Identification of novel function of vimentin for quality standard for regenerated pulp tissue.

INTRODUCTION: Pulp stem/progenitor cells have been successfully transplanted after pulpectomy in dogs and have led to pulp regeneration. The regenerated pulp tissue was investigated by the qualitative and quantitative protein expression patterns in comparison with those of normal pulp. There is an unmet need for a quality standard for regenerated pulp tissue. METHODS: Three distinct human CD105(+) stem/progenitor cells from dental pulp, bone marrow, and amnion were compared by 2-dimensional electrophoresis and the differential proteomic expression profiles. The protein identified with high confidence in pulp CD105(+) cells was examined by immunohistochemistry and real-time reverse-transcription polymerase chain reaction in regenerated pulp tissue compared with normal pulp. Its migration effect was further examined in pulp CD105(+) cells using small interfering RNA techniques to knock down the protein. RESULTS: Nine protein spots were detected solely in pulp CD105(+) cells; one of these was identified as vimentin. The expression of vimentin messenger RNA was highest in pulp tissue among a variety of human tissues and higher in pulp CD105(+) cells compared with other CD105(+) cells and unfractionated total pulp cells. Pulp cells and endothelial cells were positively stained with vimentin in regenerated pulp tissue similarly as those in normal pulp tissue. The expression of vimentin in regenerated pulp was similar to normal pulp. RNA interference knock down of vimentin expression in pulp CD105(+) cells significantly reduced the migration activity. CONCLUSIONS: The highest expression of vimentin in pulp tissue among other tissues and its migration effect in pulp stem cells suggest that it is a quality standard for pulp regeneration and pulp cell function.

Complete pulp regeneration after pulpectomy by transplantation of CD105+ stem cells with stromal cell-derived factor-1.

Loss of pulp due to caries and pulpitis leads to loss of teeth and reduced quality of life. Thus, there is an unmet need for regeneration of pulp. A promising approach is stem cell therapy. Autologous pulp stem/progenitor (CD105(+)) cells were transplanted into a root canal with stromal cell-derived factor-1 (SDF-1) after pulpectomy in mature teeth with complete apical closure in dogs. The root canal was successfully filled with regenerated pulp including nerves and vasculature by day 14, followed by new dentin formation along the dentinal wall. The newly regenerated tissue was significantly larger in the transplantation of pulp CD105(+) cells with SDF-1 compared with those of adipose CD105(+) cells with SDF-1 or unfractionated total pulp cells with SDF-1. The pulp CD105(+) cells highly expressed angiogenic/neurotrophic factors compared with other cells and localized in the vicinity of newly formed capillaries after transplantation, demonstrating its potent trophic effects on neovascularization. Two-dimensional electrophoretic analyses and real-time reverse transcription-polymerase chain reaction analyses demonstrated that the qualitative and quantitative protein and mRNA expression patterns of the regenerated pulp were similar to those of normal pulp. Thus, this novel stem cell therapy is the first demonstration of complete pulp regeneration, implying novel treatment to preserve and save teeth.

Matrix metalloproteinase-3 accelerates wound healing following dental pulp injury.

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. Angiogenesis is essential for reparative dentin formation during pulp wound healing. The mechanism of angiogenesis, however, still remains unclear. We hypothesized that certain MMPs expressed during pulp wound healing may support recovery processes. To address this issue, a rat pulp injury model was established to investigate expression of MMPs during wound healing. Real-time RT-PCR analysis showed that expression MMP-3 and MMP-9 (albeit lower extent) was up-regulated at 24 and 12 hours after pulp injury, respectively, whereas expression of MMP-2 and MMP-14 was not changed. MMP-3 mRNA and protein were localized in endothelial cells and/or endothelial progenitor cells in injured pulp in vivo. In addition, MMP-3 enhanced proliferation, migration, and survival of human umbilical vein endothelial cells in vitro. Furthermore, the topical application of MMP-3 protein on the rat-injured pulp tissue in vivo induced angiogenesis and reparative dentin formation at significantly higher levels compared with controls at 24 and 72 hours after treatment, respectively. Inhibition of endogenous MMP-3 by N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid resulted in untoward wound healing. These results provide suggestive evidence that MMP-3 released from endothelial cells and/or endothelial progenitor cells in injured pulp plays critical roles in angiogenesis and pulp wound healing.

