1 Nephrology Service 2 Biostatistical and Epidemiological Unit and 3 Autoimmune diseases Unit, Hospital Clínic, Universitat de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain
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Abstract |
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Subjects and methods. A sandwich ELISA was used to measure these soluble adhesion molecules in (i) sera from 20 patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (10 patients with Wegener's granulomatosis (WG) and 10 patients with microscopic polyangiitis (MPA)), obtained at the time of diagnosis and during the remission period; (ii) sera from 40 patients with CRF not undergoing haemodialysis.
Results. At the time of diagnosis, serum levels of sE-selectin, sICAM-1 and sVCAM-1 (88±42 ng/ml, 437±184 ng/ml, 1720±1174 ng/ml respectively) were significantly higher in patients with ANCA-associated vasculitis than in healthy controls (P<0.0001, P=0.002 and P=0.001 respectively). Serum sP-selectin values did not differ from those obtained in normal donors. In contrast, sL-selectin levels (940±349 ng/ml) were significantly lower in patients than those recorded in healthy controls (P<0.0001). A significant decrease in concentrations of sE-selectin, sP-selectin, sICAM-1, and sVCAM-1 was observed between active and remission phases (P<0.0001, P=0.002, P=0.001 and P=0.001 respectively). No significant differences were observed in sL-selectin levels between active and remission phases. sL-selectin concentrations (802±306 ng/ml) during the remission phase remained lower than those observed in healthy controls (P<0.0001). No correlation was observed between serum creatinine and sE-selectin, sP-selectin, sICAM-1 and sVCAM-1 in patients of the CRF group. A slight negative correlation was established between creatinine and sL-selectin concentration.
Conclusions. Increased serum levels of sE-selectin, sICAM-1, and sVCAM-1 and decreased levels of sL-selectin in active ANCA-associated vasculitis, and the normalization of sE-selectin, sICAM-1, and sVCAM-1 during the remission phase suggest that the concentration of soluble levels of these adhesion molecules reflects disease activity. The decrease in sP-selectin levels between active and inactive phases also suggest that this receptor may reflect clinical activity.
The lack of correlation between serum levels of sE-selectin, sP-selectin, sICAM-1, and sVCAM-1 and the degree of renal failure in patients with CRF suggests that the mechanism of clearance of these molecules is not renal.
Keywords: ANCA-associated vasculitis; disease activity; microscopic polyangiitis; soluble adhesion molecules; Wegener's; granulomatosis
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Introduction |
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The development of vascular and glomerular infiltrates requires dynamic interactions between leukocyte surface receptors and their ligands on the endothelial cell surface. This process can be divided into three steps: (i) initial interaction of leukocytes on activated endothelium mediated by selectin (E-selectin, P-selectin and L-selectin) and carbohydrate ligands; (ii) leukocyte activation with firm adhesion to endothelial cells mediated by interaction between integrins and immunoglobulin superfamily molecules (ICAM-1, VCAM-1); and finally, (iii) leukocyte extravasation into the surrounding tissue [2,3].
Circulating forms of selectins and immunoglobulin superfamily adhesion molecules can be detected in serum, plasma, and other body fluids. These molecules may derive from proteolytic cleavage of membrane-anchored forms or from alternatively spliced transcripts that lack a transmembrane domain [4]. In vitro studies have shown that these soluble forms appear in the supernatant of activated leukocytes or cytokine-stimulated endothelial cells [5,6]. The amount of soluble adhesion molecules released in vitro in activated endothelial cell supernatant correlates with their levels of cell surface expression [5].
Elevated levels of circulating adhesion molecules have been detected in disorders where leukocyte/ endothelial cell interactions play a significant role, such as infections, atherosclerosis, neoplasms, chronic inflammatory diseases and vasculitis, and have been considered a consequence of endothelial and/or immune activation [713]. Some authors have suggested a role of soluble adhesion molecules as markers for disease activity in autoimmune disease and vasculitis [1119].
