NDT Advance Access originally published online on September 15, 2006
Nephrology Dialysis Transplantation 2007 22(1):139-145; doi:10.1093/ndt/gfl509
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Renal histology in Chinese patients with anti-myeloperoxidase autoantibody-positive Wegener's granulomatosis
Renal Division and Institute of Nephrology, Peking University First Hospital, Beijing 100034, PR China
Correspondence and offprint requests to: Dr Ming-Hui Zhao, Renal Division and Institute of Nephrology, Peking University First Hospital, Beijing 100034, PR China. Email: mhzhao{at}bjmu.edu.cn
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Abstract |
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Background. Proteinase-3 antineutrophil cytoplasmic antibody (PR3-ANCA) was the serological marker for Wegener's granulomatosis (WG), while myeloperoxidase (MPO)-ANCA was the serological marker for microscopic polyangiitis (MPA). However, our previous study suggested that patients with MPO-ANCA positive WG were common in Chinese. This study aimed to analyse the renal histology of patients with MPO-ANCA positive WG.
Methods. Patients in our centre with WG were selected according to both the Chapel Hill Consensus Conference (CHCC) definition and American College of Rheumatology classification criteria. Patients with MPA were selected according to the CHCC definition. The renal histology was compared between patients with MPO-ANCA positive WG and with PR3-ANCA positive WG as well as patients with MPO-ANCA positive MPA.
Results. Sixty-one patients with WG had complete renal histological data, 39/61 with positive MPO-ANCA and 22/61 with positive PR3-ANCA. Among patients with crescents in glomeruli, those with MPO-ANCA had fewer cellular crescents and more fibrous crescents than those with PR3-ANCA (P < 0.01 and P < 0.05, respectively). Interstitial fibrosis and tubular atrophy were more prevalent and severe in patients with MPO-ANCA than in those with PR3-ANCA (P < 0.01 and P < 0.05, respectively). Compared with 44 patients with MPO-ANCA positive MPA, patients with MPO-ANCA positive WG had fewer glomeruli with crescents and more normal glomeruli (P < 0.01 and P < 0.01, respectively).
Conclusion. Patients with MPO-ANCA positive WG are common in Chinese. In renal histology, chronic lesions were more severe and prevalent in patients with MPO-ANCA positive WG than in patients with PR3-ANCA positive WG. Glomerular lesions were less severe and less prevalent in patients with MPO-ANCA positive WG than in those with MPO-ANCA positive MPA.
Keywords: ANCA; myeloperoxidase; vasculitis; Wegener's granulomatosis
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Introduction |
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Wegener's granulomatosis (WG) is a systemic vasculitis characterized by necrotizing granulomatous inflammation of the upper and lower respiratory tract and kidney. Antineutrophil cytoplasmic autoantibodies (ANCA) are useful diagnostic tools for WG, microscopic polyangiitis (MPA) and Churg–Strauss syndrome (CSS), which are categorized as ANCA-associated systemic vasculitis (AASV). Most patients with a cytoplasmic ANCA (cANCA) obtained by indirect immunofluorescence (IIF) assay have ANCA directed against proteinase-3 (PR3), as determined by antigen-specific ELISA. Patients with a perinuclear ANCA (pANCA) mostly have ANCA directed against one of a variety of antigens, such as myeloperoxidase (MPO). In literatures, cANCA/PR3-ANCA was found in a majority of patients (70–90%) with active WG and was often considered by some to be a seromarker for this disease [1–5].
In our previous study [6], however, it was found that patients with pANCA/MPO–ANCA positive WG were common, even constituting the majority in Chinese patients with WG. It was also revealed that the prevalence of renal failure at diagnosis was significantly higher in patients with MPO–ANCA than in patients with PR3-ANCA positive WG. Since renal involvement was one of the life-threatening manifestions in patients with WG, the current study investigated the renal histology of Chinese patients with MPO–ANCA positive WG.
