Journal of Histochemistry and Cytochemistry, Vol. 49, 271-278, February 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Epithelial Cell Differentiation Pathways in the Human Prostate: Identification of Intermediate Phenotypes by Keratin Expression

David L. Hudsona, Adam T. Guya, Patricia Frya, Michael J. O'Hareb, Fiona M. Wattc, and John R.W. Mastersa
a Institute of Urology and Nephrology, Research Laboratories, University College London Medical School,
b LICR/UCL Breast Cancer Laboratory, Department of Surgery, University College London Medical School
c Keratinocyte Laboratory, Imperial Cancer Research Fund, London, United Kingdom

Correspondence to: David L. Hudson, Inst. of Urology and Nephrology, Research Laboratories, University College London Medical School, 67-73 Riding House St., London WIW 7E7, UK. E-mail: d.hudson@ucl.ac.uk


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The prostate grows slowly throughout adult life, leading to benign prostatic hyperplasia (BPH), which often results in urethral obstruction in later years. The symptoms of BPH are the second most common reason for surgery in men over 65. The aim of this study was to determine the relationship between cell proliferation and cell differentiation in BPH tissue. Using multiple antibodies, simultaneously detected with different fluorophore-conjugated secondary antibodies, several subpopulations of epithelial cells were detected. In addition to K14, basal cells also expressed keratins 15, 17, and 19 in various combinations, and some of the luminal cells also expressed K19 together with K8 and K18. Co-staining for cytokeratins and Ki-67 indicated that 44% of proliferative cells expressed K14 and 36% K19, although the difference was not statistically significant. This report provides a detailed description of the relationship between keratin expression and cell proliferation in the prostate and indicates that K19-positive cells form the link between the basal and luminal layers of the epithelium. (J Histochem Cytochem 49:271–278, 2001)

Key Words: prostate, epithelium, keratin, differentiation, stem cell, benign prostatic hyperplasia


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Hyperplasia of the transition zone of the prostate and the resultant urethral obstruction are an almost inevitable accompaniment to aging in men. Benign prostatic hyperplasia (BPH) is the second most common reason for surgery in men over 65 (Oesterling 1995 ). It is not known what causes BPH to develop, but three theories have been proposed (reviewed by Isaacs and Coffey 1989 ). The first hypothesis is that the changes in the levels or ratios of androgens and/or estrogens with age stimulate prostate cell growth. The second is that alterations in epithelial–mesenchymal interactions, through a reawakening of the embryonic inductive potential of the stroma, lead to increased proliferation. The third suggests that there is an expansion of an epithelial stem cell population. The aim of this study was to determine the relationship between cell differentiation and cell proliferation in human prostate epithelium.Prostate epithelium consists of a proliferative basal layer and a luminal secretory layer (Bonkhoff and Remberger 1996 ). These layers are distinguished by the expression of differentiation specific markers. The luminal cells express prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), androgen receptor (AR), and keratins (K) 8 and 18. The basal cells express K5 and 14, CD44 (Liu et al. 1997 ), and bcl-2 (McDonnell et al. 1992 ). More recently, intermediate phenotypes expressing a mixture of basal and luminal markers, with either co-expression of K5 and K18 in the absence of K14 or of K5 together with PSA, have been described (Verhagen et al. 1992 ; Bonkhoff et al. 1994a ; Xue et al. 1998 ).

In contrast to other epithelial tissues, such as the epidermis (Jones et al. 1995 ) and the gut (Potten and Loeffler 1990 ), there is little information about stem cells in the prostate. It has been proposed that, as in the epidermis (Jones et al. 1995 ), the prostate contains a small, basally located stem cell population that gives rise to a more rapidly dividing amplifying population. This, in turn, differentiates into the luminal secretory compartment (Isaacs 1987 ; Bonkhoff and Remberger 1996 ; Bui and Reiter 1999 ). We have recently shown that a subpopulation of prostate epithelial cells show characteristics of stem cells in primary culture (Hudson et al. 2000 ). However, there are as yet no markers to identify these cells in vivo.

