AMYLOID  PLAQUE ( AND  NOT  DIFFUSE  AMYLOID )  IS  A CONDITION  FOR  NEURONAL  DYSFUNCTION

Alexei R. Koudinov,^a,CA Temirbolat T. Berezov^a and Natalia V. Koudinova^a,b

^a Berezov Academic Laboratory, Russian Academy of Medical Sciences, Timoshenko St., 38-27, Moscow, 121359 Russia;
^b Weizmann Institute of Science, Department of Biological Regulation, Rehovot, 76100 Israel.

First submitted: December 23, 1999, Published online: December, 2001

ABSTRACT INTRODUCTION EXPERIMENTAL  PROCEDURES RESULTS AND DISCUSSION: A.IMMUNOHISTOCHEMISTRY FIGURE 1: IMMUNOHISTOCHEMISTRY AND BIOCHEMISTRY:

RESULTS AND DISCUSSION: B.ELECTROPHYSIOLOGY ELECTROPHYSIOLOGY: FIGURE 2,  FIGURE 3 AND FIGURE 4 FOOTNOTES AND ACKNOWLEDGMENT REFERENCES, LETTERS TO EDITOR AND SUPPLEMENT MISCELLANEA AND KEY TAKE-HOME MESSAGES

Todate, it is not established whether the maturation of brain amyloid deposits, particularly the development of Congo Red positive neuritic plaques, is an essential event leading to neuronal dysfunction. Recent study by Naslund et al. [ 8 ] attempted to correlate the amyloid load with the cognitive decline and the severity of dementia in Alzheimer disease patients. However, the latter report estimated 55% and 40% of the study subjects without detectable plaques as the ones having readily detectable  levels of  "Ab(x-42) and Ab(x-40)" . In addition, recent reports have suggested the possible importance of factors other then brain amyloid in Alzheimer's neuronal abnormality (oxidative stress [ 9WEB+ ] or lipid metabolism [ 10WEB+, 11 ] disbalance, for example) and behavioral disturbances without amyloid deposits in mice overexpressing human APP with Flemish and Dutch mutations [ 12 ], keeping open the question on the role of neuritic plaques in the genesis of Alzheimer's neuronal dysfunction.

In our study, we utilized hippocampal slices of 16.5 and 25.5 month old transgenic mice expressing nonmutated human APP₆₉₅ [ 13 ], and age-matched non-transgenic wild type control  mice. We aimed to differentiate the separate actions of diffuse and plaque amyloid on hippocampal synaptic plasticity using immunohistochemical analysis, Congo Red staining, amyloid extraction and extracellular recording of CA1 field excitatory postsynaptic potentials ( fEPSPs ). The results indicate that amyloid plaque (and not diffuse amyloid) may represent one of the possible causes of neuronal dysfunction and synaptic plasticity failure.

Animal care and tissue collection

Transgenic and wild type mice of 16.5 and 25.5 months age groups  (n=6) were maintained on the standard diet at the animal facility of the Weizmann Institute of Science, Rehovot, Israel.  All experimental procedures were in accord with National Institutes of Health guide for the use of laboratory animals. Two or three hippocampal slices from each mouse were subjected to both electrophysiology and to immunohistochemistry.

Additionally, two transgenic and two wild type mice at the age of 16.5 months were subjected to transcardial perfusion, followed by thin sectioning of the brain and immunohistochemical analysis. In selected experiments, the hippocampal slices of 16.5 month old  transgenic and wild type mice  were subjected to the biochemical analysis of Ab.

Ex-vivo hippocampal slices

Hippocampal slices were prepared and electrophysiological analysis was performed essentially as we described previously [ 10, 14 ]. Briefly,  after  mice decapitation the hippocampus was rapidly removed and placed into cold (2⁰C) artificial cerebrospinal fluid (ACSF, pH 7.4 containing (in mM): 124 NaCl, 2.0 KCl, 1.24 KH₂PO₄, 2.0 MgSO₄, 2.5 CaCl₂, 26 NaHCO₃ and 10 D-glucose) saturated with 95 % O₂ / 5% CO₂ (flow rate 0.4 l/min) and adjusted with sucrose (7g per 600 ml of ACSF) to 320 mOsm osmolarity. The hippocampal slices  (400 m) were prepared with a McIlwain tissue slicer, USA. The slices were incubated in a recreation chamber at room temperature (25⁰C) for 1.5 h in ACSF.

