Immunohistochemical Localization of Collagen Type III and Type IV, Laminin, Tenascin and α-Smooth Muscle Actin (αSMA) in the Human Liver in Peliosis

PATHOLOGY

Original Paper

RESEARCH AND PRACTICE © Urban & Fischer Verlag http://www.urbanfischer.de/journals/prp

Immunohistochemical Localization of Collagen Type III and Type IV, Laminin, Tenascin and α-Smooth Muscle Actin (αSMA) in the Human Liver in Peliosis Maya Gulubova Department of General and Clinical Pathology, Faculty of Medicine, Thracian University, Stara Zagora 6000, Bulgaria

Summary

Introduction

The expression of collagen types III and IV, laminin, tenascin, and hepatic stellate cells (HSCs) activation marker αSMA was evaluated immunohistochemically in the liver of three patients with non-bacilar peliosis. Peliosis was attributed to tuberculosis, endometriosis treated with anabolic androgenic steroids, and to pheochromocytoma. Ultrastructural examination of the lesions of the liver revealed cavities that were sometimes lined with sinusoidal endothelial cells or hepatocytic microvilli. In liver sinusoids around cavities, cystic dilatation of the space of Disse and an abundance of amorphous matrix were observed. At this location, HSCs were transformed into transitional cells or myofibroblasts. Extracellular matrix proteins (ECM) were increased in the dilated sinusoids around cavities perisinusoidally and in the wall of cavities themselves. αSMA was also increased. Ultrastructural immunohistochemistry revealed strong intracellular deposits of collagen type IV, laminin, and αSMA in HSCs. Laminin immunoreactivity was also noted in the endocytic vesicles in the cytoplasm of a monocyte. These findings suggest that enhanced ECM accumulation and the transformation of HSCs into myofibroblasts constitute a secondary event in peliosis and an attempt of the liver to restrict and remove sinusoidal dilatation.

Peliosis hepatis is characterized by the presence of multiple blood-filled cavities within the hepatic parenchyma, which can be lined with sinusoidal endothelial cells [43]. Although its etiology is unknown, this condition may be associated with several states of disease and medications. Peliosis hepatis might result from the long-term use of gonadal steroids [2, 14], azathioprine [12] or selenium [4]. Bartonella (Rochalimaea) infection may often affect the liver and spleen, causing bacillary peliosis [18, 28]. Tuberculosis or some malignancies can also be associated with liver peliosis [43]. Liver peliosis is investigated light-microscopically in most cases. The ultrastructural features of this entity, however, have been studied less frequently [43]. The findings of perisinusoidal dilatation and accumulation of flocculent material at this site, and the report on sinusoidal cell injury [42, 43] led us to investigate the occurrence of ECM in liver peliosis. ECM of the liver is composed of fibrillar and non-fibrillar proteins, glycoconjugates, and glycosaminoglycans. It provides not only structural support for hepatocytes, but also contributes

Key words: Extracellular matrix proteins (ECM) – Peliosis – Liver – Hepatic stellate cells Pathol. Res. Pract. 198: 803–812 (2002)

Address for correspondence: Maya V. Gulubova, Department of General and Clinical Pathology, Medical Faculty, Thracian University, Armeiska str. 11, Stara Zagora 6000, Bulgaria. E-mail: [email protected] 0344-0338/02/198/12-803 $15.00/0

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to the regulation of liver functions interacting with parenchymal and nonparenchymal cells with various mediators [22, 36]. Liver sinusoidal ECM maintains the normal sinusoidal structure and function [7], and plays a key role in hepatic fibrogenesis [15]. The main ECMproducing cell type in the liver is HSC [20, 30, 32]. Alterations in the distribution of fibrillar collagen (types I and III), non-fibrillar (collagen type IV) collagen, and glycoproteins (fibronectin, laminin and, to a lesser extent, tenascin) after hepatic injury [16, 19, 31, 34, 35, 38] have been studied. The occurrence of ECM proteins in the human liver with peliosis has not yet been investigated. In the present study, we report on the distribution of some ECMproteins (collagen type III, type IV, laminin and tenascin) and of the marker of HSC activation – α-SMA in the liver with peliosis. We tried to evaluate the significance of enhanced ECM accumulation in the development of liver peliosis using the results of our investigation.