The aim of this project was to identify subpopulations of cells in the basal layer of the prostate by examining the relationship between cell proliferation and differentiation in BPH tissue. Monoclonal antibodies to keratin subunits combined with fluorescence conjugated, isotype-specific secondary antibodies allowed simultaneous detection of up to three different target proteins in the same tissue section. On the basis of the observations made, it is suggested that the K19 positive cells play a key role in prostate epithelial cell differentiation, moving between the basal and luminal layers.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Tissue Acquisition and Processing
Samples of BPH tissues from patients aged 58–77 years were confirmed histopathologically to be free of malignancy. Prostate tissue from patients who underwent transurethral prostatectomy (TURP) was snap-frozen in an isopentane bath over liquid nitrogen and stored at -80C until needed. All samples were processed within 30 min. Frozen sections were cut at a thickness of 5 µm and transferred to Vectabond-coated slides (Vector Laboratories; Peterborough, UK), air-dried for 15 min, and stored at -80C. Routinely processed paraffin-embedded blocks of prostate were selected from UCL Hospitals histopathology archives. The paraffin-embedded tissue was cut at a thickness of 3 µm and mounted on Vectabond-treated slides. A further 11 blocks were selected from patients (aged 49–74) undergoing radical prostatectomy for prostate carcinoma. The patients were diagnosed with stages T2a–T4 cancer with Gleason scores of 6–9.

Antibodies and Immunohistochemistry
Indirect multiple immunofluorescence staining was carried out on both frozen and paraffin sections using a panel of mouse monoclonal and rabbit polyclonal monospecific antibodies (Table 1). The following antibodies were generous gifts: BL18, anti-K5, LL002, anti-K14 (Purkis et al. 1990 ) and LP2K, anti-K19 (Stasiak et al. 1989 ) from Prof. E. B. Lane (Dundee, UK); LHK, anti-K15 (Waseem et al. 1999 ) and LE61, anti-K18 (Lane 1982 ) from Prof. I. M. Leigh (London, UK); and E1/2.8, anti-CD44 (Isacke et al. 1986 ) from Dr. C. Isacke (London, UK). The remaining antibodies were purchased commercially: RCK 105, anti-K7 from ICN Pharmaceuticals (Basingstoke, UK); anti-Ki-67 antigen, RCK108, anti-K19, and 35ßH11, anti-K8 from Dako (Cambridge, UK); E3, anti-K17, and HNK-1 from Sigma (Poole, UK).


 
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Table 1. Antibody reagents (see text for sources)

Frozen sections were thawed and immediately blocked with 10% FCS in PBS (v/v) for 1 hr and then incubated for 1 hr at room temperature with primary antibodies diluted to the appropriate concentration in 10% serum. After washing twice in PBS, the slides were incubated for 45 min with Ig subtype-specific anti-mouse antibodies conjugated to fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothyocyanate (TRITC) (Southern Biotechnology; Birmingham AL), or cyanine, Cy5 (Jackson ImmunoResearch Laboratories; Stratech Scientific, Luton, UK). For double staining with mouse and rabbit primary antibodies, FITC-conjugated anti-mouse and TRITC-conjugated anti-rabbit secondary antibodies were used (Dako). After two washes in PBS the sections were mounted in Gelvatol (Monsanto; St Louis, MO). To stain cell nuclei, sections were incubated before mounting with a 1 µg/ml solution of Hoechst 33258 (Sigma) for 5 min.

Paraffin-embedded sections were heated to 55C for 20 min, dewaxed by two 5-min incubations in xylene, and rehydrated through a series of graded alcohols. Antigen retrieval was carried out by microwave heating for 30 min in a citrate-based buffer (antigen unmasking solution; Vector Laboratories). Slides were allowed to cool, washed for 10 min in PBS, and then incubated for 1 hr in 10% (v/v) FCS–PBS to block nonspecific primary antibody binding. The sections were incubated with primary antibodies overnight at 4C. The staining procedure was then identical to that used for frozen sections. Primary antibodies were omitted from negative control samples.

