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Circulating anti-double-stranded DNA antibody-secreting cells in patients with systemic lupus erythematosus: a novel biomarker for disease activity
H Hanaoka, Y Okazaki, T Satoh, Y Kaneko, H Yasuoka, N Seta and M Kuwana Lupus 2012 21: 1284 originally published online 27 June 2012 DOI: 10.1177/0961203312453191 The online version of this article can be found at: http://lup.sagepub.com/content/21/12/1284

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Lupus (2012) 21, 12841293

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Circulating anti-double-stranded DNA antibody-secreting cells in patients with systemic lupus erythematosus: a novel biomarker for disease activity
H Hanaoka, Y Okazaki, T Satoh, Y Kaneko, H Yasuoka, N Seta and M Kuwana
Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Japan

Antibodies against double-stranded DNA (dsDNA) are widely used to diagnose systemic lupus erythematosus (SLE) and evaluate its activity in patients. This study was undertaken to examine the clinical utility of circulating anti-dsDNA antibody-secreting cells for evaluating SLE patients. Anti-dsDNA antibody-secreting cells quantified using an enzyme-linked immunospot assay were detected in the spleen, bone marrow and peripheral blood from MRL/lpr but not in control BALB/c mice. Circulating anti-dsDNA antibody-secreting cells were detected in 29 (22%) of 130 patients with SLE, but in none of 49 with non-SLE connectivetissue disease or 18 healthy controls. The presence of circulating anti-dsDNA antibody-secreting cells was associated with persistent proteinuria, high SLE disease activity index and systemic lupus activity measures, and a high serum anti-dsDNA antibody titre measured with an enzyme-linked immunosorbent assay. The positive predictive value for active disease was 48% for circulating anti-dsDNA antibody-secreting cells versus 17% for serum anti-dsDNA antibodies. A prospective cohort of patients with circulating anti-dsDNA antibodies and inactive SLE showed that the cumulative disease flare-free rate was significantly lower in patients with than without circulating anti-dsDNA antibody-secreting cells (p < 0.001). Circulating antidsDNA antibody-secreting cells are a useful biomarker for assessing disease activity in SLE patients. Lupus (2012) 21, 12841293. Key words: Systemic lupus erythematosus; anti-DNA antibodies; B cells; biomarker

Introduction
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies to various cellular components.1 While many autoantibody specicities are detected in SLE patients, antibodies against double-stranded DNA (dsDNA) are thought to be actively involved in tissue damage through processes such as tissue deposition and subsequent complement- and Fc receptor-mediated inammatory response activation.2 Anti-dsDNA antibodies are detected specically in patients with SLE and are included in the American College of Rheumatology (ACR) classication criteria for
Correspondence to: Masataka Kuwana, Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Email: kuwanam@z5.keio.jp Received 4 March 2012; accepted 6 June 2012
! The Author(s), 2012. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav

SLE.3 Circulating anti-dsDNA antibody levels are reported to correlate closely with SLE clinical activity.4 Thus, anti-dsDNA antibody assays are widely used to help establish a diagnosis of SLE and to evaluate disease activity in routine clinical settings.5 However, the signicance of anti-dsDNA antibodies and the extent to which they are directly involved in disease pathogenesis, rather than being mere bystanders, is under debate.6 In general, antibodies are produced by B lymphocyte-lineage plasmablasts and plasma cells in the bone marrow and the red pulp of the spleen, and, to a lesser extent, by B cells in the secondary lymphoid organs and inltrating peripheral tissues.7,8 In fact, detection of anti-dsDNA antibody-producing cells is reported in the spleen of lupus-prone mice.9,10 In systemic immune responses, antibody-secreting cells are released into the peripheral blood from secondary lymphoid organs such as spleen;11 however, it is not clear whether anti-dsDNA antibody-secreting cells are
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released into circulation, either in SLE patients or in the lupus mouse model. In this study, we developed an assay system to detect circulating anti-dsDNA antibody-secreting cells, and evaluated the clinical utility of this assay in patients with SLE.

