Integrin α5 regulates motility of human monocyte-derived Langerhans cells during immune response.

Langerhans cells (LCs) are mainly present in the epidermis and mucosa, and have important roles during skin infection. Migration of LCs to lymph nodes is essential for antigen presentation. However, due to the difficulties in isolating and culturing human LCs, it is not fully understood how LCs move and interact with the extracellular matrix (ECM) through their adhesion molecules such as integrin, during the immune responses. In this study, we aimed to investigate LC motility, cell shape and the role of integrin under inflammatory conditions using monocyte-derived Langerhans cells (moLCs) as a model. As a result, lipopolysaccharide (LPS) stimulation increased adhesion on fibronectin coated substrate and integrin α5 expression in moLCs. Time-lapse imaging of moLCs revealed that stimulation with LPS elongated cell shape, whilst decreasing their motility. Additionally, this decrease in motility was not observed when pre-treated with a neutralising antibody targeting integrin α5. Together, our data suggested that activation of LCs decreases their motility by promoting integrin α5 expression to enhance their affinity to the fibronectin, which may contribute to their migration during inflammation.

Roles of TRPM4 in immune responses in keratinocytes and identification of a novel TRPM4-activating agent.

The skin is a protective interface between the internal organs and environment and functions not only as a physical barrier but also as an immune organ. However, the immune system in the skin is not fully understood. A member of the thermo-sensitive transient receptor potential (TRP) channel family, TRPM4, which acts as a regulatory receptor in immune cells, was recently reported to be expressed in human skin and keratinocytes. However, the function of TRPM4 in immune responses in keratinocytes has not been investigated. In this study, we found that treatment with BTP2, a known TRPM4 agonist, reduced cytokine production induced by tumor necrosis factor (TNF) α in normal human epidermal keratinocytes and in immortalized human epidermal keratinocytes (HaCaT cells). This cytokine-reducing effect was not observed in TRPM4-deficient HaCaT cells, indicating that TRPM4 contributed to the control of cytokine production in keratinocytes. Furthermore, we identified aluminum potassium sulfate, as a new TRPM4 activating agent. Aluminum potassium sulfate reduced Ca2+ influx by store-operated Ca2+ entry in human TRPM4-expressing HEK293T cells. We further confirmed that aluminum potassium sulfate evoked TRPM4-mediated currents, showing direct evidence for TRPM4 activation. Moreover, treatment with aluminum potassium sulfate reduced cytokine expression induced by TNFα in HaCaT cells. Taken together, our data suggested that TRPM4 may serve as a new target for the treatment of skin inflammatory reactions by suppressing the cytokine production in keratinocytes, and aluminum potassium sulfate is a useful ingredient to prevent undesirable skin inflammation through TRPM4 activation.

Family-Centered Care in Neonatal Intensive Care Units: Combining Intensive Care and Family Support.

Advances in treatment in neonatal intensive care units (NICU) for preterm and sick newborns have improved the mortality rate of patients, but admission to the NICU may disrupt parent-infant interaction, with adverse consequences for infants and their families because of physical, psychological, and emotional separation. The concept of family centered care (FCC), in which family members are part of the care team and infants are close to the family, is important and has become popular in NICU. In 2013, we created a team called "Kodomo-Kazoku Mannaka" to promote FCC in Japan, and visited the NICU at Uppsala University Hospital in Sweden, which is internationally famous for FCC. Since this fruitful visit, we have been promoting FCC in Japan by exhibitions and presentations of the FCC ideas at academic conferences and using internet services. A questionnaire survey conducted in 2015 revealed that the importance and the benefits of FCC in NICU are recognized, although there are some barriers to FCC in each facility. It is hard to change facilities and social systems right away, but it is easier and more important to change people's minds. Our role is to spread the concept of FCC and to help each facility find its own way to adopt it. We will continue to make efforts encourage to promote FCC in Japan.

Tangent field technique of TomoDirect improves dose distribution for whole-breast irradiation.

