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Human Molecular Genetics, 2003, Vol. 12, No. 24 3269-3276
DOI: 10.1093/hmg/ddg356
© 2003 Oxford University Press

Abnormalities of the vitreoretinal interface caused by dysregulated Hedgehog signaling during retinal development

Graeme C.M. Black1,2,3,{dagger}, Chantal J. Mazerolle4,{dagger}, Yaping Wang4, Katrina D. Campsall4, Dino Petrin5, Brian C. Leonard5, Karim F. Damji5, D. Gareth Evans1, David McLeod2 and Valerie A. Wallace4,5,*

1Academic Unit of Medical Genetics and Regional Genetic Service, St Mary's Hospital, Hathersage Road, Manchester, UK, 2Academic Department of Ophthalmology, Manchester Royal Eye Hospital, Oxford Road, Manchester, UK, 3Centre for Molecular Medicine, Stopford Building, Oxford Road, Manchester, UK, 4Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada and 5University of Ottawa Eye Institute, 501 Smyth Road, Ottawa, Ontario, Canada

Received July 10, 2003; Accepted October 6, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in Patched (PTCH), encoding the Hedgehog (Hh) receptor, underlie Basal Cell Naevus syndrome (BCNS) and, in addition to tumor predisposition, are associated with a wide range of ‘patterning’ defects. The basis for the underlying patterning problems in Hh-dependent tissues in BCNS and their long-term consequences on tissue homeostasis are, however, not known. Hh signaling is required for normal growth and organization of the mammalian retina and we show that PtchlacZ+/- mice exhibit vitreoretinal abnormalities resembling those found in BCNS patients. The retinas of PtchlacZ+/- mice exhibit abnormal cell cycle regulation, which culminates in photoreceptor dysplasia and Müller cell-derived gliosis. In BCNS, the intraretinal glial response results in epiretinal membrane (ERM) formation, a proliferative and contractile response on the retinal surface. ERMs are a cause of significant visual loss in the general, especially elderly, population. We hypothesize that alteration of Müller cell Hh signaling may play a role in the pathogenesis of such age-related ‘idiopathic’ ERMs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Basal Cell Naevus syndrome or Gorlin syndrome (BCNS, MIM#109400) is an autosomal dominant disorder characterized by birth defects (dental, skeletal and radiographic abnormalities including Falx calcification, bifid/fused ribs and altered vertebral segmentation), as well as a predisposition to tumor development [including early-onset basal cell carcinomata (BCCs), medulloblastoma (~5%) and rhabdomyosarcoma]. BCNS results from mutations of the PTCH gene on chromosome 9q23.1 (1,2). The human homologue of the Drosophila patched gene, PTCH is a transmembrane protein that functions as the receptor for members of the Hedgehog (Hh) family of intercellular signaling molecules (3,4). Sonic hedgehog (Shh), the most widely represented member of this family, is required for many aspects of developmental patterning in the vertebrate embryo (5).

Ptch normally functions to block Hh signaling by antagonizing the activity of Smoothened, a seven domain transmembrane protein that is required for transmission of the Hh signal to the nucleus via cubitus interruptus/Gli transcription factors; this results in the activation of target genes including Ptch and Gli (6–11). The developmental abnormalities observed in BCNS patients, and in mice that are heterozygous for the Ptch gene (PtchlacZ+/- mice) (12,13), indicate that Hh pathway activation is sensitive to Ptch gene dosage. Mutations in PTCH have also been documented in sporadic BCCs and medulloblastomas, indicating that the gene also functions as a tumor suppressor (14).

Of the various developmental abnormalities described in BCNS, the ocular features remain poorly characterized. Estimated to be present in between 15 and 25% of patients (15,16), previous reports include defects of organogenesis (microphthalmia, coloboma), as well as both anterior segment (cataract) and posterior segment abnormalities, the latter including inappropriate retinal myelination and retinoschisis (abnormal splitting of the retina) (17–20). Hh pathway activation is known to play a role in mammalian visual system development. Shh is expressed in the retinal ganglion cells (RGC), the first neurons to differentiate in the retina, and Ptch and Gli are expressed in retinal neuroblasts, as well as astrocyte precursor cells in the optic nerve (21–23 and summarized in Fig. 1B). RGC-derived Shh expression is required for Hh target gene induction in the retina and optic nerve and plays a role in precursor cell proliferation, photoreceptor differentiation and normal cellular organization in the rodent retina (21–25).