Materials and Methods Tissue specimens We examined three patients (2 men and 1 woman between 40 and 43 years of age) for whom the diagnosis of peliosis hepatis had been rendered on sections of paraffin-embedded liver tissue. The main clinical and laboratory data are given in Table 1. Peliosis was attributed to tuberculosis in patient 1. Patient 2 had adrenal pheochromocytoma, detected 1 year after the diagnosis of peliosis had been made. Patient 3 suffered from arterial hypertension and endometriosis; before death, she had developed iron deficiency anemia. She was treated with anabolic androgenic steroid hormones for the endometriosis. Patients 1 and 2 had had permanent dull pain in the right hypochondrium, lasting for 1–2 months before admission to the hospital. Ultrasonography and computed to-

mography showed enlarged livers without any specific lesions. Patient 3 had asymptomatic peliosis, diagnosed occasionally on autopsy. Patient 3 died from cerebral hemorrhage. Wedge-like liver surgical biopsies were obtained from patients with peliosis for diagnostic purpose. In addition, 3 patients (1 man and 2 women between 39 and 61 years of age) were used as controls. They underwent explorative laparotomy, and liver calcification (2 patients) was an occasional finding during surgery. Controls had histologically normal liver. None of the patients had had a history of previous liver diseases, i.e. viral hepatitis, diabetes mellitus, hemochromatosis, hemotransfusions, or alcohol abuse (< 20 g/per day). Informed consent was obtained from each patient. Surgical biopsies of approximately 16 × 15 × 8 mm were taken from the lower border of the liver. Each of the biopsies was divided in 3 pieces and processed as follows. Routine histology Tissue samples of 12 × 6 × 8 mm were fixed in 10% neutrally buffered formalin for routine histology. Paraffin sections were stained with hemalaun and eosin and Van Gieson staining. All three cases were investigated histologically. Electron microscopy Liver tissue from patient 1 and patient 2 was investigated using electron microscopy. Liver tissue samples of 4 × 12 × 4 mm were divided into four pieces, each 4 × 3 × 4 mm, fixed in 4% glutaraldehyde in 0.1 mol/l phosphate buffer (PB), pH 7.4, and postfixed in 1% osmium tetroxide in the same buffer. Semithin sections were stained using the Humphrey & Pittman [21] procedure for the assessment of fibrosis and transformation of HSCs. Ultrathin sections were cut from liver lobule zones with dilated sinusoids around cavities and from the cavities themselves. Sections were contrasted with uranyl acetate and lead citrate.

Table 1. Clinical and laboratory data in three patients with peliosis hepatis and of control patients.

Sex Age (years) Jaundice Portal hypertension Total serum Bilirubin (mmol/l) GOT/GPT (U/L)

Patient 1 tuberculosis

Patient 2 pheochromocytoma

Patient 3 iron def. anemia

Control 1

Control 2

Control 3

male 40 no no 15.2 21/26

male 43 no no 12.2 15/17

female 40 no no 12.6 26/34

male 40 no no 11 8/10

female 48 no no 12 9/12

Female 61 No No 11.8 8/11

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Light microscopical immunohistochemistry All three liver samples were investigated using this method. The procedure was done as described previously [17]. In brief, cryostat sections (5 µm thick) of the liver were incubated with primary antibodies against collagen type III and type IV, laminin, tenascin, and αsmooth muscle actin for 24 hrs at room temperature. Afterwards, they were reacted with biotinylated antimouse antibodies for 4 hrs at room temperature. Peroxidase activity was demonstrated using a freshley-prepared solution of 3-amino-9-ethyl-carbasole and 3,3′-diaminobenzidine (DAB). Electron microscopical immunohistochemistry Only patient 1 and patient 2 were investigated using this method. The method applied was described previously [11, 17]. The procedure was carried out simultaneously using light microscopical immunohistochemistry. Samples (12 × 62 × 8 mm) were immersion-fixed in a twostep procedure, thawed in 0.1 M PB, pH 7.3, containing 4% paraformaldehyde, for 1 h at room temperature, and then fixed overnight at 2 °C in a 0.1 M disodium hydrogene phosphate, pH 9, containing 4% paraformaldehyde. Thirty per cent sucrose solution was used for cryoprotection. Free-floating frozen cryostat sections were cut at 50–90 µm and thawed in 0.1M phosphate buffered saline (PBS), pH 7.4, overnight. To block endogenous peroxidase activity, the sections were incubated in 1.2% H2O2 in methanol for 30 min, followed by a 15-min wash in PBS. Non-specific protein binding was inhibited by a 20-min incubation in 5% goat serum. Primary antibodies (anti-collagen type III; anti-collagen type IV; anti-laminin, 1: 100; anti tenascin; anti-α-smooth muscle actin) were incubated overnight at room temperature, followed by a 15-min wash in PBS. The slides were then treated with the linking reagent (biotynilated

