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Keywords:

  • erythropoietin receptor;
  • autoantibodies;
  • autoimmune diseases;
  • anaemia;
  • erythropoiesis

Summary

  1. Top of page
  2. Summary
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interests
  8. References

The prevalence, clinical associations and pathogenic role of newly identified autoantibodies to the erythropoietin receptor (EPOR) in patients with anaemia were investigated. Sera from 203 patients with immune-related or chronic kidney diseases were screened for anti-EPOR antibodies by enzyme-linked immunosorbent assay, and antibody specificity was evaluated by immunoprecipitating EPOR from AS-E2 cells using purified immunoglobulin (Ig) fractions. In addition, the pathogenic role of anti-EPOR antibodies was determined by examining their inhibitory effects on AS-E2 cell proliferation. Clinical findings were compared between patients with and without anti-EPOR antibodies, in all patients and those with systemic lupus erythematosus (SLE). Serum anti-EPOR antibodies were detected in 52 patients. Purified IgG or IgM fractions from anti-EPOR antibody-positive sera immunoprecipitated EPOR and inhibited the EPO-dependent proliferation of AS-E2 cells in a dose-dependent manner. Anti-EPOR antibodies were associated with low haemoglobin concentrations and reticulocytopenia in all patients enrolled and those with SLE. Further, there was a negative correlation between the levels of anti-EPOR antibodies and the number of bone marrow erythroblasts in patients who underwent bone marrow examinations. These findings suggest that EPOR autoantibodies are present in a subset of patients with anaemia and that impaired erythropoiesis can be mediated by anti-EPOR antibodies, which functionally neutralize EPO activity.

Erythropoietin (EPO) is a 30·4-kDa glycoprotein produced mainly in the adult kidney under the control of an oxygen-sensing mechanism that regulates red cell production by stimulating the differentiation of erythroid progenitor cells in the bone marrow (Mole & Ratcliffe, 2009). Acquired EPO deficiency or resistance to its action for pathogenic reasons leads to impaired erythropoiesis, resulting in the development of renal anaemia, anaemia of chronic disease (ACD), and, less frequently, pure red cell aplasia (PRCA) (Weiss & Goodnough, 2005; Mole & Ratcliffe, 2009).

PRCA is a severe form of this type of anaemia, mostly attributable to immunological interactions (Young et al, 2000). It is also associated with rheumatological diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), lymphoma, thymoma and the use of certain drugs (Young et al, 2000). Sera from PRCA patients contain autoantibodies that are selectively cytotoxic to bone marrow erythroid cells or specific against EPO (Casadevall et al, 1996; Young et al, 2000), while the possible alternative target of the EPO receptor (EPOR) has not been addressed previously.

In the present study, we hypothesized that autoantibodies that interfere with the EPO–EPOR interaction, especially those against EPOR, may be present in anaemic patients with immune-mediated diseases. To test this, enzyme-linked immunosorbent assays (ELISAs) were carried out to detect serum anti-EPOR antibodies and screen patient sera. Autoantibody specificity and biological activity were also examined. Finally, we evaluated the clinical characteristics associated with the presence of anti-EPOR antibodies and the pathogenic roles of these antibodies in all patients and in the SLE subset. We report the presence and function of EPOR autoantibodies, and show that they are associated with anaemia and are accompanied by reduced reticulocyte and bone marrow erythroblast numbers in clinical settings.

Patients, materials and methods

  1. Top of page
  2. Summary
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interests
  8. References

Patients and controls

Patients (= 203) aged 7–87 years old, diagnosed with various autoimmune diseases and followed up at Kanazawa University Hospital, Kanazawa, Japan were enrolled in this study. These included patients with SLE (= 60), anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (= 32), RA (= 16), systemic sclerosis (= 9), lymphoproliferative disorders (LPDs; = 9) and other autoimmune diseases. In addition, 35 anaemic patients with chronic kidney disease (CKD) were enrolled in the study, and 40 healthy individuals without anaemia were included as a control group. Blood samples were obtained after the patients and control subjects had given their written informed consent, as approved by the Kanazawa University Institutional Review Board.