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  		Figure 1. General eye anatomy and summary of Hh pathway gene expression in the retina. (A) Adult mouse eye stained with hematoxylin and photographed at low magnification. RPE, retinal pigment epithelium; NR, neural retina; ON, optic nerve; per and cen refer to the peripheral and central retina, respectively. (B) Diagram of the developing and adult mouse retina. At late stages of embryogenesis, the retina consists of two layers, the retinal ganglion cell (RGC) layer and the neuroblast (NB) layer, which contains proliferating precursor cells. RGC axons are located on the surface of the retina and exit the eye at the optic disc to form the optic nerve. The adult retina is organized into three cellular layers: RGC, inner nuclear layer (INL), and the rod and cone-containing outer nuclear layer (ONL). The nuclear layers are separated by the inner and outer plexiform layers (IPL, OPL), which contain neuronal processes. Müller cells, radial-type glial cells, have processes that span the width of the retina from the RGC to the ONL and cell bodies that are located in the middle of the INL. In the embryonic and adult retina Shh is expressed in RGCs and Ptch is expressed by dividing precursor cells in the neuroblast layer (embryonic) and Müller cells (adult). The diagram on the far right depicts a photoreceptor rosette in the ONL, and an epiretinal membrane (ERM) at the vitreoretinal interface where the invasion of Müller cell processes into the vitreous is associated with the recruitment of contractile cells, which in some instances can pull on the retina causing distortion of the blood vessels and retinal detachments.

 
Given the importance of the Hh signaling pathway in eye morphogenesis and retinal development, we reasoned that dysregulation of this pathway caused by haploinsufficiency for a key regulatory component, the Ptch receptor, could cause ocular defects. We studied 30 patients with BCNS and documented a wide range of ocular abnormalities. Amongst these were defects of retinogenesis including fibroglial epiretinal membrane (ERM) formation (Fig. 1B) and abnormal ganglion-cell axon myelination. To understand the basis for these retinal abnormalities in BCNS patients, we undertook a histological analysis of the retinas of PtchlacZ+/- mice (12). Approximately 50% of adult PtchlacZ+/- mice exhibited dysplastic foci in the retina that were associated with an abnormal Müller cell-derived gliotic response. Analysis of perinatal PtchlacZ+/- mice revealed ectopic proliferation and delayed differentiation. Our findings confirm a role for Ptch/Shh signaling in retinal histogenesis and implicate this pathway in glial cell homeo-stasis. Furthermore, our findings indicate that there is an underlying developmental basis for ERM formation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Abnormal retinal phenotypes associated with Basal Cell Naevus syndrome
Thirty BCNS patients were examined and, in keeping with previous reports, we documented a wide range of ocular abnormalities. The frequency and range of non-ocular manifestations, as previously described, was in keeping with those expected for BCNS (15). Since BCCs around the eye are a general feature of BCNS they were not included in this study. The ocular abnormalities included: squint (9/30), microphthalmos (1/30) and defects of both anterior segment development (Peters' anomaly 1/30, cataract 7/30) and posterior segment development. In total, 9/30 patients had early-onset unilateral visual reduction as a direct result of a structural ocular abnormality that could be attributed to BCNS. There were no cases of bilateral visual reduction.

Examination of the posterior segment revealed retinal and vitreoretinal abnormalities in 11 patients. The range of retinal and vitreous abnormalities is shown in Figure 2 and may be classified into two groups:



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  		Figure 2. Surface retinal anomalies in BCNS patients. Retinal photographs of normal (A) and BCNS patients (B–F). (A) Normal retina indicating the position of the optic disc (OD), the exit point for RGC axons and the entry point for the major retinal blood vessels (BV). The macula, the region of the retina required for high acuity visual tasks, is outlined by the dashed circle. (B) Retinal myelination is visible as an arcuate opaque region above the macula. Surface retinal (epiretinal) membrane formation results in wrinkling of the retinal surface and is visible as a fan of lines radiating from its epicenter (arrowed). (C) Discrete opaque nodule, consistent with astrocytic proliferation, embedded in the retina (arrowed n). As in several other cases, an associated epiretinal membrane was located close to retinal arteries (arrowed e). (D) Abnormal glial proliferation around optic disc. (E, F) Premacular isolated vitreous cysts. In (E) the cyst is situated close to the macula, while in (F), in a second patient the cyst was associated with a full-thickness retinal detachment at the macula (macular hole) (arrowed).