Electrophysiology

The slices were transferred to a recording chamber (held at constant temperature of 32⁰C) and submerged slices were superfused with ACSF at a flow rate of ~1.5 ml/min.  Extracellular electrodes (~4 MW, 0.75 mM NaCl) were guided by micromanipulator into stratum radiatum of CA1 (200 m deep) under binocular. Bipolar (A. Koudinov fabricated) tungsten (50 m wire size) stimulating electrode was also placed into CA1 stratum radiatum. The stimulations were delivered every 30 s at 50 ms pulse duration yielding fEPSP waveforms. After stable baseline responses were established and input /output (I/O) curve values recorded, tetanus induced LTP was induced by delivering a 100 Hz, 1 sec stimuli train through the stimulation electrode at the baseline test stimulus intensity. For long term depression (LTD) study slices were stimulated every half a second (2 Hz) for 5 min at the test stimulus intensity.

Data were collected, stored and analyzed on a PC using Asyst 3.1 and GraphPad Prizm 2.0 data acquisition and analysis software. The I/O relationship, LTP and LTD were expressed as a fEPSP amplitude and slope change versus stimulus intensity and time, respectively. Data were normalized with respect to the steady baseline values and expressed as mean±SEM. Non-parametric unpaired Mann-Whitney test was used for determining significant differences between potentiation/depression levels of trangenic and wild type slices at the indicated time points. A probability of 0.05 (one tailed) or less was accepted as statistically significant.

Immunohistochemistry and Congo Red staining for amyloid

The hippocampal slices from 25.5 month old mice used for synaptic plasticity study, were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS), pH=7.4 for 72 hrs [ 10, 14 ]. We also used hippocampal sections from 16.5 month old mice killed by transcardial perfusion with PBS and 4%  paraformaldehyde in PBS [ 11, 14 ]. For immunohistochemistry, 400 m-thick fixed slices were cutted with a microtome  to a 40 m  sections. Free floating sections were washed in PBS (2x) and then incubated with 3% H202 (prepared on PBS, containing 10% methanol) for 20 min to remove endogenous peroxidase activity. Sections were then washed (4x 10 min each) with PBS and blocked for 24 hrs at 4⁰C in 10% fetal calf serum and 1% glycine in PBS (blocking solution), followed by 14 hrs incubation with 4G8 or 6E10 (1:1000, Senetek, PLC.) monoclonal anti-Ab antibodies in the blocking solution at 4⁰C. Tissue sections were washed (5x 40 min each) in blocking solution to remove unreacted primary antibodies. Secondary biotinylated goat anti-mouse antibody (1:750) were added for 1.5 h at 25⁰C, followed by washing with the blocking buffer (3x 40 min each).  After washing, ABC solution (Vector Elite kit, 1:300 of reagents A and B in blocking solution) was added for 40 min, followed by section washing in blocking solution and then in PBS (2x 10 min each). For visualization, immunostained sections were reacted with 3',3-diaminobenzidine tetra-hydrochloride (Sigma Tablet kit, USA) and washed in PBS.

For Congo Red staining thin sections were stained with 0.5% Congo Red in 50% alcohol for 30 min, followed by one minute treatment with 0.2% KOH in 80% alcohol and water. Sections were mounted on gelatinized slides, air dried, dehydrated in serially diluted ethanol (50, 70, 90, 95 and 100%), cleared with Xylenes, and coverslipped with cover glass and Permount, USA.

To control the specificity of 4G8 and 6E10 immunostaining, antibody solutions were preadsorbed with the access of synthetic peptide Ab1-40 (1 mg /0.5 ml) prior to the incubation with the sections.

Fluorescence of plaque-like amyloid labeled by Congo Red was obtained with 488 nm of excitation using a confocal microscope LSM 510 (Zeiss, Germany) equipped with an argon laser [ 1S ].