anti-mouse immunoglobulins) and the peroxidase-conjugated streptavidin label, respectively, for 4 hrs at room temperature. Peroxidase activity was developed in a mixture of 3 mg, in DAB, in 15 ml 0.05 M Tris-HCL buffer, pH 7.5, and 36 µl 1% hydrogen peroxide for 10–20 min, and rinsed in 0.1 M PBS, pH 7.4. The sections were postfixed in PB containing 2% osmium tetroxide for 15–30 min at 2 °C, followed by a rinse in 0.1 M PB, pH 7.4. Finally, sections were dehydrated in graded concentrations of ethanol and propylene oxide, and flat-embedded with Durcupan, between cellophane sheets. The ultrathin sections were counterstained with uranyl acetate, and examined and photographed using an OPTON EM 109 electron microscope at 50 kV. Negative controls Control sections were incubated with non-immune sera instead of the primary antibodies. Immunochemicals The following antibodies were used: monoclonal mouse anti-human laminin (M0638) diluted 1:100 (Dako, Glostrup, Denmark); monoclonal mouse antihuman collagen type III (MA167-5C); monoclonal mouse anti-human collagen type IV (MA079-5C); monoclonal mouse anti-human α-smooth muscle actin (AM128-5M); monoclonal mouse anti-human tenascin (AM295-5M); mouse anti-CD31 (PECAM-1) (AM2415M) and the detection system of the immunostaining kit StrAviGen SS mouse (AD000-5M) (BioGenex Laboratories, San Ramon, CA, USA). The chromogens used for ultrastructural and light microscope immunohistochemistry were DAB (Sigma, St. Louis, MO, USA) and 3-amino-9-ethylcarbazole (BioGenex Laboratories), respectively.

Results Histology

Hemalaun- and eosin-stained sections of the liver showed round to oval intralobular cavities that had no preference for a certain location within the lobule (Fig. 1). Some cavities were lined with endothelial cells or hepatocytes. They communicated with sinusoids, which were dilated and contained various numbers of blood cells. There was no evidence of hepatocellular necrosis, but hepatocytes were often atrophic or compressed. Portal tracts were normal. Electron microscopy

Fig. 1. Round to oval intralobular cavities lined with endothelial cells and hepatocytes. Magnification ×125.

In normal liver, the space of Disse contained a small quantity of amorphous matrix, regularly arranged hepatocyte microvilli, and HSC processes (Fig. 2a).

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Peliosis manifested in cavities lined by endothelial cells, or hepatocytes (Fig. 2b). The endothelial cells did not show any noticeable cytoplasmic or nuclear abnormalities, except for enlargement of their fenestrae. Hepatocytic microvilli were chaotically orientated to the sinusoidal lumen. The space of Disse was enormously dilated and contained red blood cells, cellular debris, and a large quantity of amorphous matrix (Fig. 2b). Basement membrane structures were almost not visible. In the dilated sinusoids around cavities, the space of Disse was also enormously dilated and contained amorphous matrix, collagen fibers, and many cellular processes of hepatocytes and HSCs. The sinusoidal endothelial cells contained lipofuscin granules in their cytoplasm, and were lined by basement membrane structures. Their fenestrae were almost normal. Kupffer cells contained many lysosomes and cytoplasmic digitations. HSCs were transformed into transitional cells (with well-developed rough endoplasmic reticulum, indented nuclei, and single lipid droplets) and sometimes into myofibroblasts (with bundles of intermediate filaments and many pinocytic vesicles) (Figs. 2c,d). HSCs had strongly branched processes (Fig. 2c). Light microscopical immunohistochemistry

