Differences in Elderly and Non-Elderly Outpatient Subjective Evaluation of "Easy-to-Eat Meals" after Dental Treatment.

Dental treatment improves the experience of eating by healing illnesses in the oral cavity or through the installation of special devices. However, mastication can often prove difficult for short periods of time after dental treatment, potentially limiting the types of food that can be consumed. Therefore, we proposed a highly nutritious meal strategy for dental outpatients (hereafter, "easy-to-eat meals"). We previously reported patients' subjective assessment of these easy-to-eat meals as determined through a questionnaire survey. The purpose of the present study was to investigate how differences in age affected such assessments. The study participants comprised patients scheduled to undergo dental treatment. They were divided into 2 groups: one of patients aged above and one of those aged below 70 years. All were required to consume provided easy-to-eat meals at the dental hospital directly after treatment and then answer a questionnaire. The questionnaire included items on patient satisfaction with the meals, taste, portion size, convenience, reduction in discomfort, and whether they would consume them again. The format of the questionnaire was a visual analog scale (VAS), ranging from 0 (negative) to 10 (positive). Portion size was to be rated on a scale from 0 ("Not enough") to 10 ("Too much"), with 5 being "Just right". Correlations between the questionnaire items were investigated to determine how they influenced each other. The VAS average for "Reduction in discomfort" was 8.45±1.39 in the non-elderly group and 6.07±2.92 in the elderly group, and the difference was significant (p=0.02); the VAS average for "Taste" was 6.49±2.32 in the non-elderly group and 4.91±0.98 in the elderly group, and the difference was significant (p=0.04). The results of this study suggest that providing such meal plans as nutritional guidance after dental treatment can influence quality of life in elderly patients.

Molecular mechanisms of Streptococcus pneumoniae-targeted autophagy via pneumolysin, Golgi-resident Rab41, and Nedd4-1-mediated K63-linked ubiquitination.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

"Easy-to-eat Meals" for Outpatients Following Dental Treatment.

Patients often experience temporary difficulty in masticating during the period immediately following dental treatment. The purpose of this study was to investigate subjectively assessed satisfaction with a specially designed diet for such patients by means of a questionnaire. These "easy-to-eat meals" were planned and provided by this hospital in Japan, and comprised a combination of commercially available and nutritionally rich soft foods, jellied foods, drinks, and other items. The patients were required to commence consuming them immediately following dental treatment. The questionnaire contained 6 categories -Satisfaction, Taste, Meal completion, Convenience, Reduction in discomfort, and Likelihood of reuse - to be evaluated on a 10-cm visual analog scale (VAS). The overall response was positive in all 41 completed questionnaires, with an overall score of 6 or higher for every category. Orthodontics achieved the highest VAS score in every category, followed by oral implantology, prosthodontics, and conservative Original Article doi:10.2209/tdcpublication.2018-0055 dentistry. A correlation was observed between Satisfaction and each of the 5 remaining questionnaire categories (Taste: |r|=0.70, p≤0.00; Meal completion: |r|=0.60, p≤0.00; Convenience: |r|=0.56, p≤0.00; Reduction in discomfort: |r|=0.48, p=0.00; and Likelihood of reuse: |r|=0.79, p≤0.00). An acceptable level of convenience was obtained with these meals, as they were reported to be useful during the period immediately following treatment, when eating out or preparing meals was physically and/or psychologically difficult.

Preferential capture of EpCAM-expressing extracellular vesicles on solid surfaces coated with an aptamer-conjugated zwitterionic polymer.

Extracellular vesicles (EVs) collectively represent small vesicles that are secreted from cells and carry biomolecules (e.g., miRNA, lncRNA, mRNA, proteins, lipids, metabolites, etc.) that originate in those cells. Body fluids, such as blood and saliva, include large numbers of EVs, making them potentially a rich source of diagnostic information. However, these EVs are mixtures of vesicles released from diseased tissues as well as from normal cells. This heterogeneous nature therefore blurs the clinical information obtainable from EV-based diagnosis. Here, we synthesized an EpCAM-affinity coating agent, which consists of a peptide aptamer for EpCAM and a zwitterionic MPC polymer, and have shown that this conjugate endowed the surfaces of inorganic materials with the preferential affinity to EpCAM-expressing EVs. This coating agent, designated as EpiVeta, could be useful as a coating for various diagnostic devices to allow concentration of cancer-related EVs from heterogeneous EV mixtures.

Synergistic Costimulatory Effect of Chlamydia pneumoniae with Carbon Nanoparticles on NLRP3 Inflammasome-Mediated Interleukin-1ß Secretion in Macrophages.