Image Capture and Microscopy
The sections were examined under an Hg-arc Zeiss Axiophot fluorescence microscope fitted with a bandpass filter for optimal FITC–TRITC–Cy5 separation. This was coupled to a Coolview 12 cooled charge-couple device (CCD) camera (1024 x 1024, 12-bit pixels; Photonic Science, Robertsbridge, UK) controlled by Image Pro-Plus software (v3.0; Media Cybernetics, Rockville, MD). The image obtained with each antigen was stored separately as a data file. To generate the coincident multicolored images, files were merged and given computer-generated colors using Adobe PhotoShop (v5.0; Adobe Systems, San Jose, CA).

Quantitation of Keratin Distribution and Cell Proliferation
The extent of keratin staining was assessed for each sample by counting the total number of cross-sections of ducts or acini within the section and then recording the number that stained positively for each individual keratin.

To assess the distribution of proliferative cells in basal and luminal epithelial populations, all cell nuclei were stained with Hoechst. The basal cells were distinguished from the luminal cells by staining for CD44, a marker of basal cells (Liu et al. 1997 ). The Ki67-positive cells in each population were scored. The proliferative index of the basal and luminal cell populations was estimated by photographing, at x400 magnification, five randomly selected fields triple-stained with Hoechst and antibodies to CD44 and Ki67. The number of Ki67-positive basal and luminal cells in each cross-section was counted from the photographs. The proliferation of cells expressing individual keratins was determined by scoring sections that had been double-stained for Ki67 together with each of the basally expressed keratins. The total number of epithelial Ki67-positive nuclei within the section was counted and cells checked individually for keratin staining. The proliferative index for cells expressing each basal keratin was assessed by counting the number of cells positive for each keratin within a field of view and scoring those that were Ki67-positive. The number of Ki67-positive proliferative cells was then expressed as a percentage of the total keratin-positive cells. Fields were chosen on the basis of countable keratin-positive cells independently of the presence of Ki67 staining, and a minimum of 1000 keratin-positive cells were counted for each keratin in each tissue (between 15 and 20 fields at x630 under oil).


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Keratin Staining
Ten BPH samples were stained for keratins with a panel of antibodies and the sections scored for distribution of staining. Staining patterns were consistent between frozen and paraffin-embedded tissue, with only the antibody to K18 failing to stain paraffin-embedded tissue. Initial keratin 19 staining was carried out using both LP2K and RCK108. Similar results were obtained for each antibody, although slightly stronger basal cell staining was seen with LP2K and this antibody was used for all the data presented. As a control for damage during BPH tissue collection, sections from 11 radical prostatectomy whole-mount specimens were double stained for K14 and K19.

Heterogeneous Keratin 14 Expression in the Basal Epithelial Cell Layer
Staining for K5, the heterodimer partner of K14, revealed a virtually complete basal cell layer in all samples (Fig 1A). This was clearly separate from the K5-negative luminal layer that was strongly positive for the keratin pair of 8 and 18 (Fig 1B). The presence of two complete and distinct cell layers was also demonstrated by staining with antibodies against CD44 and CD57 which, as described previously (Liu et al. 1997 ), localized to the basal and luminal layers, respectively (data not shown).



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Figure 1. Immunofluorescent staining of paraffin embedded (A,G–M) or frozen (B–F) BPH tissue sections single-, double-, or triple-stained with antibodies to the following proteins: (A) K5. (B,C) Two views of a single triple-stained section; K14 (green), K8 (red), K17 (blue). (D,E) Serial sections stained for K14 (D) and K19 (E). (F) Triple-staining for K8 (blue), K14 (green), and K17 (red). (G) Double-staining for K14 (green) and K15 (red). (H) Double-staining for K15 (green) and K19 (red). Arrows indicate basal cells co-expressing both keratins (yellow). (I) Double-staining for K14 (green) and K19 (red). (J) Double staining for K19 (green) and K8 (red). K19-positive cells are found basally and in a position intermediate between the basal and the luminal K8-positive layer. Some of these cells are maintaining contact with the basal layer (asterisk). (K–M) Enlargement of region expressing luminal K8 (K) and basal and luminal K19 (L). (M) Merged view of K and L, showing co-expression of K8 and 19 in luminal cells but not in the basal layer. Original magnifications: A–C,F,H x 200; D,E,G,I–M x 400. Bar = 100 µm.