 
Inner retinal abnormalities.
ERM formation, which is manifest as a cellular proliferation in the surface of the retina, was observed in eight eyes and in six eyes there was evidence of retinal myelination (Fig. 2B). In a single eye the abnormalities at the interface between the retina and vitreous were associated with a discrete opaque nodule that was consistent with astrocytic proliferation (Fig. 2C). In other eyes there were focal retinal abnormalities located close to retinal arteries or at sites where retinal arteries and veins cross. Finally in one eye there was an extensive fibroglial ERM encompassing the optic nerve head associated with abnormal retinal vessels (Fig. 2D).

Developmental vitreous abnormalities.
Three eyes showed persistence of the fetal hyaloid system. In a further three eyes isolated vitreous cysts were present. In one, this was free floating in the vitreous humor. In two cases (Fig. 2E and F) the cysts were close to the macular region of the retina. In one case there was an associated full-thickness retinal (macular) hole (Fig. 2F). The site and appearance of the surface retinal pathology strongly suggested recruitment of contractile (fibroglial) elements. Furthermore they were focal and indicative of developmental disturbances.

Retinal dysplasia and gliosis associated with Ptch mutation in mice
The basis for these retinal abnormalities in BCNS patients is unclear, therefore we examined the retinas of mice that are heterozygous for the Ptch gene, PtchlacZ+/- mice (12). External ocular examination, fundoscopy and electroretinography detected no gross abnormalities in 8–12-week-old PtchlacZ+/- mice compared with wildtype littermates and age matched C57Bl/6 mice (data not shown). Histological analysis of retinas from adult (3–6 months) PtchlacZ+/- mice revealed that 50% (n=7/14) exhibited foci of dysplasia (including rosetting or clustering of photoreceptor nuclei around a central lumen as illustrated in Fig. 1B) involving photoreceptors in the outer nuclear layer (ONL) compared with 10% of controls (n=1/9) (Fig. 3B and C). Since these types of abnormalities are usually associated with gliosis or activation of the Müller glial cells (Fig. 1B), we stained companion sections with antibodies against glial fibrillary acidic protein (GFAP), which is normally not expressed by Müller cells, but is induced when they are activated. Our analysis revealed that the dysplastic regions contained reactive Müller cells, as indicated by exaggerated GFAP and glutamine synthetase (a Müller cell marker) staining (Figs 3B, C and 4A, B). In some cases the Müller cell gliotic response was confined to the inner and outer plexiform layers and was not associated with retinal dysplasia (Fig. 4A and B and data not shown). Dysplastic lesions were observed as early as postnatal day 21 (P21) in 4/7 of PtchlacZ+/- mice and in none of their littermates (n=6) (Fig. 4C and D); these dysplastic regions were more numerous and larger than those in adult PtchlacZ+/- mice, suggesting that they arose early and often resolved before adulthood. At P21 they were sometimes associated with an accumulation of photoreceptor cell bodies outside the outer limiting membrane (Fig. 4C and D); this was never observed in adult PtchlacZ+/- mice. GFAP staining associated with the dysplastic foci was weak or absent at P21, suggesting that the induction of Müller cell-derived gliosis that we observed in adult PtchlacZ+/- mice occurs as a consecutive or secondary response to the outer retinal dysplasia (Fig. 4C and D).