The obligate intracellular bacterium Chlamydia pneumoniae is not only a causative agent of community-acquired pneumonia but is also associated with a more serious chronic disease, asthma, which might be exacerbated by air pollution containing carbon nanoparticles. Although a detailed mechanism of exacerbation remains unknown, the proinflammatory cytokine interleukin-1ß (IL-1ß) is a critical player in the pathogenesis of asthma. C. pneumoniae induces IL-1ß in macrophages via NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activation and Toll-like receptor 2/4 (TLR2/4) stimulation. Carbon nanoparticles, such as carbon nanotubes (CNTs), can also evoke the NLRP3 inflammasome to trigger IL-1ß secretion from lipopolysaccharide-primed macrophages. This study assessed whether costimulation of C. pneumoniae with CNTs synergistically enhanced IL-1ß secretion from macrophages, and determined the molecular mechanism involved. Enhanced IL-1ß secretion from C. pneumoniae-infected macrophages by CNTs was dose and time dependent. Transmission electron microscopy revealed that C. pneumoniae and CNTs were engulfed concurrently by macrophages. Inhibitors of actin polymerization or caspase-1, a component of the inflammasome, significantly blocked IL-1ß secretion. Gene silencing using small interfering RNA (siRNA) targeting the NLRP3 gene also abolished IL-1ß secretion. Other inhibitors (K(+) efflux inhibitor, cathepsin B inhibitor, and reactive oxygen species-generating inhibitor) also blocked IL-1ß secretion. Taken together, these findings demonstrated that CNTs synergistically enhanced IL-1ß secretion from C. pneumoniae-infected macrophages via the NLRP3 inflammasome and caspase-1 activation, providing novel insight into our understanding of how C. pneumoniae infection can exacerbate asthma.

A dominant negative form of inositol 1,4,5-trisphosphate receptor induces metacyclogenesis and increases mitochondrial density in Trypanosoma cruzi.

Inositol 1,4,5-trisphosphate receptor (IP3R) is a key regulator of intracellular Ca(2+) concentration that release Ca(2+) from Ca(2+) stores in response to various external stimuli. IP3R also works as a signal hub which form a platform for interacting with various proteins involved in diverse cell signaling. Previously, we have identified an IP3R homolog in the parasitic protist, Trypanosoma cruzi (TcIP3R). Parasites expressing reduced or increased levels of TcIP3R displayed defects in growth, transformation, and infectivity. In the present study, we established parasitic strains expressing a dominant negative form of TcIP3R, named DN-TcIP3R, to further investigate the physiological role(s) of TcIP3R. We found that the growth of epimastigotes expressing DN-TcIP3R was significantly slower than that of parasites with TcIP3R expression levels that were approximately 65% of wild-type levels. The expression of DN-TcIP3R in epimastigotes induced metacyclogenesis even in the normal growth medium. Furthermore, these epimastigotes showed the presence of dense mitochondria under a transmission electron microscope. Our findings confirm that TcIP3R is crucial for epimastigote growth, as previously reported. They also suggest that a strong inhibition of the IP3R-mediated signaling induces metacyclogenesis and that mitochondrial integrity is closely associated with this signaling.

High-temperature adapted primitive Protochlamydia found in Acanthamoeba isolated from a hot spring can grow in immortalized human epithelial HEp-2 cells.

To elucidate how ancient pathogenic chlamydiae could overcome temperature barriers to adapt to human cells, we characterized a primitive chlamydia found in HS-T3 amoebae (Acanthamoeba) isolated from a hot spring. Phylogenetic analysis revealed the primitive species to be Protochlamydia. In situ hybridization staining showed broad distribution into the amoebal cytoplasm, which was supported by transmission electron microscopic analysis showing typical chlamydial features, with inclusion bodies including both elementary and reticular bodies. Interestingly, although most amoebae isolated from natural environments show reduced growth at 37°C, the HS-T3 amoebae harbouring the Protochlamydia grew well at body temperature. Although infection with Protochlamydia did not confer temperature tolerance to the C3 amoebae, the number of infectious progenies rapidly increased at 37°C with amoebal lysis. In immortalized human epithelial HEp-2 cells, fluorescence microscopic study revealed atypical inclusion of the Protochlamydia, and quantitative real-time polymerase chain reaction analyses also showed an increase in 16S ribosomal RNA DNA amounts. Together, these results showed that the Protochlamydia found in HS-T3 amoebae isolated from a hot spring successfully adapted to immortalized human HEp-2 cells at 37°C, providing further information on the evolution of ancient Protochlamydia to the present pathogenic chlamydiae.

