Cholesterol Accumulation Is Associated with Lysosomal Dysfunction and Autophagic Stress in Npc1−/− Mouse Brain
2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.070052
ISSN1525-2191
AutoresGuanghong Liao, Yueqin Yao, Jihua Liu, Yu Zhang, Simon Cheung, Ang Xie, Xiaoli Liang, Xiaoning Bi,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoNiemann-Pick type C (NPC) disease is an autosomal recessive disorder caused by mutations of NPC1 and NPC2 genes. Progressive neurodegeneration that accompanies NPC is fatal, but the underlying mechanisms are still poorly understood. In the present study, we characterized the association of autophagic-lysosomal dysfunction with cholesterol accumulation in Npc1−/− mice during postnatal development. Brain levels of lysosomal cathepsin D were significantly higher in mutant than in wild-type mice. Increases in cathepsin D occurred first in neurons and later in astrocytes and microglia and were both spatially and temporally associated with intracellular cholesterol accumulation and neurodegeneration. Furthermore, levels of ubiquitinated proteins were higher in endosomal/lysosomal fractions of brains from Npc1−/− mice than from wild-type mice. Immunoblotting results showed that levels of LC3-II were significantly higher in brains of mutant than wild-type mice. Combined LC3 immunofluorescence and filipin staining showed that LC3 accumulated within filipin-labeled cholesterol clusters inside Purkinje cells. Electron microscopic examination revealed the existence of autophagic vacuole-like structures and multivesicles in brains from Npc1−/− mice. These results provide strong evidence that cholesterol accumulation-induced changes in autophagy-lysosome function are closely associated with neurodegeneration in NPC. Niemann-Pick type C (NPC) disease is an autosomal recessive disorder caused by mutations of NPC1 and NPC2 genes. Progressive neurodegeneration that accompanies NPC is fatal, but the underlying mechanisms are still poorly understood. In the present study, we characterized the association of autophagic-lysosomal dysfunction with cholesterol accumulation in Npc1−/− mice during postnatal development. Brain levels of lysosomal cathepsin D were significantly higher in mutant than in wild-type mice. Increases in cathepsin D occurred first in neurons and later in astrocytes and microglia and were both spatially and temporally associated with intracellular cholesterol accumulation and neurodegeneration. Furthermore, levels of ubiquitinated proteins were higher in endosomal/lysosomal fractions of brains from Npc1−/− mice than from wild-type mice. Immunoblotting results showed that levels of LC3-II were significantly higher in brains of mutant than wild-type mice. Combined LC3 immunofluorescence and filipin staining showed that LC3 accumulated within filipin-labeled cholesterol clusters inside Purkinje cells. Electron microscopic examination revealed the existence of autophagic vacuole-like structures and multivesicles in brains from Npc1−/− mice. These results provide strong evidence that cholesterol accumulation-induced changes in autophagy-lysosome function are closely associated with neurodegeneration in NPC. Niemann-Pick type C disease (NPC) is a fatal neurodegenerative disorder that mainly affects children. 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Levels of lysosomal cathepsin D in neurons are increased in AD vulnerable regions before the onset of major pathology.18Cataldo AM Paskevich PA Kominami E Nixon RA Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease.Proc Natl Acad Sci USA. 1991; 88: 10998-11002Crossref PubMed Scopus (240) Google Scholar Cathepsin D up-regulation correlates on a cell-by-cell basis with other markers of early-stage AD, including decreased levels of the synaptic vesicular protein synaptophysin and increased levels of intraneuronal neurofibrillary tangles.19Ginsberg SD Hemby SE Lee VM Eberwine JH Trojanowski JQ Expression profile of transcripts in Alzheimer's disease tangle-bearing CA1 neurons.Ann Neurol. 