Materials and methods

Mice Female MRL/lpr (lupus-prone) and BALB/c mice (control) were purchased from Sankyo Lab Service (Tokyo, Japan). Peripheral blood, spleen and bone marrow were obtained from mice at three, ve, seven and 16 weeks of age. All experimental protocols were approved by the Keio University Ethics Committee for Animal Experiments. Patients and controls We studied 130 patients with SLE fullling the ACR classication criteria.3 Fifty-seven patients with non-SLE connective-tissue disease were used as a disease control, including 32 with rheumatoid arthritis who met the ACR criteria,12 15 with primary Sjo grens syndrome who met the revised European criteria13 without any other connectivetissue disease, and 10 with systemic sclerosis who met the ACR criteria.14 Eighteen healthy volunteers were also used as a control. All blood samples were obtained after the subjects gave informed, written consent, as approved by the Institutional Review Board. Clinical features Through a retrospective chart review conducted at the same time as blood sampling, we recorded the demographic features, treatment regimens and individual items of the classication criteria for SLE,3 the disease activity index (SLEDAI)15 and systemic lupus activity measures (SLAM)16 for all the SLE patients. We also recorded laboratory ndings, including leukocyte count, lymphocyte count, haemoglobin, haematocrit, platelet count, erythrocyte sedimentation rate, serum albumin, 50% complement haemolytic activity (CH50), serum creatinine and urinary sediment, and calculated the SLEDAI and SLAM for each patient. Active disease was dened as SLEDAI ! 5, whereas