TomoDirect (TD) is an intensity-modulated radiotherapy system that uses a fixed gantry angle instead of rotational beam delivery. Here, we investigated the effect of the multiple beam technique of TomoDirect on dose distribution compared with commonly-used tangential beams. We included 45 consecutive patients with right breast cancer who underwent postoperative radiotherapy in our institute in the present study. Clinical target volume (CTV) was the whole right breast. The planning target volume (PTV) was created by expanding the CTV by a 0.5 cm margin. Paired TD plans were generated for each patient; a two-beam plan using paired tangential beams and a six-beam plan with four additional beams with modified gantry angles of ± 5° from the original tangential beam set. A prescribed dose of 50 Gy was defined for 50% isodoses of the PTV. The six-beam plan delivered significantly more homogeneous doses to the PTV than the two-beam plan; and the mean dose to the PTV in the six-beam plan more closely reflected the prescribed dose. V20Gy and mean dose to the right lung and mean dose to the whole body were also significantly decreased in the six-beam plan. However, duration of radiation exposure was 1 min longer in the six-beam plan than in the two-beam plan. The dose distribution to the target and organs at risk were improved with the six-beam plan relative to the two-beam plan without increasing the whole-body radiation dose. The six-beam plan using TD is a simple technique that can be routinely applied to whole-breast irradiation in clinical practice.

Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation.

Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP(+) to NADPH. IDH1 mutations are frequently detected in grades 2-4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to D-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-L-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

The accumulation pattern of ferruginol in the heartwood-forming Cryptomeria japonica xylem as determined by time-of-flight secondary ion mass spectrometry and quantity analysis.

BACKGROUND AND AIMS: Heartwood formation is a unique phenomenon of tree species. Although the accumulation of heartwood substances is a well-known feature of the process, the accumulation mechanism remains unclear. The aim of this study was to determine the accumulation process of ferruginol, a predominant heartwood substance of Cryptomeria japonica, in heartwood-forming xylem. METHODS: The radial accumulation pattern of ferruginol was examined from sapwood and through the intermediate wood to the heartwood by direct mapping using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The data were compared with quantitative results obtained from a novel method of gas chromatography analysis using laser microdissection sampling and with water distribution obtained from cryo-scanning electron microscopy. KEY RESULTS: Ferruginol initially accumulated in the middle of the intermediate wood, in the earlywood near the annual ring boundary. It accumulated throughout the entire earlywood in the inner intermediate wood, and in both the earlywood and the latewood in the heartwood. The process of ferruginol accumulation continued for more than eight annual rings. Ferruginol concentration peaked at the border between the intermediate wood and heartwood, while the concentration was less in the latewood compared with the earlywood in each annual ring. Ferruginol tended to accumulate around the ray parenchyma cells. In addition, at the border between the intermediate wood and heartwood, the accumulation was higher in areas without water than in areas with water. CONCLUSIONS: TOF-SIMS clearly revealed ferruginol distribution at the cellular level. Ferruginol accumulation begins in the middle of intermediate wood, initially in the earlywood near the annual ring boundary, then throughout the entire earlywood, and finally across to the whole annual ring in the heartwood. The heterogeneous timing of ferruginol accumulation could be related to the distribution of ray parenchyma cells and/or water in the heartwood-forming xylem.

Seroprevalence of SARS-CoV-2 antibodies among Japanese healthcare workers from 2020 to 2022 as assayed by two commercial kits.