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  		Figure 3. Extensive dysplasia and gliosis in the retinas of adult PtchlacZ+/- mice. GFAP (A, B), glutamine synthetase (GS) staining, (C) and ß-galactosidase activity (blue stain) (D–F) in the retinas of adult PtchlacZ+/- mice. Retinal cross sections were stained with the indicated antibodies (brown) and nuclei were counterstained blue with hematoxylin. GFAP staining is normally confined to astrocytes located in the nerve fibre layer, the layer of RGC axons on the retinal surface (A), however gliosis is indicated by the extension of GFAP+ processes into the retina (B). GS staining, which marks Müller cells, of a companion section to (B) reveals that Müller cells are involved in the gliotic response. Nuclear staining with hematoxylin reveals foci of dysplasia of cells in the outer nuclear layer of the retina, note rosettes in (B) and (C). X-gal staining reveals that ß-galactosidase activity is reduced in dysplastic, red box in (D), compared with normal, green box in (D), regions of the retina. (E) and (F) represent higher magnification views of the areas in (D) indicated by the green and red boxes, respectively. Please see Figure 1 for a definition of the abbreviations.

 


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  		Figure 4. Dysplasia is an early feature in retinal histogenesis in PtchlacZ+/- mice. GFAP (A, C, D) and GS (B) staining in the retinas of postnatal day 21 (P21) PtchlacZ+/- mice. Retinal cross sections were stained with the indicated antibodies (brown) and nuclei were counterstained blue with hematoxylin. Staining of serial sections for GFAP and GS reveals the Müller cell contribution to gliosis in the inner nuclear layer [the region between the arrows in (A) and (B)]. (C, D) Examples of dysplastic lesions involving photoreceptor nuclei located in the outer nuclear layer. Note in (C) the extension of photoreceptor nuclei past the outer limiting membrane at P21, which was never observed in adult PtchlacZ+/- mice. The difference in the severity of the dysplasia in young versus old PtchlacZ+/- mice is consistent with the possibility that some of these abnormalities are corrected by cell death. The dysplasia at P21 was not always associated with gliosis, as indicated by the lack of GFAP staining in (C), suggesting that gliosis occurs as a secondary response to an initial dysplastic lesion in the PtchlacZ+/- retina.

 
To determine whether the Müller cell activation in the dysplastic regions of PtchLacZ+/- mice was associated with changes in Hh pathway activation, we also examined ß-gal activity in these regions. The Ptch locus in PtchlacZ+/- mice is disrupted by the insertion of the lacZ gene, thus ß-gal activity is a convenient readout for Ptch gene expression in this mouse strain. In the apparently normal regions of the PtchlacZ+/- retina (i.e. those areas where dysplasia and gliosis were absent) ß-gal+ cells were localized to the INL (i.e. in Müller cell nuclei) and in a subset of astrocytes in the nerve fiber layer (Fig. 3D and E). In dysplastic regions, however, the density of ß-gal+ cells was reduced (Fig. 3F), indicating that these regions contain fewer Müller cells or that those cells that normally express the gene do so at a reduced level.

Since the retinal dysplasia that we observed in PtclacZ+/- mice is similar to that resulting from dysregulated expression of cell cycle components in the retina (26–30), we sought to determine whether defective cell cycle regulation could underlie the retinal abnormalities in PtchlacZ+/- mice. Retinal maturation proceeds from center to periphery such that, by P5, proliferation (as assessed by BrdU incorporation) has ceased in the central retina and cell division is confined to the retinal periphery (Fig. 5C and D). In all four PtclacZ+/- mice examined, however, we observed BrdU+ cells in the central retina and a greater extent and intensity of BrdU labeling in the peripheral retina compared with in littermate controls (Fig. 5A and B). There were associated abnormalities of photoreceptor and horizontal cell maturation; the intensity of both rhodopsin staining in the ONL (Fig. 5 compare I and K, J and L) and syntaxin staining at the border of the developing outer plexiform layer (Fig. 5 compare E and G and F and H) being reduced in PtclacZ+/- mice compared with littermate controls. Retinal maturation in PtclacZ+/- mice is likely to have been delayed rather than permanently disrupted since we did not observe differences in adult PtclacZ+/- mice in staining for a variety of retinal markers (data not shown) and, aside from the dysplastic foci, normal lamination was established.