2000; 48: 77-87Crossref PubMed Scopus (292) Google Scholar, 20Callahan LM Vaules WA Coleman PD Quantitative decrease in synaptophysin message expression and increase in cathepsin D message expression in Alzheimer disease neurons containing neurofibrillary tangles.J Neuropathol Exp Neurol. 1999; 58: 275-287Crossref PubMed Scopus (115) Google Scholar Experimentally induced lysosomal dysfunction is associated with rapid formation of neurofibrillary tangles in hippocampal slices cultured from apolipoprotein E knockout mice.21Bi X Yong AP Zhou J Ribak CE Lynch G Rapid induction of intraneuronal neurofibrillary tangles in apolipoprotein E-deficient mice.Proc Natl Acad Sci USA. 2001; 98: 8832-8837Crossref PubMed Scopus (40) Google Scholar Cytoplasmic presence of cathepsin D can induce release of cytochrome c from mitochondria and activation of proapoptotic factors, which leads to caspase-dependent apoptosis, also referred to as type 1 programmed cell death.22Bidère N Lorenzo HK Carmona S Laforge M Harper F Dumont C Senik A Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis.J Biol Chem. 2003; 278: 31401-31411Crossref PubMed Scopus (370) Google Scholar, 23Johansson AC Steen H Ollinger K Roberg K Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine.Cell Death Differ. 2003; 10: 1253-1259Crossref PubMed Scopus (181) Google Scholar, 24Cirman T Oresic K Mazovec GD Turk V Reed JC Myers RM Salvesen GS Turk B Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins.J Biol Chem. 2004; 279: 3578-3587Crossref PubMed Scopus (412) Google ScholarLysosomes also participate in type 2 programmed cell death, referred to as autophagic cell death, which is defined by the presence of autophagic morphology.25Schweichel JU Merker HJ The morphology of various types of cell death in prenatal tissues.Teratology. 1973; 7: 253-266Crossref PubMed Scopus (558) Google Scholar, 26Uchiyama Y Autophagic cell death and its execution by lysosomal cathepsins.Arch Histol Cytol. 2001; 64: 233-246Crossref PubMed Scopus (211) Google Scholar Neuronal death with features of autophagy has been observed during normal development27Clarke PG Developmental cell death: morphological diversity and multiple mechanisms.Anat Embryol (Berl). 1990; 181: 195-213Crossref PubMed Scopus (1528) Google Scholar and in pathological conditions, such as in AD28Stadelmann C Deckwerth TL Srinivasan A Bancher C Bruck W Jellinger K Lassmann H Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer's disease: evidence for apoptotic cell death.Am J Pathol. 1999; 155: 1459-1466Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 29Nixon RA Wegiel J Kumar A Yu WH Peterhoff C Cataldo A Cuervo AM Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study.J Neuropathol Exp Neurol. 2005; 64: 113-122Crossref PubMed Scopus (1122) Google Scholar and in Parkinson's disease.30Anglade P Vyas S Javoy-Agid F Herrero MT Michel PP Marquez J Mouatt-Prigent A Ruberg M Hirsch EC Agid Y Apoptosis and autophagy in nigral neurons of patients with Parkinson's disease.Histol Histopathol. 1997; 12: 25-31PubMed Google Scholar On the other hand, neuroprotective function of autophagy has also been implicated in certain neurodegenerative diseases, such as Huntington's disease. A recent study reported the existence of autophagic features in Purkinje cells in Npc1−/− mice.31Ko DC Milenkovic L Beier SM Manuel H Buchanan J Scott MP Cell-autonomous death of cerebellar Purkinje neurons with autophagy in Niemann-Pick type C disease.PLoS Genet. 