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From the Department of Neurology,* Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, Massachusetts; the Department of Neurology, Genetics and Aging Research Unit, Center for Aging, Genetics, and Neurodegeneration, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; and the Mayo Clinic Jacksonville, Jacksonville, Florida
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Abstract |
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15 to 30% deficit in the degradation of both insulin and Aß. Endogenously secreted Aß40 and Aß42 are significantly elevated in primary neuronal cultures from animals with the IDE mutations, but there is no increase in steady-state levels of rodent Aß in the brain up to age 14 months. We conclude that naturally occurring, partial loss-of-function mutations in IDE sufficient to cause DM2 also impair neuronal regulation of Aß levels, but the brain can apparently compensate for the partial deficit during the life span of the rat. Our findings have relevance for the emerging genetic evidence suggesting that IDE may be a late-onset AD-risk gene, and for the epidemiological relationships among hyperinsulinemia, DM2, and AD.
Several proteases have been shown capable of degrading Aß, including insulin-degrading enzyme (IDE, insulysin; EC 3.4.24.56),4-8
neprilysin (NEP),9,10
endothelin-converting enzyme,11
the uPA/tPA/plasminogen system,12,13
and angiotensin-converting enzyme.14
Mice with experimental deletions of the NEP,15
endothelin-converting enzyme,16
or IDE17
genes have elevated levels of endogenous cerebral Aß of similar magnitude (60 to 100%). These animal models validate the role of proteolysis in the regulation of cerebral Aß levels in vivo.
IDE, an 110-kd thiol zinc metalloendopeptidase located in cytosol, peroxisomes, endosomes, and on the cell surface,8,18-20
cleaves small proteins of diverse sequence, several of which share a propensity to form ß-pleated sheet-rich amyloid fibrils, including insulin, Aß, amylin, atrial natriuretic factor, and calcitonin.21,22
An unbiased screen of various cell lines for Aß-degrading activity revealed that the major such activity was attributable to IDE.6,7
Direct comparison of IDE and NEP activity by transfecting their respective cDNAs into stable human APP-expressing cell lines showed that IDE markedly reduced the levels of soluble and insoluble intracellular Aß, whereas NEP lowered the levels of only insoluble intracellular Aß.23
It is not yet fully established which pool of Aß is more pathogenic, although recent studies suggest that cognitive dysfunction in AD patients correlates more tightly with soluble than insoluble levels of cortical Aß.24,25
It has been demonstrated that proteolysis of Aß by IDE eliminates the neurotoxic effects of the peptide in rat primary neuronal cultures and prevents the deposition of Aß onto synthetic amyloid aggregates.26
Recently, it has been reported that levels of IDE protein and transcripts are reduced in the hippocampi from AD patients with an apolipoprotein E-
4 allele compared to either AD patients without this AD-risk allele or normal patients (with or without the allele).27
Consistent with a possible role for Aß-degrading proteases in human disease, the IDE region of chromosome 10q has been shown to have genetic linkage to late-onset (conventional) AD28
and to age of disease onset in AD families.29
Some studies have also reported allelic association in or near the IDE gene with late-onset AD,28,30,31
whereas others have not.32,33
In separate reports, the occurrence of AD34
and elevated plasma Aß42 levels35
were each linked to a region of chromosome 10q 40 Mb centromeric to the linkage peaks over the IDE region. It remains unclear whether these results signify the same underlying locus or two separate loci.
The IDE region of chromosome 10q has also been genetically linked to type 2 diabetes mellitus (DM2)36-38
and to fasting glucose levels (20-year means).38,39
In this regard, it was shown that transferring a 3.7-cM chromosomal region containing the IDE gene from an inbred rat model of DM2 [Goto-Kakizaki (GK) rat] to a normoglycemic rat recapitulated several features of the diabetic phenotype, including hyperinsulinemia and postprandial hyperglycemia.40
The GK allele of IDE in this chromosomal region was found to bear two missense mutations that, when transfected into COS-1 cells, resulted in 31% less insulin degradation by these cells than by cells transfected with the wild-type (WT) allele. Targeted homozygous deletion of the IDE gene in mice, in addition to elevating cerebral levels of Aß, also produced hyperinsulinemia and glucose intolerance in the animals.17
Thus, although the gene(s) responsible for the linkage and association of AD and DM2 to chromosome 10q has not been identified, the IDE gene is a leading candidate in both diseases.37,41
In this regard, there is growing evidence that DM2 and, in particular, hyperinsulinemia are associated with an increased risk of developing AD (see Discussion).