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  		Figure 5. Ectopic proliferation and delayed differentiation in the retinas of perinatal PtchlacZ+/- mice. Cross sections of the retinas of PtchlacZ+/- (A, B, E, F, I, J) and wildtype (C, D, G, H, K, L) littermates at P5 stained with (A–D) BrdU, (E–H) anti-syntaxin and (I–L) anti-rhodopsin (all brown signals). Comparison of BrdU incorporation in the retinas of PtchlacZ+/- and wildtype mice in a (A, C) low magnification view of the peripheral retina and a (B, D) high magnification view of the central retina at the optic disc (asterisk) reveals ectopic BrdU incorporation, as indicated by the extension of BrdU+ cells beyond the bracket in the peripheral retina [compare (A) versus (C)], and the presence of BrdU+ cells in the central retina in PtchlacZ+/- mice [compare (B) and (D)]. Immunostaining with anti-syntaxin antibodies, which identify amacrine neurons in the inner nuclear layer [arrows in (F)] and horizontal cells at the border of the outer plexiform layer (OPL) [arrowheads in (F)], reveals that horizontal cell differentiation and the establishment of the OPL are delayed in PtchlacZ+/- mice (compare straight line of syntaxin+ cells that extends from the central to the peripheral retina in wildtype mice [(G) top and bottom, (H)] with the disorganized line of syntaxin+ cells that is restricted to the central retina of PtchlacZ+/- mice [(E) compare top versus bottom, (F)]. Similarly, staining with anti-rhodopsin antibodies reveals that rod photoreceptor differentiation, as assessed by intensity of rhodopsin staining in the ONL [bracketed in (J)], is delayed in central and peripheral regions of the retina in PtchlacZ+/- mice compared wildtype littermates [compare (I), (K) and (J), (L)]. cen, central retina; per, peripheral retina.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our analysis of the retinas of BCNS patients and PtchlacZ+/- mice revealed defects in retinal histogenesis and glial cell function that were developmental in origin and focal in nature. The similarities in the retinal abnormalities between BCNS patients and PtchlacZ+/- mice also suggest that the latter represent a true model of the human disorder. Our findings indicate that retinal histogenesis is sensitive to Ptch gene dosage and support the hypothesis for a developmental basis for ERM formation.

We have shown previously that the Hh pathway is involved in precursor cell proliferation in the retina (21,22). Our observation that proliferation is extended in the central retina of PtchlacZ+/- mice is consistent with previous reports showing that other Hh-dependent processes are sensitive to Ptch gene dosage (13,31). Hh pathway activation has been directly linked to transcriptional activation of cell cycle genes (32–35), which raises the possibility that the reduction in Ptch expression levels in Ptch-LacZ+/- mice could result in an increase in the expression of cell cycle genes, thus predisposing cells to higher rates of proliferation. However, our preliminary analyses indicate that retinal precursor cells from Ptch-LacZ+/- mice do not appear to have overall a higher rate of proliferation in response to recombinant Shh (data not shown).

Our results are consistent with the possibility that focal changes in retinal precursor cell proliferation account for the localized dysplasias that we observed in Ptch-LacZ+/- mice and that we infer to have occurred in BCNS patients. That cell cycle dysregulation in the retina can result in a delay in differentiation and retinal dysplasia is supported by the phenotypes of p27 mutant mice or transgenic mice expressing cell cycle promoting genes (26–30). It is unclear, however, whether the retinal dysplasia that we observed in the PtchlacZ+/- mice is associated with loss of expression of the wild type Ptch allele, as is the case in medulloblastomas derived from these mice (36). In contrast to the cerebellum, ectopic proliferation in Ptc-LacZ+/- mouse retina does not result in tumorigenesis, perhaps because of the increased propensity of retinal cells to undergo apoptosis (29,30,37).

In both murine and human cases we have demonstrated focal areas of dysplasia involving cells in the outer nuclear layer, which in adults was associated with reactive Müller cells (gliosis). Hh responsiveness in the adult mouse retina, as defined by Ptch expression, is largely confined to Müller cells (23). Müller cells play a key role in retinogenesis and, in vitro promote the establishment of retinal lamination and counteract rosette formation (38). Thus, it may be possible that some of the retinal abnormalities that we observed in BCNS patients and PtchlacZ+/- mice result, at least in part, from a signaling defect at the level of the Müller cell. However, such a defect is likely to be a late event in the disease process, as our analysis of 3-week-old PtchlacZ+/- mice indicates that the gliosis likely occurs after the development of retinal dysplasia and the gliotic lesions in adult mice were not associated with an increase in ß-galactosidase activity, an indicator of Hh pathway activation in PtchlacZ+/- mice.