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Patients
From the patients with AASV, diagnosed from 1997 to 2006 in the Institute of Nephrology, Peking University First Hospital, those who fulfilled both the following criteria of WG were recruited in this retrospective study: (i) Chapel Hill Consensus Conference (CHCC) definition [7]: granulomatous inflammation involving the respiratory tract, and necrotizing vasculitis affecting small to medium-sized vessels, e.g. capillaries, venules, arterioles and arteries; necrotizing glomerulonephritis is common. Patients were classified as WG if they had systemic vasculitis and the presence of granulomatous inflammation in a biopsy specimen of the respiratory tract or the presence of clinical signs strongly suggestive of granulomatous disease in the respiratory tract, which comprised involvement of the upper respiratory tract with nasal inflammation (purulent/bloody nasal discharge), sinusitis or otitis media or lower respiratory tract manifestion with pulmonary nodules, cavities or fixed infiltrate. (ii) American College of Rheumatology (ACR) classification criteria of WG [8], a patient is diagnosed to have WG if at least two of the following four criteria are present: nasal or oral inflammation (development of painful or painless oral ulcers or purulent or bloody nasal discharge); abnormal chest radiograph (chest radiograph showing the presence of nodules, fixed infiltrates, or cavities); urinary sediment (microhaematuria or red cell casts in urine sediment); granulomatous inflammation on biopsy (histological changes showing granulomatous inflammation within the wall of an artery or in the perivascular or extravascular area).
The diagnosis of MPA was based on the CHCC definition [7]: necrotizing vasculitis with few or no immune deposits affecting small vessels, i.e. capillaries, venules, or arterioles; necrotizing arteritis involving small and medium-sized arteries may be present; necrotizing glomerulonephritis is very common; pulmonary capillaritis often occurs. Patients were classified as having MPA if they had systemic vasculitis, and the absence of granuloma formation in a biopsy specimen and the absence of clinical signs compatible with WG, which is strongly suggestive of granulomatous disease. Among the patients classified as MPA, only those with positive MPO–ANCA were recruited in the current study.
Furthermore, patients with secondary vasculitis or with anti-glomerular basement membrane antibodies were excluded.
IIF assay to detect ANCA
Standard IIF assays were performed according to the manufacturer [EUROIMMUN, Lübeck, Germany]. Ethanol-fixed human polymorphonuclear leucocytes were used to detect ANCA, and monkey liver sections were used to exclude anti-nuclear antibodies (ANA). cANCA and pANCA were distinguished according to staining patterns by two experienced technicians.
Antigen-specific ELISAs
Two highly purified known ANCA antigens, PR3 and MPO, purified as previously reported [9], were used as solid phase ligands in ELISA. Antigens were diluted to 1
2 µg/ml with 0.05 M bicarbonate buffer (pH 9.6) and were coated onto the wells of one half of a Coatar microtitre plate, the wells in the other half being coated with coating buffer alone and acting as antigen-free wells. The volumes of this step and subsequent steps were 100 µl; all incubations were carried out at 37°C for 1 h, and plates were washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST) between stages. Test serum samples were diluted 1 : 50 with PBST and coated in duplicate on both antigen-coated wells and antigen-free wells; every plate contained positive, negative and blank (PBST) controls. The binding was detected with horseradish peroxidase conjugated goat anti-human IgG (Gibco BRL) 1 : 5000 in PBST. The horseradish peroxidase substrate o-phenylenediamine was used at 0.4 mg/ml in 0.1 mol/l citrate phosphate buffer (pH 5.0). The reaction was stopped by 2.0 mol/l H2SO4 and the results were recorded as the net A490 nm (average value of antigen wells minus average value of antigen-free wells), and expressed as percentage of the known positive controls. Samples were considered positive if they exceeded the mean +3 SD from 100 normal blood donors.