Transcriptome profiling of brain edemas caused by influenza infection and lipopolysaccharide treatment.

Influenza A virus-associated encephalopathy triggered by influenza virus infection often occurs in children aged five and younger in Japan. However, the mechanisms behind Influenza A virus-associated encephalopathy are not yet well understood. This study developed an Influenza A virus-associated encephalopathy-like model using mice infected with Influenza A virus and given lipopolysaccharide treatment. The results showed that the mice used in the model suffered from brain edemas nearly three times more severe, as well as having higher cytokine levels in sera compared to those of the control groups. Using gene expression profiling, cytokine-related genes were found not to be up-regulated in the brain in situ, while protein coding genes, which are known to be involved in blood-brain barrier disruption, were up-regulated. Categorizing the functional groups using gene ontology revealed the terms "ion channels," "calcium oscillation," and "membrane transporter activities." The blood-brain barrier disruption found in this Influenza A virus-associated encephalopathy model can therefore be assumed to be due to a cellular electrolyte imbalance of the neuronal tissue, in addition to a cytokine storm.

A domino-like chlamydial attachment process: concurrent Parachlamydia acanthamoebae attachment to amoebae is required for several amoebal released molecules and serine protease activity.

Parachlamydia acanthamoebae is an obligate intracellular bacterium that infects free-living amoebae (Acanthamoeba), and is a potential human pathogen associated with hospital-acquired pneumonia. The attachment mechanism of this bacteria to host cells is crucial in bacterial pathogenesis, yet remains undetermined. Hence, we obtained monoclonal antibodies (mAbs) specific to either P. acanthamoebae or amoebae in an attempt to elucidate the attachment mechanism involved. Hybridomas of 954 clones were assessed, and we found that four mAbs (mAb38, mAb300, mAb311, mAb562) that were reactive to the amoebae significantly inhibited bacterial attachment. All mAbs recognized amoebal released molecules, and mAb311 also recognized the amoebal surface. mAbs reacted with the bacteria not only within amoebae, but also when they were released from amoebae (except mAb311). Furthermore, a serine protease inhibitor had an inhibitory effect on the bacterial attachment to amoebae, although none of the mAbs had any synergistic effect on the inhibition of attachment by the protease inhibitor. Taken together, we conclude that concurrent P. acanthoamebae attachment to amoebae is required for several amoebal released molecules and serine protease activity, implying the existence of a complicated host-parasite relationship.

Ciliates expel environmental Legionella-laden pellets to stockpile food.

When Tetrahymena ciliates are cultured with Legionella pneumophila, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmental L. pneumophila, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human L. pneumophila isolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental L. pneumophila for stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance.

Chlamydia trachomatis serovar L2 infection model using human lymphoid Jurkat cells.

Chlamydia trachomatis L2 invasively attacks lymphatic and subepithelial tissues of the genital tract during the formation of primary lesions. This subsequently results in lymphadenopathy, and suggests a greater propensity for systemic dissemination. However, whether lymphocytes are a potential vehicle cell for the dissemination of this infection remains unknown. We therefore assessed the growth properties of C. trachomatis L2 in lymphoid Jurkat cells compared with those observed in epithelial HeLa cells. Both cells supported the growth of C. trachomatis with a similar increase in infective progenies. Enriched human-blood lymphocytes also supported the C. trachomatis growth as well as Jurkat cells. Bacteria infecting the Jurkat cells were more susceptible to antibiotics (doxycycline, azithromycin, ofloxacin) than those in HeLa cells. Of the sphingomyelin biosynthesis inhibitors tested, both myriocin and fumonisin B1 significantly inhibited bacterial growth in both cells types. A Jurkat cell mutant that impaired bacterial growth was established using ethylmethanesulfonate treatment. DNA microarray analysis with real-time reverse transcription-polymerase chain reaction revealed that the mutant cells over-expressed granzyme K gene. Immunofluorescence staining also indicated that granzyme K irregularly over-expressed among the mutant cells as compared with that of the wild cells, suggesting a possible mechanism refractory to C. trachomatis infection. Thus, we concluded that C. trachomatis L2 could infect Jurkat cells with lymphoid properties, providing a new tool for studying C. trachomatis dissemination to tissues via lymphocyte movement.

Significance of broad distribution of electron-dense deposits in patients with IgA nephropathy.