Detection of EPOR antibodies

The detection of anti-EPOR antibodies was carried out by ELISA using recombinant human EPOR (R & D Systems, Minneapolis, MN, USA) as an antigen. Briefly, polyvinyl 96-well microtitration plates (Nunc International, Tokyo, Japan) were coated with recombinant human EPOR at 5 μg/ml diluted in 0·2 mol/l NaHCO3 buffer at 4°C for 24 h. The remaining free binding sites were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at 4°C. After washing with Tween 20-Tris-buffered saline (TBS), samples were added in duplicate at 1:1 000 dilution to 1% BSA in PBS for 20 h at 4°C. Plates were washed four times with the same buffer and incubated with rabbit anti-human Ig conjugated with horseradish peroxidase (Zymed Laboratories, San Francisco, CA, USA) at 1:5 000 dilution for 1·5 h at room temperature. The substrate tetramethylbenzidine (TMB) (Dako, Carpinteria, CA, USA) was added and the reaction was stopped by the addition of 2 N sulphuric acid. The optical density at 450 nm (OD450) was determined with an automatic plate reader and the antibody considered positive when the ratio of patient serum OD450 to that of control sera was ≥1·5.

Purification of immunoglobulin fractions

Sera from patients and control subjects were clarified by centrifugation at 1,500 × g for 20 min (500 μl) and filtration through 0·45 μm filters (Millipore, County Cork, Ireland). Immunoglobulin (Ig) G fractions were prepared using a MAbTrap Kit (GE Healthcare, Tokyo, Japan) according to the manufacturer's instructions. IgM fractions were also purified with HiTrap IgM Purification HP (GE Healthcare) following the manufacturer's instructions. Purified IgG and IgM fractions were concentrated using Centriprep centrifugal filters (Millipore) and stored at 4°C until required.

Cell culture and growth factors

The human EPO or stem cell factor (SCF)-dependent cell line AS-E2 was grown as previously described (Miyazaki et al, 1997). High-purity recombinant human EPO (1 mg/ml) was a gift from Chugai Pharmaceutical Ltd. (Tokyo, Japan). Recombinant human SCF was purchased from R & D Systems.

Immunoprecipitation and Western blotting analysis of cell-surface EPOR

Immunoprecipitation of cell-surface EPOR was performed to clarify specific antibody binding to EPOR. AS-E2 cells were lysed with TNE buffer (10 mmol/l Tris-HCl, pH 7·8, 1% Nonidet P40, 0·15 mol/l NaCl, 1 mmol/l EDTA) and centrifuged at 10,000 × g to obtain EPOR-containing membrane fractions. The obtained lysate was divided equally into aliquots of 1 ml and each lysate was immunoprecipitated with 10 μg of IgG or IgM fractions using Protein A Sepharose (GE Healthcare) or anti-human IgM agarose (EY Laboratories, San Mateo, CA, USA). Immunoprecipitates were then eluted in an equal amount of gel sample buffer (TEFCO Co. Ltd, Tokyo, Japan), boiled for 5 min and cooled on ice. Proteins were run on 10% polyacrylamide/sodium dodecyl sulphate gels and electroblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore). The membranes were blocked with BSA-containing TBST buffer (20 mmol/l Tris, pH 7·6, 140 mmol/l NaCl, 0·1% Tween 20), and then incubated with biotinylated anti-human EPOR antibody (R & D Systems). To visualize the signals, the membranes were incubated with streptavidin-horseradish peroxidase complex (Zymed Laboratories) and the TMB-stabilized substrate (Promega, Madison, WI, USA) was added.

EPO or SCF-dependent growth assay

AS-E2 cells were grown in EPO medium as previously described (Miyazaki et al, 1997). To assay factor-stimulated growth, cells were washed three times in plain medium (Iscove's modified Dulbecco's medium + 10% FBS with no supplemental growth factor) and resuspended in plain medium. Cells (1 × 104 cells/well) were seeded into each well of 96-well plates (Costar Inc., Temecula, CA, USA). Either supplemental EPO or SCF at a final concentration of 10 ng/ml in the presence of different concentrations of purified Ig fractions (0·625–20%) was added and growth was measured after 7 d by the Premix WST-1 proliferation system (Takara Bio Inc., Shiga, Japan).