K14 was localized exclusively to the basal layer in a similar pattern to its partner K5 (Fig 1A and Fig 1B). There was staining of the majority of cross-sections in seven of 10 samples. In two samples there were lower levels of expression, with over a third of all cross-sections being negative. One sample showed no immunoreactivity for K14 despite being positive for K5. From a total of 616 cross-sections scored from 10 BPH samples, 81% (496) were positive. In most samples there were gaps in K14 staining of the basal layer with cells in these regions expressing other basal keratins (Fig 1). Gaps were often found at branching points, with strongest K14 staining at the tips of the acini and with weaker or absent staining between.

To identify other basal keratins expressed by K14-negative cells, sections were stained for K14 together with antibodies to keratin 15, 17, or 19 (Fig 1C, Fig 1F, Fig 1G, and Fig 1I), or separately on serial sections (Fig 1D and Fig 1E). Co-expression data are summarized in Table 2. The K14-negative regions indicated by arrows stained strongly for K17 (Fig 1B and Fig 1C) and K19 (Fig 1D and Fig 1E). K17 was expressed in 22% of cross-sections in nine of 10 samples (137 of 616) and, in addition to the small K14-negative patches, was also strongly expressed in long K14-negative regions, which may be representative of ducts (Fig 1F). K19 was found in the basal layers of all samples examined but at a lower frequency than K14, with only 55 (14%) of 389 cross-sections counted being positive. K15 was expressed to various degrees in all samples in 138 (35%) of cross-sections and, although it was often co-expressed with K14 (Fig 1G), there were also regions that stained for K15 alone. These K14 weak, K15 strong patches were often at the base of acini (Fig 1G, arrowheads), in a pattern similar to that seen for K17 and K19. In addition to staining the K14-negative regions, 60% of K15, 42% of K19, and 50% of K17 staining was co-expressed with K14 in some of the samples.


 
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Table 2. Keratin co-expression in prostate tissuea

Keratin 19 Is Expressed in Both Basal and Luminal Cells
In addition to the expression of keratins 8 and 18 in all luminal cells, some staining for keratins 7 and 19 was also observed. K7 was detected in occasional cells in half the samples, co-expressed mainly with K19 (data not shown). K19 was detected in luminal cells in nine samples, with distribution ranging from occasional cells to the complete luminal cell layer. Six cases had co-localization with K8, but in the other three occasional K19-positive cells were found that failed to stain for K8 despite being in a luminal location.

Co-expression patterns of K19 are shown in Fig 1H–1M. As shown earlier, K19 was often expressed most strongly in areas of the basal layer which are negative for K14 (Fig 1I, asterisks) and weakest in areas with high levels of K14. K19-positive cells were also seen overlying K14 cells but below the luminal cells (arrows in Fig 1I). This is more clearly shown in Fig 1J, where double staining for K19 and K8 shows two layers of K19 expressing cells under the luminal layer (arrowhead). The cells that appear to be leaving the basal layer are larger and more elongated than the small, more triangular K14-expressing cells. K19 can be co-expressed with K15 (Fig 1H), although not all K15-positive cells expressed K19. This figure also shows a distinctive concentration of K19 in a band appearing to link the apical tips of the luminal cells, which also stained for K8 (Fig 1K). Fig 1K–1M show a tubular region with K19 expression in both layers, while K8 is present only in luminal cells.

To confirm that keratin staining in the BPH tissues was not affected by the TURP procedure, 11 sections of whole-mount sections from radical prostatectomies were examined. These were double stained for keratins 14 and 19 and the alternating expression patterns reported above were found to be present in eight of the samples. The regions in which the staining was found were classified on the basis of orientation of the urethra, relationship with the boundary of the prostate, and cell morphology, both stromal and epithelial. From this, it was determined that although the K14/19 pattern was found in the transitional zone in all cases, it was also present in the peripheral zone of four samples and the central zone of two.