In this study we measured circulating levels of soluble E-selectin, P-selectin, L-selectin, ICAM-1 and VCAM-1 in patients with ANCA-associated vasculitis such as Wegener's granulomatosis and microscopic polyangiitis, as well as in patients with chronic renal failure (CRF), in order to determine: (i) whether changes in soluble adhesion molecules reflect disease activity; (ii) whether serum levels of the adhesion molecules studied are related to the degree of renal failure in a group of patients with stable-phase CRF.
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Subjects |
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Serum from the 20 patients was obtained at the time of diagnosis (pre-treatment) and during the remission period (between 3 and 6 months after diagnosis). Two patients required haemodialysis during the active phase. The serum samples were obtained prior to start this treatment. Whenever serum samples were obtained, the absence of any other disease, such as infection or neoplasia, was confirmed.
CRF group
In order to ascertain whether there was any link between soluble forms of adhesion molecules and degree of renal failure as measured by creatinine levels, we examined forty patients with CRF, not undergoing haemodialysis (22 male and 18 female, mean age 64.85±9.55 years (range 44-79 years). The group consisted of 10 patients with creatinine levels of between 1.3 and 3 mg/dl, 10 with levels of between 3.1 and 5 mg/dl, 10 with levels of between 5.1 and 7 mg/dl, and finally 10 with levels of between 7.1 and 9 mg/dl. The aetiology of CRF in these patients was nephroangiosclerosis (27, 67.5%), diabetic nephropathy (three, 7.5%), tubulointerstitial nephropathy (six, 15%), and non-ANCA-related glomerular nephropathy (four, 10%). None of these patients had neoplasia, liver disease, or concomitant infections.
Control group
Control serum samples were obtained from 20 healthy volunteers, eight men and 12 women, with a mean age of 43.25±12.51 years (range 2565 years). All subjects had normal hepatic and renal function.
In all cases, serum was aliquoted, frozen at -70°, and stored until measurement of adhesion molecules. All samples were obtained with the prior consent of patients.
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Methods |
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Remission was defined as the absence of clinical activity using the BVAS list (score below 5), supported by normal CRP levels. Renal remission was defined as the absence of microhaematuria or haematic cell cast, together with improved or stable renal function [21].
Measurement of soluble adhesion molecules
Levels of sE-selectin, sL-selectin, sP-selectin, sICAM-1, and sVCAM-1 were analysed using sandwich ELISA following manufacturer's instructions, using commercially available kits from R&D Systems, Avingdon, UK. All measurements were performed in duplicate. Briefly, a horseradish peroxidase-conjugated monoclonal antibody against adhesion molecules (sE-selectin, sP-selectin, sL-selectin, sICAM1, and sVCAM1) was added to microtitre plates coated with murine monoclonal IgG antibody recognizing a different epitope of the corresponding molecule. After incubation with patient and control samples and standards at appropriate dilutions, the colour reaction was developed with tetramethylbenzidine, and plates were read on an automated multiscanner at 450 nm (reference wavelength 600 nm).
The calculated intra-assay coefficient of variation was 2.73% for s-E-selectin, 1.6% for sP-selectin, 0.92% for sL-selectin, 2% for sICAM-1 and 3.27% for sVCAM-1.
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Other laboratory studies |
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Results were expressed as arbitrary units by reading off a standard curve, and the normal range was determined by the mean plus 2 SD obtained from 50 normal blood donors. The quantitative anti-PR3 ELISA was performed using a commercial kit (Diagnostika GMBH, Hamburg, Germany). Sera below 10 IU were considered negative.
Measurement of CRP
Serum CRP concentrations were measured by standard nephelometry. Values below 0.8 mg/dl were considered normal.