Amyloid extraction

Hippocampal slices  were homogenized with the cold PBS (0.5 ml per 20 mg of tissue) containing protease inhibitors [ 15 ], and subjected to centrifugation in a Beckman TiL 100.2 rotor at 100,000 g for 3 hrs at 4⁰C. The supernatant fraction and the pellet were dialyzed against water with 1,000 Da cut-off membrane (Spectrum, USA), lyophilized and subjected to Ab extraction with 20% and then with 80% acetonitrile in 0.1%  trifluoroacetic acid [ 16 ]. Both acetonitrile soluble fractions were combined, lyophilized and subjected to 13% TRIS-Tricine SDS/PAGE [ 17, 18 ] and immunoblot analysis on Immobilon P membranes (Wattman, USA) with 6E10 and 4G8 anti-Ab monoclonal antibodies and with the monoclonal antibody against APP (Zymed, USA), followed by ECL (Amersham) essentially as previously reported [ 15, 18 ].

Immunohistochemistry of slices from aged animals (25.5 months) with 4G8 and 6E10 antibodies (anti-human/mouse-Ab and anti-human-Ab, respectively, see antibody specificity scheme, Figure 1G ) revealed extracellular (staining with no triton) hippocampal immunoreactivity of mouse Ab in transgenic mice (Fig. 1A) and in wild types (Fig. 1B), and verified extracellular deposits of human Ab in the transgenic mice hippocampus (Fig. 1C). Essentially identical results were obtained under the condition of membrane permeabilization with 0.1% Triton X-100 in the blocking buffer (not shown). The specificity of immunostaining was confirmed by the preadsorption of antibodies with the access of synthetic peptide Ab(1-40).

Staining for amyloid showed Congo Red fluorescence specifically in aged transgenic mice  (and not in aged wild type mice) hippocampal sections (Fig. 1E). Congo red stains specifically amyloid plaques (but not diffuse amyloid) due to the binding to the b-pleated sheet secondary structure of Ab protein in amyloid fibrils [ 1, 2 ]. The latter observation indicates that expression of non-mutated human Ab in aged transgenic mice leads to a mature Alzheimer's plaque-like amyloid and that  Ab deposits in wild type mice have a nonmature diffuse nature.

In contrast to the 25.5 month aged animals, the 16.5 month old transgenic  and wild type mice expressed neither human nor mouse Ab immunoreactivity (not shown). To confirm this observation we analyzed 16.5 month old transgenic and wild type mice for soluble and aggregated Ab by immunoblot analysis of the acetonitrile extracts of ultracentrifugation supernatant- and pellet-fractions of hippocampal homogenates. We did not recover human transgenic (Fig. 1H) or mouse (not shown) Ab immunoreactivity in the transgenic and wild type hippocampus. However, anti-human Ab antibody 6E10 labeled a protein of high molecular weight (in the range of 96 to 200 kDa) in the trangenic mice. This protein band was also stained with the antibody against APP aminoterminus (not shown) confirming human APP expression in the transgenic hippocampus.

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FIGURE 1 
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Immunochemical analysis of APP transgenic mice.
A-D, Comparison of extracellular Ab immunoreactivity in aged (25.5 months) human non-mutated APP₆₉₅ transgenic mice and age-matched non-transgenic wild type control mice hippocampus. Immunohistochemistry was performed with no triton and 4G8 and 6E10 monoclonal antibodies (1:1000).  The presented fields are CA1 areas of ex-vivo slices from the batches used for extracellular recording;  Bar, 20 m. Similar Ab pattern was observed in the dentate gyrus (not shown). Alzheimer's-like plaque amyloid in transgenic mice (E), versus wild type mice (F), was visualized by confocal fluorescent microscopy of Congo Red stained sections; Bar, 100 m. (H) 16.5 month old wild type (control, lanes 1 and 2) and transgenic mice (lanes 3 and 4) were analyzed for the soluble and/or aggregated human Ab in the acetonitrile extracts of the supernatant (lanes 1 and 3) and pellet (lanes 2 and 4) fractions of the hippocampal homogenates, respectively, by immunoblot analysis with 6E10 and 4G8 (not shown) monoclonal antibody. While there were no detectable levels of Ab present in transgenic hippocampus, we recovered 6E10-positive high molecular weight human APP immunoreactivity in the supernatant fraction (lane 3), and confirmed it by immunostaining with the antibody against APP aminoterminus. Lane 5, 5 ng of synthetic Ab1-40 (positive control). Molecular weight markers (in kDa) are shown on the left. Scheme (G) represents human and rodent Ab1-40 amino acid sequence differences and the sequence specificity of anti-Ab monoclonal antibodies used in this study.