Clinical features and routine laboratory tests

Demographic and clinical features were evaluated for each SLE patient at the time of serum collection. Fifty-eight clinical and laboratory findings were recorded, including American College of Rheumatology criteria (Tan et al, 1982) and the SLE Disease Activity Index (SLEDAI) (Bombardier et al, 1992). SLE patients were divided into three groups according to their Hb level (Tzioufas et al, 1997): Group A, patients with severe anaemia (Hb <100 g/l); Group B, patients with moderate anaemia (Hb 100–120 g/l); Group C, patients with mild or no anaemia (Hb l >120 g/l). Anti-double-stranded DNA (anti-dsDNA), anti-Sm, anti-U1 RNP, anti-SSA/Ro, anti-SSB/La and anti-β2-glycoprotein I (anti-β2GPI) antibodies and lupus anticoagulant were identified as described elsewhere (Kuwana et al, 2002).

Bone marrow images of patients

Bone marrow aspiration was performed in 27 patients with anaemia. The indications for bone marrow examination were 1) bicytopenia or pancytopenia including anaemia, or 2) non-haemolytic anaemia that was not readily diagnosed as iron deficiency, vitamin B12 deficiency, folate deficiency, or another type of anaemia defined by blood cell examination and supporting laboratory tests (Ryan & Felgar, 2006). Bone marrow samples were obtained only when patients with these conditions had given their written informed consent. The proportion and the number of erythroblasts in bone marrow nucleated cells were evaluated on bone marrow cell smears. At least 1,000 nucleated cells were counted in each sample. A myeloid to erythroid (M/E) ratio <2:1 was regarded as ‘increased’, and a ratio >4:1 was regarded as ‘decreased’ (Ryan & Felgar, 2006).

Statistical analysis

All comparisons for statistical significance between two patient groups were performed using the χ2 test, Student's t test and Mann–Whitney U test. Analysis of variance (anova) regression analysis was used to correlate the OD450 of anti-EPOR antibodies measured by ELISA with reticulocyte counts and the proportion and number of erythroblasts. In all analyses, < 0·05 was considered significant.

Results

  1. Top of page
  2. Summary
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interests
  8. References

Prevalence of anti-EPOR antibodies

Serum samples from 203 patients and 40 healthy individuals were analysed by ELISA. Anti-EPOR antibodies were detected in 22 SLE patients, six with RA, four with ANCA-associated vasculitis, three with mixed connective tissue disease, two with IgG4-related disease, one with primary anti-phospholipid syndrome, one with systemic sclerosis, three with LPDs, four with other autoimmune diseases and six with CKD, but in none of the other diseases examined or in healthy controls (Fig. 1). The distribution of anti-EPOR antibodies in patients with SLE was as follows: 58% (14/24) from Group A, 25% (6/24) from Group B and 17% (2/12) from Group C (Fig. 1). Anti-EPOR antibodies were significantly more frequent in Group A than groups B or C (= 0·05).

image

Figure 1. Serum anti-erythropoietin receptor (EPOR) antibodies determined by enzyme-linked immunosorbent assay Systemic lupus erythematosus (SLE) patients were divided into three groups according to their haemoglobin level. The broken line denotes the positive cut-off point, which is expressed as a ratio of serum optical density:control sera of 1·5. LPDs, lymphoproliferative disorders.

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Specificity of anti-EPOR antibodies

The specificity of EPOR autoantibodies was examined by immunoprecipitation of the cell-surface human EPOR extracted from AS-E2 cells. Purified Ig fractions with anti-EPOR autoantibodies immunoprecipitated a single band of 66 kD (Fig. 2A and B, arrow), consistent with the size of the human EPOR described previously (D'Andrea et al, 1993). Notably, we identified an IgM antibody against EPOR in one SLE patient (Fig. 2B).