inactive disease was dened as SLEDAI<5, according to the previous report.17 In some instances, SLE patients with inactive disease and high-titre serum anti-dsDNA antibodies were prospectively followed for up to 25 months after the anti-dsDNA antibody-secreting cell measurement to assess whether the disease ared or remained inactive. A disease are was recorded if the patient experienced an exacerbation of SLE that met the standard of active disease and required treatment modication, such as increased corticosteroid dosage or the introduction of immunosuppressants. Preparation of mononuclear cells (MNCs) Mouse MNCs were isolated from the peripheral blood, spleen and bone marrow using Lymphosepar II (Immuno-Biological Laboratories, Gunma, Japan) density-gradient centrifugation. Human MNCs were isolated from heparinized venous blood by Lymphoprep (Nycomed Pharma AS, Oslo, Norway) densitygradient centrifugation. MNCs were counted and resuspended in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin and 50 mg/mL streptomycin. Quantification of anti-dsDNA antibody-secreting cells Anti-dsDNA antibody-secreting cells were quantied by an enzyme-linked immunospot (ELISPOT) assay based on a previously reported strategy,18 with some modications. Briey, lambda phage DNA (Sigma, St. Louis, MO, USA) was treated with S1 nuclease (1,000 U/mL; Thermo Fisher Scientic, Waltham, MA, USA) to eliminate contaminating single-stranded DNA. Polyvinylidene diouride-coated 96-well microtitre plates (Millipore-Amicon, Bedford, MA, USA) were incubated with 0.1% methylated bovine serum albumin (BSA) (Sigma), then with the nuclease-treated lambda phage DNA (100 mg/mL), followed by blocking with 1% BSA (Sigma). MNCs (5 105 cells/well) were then cultured in the dsDNAcoated plates at 37 C in a humidied atmosphere. After four hours of culture, the cells were removed and the membranes were incubated with alkaline phosphatase-conjugated goat anti-mouse or antihuman IgG (ICN/Cappel, Aurora, OH, USA). Finally, antibodies bound to the membrane were visualized as spots by incubation with nitro blue
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tetrazolium/5-bromo-4-chloro-indolyl phosphate. Each experiment was carried out in ve independent wells, and the results were expressed as the mean of the ve values. The cut-o level of antidsDNA antibody-secreting cells was set at two cells per 5 105 MNCs, according to our previous study.18 In some experiments, MNCs depleted of CD19 or CD138 cells were applied to the ELISPOT assay as described previously;19 the cells were depleted with an anti-CD19 or antiCD138 monoclonal antibody (Beckman-Coulter, Fullerton, CA, USA) combined with magnetic bead-coupled anti-mouse IgG antibody (Miltenyi Biotech, Bergisch Gladbach, Germany), followed by separation on a MACS column. Mock treatment used a control monoclonal antibody to an irrelevant antigen. Flow cytometric analysis revealed that CD19 or CD138 cells were nearly absent in the treated fractions (<0.5%). Results were expressed as the relative number of antidsDNA antibody-secreting cells, calculated as the ratio of spots obtained in depleted cell cultures to those in mock-treated cultures. Serum anti-dsDNA antibody measurement Serum anti-dsDNA antibodies were measured using a commercial enzyme-linked immunosorbent assay kit (MESACUP DNA-II test, MBL, Nagoya, Japan) according to the manufacturers instructions. The nuclease-treated lambda phage DNA used in this kit was identical to the antigen used in the ELISPOT assay to detect anti-dsDNA antibody-secreting cells. The cut-o level was 12 IU/mL. The maximum reliable range of the antibody titre was 400 IU/mL, which was twice the concentration of Wo/80, a worldwide standard antidsDNA antibody serum.20 In some experiments, high-titre anti-dsDNA antibodies were dened as having the antibody level >100 IU/mL. Statistical analysis Continuous values were shown as the mean standard deviation (SD). Dierences between two groups were compared using the non-parametric Mann-Whitney U test. Correlation coecients were obtained by Spearmans correlation analysis. Multivariate analysis using a logistic regression model was conducted to determine independent parameters associated with the presence of circulating anti-dsDNA antibody-secreting cells. Cumulative disease are-free rates were calculated using the Kaplan-Meier method, and dierences between two groups were tested with a log rank test.
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Results
Detection of anti-dsDNA antibody-secreting cells in lupus-prone mice We rst used the MRL/lpr lupus model mouse to assess our ELISPOT assay system for detecting anti-dsDNA antibody-secreting cells. In images of anti-dsDNA antibody-secreting cells from the peripheral blood, bone marrow and spleen of MRL/lpr and control BALB/c 16-week-old mice (Figure 1A), typical round spots with dark, slightly fuzzy-edged centres were detected specically in the MRL/lpr mouse samples. We tested dierent culture periods, including one, two, four, eight and 24 hours, and found that the clear spots were consistently obtained when the cells were cultured for four hours. The frequency of anti-dsDNA antibodysecreting cells was signicantly greater in the spleen than in the bone marrow or peripheral blood (p 0.02 for both comparisons) (Figure 1B). Further evaluation of MRL/lpr mice at three, ve, seven and 16 weeks of age revealed antidsDNA antibody-secreting cells in the bone marrow and spleen as early as three weeks of age, and in the peripheral blood of mice older than ve weeks of age (data not shown). Circulating anti-dsDNA antibody-secreting cells in patients with SLE We next evaluated the anti-dsDNA antibodysecreting cells in the peripheral blood from control subjects and patients with SLE using the ELISPOT assay, in which the secondary anti-mouse IgG antibody was replaced by an anti-human IgG antibody. As shown in Figure 2, anti-dsDNA antibodysecreting cells were specically detected in 29 (22%) patients with SLE, and the levels of these cells were signicantly higher in the SLE patients than in the disease or healthy control groups (p < 0.001 for both comparisons). Despite the high clinical specicity, the clinical sensitivity of circulating anti-dsDNA antibody-secreting cells for SLE was only 22%, which was markedly inferior to that of serum anti-dsDNA antibodies measured simultaneously (79%). To determine the cell types secreting IgG antidsDNA antibodies in our assay system, peripheral blood MNCs depleted of CD19 or CD138 cells were prepared and subjected to the ELISPOT assay (Figure 3A). Depleting the CD19 or CD138 cells reduced but did not completely suppress the frequency of anti-dsDNA antibody-secreting cells (Figure 3B), indicating that the circulating cells