2005; 1: 81-95Crossref PubMed Scopus (0) Google Scholar To investigate further the roles of autophagy-lysosome system in neurodegeneration in NPC, the present study determined levels and localization of the lysosomal enzyme cathepsin D and of autophagic activity and the potential association of autophagic-lysosomal dysfunction with accumulation of cholesterol and neurodegeneration in brains of Npc1−/− mice.Materials and MethodsMiceBreeding pairs of BALB/cNctr-npc1NIH mice heterozygous for Npc1 (+/−) were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in our animal facility in accordance with National Institutes of Health guidelines and protocols approved by the Institutional Animal Care and Use Committee with care to minimize distress to the animals. Mouse breeding and genotyping were performed as previously described.32Baudry M Yao Y Simmons D Liu J Bi X Postnatal development of inflammation in a murine model of Niemann-Pick type C disease: immunohistochemical observations of microglia and astroglia.Exp Neurol. 2003; 184: 887-903Crossref PubMed Scopus (141) Google Scholar Animals were sacrificed at postnatal weeks 1, 2, 4, and 8 (four to eight animals for each age group) under deep anesthesia (100 mg/kg sodium pentobarbital) by perfusion for immunohistochemical and histological studies or by decapitation for biochemical analyses. For histological studies, animals were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Brains were removed and incubated with 15% sucrose followed by 30% sucrose before being sectioned at 25 μm with a microtome. Coronal sections were stored in a cryoprotective solution at −20°C before being processed for immunohistochemical studies.Subcellular FractionationBrains from Npc1−/− and their wild-type littermates were dissected in ice-cold artificial cerebrospinal fluid and homogenized in homogenization buffer [homogenization buffer-EDTA: 3 mmol/L imidazole, 250 mmol/L sucrose, and 1 mmol/L ethylenediamine tetraacetic acid (EDTA), pH 7.4] containing protease inhibitors (Sigma-Aldrich, St. Louis, MO); homogenates were centrifuged for 10 minutes at 1500 × g. The sucrose concentration of the collected postnuclear supernatant was adjusted to 40.6% by the slow addition of 62% sucrose in homogenization buffer-EDTA. Postnuclear supernatant was then carefully overloaded with 1.5 ml of 35% and 1.0 ml of 25% sucrose in homogenization buffer-EDTA, and the samples were centrifuged in an SW 55 rotor (Beckman Instruments, Inc., Palo Alto, CA) at 14,000 × g for 90 minutes at 4°C. Subcellular fractions were collected from the top of the tube. The late endosome/lysosome-enriched fraction was localized in the upper interface, containing 25% sucrose and homogenization buffer, and the early endosome-enriched fraction in the middle interface containing 35 and 25% sucrose. The lower interface containing 40.6 to 35% sucrose was enriched in plasma membranes and other heavy membrane compartments.Western BlotsElectrophoresis and immunoblotting were performed following conventional procedures. In brief, after protein concentration was determined, proteins (40 to 60 μg) of postnuclear supernatant from different brain regions [cerebellum, brainstem (including interbrain, midbrain, and hindbrain), hippocampus, and cortex] or of other subcellular fractions were denatured by boiling for 5 minutes in a sample buffer (2% sodium dodecyl sulfate, 50 mmol/L Tris-HCl pH 6.8, 10% 2-mercaptoethanol, 10% glycerol, and 0.1% bromphenol blue) and separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (12%), after which proteins were transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with primary antibodies for 12 to 16 hours at 4°C; immunoreactivity was visualized by using enhanced chemiluminescence (ECL Plus kit and reagents; Amersham Pharmacia Biotech, Piscataway, NJ). Antibodies used included anti-cathepsin D (1:1000; EMD Biosciences, San Diego, CA), anti-cathepsin B (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-rab7 (1:1000; Santa Cruz Biotechnology), anti-ubiquitin (1:500; Zymed, Carlsbad, CA), and anti-LC3 serum (gift from T. Yoshimori, National Institute of Genetics, Mishima, Shizuoka, Japan33Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T Kominami E Ohsumi Y Yoshimori T LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5361) Google Scholar). Levels of different bands were analyzed by using the National Institutes of Health Image program (Bethesda, MD). Statistical significance was determined