Although it is now clear that experimental deletion of the IDE gene results in acute elevations of cerebral Aß, it is not known whether naturally occurring, partial loss-of-function mutations in IDE can affect Aß catabolism, and, if they do, whether the effect is subtle enough to be compatible with the genetic evidence that IDE may be a risk-conferring gene for late-onset AD. It is possible that, in the pertinent neuronal compartments, the protease is in sufficient excess relative to its Aß substrate so that mild hypofunction of IDE will not alter Aß turnover, or that there is a compensatory increase in the levels of the hypofunctional IDE or another Aß-degrading protease such that there is no net effect on Aß catabolism. For IDE to remain a valid candidate gene for late-onset AD risk, as suggested by the emerging genetic evidence, not only must it be shown that partial loss of IDE function is physiologically relevant, but also that it does not cause an early and severe elevation of Aß levels. The magnitude of IDE hypofunction in humans would be expected to be significantly less severe than that of the IDE gene-deleted mice, with their complete absence of IDE function and >60% increases in cerebral Aß by only 12 weeks of age.
In this study, we take advantage of the GK rat to ascertain whether, and to what extent, naturally occurring, partial loss-of-function mutations in IDE that are sufficient to cause features of the DM2 phenotype40 can also affect Aß turnover and alter levels of Aß in neuronal cultures and brain. Based on our findings, we discuss the potential relationship among mutations in IDE, DM2, and AD.
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Materials and Methods |
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GK and Wistar rats were purchased from Taconic M&B (Denmark) and Charles River Labs (Wilmington, MA), respectively. IDE cDNA was amplified from rat liver by reverse transcriptase-polymerase chain reaction with gene-specific primers, ligated into a pcDNA5/FRT (Invitrogen, Carlsbad, CA) vector, and sequenced using an ABI PRISM 377 sequencer.
Preparation and Immunoblotting of Brain Fractions
Fresh-frozen rat cerebral hemispheres were homogenized in 8 vol (w:v) of 250 mmol/L sucrose in 50 mmol/L Tris-HCl (pH 7.4) in a Potter-Elvehjem homogenizer. After pelleting nuclei and unbroken cells, the supernatant was spun again at 100,000 x g for 1 hour. This resulting supernatant was saved as the soluble fraction, and the membrane pellet was washed in 100 mmol/L Na2CO3 (pH 11.3) to linearize microsomes and strip loosely associated proteins.42 The membranes were pelleted again, resuspended in 50 mmol/L Tris-HCl (pH 7.4), and sonicated. Protein concentrations were determined using a bicinchoninic acid-based protein assay (Pierce, Rockford, IL). For immunoblotting, membranes were probed with antibodies raised to: IDE (IDE-1),8 NEP (56C6; Novacastra, Newcastle, UK); amyloid precursor protein and its proteolytic derivatives C83, C99 (antibody C8),43 and the secreted APP fragment (22C11; Chemicon, Temecula, CA); presenilin N-terminal (Ab14, gift from S. Gandy, Thomas Jefferson University) and C-terminal (4627)44 fragments; BACE (Ab-2; Oncogene), and ADAM10 (AB19026, Chemicon).
Primary Cell Cultures
For primary fibroblasts, tissue from the abdominal wall and penis of adult rats were minced and partially digested with 0.25% trypsin in Hanks’ balanced salt solution. The dissociated cells were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. For primary cortical neuronal cultures, neocortices and hippocampi from postnatal day 0 rat litters were partially digested in 0.025% trypsin in Hanks’ balanced salt solution, 10 mmol/L HEPES, 10 mmol/L glucose, and 10 mmol/L sucrose, mechanically dissociated, and plated at varying densities onto poly-L-lysine-coated dishes. Cells were maintained in a neuronal-selecting medium (Neurobasal medium with the growth supplement B27; Invitrogen).