image

Figure 2. Immunoprecipitation of cell-surface EPOR and dose-dependent inhibition of EPO-induced AS-E2 cell growth by purified immunoglobulins from serum (A) The membrane fraction of AS-E2 cells expressing the human EPOR polypeptide was extracted and an equal amount of cell lysate was immunoprecipitated with purified immunoglobulin (Ig) G from normal or patient sera that were positive or negative for anti-EPOR antibodies by ELISA. Lane 1, normal control; lanes 2 and 3, anti-EPOR-negative SLE; lanes 4–6, anti-EPOR-positive. (B) AS-E2 lysate was immunoprecipitated with purified IgM from normal or patient sera. Lane 1, anti-EPOR-positive; lane 2, normal control. AS-E2 cells were incubated in EPO (10 ng/ml) or SCF (10 ng/ml) in the presence of various concentrations of purified immunoglobulins. After 7 d, the WST-1 assay was performed. The results are expressed as the means of triplicate values. Symbols represent AS-E2 incubated in EPO (closed circles) and CF (open circles). Purified immunoglobulins were IgG from normal control (C), IgG from an anti-EPOR-negative SLE patient (D), IgG from an anti-EPOR-positive SLE patient (E) and IgM from an anti-EPOR-positive SLE patient (F).

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Inhibition of EPO-dependent proliferation of AS-E2 cells by anti-EPOR antibodies

To investigate the bioactivity of anti-EPOR antibodies, we evaluated the effects of various concentrations of purified Ig fractions with anti-EPOR antibodies on EPO- or SCF-dependent cell growth. The preliminary experiment confirmed that cultured AS-E2 cells had similar EPO- and SCF-dependent growth rates at 2·5–10 ng/ml of each growth factor in 10% FBS-added IMDM (data not shown). With a 10 ng/ml concentration of EPO or SCF, IgG purified from normal or disease control sera did not alter AS-E2 cell proliferation (Fig. 2C and D); however, both IgG and IgM antibodies from sera of SLE patients positive for anti-EPOR antibodies dose-dependently inhibited EPO-mediated growth of AS-E2 cells, but had no effect on SCF-mediated growth (Fig. 2E and F).

Characteristics of anaemia in overall patients

The extent of anaemia and the number of reticulocytes were compared between patients with and without anti-EPOR antibodies. CKD patients receiving erythropoiesis-stimulating agents (ESA) were excluded from the analysis. The haemoglobin level and the number of reticulocytes were lower in patients with than in those without anti-EPOR antibodies (Fig. 3A and B). In addition, there was a negative correlation between optical density of anti-EPOR antibodies by ELISA and the number of reticulocytes (< 0·05) (Fig. 3C).

image

Figure 3. Comparison of haemoglobin levels and reticulocyte counts between patients with and without anti-EPOR antibodies (A) Haemoglobin concentration and (B) reticulocyte counts. (C) The reticulocyte count (x 109/l) was plotted against OD450 of anti-EPOR antibodies determined by ELISA.

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Evaluation of bone marrow erythroblasts in enrolled patients

Twenty-seven patients with anaemia in whom bone marrow aspiration was performed were examined to evaluate the status of the bone marrow erythroid series. As before, CKD patients treated with ESA were excluded from the analysis. The Mann–Whitney U test indicated that the proportion of bone marrow erythroblasts was lower in patients with than in those without anti-EPOR antibodies (Fig. 4A). In addition, the number of erythroblasts was decreased in patients with anti-EPOR antibodies (Fig. 4B), and the number of erythroblasts was inversely correlated to the optical density of anti-EPOR antibodies measured by ELISA (Fig. 4C).

image

Figure 4. Comparison of the proportion and number of erythroblasts in bone marrow between patients with and without anti-EPOR antibodies (A) Proportion of bone marrow erythroblasts. Increased, myeloid:erythroid (M/E) ratio <2:1; decreased, M/E ratio >4:1. (B) Erythroblast counts. (C) Relationship between bone marrow erythroblasts and OD450 of anti-EPOR antibodies. The erythroblast count was plotted against OD450 of anti-EPOR antibodies determined by ELISA.

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Clinical characteristics of SLE patients with anti-EPOR antibodies

Demographic and clinical findings as well as coexisting autoantibodies were compared between SLE patients with and without anti-EPOR antibodies; selected findings are shown in Table 1. The Hb level and the number of reticulocytes were lower in patients with than in those without anti-EPOR antibodies. However, the SLEDAI score was the same for both groups, indicating that the presence of anti-EPOR antibodies was not associated with disease activity. With respect to other autoantibodies, anti-U1 RNP was more frequent in patients with than in those without anti-EPOR antibodies.