by two-tailed Student's t-test.Activity Assay of Cathepsins B and DWhole homogenates of brainstem, cerebellum, or hippocampus from Npc1−/− and wild-type mice were used to analyze the activity of cathepsins B and D using fluorogenic immunocapture activity assay kits (EMD Biosciences) according to the kit instructions.ImmunohistochemistrySagittal sections from cerebellum and coronal sections from the rest of the brain of animals from different ages were simultaneously processed for immunostaining. Immunohistochemistry was performed using the avidin-biotin horseradish peroxidase complex method. In brief, free-floating sections were first incubated in 10% normal horse serum (for monoclonal antibodies) or 3% normal goat serum (for polyclonal antibodies) diluted in PBS with 0.1% Triton X-100 for 1 hour at room temperature, followed by incubation with primary antibodies overnight at 4°C. Antibodies used were anti-cathepsin D (1:500; EMD Biosciences) and anti-cathepsin B (1:100; Santa Cruz Biotechnology). After three washes in PBS, sections were incubated with corresponding biotinylated secondary antibodies (1:400; Vector Laboratories, Burlingame, CA) in 5% normal horse serum or 1.5% normal goat serum solution for 2 to 3 hours, then in avidin-biotin horseradish peroxidase complex diluted in PBS for 45 minutes. Peroxidase reaction was performed with 3,3′-diaminobenzidine tetrahydrochloride (0.05% in 50 mmol/L Tris-HCl buffer, pH 7.4) as chromogen and 0.03% H2O2 as oxidant. Free-floating sections were mounted on precoated slides (SuperPlus; Fisher Scientific International Inc.) and air-dried. Sections were then dehydrated in graded ethanol and finally covered with Permount (Fisher Scientific).Double-labeling immunohistochemistry was done with sections first incubated with primary antibodies [rabbit anti-cathepsin D in combination with either rat anti-F4/80 (1:1000; Serotec, Raleigh, NC) or mouse anti-calbindin (1:1000; Abcam, Cambridge, MA)], then with corresponding secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594. Both secondary antibodies were purchased from Molecular Probes, Eugene, OR.Filipin StainingFilipin has been demonstrated to specifically stain free cholesterol because treatment with cholesterol oxidase results in a complete loss of fluorescence.34Bornig H Geyer G Staining of cholesterol with the fluorescent antibiotic “filipin.”.Acta Histochem. 1974; 50: 110-115PubMed Google Scholar Brain tissue sections were washed with phosphate-buffered saline and incubated in the dark with 125 μg/ml filipin in PBS for 3 hours under agitation at room temperature. After washing in PBS, some sections were further processed for immunostaining with anti-calbindin or -LC3 (1:3000; Abgent, San Diego, CA) antibodies and corresponding secondary antibodies conjugated with Alexa Fluor 594.Images of immunostained sections from different brain regions were visualized using a Zeiss microscope (Axioskop 2 Mot Plus) and digitized via a Zeiss digital photo camera (AxioCam Hrc) and the Axiovision program, version 3.1 (Zeiss), was used to capture and save digitized images. Digitized images were then assembled in Photoshop (version 7; Adobe Systems, Mountain View, CA) with only the brightness adjusted to match other panels in a given figure. Images of double fluorescent labeled sections were acquired by using a Nikon confocal microscope (Nikon TE 2000U with D-Eclipse C1 system; Melville, NY).Electron Microscopy AnalysisElectron microscopy analysis was performed as previously described.35Bednarski E Ribak CE Lynch G Suppression of cathepsins B and L causes a proliferation of lysosomes and the formation of meganeurites in hippocampus.J Neurosci. 