Antibody tests are used as surveillance tools for informing health policy making. However, results may vary by type of antibody assay and timing of sample collection following infection. Long-term longitudinal cohort studies on antibody assay seropositivity have remained limited, especially among Asian populations. Using blood samples obtained at health physicals (2020-2022) of healthcare workers (mass vaccinated with mRNA COVID-19 vaccines) at a Japanese medical center, we measured N-specific antibodies using two commercially available systems. Roche Elecsys Anti-SARS-CoV-2 measures total antibodies and Abbott Alinity SARS-CoV-2 IgG measures only IgG. Among 2538 participants, seroprevalence was found to be 16.6% via total antibody assay versus 12.9% by IgG-only (including grayzone) by mid-June 2022. For 219 cases with a previous PCR-confirmed infection, positivity was 97.3% using total antibody assay versus 76.3% using IgG-only assay at the 2022 health physical. Using PCR positive test date as day 0, while the positivity of the total antibody assay was retained for the entire study period (until more than 24-months post-infection), the IgG-only assay's positivity declined after month 4. The Mantel-Haenszel test found a significant difference in the two assays' seropositivity, between stratified groups of "within 3 months" and "4 months or more" from infection (P < 0.001). Our study found significant differences in seropositivity over time of total antibody versus IgG-only assays, suggesting an optimal assay for retaining sensitivity over the entire infection period when designing seroprevalence studies.

Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry.

Lignin, one of the main structural polymer of plant cell walls, varies in amount and monomeric composition among tissue and cell types, as well as among plant species. However, few analytical methods are available that can conveniently and accurately determine the morphological distribution of lignin units at the cellular level. In this report, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly map guaiacyl (G) and syringyl (S) lignin units in several successive growth rings of the maple xylem. TOF-SIMS imaging and a semiquantitative approach revealed clear difference in the annual distribution of lignins between the fiber and vessel. While the vessel walls were constantly G-rich with varied S/G ratios through a growth ring, the fibers showed fairly regular annual distribution of lignins in which the earlywood was S-rich with an almost constant S/G ratio and the latewood was G-rich resulting from a decrease of the S unit. The reliability of TOF-SIMS results was demonstrated by its high correlation with the results of thioacidolysis on radial distribution of the S/G ratio in several contiguous tree rings and also in the latewood and earlywood of each ring. These results indicate that TOF-SIMS allows direct visualization of lignin composition in plant tissues.

Anti-Inflammatory Role of TRPV4 in Human Macrophages.

The pathology of skin immune diseases such as atopic dermatitis is closely related to the overproduction of cytokines by macrophages. Although the pathological functions of macrophages in skin are known, mechanisms of how they detect the tissue environment remain unknown. TRPV4, a nonselective cation channel with high Ca2+ permeability, is activated at physiological temperatures from 27 to 35°C and involved in the functional control of macrophages. However, the relationship between TRPV4 function in macrophages and skin immune disease is unclear. In this study, we demonstrate that TRPV4 activation inhibits NF-κB signaling, resulting in the suppression of IL-1ß production in both human primary monocytes and macrophages derived from human primary monocytes. A TRPV4 activator also inhibited the differentiation of human primary monocytes into GM-CSF M1 macrophages but not M-CSF M2 macrophages. We also observed a significant increase in the number of inducible NO synthase-positive/TRPV4-negative dermal macrophages in atopic dermatitis compared with healthy human skin specimens. Our findings provide insight into the physiological relevance of TRPV4 to the regulation of macrophages during homeostasis maintenance and raise the potential for TRPV4 to be an anti-inflammatory target.

Performance evaluation of the Ortho VITROS SARS-CoV-2 Spike-Specific Quantitative IgG test by comparison with the surrogate virus neutralizing antibody test and clinical assessment.

