide epitope, the resulting particles had a larger sized distribution compared with the peptide alone. These experiments provided information which can be applied when formulating these peptides as drugs.

Design of three-component vaccines against group A streptococcal infections: importance of spatial arrangement of vaccine components.

Immunological assessment of group A streptococcal (GAS) branched lipopeptides demonstrated the impact of spatial arrangement of vaccine components on both the quality and quantity of their immune responses. Each lipopeptide was composed of three components: a GAS B-cell epitope (J14), a universal CD4(+) T-cell helper epitope (P25), and an immunostimulant lipid moiety that differs only in its spatial arrangement. The best systemic immune responses were demonstrated by a lipopeptide featuring the lipid moiety at the lipopeptide C-terminus. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side chain. The former candidate features α-helical conformation required to produce protective J14-specific antibodies. Our results highlight the importance of epitope orientation and lipid position in the design of three-component synthetic vaccines.

Structure-activity relationship of lipopeptide Group A streptococcus (GAS) vaccine candidates on toll-like receptor 2.

Incorporation of lipoamino acids (LAAs) into peptide structures effectively imparts self-adjuvanting activity onto otherwise ineffective immunogens. Our fully synthetic lipopeptide vaccine candidates against group A streptococcus (GAS) were composed of J14 as a target GAS B-cell epitope alongside a universal helper T-cell epitope (P25) and a LAA-based lipid moiety. In the current study, we investigated the ability of our lipopeptides to activate nuclear factor-kappaB (NF-kappaB) in a toll-like receptor-2 (TLR2)-dependent manner as the possible mode of action and reported the structure-function requirements for novel TLR2 targeting lipopeptides based on LAAs. The NF-kappaB activation was dependent on the dose and the length of the alkyl chains of the incorporated lipid moieties with the hierarchy LAA 3 (16 carbons)>LAA 2 (14 carbons)>LAA 1 (12 carbons). The position of the lipid moiety (C-terminus vs. N(epsilon)-terminus of the central lysine residue) does not significantly affect NF-kappaB activation. Lipopeptides containing different copies of LAA 3 were synthesized and the di-lipidated analogue was the most effective in NFkappaB activation.

Investigation toward multi-epitope vaccine candidates using native chemical ligation.

We applied native chemical ligation (NCL) method to the synthesis of highly pure lipid-core peptide (LCP) vaccines to attach various peptide epitopes. In the case of the synthesis of LCP vaccine with two different peptide epitopes, LCP moieties having two free Cys and two protected Cys derivatives (S-acetamidemethyl-Cys, (Cys(Acm)), N-methylsulfonylethyloxycarbonyl-Cys (Msc-Cys), or 1,3-thiazolidine-4-carboxylic acid (Thz)) on oligolysine branches were prepared in order to couple two different epitopes by stepwise NCL. It was found that the difficulty in NCL of first two peptide antigen was associated with the steric hindrance. Using Thz instead of Cys(Acm) and Msc-Cys was important to reduce the steric hindrance and improve NCL yield.

Synthesis and immunological evaluation of self-adjuvanting glycolipopeptide vaccine candidates.

Synthesis of four glycolipids with different number of lauroyl groups on glucose or cellobiose as scaffolds is described. Their immunological evaluations either admixed with or covalently linked to J8, a peptide antigen derived from the C-terminus of the antiphagocytic M-protein of group A streptococcus, are also investigated. Administration of mixtures of J8 and glycolipids to B10BR (H-2(k)) mice induced low-levels of J8-specific IgG antibodies. While glycolipopeptides, in which J8 was covalently linked to the synthetic glycolipids, demonstrated high-levels of antibody titers comparable with the co-administration of these glycolipopeptides with complete Freund's adjuvant, suggesting clearly the strong potency of the synthesized glycolipids as self-adjuvanting moieties.

Synthesis and in vivo studies of carbohydrate-based vaccines against group A streptococcus.

Carbohydrates, as carriers, providing numerous attachment points for the conjugation of peptide antigens and their optimal orientation for the recognition by cells of the immune system, reducing degradation of the attached peptide antigens and many other advantages make carbohydrate-based vaccine highly promising approach. Multiple copies of a single group A streptococcal (GAS) M protein derived specific peptide antigens (J8 or J14) were coupled onto carbohydrate cores (D-glucose and D-galactose) linked to lipophilic amino acids to produce a self-adjuvanting liposaccharide vaccine against GAS strains. In vivo experiments showed high serum IgG antibody titers against each of the incorporated peptide epitopes, J8 or J14.