Table 1. Selected clinical and serological characteristics of SLE patients with and without serum anti-EPOR antibodies
Clinical or laboratory findingsAnti-EPOR positive (= 22)Anti-EPOR negative (= 36) P
  1. SLE indicates systemic lupus erythematosus; EPOR, erythropoietin receptor; SLEDAI, SLE Disease Activity Index; anti-dsDNA, anti-double strand DNA; and anti-β2GPI, anti-β2-glycoprotein I.

  2. a

    Data are presented as mean ± SD.

Male/female, n3/193/330·66
Age at examination, yearsa41·5 ± 19·239·5 ± 17·10·68
Disease duration, monthsa65·9 ± 105·6112·2 ± 123·90·20
SLEDAI scorea12·2 ± 7·412·2 ± 7·60·99
Renal disorder, %40·968·60·07
Neurological disorder, %10·05·90·62
Leucopenia (<4 × 109/l), %63·634·30·06
Anaemia, %90·968·60·05
Hb, g/l96 ± 17108 ± 21<0·05
Reticulocyte, ×109/la28·6 ± 20·549·8 ± 27·0<0·01
Thrombocytopenia (<100 × 109/l), %36·417·10·18
Hypocomplementaemia, %72·782·90·51
Anti-dsDNA antibody, %77·360·00·25
Anti-Sm antibody, %47·437·50·69
Anti-RNP antibody, %75·037·9<0·05
Anti-SS-A antibody, %66·760·70·92
Anti-SS-B antibody, %27·834·60·75
Coombs test, %38·515·00·21
Anti-β2GPI antibody, %9·19·40·99
Lupus anticoagulant, %57·139·40·32

Clinical course of SLE patients with anaemia and anti-EPOR antibodies

Table 2 shows Hb concentrations, number of reticulocytes, and serum anti-EPOR antibody levels over time in two SLE patients with anaemia. Both patients were newly diagnosed with SLE and had haematological manifestations at the time of their first admission to our hospital. Bone marrow examination in Patient 2 revealed erythroid hypoplasia. Before-treatment sera from each patient were anti-EPOR antibody-positive when first tested, but after corticosteroid therapy, the Hb and reticulocyte levels increased and the serum became negative for anti-EPOR antibodies. The clinical courses of these patients further supported the association between impaired erythropoiesis and anti-EPOR antibodies.

Table 2. Serial measurement of haemoglobin concentrations, reticulocyte count and serum anti-EPOR antibody levels in SLE patients with anaemia
  1. EPOR, erythropoietin receptor; SLE, systemic lupus erythematosus; Hb, haemoglobin; ND, not determined.

  2. a

    Normal ratio <1·5.

Patient 1
Date4·1111·1117·1124·111·12
Hb concentration, g/l898785102106
Reticulocyte count, × 109/l22ND365065
Anti-EPOR antibodiesa1·861·571·821·410·99
Patient 2
Date13·12·29·217·224·2
Hb concentration, g/l86829299114
Reticulocyte count, × 109/l57ND113ND91
Anti-EPOR antibodiesa3·712·071·871·591·31

Discussion

  1. Top of page
  2. Summary
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interests
  8. References

This is the first report to document the presence of EPOR autoantibodies in pathological states of anaemia with erythroid hypoplasia. We showed that purified IgM and IgG fractions with anti-EPOR antibodies bound specifically to cell-surface human EPOR, and that these antibodies inhibited the EPO-induced proliferative response in vitro but did not block SCF-driven cell proliferation. Clinically, the presence of anti-EPOR antibodies was associated with anaemia, reticulocytopenia and marrow erythroblastopenia. Further, the optical density of anti-EPOR antibodies was negatively correlated with the numbers of reticulocytes and marrow erythroblasts. These findings suggest that anti-EPOR antibodies may be involved in the pathophysiology of immune-mediated anaemia via the inhibition of EPO-induced erythropoiesis.

