Mamoru Noda

Components of Dentinal Adhesives Modulate Heat Shock Protein 72 Expression in Heat-stressed THP-1 Human Monocytes at Sublethal Concentrations

Few studies have investigated the ability of dental resins to induce cellular stress at sublethal concentrations. Cellular stress, especially in immune cells such as monocytes, may modulate the biological response to materials or the hosts ability to respond to bacterially mediated inflammation. The current study examined the ability of sublethal concentrations of 2-hydroxylethylmethacrylate (HEMA) and triethyleneglycol dimethacrylate (TEGDMA) to induce heat shock protein 72 (HSP72) in human monocytes. HEMA and TEGDMA significantly suppressed heat-induced HSP72 expression, even at sublethal levels, but did not induce HSP72 by themselves. The results of the current study suggest that components released from dental resin could modulate the HSP stress response without altering cellular metabolic activity.

Detection of norovirus and sapovirus infection among children with gastroenteritis in Ho Chi Minh City, Vietnam

Summary.This report describes norovirus (NoV) and sapovirus (SaV) infections in hospitalized children with acute sporadic gastroenteritis in Ho Chi Minh City, Vietnam. Stool specimens collected between December 1999 and November 2000 were examined for NoV and SaV using reverse transcription-PCR and phylogenetic analysis. NoVs were detected in 72 of 448 rotavirus-negative specimens, counted as part of an overall annual detection rate of 5.4% (72 of 1,339 children). This included four NoV genogroup I (GI) strains and 68 NoV GII strains. Only one SaV GI strain was detected in the rotavirus-negative specimens. Over 73% of the NoV sequences belonged to GII/4 (Lordsdale cluster) and were detected in all months except March. We also detected GII/3 strains (Saitama U201 cluster), a naturally occurring recombinant NoV, between January 2000 and March 2000 but not after this period. Other NoV strains belonging to GI/4, GI/8, GII/1, and GII/7 were also detected but were infrequent. In addition, two almost identical NoV GII strains (strains 026 and 0703) collected six months apart were classified into a new genotype that includes the Mc37 strain, which was previously shown to be a recombinant NoV. During this one-year study, the NoV prevailed at the end of the rainy season and the beginning of the dry season. Further epidemiological studies may be necessary to determine whether the GII/4 strains continue to dominant in this region.

The in vitro cytotoxicity of eluates from dentin bonding resins and their effect on tyrosine phosphorylation of L929 cells.

OBJECTIVES The aim of this study was to examine the relationship between the monomers eluted from dentin-bonding systems and their cytotoxicities, and to investigate the biochemical effect of the monomers on tyrosine phosphorylation, especially relating to the cell growth activity, of L929 cells in vitro. METHODS The primers, uncured or cured adhesives (3M and Kuraray) were tested to determine the cytotoxicity of confluent L929 cells cultured by Eagles MEM medium supplemented with 10% FCS. The area of cells affected by the eluted monomers were evaluated with an image analyzer and the concentrations of monomers eluted into the medium were measured with high performance liquid chromatography (HPLC) after 24h incubation. The protein composition of the stimulated cells was compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tyrosine phosphorylation was detected by Western blot. RESULTS The primer and uncured adhesives revealed variable cytotoxicities. 2-hydroxyethyl-methacrylate (HEMA) was the major component eluted from uncured primers and adhesives. Small amounts of triethylene glycol dimethacrylate (TEGDMA) were also detected from the uncured adhesives. The cytotoxicities of the adhesives decreased as photo activation time increased. The amount of monomers eluted from the cured adhesives was almost undetectable and did not reach a sufficient concentration to suppress cell viability or cell growth. The cytotoxicities of the primers and adhesives correlated well with the amounts of either HEMA or TEGDMA eluted. Moreover, a high concentration of HEMA (4 mg/ml medium) affected intracellular tyrosine phosphorylation, which is related to cellular activities. SIGNIFICANCE Although the monomers present in dentin bonding resins are cytotoxic to L929 cells, the amount from cured bonding resin is very small and does not provide a cytotoxic dose. This data does however suggest that clinical exposure to the uncured primers and adhesives of dentin bonding resins should be minimized.

Sublethal, 2-week exposures of dental material components alter TNF-α secretion of THP-1 monocytes

OBJECTIVES The aim of this study was to investigate the hypothesis that dental material components alter cytokine secretion from monocytes if applied for several weeks at sublethal doses. The current study significantly extended exposure times of monocytes to the components over times published in previous studies. These exposure times approached the estimated average life span of monocytes in the bloodstream. METHODS Human THP-1 monocytes were exposed to 2-hydroxyethylmethacrylate (HEMA, 0-1.2mmol/l), triethyleneglycoldimethacrylate (TEGDMA, 0-0.75mmol/l), Hg(2+) (0-2 micromol/l), or Ni(2+) (0-20 micromol/l) for 2 weeks. The cells were then collected and additionally incubated for 24h, with or without bacterial lipopolysaccharide (LPS), a common component of dental plaque. TNF-alpha secretion from THP-1 was determined using by enzyme-linked immunosorbent assay. RESULTS None of the dental material components induced TNF-alpha from THP-1 by themselves, but LPS alone strongly induced TNF-alpha secretion as expected. HEMA and TEGDMA significantly suppressed (40-70%) TNF-alpha secretion from cells stimulated with LPS. Hg(2+) at 2.0 micromol/l doubled TNF-alpha secretion from THP-1s stimulated with LPS over LPS alone. Ni(2+) did not significantly affect TNF-alpha secretion, with or without LPS exposure. Significance. The results in this study suggest that sublethal, 2-week exposures of some dental material components may alter TNF-alpha secretion from THP-1 monocytes when the cells are challenged. These alterations may influence the biological response of tissues to materials in an inflammatory intraoral environment.