BACKGROUND: Despite the worldwide campaigns of COVID-19 vaccinations, the pandemic is still a major medical and social problem. The Ortho VITROS SARS-CoV-2 spike-specific quantitative IgG (VITROS S-IgG) assay has been developed to assess neutralizing antibody (NT antibody) against SARS-CoV-2 spike (S) antibodies. However, it has not been evaluated in Japan, where the total cases and death toll are lower than the rest of the world. METHODS: The clinical performance of VITROS S-IgG was evaluated by comparing with the NT antibody levels measured by the surrogate virus neutralizing antibody test (sVNT). A total of 332 serum samples from 188 individuals were used. Of these, 219 samples were from 75 COVID-19 patients: 96 samples from 20 severe/critical cases (Group S), and 123 samples from 55 mild/moderate cases (Group M). The remaining 113 samples were from 113 healthcare workers who had received 2 doses of the BNT162b2 vaccine. RESULTS: VITROS S-IgG showed good correlation with the cPass sVNT assay (Spearman rho = 0.91). Both VITROS S-IgG and cPass sVNT showed significantly higher plateau levels of antibodies in Group S compared to Group M. Regarding the humoral immune responses after BNT162b2 vaccination, individuals who were negative for SARS-CoV-2 nucleocapsid (N)-specific antibodies had statistically lower titers of both S-IgG and sVNT compared to individuals with a history of COVID-19 and individuals who were positive for N-specific antibodies without history of COVID-19. In individuals who were positive for N-specific antibodies, S-IgG and sVNT titers were similar to individuals with a history of COVID-19. CONCLUSIONS: Although the automated quantitative immunoassay VITROS S-IgG showed a reasonable correlation with sVNT antibodies, there is some discrepancy between Vitros S-IgG and cPass sVNT in milder cases. Thus, VITROS S-IgG can be a useful diagnostic tool in assessing the immune responses to vaccination and herd immunity. However, careful analysis is necessary to interpret the results.

Increased SARS-CoV-2 seroprevalence and spread of infection without awareness among healthcare workers through 2020-2022 in a Japanese medical center.

Despite Japan's high vaccination coverage, daily numbers of new COVID-19 cases have been high. However, studies on the seroprevalence among Japanese people and the causative factors for rapid spread have remained limited. In this study, we aimed to examine the seroprevalence and associated factors in healthcare workers (HCWs) of a medical center in Tokyo using blood samples drawn at annual check-ups from 2020 to 2022. We found that of the 3,788 HCWs in 2022 (by mid-June), 669 were seropositive for N-specific antibodies (tested by Roche Elecsys Anti-SARS-CoV-2 assay); the seroprevalence surged from 0.3% in 2020 and 1.6% in 2021 to 17.7% in 2022. Notably, our study found 325 (48.6%; 325/669) cases were infected without awareness. Among those with a previously PCR-confirmed SARS-CoV-2 infection during the past three years, 79.0% (282/357) were found after January 2022, after the Omicron variant was first detected in Tokyo at the end of 2021. This study indicates the fast spread of the SARS-CoV-2 among HCWs during the Omicron surge in Japan. The high percentage of infection without awareness may be a key driving factor causing rapid person-to-person transmission, as shown in this medical center with high vaccination coverage and strict infection control measures.

Reconstituted ovaries self-assemble without an ovarian surface epithelium.

Three-dimensional (3D) stem cell models of the ovary have the potential to benefit women's reproductive health research. One such model, the reconstituted ovary (rOvary) self-assembles with pluripotent stem cell-derived germ cells creating a 3D ovarian mimic competent to support the differentiation of functional oocytes inside follicles. In this study, we evaluated the cellular composition of the rOvary revealing the capacity to generate multiple follicles surrounded by NR2F2+ stroma cells. However, the rOvary does not develop a surface epithelium, the source of second-wave pre-granulosa cells, or steroidogenic theca. Therefore, the rOvary models represent the self-assembly of activated follicles in a pre-pubertal ovary poised but not yet competent for hormone production.

Evaluation of bone marrow aspirates using the automated hematology analyzer Sysmex XN-3000.

