Publications by authors named "Masato Yamauchi"

18 Publications

  • Page 1 of 1

Evidence of Artemisinin-Resistant Malaria in Africa.

N Engl J Med 2021 Sep;385(13):1163-1171

From the Department of Tropical Medicine and Parasitology, School of Medicine (B.B., N.F., M.I., S.-I.T., M.Y., S.E., T.M.), and the Atopy Research Center, Graduate School of Medicine (B.B.), Juntendo University, Tokyo, the School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki (E.K.), and the Department of Malaria Vaccine Development, Research Institute for Microbial Diseases, Osaka University, Osaka (N.M.Q.P., T.H.) - all in Japan; and Mildmay Uganda, Nazibwa Hill, Kampala (O.T.K.), and St. Mary's Hospital Lacor (W.O., M.O.) and the Faculty of Medicine, Gulu University (D.A.A., E.I.O.-A.), Gulu - all in Uganda.

Background: In the six Southeast Asian countries that make up the Greater Mekong Subregion, has developed resistance to derivatives of artemisinin, the main component of first-line treatments for malaria. Clinical resistance to artemisinin monotherapy in other global regions, including Africa, would be problematic.

Methods: In this longitudinal study conducted in Northern Uganda, we treated patients who had infection with intravenous artesunate (a water-soluble artemisinin derivative) and estimated the parasite clearance half-life. We evaluated ex vivo susceptibility of the parasite using a ring-stage survival assay and genotyped resistance-related genes.

Results: From 2017 through 2019, a total of 14 of 240 patients who received intravenous artesunate had evidence of in vivo artemisinin resistance (parasite clearance half-life, >5 hours). Of these 14 patients, 13 were infected with parasites with mutations in the A675V or C469Y allele in the gene. Such mutations were associated with prolonged parasite clearance half-lives (geometric mean, 3.95 hours for A675V and 3.30 hours for C469Y, vs. 1.78 hours for wild-type allele; P<0.001 and P = 0.05, respectively). The ring-stage survival assay showed a higher frequency of parasite survival among organisms with the A675V allele than among those with the wild-type allele. The prevalence of parasites with mutations increased significantly, from 3.9% in 2015 to 19.8% in 2019, due primarily to the increased frequency of the A675V and C469Y alleles (P<0.001 and P = 0.004, respectively). Single-nucleotide polymorphisms flanking the A675V mutation in Uganda were substantially different from those in Southeast Asia.

Conclusions: The independent emergence and local spread of clinically artemisinin-resistant has been identified in Africa. The two mutations may be markers for detection of these resistant parasites. (Funded by the Japan Society for the Promotion of Science and others.).
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http://dx.doi.org/10.1056/NEJMoa2101746DOI Listing
September 2021

Fitness of sulfadoxine-resistant Plasmodium berghei harboring a single mutation in dihydropteroate synthase (DHPS).

Acta Trop 2021 Oct 15;222:106049. Epub 2021 Jul 15.

Department of Tropical Medicine and Parasitology, Faculty of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. Electronic address:

Genetic changes conferring drug resistance are generally believed to impose fitness costs to pathogens in the absence of the drug. However, the fitness of resistant parasites against sulfadoxine/pyrimethamine has been inconclusive in Plasmodium falciparum. This is because resistance is conferred by the complex combination of mutations in dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), which makes it difficult to separately assess the extent and magnitude of the costs imposed by mutations in dhps and dhfr. To assess the fitness costs imposed by sulfadoxine resistance alone, we generated a transgenic rodent malaria parasite, P. berghei clone harboring an A394G mutation in dhps (PbDHPS-A394G), corresponding to the causative mutation for sulfadoxine resistance in P. falciparum (PfDHPS-A437G). A four-day suppressive test confirmed that the PbDHPS-A394G clone was resistant to sulfadoxine. PbDHPS-A394G and wild-type clones showed similar growth rates and gametocyte production. This observation was confirmed in competitive experiments in which PbDHPS-A394G and wild-type clones were co-infected into mice to directly assess the survival competition between them. In the mosquitoes, there were no significant differences in oocyst production between PbDHPS-A394G and wild-type. These results indicate that the PbDHPS-A394G mutation alters the parasites to sulfadoxine resistance but may not impose fitness disadvantages during the blood stages in mice and oocyst formation in mosquitoes. These results partly explain the persistence of the PfDHPS-A437G mutant in the natural parasite populations.
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http://dx.doi.org/10.1016/j.actatropica.2021.106049DOI Listing
October 2021

Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in Northern Uganda.