Electrophysiological analysis revealed that 25.5 month old transgenic mice had lower input-stimulus/output-response (I/O) characteristics (Figure 2A). It is generally accepted that in the CA1 area of the hippocampus EPSP consists of two major elements that depend on the activation of N-methyl-D-aspartate (NMDA) and non-NMDA (mainly AMPA) subtypes of ionotropic glutamate receptors [ 19WEB+ ]. The 50 ms stimulation pulse duration (employed here to evoke fEPSP) generates significant AMPA responses and it is thus possible that this particular component of the EPSP is responsible for lower I/O characteristics of aged transgenic mice [ 19WEB+ ].

Aged transgenic mice were impaired in both the amount of initial post-tetanic potentiation (137.2±9.3%, n=9, against 201.8±20.8% of the wild type, n=10, p=0.014) and in the maintenance of the LTP, sampled 4 smin (99.53±9.78%, n=9, and 148.6±15.01%, n=10, p=0.0318) and 10 min (92.64±7.02%, n=9, and 133.2±9.29%, n=10, p=0.0357) after the tetanic stimulation. There  were no significant differences in induction (p=0.2403) and maintenance (p=0.0649) of the LTP in wild type slices taken from 16.5 and 25.5 months old mice (Fig. 2 and Figure 3A) despite the development of diffuse mouse Ab deposits in wild type hippocampus over the indicated age (indicating the lack of importance of diffuse amyloid for synaptic plasticity failure). On the other hand,  transgenic slices expressing  plaque-like amyloid at age 25.5 months showed a significant decline in both induction (p=0.0047) and maintenance (p=0.0048) of the LTP compared to the 16.5 month old mice.

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FIGURE 2 
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Electrophysiological analysis of aged (25.5 months) APP transgenic mice
(A) Input/output (I/O) relationship in APP transgenic (squares) and wild type non-transgenic (WT, triangles) hippocampal slices. (B) Field synaptic responses obtained at the baseline (1) and high stimulus intensity (2) recordings as well as immediately after (3) and 3 (4) and 13 min after (5) the high-frequency train of stimuli. (C) Impairment of tetanic LTP in CA1 of ex-vivo slices from APP transgenic versus WT hippocampus. In all cases n=9 and n=10 slices for APP transgenic and WT mice, respectively. Arrow indicates time of tetanus.

In contrast to the aged 25.5 month old mice, transgenic mice of the younger age (16.5 months) expressed no amyloid and did not differ from the wild type mice in the induction and maintenance of the LTP (Fig. 3A). There were, however, differences in the LTD (Fig. 3B), another important parameter of neuronal plasticity. It was shown previously [ 20 ] that bath application of the soluble APP (100 nM, 1 h) adsorbed the ability of rodent hippocampal slices to maintain LTD. It is thus conceivable that mild modulation of the hippocampal LTD in adult transgenic mice (16.5 months) is due to the human APP expression in the transgenic mice hippocampus (Fig. 1H). Another study by Larson et al. [ 21 ], however, suggests a more striking modulation of hippocampal physiology by human mutant (V717F)APP in transgenic mice at age 4-5 months. Although the experimental protocol of this report (specifically, maintaining slices at 36⁰C; differences in the media recipe, particularly including ascorbate, known to modulate EPSP [ 22 ] in ACSF) does not match the one employed here and in the above cited report by Ishida et al. [ 20 ], it indicates that  APP mutations (yet representing very small cohort of all Alzheimer disease cases) may exacerbate additional abnormalities in synaptic and behavioral plasticity "prior to the formation of amyloid beta peptide deposits." The transgenic mice that we used in this study mild overexpress non-mutated human APP and in our view offer more relevant system  to model non-mutated human APP expression [ 13 ].

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FIGURE 3 
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Electrophysiological analysis of 16.5 month old  APP transgenic mice

APP transgenic and wild type non-transgenic (WT) mice expressed similar tetanus (arrow) induced LTP (A) and were different in the amount of the long term depression (B). However, depression values probed 10 min after the low frequency stimulation sequence did not reach statistical significance (98.95±10.34%, n=7, and 79.3±12.8%, n=6 in the APP-TG and WT, respectively, p=0.0625, one-tailed).