The presence of autoantibodies to various self-proteins is a known immunological feature of patients with SLE (Peeva et al, 2002). In the present study, we found that more than 25% of enrolled patients, especially those with SLE, had EPOR antibodies in their sera. Among the autoantibodies interfering with the EPO-EPOR system, antibodies to endogenous EPO have previously been shown to be involved in the pathogenesis of anaemia in SLE (Tzioufas et al, 1997; Voulgarelis et al, 2000; Giannouli et al, 2006; Hara et al, 2008). In SLE patients enrolled in previous studies, the frequencies of anti-EPO antibodies were 15% (Tzioufas et al, 1997) and 46% (Schett et al, 2001), while that of anaemia was 21% (Voulgarelis et al, 2000). Although non-EPO autoantibodies to the erythropoietic system were suggested to be involved in the pathogenic process in some patients with anaemia (Krantz, 1974; Dessypris et al, 1984; Mangan et al, 1984), no anti-EPOR antibodies had been detected in previous studies. Our results indicated the presence of EPOR autoantibodies in the sera of SLE patients at a similar frequency to that of anti-EPO antibodies.

Six of 35 enrolled CKD patients with or without ESA therapy were positive for anti-EPOR antibodies. The development of antibodies to recombinant human EPO (rHuEPO) and concomitant PRCA have been reported after administration of the drug in patients with CKD undergoing haemodialysis (Peces et al, 1996; Prabhakar & Muhlfelder, 1997; Casadevall et al, 2002; Praditpornsilpa et al, 2011). Not all of these patients with CKD who were treated with rHuEPO and developed a sudden loss of efficacy had anti-rHuEPO antibodies (Praditpornsilpa et al, 2011). This suggested the presence of anti-EPOR antibodies in this subgroup of patients with CKD. Although it remains to be determined whether the production of anti-EPOR antibodies is induced by ESA therapy, anti-EPOR antibodies can be present in some patients with CKD.

We observed an inhibitory effect of purified IgG and IgM fractions from patients with anti-EPOR antibodies on the EPO-dependent growth of AS-E2 cells, as well as showing its specific binding to cell-surface EPOR. In view of antibody functions, the Ig fractions may contain other inhibitory IgGs or IgMs against AS-E2 cells. We excluded the possibility of the inhibitory effects of anti-EPO antibody on cell proliferation by ELISA (data not shown). Although we could not completely exclude the possibility of other inhibitory Igs at present, the biological activities of anti-EPOR IgG and IgM may include the inhibition of in vitro erythropoiesis.

The present study showed a negative correlation between serum anti-EPOR antibodies and the number of reticulocytes and erythroblasts in bone marrow, indicating that anti-EPOR antibodies play a functional role in inducing anaemia. In addition, the disappearance of the antibodies after immunosuppressive treatment and following an increase in the number of reticulocytes and Hb levels further indicated the involvement of these antibodies in the development of anaemia. On the other hand, in the present study, not all sera that were positive for anti-EPOR antibodies by ELISA were examined by cell growth inhibition assay or further biochemical characteristics, which may be a limitation of this study. Therefore, we cannot exclude the possibility that some anti-EPOR antibodies may not have inhibitory activity against erythroid progenitors. In fact, with respect to the in vivo function of EPO antibodies, they were previously shown not to be clinically associated with increased severity of SLE-associated anaemia (Schett et al, 2001). Despite this limitation, these results suggest that some anti-EPOR antibodies interfere with the differentiation of erythroid progenitor cells in bone marrow, leading to anaemia with erythroid hypoplasia.

Acknowledgements

  1. Top of page
  2. Summary
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interests
  8. References

This work was supported by a grant from the Kidney Foundation, Japan (JKFB08-13) and the Ministry of Education, Science, Sports, and Culture, Japan.

A.H. performed the experiments and was involved in the interpretation of the results and preparation of the manuscript. K.F. assisted in the analysis. M.H. performed the experiments and assisted in the analysis. Y.I. and N.S. assisted in the analysis and were involved in the interpretation of results. S.K. assisted in the analysis and interpretation of the data. T.W. initiated, organized and designed the study, contributed to analysis and interpretation of the data and wrote the manuscript.

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  2. Summary
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Conflict of interests
  8. References
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