Parasitol Int 2021 Apr 25;81:102277. Epub 2020 Dec 25.

Department of Tropical Medicine and Parasitology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address:

In Uganda, artemether-lumefantrine was introduced as an artemisinin-based combination therapy (ACT) for malaria in 2006. We have previously reported a moderate decrease in ex vivo efficacy of lumefantrine in Northern Uganda, where we also detected ex vivo artemisinin-resistant Plasmodium falciparum. Therefore, it is necessary to search for candidate partner alternatives for ACT. Here, we investigated ex vivo susceptibility to four ACT partner drugs as well as quinine and chloroquine, in 321 cases between 2013 and 2018. Drug-resistant mutations in pfcrt and pfmdr1 were also determined. Ex vivo susceptibility to amodiaquine, quinine, and chloroquine was well preserved, whereas resistance to mefloquine was found in 45.8%. There were few cases of multi-drug resistance. Reduced sensitivity to mefloquine and lumefantrine was significantly associated with the pfcrt K76 wild-type allele, in contrast to the association between chloroquine resistance and the K76T allele. Pfmdr1 duplication was not detected in any of the cases. Amodiaquine, a widely used partner drug for ACT in African countries, may be the first promising alternative in case lumefantrine resistance emerges. Therapeutic use of mefloquine may not be recommended in this area. This study also emphasizes the need for sustained monitoring of antimalarial susceptibility in Northern Uganda to develop proper treatment strategies.
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http://dx.doi.org/10.1016/j.parint.2020.102277DOI Listing
April 2021

Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea.

PLoS Pathog 2020 12 15;16(12):e1009133. Epub 2020 Dec 15.

Department of Tropical Medicine and Parasitology, Juntendo University Faculty of Medicine, Tokyo, Japan.

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants' genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites' drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.
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http://dx.doi.org/10.1371/journal.ppat.1009133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771869PMC
December 2020

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda.

Malar J 2020 Feb 18;19(1):76. Epub 2020 Feb 18.

Department of Tropical Medicine and Parasitology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Background: Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods.

Methods: Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed.

Results: Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC values for chloroquine were persistently low throughout the study period (17.4-24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower ICs to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018.

Conclusion: This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.
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http://dx.doi.org/10.1186/s12936-020-03157-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026951PMC
February 2020

Lack of significant recovery of chloroquine sensitivity in Plasmodium falciparum parasites following discontinuance of chloroquine use in Papua New Guinea.

Malar J 2018 Nov 26;17(1):434. Epub 2018 Nov 26.

Department of Tropical Medicine and Parasitology, Juntendo University, Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.

Background: Chloroquine treatment for Plasmodium falciparum has been discontinued in almost all endemic regions due to the spread of resistant isolates. Reversal of chloroquine susceptibility after chloroquine discontinuation has been reported in dozens of endemic regions. However, this phenomenon has been mostly observed in Africa and is not well documented in other malaria endemic regions. To investigate this, an ex vivo study on susceptibility to chloroquine and lumefantrine was conducted during 2016-2018 in Wewak, Papua New Guinea where chloroquine had been removed from the official malaria treatment regimen in 2010. Genotyping of pfcrt and pfmdr1 was also performed.

Results: In total, 368 patients were enrolled in this study. Average IC values for chloroquine were 106.6, 80.5, and 87.6 nM in 2016, 2017, and 2018, respectively. These values were not significantly changed from those obtained in 2002/2003 (108 nM). The majority of parasites harboured a pfcrt K76T the mutation responsible for chloroquine resistance. However, a significant upward trend was observed in the frequency of the K76 (wild) allele from 2.3% in 2016 to 11.7% in 2018 (P = 0.008; Cochran-Armitage trend test).

Conclusions: Eight years of chloroquine withdrawal has not induced a significant recovery of susceptibility in Papua New Guinea. However, an increasing tendency of parasites harbouring chloroquine-susceptible K76 suggests a possibility of resurgence of chloroquine susceptibility in the future.
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http://dx.doi.org/10.1186/s12936-018-2585-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260888PMC
November 2018

Rapid selection of sulphadoxine-resistant Plasmodium falciparum and its effect on within-population genetic diversity in Papua New Guinea.