Immunoglobulin A nephropathy (IgAN) is characterized by mesangial cell proliferation and mesangial expansion with mesangial depositions of IgA. We have found that electron-dense deposits (EDD) are often observed in areas other than paramesangial areas in glomeruli. To compare electron microscopic findings with light microscopic findings and clinical data, we examined the biopsies from 178 patients with IgAN. Patients were divided into two groups: group A had only paramesangial deposits and group B had deposits not only in paramesangial areas but also in other areas. All patients examined in this study had EDD in glomerular paramesangial areas. Thirty-six patients were included in group B. Cellular crescent formation in glomeruli and urinary protein in group B were significantly higher than those in group A (P < 0.01). Serum albumin and estimated glomerular filtration rate (eGFR) in group B were significantly lower than those in group A (P < 0.05). Group B showed a significant positive correlation with histological severity, which is defined in the Japanese Clinical Guidelines on IgAN. In patients with broad distribution of EDD, urinary protein was significantly increased (P < 0.05). Detailed observation of EDD distribution has an impact on evaluation of the disease activity of IgAN.

Impact of anaerobic and oligotrophic conditions on survival of Alloiococcus otitidis, implicated as a cause of otitis media.

The survival of Alloiococcus otitidis (NCFB2890) with different nutritional supplements, including brain-heart infusion broth (BHI), phosphate-buffered saline (PBS), distilled water (DW), and middle ear effusion (MEE), as well as various atmospheres (aerobic, microaerobic, anaerobic), was compared using cultures, LIVE/DEAD staining, and transmission electron microscopy. The bacterial morphological traits and viability were maintained in BHI and MEE under aerobic conditions but were rapidly lost in PBS and DW. In contrast, anaerobic conditions did not support viability at all. Thus, the bacteria critically required an aerobic atmosphere for its survival as well as the appropriate nutrients, implying that culture of this pathogen from clinical specimens would become more difficult through oxygen depletion depending on a slight change in the middle ear atmosphere.

Immunohistochemical Detection of Aflatoxin in Lesions of Aflatoxin-producing Aspergillus flavus Infection.

Aflatoxin produced by Aspergillus flavus is known to be strongly related to liver injury (hepatocellular carcinoma) and immune system damage involving leukocytes. This toxin suppresses both the cell-mediated immune system and macrophage function, and decreases the production of complement and interferon molecules. PURPOSE: To evaluate the presence of aflatoxin in infectious lesions as well as how the toxin is taken up by leukocytes. METHOD: Pathological specimens from a patient who died from aspergillosis caused by aflatoxin-producing A. flavus were used. Anti-aflatoxin B1 antibody was reacted with paraffin-embedded lesion specimens from the heart, kidney, and thyroid gland of the patient and observed microscopically. RESULT: Positive reactions were detected in fungal elements and leukocytes (neutrophils and macrophages) in inflammatory lesions. CONCLUSION: Within the patient's body, A. flavus likely produced aflatoxin, which then was taken up by neutrophils and macrophages.These results suggest that leukocyte function and the immune mechanism are locally suppressed by aflatoxin.

Streptococcus pneumoniae triggers hierarchical autophagy through reprogramming of LAPosome-like vesicles via NDP52-delocalization.

In innate immunity, multiple autophagic processes eliminate intracellular pathogens, but it remains unclear whether noncanonical autophagy and xenophagy are coordinated, and whether they occur concomitantly or sequentially. Here, we show that Streptococcus pneumoniae, a causative of invasive pneumococcal disease, can trigger FIP200-, PI3P-, and ROS-independent pneumococcus-containing LC3-associated phagosome (LAPosome)-like vacuoles (PcLVs) in an early stage of infection, and that PcLVs are indispensable for subsequent formation of bactericidal pneumococcus-containing autophagic vacuoles (PcAVs). Specifically, we identified LC3- and NDP52-delocalized PcLV, which are intermediates between PcLV and PcAV. Atg14L, Beclin1, and FIP200 were responsible for delocalizing LC3 and NDP52 from PcLVs. Thus, multiple noncanonical and canonical autophagic processes are deployed sequentially against intracellular S. pneumoniae. The Atg16L1 WD domain, p62, NDP52, and poly-Ub contributed to PcLV formation. These findings reveal a previously unidentified hierarchical autophagy mechanism during bactericidal xenophagy against intracellular bacterial pathogens, and should improve our ability to control life-threating pneumococcal diseases.

Isolation of Extracellular Vesicles in Saliva Using Density Gradient Ultracentrifugation.