Trichloroacetic Acid (TCA) Precipitation Degradation Assays
To measure proteolysis by brain fractions, 100 pmol/L synthetic 125I-Aß1-40 (human or rodent) (Amersham, Arlington Heights, IL), or 100 pmol/L recombinant human 125I-insulin (Amersham) was incubated at 37°C with 100 µg/ml and 50 µg/ml of protein, respectively. To determine rates of proteolysis by intact primary fibroblast (22,000 cells/cm2) and neuronal cultures (80,000 and 1,600,000 cells/cm2), cells were washed with serum-free and supplement-free medium (Dulbecco’s modified Eagle’s medium and Neurobasal, respectively) containing 0.1% bovine serum albumin, then incubated in the same medium with 40 pmol/L of either 125I-insulin or 125I-Aß1-40. At each time point, an aliquot of the sample was added to an equal volume of 15% TCA to allow the uncleaved peptides to precipitate. After centrifugation, gamma counts in the TCA-insoluble pellet (undegraded peptide) and TCA supernatant (degraded peptide fragments) were determined, and the percentage of radiolabeled substrate degraded was calculated.
Quantification of Endogenous Aß
After primary neurons were allowed to condition Neurobasal media with B27 supplement, the media were removed, 1,10-phenanthroline and a protease inhibitor cocktail (Sigma, St. Louis, MO) were added, and the samples were centrifuged to remove any cellular material. Aß was extracted from brain using either the previously described diethylamine protocol2,45 or Tris-buffered saline (TBS)/guanidine HCl. In brief, we homogenized cerebral hemispheres in either 9 vol (w/v) 0.2% diethylamine in 50 mmol/L NaCl or 5 vol (w/v) of TBS with the above protease inhibitors using a Potter-Elvehjem homogenizer. Homogenates were centrifuged at 100,000 x g for 1 hour. Supernatants from the diethylamine extractions were neutralized with 1/10 volume 500 mmol/L Tris-HCl (pH 6.8), and the pellets were discarded. Supernatants from the TBS extraction were saved as soluble Aß, while the associated pellets were resuspended in a 5-mol/L guanidine HCl and 50-mmol/L Tris-HCl (pH 8.0) buffer and saved as insoluble Aß. Aß enzyme-linked immunosorbent assays (ELISAs) on neuronal media and DEA brain extracts were performed using the well-characterized BNT-77/BA-27 (Aß X-40) and BNT-77/BC-05 (Aß X-42) system.46 ELISAs on the guanidine HCl and TBS brain extracts and confirmatory ELISAs on the neuronal media were done with the 2G3/4G8 (Aß X-40) and 21F12/4G8 ELISA system (Aß X-42).47
Transfection of CHO Cells
Equal numbers CHO cells stably expressing human APP751 containing the V717F AD-causing missense mutation were transiently transfected with empty vector or vectors containing either the GK or WT IDE allele (as generated above) using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were washed and allowed to condition fresh media for 24 hours (OptiMEM, Invitrogen). The media was then treated identically to the neuronal conditioned media as above, except a different Aß ELISA system was used.48,49 Thirty-six hours after transfection, cell lysates were made from duplicate dishes of the three transfected constructs, and IDE overexpression was confirmed by immunoblotting.
IDE Immunodepletion
Brain-soluble fractions were prepared as above without protease inhibitors. For each sample, 400 µg of brain-soluble protein were first precleared with protein G-agarose resin (Roche, Indianapolis, IN), then incubated with fresh resin and either no immunoglobulin (sham immunodepletion controls) or the anti-IDE monoclonal antibody 9B12 (titer of 1:100) (gift from R. Roth, Stanford University) at 4°C for 12 hours. The resin was then pelleted by centrifugation, and the IDE-immunodepleted supernatant was saved for Aß degradation assays.
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Results |
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CGC at codon 18 (H18R), GCGGTC at codon 890 (A890V), and GATGAC at codon 934 (silent). The WT Wistar sequence was identical to that in GenBank (accession no. NM 013159). Protein levels of IDE, neprilysin, APP, the APP proteolytic derivatives (C83, C99, secreted APP fragment, and AICD), presenilin NTF and CTF, BACE, and ADAM 10 were indistinguishable by immunoblotting in GK and control rat brain fractions and primary neuronal lysates (not shown).