1997; 17: 4006-4021Crossref PubMed Google Scholar In brief, animals were perfused with an ice-cold solution of 0.1 mol/L phosphate buffer, pH 7.4, containing 1.5% paraformaldehyde and 1.5% glutaraldehyde. Cerebellum blocks were transferred to 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, at 4°C for 24 hours, rinsed overnight in the phosphate buffer, postfixed with 1% osmium tetroxide in phosphate buffer for 2 hours, followed by dehydration and embedded in epoxy resin. Ultrathin sections were prepared using a Reichert ultramicrotome, contrasted with uranyl acetate and lead citrate, examined under a Philips CM120 transmission electron microscope at 80 kV.ResultsIncreased Levels of Cathepsin D in Brains of Npc1−/− Mice during Postnatal DevelopmentCathepsin D is synthesized as an inactive 52- to 53-kd proenzyme; cathepsin D activation produces a 48-kd (single chain) intermediate and mature forms at 34 and 14 kd (heavy and light chains, respectively).36Liaudet-Coopman E Beaujouin M Derocq D Garcia M Glondu-Lassis M Laurent-Matha V Prebois C Rochefort H Vignon F Cathepsin D: Newly discovered functions of a long-standing aspartic protease in cancer and apoptosis.Cancer Lett. 2006; 237: 167-179Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 37Follo C Castino R Nicotra G Trincheri NF Isidoro C Folding, activity and targeting of mutated human cathepsin D that cannot be processed into the double-chain form.Int J Biochem Cell Biol. 2007; 39: 638-649Crossref PubMed Scopus (17) Google Scholar Immunoblotting studies using anti-cathepsin D antibodies revealed an early-onset increase in levels of cathepsin D (both single chain and heavy chain) in all brain regions tested. At 2 weeks postnatal, levels of single chain cathepsin D (Figure 1A, arrows) in brainstem, cerebellum, cerebral cortex, and hippocampus of Npc1−/− mice were 274 ± 7%, 190 ± 5%, 176 ± 12%, and 199 ± 5% of those measured in Npc1+/+ mice, respectively (means ± SEM, n = 5, P < 0.001; Figure 1B). Levels of single chain-cathepsin D remained elevated at 4 weeks with further increase being only evident in cerebellum (Figure 1B). Changes in heavy chain-cathepsin D (Figure 1A, −) were similar to those observed for the single chain isoform (Figure 1, A and B).Immunohistochemical results revealed significant increases in cathepsin D immunoreactivity throughout the brain in 1-week-old Npc1−/− mice. In contrast to the pattern observed in wild-type mice, numerous darkly labeled cells were found in cerebellum of Npc1−/− mice, and most of them were located in white matter (compare Figure 2, B with A), suggesting that they were glial cells. High magnification examination showed that cathepsin D immunoreactivity was also moderately increased in Purkinje cells in Npc1−/−(Figure 2D) compared with wild-type mice (Figure 2C). The number of cathepsin D-immunoreactive cells was also increased in the ventral posterior nuclei of the thalamus at 1 week (Figure 2F) compared with that in Npc1+/+ mice (Figure 2E), and it was further increased by 4 weeks. By 8 weeks, the ventral posterior nuclei of the thalamus were filled with anti-cathepsin D-immunopositive cells (Figure 2H). Higher magnification images showed that although cathepsin D-immunoreactive products existed in small granules that were scattered in cell bodies in the ventral posterior nuclei of the thalamus of Npc1+/+ mice (Figure 2I), those in Npc1−/− mice were present in larger punctates that often clustered together (Figure 2J). Furthermore, higher cytoplasmic levels of cathepsin D immunoreactivity were observed around these punctates. Immunohistochemical and immunoblotting results showed that levels of another lysosomal hydrolase, cathepsin B, were also increased in mutant mice as compared with wild-type mice (Figure 2, K–M). Cathepsin D activity in homogenates from brainstem of 4-week-old Npc1−/− mice was about 2.4 times higher than that from Npc1+/+ mice (n = 3 for Npc1+/+ and n = 4 for Npc1−/− mice; P < 0.01). Cathepsin B activity also increased in samples from hippocampus (295 ± 8%; P < 0.01) and cerebellum (187 ± 3%; P < 0.01) of 8-week-old Npc1−/− mice (n = 5) compared with Npc1+/+ mice (n = 5).Figure 2Distribution of cathepsin D and B in cerebellum and thalamus of Npc1−/− mice during postnatal development. Cerebellar (A–D) and thalamic (E–L) tissue