In normal human liver, type III collagen was predominantly present in the portal stroma. Although sinusoidal walls were mostly devoid of collagen type III, occasional thin delicate strands were present in the lobule (Fig. 3a). Collagen type IV was found in basement membranes: vascular, ductal, and neural in portal tracts. In addition, collagen type IV was observed along most sinusoids (Fig. 3c). The basement membrane component laminin was found exclusively in the portal basement membranes (vascular, ductal, and neural). Laminin was very faint along sinusoids (Fig. 3e). The occurrence of tenascin at perisinusoidal location was faint and discontinuous. In portal tracts, tenascin staining ranged from negative to weakly positive (Fig. 3g). Vascular smooth muscle cells, pericytes, and some stromal cells around bile ducts were positive for α-SMA (Fig. 3 i). PECAM-1 immunoreactivity was detected on the endothelium of portal vessels. In the liver of patients with peliosis, collagen type III immunoreactivity was increased along the dilated sinusoids in a continuous pattern. Collagen type III was focally deposited beneath the endothelium, covering the cavities (Fig. 3b). The deposition of type IV collagen was intense and continuous along the dilated sinusoids and beneath the endothelial layer of the cavities (Fig. 3d). Laminin became more prominent perisinusoidally as compared with controls. It was present in an almost continuous pattern in dilated sinusoids (Fig. 3f). A strong and preferential accumulation of tenascin was seen around cavities and in the wall of dilated sinusoids

(Fig. 3h). α-SMA-positive cells greatly increased in number perisinusoidally in the wall of dilated sinusoids and around cavities (Fig. 3j). PECAM-1 was expressed on the endothelial cells covering some cavities (Fig. 3k). Electron microscopical immunohistochemistry

In the liver of patients with peliosis type III, collagen fibers were found increased in the subendothelial spaces in sinusoids (data not shown). Amorphous reaction product of type IV collagen was intensely and continuously present throughout the rough endoplasmic reticulum of transformed HSC situated near collagen fiber bundles (Figs. 4a, b). Strongly stained laminin immunodeposits were present mainly in HSCs (Fig. 4c) with long cytoplasmic processes. In contrast, a faint laminin reaction product was observed in the space of Disse. Interestingly, intensely immunostained laminin deposits were present in vesicles in the cytoplasm of some cells (Fig. 4d), observed in the dilated sinusoids. An abundance of morphous reaction products of tenascin were present in the space of Disse of dilated sinusoids (Fig. 4e). Tenascin immune deposits were observed around activated HSCs, and between collagen fibers perisinusoidally (Fig. 4f). The α-SMA-positive HSCs had long processes. Electron microscopy revealed an increase in microfilaments in the peripheral cytoplasm, with an arrangement parallel to the long axis of the cell. Activated HSCs contained single lipid droplets, and had well-developed rough endoplasmic reticulum (Fig. 4g).

Discussion Light microscopically investigated lesions of peliosis hepatis have been thoroughly discussed in the literature [1, 3, 28, 40, 43]. They are characterized by the presence of cavities randomly distributed in the liver lobule. Our histological findings are in line with those previously described. The ultrastructural lesions of peliosis hepatis, however, have been investigated less frequently [1, 13, 40, 43]. Our results seem to confirm and add to the majority of their findings [43]. The main lesion of our study consisted of marked dilatation of the space of Disse and the sinusoidal lumen. A distinct enlargement of the sinusoids around cavities was also observed. Similar results have already been reported in human liver peliosis attributed to Hodgkin’s disease, anemia, light-chain deposition disease, and tuberculosis [43]. Two of our patients suffered from tuberculosis (case 1) and endometriosis treated with androgenic steroids (case 3). The presence of strongly branched HSCs with morphologic (well-developed rough endoplasmic reticulum and Golgi apparatus, and a decreased number of lipid droplets) and im-