Statistical analysis
All data are expressed as mean±SD. The normality hypothesis of the variables was tested using the KolmogorovSmirnov test. Comparisons of serum adhesion molecule levels between WG and MPA groups in active and inactive phases were analysed by covariance analysis (ANCOVA). A paired Student test was used to compare differences between serum adhesion levels during active and inactive phases. Differences between two independent groups were analysed by Student t-test for independent samples. Correlation between variables were evaluated by Pearson's correlation coefficient. The cut-off point between active and inactive phases was calculated by ROC curve. The sensitivity and specificity of the cut-off point were estimated using Stata program version 5.0 (Stata, Santa Mónica, California, USA).
Statistical analyses were performed using SPSS program version 6.0 (SPSS Inc. Chicago, USA). Differences were taken to be significant at P<0.05.
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Results |
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Circulating soluble adhesion molecules in unified vasculitis group
sE-selectin
Mean sE-selectin levels during the active phase were significantly higher than those recorded in healthy controls (t=4.10, P<0.0001). These levels fell significantly on clinical remission (t=4.94, P<0.0001). Mean decrease between phases was 37.157 ng/ml (95% CI:21.42; 52.89). No significant differences were observed in sE-selectin levels between patients in remission and healthy controls (P=0.951) (Figure 2).
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sL-selectin
Mean sL-selectin levels in vasculitis patients during the active phase were significantly lower than those recorded in healthy controls (t=6.99, P<0.0001). No significant difference was observed in sL-selectin levels between active and inactive phases (t=1.88, P=0.075). Concentrations during the remission phase remained significantly lower than those observed in controls (t=8.71, P<0.0001; Figure 2).
sP-selectin
sP-selectin levels were significantly higher in the active than in the inactive phase (t=3.55, P=0.002). Mean decrease between phases was 56.97 ng/ml (95% CI, 23.36; 90.57). No significant difference in concentrations was observed, however, between active phase and controls (t=-1.01, P=0.322) or between inactive phase and controls (t=1.42, P=0.164; Figure 2).
sICAM-1
Mean sICAM-1 levels during the active phase were significantly higher than those recorded in healthy controls (t=-3.42, P=0.002). A significant decrease in concentration was recorded between active and inactive phases (t=3.83, P=0.001). Mean decrease between phases was 127.45 ng/ml (95% CI, 57.72; 197.18). No significant differences were observed for this adhesion molecule between patients in remission and healthy controls (t=-0.76, P=0.454; Figure 2).
sVCAM-1
Mean sVCAM-1 levels during the active phase were significantly higher than those recorded in healthy controls (t=3.70, P=0.001). A significant decrease in concentration was recorded between active and inactive phases (t=4.02, P=0.001). Mean decrease between phases was 496.48 ng/ml (95% CI, 238.17; 754.80). No statistically significant differences were observed for this adhesion molecule between inactive-phase patients and healthy controls (t=-2.18, P=0.054; Figure 2).
ROC curves
Area of the ROC curve for sE-selectin was 0.786 (standard error 0.073). The proposed cut-off point was 78.6 ng/ml, yielding a sensitivity of 55% (IC 95% (31.6-76.9%)) and a specificity of 90% (IC 95% (68.3-98.5%)). For sICAM-1, the area of the ROC curve was 0.740 (standard error 0.079). The proposed cut-off point of 269.2 ng/ml gave a sensitivity of 95% (IC 95% (75.1-99.2%)) and a specificity of 50% (IC 95% (27.2-72.8%)).
Correlation between serum adhesion molecule levels and renal function, ANCA, CRP, and BVAS
Renal function
A positive correlation was recorded between VCAM-1 and creatinine levels during the active phase (r=0.5171, P=0.02), though not between other adhesion molecules and this variable.
To determine the possible link between the fall in adhesion molecule levels (sE-selectin, sP-selectin, sICAM-1, and sVCAM-1) from active to inactive phase and the decrease in creatinine concentrations, Pearson's correlation coefficient was calculated for decreases in adhesion molecules (active-phase adhesion molecule concentrations minus inactive-phase adhesion molecule concentrations) and decreases in creatinine in the same phases (active-phase creatinine minus inactive-phase creatinine). Results failed to suggest any link between the two decreases.