Renal histopathology
Renal specimens were evaluated using direct immunofluorescence (for immunoglobulins and complement components), light and electron microscopy. For light microscopy, paraffin sections were stained with silver, periodic acid-Schiff, haematoxylin and eosin and trichrome and were forwarded to two pathologists. Both pathologists scored the biopsies separately, blinded to patients’ data and the scores of the other observer, according to a previously standardized protocol for scoring renal biopsies of patients with AASV [10–12]. In short, each glomerulus was scored separately on the presence of fibrinoid necrosis, crescents (cellular/fibrous and segmental/circumferential), glomerulosclerosis (local/segmental/global), granulomatous reactions, as well as a number of other lesions. The presence of glomerular lesions was calculated as the percentage of the total number of glomeruli in a biopsy. Interstitial and tubular lesions were scored semiquantitatively on the basis of the percentage of the tubulointerstitial compartment that was affected: interstitial infiltrates (‘–’ for 0%, ‘+’ for 0–20%, ‘++’ for 20–50% and ‘+++’ for >50%), interstitial fibrosis (‘–’ for 0%, ‘+’ for 0–50%, ‘++’ for >50%) and tubular atrophy (‘–’ for 0%, ‘+’ for 0–50%, ‘++’ for >50%). Vascular lesions were scored as present or absent. Differences in scoring between the two pathologists were resolved by re-reviewing the biopsies and coming to consensus.
Statistics
Differences of quantitative parameters between groups were assessed with the t-test (for normally distributed data) or non-parametric test (for non-normally distributed data). Differences of semiquantitative results were tested with the Mann–Whitney U-test. Differences of qualitative results were compared by means of the chi-square test. The difference was considered significant if the P-value was <0.05. Analyses were performed with SPSS statistical software package (version 11, Chicago, Ill, USA).
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Results |
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Demographic and clinical data
Sixty-one patients with complete renal histological data fulfilled both the CHCC and ACR criteria of WG. Of the 61, 39 (63.9%) were pANCA positive, all could recognize MPO in antigen specific ELISA, and 22 (36.1%) were cANCA positive and all the sera recognized PR3. Among the 61 patients with WG, granulomatous inflammation could be detected in pathology in 26 patients. Five were in the respiratory tract biopsy, 3/5 were MPO–ANCA positive and 2/5 were PR3–ANCA positive. Twenty-one were in the renal biopsy, in which granulomatous inflammation could be detected in renal interstitium; only seven of the 21 (33.3%) were PR3–ANCA positive and 14 of 21 (66.7%) were MPO–ANCA positive. Among the 39 patients with MPO–ANCA, 18 were men and 21 were women. The average age at onset of the disease (defined as ‘patients had renal or extra-renal signs and symptoms of vasculitis, or abnormalities related to vasculitis were detected by various examinations’) was 55.0(17–80) years old. The mean and median interval between onset of the disease and renal biopsy was 9.6 and 3.0 (ranging from 1–120) months, respectively.
Forty-four MPO–ANCA positive patients with complete renal histological data fulfilled the CHCC criteria of MPA, all of whom were pANCA and MPO–ANCA positive.
There was no significant difference in the intervals between onset of disease and renal biopsy between patients with MPO–ANCA and PR3–ANCA positive WG and between patients with MPO–ANCA positive WG and MPA (Figure 1).
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Of the 39 cases with MPO–ANCA positive WG, the median and mean level of serum creatinine (Scr) was 257.5 µmol/l and 360.1 µmol/l, respectively (ranging from 55 µmol/l to 890 µmol/l). Thirty-three cases (33/39, 84.6%) had renal insufficiency at diagnosis. The level of Scr was significantly lower in patients with MPO–ANCA positive WG than in patients with MPO–ANCA positive MPA (P < 0.05, by non-parametric analysis). The prevalence of renal insufficiency at diagnosis was significantly higher in patients with MPO–ANCA positive WG than in patients with PR3–ANCA positive WG (84.6% vs 59.1%,
2 = 4.94, P < 0.05) (Table 1). Other organ involvements are also shown in Table 1.
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Glomerular lesions
In each renal biopsy specimen, an average of 23.5 ± 12.8 glomeruli could be seen.