Two major components contributing to the tetanus induced LTP are NMDA LTP and non-NMDA (dependent on a rise in intracellular calcium concentration) LTP [ 23WEB+ ]. Fast onset NMDA- and developing slow non- NMDA-LTP can be isolated by using a specific tetanic stimulation paradigm in the presence of 30 mM nifedepine, a blocker of voltage-gated calcium channels, and 25 mM D,L-2-amino-5-phosphonovaleric acid, an NMDA antagonist, respectively [ 23WEB+, 24 ]. Moreover, another type of slow onset LTP was described, a muscarinic  LTP, which can be evoked by the application of 0.25-2.0 mM carbachol in the absence of tetanic stimulation [ 23WEB+, 24 ]. Non-NMDA-LTP and muscarinic LTP share similar lack of expression in adult transgenic mice expressing human Cu/Zn-superoxide dismutase, SOD1 [ 24 ] in very old (24-30 months) and in adult Wistar rat hippocampal slices treated with low (~30 mM) dose of H₂O₂ [ 23 ].

Regardless of the deficit of specific receptor machinery hippocampal slices from aged transgenic mice may be different from wild type controls in their ability to regulate second messenger pathways and/or generate the action potential. Thus,  transgenic mice may be impaired in the phosphorylation of the nuclear cAMP responsive element binding protein (abbreviated as CREB), modulated by micromolar concentrations of Ab [ 25 ] and known as a necessary event in neuronal plasticity [ 26 ]. Transgenic mice may be also impaired in metabotropic glutamate receptors (mGluR) and associated second messenger machinery, activation which was shown to be coupled to the APP processing [ 27 ] and is essential for the priming of the LTP [ 28 ]. It is also possible that in transgenic  mice neurons are depolarized relative to the control slices, yielding reduction of their fEPSPs and impairment of their ability to express larger fEPSPs following tetanic stimulation.

Finally, transgenic mice may have deteriorated neuronal spike propagation machinery due to the tunneling amyloid plaques (Figure 4A). This is supported by the study on the disruption of neural networks in Alzheimer's disease [ 29 ]. This report modeled the electrophysiological effect of the changed neuronal processes that cross through Ab plaque deposit and foretold the delay of several milliseconds over an average plaque. Our comparison of individual fEPSPs revealed this predicted change of few milliseconds in initial post-tetanic traces in  transgenic versus wild type slices (Fig. 4B), confirming importance of plaque amyloid for neuronal spike propagation.

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FIGURE 4 
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Schematic matching of the positioning of the recording (rec) and stimulating (stim) electrodes

for the employed in the study extracellular recording in the CA1 and the Congo Red fluorescence, observed in the APP transgenic hippocampal slice (A). (B) Individual fEPSPs recorded after the high frequency (100 Hz/1 s) train of stimuli revealed 1.5-2.0 msec delay in the onset of the evoked synaptic responses in the APP transgenic (arrow) compared to the wild type non-transgenic slices.

Our data provide evidence that one of the possible causes of Alzheimer's-like neuronal dysfunction and synaptic plasticity deficit is senile plaque (and not diffuse) amyloid.  Our data are in dispute with the paper in JAMA [ 8 ] and the accompanying commentary [ 30WEB+ , see also 31WEB+ ] proposing that Ab peptide not incorporated into histologically visible plaques is an early neurochemical hallmark of dementia [ 2S, 3S ].

Our results also suggest that in Down syndrome (characterized by diffuse amyloid deposition in early life) and in pre-plaque stages of Alzhemer disease, the other factors (such as brain cholesterol and other lipid metabolism misregulation [ 10WEB+, 11 , LE1, LE2, LE3, LE4, LE5 ] or oxidative stress disbalance [ 9WEB+, 24 ]) may contribute to the neuronal and behavioural [ 2S, 3S ] dysfunction.

The precise molecular mechanism of amyloid-plaque-mediated synaptic plasticity deficit, however, remains to be investigated.

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ACKNOWLEDGMENT 

While this paper was under submission another key contribution on this subject was published in December 2000 [ 4S ]. We are indebted to Sh. Halav and J. Hermesh for excellent animal care. See Ref. 10 for support and funding itemization. The preliminary account of this report has been published in the abstract form and presented at the 10th Meeting of the European Neurological Society, 18-21 June 2000, Jerusalem, Israel [ 5S ], and at World Alzheimer congress 2000, Washington, DC, July 9-12, 2000 [ 6S ]. The earlier version of this article was first submitted as research letter (for letter text see [ 5S ]) during December 23, 1999. In the present form this contribution was submitted to Science, J Neurosci and to Neurosciences. Our research is dedicated to our parents.