Correlation between differing creatinine values and:
ANCA
A positive correlation was recorded between and sE-selectin, s-Pselectin, ICAM-1, and sVCAM-1 and ANCA levels (r=0.47, r=0.34, r=0.55, r=0.36 respectively)
CRP/BVAS
No correlation was observed between CRP and serum adhesion molecule levels during the active phase, or between molecule levels and the BVAS score.
Correlation between different adhesion molecules
No correlation was observed between serum levels of different adhesion molecules during active-phase vasculitis.
CRF group
Mean serum levels of sE-selectin, sICAM-1 and sVCAM-1 were significantly higher than those recorded in healthy controls (t=3.11, P=0.003; t=4.54, P=0.0001; t=7.06, P=0.0001 respectively). No significant differences were observed in sP-selectin levels between the CRF group and the healthy control group (t=0.87;P=0.388). Finally, mean sL-selectin levels in this group were significantly lower than those observed in the healthy control group (t=2.35, P=0.022).
No correlation was observed between serum creatinine and the variables sE-selectin (r=-0.06, P=0.71), sP-selectin (r=-0.32, P=0.06), sICAM-1 (r=0.08, P=0.61) and sVCAM-1 (r=0.33, P=0.061). A negative correlation was established between creatinine and sL-selectin (r=-0.32, P=0.04).
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Discussion |
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ICAM-1 is basally expressed in significant amounts in a limited number of cell types, including monocytes and endothelial cells and it is widely inducible, or upregulated, on many cells including lymphocytes, monocytes, dendritic cells, epithelial cells, and endothelial cells under appropriate stimuli [23]. VCAM-1 has a more restricted distribution, mainly in the endothelium, and E-selectin is exclusively expressed by endothelial cells [24].
Increased circulating levels of adhesion molecules ICAM-1, VCAM-1 and E-selectin, have been considered a consequence of endothelial cell activation, particularly in the case of E-selectin [5,25]. Both ICAM-1, and VCAM-1 are also expressed and released by activated lymphocytes and macrophages; therefore, their increase may also reflect immune activation.
Elevated soluble adhesion molecule levels have been demonstrated by ELISA in the sera of patients with vasculitis [26]. Some initial studies included patients with a variety of diseases and are therefore difficult to interpret. [8,11,27]. In giant-cell arteritis, high levels of ICAM-1 and normal values of E-selectin, and VCAM-1 are reported during the active phase. Circulating ICAM-1 concentrations clearly correlate with clinical activity [13]. Increased levels of ICAM-1, VCAM-1, and E-selectin have been shown in patients with active PAN [12]. Increased ICAM-1 and E-selectin levels also have been demonstrated in Kawasaki disease [28,29], with higher levels of ICAM-1 in patients with coronary-artery lesions. Furukawa et al. [28] suggest that serum ICAM-1 level is an important predictor for the severity of vascular damage in Kawasaki disease. In Wegener's granulomatosis, ICAM-1 and VCAM-1 levels, but not E-selectin levels, are significantly elevated and correlate with disease activity. [10]. Tesar et al. [30] show that patients with MPA in active phase (n=5) had elevated levels of ICAM-1 and VCAM-1.
Histological examination of the kidney confirms that adhesion molecules ICAM-1 and VCAM-1 are over-expressed in patients with ANCA-positive, small-vessel vasculitis. [31] and there is some evidence that suggests a high degree of correlation between histological expression of ICAM-1 and its serum concentrations [32].
In our study, serum concentrations of sE-selectin, sICAM-1, and sVCAM-1 in patients with active phase of ANCA-positive, small-vessel vasculitis were higher than those recorded in controls. Concentrations of these molecules fell sharply on clinical remission. This behaviour is consistent when the two groups (WG and MPA) are viewed separately. Given the sample size used here, it cannot be stated unequivocally that the two groups are the same in term of soluble adhesion molecules, although descriptive statistics and inter-group correlations strongly support a high degree of similarity.