This chapter describes a method for isolating human salivary extracellular vesicles (EVs) using density gradient ultracentrifugation. Standard protocols established for isolation of EVs from blood or a conditioned medium of cultured cells do not work for whole saliva, due to its viscosity. Therefore, procedures including a pretreatment step and utilizing iodixanol as a gradient material enable EVs to be concentrated to a 1.1 g/ml density. This protocol is compatible with both swing and angle rotors. By employing an angle rotor, which enables high g-force, the centrifugation time was reduced to 4 h from the 17 h required when using a swing rotor.

Leucine-rich α2-glycoprotein overexpression in the brain contributes to memory impairment.

We previously reported increase in leucine-rich α2-glycoprotein (LRG) concentration in cerebrospinal fluid is associated with cognitive decline in humans. To investigate relationship between LRG expression in the brain and memory impairment, we analyzed transgenic mice overexpressing LRG in the brain (LRG-Tg) focusing on hippocampus. Immunostaining and Western blotting revealed age-related increase in LRG expression in hippocampal neurons in 8-, 24-, and 48-week-old controls and LRG-Tg. Y-maze and Morris water maze tests indicated retained spatial memory in 8- and 24-week-old LRG-Tg, while deteriorated in 48-week-old LRG-Tg compared with age-matched controls. Field excitatory postsynaptic potentials declined with age in LRG-Tg compared with controls at 8, 24, and 48 weeks. Paired-pulse ratio decreased with age in LRG-Tg, while increased in controls. As a result, long-term potentiation was retained in 8- and 24-week-old LRG-Tg, whereas diminished in 48-week-old LRG-Tg compared with age-matched controls. Electron microscopy observations revealed fewer synaptic vesicles and junctions in LRG-Tg compared with age-matched controls, which became significant with age. Hippocampal LRG overexpression contributes to synaptic dysfunction, which leads to memory impairment with advance of age.

ATP bioluminescence values are significantly different depending upon material surface properties of the sampling location in hospitals.

BACKGROUND: Our previous study into assessing hospital cleanliness in Japan by two common methods, ATP bioluminescence and the stamp agar method, revealed considerable variability in the data of both methods (BMC Research Notes, 7: 121, 2014). To investigate the reason(s) for the variability, we reanalyzed the data (n = 752) from the point of view of the material surface properties of sampling sites. METHODS: Data obtained from surfaces with unknown properties and different purposes such as floor were omitted, and the remaining data (n = 488) were used for this study. The material surface properties on sampling sites were divided into six categories: melamine coated (n = 216), vinyl chloride (n = 16), stainless steel (n = 144), wood (n = 63), and acrylonitrile-butadiene styrene resin coated (n = 48). The data between individual material properties were compared. RESULTS: The ATP values of high-touch places were significantly different depending on the type of surface, but no significant difference in stamp values between material properties was seen, indicating that in contrast to stamp values, ATP-accumulation more depends on the physical properties of the material surface such as electronic charges or roughness. To confirm this, we assessed a degree of roughness on vinyl chloride material surface (disutilized floor samples actually used for each of the hospitals) by observation with scanning electron microscope (SEM). As a result, SEM observation similarly revealed considerable roughness on the materials, which may allow microbes to contaminate the materials without noticing it. CONCLUSION: Material properties must be considered when evaluating hospital cleanliness with ATP values, and provide a strong warning into evaluating hospital cleanliness.

Amoebal endosymbiont Parachlamydia acanthamoebae Bn9 can grow in immortal human epithelial HEp-2 cells at low temperature; an in vitro model system to study chlamydial evolution.

Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37 °C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30 °C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30 °C compared to at 37 °C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells.

Design and synthesis of 4,6-di-tert-butyl-2,3-dihydro-5-benzofuranols as a novel series of antiatherogenic antioxidants.

Antioxidants have been considered as potential antiatherogenic agents by inhibiting oxidation of low-density lipoprotein (LDL), albeit vitamin E, a natural antioxidant, has failed to show reduction on atherosclerosis in clinical trials. We have rationally designed and synthesized a novel series of antioxidants, 4,6-di-tert-butyl-2,3-dihydro-5-benzofuranols, to overcome the clinical limitation of vitamin E. In vitro, the compounds showed a potent inhibitory effect on lipid peroxidation detected as 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA)-dependent chemiluminescence in linoleic acid autoxidation. They also inhibited the LDL oxidation induced by Cu(2+), and the inhibition is more potent than that of vitamin E and probucol. In vivo, 4,6-di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653, 1f), an optimal compound, showed the highest concentration in plasma and LDL fraction in Watanabe heritable hyperlipidemic rabbits, due to its high affinity to LDL. The isolated LDL samples from the 1f-treated rabbits showed potent resistibility to LDL oxidation. Compound 1f has been taken into clinical trials.