The discrepant severity of external and internal injuries in a traffic accident: The cushioning effect via a human body against direct impact: Autopsy cases.

Traffic accidents cause unexpectedly severe injuries of internal organs despite tiny injuries observed on the external body. A 51-year-old woman (subject 1) and a 54-year-old man (subject 2) were found dead on a road. Subject 1 had subcutaneous and intramuscular bleeding with décollement on the posterior aspect of her body, including upper cervical spine dislocation. Subject 2 did not exhibit any apparent findings on autopsy that were indicative of a direct injury by a motor vehicle, but had severe internal organ injuries, including the transection at the pontomedullary junction. We surmise that subjects 1 and 2 were walking in line with the vehicle which collided with them from behind, and then the body of subject 1 cushioned the direct impact of the vehicle against subject 2. This report illustrates the need of forensic autopsy for victims with no severe external injuries.

Ethanol-induced JNK activation suppressed via active Akt in hepatocytes.

Ethanol induces c-Jun N-terminal kinase (JNK) activation leading to cell death in hepatocytes. However, acute alcohol exposure does not induce remarked cell death in hepatocytes. We hypothesized that active Akt may suppress JNK activation. To clarify this point, we evaluated the role of active Akt in JNK activation under treatment with hepatocyte growth factor (HGF) and compared it with ethanol treatment. Primary rat hepatocytes were treated with 10 ng/ml HGF. 10 min after that, 5 microM insulin, an activator of the Akt pathway, and/or 5 microM LY294002, an inhibitor of the pathway, were added. Hepatocytes were treated with 100 mM ethanol and LY294002. HGF treatment increased JNK activities in hepatocytes. This JNK activation was accumulated by addition of LY294002. These finding suggest that active Akt suppresses JNK activation induced by HGF. On the other hand, addition of insulin did not decrease the JNK activity, showing that insulin-induced Akt activation may rather increase JNK activity. Ethanol also induced JNK activation and this JNK activation was enhanced by LY294002 similar to HGF treatment. We found that active Akt suppressed JNK activation induced by ethanol as well as HGF in hepatocytes. JNK activation may be suppressed by prolonged active Akt or basal active Akt, rather than peaked activation of Akt induced by insulin stimulation. Our results suggest that the suppression of JNK by active Akt may prevent cell death in acute alcohol intoxication.

Saturated and monounsaturated fatty acids increase interleukin-10 production in rat hepatocytes.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, but it still remains unknown whether saturated or unsaturated fatty acid affects IL-10 production in hepatocytes that contribute to lipid metabolism. Primary rat hepatocyte cultures were treated with different fatty acids (18:0 stearic acid, 18:1 oleic acid; 18:2 linoleic acid, 18:3 linolenic acid) at 300 microM for 24 hours. The concentrations of IL-2, IL-10, GM-CSF and IFN-gamma in the medium were detected by multiplex cytokine array. IL-10 was significantly increased with treatment of stearic acid and oleic acid. Production of IL-10 by saturated and monounsaturated fatty acids in hepatocytes may be one of the reasons why the lard oil had less inflammation in the hepatic steatosis animal models.

A degloving foot injury in a traffic accident.

An unusual human remain found on a road became one key to reconstruct the traffic accident. A woman was found dead on a snow-covered road. Her left foot showed a large deficit of skin and underlying fat tissue. The detached skin and tissue were found into her left sock and shoe lying at a distance of 23 m from the body. There were multiple fractures on her back and occipital cranial bone. Severe injuries revealed in heart, liver, and brain. The neck remained intact. These findings suggested that her back has been strongly hit by the object with the flat surface, for example, the front side of a cab-over or truck, from behind at a high speed. Furthermore, the degloving injury in her left foot suggested that a vehicle ran over the foot which faced the vehicle. We reconstructed the accident as follows. Firstly the wheel of the vehicle ran over the victim's foot, and then, the victim turned away from the vehicle. Finally, the front side hit her twisted body from behind, resulting in a traumatic degloving injury.