A role for Hh signaling in the adult retina is indicated by sustained Shh expression in both RGCs and a subset of inner nuclear layer amacrine cells, and of Ptch expression in Müller cells (21,23). Moreover, we have detected Shh and Ptch expression by RT–PCR analysis of adult human retinas (data not shown). ERM formation from glial activation in BCNS patients and intraretinal gliosis in PtchlacZ+/- mice represent the retinal response to dysregulation of this pathway and raise the possibility that inappropriate activation of this pathway could underlie non-genetic disorders with these features. In the general population idiopathic ERMs are seen in 25% of post-mortem eyes after the age of 75 years and are a well-recognized, predominantly unilateral, cause of visual loss in later life (39,40). The ERMs have an initial glial component derived from extension of Müller processes through and over the inner limiting lamina (41), but their pathogenesis remains undefined. In the light of our findings, we speculate that altered Müller cell responsiveness to Shh signaling, for example a localized reduction in Müller cell Ptch expression, could represent one such mechanism. Such epiretinal gliotic lesions then recruit contractile fibroblastic cells, with vision threatening consequences, such as macular retinal distortion and macular hole formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Clinical details
Patients with Gorlin syndrome known to the North-West Regional Genetics Service were ascertained according to guidelines approved by the North-West Region Ethics Committee. A thorough medical history was obtained and examinations were performed seeking evidence of developmental, dermatological, and dental problems. Thirty individuals underwent a complete eye examination, including slit-lamp biomicroscopy, applanation tonometry and dilated fundus examination.

Transgenic mice and immunohistochemistry
PtchlacZ+/- mice (12) were purchased from Jackson Laboratories (Bar Harbor, Maine) and maintained on a C57Bl/6 background. To harvest tissues, an anesthetic overdose (euthanyl) was administered intraperitoneally and the animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4. Eyes were enucleated and the lens removed. The posterior segment tissues were then cryoprotected in 30% sucrose/PBS and embedded in a 1 : 1 mixture of 30% sucrose : OCT (Tissue Tek compound). Serial sections of the eye were cut at 9–14 µm and collected in series of four slides. To assess the retinal architecture, every 4th slide was processed for immunostaining with anti-GFAP antibodies using established protocols, as described previously (21), followed by counterstaining with hematoxylin. Other slides in each series were stained with anti-glutamine synthetase antibodies (BD Pharmingen) to identify Müller cells. To detect cells in S-phase, postnatal day 5 PtchlacZ+/- mice were given two intraperitoneal injections 2 h apart with 30 µl of a 16 mg/ml solution of BrdU (Sigma Aldrich) in MEM (ICN Biomedicals cat#12-104-54). Two hours after the last injection the tissues were harvested, as described above, and processed for immunohistochemistry with anti-BrdU antibodies (Becton Dickinson), as previously described (22), anti-rhodopsin [B630, (42)] to detect rod photoreceptors and anti-syntaxin antibodies (HPC-1, Sigma Biosciences) to identify horizontal and amacrine cells. Primary antibodies were detected with the appropriate horseradish peroxidase conjugated secondary antibodies and developed using DAB. Sections were analyzed on a Zeiss Axioplan microscope and digital images were captured using an AxioVision 2.05 (Zeiss) camera and processed with Adobe® Photoshop.


   ACKNOWLEDGEMENTS
 
We thank M. Raff for antibodies, and R. Bremner, D. Picketts and C. C. Hui for criticism of the manuscript. V.A.W's laboratory was supported by Canadian Institutes of Health Research and the National Cancer Institute of Canada. V.A.W. has a Canadian Institutes of Health Research Scholarship. G.C.M.B. is a Wellcome Trust Senior Research Fellow in Clinical Science.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada. Tel: +1 6137378234; Fax: +1 6137378803; Email: vwallace{at}ohri.ca Back

{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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