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Fig. 2. a: The space of Disse in the normal liver containing a small quantity of amorphous matrix, regularly arranged hepatocytic microvilli, and an endothelial cell (E). Hepatocyte (H), sinusoid (S). b: Part of a peliotic cavity lined by remnants of endothelial cells with large fenestrae and chaotically orientated hepatocytic microvilli. The space of Disse is filled with amorphous matrix, erythrocytes and large blebs from disintegrated cellular cytoplasm. c: Dilated sinusoid (S) with enormously dilated space of Disse (D) containing abundant amorphous matrix and many cellular processes of hepatocytes and HSCs (small star). Endothelial cells (large star) have small fenestrae. d: Dilated sinusoid (S) containing HSC transformed into myofibroblastic (m) cell in the space of Disse. Magnifications: a) ×12000, b) ×4400, c) ×12000, d) ×3000.

munohistochemical (α-SMA positivity) signs of activation were frequently observed in our study. The increase in the number of HSCs in peliosis has already been detected [43], but the signs of their activation have not been noticed. HSCs activation together with sinusoidal cell fenestrae enlargement and the formation of cavities seem to be the most prominent features of sinusoidal cell deterioration in peliosis as detected at the electron microscopical level. The finding of activated HSCs suggests the presence of a stimulus for fibrogenesis in the peliotic liver. Probably, the inability of sinusoidal endothelial cell fenestrae to contract [6] and the entry of plasma and blood cells into the space of Disse initiate HSCs activation and protein synthesis. The accumula-

tion of sinusoidal ECM is known to modulate the diameter of sinusoidal fenestrae [10]. To the best of our knowledge, this study is the first to describe ECM accumulation in human liver with peliosis. It has already been shown that increased perisinusoidal expression of collagen types I, III and IV, laminin and fibronectin occurs in hypervitaminosis A [5], a rare cause of peliosis [42]. In the present study, we demonstrated that non-bacillar liver peliosis is characterized by an increased perisinusoidal deposition of collagen types III and IV, laminin, tenascin and α-SMA. The de novo appearance of laminin in liver sinusoids around cavities and of PECAM-1 on endothelial lining of cavities leads to the development of sinusoidal “capillarization”. It is

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Fig. 3. a: Single thin delicate strands of collagen type III in the liver sinusoids and stronger immune reaction around a central vein in the liver of a control patient. b: Enhanced collagen type III immunoreactivity perisinusoidally and in the wall of cavities in the liver of a patient with peliosis. c: Discontinuous immune deposits of collagen type IV along sinusoids in the liver of a control patient. d: Intense and continuous collagen type IV immune reaction along sinusoids and in the wall of cavities in the liver of a patient with peliosis. e: Faint laminin immune reaction in the wall of sinusoids in the liver of a control patient. f: Continuous perisinusoidal laminin immune reaction near a cavity in the liver of a patient with peliosis. g: Faint and discontinuous tenascin immune reaction in the sinusoids in the liver of a control patient. h: Strong accumulation of tenascin in the wall of cavities and in the dilated sinusoids around them in the liver of a patient with peliosis. i: α-SMA-positive vascular smooth muscle cells in the portal tract in the liver of a control patient. j: Enhanced α-SMA immunoreactivity in the sinusoids and cavities in the liver of a patient with peliosis. k: PECAM-1 immunoreactivity on the endothelial lining covering the wall of cavities. Magnifications: a) ×100, b) ×125, c) ×125, d) ×125, e) ×100, f) ×100, g) ×160, h) ×100, I) ×100, j) ×100, k) ×100.

known that sinusoidal capilarization is initiated by laminin deposition [8, 29, 38] and by the induction of PECAM-1 expression [9]. Using ultrastructural immunohistochemistry, we demonstrated activated HSCs overloaded with laminin and collagen type IV in their cytoplasm in liver sinusoids in peliosis. Similar HSCs were detected in the areas of piecemeal necroses [38], although their intracellular DAB-labeled immunodeposits were considerably weaker than in our case. This means that HSCs in peliosis are strongly and constantly stimulated to synthesize ECM, and that this ECM syn-

thesis and secretion are abnormal. It is generally accepted that predominantly HSCs and, to a lesser extent, sinusoidal endothelial cells and hepatocytes participate in the process of ECM restoration in response to an inappropriate sinusoidal environment. In liver peliosis, we observed laminin immunoreactivity in cytoplasmic vesicles of monocytes or leukocytes within the dilated sinusoids. Similar vesicles positive for ECM proteins were described in rat Kupffer cells under experimental conditions, leading to liver fibrosis [8]. The ECM-positive vesicles have been ascribed to endocytosis. It would be