Sci Rep 2018 04 3;8(1):5565. Epub 2018 Apr 3.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

The ability of the human malarial parasite Plasmodium falciparum to adapt to environmental changes depends considerably on its ability to maintain within-population genetic variation. Strong selection, consequent to widespread antimalarial drug usage, occasionally elicits a rapid expansion of drug-resistant isolates, which can act as founders. To investigate whether this phenomenon induces a loss of within-population genetic variation, we performed a population genetic analysis on 302 P. falciparum cases detected during two cross-sectional surveys in 2002/2003, just after the official introduction of sulphadoxine/pyrimethamine as a first-line treatment, and again in 2010/2011, in highly endemic areas in Papua New Guinea. We found that a single-origin sulphadoxine-resistant parasite isolate rapidly increased from 0% in 2002/2003 to 54% in 2010 and 84% in 2011. However, a considerable number of pairs exhibited random associations among 10 neutral microsatellite markers located in various chromosomes, suggesting that outcrossing effectively reduced non-random associations, albeit at a low average multiplicity of infection (1.35-1.52). Within-population genetic diversity was maintained throughout the study period. This indicates that the parasites maintained within-population variation, even after a clonal expansion of drug-resistant parasites. Outcrossing played a role in the preservation of within-population genetic diversity despite low levels of multiplicity of infection.
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http://dx.doi.org/10.1038/s41598-018-23811-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882878PMC
April 2018

Artemisinin-Resistant Plasmodium falciparum with High Survival Rates, Uganda, 2014-2016.

Emerg Infect Dis 2018 04;24(4):718-726

Because ≈90% of malaria cases occur in Africa, emergence of artemisinin-resistant Plasmodium falciparum in Africa poses a serious public health threat. To assess emergence of artemisinin-resistant parasites in Uganda during 2014-2016, we used the recently developed ex vivo ring-stage survival assay, which estimates ring-stage-specific P. falciparum susceptibility to artemisinin. We conducted 4 cross-sectional surveys to assess artemisinin sensitivity in Gulu, Uganda. Among 194 isolates, survival rates (ratio of viable drug-exposed parasites to drug-nonexposed controls) were high (>10%) for 4 isolates. Similar rates have been closely associated with delayed parasite clearance after drug treatment and are considered to be a proxy for the artemisinin-resistant phenotype. Of these, the PfKelch13 mutation was observed in only 1 isolate, A675V. Population genetics analysis suggested that these possibly artemisinin-resistant isolates originated in Africa. Large-scale surveillance of possibly artemisinin-resistant parasites in Africa would provide useful information about treatment outcomes and help regional malaria control.
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http://dx.doi.org/10.3201/eid2404.170141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5875287PMC
April 2018

Morphological classification and comparison of suboccipital muscle fiber characteristics.

Anat Cell Biol 2017 Dec 29;50(4):247-254. Epub 2017 Dec 29.

Department of Anatomy, Tokyo Dental College, Tokyo, Japan.

In an attempt to clarify the function of the suboccipital muscles, we performed morphological observation of the suboccipital muscles for variations in the muscle belly and compared the morphology of their muscle fibers in terms of cross-sectional area by immunostaining with anti-myosin heavy chain antibodies. The cadavers of 25 Japanese individuals were used: 22 for morphological examinations and three for histological examinations. Among samples of the rectus capitis posterior major muscle (RCPma) and rectus capitis posterior minor muscle (RCPmi), 86.4% had a typical muscle appearance with a single belly, and 13.6% had an anomalous morphology. None of the samples of the obliquus capitis superior (OCS) or obliquus capitis inferior (OCI) muscles had an anomalous appearance. Measurement of cross-sectional area revealed that fast-twitch muscle fibers in the RCPma and OCI had a significantly greater cross-sectional area than those of the RCPmi and OCS. The cross-sectional area of intermediate muscle fibers was also significantly greater in the OCS than in the RCPma, RCPmi, and OCI. The cross-sectional area of slow-twitch muscle fibers was significantly greater in the OCS than in the RCPma, RCPmi, and OCI, and the RCPmi showed a significantly greater cross-sectional area for slow-twitch muscle fibers than did the RCPma, and OCI. Our findings indicate that the RCPmi and OCS exert a greater force than the RCPma and OCI, and act as anti-gravity agonist muscles of the head. Prolonged head extension in individuals with anomalous suboccipital muscle groups could result in dysfunction due to undue stress.
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http://dx.doi.org/10.5115/acb.2017.50.4.247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768560PMC
December 2017

Anatomic and Histological Study of Lingual Nerve and Its Clinical Implications.