sections were prepared from Npc1+/+ (A, C, E, G, I, and K) and Npc1−/− (B, D, F, H, J, and L) mice at postnatal week 1 (A–F), 4 (K and L), and 8 (G–J) and were immunostained with anti-cathepsin D (A–J) or anti-cathepsin B (K and L) antibodies. Higher magnification images show that in Npc1+/+ mice (I) cathepsin D immunoreactive products are mainly located in small-sized granules, whereas in Npc1−/− mice they are present in larger puncta and their surrounding cytoplasmic structures (J). M shows immunoblots of cathepsin B-labeled samples from brainstem of 4-week-old mice. ml, molecular layer; pl, Purkinje layer; gl, granular layer; VPT, ventral posterior nucleus of thalamus. Scale bar = 50 μm (A and B); 12.5 μm (C–H and K and L).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Double immunofluorescence staining was used to determine the cellular and subcellular localization of cathepsin D in mutant mice. At 4 weeks, cathepsin D-immunoreactive granules were observed in cell bodies of calbindin-immunopositive Purkinje cells in Npc1−/− mice (Figure 3A, the asterisk in bottom panels), whereas very few cathepsin D positive granules were found in the cerebellum of Npc1+/+ mice (Figure 3A, top panels). In the cerebellum of 4-week-old Npc1−/− mice, cathepsin D immunoreactivity was also found in reactive microglia identified with antibodies against the macrophage marker F4/80 antigen (data not shown). By 8 weeks, cathepsin D-labeled granules accumulated mainly in the apical processes of small cells dispersed among Purkinje cells in wild-type mice; from their position and morphology, these cells resembled Bergman glia (Figure 3B, arrows). Cathepsin D immunoreactivity in Bergmann glia in Npc1−/− mice was similar to that in wild-type mice (Figure 3B). In the cerebellum of 8-week-old Npc1−/− mice, cathepsin D immunoreactivity was also observed in F4/80-labeled reactive microglia (Figure 3B, mg); at this postnatal age, microglia became larger and rounder and invaded both the Purkinje cell layer and the molecular layer.Figure 3Cellular localization of cathepsin D in cerebellar cortex at 4 and 8 weeks postnatal. A: Double immunofluorescence staining using antibodies against cathepsin D (red) and calbindin (green) in cerebellum of 4-week-old Npc1+/+ (top panels) and Npc1−/− (bottom panels) mice. DAPI (blue) was included in the mounting medium to label nuclei. B: Double immunofluorescence staining using antibodies against cathepsin D (red) and F4/80 (green; a marker for microglia) in cerebellum of 8-week-old Npc1+/+ (top panels) and Npc1−/− (bottom panels) mice. *, Purkinje cells; arrows, Bergmann glia; mg, microglia. Scale bar = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Abnormal Subcellular Protein Distribution in Brains of Npc1−/− MiceThe subcellular localization of various proteins was determined by combining subcellular fractionation and immunoblotting analysis. Cathepsin D levels were markedly higher in the late endosomal/lysosomal fractions in mutant compared with wild-type mice (Figure 4, top panel). Levels of the small GTP-binding protein Rab7, which participates in the maturation of autophagic vacuoles,38Jäger S Bucci C Tanida I Ueno T Kominami E Saftig P Eskelinen EL Role for Rab7 in maturation of late autophagic vacuoles.J Cell Sci. 2004; 117: 4837-4848Crossref PubMed Scopus (681) Google Scholar, 39Gutierrez MG Munafo DB Beron W Colombo MI Rab7 is required for the normal progression of the autophagic pathway in mammalian cells.J Cell Sci. 2004; 117: 2687-2697Crossref PubMed Scopus (486) Google Scholar were higher in the late endosomal/lysosomal fractions but lower in the early endosomal fractions in Npc1−/− compared with wild-type mice. As a close link between autophagy and protein ubiquitination has previously been reported,40Mariño G Lopez-Otin C Autophagy: molecular mechanisms, physiological functions and relevance in human pathology.Cell Mol Life Sci. 2004; 61: 1439-1454Crossref P
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