Department of Internal Medicine I, University of Ulm, 89081 Ulm, Germany
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ABSTRACT |
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The pancreas
morphology of transgenic mice that overexpress transforming growth
factor-1 (TGF-
1) in the pancreas resembles partially
morphological features of chronic pancreatitis, such as progressive
accumulation of extracellular matrix (ECM). Using this transgenic mouse
model, we characterized the composition of pancreatic fibrosis and
involved fibrogenic mediators. On day 14 after birth,
fibrotic tissue was mainly composed of collagen type I and III. At this
time, mRNA levels of TGF-
1 were increased. On day 70, the
ECM composition was expanded by increased deposition of fibronectin,
whereas connective tissue growth factor, fibroblast growth factor
(FGF)-1, and FGF-2 mRNA expression levels were elevated in addition to
TGF-
1. In parallel, the number of pancreatic stellate cells (PSC)
increased over time. In vitro, TGF-
1 stimulated collagen type I
expression but not fibronectin expression in PSC, in contrast to FGF-2,
which stimulated both. This confirms that TGF-
1 mediates pancreatic
fibrosis through activation of PSC and deposition of collagen type I
and III at early time points. Furthermore, this points to an indirect
mechanism in which TGF-
regulates pancreatic ECM assembly by
induction of additional growth factors.
transforming growth factor-; connective tissue growth factor; fibroblast growth factor-2; chronic pancreatitis; pancreatic stellate
cells
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INTRODUCTION |
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IN CHRONIC
PANCREATITIS, exogenous and endogenous function is disturbed due
to increased deposition of extracellular matrix (ECM) proteins. The
development of pancreatic fibrosis in chronic pancreatitis is poorly
understood (11, 16, 17). The comprehension of diseases
dominated by ECM was expanded by identifying ECM-producing cells. It is
well known that fibroblasts produce and maintain ECM architecture
(8). However, recent evidence demonstrates that
fat-storing cells significantly contribute to ECM production (13,
19). These cells, named stellate cells, are characterized by
their ability to store vitamin A. Following activation, they change
into myofibroblast-like cells with positive staining for -smooth
muscle actin and desmin. Recently, cells with similar characteristics
were identified in the pancreas and named pancreatic stellate cells
(PSC) (1, 3). They are shown to produce ECM proteins such
as collagen type I and III and fibronectin. Among other growth factors,
they are mainly activated by transforming growth factor (TGF)-
1
(2, 3).
Members of the TGF- superfamily play a role in cell proliferation
and differentiation, tissue repair, and recycling (25). TGF-
1 was found to be overexpressed in inflammatory pancreatic diseases (6, 34). Several groups showed an upregulation of TGF-
1 during regeneration of acute pancreatitis in humans and in
animal models (12, 27, 34, 35). ECM deposition and fibroblast proliferation were paralleled by an increase of TGF-
1 in
cerulein-induced pancreatitis in the rat (12). This effect could be partially reversed by antibodies against TGF-
1
(27). Moreover, repeated injections of recombinant
TGF-
1 into mice after recurrent episodes of acute pancreatitis led
to pancreatic fibrosis (35). This suggests that TGF-
1
is involved in the development of pancreatic fibrosis.
Other growth factors, such as connective tissue growth factor (CTGF),
were also found to be overexpressed in inflammatory pancreatic diseases
(6, 36). CTGF is a cysteine-rich mitogenic peptide leading
to cell proliferation and ECM production in fibroblasts. Connective
tissue cells secrete CTGF after activation by TGF-1, and therefore
CTGF acts downstream of TGF-
1 (14). However, it is not
known how CTGF and TGF-
1 interact with each other during the
development of pancreatic fibrosis.
Transgenic mice overexpressing active TGF-1 selectively in
the pancreas show enhanced proliferation of fibroblasts, deposition of
ECM, and inhibition of acinar and centroacinar cell proliferation (23, 30). In the present study, we investigated the
development of pancreatic fibrosis in the TGF-
1 transgenic mouse
model generated by Sanvito and colleagues (30). We
demonstrated an early increase in the number of PSC in the pancreas of
TGF-
1 transgenic mice. This was paralleled by deposition of collagen
type I and III. At this time, TGF-
1 was the only growth factor found
to be overexpressed. Later, on day 70, when fibronectin was
added to the collagen deposition, other growth factors, such as CTGF,
fibroblast growth factor (FGF)-1, and FGF-2, were also increased. In
vitro, FGF-2 stimulated fibronectin and collagen type I mRNA expression
in PSC, whereas TGF-
1 was only capable of enhancing collagen type I
expression. This suggests a cooperation of TGF-
1, CTGF, FGF-1, and
FGF-2 in the development of pancreatic fibrosis.