Bull Tokyo Dent Coll 2017 ;58(2):95-101

Department of Anatomy, Tokyo Dental College.

Although the risk of injuring the lingual nerve in the mandibular molar area during dental treatment is high, it can be repaired by nerve grafting. However, from the perspective of clinical dentistry, the pathway and histomorphometric characteristics of this nerve remain to be documented in detail. The purpose of the present study was to morphologically elucidate the pathway of the lingual nerve to clarify its significance in a clinical setting. A histomorphometric analysis was also performed in consideration of nerve grafting. The vertical distance between the occlusal plane and the superior margin of the lingual nerve showed a gradual decrease from the premolar toward the distal molar area. This suggests that the risk of injuring the lingual nerve increases gradually toward the distal area. The average total fascicular area of the lingual nerve was 1.90 mm, which was larger than that of the sural nerve. It is the first-choice donor nerve for grafting. Therefore, even though the total fascicular area of the donor nerve is a little smaller than that of the recipient nerve, nerve grafting should be successful.
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http://dx.doi.org/10.2209/tdcpublication.2016-0010DOI Listing
June 2018

Measuring the physical and economic impact of filarial lymphoedema in Chikwawa district, Malawi: a case-control study.

Infect Dis Poverty 2017 Apr 3;6(1):28. Epub 2017 Apr 3.

Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, UK.

Background: Lymphatic filariasis (LF) is one of the primary causes of lymphoedema in sub-Saharan Africa, and has a significant impact on the quality of life (QoL) of those affected. In this paper we assess the relative impact of lymphoedema on mobility and income in Chikwawa district, Malawi.

Methods: A random sample of 31 people with lymphoedema and 31 matched controls completed a QoL questionnaire from which both an overall and a mobility-specific score were calculated. Two mobility tests were undertaken, namely the 10 m walking test [10MWT] and timed up and go [TUG] test, and a subset of 10 cases-control pairs wore GPS data loggers for 3 weeks to measure their mobility in a more natural setting. Retrospective economic data was collected from all 31 case-control pairs, and each participant undertaking the GPS activity recorded daily earnings and health expenditure throughout the observation period.

Results: Cases had a significantly poorer overall QoL (cases = 32.2, controls = 6.0, P < 0.01) and mobility-specific (cases = 43.1, controls = 7.4, P < 0.01) scores in comparison to controls. Cases were also significantly slower (P < 0.01) at completing the timed mobility tests, e.g. mean 10MWT speed of 0.83 m/s in comparison to 1.10 m/s for controls. An inconsistent relationship was observed between mobility-specific QoL scores and the timed test results for cases (10MWT correlation = -0.06, 95% CI = (-0.41, 0.30)), indicating that their perceived disability differed from their measured disability, whereas the results were consistent for controls (10MWT correlation = -0.61, 95% CI = (-0.79, -0.34)). GPS summaries indicated that cases generally walk shorter distances at slower speeds than control, covering a smaller geographical area (median area by kernel smoothing: cases = 1.25 km, controls = 2.10 km, P = 0.16). Cases reported earning less than half that earned by controls per week (cases = $0.70, controls = $1.86, P = 0.064), with a smaller proportion of their earnings (16% vs 22%, P = 0.461) being spent on healthcare.

Conclusions: Those affected by lymphoedema are at a clear disadvantage to their unaffected peers, experiencing a lower QoL as confirmed by both subjective and objective mobility measures, and lower income. This study also indicates that objective measures of mobility may be a useful supplement to self-assessed QoL questionnaires when assessing the future impact of lymphoedema management interventions.
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http://dx.doi.org/10.1186/s40249-017-0241-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376674PMC
April 2017

Sympathetic and parasympathetic neurons are likely to be absent in the human vestibular and geniculate ganglia: an immunohistochemical study using elderly cadaveric specimens.

Okajimas Folia Anat Jpn 2016 ;93(1):1-4

Department of Anatomy, Tokyo Dental College.