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reasonable to assume that in peliosis ECM synthesis is very intensive, and that some excessive protein deposits are engulfed by macrophageal elements in the sinusoids. Tenascin is an ECM component that is strongly expressed during tissue restructuring. Thus, this component is believed to mediate diverse morphogenic events, including cell proliferation and migration [23]. Tenascin positivity in dilated liver sinusoids and peliotic cavities suggests an intensive interplay between actively growing cells (epithelial or mesenchymal) and their surrounding ECM in the sinusoid in peliosis. The most prominent tenascin accumulation in liver diseases has been found around proliferating bile ductules and in the areas of piecemeal necrosis [31, 41], i.e. in the zone of intense epithelial proliferation or immunologic conflict. Therefore, accumulation of tenascin in dilated sinusoids and around cavities in peliosis might be responsible for cell proliferation or migration of HSCs or sinusoidal endothelial cells. Enhanced ECM deposition in sinusoids might represent an additional factor explaining the early disruption of sinusoidal endothelium, resulting in sinusoidal dilatation [9]. From our findings, we cannot state with any certainty whether the increased ECM deposition in liver sinusoids precedes sinusoidal dilatation or whether it is a result of this process. Probably, sinusoids around cavities transform into capillary vessels in an attempt to contract this dilated part of the microvascular bed. The reason for sinusoidal dilatation in peliosis has not yet been elucidated. It has been speculated that long-term steroid therapy could lead to the development of vascular aneurysms [39]. The application of tuberculostatics (Rimicid, Tubocin) is considered a cause of hepatocellular damage [37, 43], thus acting as an indirect stimulus for ECM synthesis. The development of peliosis in two of our cases (1 and 3) could be explained in that way. In case 2, we did not establish the serum levels of adrenalin and noradrenalin (the patient refused medical help). One might believe that neuromediators, released from the tumor, stimulate liver adrenoreceptors [33]. It has been reported earlier that the human liver shows a high density of parenchymal adrenergic nerves [24, 26]. Activation of adrenergic nerves results in sinusoidal narrowing only in the initial period (30–40 s) of adrenergic stimulation [27]. Therefore, sinusoidal dilatation could

be assumed to be caused by defective nerve function, resulting in an endothelial cell injury. Probably, adrenergic nerves and activated HSCs showing increased α-SMA immunoreactivity are an attempt of the liver to restrict and remove sinusoidal dilatation. It is known that activated HSCs (with many intermediate filaments) can be stimulated by vasoconstrictor nerves [25] and modulate sinusoidal tone. Therefore, we conclude that activation of HSCs, the increase of their α-SMA content, enhanced perisinusoidal collagen type III and type IV, and laminin accumulation in peliotic liver are rather a result than a cause of sinusoidal dilatation in peliosis, and also an attempt to correct the deteriorated sinusoidal architecture. In conclusion, liver peliosis is characterized by increased ECM accumulation in dilated sinusoids and around cavities, and by developing sinusoidal “capillarization”.

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b Fig. 4. a: Amorphous reaction product of collagen type IV in the rough endoplasmic reticulum of transformed HSC containing pinocytic vesicles. A patient with peliosis. b: Intense collagen type IV immune deposit in the cytoplasm of HSC. A patient with peliosis. c: Strong laminin immune reaction in the cytoplasm of HSC in the wall of a dilated sinusoid. d: Laminin immune deposits in vesicles in the cytoplasm of a mononuclear blood cell. e: Amorphous reaction product of tenascin in the space of Disse in the wall of dilated sinusoids around cavities. f: Tenascin immune deposits surrounding part of the cytoplasm of transformed HSC. g: α-SMA-positive intermediate filaments in the peripheral cytoplasm of transformed HSC. Magnifications: a) ×20000, b) ×20000, c) ×3000, d) ×30000, e) ×12000, f) ×20000, g) ×12 000.

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Received: April 10, 2002 Accepted in revised version: November 25, 2002