MISCELLANEA 

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THIS ARTICLE IS BASED ON: Authors near 10 years expertise on the synthesis of research on soluble Ab, lipids and Alzheimer's disease, and 5 years of basic neuroscience research. 31 web references, 18 WEB+ citations, plus supplementary references.

KEY TAKE-HOME MESSAGES  Todate, it is not established whether  the maturation of brain amyloid deposits, particularly the development of amyloid neuritic plaques, is an essential event leading to neuronal dysfunction in Alzheimer's patients. Our data provide evidence that amyloid plaque (and not diffuse amyloid) impairs neuronal function and synaptic plasticity. Our current results and recent discussion suggest that in pre-plaque stages of Alzhemer disease and in Down syndrome other factors (such as brain cholesterol/lipid metabolism misregulation or oxidative stress disbalance) have primary causative role for the development of neuronal dysfunction. Amyloid plaque buildup is not causative for the sporadic Alzheimer's disease and unlikely represents the proper target for Alzheimer's therapy.

FOOTNOTES   CACorresponding author: Alexei R. Koudinov, M.D., Ph.D., P.O.Box 1665, Rehovot 76100 Israel; Tel:  (972 8) 947-1803; Fax: (972 8) 934-4116; E.contact: amyloidbeta@hotmail.com ; koudin@imb.ac.ru ; Internet Office ABBRAbbreviations: AD, Alzheimer's disease; APP, amyloid precursor protein; Ab, amyloid b protein; DS, Down syndrome; LTP, long-term potentiation; LTD, long-term depression; NMDA,  N-methyl-D-aspartate;  PBS, phosphate buffered saline;  SOD, superoxide dismutase; TG, transgenic mice; WT, wild-type non-transgenic mice. Keywords: Alzheimer's disease, amyloid beta protein precursor, cholesterol, Down syndrome, etiology, extracellular recording, EPSP, hippocampus, learning, lipid, long term potentiation and depression, LTP, LTD, memory, neurodegeneration marker, neurofilament, oxidative stress cascade, PHF NFT tau phosphorylation, phospholipids, plaque, secretase, synaptic plasticity, therapy

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LE1. Koudinov AR, Koudinova NV. Alzheimer's pathogenesis: tau and amyloid - a consensus or a challenge for a third party quest ? Br Med J. E.letter published online September 4, 2001 [ Read the letter ].

LE2. Koudinov AR, Koudinova NV. Cholesterol, synaptic function and Alzheimer's disease. Br  Med J. E.letter published online October 16, 2001 [ Read the letter ].

LE3. Koudinov AR, Koudinova NV. Dementia, cholesterol and the soft science of dietary fat. Br  Med J. E.letter published online July 27, 2001 [ Read the letter ].

LE4. Koudinov AR, Koudinova NV. Looking forward for a historic issue, or: Is there anything besides amyloid? Br  Med J. E.letter published online October 19, 2001 [ Read the letter ].

LE5. Koudinov AR, Koudinova NV. Cholesterol supply and synaptic plasticity. Submitted to Science  on November 9, 2001 [ Read the letter ].

LE6. Koudinov AR, Koudinova NV. Cholesterol misregulation causes Alzheimer's disease: diet likely involved. Br  Med J. E.letter published online December 1, 2001 [ Read the letter ].

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5S. Koudinov AR, Berezov TT. Alzheimer's amyloid plaque formation is a condition for neuronal dysfunction. 10th European Neurological Society Meeting, 18 June, 2000, Jerusalem, Israel. Abstract book. P534 (2000) [ Abstract ].

6S. Koudinov AR, Koudinova, NV, Berezov TT. Is Alzheimer's amyloid plaque formation a condition for neuronal dysfunction? Neurobiol. Aging 21, S155 (2000) [ Abstract ].
 

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Citation example for this article: Koudinov AR, Berezov TT, Koudinova NV. Amyloid plaque (and not diffuse amyloid) is a condition for neuronal dysfunction. Clin. Med. Health Res. published online December, 2001, clinmed/2001110002, http://clinmed.netprints.org/cgi/content/full/2001110002v1 You may also wish to cite year 2000 preliminary accounts of this article [ 5S or 6S ].