INTRODUCTION: This study evaluated the feasibility of the Sysmex XN-3000 automated hematology analyzer for the assessment of total nucleated cells (TNC) and bone marrow (BM) cell density in routine bone marrow aspiration (BMA) samples. METHODS: A total of 54 BMA samples from 39 hematological patients were evaluated. The number of megakaryocytes was calculated by a specific gating algorithm using the body fluid mode of the WBC differential (WDF) channel. Lipid contents were calculated through a newly developed algorithm utilizing the WDF channel. The ratio of lipid particles over TNCs by the WNR channel was compared with the BM cellularity assessed by the BM biopsy. The myeloid/erythroid (M/E) ratio was calculated by measuring the number of myeloid cells in the WDF channel and the number of nucleated red blood cells (NRBCs) in the WNR channel. RESULTS: XN-3000 counts and microscopic results showed a linear correlation in TNC (R2  = .98, p < .001), megakaryocytes (R2  = .59, p = .002), NRBC (R2  = .84, p < .001), and M/E ratio (R2  = .59, p < .001). There were significant differences in the lipid/TNC ratios of hypercellular, normocellular, and hypocellular BMs measured by XN-3000 (p < .001). Receiver-operating characteristic analysis detected cut-off values of the lipid/TNC ratio of >0.4054 for hypoplasia and <0.157 for hyperplasia. The sensitivity and specificity for hypoplasia were 100% and 88%, and for hyperplasia were 89% and 86%, respectively. CONCLUSION: XN-3000 provides a quantitative assessment of BM cellularity, supporting the qualitative assessment by myelogram and BM biopsy.

Assessment of antibody dynamics and neutralizing activity using serological assay after SARS-CoV-2 infection and vaccination.

The COVID-19 antibody test was developed to investigate the humoral immune response to SARS-CoV-2 infection. In this study, we examined whether S antibody titers measured using the anti-SARS-CoV-2 IgG II Quant assay (S-IgG), a high-throughput test method, reflects the neutralizing capacity acquired after SARS-CoV-2 infection or vaccination. To assess the antibody dynamics and neutralizing potency, we utilized a total of 457 serum samples from 253 individuals: 325 samples from 128 COVID-19 patients including 136 samples from 29 severe/critical cases (Group S), 155 samples from 71 mild/moderate cases (Group M), and 132 samples from 132 health care workers (HCWs) who have received 2 doses of the BNT162b2 vaccinations. The authentic virus neutralization assay, the surrogate virus neutralizing antibody test (sVNT), and the Anti-N SARS-CoV-2 IgG assay (N-IgG) have been performed along with the S-IgG. The S-IgG correlated well with the neutralizing activity detected by the authentic virus neutralization assay (0.8904. of Spearman's rho value, p < 0.0001) and sVNT (0.9206. of Spearman's rho value, p < 0.0001). However, 4 samples (2.3%) of S-IgG and 8 samples (4.5%) of sVNT were inconsistent with negative results for neutralizing activity of the authentic virus neutralization assay. The kinetics of the SARS-CoV-2 neutralizing antibodies and anti-S IgG in severe cases were faster than the mild cases. All the HCWs elicited anti-S IgG titer after the second vaccination. However, the HCWs with history of COVID-19 or positive N-IgG elicited higher anti-S IgG titers than those who did not have it previously. Furthermore, it is difficult to predict the risk of breakthrough infection from anti-S IgG or sVNT antibody titers in HCWs after the second vaccination. Our data shows that the use of anti-S IgG titers as direct quantitative markers of neutralizing capacity is limited. Thus, antibody tests should be carefully interpreted when used as serological markers for diagnosis, treatment, and prophylaxis of COVID-19.

Structural characterization of a carbon monoxide adduct of a heme-DNA complex.

The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3'-terminal G-quartet of the DNA though a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π-π stacking interaction between the pseudo-C2-symmetric heme and the C4-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C2 axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe-CO bond in the complex is similar to that of the Fe-CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.

Interaction between the heme and a G-quartet in a heme-DNA complex.