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Figure 1 ELISPOT assays to detect anti-dsDNA antibody-secreting cells in MRL/lpr and BALB/c mice. Anti-dsDNA antibodysecreting cells in (A) peripheral blood, bone marrow and spleen from a representative MRL/lpr or BALB/c mouse at 16 weeks of age; (B) MNCs from peripheral blood, bone marrow and spleen from MRL/lpr and BALB/c mice at 16 weeks of age (n 4). Comparisons between two groups were made using the Mann-Whitney U test. MNCs: mononuclear cells; PB: peripheral blood; BM: bone marrow; SP: spleen.

secreting anti-dsDNA antibodies consisted of both CD19 B cells and CD138 plasmablasts or plasma cells. Correlation between circulating anti-dsDNA antibody-secreting cells and serum anti-dsDNA antibodies The frequency of circulating anti-dsDNA antibodysecreting cells was correlated with the serum antidsDNA antibody titre in 130 patients with SLE (Figure 4) (r 0.48, p < 0.001). Further analysis of the concordance between circulating anti-dsDNA antibody-secreting cells and serum anti-dsDNA antibodies found an analytical sensitivity and

specicity of 27% and 100%, respectively. Thus, SLE patients with anti-dsDNA antibody-secreting cells in the circulation represented only a small subset of the patients with serum anti-dsDNA antibodies. Clinical features of SLE patients with circulating anti-dsDNA antibody-secreting cells The demographic and clinical features were compared between SLE patients with and without circulating anti-dsDNA antibody-secreting cells; the results are listed in Table 1. Patients with circulating anti-dsDNA antibody-secreting cells were predominantly males and had a high frequency of
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Figure 2 ELISPOT assay of circulating anti-dsDNA antibody-secreting cells in SLE patients and disease or healthy controls. Disease controls included patients with rheumatoid arthritis, primary Sjo grens syndrome and systemic sclerosis. Comparisons between two groups were made using the Mann-Whitney U test. Dotted line: cut-off level (two per 5 105 MNCs).

persistent proteinuria (p < 0.001 for both comparisons). Patients with circulating anti-dsDNA antibody-secreting cells scored worse than those without on clinical disease activity assessments, including the SLEDAI and SLAM (p 0.004 and 0.02, respectively). In fact, the number of circulating anti-dsDNA antibody-secreting cells correlated with SLEDAI (r 0.58, p 0.007). In addition, we found active disease in nearly half of the patients with circulating anti-dsDNA antibody-secreting cells, a much larger proportion than in those without circulating anti-dsDNA antibody-secreting cells (48% versus 5%; p < 0.001). Finally, the serum anti-dsDNA antibody titres were higher in patients with circulating anti-dsDNA antibodysecreting cells than in those without (p 0.001), and the prednisolone dosage was greater in patients with circulating anti-dsDNA antibody-secreting cells (p 0.02). Multivariate analysis revealed that SLEDAI and anti-dsDNA antibody titres were identied as independent parameters associated with the presence of circulating anti-dsDNA
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antibodies-secreting cells (p 0.01 and p < 0.001, respectively), but the association of gender with circulating anti-dsDNA antibodies-secreting cells was lost. As shown in Figure 4, SLE patients with active disease were distributed mainly in the group positive for both circulating anti-dsDNA antibodysecreting cells and serum anti-dsDNA antibodies. The positive predictive value for active disease in SLE patients was 48% for circulating anti-dsDNA antibody-secreting cells and 17% for serum antidsDNA antibodies, while the negative predictive value was almost the same for these assays (95% and 96%, respectively).
Prospective cohort of SLE patients with inactive disease and positive for serum anti-dsDNA antibody in the presence or absence of circulating anti-dsDNA antibodysecreting cells

Despite a strong association between the presence of circulating anti-dsDNA antibody-secreting cells and active SLE, almost half of the patients with