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MATERIALS AND METHODS |
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Materials.
Polyclonal antiserum against collagen type I was obtained from Chemicon
International (Temecula, CA). Antisera against collagen type
III and IV were acquired from Rockland (Gilbertsville, PA). Antisera
against fibronectin was supplied by Biomol (Hamburg, Germany).
Monoclonal antibodies against -smooth muscle actin,
-actin, and
desmin were purchased from Sigma Chemical (St. Louis, MO), and those
against FGF-2 were received from Transduction Laboratories (Lexington,
KY). The polyclonal antibody against CTGF, fisp12, and its
cDNA clone were kindly provided by C. Wenger (University of Ulm).
TGF-
1 was obtained from Pepro Tech (Rocky Hill, NY), and FGF-2 was
purchased from Boehringer Mannheim (Mannheim, Germany). cDNA
clones for
1(I),
1(III), and
1(IV) collagen and for
fibronectin were purchased from the American Type Culture Collection
(ATCC nos. 61322, 61234, 65036, and 61038; Rockville, MD). The cDNA clone for 18S mRNA was made available by T. Gress (University of Ulm).
Animals.
TGF-1 transgenic mice, expressing TGF-
1 under control of a rat
insulin II gene promoter, were a generous gift from Sanvito et al.
(30). They were crossbred to C57BL/6 mouse strain (Charles River, Sulzfeld, Germany) and kept as heterozygotes for experiments. All experiments were performed according to the guidelines of local
Animal Use and Care Committees. Transgenesis was determined on tail DNA
(22) by PCR and Southern blot analysis. Primers for PCR
analysis and the TGF-
/human growth hormone cDNA probes for
the transgene for Southern blot analysis were used as described by
Sanvito et al. (30).
Protein studies. For protein analysis, samples were homogenized in protein lysis buffer (0.5 g tissue/ml) according to Laemmli (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 5% mercaptoethanol, and 0.2% bromphenol blue) containing 5 µM aprotinin (Bayer-Leverkusen, Leverkusen, Germany), 1 mM pefabloc, 10 mM leupeptin, 10 µm pepstatin, and 5 mM soybean trypsin inhibitor (all Boehringer-Mannheim). Thereafter, probes were incubated for 10 min at 95°C, and 15 µl of each probe were analyzed by SDS-PAGE according to standard procedures (21). Quantity of protein was equalized by Coomassie blue staining of the gel. The fractionated proteins were blotted by semidry technique onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). For immunodetection, blots were incubated for 1 h at room temperature with first antibody as indicated and then incubated for 1 h with secondary peroxidase-coupled antiserum (Pierce, Rochester, NY). Antibody detection was carried out using an enhanced chemiluminescence reaction system (Pierce).
RNA studies.
RNA was extracted according to Chomczynski and Sacchi (4).
For Northern blots, 30 µg of total RNA were transferred on Hybond-N membranes by capillary blotting. Blots were hybridized with
[32P]dCTP-labeled purified cDNA probes as described
earlier (27). Membranes were exposed to X-ray films (Kodak
XAR, Rochester, NY) at 70°C for 7-10 days. The mRNA expression
was evaluated using quantitative RT-PCR analysis (TaqMan; PE Applied
Biosystems, Norwalk, CT) (10). RT of 2 µg total RNA (20 mM Tris · HCl, 50 mM KCl, 2.5 mM MgCl2, 10 mM
dithiothreitol, 0.5 µM random hexamer primer, 0.5 mM each dNTP, and
200 units Superscript II RT; GIBCO BRL, Rockville, MD) was carried out
in duplicate and further processed independently. PCR was performed in
triplicate using the primer combinations listed below (Sybr Green PCR
core reagents; PE Applied Biosystems) and normalized to the endogenous
18S mRNA level for each reaction (ribosomal control reagent; PE Applied
Biosystems). Relative quantification of the target cDNA in transgenic
mice compared with littermate controls was established using the
-
-Cycle Threshold (CT) method. Results were expressed as
means ± SD. Specific amplification was confirmed by
electrophoresis on a 4% low-melting agarose gel, resulting in bands of
the predicted size.
Primer design.