Proliferation and Keratin Expression
To assess the proliferative status of the basal and luminal compartments, the distribution of expression of the Ki67 antigen in the two cell layers was measured. Sections were triple-stained with anti-Ki67, anti-CD44, and the nuclear stain Hoechst. Every cross-section in each tissue section was examined by fluorescence microscopy and each Ki67-positive epithelial cell compared to the staining for CD44 to determine whether it was basal or luminal.

Between 74 and 85% of cycling cells were in the basal layer, with a mean of 80.7% compared to 19.3% in the luminal layer (Table 3). To take account of the higher proportion of cells in the luminal layer, the proliferative index for each compartment was determined. Five images were captured from each tissue for each of the three markers, and from this the total number of cells in each epithelial compartment within a field of view were counted, along with the number of Ki67-positive cells. Ki67-positive cells in the stroma were also scored. The proliferative index of the basal layer was 1.66%, whereas the luminal layer had an index of only 0.14%, similar to the 0.16% found in the stromal cells. The ratio of luminal to basal cells was 2.7:1.0.


 
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Table 3. Distribution of Ki67 staining in prostate cell compartments (paraffin sections)

To determine which keratins are expressed by proliferative cells, sections were double stained for Ki67 with individual keratins. Almost half of all Ki67-positive cells expressed K14 (44%; range 30–57%) and a high proportion (35.8%; range 20–48%) expressed K19. Cycling cells were also found that were K15- and K17-positive but in lower numbers, at 15% and 25%, respectively. Cells expressing luminal keratins had the lowest level of cell division, with only 9.4 and 12.4% of cycling cells expressing K18 and K8, respectively.

To determine the proliferative index of each keratin-expressing cell type, the number of Ki67-positive cells was determined for each of the basal keratin partners of K5. The results are shown in Table 4. The highest proliferative index was seen to be for K19 at 1.37% and K15 at 1.2% and the lowest for K17, 0.96% and K14, 0.84%. The 95% confidence intervals indicate that, for these samples, the differences in proliferation between keratins are not significant.


 
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Table 4. Keratin expression of proliferative cells (paraffin-embedded)a


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The prostate is the site of two of the most common diseases in elderly men, benign prostatic hyperplasia and prostate cancer. Both these conditions are disorders of cell differentiation and cell proliferation. Previous studies examining the heterogeneity of prostate epithelial cells have identified phenotypes intermediate between those of the basal and luminal layers, cells that lack K14 expression but still express K5, with some expressing K8 (Verhagen et al. 1992 ; Xue et al. 1998 ). These intermediate cell phenotypes are believed to represent the amplifying population. Other recently described markers for a subset of basal cells thought to be the amplifying population are positive staining for PSCA (Reiter et al. 1998 ) and absence of p27kip1 (De Marzo et al. 1998 ).

In this study we have shown that K19 is also expressed by some of the K14-negative basal population, with continued expression into the luminal layer. It may therefore be a useful marker for the amplifying non-stem-cell basal population. To examine whether these different keratin expression patterns represent cells with differing proliferative abilities, we measured the proliferation rate of the basal and luminal cell compartments and for each basal keratin phenotype. The basal layer was the main proliferative compartment, with a tenfold higher proliferative index than the luminal layer, confirming previous studies (Bonkhoff et al. 1994b ). Determination of the proliferative index of cells expressing either K14, 15, 17, or 19 showed that all were capable of cell division. K19 had the highest proliferation index, as would be expected for an amplifying population, although for these samples the differences were not statistically significant.

In addition to the immunohistological data, there is also evidence from in vitro studies for intermediate cell populations. Robinson et al. 1998 demonstrated the expansion in culture of a population of cells expressing both K14 and K18 as well as cells expressing K19 and K18. K19 expression as a marker of more differentiated cells has also been recently demonstrated in two culture systems. We found co-staining of K19 with a subset of both K14- and K8-positive cells in monolayer culture (Fry et al. 2000 ). In a three-dimensional culture system of putative stem cells, we found loss of expression of K14 in cells that expressed K19 and K8 but that also stained for K5 (Hudson et al. 2000 ).