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Figure 3 Anti-dsDNA antibody-secreting cells in peripheral blood MNCs depleted of CD19 or CD138 cells. (A) Representative images of anti-dsDNA antibody-secreting cells from an SLE patient, obtained from ELISPOT assays of peripheral blood MNCs with mock treatment or depletion of CD19 or CD138 cells. (B) Relative frequency of anti-dsDNA antibody-secreting cells in peripheral blood samples after CD19 or CD138 cell depletion (n 4), calculated as the frequency of spots obtained with depletion relative to that with mock treatment (100%). Comparisons between two groups were made using the Mann-Whitney U test. MNCs: mononuclear cells; ELISPOT: enzyme-linked immunospot.

circulating anti-dsDNA antibody-secreting cells were inactive at the time of examination. To examine if these patients would experience a disease are during follow-up, we prospectively followed all 28 patients with inactive disease who had high-titre serum anti-dsDNA antibodies (>100 IU/mL) (Figure 5). Of these patients, eight (29%) also had circulating anti-dsDNA antibody-secreting cells. Patients with circulating anti-dsDNA antibodysecreting cells had signicantly lower cumulative rates for remaining disease are-free than did patients without these cells in the circulation (p < 0.001). Nearly all the patients with circulating anti-dsDNA antibody-secreting cells relapsed within 12 months, with the main disease are manifestations being serositis in two patients, and lupus nephritis, immune thrombocytopaenia and thrombotic thrombocytopaenic purpura in one patient each.

Discussion
In this study, we successfully developed an ELISPOT assay system to detect and quantify anti-dsDNA antibody-secreting cells in lupusprone mice as well as in SLE patients. The detection of circulating anti-dsDNA antibody-secreting B cells was highly specic for SLE patients, but its sensitivity was quite low, limiting the assays utility as a sole test for diagnosing SLE. In contrast, the presence of circulating anti-dsDNA antibodysecreting cells in SLE patients was strongly associated with active disease, and its positive predictive value for active disease was much better than that of serum anti-dsDNA antibodies. In addition, our prospective cohort demonstrated that the detection of circulating anti-dsDNA antibody-secreting cells predicts a future disease are. These ndings indicate that circulating anti-dsDNA antibodyLupus

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Figure 4 Correlation between circulating anti-dsDNA antibody-secreting cells and serum anti-dsDNA antibody titres in SLE patients. Dotted lines indicate cut-off levels (two per 5 105 PBMCs for anti-dsDNA antibody-secreting cells, and 12 IU/mL for serum anti-dsDNA antibodies). A fitted line was obtained from 130 patients with SLE. Open circles represent patients with active disease; filled circles represent those with inactive disease. Correlation coefficients were obtained by Spearmans correlation analysis.

Table 1 Clinical features of SLE patients with and without circulating anti-dsDNA antibodysecreting cells
Anti-dsDNA antibody-secreting cells Demographic and clinical features Gender (% female) Age (years) Persistent proteinuria (%) SLEDAI SLAM Active disease (SLEDAI ! 5) (%) CH50 (U/mL) Serum anti-dsDNA antibody (IU/mL) Dosage of prednisolone (mg/day) Present (n 29) 58 44.2 10.7 52 5.3 2.1 5.7 2.5 48 28.2 13.8 265 147 12.7 12.9 Absent (n 101) 86 45.1 15.8 21 2.4 2.1 3.3 2.5 5 34.1 12.8 71 94 8.1 9.4 p <0.001 0.6 <0.001 0.004 0.02 <0.001 0.4 0.001 0.02

SLE: systemic lupus erythematosus; SLEDAI: SLE disease activity index; SLAM: systemic lupus activity measures.

secreting cells are a novel biomarker for assessing disease activity in SLE patients. Serum anti-dsDNA antibody is well-known as a useful biomarker to evaluate disease activity in SLE patients, but anti-dsDNA antibody titres do not always correlate with disease activity.5,6 In
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our study, the positive predictive value of serum anti-dsDNA antibodies for active disease in SLE patients was only 17%. In general, a rise in antidsDNA antibody levels during follow-up predicts disease are, but Ho and colleagues reported that this rise is followed by a fall before the are