Weight ratio of the lungs and pleural effusion to the spleen in the diagnosis of drowning.

An accurate and reliable method of diagnosing death by drowning is an important requirement in forensic autopsies. In this study, we compared the weight ratio of the lungs and pleural effusion to the spleen for 55 cases of drowning (37 males, 18 females), 36 cases of mechanical asphyxiation (16 males, 20 females), and 26 cases of acute cardiac death (19 males, 7 females). In the case of the males, there were significant differences in the weight of the spleen and the total weight of the lungs and pleural effusion between drowning and the other causes of death; however, there was no such significant difference in the females. We observed significant differences in the lungs and pleural effusion/spleen weight ratio between drowning and the other causes of death for both sexes. Therefore, these findings suggest that the ratio may be a useful index to accurately diagnose death by drowning, while ruling out mechanical asphyxiation and acute cardiac death in forensic autopsies.

A new HLA-DRB1 genotyping method using single nucleotide polymorphism (SNP) analysis with multiplex primer extension reactions and its application to mixed samples.

We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine.

A new 39-plex analysis method for SNPs including 15 blood group loci.

A novel 39-plex typing system for single nucleotide polymorphisms (SNPs) has been developed. This multiplex approach has the advantage of being able to type 38 autosomal SNPs and one sex-discriminating base exchange site on the X and Y chromosomes rapidly and simultaneously. The SNP loci on the autosomes, which we examined, contain 15 loci distributed on blood type genes: three on RhCE, two each on Km and Gc, and one each on Duffy, AcP1, Tf, MN, GPT, EsD, PI, and Kidd genes. Thirty-seven genomic DNA fragments containing a total of 38 SNPs and one sex-discriminating site were amplified in one multiplex PCR reaction. Following the reaction, single nucleotide primer extension reaction was performed by dividing these SNP loci into five groups. The SNP type of each of the 39 loci was determined at one time by capillary electrophoresis using the newly designed multi-injection method. The combined PD (power of discrimination) of this typing system was (1-1.1) x 10(-14), and the MEC (mean exclusion chance) was 0.9990. We applied this system to forensic cases, including 16 paternity testing cases (13 non-exclusion and three exclusion cases) and one personal identification case. For the paternity testing cases, the highest Essen-Möller's W-value was 0.9999995. The pM (matching probability) of the personal identification case was 2.22 x 10(-17). These data showed that this system was an excellent tool for use in forensic cases of paternity testing and personal identification.

Analysis of short tandem repeat (STR) polymorphisms by the powerplex 16 system and capillary electrophoresis: application to forensic practice.

Allele and genotype frequencies for 15 short tandem repeat (STR) polymorphisms--D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA--in a Japanese population were estimated. No deviations of the observed allele frequency from Hardy-Weinberg equilibrium expectations were found for any of the systems studied. Between 2 new pentanucleotide STR loci, Penta E and Penta D, for which there is only limited data regarding the allelic distribution in Japanese, the Penta E locus was found to be highly polymorphic and exhibited a tri- or tetra-modal distribution pattern having allelic peaks with 5, 11, 15 and 20 repeats. The distribution was significantly different from that of the other ethnic groups. Statistical parameters of forensic importance, the power of discrimination (PD), observed and expected heterozygosity values (H), polymorphism information content (PIC), power of discrimination (PD), matching probability (pM), power of exclusion (PE), and typical paternity index (PI), were calculated for the loci. These parameters indicated the usefulness of the loci in forensic personal identification and paternity testing among Japanese. The systems Penta E, FGA, D18S51 and D8S1179 were the most informative. This method was successfully applied to forensic personal identification and paternity testing among Japanese, thereby confirming its efficacy for forensic practice.