The vestibular and geniculate ganglia of the ear in experimental animals carry both of the tyrosine hydroxylase (TH)-positive sympathetic neurons and the neuronal nitric oxide synthase (nNOS)-positive parasympathetic neurons. With an aid of immunohistochemistry, we examined these ganglia as well as the horizontal part of the facial nerve using specimens from 10 formalin-fixed elderly cadavers. The submandibular ganglion from the same cadavers was used for the positive control for both markers. Although there was a nonspecific reaction in nuclei for the present antibody of nNOS, these ganglia were unlikely to contain either nNOS- or TH-positive neurons. However, we did not deny a possibility that the absence was a result of degeneration with aging. In contrast, the facial nerve horizontal part consistently contained both of TH-positive- and nNOS-positive fibers. These fibers might regulate blood supply to the facial nerve and the dysregulation leads to edema to elevate pressure on the nerve within its osseous canal.
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http://dx.doi.org/10.2535/ofaj.93.1DOI Listing
April 2017

Radiation dose evaluation in head and neck MDCT examinations with a 6-year-old child anthropomorphic phantom.

Pediatr Radiol 2010 Jul 12;40(7):1206-14. Epub 2010 Jan 12.

School of Health Sciences, Nagoya University, Daikominami, Higashi-ku, Nagoya 461-8673, Japan.

Background: CT examinations of the head and neck are the most commonly performed CT studies in children, raising concern about radiation dose and their risks to children.

Objective: The purpose of this study was to clarify radiation dose levels for children of 6 years of age undergoing head and neck multidetector CT (MDCT) examinations.

Materials And Methods: Radiation doses were measured with small-sized silicon-photodiode dosimeters that were implanted at various tissue and organ positions within a standard 6-year-old anthropomorphic phantom. Organ and effective doses of brain CT were evaluated for 19 protocols in nine hospitals on various (2-320 detector rows) MDCT scanners.

Results: The maximum value of mean organ dose in brain CT was 34.3 mGy for brain. Maximum values of mean doses for the radiosensitive lens and thyroid were 32.7 mGy for lens in brain CT and 17.2 mGy for thyroid in neck CT. seventy-fifth percentile of effective dose distribution in brain CT was approximately the same as the diagnostic reference level (DRL) in the 2003 UK survey.

Conclusion: The results of this study would encourage revision of MDCT protocols in pediatric head and neck CT examinations for dose reduction and protocol standardization.
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http://dx.doi.org/10.1007/s00247-009-1495-zDOI Listing
July 2010

Evaluation of radiation doses from MDCT-imaging in otolaryngology.

Radiat Prot Dosimetry 2009 Aug 18;136(1):38-44. Epub 2009 Jul 18.

School of Health Sciences, Nagoya University, 1-1-20, Daikominami, Higashi-ku, Nagoya, Japan.

The purpose of this study was to clarify patient doses in the current otolaryngological multi-detector row computed tomography (MDCT) examinations. Patient doses were measured with an in-phantom dosimetry system which was composed of 48 photodiode dosimeters embedded within an anthropomorphic phantom. Organ and effective doses were evaluated according to the International Commission on Radiological Protection Publication 103. In neck CT, doses for salivary glands and for thyroid were high, 7.6-29.9 and 13.4-60.3 mGy, respectively. In sinus CT, brain and lens doses were high, 7.6-24.6 and 10.6-32.0 mGy, respectively, and in inner ear CT, lens dose was 8.0-35.3 mGy. Effective doses were 1.8-6.6 mSv in neck CT, 0.5-0.9 mSv in sinus CT and 0.3-0.6 mSv in inner ear CT. The present dose data would be used to estimate radiation risks for patients undergoing otolaryngological MDCT examinations.
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http://dx.doi.org/10.1093/rpd/ncp138DOI Listing
August 2009

An in vivo model to study osteogenic gene regulation: targeting an avian retroviral receptor (TVA) to bone with the bone sialoprotein (BSP) promoter.

J Bone Miner Res 2005 Aug 21;20(8):1403-13. Epub 2005 Mar 21.

Division of Oral Biology, Tufts School of Dental Medicine, Boston, Massachusetts 02111, USA.

Unlabelled: To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene.

Introduction: Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation.

Materials And Methods: We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice.

Results And Conclusion: Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased approximately 15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo.
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http://dx.doi.org/10.1359/JBMR.050316DOI Listing
August 2005

Autoregulation of bone sialoprotein gene in pre-osteoblastic and non-osteoblastic cells.

Biochem Biophys Res Commun 2004 Apr;316(2):461-7

Division of Oral Biology, Department of General Dentistry, Tufts University School of Dental Medicine, Boston, MA 02111, USA.