Of the 39 cases with MPO–ANCA positive WG, necrotizing glomerulonephritis with crescent formation was observed in 12 cases (30.8%), glomerulonephritis with crescent formation but without fibrinoid necrosis was present in 24 cases (61.5%). One case (2.6%) had mild lesions, and two cases (5.1%) showed diffuse global glomerulosclerosis. Further analysis revealed that 36.7 ± 29.8% of the glomeruli were normal and 46.9 ± 30.9% of the glomeruli had crescents. The mean and median percentage of global sclerosis was 9.34 and 0.0% (ranging from 0 to 100%), respectively. The mean and median percentage of fibrinoid necrosis of glomeruli tuft was 3.24 and 0.0% (ranging from 0 to 27.0%), respectively.
The prevalences of glomeruli with crescents were similar in patients with MPO–ANCA positive WG and patients with PR3–ANCA positive WG (46.9 ± 30.9% vs. 52.1 ± 33.2%). However, among the patients with crescents in glomeruli, those with MPO–ANCA positive WG had a lower proportion of cellular crescents and a higher proportion of fibrous crescents than those with PR3–ANCA positive WG (41.9 ± 34.8% vs 71.0 ± 34.4%, P < 0.01; 30.7 ± 31.4% vs 12.1 ± 24.0%, P < 0.05, respectively) (Table 2).
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Compared with the 44 patients with MPO–ANCA positive MPA, patients with MPO–ANCA positive WG had a lower proportion of glomeruli with crescents and a higher proportion of normal glomeruli (46.9 ± 30.9% vs 64.1 ± 24.4%, P < 0.01; 36.7 ± 29.8% vs 20.4 ± 25.8%, P < 0.01, respectively) (Table 2).
Tubulointerstitial lesions
Of the 39 cases with MPO–ANCA positive WG, 36/39 (92.3%) had interstitial infiltrate, 7/39 (17.9%), 23/39 (59.0%) and 6/39 (15.4%) scored as (+),(++) and (+++), respectively. Of the 39, cases 26 (66.7%) had interstitial fibrosis, 6/39 (15.4%) and 20/39 (51.3%) scored as (+) and (++), respectively. Tubular atrophy was present in 37/39 (94.9%) biopsies; 8/39 (20.5%) and 29/39 (74.4%) scored as (+) and (++), respectively. Interstitial granulomatous inflammation was present in 14/39 (35.9%) biopsies.
Interstitial fibrosis and tubular atrophy were more prevalent and severe in patients with MPO–ANCA positive WG than in those with PR3-ANCA positive WG (P < 0.01 and P < 0.05, respectively). There was no significant difference of interstitial infiltrates, interstitial fibrosis and tubular atrophy between patients with MPO–ANCA positive WG and patients with MPO–ANCA positive MPA (Figure 2).
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Vascular lesion
Of the 39 cases with MPO–ANCA positive WG, arteriolosclerosis was present in 35/39 (89.7%) cases. Necrotizing lesions of arteriole was not found, but necrotizing lesions of interlobular arteries could be found in four cases (4/39, 10.3%).
No significant differences of vascular lesion could be found among patients with MPO–ANCA positive WG, patients with PR3–ANCA positive WG and patients with MPO–ANCA positive MPA (Table 3).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionAcknowledgementsReferences |
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In renal histology, WG was characterized by necrotizing crescentic glomerulonephritis and granulomatous inflammation. In most of the studies, PR3–ANCA was found in a majority of patients with active WG. MPO–ANCA, being more common in MPA, CSS and renal-limited vasculitis, was observed in a minority (only about 5–10%) of patients with WG, far fewer than PR3–ANCA [5]. Clinical characteristics of patients with MPO–ANCA positive WG had not been fully investigated except for a few studies [13–15], let alone their renal histology [10–12]. However, our previous studies showed that MPO–ANCA were much more common than PR3–ANCA (about 5:1) in Chinese patients with AASV [16–18], and even in patients with WG, there was a predominance of MPO–ANCA over PR3-ANCA [6]. This finding agreed with the report by Tervaert et al. [14]. In order to gain further insight into patients with MPO–ANCA positive WG, the current study investigated their renal histology, and a comparison was made between patients with MPO–ANCA and PR3–ANCA positive WG, as well as between patients with MPO–ANCA positive WG and MPA.