To initially assess degradative capacity for insulin and Aß in intact, living cells of the GK rat, we incubated primary cultured fibroblasts of GK and WT rats with physiological levels (40 pmol/L) of radioiodinated human insulin or human Aß1-40 in serum-free medium for 0, 3, 6, and 24 hours and then performed TCA precipitation assays at each time point as described in Materials and Methods. We found that the GK fibroblasts degraded insulin 24% less well than the controls (P < 0.005) (Figure 1A)
, in close agreement with the deficit in insulin degradation (21%) reported in isolated muscle fibers from rats with the GK IDE allele.40
The GK fibroblasts also degraded Aß 20% less than controls (P < 0.01) (Figure 1B)
. Unlabeled insulin (10 µmol/L), a potent and relatively selective competitive inhibitor of IDE, blocked 90% of Aß degradation in both the GK and control cultures, indicating that virtually all Aß proteolysis by these intact cells is attributable to IDE.
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30% decrement in the degradation of both substrates at both time points. At 1 and 3 hours, insulin degradation in the WT rats was 37% and 60%, respectively, whereas in the GK rats, it was 24% and 43% (P < 0.01 at 1 hour, P < 0.001 at 3 hours) (Figure 2A)
. At 3 and 8 hours, the WT Aß proteolysis was 35% and 59%, respectively, while in the GK it was 25% and 40% (P < 0.00001 for each time point). The GK brain-soluble fraction also showed degradation deficits. Insulin proteolysis was decreased 26% at 15 minutes (P < 0.01) and 19% at 30 minutes (P < 0.0001), whereas parallel Aß assays showed a 14%, highly significant (P < 0.00001) decrease at both 30 and 60 minutes (Figure 2B)
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. In washed brain membranes, insulin inhibited 77% of control and 71% of GK-mediated degradation, resulting in identical residual proteolysis in the two genotypes, as would be expected if the GK deficit is because of IDE. Glucagon (10 µmol/L), a less avid competitive inhibitor of IDE than insulin, prevented 60% of the Aß degradation. Thiorphan (1 mmol/L), a potent inhibitor of NEP and angiotensin-converting enzyme, and an inhibitor cocktail containing only serine and cysteine protease inhibitors (Roche) (not shown) each blocked only 15% of the degradation. In the presence of thiorphan and the serine and cysteine protease inhibitors, the GK membrane fraction continued to show a significant decrease in Aß proteolysis, again supporting the attribution of this deficit to IDE. 1,10-Phenanthroline (2 mmol/L), a potent zinc metalloprotease inhibitor that can inhibit both IDE and NEP, prevented >80% of the Aß proteolysis. In the brain-soluble fraction, Aß degradation was inhibited >96% by either insulin or 1,10-phenanthroline, 75% by glucagon, <5% by thiorphan, and not at all by the serine and cysteine protease inhibitor cocktail (data not shown). Degradation assays done in brain membranes and soluble fractions using rodent 125I-Aß40 iodinated at two different sites than the human peptide used above (ie, either His6 or His14 instead of Tyr10) yielded similar degradation deficits in the GK rats (not shown). These results indicate that the GK Aß degradation deficit extends to the rodent peptide, with its three amino acid differences compared with human Aß, and that the site of iodination does not affect the results of our Aß degradation assays.