Previous studies have described K19 distribution in prostate tissue. One study suggested that K19 expression is restricted to luminal cells (Cussenot et al. 1994 ), whereas, in common with our study, two others describe K19 expression in both the basal and luminal layers (Nagle et al. 1991 ; Peehl et al. 1996 ). Nagle et al. showed basal expression of K19 to be restricted to tubular portions of distal regions and in proximal ducts, while the distal alveoli lacked K19 altogether. This is consistent with our observations of lowest expression of K19 in acini, with onset of expression at sites of branching and with expression in both cell layers in tubular regions.

K19 has been implicated in the differentiation of many different epithelial tissues in which there is a transition between differentiated phenotypes (Stasiak et al. 1989 ). Examples include oral epithelium, mammary gland ducts, and the outer root sheath of the hair follicle. K19 is structurally unique among keratins in that it has a truncated tail that results in weaker bonds with its partners. It is postulated to be a neutral switch keratin that permits the changeover from one type of cytoskeleton to another (Stasiak et al. 1989 ). Co-expression of K15 and K17 with K19 in areas lacking K14 indicates that K15 and K17 may also be markers of intermediate stages in the differentiation process. A theoretical pathway for this complex pattern of differentiation is proposed in Fig 2 for the glandular epithelium. The least-differentiated population, containing the stem cells, expresses only K5/14, whereas K15, 17, and 19 are expressed as the cells differentiate, initially with K14 but then without. K15 and 17 are restricted to intermediate basal cells but K19 continues to be expressed as the cells become K8/18-positive. The process ends with the loss of K19, leaving only K8/18 in the fully differentiated secretory population.



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Figure 2. Hypothetical differentiation pathway for human prostate epithelial cells based on patterns of keratin staining. Basal stem cells (K5 and 14 only) give rise to an intermediate transit amplifying population expressing K19. These cells differentiate into luminal cells with transient co-expression of K19, 8, and 18 before complete differentiation into cells expressing K8/18 only.

There was wide variation in some of the staining patterns, particularly in relation to the extent of staining of particular keratins in individual cross-sections. This may reflect disruption of the normal architecture as a result of BPH. Studies of normal prostates from young men may determine whether any of these variations are symptomatic of disease progression.

Because prostate disease, both malignant and benign, involves inappropriate cell division and differentiation, it is important to identify the stem cell and amplifying populations. All carcinomas express keratin 8 and 18 and most, including prostatic intraepithelial neoplasia (PIN) lesions, express K19 (Nagle et al. 1991 ; Verhagen et al. 1992 ) and not K14. It is therefore possible that these tumors arise from a K14-negative, K19-positive population. The number of cells expressing K14 alone in our study is greater than expected for a stem cell population (Hudson et al. 2000 ). In the epidermis, stem cells are estimated to make up approximately 10% of basal cells (Potten and Morris 1988 ), where all cells of the basal layer are K14-positive. It is therefore likely that there will be other markers which will identify a subpopulation of stem cells within this compartment. Studies to examine the relative proportions of these basal subpopulations in normal and aberrant growth conditions should help develop an understanding of the role played by stem cells and differentiation in prostate disease.


  Footnotes

1 Laboratory in which work was carried out.


  Acknowledgments

DLH was supported by National Institutes of Health Grant AG14960-02, PF by the Wellcome Trust, and ATG by the Covent Garden Cancer Research Trust.

We wish to thank all those who kindly donated antibodies to this study, especially Birgitte Lane, Trish Purkis, and Irene Leigh for keratin antibodies. Thanks also to those who provided tissue, to Constance Parkinson (UCLH Dept. of Histopathology, London, UK) for invaluable advice, Alan Entwistle of the Ludwig Institute for help and advice on microscopy and image capture, and Liz Rugg (Royal London Hospital, London, UK) for helpful discussion.

Received for publication June 13, 2000; accepted October 5, 2000.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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