Regulation of the bone sialoprotein (BSP) gene is important in the differentiation of osteoblasts, in bone matrix mineralization, and in tumor metastasis. We investigated BSP gene transcription by performing functional analysis of the 9256bp of the 5' flanking region of the murine BSP gene containing its promoter. We found that the forced expression of BSP stimulated mouse BSP promoter activity in a dose-dependent manner in both MC3T3-E1 preosteoblast and HEK-293 cell lines, which was transcriptional factor Cbfa1 independent. Co-culture of cells separately expressing BSP promoter reporter and BSP failed to mediate the BSP autoregulation, suggesting that the event might happen intracellularly. Deletion analysis of the BSP promoter indicated that the proximal promoter (110bp) was sufficient to confer this autoregulation. We conclude that the BSP gene is autoregulated in part by a positive feedback on its own promoter.
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http://dx.doi.org/10.1016/j.bbrc.2004.02.068DOI Listing
April 2004

Progenitor cells from dental follicle are able to form cementum matrix in vivo.

Connect Tissue Res 2002 ;43(2-3):406-8

Department of Operative Dentistry and Endodontics, Kanagawa Dental College, 82 Inaokacho, Yokosuka-city, Kanagawa, Japan 238-8580.

To address the molecular mechanisms of cementogenesis, we have isolated dental follicle cells and examined them to see if they contain cementoblast progenitors. Dental follicle tissue was dissected from the root surface of bovine tooth germ and cells were released by digestion with bacterial collagenase. The released cells were maintained as a bovine dental follicle cells (BDFC). To elucidate the differentiation capacity of BDFC, they were transplanted into severe combined immunodeficiency (SCID) mice for 4 weeks. Transplanted BDFC formed cementumlike matrix; in contrast, bovine alveolar osteoblast (BAOB) transplants formed bonelike matrix, and bovine periodontal ligament cells (BPDL) formed a small amount of the cementumlike matrix. Immunohistochemical analysis showed that cementumlike matrix was positive for anti-cementum attachment protein monoclonal antibody, whereas bone-like matrix was negative. These results indicated that the BDFC contained cementoblast progenitors that were able to differentiate to cementoblasts in vivo. They also indicated that the BDFC are phenotypically distinct from BAOB and BPDL, and provide a useful model for investigating molecular mechanisms of cementogenesis.
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http://dx.doi.org/10.1080/03008200290001023DOI Listing
April 2003

An L1 element disrupts human bone sialoprotein promoter: lack of tissue-specific regulation by distalless5 (Dlx5) and runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements.

Gene 2002 Oct;299(1-2):205-17

CIHR Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Room 239 FitzGerald Building, 150 College Street, Toronto, Ontario M5S 3E2, Canada.

Bone sialoprotein (BSP) is a phosphorylated and sulphated glycoprotein with hydroxyapatite nucleating properties that is specifically expressed in association with physiological and pathological mineralization. Although previous studies have indicated that tissue-specific expression of murine BSP is regulated through a proximal homeodomain element that binds distalless5 (Dlx5) transcription analysis of the homologous human promoter revealed modest enhancement in osteogenic cells. Moreover, whereas forced expression of an antisense Dlx5 vector increased transcription, Dlx5 expression did not alter transcription significantly. Since extended promoter sequences are required to confer absolute tissue-specific expression of BSP in vivo, we characterized the upstream region of the human BSP gene. In contrast to the rat and mouse promoters, which show conserved sequences extending several kbs upstream, analysis of approximately 3 kb of the human promoter showed no sequence conservation beyond -0.99 kb. Southern blot analysis of genomic DNA from four different BAC clones showed that this sequence was not an aberration in the human genomic library used to isolate the BSP gene. Using clone BAC H-NH0811I08, the human BSP promoter sequence was extended approximately 8 kb upstream from which the non-homologous region was characterized as a 3.48 kb insert coding for an L1 retrotransposon element. Transcriptional analyses of chimeric promoter constructs revealed that the retrotransposon element suppresses transcription <80%. Upstream of the inserted DNA several regions, varying in length from 26 to 161 bps, were conserved within the mouse and human promoters. One of these conserved regions included a runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements consensus element in reverse orientation. Whereas a multimeric form of the element was transcriptionally active in response to Runx2/Cbfa1 when ligated to the BSP basal promoter, the single element in the context of the extended promoter was unresponsive. These studies have characterized the upstream promoter of the human BSP gene, which is interrupted by a unique high-frequency DNA insert that suppresses BSP gene transcription.
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http://dx.doi.org/10.1016/s0378-1119(02)01074-0DOI Listing
October 2002
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