In the current study, it was found that fibrous crescents, interstitial fibrosis and tubular atrophy were more abundant in patients with MPO–ANCA positive WG than in those with PR3–ANCA positive WG, whereas the percentage of cellular crescents was higher in patients with PR3–ANCA positive WG than in those with MPO–ANCA positive WG. In other words, it seemed that chronic lesions were more common in patients with MPO–ANCA positive WG than those with PR3–ANCA positive WG and vice versa. This result partially agreed with the investigation conducted by the European Vasculitis Study Group (EUVAS), which compared renal histology in AASV according to diagnostic and serological subgroups. It was revealed that both active and chronic lesions were more abundant in patients with MPO–ANCA than in patients with PR3–ANCA [12]. However, the study of EUVAS was focused on AASV rather than WG. There might be two explanations for the results of the current study. Firstly, it was suggested in our previous study that the prevalences of arthagia, skin rash, ophthalmic and ear involvement were significantly lower in patients with MPO–ANCA positive WG than those in patients with PR3–ANCA positive WG [6], which would probably result in a late diagnosis. However, there was no significant difference of the intervals between onset of disease and renal biopsy between patients with PR3–ANCA positive and MPO–ANCA positive WG. Secondly, it was speculated that the difference between MPO–ANCA positive and PR3–ANCA positive WG might be associated with different mechanisms in the pathogenesis of WG. The role of different ANCA specificities played in the pathogenesis of WG needed further investigations.
It was also demonstrated in the current study that patients with MPO–ANCA positive WG had a lower proportion of glomeruli with crescents and a higher proportion of normal glomeruli than those with MPO–ANCA positive MPA. This result was partially different from the study of EUVAS, which found that normal glomeruli were more common in WG than in MPA, and glomerulosclerosis was more prominent in MPA than in WG, but the percentages of crescent were similar between these two groups of patients [12]. The current study raised an interesting issue that patients with the same seromarker, MPO-ANCA, could develop into two different clinical entitities (WG and MPA), which suggested that the pathogenesis involved in MPO–ANCA positive WG and MPO–ANCA positive MPA might be different. Whether there are different mechanisms other than ANCA involved in the pathogenesis of these two entities needs further investigation.
The proportion of fibrinoid necrosis in the current study was much lower than others [19]; this might be due to the number of sections cut in specimens. It could also not be excluded that sample errors might reflect some of the differences found in the study, since not all patients with AASV in our centre received renal biopsy.
In conclusion, patients with MPO–ANCA positive WG are common in Chinese. Chronic lesions were more abundant in patients with MPO–ANCA positive WG than that in those with PR3–ANCA positive WG. Glomerular lesion was less severe and less prevalent in patients with MPO–ANCA positive WG than in those with MPO–ANCA positive MPA.
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Acknowledgements |
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We are very grateful to Dr Jie E and Xin Zheng for assistance in collecting pathological data of the patients.
Conflict of interest statement. None declared.
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References |
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- van der Woude FJ, Rasmussen N, Lobatto S, et al. (1985) Autoantibodies against neutrophils and monocytes: a new tool for diagnosis and marker of disease activity in Wegener's granulomatosis. Lancet 1:425–429.[Medline]
- Tervaert JW, van der Woude FJ, Fauci AS, et al. (1989) Association between active Wegener's granulomatosis and anticytoplasmic antibodies. Arch Intern Med 149:2461–2465.