Next, we quantified insulin and Aß proteolysis in intact primary cortical neurons. Neocortical cells from two GK and two WT litters were incubated with 40 pmol/L of 125I-insulin or 125I-Aß40 in serum-free medium for 0, 3, 6, and 24 hours and TCA precipitation assays were performed. Overall, 125I-insulin proteolysis ranged from 45% at 3 hours to 85% at 24 hours, whereas 125I-Aß degradation ranged from 10% at 3 hours to 65% at 24 hours. The GK neurons demonstrated less insulin and Aß proteolysis than WT neurons at each time point, with the differences at 6 and 24 hours being statistically significant (6-hour values in Figure 3
). Insulin degradation (Figure 3A)
in the GK neurons at 3, 6, and 24 hours was 12% (P = 0.12), 15% (P = 0.005), and 4% (P = 0.03) less than WT, respectively. The Aß proteolytic deficit (Figure 3B)
was 8% (P = 0.07), 15% (P = 0.0008), and 7% (P = 0.0007) at 3, 6, and 24 hours, respectively. Unlabeled insulin prevented >90% of the neuronal Aß degradation (P < 0.01), leaving identical amounts of residual activity in the GK and controls (Figure 3B)
, whereas glucagon blocked >50% of Aß proteolysis (P < 0.05) (not shown). To determine whether the GK Aß-degradation deficit documented in primary neurons occurs for the naturally produced, endogenous peptide, we measured Aß levels in the neuronal conditioned media by ELISA. Neocortical cells from two GK and two WT postnatal day 0 litters plated at a density of 40,000 cells/cm2 were conditioned for 4 or 7 days. Compared to WT conditioned media, Aß X-40 levels in the GK neuronal media were increased 84% after 4 days (P < 0.0005) and 162% after 7 days (P < 0.005) (Figure 4A)
. Aß X-42 levels were also elevated in the GK neuronal media, increasing by 120% after 4 days (P < 0.0005), and 138% by 7 days (P < 0.05) (Figure 4B)
. In confirmatory experiments using a different ELISA system (see Materials and Methods), conditioned media were analyzed from primary neurons plated at 80,000 cells/cm2 (twice the density of the first set), and again, the GK media had highly significant elevations of both Aß X-40 (60% after 4 days, P < 0.005; 57% after 7 days, P < 0.01) and Aß X-42 (85% at 4 days, P < 0.001; 107% at 7 days (P < 0.05) (data not shown).
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. Aß X-42 values were below the level of detection in the media of cells transfected with either IDE allele.
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80% of the enzyme from each sample, as determined by immunoblotting (not shown) and removed 85% of the Aß-degrading activity from the WT samples and 80% from the GK samples (Figure 5B)
. Before IDE immunodepletion (pre-ID), WT and GK Aß degradation at 30 minutes was significantly different at 33% and 29%, respectively (P < 0.001), whereas after IDE removal (post-ID), residual Aß degradation was identical at 5.2% (P = 0.49). Thus, the consistent difference observed in Aß proteolysis between the GK and WT brain samples was eliminated by specific removal of IDE.
To determine whether the IDE-mediated deficit in Aß degradation that elevates Aß in neuronal cultures also increases steady-state levels of Aß, in the brain, we quantified levels of the peptide in GK and WT brains by ELISA. Cerebral Aß was extracted using two separate protocols, which yielded three distinct pools of Aß. The diethylamine protocol, which extracts the aqueous soluble and a portion of the aqueous insoluble Aß, was performed on one cerebral hemisphere from each of 34 rats divided into two age groups. The other hemisphere from a subset of these animals (19 rats) was first extracted in TBS to acquire the soluble Aß, and then the TBS-insoluble material was extracted in guanidine HCl to obtain insoluble Aß. There were no statistically significant differences between GK and WT brain Aß X-40 and Aß X-42 levels in any of the three types of extracts at either 5 to 7 months or 10 to 14 months of age (Table 1)
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15% relative to WT, which is in good agreement with the data presented above. |
Discussion |
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Although the relatively small (15 to 30%) decrement in Aß proteolysis led to considerable increases in endogenous Aß levels in neuronal cultures, we detected no quantifiable rise in steady-state levels of the peptide in brains from rats up to age 14 months. In the intact brain, microglia,6,7
astrocytes,53
and transport into the circulation could each play roles in Aß clearance,54
and these may compensate for the relatively small decrease in IDE function. This is in sharp contrast to full loss of IDE function by homozygous deletion of the gene in mice, which was found to lead to a 65% elevation of endogenous cerebral Aß40 levels by 3 months of age.17
Combining the results from these two different animal models strengthens the hypothesis, based on the recent human genetic data that IDE mutations could serve as a risk factor for late-onset AD. Whereas the gene-deleted mice provide proof-of-principle that IDE dysfunction can significantly elevate cerebral Aß levels in vivo, the GK rats demonstrate that naturally occurring, subtle loss-of-function mutations in IDE are physiologically relevant (decreasing the catabolism of Aß in intact neurons), but not so severe as to cause significant elevations of rodent Aß in the brain during the 14-month life span of our rats. In late-onset AD patients, the imbalance between rates of Aß production and clearance is such that the presymptomatic accumulation of cerebral Aß occurs very slowly throughout decades, and the symptomatic phase of the disease is measured in years. If IDE turns out to be a genetic risk factor for late-onset AD, the magnitude of IDE hypofunction in humans would be expected to resemble that of the GK rat more than that of the IDE gene-deleted mice. It is possible that a modest decrease in neuronal Aß clearance would only lead to detectably elevated cerebral Aß levels after many years. We speculate that a subtle decrease in IDE function resulting in elevated levels of monomeric Aß could lead to more accumulation of Aß in humans than rodents, because the human peptide is far more prone to form stable aggregates that resist degradation by proteases than is the rodent form.