The structure of a complex between heme(Fe(3+)) and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized by (1)H NMR. The study demonstrated that the heme(Fe(3+)) is sandwiched between the 3'-terminal G-quartets of the G-quadruplex DNA. Hence, the net +1 charge of the heme(Fe(3+)) in the complex is surrounded by the eight carbonyl oxygen atoms of the G-quartets. Interaction between the heme Fe(3+) and G-quartets in the complex was clearly manifested in the solvent (1)H/(2)H isotope effect on the NMR parameters of paramagnetically shifted heme methyl proton signals, and interaction of the heme Fe(3+) with the eight carbonyl oxygen atoms of the two G-quartets was shown to provide a strong and axially symmetric ligand field surrounding the heme Fe(3+), yielding a heme(Fe(3+)) low-spin species with a highly symmetric heme electronic structure. This finding provides new insights as to the design of the molecular architecture and functional properties of various heme-DNA complexes.

Assessment of an immunochromatographic kit for detection of severe acute respiratory syndrome coronavirus 2 and influenza viruses.

An immunochromatographic kit was developed to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses (A and B) on two detection positions of a single strip. The sensitivity and specificity for SARS-CoV-2 were 97.4 % and 100 %, respectively, and those for influenza viruses were 100 %, respectively.

Antibody response and seroprevalence in healthcare workers after the BNT162b2 vaccination in a University Hospital at Tokyo.

In 2020, we reported a low seroprevalence of N-specific antibodies in 4147 health care workers (HCWs) at a frontline hospital in Tokyo, Japan. In Japan, a vaccine campaign was launched in early 2021. We re-evaluated seroprevalences of N- and S-specific antibodies in 2202 HCWs who took two doses of the BNT162b2 vaccine. In 2021, N-specific seroprevalence remains as low as 1.59%. The seroprevalences were comparable among all HCWs regardless of exposure levels. Almost all of the HCWs elicited S-specific antibodies after vaccination. However, the HCWs who had COVID-19 elicited higher S-specific antibody titers than those who did not have COVID-19. In the HCWs without a history of COVID-19, 1.1% (23 out of 2185) were seropositive with N-specific antibodies, indicating the existence of asymptomatic infections. Also, S-specific antibody titers were higher in females and younger HCWs, and in those who had severe side effects. However, S-specific antibody titers were lower depending on the number of days after the second dose of vaccination specifically in elderly individuals. In conclusion, this study indicates N-specific seroprevalence remains low in HCWs at a frontline hospital in Tokyo. The mRNA vaccine elicited S-specific antibody in HCWs, however, the titers decreased as the days proceeded.

Variation in blood pressure and heart rate of radiological technologists in worktime tracked by a wearable device: A preliminary study.

The aim of this preliminary study was to measure the systolic BP (SBP) and diastolic BP (DBP) and heart rate (HR) of radiological technologists by WD, and evaluate variation among individuals by worktime, day of the week, job, and workplace. Measurements were obtained using a wristwatch-type WD with optical measurement technology that can measure SBP and DBP every 10 minutes and HR every 30 minutes. SBP, DBP, and HR data obtained at baseline and during work time were combined with the hours of work, day of the week, job, and workplace recorded by the participants in 8 consecutive weeks. We calculated the mean, the ratio to baseline and coefficient of variation [CV(%)] for SBP, DBP, and HR. SBP, DBP, and HR values were significantly higher during work hours than at baseline (p<0.03). The ratio to baseline values ranged from 1.02 to 1.26 for SBP and from 1.07 to 1.30 for DBP. The ratio to baseline for SBP and DBP showed CV(%) of approximately 10% according to the day of the week and over the study period. For HR, ratio to baseline ranged from 0.95 to 1.29. The ratio of mean BP to baseline was >1.2 at the time of starting work, middle and after lunch, and at 14:00. The ratio to baseline of SBP were 1.2 or more for irradiation, equipment accuracy control, registration of patient data, dose verification and conference time, and were also working in CT examination room, treatment planning room, linac room, and the office. CV(%) of BP and HR were generally stable for all workplaces. WD measurements of SBP, DBP, and HR were higher during working hours than at baseline and varied by the individuals, work time, job, and workplace. This method may enable evaluation of unconscious workload in individuals.