[Abstract/Free Full Text] - Nolle B, Specks U, Ludemann J, et al. (1989) Anticytoplasmic autoantibodies: their immunodiagnostic value in Wegener granulomatosis. Ann Intern Med 111:28–40.[Web of Science][Medline]
- Russell KA, Wiegert E, Schroeder DR, et al. (2002) Detection of anti-neutrophil cytoplasmic antibodies under actual clinical testing conditions. Clin Immunol 103:196–203.[CrossRef][Web of Science][Medline]
- Hagen EC, Daha MR, Hermans J, et al. (1998) Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis. EC/BCR Project for ANCA Assay Standardization. Kidney Int 53:743–753.[CrossRef][Web of Science][Medline]
- Chen M, Yu F, Zhang Y, et al. (2005) Characteristics of Chinese patients with Wegener's granulomatosis with anti-myeloperoxidase autoantibodies. Kidney Int 68:2225–2229.[CrossRef][Web of Science][Medline]
- Jennette JC, Falk RJ, Andrassy K, et al. (1994) Nomenclature of systemic vasculitides: the proposal of an international consensus conference. Arthritis Rheum 37:187–192.[Web of Science][Medline]
- Leavitt RY, Fauci AS, Bloch DA, et al. (1990) The American College of Rheumatology 1990 criteria for the classification of Wegener's granulomatosis. Arthritis Rheum 33:1101–1107.[Web of Science][Medline]
- Zhao MH and Lockwood CM. (1996) A comprehensive method to purifiy three major ANCA antigens: proteinase 3, myeloperoxidase and bactericidal/permeability- increasing protein from human neutrophil granule acid extract. J Immunol Meth 197:121–130.[CrossRef][Web of Science][Medline]
- Bajema IM, Hagen EC, Hansen BE, et al. (1996) The renal histopathology in systemic vasculitis: an international survey study of inter- and intra-observer agreement. Nephrol Dial Transplant 11:1989–1995.
[Abstract/Free Full Text] - Bajema IM, Hagen EC, Hermans J, et al. (1999) Kidney biopsy as a predictor for renal outcome in ANCA-associated necrotizing glomerulonephritis. Kidney Int 56:1751–1758.[CrossRef][Web of Science][Medline]
- Hauer HA, Bajema IM, van Houwelingen HC, et al. (2002) Renal histology in ANCA-associated vasculitis: differences between diagnostic and serologic subgroups. Kidney Int 61:80–89.[CrossRef][Web of Science][Medline]
- Schonermarck U, Lamprecht P, Csernok E, Gross WL. (2001) Prevalence and spectrum of rheumatic diseases associated with proteinase 3-antineutrophil cytoplasmic antibodies (ANCA) and myeloperoxidase-ANCA. Rheumatology 40:178–184.
[Abstract/Free Full Text] - Tervaert JW, Goldschmeding R, Elema JD, et al. (1990) Association of autoantibodies to myeloperoxidase with different forms of vasculitis. Arthritis Rheum 33:1264–1272.[Web of Science][Medline]
- Bajema IM, Hagen EC, Ferrario F, et al. (1997) Renal granulomas in systemic vasculitis. EC/BCR Project for ANCA-Assay Standardization. Clin Nephrol 48:16–21.[Web of Science][Medline]
- Wang Y, Zhao MH, Yu J, et al. (2004) The clinical and pathological characters of Chinese elderly patients with anti-neutrophil cytoplasmic autoantibodies associated small vasculitis. Exp Gerontol 39:1401–1405.[CrossRef][Web of Science][Medline]
- Xin G, Zhao MH, Wang HY. (2004) Detection rate and antigenic specificities of antineutrophil cytoplasmic antibodies in Chinese patients with clinically suspected vasculitis. Clin Diagn Lab Immunol 11:559–562.
- Chen M, Yu F, Zhang Y, Zhao MH. (2005) Clinical and pathological characteristics of Chinese patients with antineutrophil cytoplasmic autoantibody associated systemic vasculitides: a study of 426 patients from a single center. Postgrad Med J 81:723–727.
[Abstract/Free Full Text] - Hauer HA, Bajema IM, de Heer E, Hermans J, Hagen EC, Bruijn JA. (2000) Distribution of renal lesions in idiopathic systemic vasculitis: a three-dimensional analysis of 87 glomeruli. Am J Kidney Dis 36:257–265.[Web of Science][Medline]
- Luqmani RA, Bacon PA, Moots RJ, et al. (1994) Birmingham vasculitis activity score (BVAS) in systemic necrotizing vasculitis. Q J Med 87:671–678.[Web of Science]
Accepted in revised form: 31. 7.06
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