Divergent Evolution of Norovirus GII/4 by Genome Recombination from May 2006 to February 2009 in Japan

ABSTRACT Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P ≤ 0.000001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII/4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsids were preferentially positioned on the outer surface loops of the protruding P2 domain and more abundant in the dominant subtypes. These findings suggest that intersubtype genome recombination at the ORF1/2 boundary region is a common mechanism that realizes independent and concurrent changes on the virion surface and in viral replication proteins for the persistence of norovirus GII/4 in human populations.

HPLC analysis of dental resin composites components

Five uncured commercial dental resin composites (two bis-glycidyl methacrylate based products and three non-bis-glycidyl methacrylate based products) were examined for contamination with bisphenol A, which is a known xenobiotic. After the samples were processed with acetonitrile for extraction of their components, high performance liquid chromatography analysis was performed and the eluted peaks were fractionated for comparison using UV spectra. The results suggested that all the resin composites tested were contaminated with bisphenol A or its derivatives. Theoretically, bisphenol A is not a component of dental resin composite, but it could remain as an impurity of the composite during the synthesis of Bis-GMA. The results suggest that it is necessary to investigate the ability of this impurity and its derivatives in dental resin composites to cause estrogenic effects, as well as to evaluate the release of the impurity from cured resin composites.

Statistical analysis of attack rate in norovirus foodborne outbreaks

Norovirus (NoV), which causes foodborne gastroenteritis outbreaks, is one of the important viruses in public health. We statistically analyzed the attack rate in foodborne outbreaks caused by NoV. The attack rate in 95 oyster-associated outbreaks was significantly higher than that in 195 food handler-associated outbreaks (P=0.007). The difference in the number of NoV genotypes implicated is considered to be an important factor for this difference. The attack rate in 20 outbreaks associated only with GII/3 was higher than that in 143 other outbreaks (P=0.247), while the attack rate in 27 outbreaks associated only with GII/4 was lower than that in 136 other outbreaks (P=0.004), suggesting that GII/4 NoVs cause asymptomatic infection more frequently than do other NoV genotypes. Our results suggest that differences in implicated foods, susceptibility of the host to NoV infection, and pathogenicity of NoVs may influence the attack rate in NoV foodborne outbreaks.

In vitro biological response to core and flowable dental restorative materials.

OBJECTIVES In vitro cytotoxicities of commercially available core and flowable dental restorative materials were assessed and compared to traditional resin composites. Our hypothesis was that the increased resin diluents added to achieve higher flow in flowables would increase cytotoxicities, whereas the higher filler content of core materials would decrease cytotoxicities relative to traditional resin composites. METHODS Specimens were made under aseptic conditions, then extracted into an artificial saliva solution for 0-4 weeks, to assess the effect of aging on cytotoxicity. After extraction, specimens were tested for cytotoxicity in vitro using Balb/c fibroblasts in direct contact format. Cells were exposed to the materials for 48h, after which the mitochondrial activity of the cells was measured (MTT method). Cellular activity was normalized to Teflon negative controls. RESULTS Core materials were uniformly and severely (<50% of Teflon cellular activity) cytotoxic initially, but several materials (Corepaste, Definite core) improved somewhat with aging in artificial saliva. Flowable materials were uniformly and severely cytotoxic with no trend toward improvement with aging. The Definite-flow was the least cytotoxic of the flowable materials, but it too was severely cytotoxic. SIGNIFICANCE Commercially available core and flowable restorative materials showed severe in vitro cytotoxicities that are worse than some traditional composites and most dental casting alloys and amalgams used today. Of particular note was the persistent cytotoxicity of these materials after 4 weeks of extraction with artificial saliva. These cytotoxicities indicate a continuing release of mass from these materials at levels that have biological relevance in vitro. In vivo relevance of these cytotoxicities is less clear, but these results indicate a higher biological risk for these materials compared to traditional materials that exhibit less initial toxicity and improve with aging time.

Detection of sapoviruses and noroviruses in an outbreak of gastroenteritis linked genetically to shellfish

Norovirus (NoV) and sapovirus (SaV) are important pathogens of human gastroenteritis. Compared to NoV, the transmission route of SaV is unclear. An outbreak of gastroenteritis occurred at a restaurant in June 2008, and SaV and NoV were detected in fecal specimens from 17 people who ate at the restaurant and one asymptomatic food handler and also in stripped shellfish and liquids remaining in the shellfish packages by reverse transcription‐polymerase chain reaction (RT‐PCR) and/or real‐time RT‐PCR. Nucleotide sequencing analysis of the RT‐PCR products corresponding to the partial capsid region revealed 99.3–100% identities for SaV and 98.6–99.3% identities for NoV among the digestive diverticulum of the frozen stripped shellfish (Ruditapes philippinarum), “Asari,” the package liquid, and feces from symptomatic or asymptomatic guests. These results suggested a link between the consumption of contaminated shellfish and clinical features in the patients. While the transmission of NoV by shellfish has been reported, this report shows that SaV can also be transmitted by shellfish. J. Med. Virol. 82: 1247–1254, 2010.

Human sapovirus in clams, Japan.

Human sapovirus was detected in 4 of 57 clam packages by reverse transcription–PCR and sequence analysis. This represents the first finding of sapovirus contamination in food. Closely matching sequences have been detected in stool specimens from patients with gastroenteritis in Japan, which indicates a possible food-to-human transmission link.