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Figure 5 Cumulative SLE flare-free rates with inactive disease and anti-dsDNA biomarkers. Rates of remaining disease flare-free are shown for 28 SLE patients with inactive disease and positive high-titre serum anti-dsDNA antibodies in the presence or absence of circulating anti-dsDNA antibody-secreting cells. Cumulative flare-free rates in two groups were compared using a log rank test. Marks on the lines indicate the end of observation.

becomes evident.21 One potential explanation for this phenomenon is that the circulating antidsDNA antibody levels drop as pathogenic antibodies are deposited in the kidneys or other aected tissues. In addition, it is widely recognized that some SLE patients have persistently high antidsDNA antibody titres without disease activity, suggesting that not all anti-dsDNA antibodies are pathogenic, a fact that has also been noted in the mouse lupus model.22 On the other hand, our ELISPOT assay can detect circulating B cells and plasmablasts that are capable of producing antidsDNA antibodies, which may not reect the gross anti-dsDNA antibody production, since plasma cells residing in the secondary lymphoid organs are presumed to be a major producer of anti-dsDNA antibodies. In addition, the ELISPOT assay is theoretically not inuenced when pathogenic anti-dsDNA antibodies are consumed by binding to the aected tissues. It is therefore likely that circulating anti-dsDNA antibodysecreting cells and circulating anti-dsDNA antibodies provide insight into dierent aspects of the

SLE pathogenic processes. Thus, it is preferable to measure these two parameters simultaneously in routine clinical practice to accurately evaluate the disease activity in patients with SLE. The ELISPOT assay is somewhat cumbersome, but an interferon-g release assay by applying this system is now used routinely for the diagnosis of latent tuberculosis infection.23 Further prospective studies involving a large number of SLE patients are necessary to conrm the usefulness of the anti-dsDNA antibody-secreting cell measurement as a biomarker. In this study, nearly all the SLE patients with serum anti-dsDNA antibodies, but without circulating anti-dsDNA antibody-secreting cells, had inactive disease. This suggests that the emergence of anti-dsDNA antibody-producing cells into the circulation may be involved in the SLE pathogenic processes, but the mechanisms that recruit these cells into the circulation remain unclear. In this regard, it was reported that large quantities of cells that secrete antibodies to tetanus toxoid are released into the peripheral blood after secondary
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tetanus toxoid vaccination.11 Thus, it is likely that the emergence of anti-dsDNA antibody-secreting cells into the circulation results from the ongoing autoantigenic stimulation associated with the SLE pathogenesis. On the other hand, it is increasingly appreciated that B-lineage cells are functionally altered and are involved in SLE pathogenesis through both autoantibody-dependent and -independent mechanisms.24 Circulating na ve and memory B-cell subsets are markedly reduced in patients with active SLE, while plasmablasts are expanded in the peripheral blood,25,26 suggesting that plasmablasts secreting pathogenic autoantibodies, including anti-dsDNA antibodies, are released into the circulation. This process may be mediated through the dysregulated chemokine and chemokine receptor expression reported in SLE patients.27,28 Male gender was associated with the presence of circulating anti-dsDNA antibody-secreting cells, but this association was lost by multivariate analysis. This may be explained by a primary association between male gender and high disease activity in our cohort. In this regard, it has been reported that male patients with SLE have more severe organ damage, including renal, neuropsychiatric, cardiovascular and peripheral vascular involvement, than do female patients with SLE.29,30 In summary, this ELISPOT assay for detecting circulating anti-dsDNA antibody-secreting cells is a convenient method for identifying SLE patients with active disease and should be used as a biomarker in routine clinical practice.

References
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Acknowledgment
The authors thank Yuichiro Shirai for assisting in statistical analysis.

Funding
This work was supported by a research grant for intractable diseases from the Japanese Ministry of Health, Labour and Welfare.

Conflict of interest
None declared.
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