The restriction of E-selectin to activated endothelium makes this adhesion receptor an attractive marker of endothelial cell activation. Initial studies in vasculitis patients have yielded conflicting results. While some authors report high levels of E-selectin in various types of vasculitis [13,33], others find normal levels [10]. For example, Stegeman et al. [10] report normal mean soluble E-selectin levels in a group of 22 active-phase WG patients. This study shows that patients with generalized WG had significantly higher E-selectin levels than patients with localized WG. No patients with localized WG were included in the present study, which might account for the high E-selectin levels recorded in the active phase. Other authors report a progressive increase in serum E-selectin concentrations prior to the onset of clinical relapse [10]. Our results suggest a clear relationship between serum E-selectin levels and clinical activity in both WG and MPA.
Previous studies of WG, and the present study of MPA and WG show that ICAM-1 levels mirror the clinical course of the disease. The successful prevention of crescentic glomerulonephritis with anti-ICAM-1 antibodies has been demonstrated experimentally [34]. These findings suggest that this adhesion molecule may play a major role in the development of inflammatory lesions, and thus may in future furnish helpful markers of disease activity in patients with ANCA vasculitis. The results obtained with ROC curves here indicate that sE-selectin levels and sICAM-1 levels may provide a valuable tool for differentiating between active- and inactive-phase patients.
sVCAM-1 levels also showed good correlation with clinical activity. A positive correlation was found here between serum creatinine levels in active-phase patients and serum VCAM-1 levels. At the same time, no correlation was observed between VCAM-1 and creatinine in the CRF group. It is felt that this molecule may be a useful marker of histological activity in tissue [31]. If the present results are confirmed, serum VCAM levels may be valuable as an indicator of the severity of renal vasculitis.
After starting immunosuppressive treatment, when patients are in the inactive phase, E-selectin, ICAM-1, and VCAM-1 levels were significantly lower than those recorded during the active phase, and no differences were observed between inactive-phase patients and healthy controls. This decrease in adhesion molecule levels may be linked to the treatment; it has been shown that corticoids decrease the production of cytokines, thus possibly reducing adhesion molecule expression or function [35]. It has been demonstrated in vitro that corticosteroids directly decrease endothelial ICAM-1 and E-selectin expression [36].
In some patients, inactive-phase adhesion molecule levels, though significantly lower than those recorded during the active phase, were still considerably higher than control values (particularly VCAM-1). Preliminary results for various groups suggest that although adhesion molecules decrease on clinical remission, they may not actually return to normal [12]. These findings might reflect a persistent exposure of endothelial cells to a mild remaining inflammatory microenvironment.
As indicated earlier, there was a good correlation between clinical activity and levels of circulating soluble adhesion molecules. However, there was no evidence of correlation between CRP values and BVAS score with levels of soluble adhesion molecules. In contrast, a strong correlation was noted between ELISA-measured ANCA levels and concentrations of the five soluble adhesion molecules.
P-selectin is a membrane glycoprotein located in alpha granules of platelets and WeibelPalade bodies of endothelial cells. When platelets or endothelial cells are activated by agonists such as thrombin or histamine, P-selectin is rapidly translocated to the cell surface and can be a receptor for leukocytes at sites of inflammation [37].
P-selectin has not been fully studied in human glomerulonephritis. Segawa et al. [38] showed that P-selectin was associated with both glomerular and interstitial leukocyte accumulation in human proliferative glomerulonephritis, and might be expressed by two distinct mechanisms that are the activated platelets in glomeruli and de novo expression in the interstitial lesions. Soluble sP-selectin may be useful marker for predicting in situ P-selectin expression and could reflect vascular damage and local platelet activation [38]. This is the first report about serum sP-selectin levels in patients with ANCA-associated vasculitis. We showed that sP-selectin levels were higher in the active phase of vasculitis than in the control group but the difference was not significant. P-selectin probably plays a role in the very early stages of leukocyte/endothelial cell interaction, and at the time of diagnosis all patients had well-established lesions of several weeks standing [37,39]. A significant decrease in concentrations of this adhesion molecule was recorded between the active and the inactive phase of the disease. This fact suggests that sP-selectin may correlate with clinical activity.