We have extended the finding that muscle fibers from rats with the GK allele degrade insulin less well than controls40 by showing that these diabetic rats also have a deficit in insulin proteolysis in primary fibroblasts, brain fractions, and intact neurons. The consequences of having chronic insulin-degrading deficits in the brain are unclear at present, but there is mounting evidence that insulin plays numerous roles in the central nervous system.55 When Fakhrai-Rad and colleagues40 transfected COS cells with the GK and IDE alleles, they were able to detect the GK insulin-degrading defect in intact cells, but not in lysates of the same cells. They hypothesized that the IDE defect is coupled to receptor-mediated internalization of insulin or other activities that require intact cell structures. In contrast, we clearly detected the GK IDE defect not only in untransfected intact fibroblasts and neurons, but also in tissue fractions, providing the simpler explanation that the IDE missense mutations directly decrease the ability of the protease to cleave insulin, independent of other processes. Furthermore, we have begun to characterize the kinetic effects of these insulin- and Aß-elevating mutations, showing that they lower the Vmax without changing the Km of the protease, resulting in a modest but significant decrease in catalytic efficiency.
By demonstrating faulty Aß degradation in a well-characterized animal model of DM2, our findings are directly relevant to emerging evidence that diabetes and, in particular, hyperinsulinemia, are associated with a heightened likelihood of developing AD. It is well known that DM2 is a risk factor for vascular diseases, including vascular dementia, but the relationship between the occurrence of DM2 and AD has only recently been addressed in large-scale epidemiological studies. Two of these studies in which other causes of dementia were carefully excluded showed that individuals with DM2 were approximately twice as likely to develop a clinical diagnosis of AD compared to normals, independent of vascular factors,56,57 whereas a third group found that diabetic patients had a 60% increased risk of developing either frank AD or mild cognitive impairment (a clinical state frequently progressing to AD).58 Another population-based study of Japanese-American men reported no association between AD and midlife diabetes,59 but a more recent report based on the same cohort concluded that diabetic patients’ relative risk of AD was 1.8 and that this increased risk was not related to vascular disease.60 The latter report hypothesized that the small number of diabetic patients included in first analysis limited its ability to find an association between DM2 and AD, but as the study population aged, the prevalence of DM2 more than doubled, giving the second study more power. Although it is not clear from the above epidemiological studies which particular aspect of the DM2 phenotype (eg, hyperglycemia, hyperinsulinemia) is most associated with an increased risk of AD, elevated plasma insulin levels in nondiabetic patients have been linked to dementia61 even after adjustment for vascular disease, and specifically AD.62,63 Supporting a role for IDE in these findings is evidence that the exaggerated insulin response to administered glucose seen in AD patients is secondary to an insulin clearance defect, rather than to overproduction of the hormone.64
Thus, although the mechanisms underlying the associations of DM2 and hyperinsulinemia with AD are not yet clear, the demonstration that naturally occurring partial loss-of-function mutations in IDE affect the catabolism of both insulin and Aß, combined with the genetic linkage of both DM2 and AD to the IDE region of chromosome 10q, recommend IDE hypofunction as an intriguing explanation. Our results have attendant therapeutic implications in that compounds that subtly increase IDE activity, either directly or via blocking a natural IDE inhibitor, could chronically decrease Aß levels in the human brain.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grant AG12479 to W.F., S.M., M.A.L., D.J.S.), the Mayo Alzheimer’s Disease Research Center (to C.E. and E.E.), the Alzheimer’s Association (to C.E. and E.E.), and the Smith Fellowship (to E.E.).
Accepted for publication December 15, 2003.
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 Papers of Note Sci. Aging Knowl. Environ., March 31, 2004; 2004(13): nw12 - nw12. [Full Text]
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