L-selectin is a cell surface receptor on granulocytes, lymphocytes, and monocytes, which is responsible for the initial contact of leukocytes with the endothelium [40]. The extracellular domain of L-selectin is proteolytically shed from leukocytes following cellular activation in vitro [41]. The shed form is functionally active, and at high concentrations can inhibit attachment to the endothelium [42]. Patients with sepsis and HIV infection showed markedly elevated L-selectin levels in serum [6]. In contrast to these diseases, patients who progressed to adult respiratory distress syndrome (ARDS) had significantly lower plasma L-selectin levels, and a good correlation was found between low values of L-selectin and indices of subsequent lung injury and mortality [43]. This is the first report about serum levels of L-selectin in patients with ANCA-associated vasculitis. We showed that patients with MPA and WG (active and inactive phases) had low levels of sL-selectin. This apparent paradox has also been observed in Kawasaki disease [6], systemic sclerosis, and other vasculitides [44].
Low levels of sL-selectin in patients with vasculitis may be due to reduced expression in leukocytes, reduced shedding or increased internalization of cell bound L-selectin. sL-selectin may also bind with stronger than usual affinity to possible up-regulated levels of its ligand(s) on the endothelial cell (i.e. gly-CAM-1 and/or MAdCAM-1). The implications of the present findings (low levels of sL-selectin in vasculitis) are unclear. sL-selectin retains functional activity [42], so that if the soluble molecule does have a physiological role in inhibiting adhesion, it may be speculated that low levels indirectly promote the adhesion process, and this may be related to the presence of leukocytes in the vessel wall and/or injury to the endothelium.
In the present study-group, inflammatory disease was concurrent with renal failure. The question of whether elevated levels of adhesion molecules in the blood are due to an increase in production and/or to a reduction in elimination remains at present unanswered, and the mechanisms of clearance of these molecules are largely unknown.
Like other authors [11,32], we observed no significant correlation between creatinine levels and serum levels of sE-selectin, sP-selectin, sICAM-1, and sVCAM-1 in patients with CRF. The lack of correlation between the degree of renal failure and sE-selectin, sICAM-1 and sVCAM-1 levels may be due to the high molecular weight (approximately 100 kDa) of these proteins.
Patients with active vasculitis had worse renal function than inactive patients, but no relationship was observed between serum concentrations of adhesion molecules and the degree of renal failure in these patients, except for active-phase VCAM-1. No statistically significant correlation was observed between the degree of improvement of renal function in these patients and the decrease in levels of adhesion molecules (sE-selectin, sP-selectin, sICAM-1, and sVCAM-1) observed between active phase and remission.
Low levels of sL-selectin were observed in active and inactive phases of vasculitis, and normal values of E-selectin, P-selectin, ICAM-1, and VCAM-1 were observed in remission patients although some of these patients had impaired renal function.
Only three patients with active vasculitis and normal renal function were included in this study and all of them had elevated levels of sE-selectin, sICAM-1 and sVCAM-1 in the active phase and normal values of these soluble adhesion molecules in the remission period.
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Conclusions |
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The increase in E-selectin, ICAM-1 and VCAM levels in active-phase vasculitis and the return to normal values during the inactive phase suggest that the serum concentration of soluble forms of these adhesion molecules reflect disease activity. The difference in serum sP-selectin levels between active and inactive phases also suggest that this protein may reflect clinical activity.
In patients with stable-phase CRF, no relationship was observed between serum E-selectin, P-selectin, ICAM-1, and VCAM-1 levels and the degree of renal failure as measured by serum creatinine.
Further research is required to determine whether there is any correlation between expression of adhesion molecules in tissue and serum levels of those molecules, and to ascertain the value of serum concentrations in monitoring activity and identifying patients with low activity and no clinical symptoms.
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Acknowledgments |
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Notes |
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References |
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