If possible, primers were designed spanning exon-intron borders
according to the published murine GenBank sequences. Regions of high
similarity between the individual genes were excluded. Furthermore,
each primer was checked in a similarity search for possible
cross-recognition of related or unrelated genes. Optimal primer
concentrations were established in previous experiments. The following
forward (FP) and reverse (RP) primers and primer concentrations were
used: TGF-1-FP (300 nM), 5'-GTACAGCAAGGTCCTTGCCCT-3'; TGF-
1-RP
(300 nM), 5'-TAGTAGACGATGGGCAGTGGC-3'; TGF-
2-FP (300 nM),
5'-GCAGAGTTCAGGGTCTTCCG-3'; TGF-
2-RP (300 nM),
5'-CAGCGTCTGTCACGTCGAA-3'; TGF-
3-FP (50 nM),
5'-TGACCCACGTCCCCTATCA-3'; TGF-
3-RP (900 nM), 5'-TCTCCTGAGTGCAGCCTTCC-3'; fisp12-FP (300 nM),
5'-GTGTGCACTGCCAAAGATGGT-3'; fisp12-RP (300 nM),
5'-ACACCCACTCCTTGCAGCATT-3'; FGF-1-FP (300 nM),
5'-TATACGGCTCGCAGACACCAA-3'; FGF-1-RP (50 nM),
5'-AACCAGTTCTTCTCCGCATGC-3'; FGF-2-FP (300 nM),
5'-AGCGACCACACGTCAAACTAC-3'; FGF-2-RP (300 nM),
5'-CAGCCGTCCATCTTCCTTCATA-3'; platelet-derived growth factor (PDGF)-A-chain-FP (300 nM), 5'-CCCATTCGCAGGAAGAGAAGTA-3';
PDGF-A-chain-RP (900 nM), 5'-TTGACGCTGCTGGTGTTACAA-3'; PDGF-B-chain-FP
(300 nM), 5'-GCAAGAGTGTGGGCAGGGTTAT-3'; PDGF-B-chain-RP (300 nM),
5'-GAATCAGGCATCGAGACAGACG-3'; insulin-like growth factor
(IGF)-I-FP (900 nM), 5'-AGATGTACTGTGCCCCACTGAA-3'; IGF-I-RP (900 nM), 5'-CTTCCTTCTGAGTCTTGGGCAT-3'; TGF-
-FP (300 nM), 5'-GGCTGCCAGCCAGAAGAA-3'; TGF-
-RP (300 nM),
5'-ACAGGTGATAATGAGGACAGCCA-3'.
Morphological studies. Specimens of pancreatic tissue were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 µm) were deparaffinized and stained with hematoxylin and eosin for light microscopic evaluation. Other sections were deparaffinized, pretreated in the microwave in citrate buffer (10 mM, pH 6.0), and blocked with calcium- and magnesium-free (CMF)-PBS containing 3% BSA for 1 h at room temperature for immunofluorescence. The first antibody was diluted in CMF-PBS plus 0.3% BSA and incubated for 1 h at room temperature. The staining was visualized by a second Cy3-conjugated antibody (1 h at room temperature), and examination was performed by use of an Axiophot microscope (Zeiss, Oberkochen, Germany) or Leica confocal microscope TCS-4 (Leica, Wetzlar, Germany).
To estimate collagen and total protein content in histological sections, we stained 10-µm sections with fast green and sirius red in accordance with the method of López-de León and Rojkind (24). Dye was eluted by washing with NaOH-methanol, and concentrations were determined in a spectrophotometer at 605 and 540 nm. Collagen and total protein content were mathematically determined from absorbance at 605 and 540 nm (33).Cell isolation.
PSC were isolated by outgrowth techniques from mouse pancreas as
described previously (3). Briefly, pancreatic tissue of TGF- transgenic as well as wild-type mice was mechanically
dissociated and seeded in uncoated culture wells in the presence of
DMEM containing 10% FCS. After incubation at 37°C in a 10%
CO2-air humidified atmosphere, tissue blocks were removed
and culture medium was changed. Outgrown cells were trypsinized and
transferred to new culture plates. For immunocytochemical
characterization, nonspecific binding was blocked with CMF-PBS
containing 3% BSA for 1 h at room temperature. The first antibody
was diluted in CMF-PBS plus 0.3% BSA incubated for 1 h at room
temperature. The staining was visualized by a second Cy3-conjugated
antibody (1 h at room temperature), and examination was performed by
use of an Axiophot microscope.
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RESULTS |
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Compared with the pancreas of wild-type mice (Fig.
1A), we found a progressive
accumulation of ECM in TGF-1 transgenic mice. On day 14 after birth, ECM deposition started around endocrine cells (Fig.
1B). The amount of ECM was increasing and continuously replaced exocrine tissue (Fig. 1C). In 330-day-old
transgenic mice, most of the acini were replaced by fibrotic tissue
(Fig. 1D).
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Time course of ECM deposition.
We analyzed the time course of different members of the ECM on protein
and mRNA level in TGF-1 transgenic mice compared with the wild type.
As shown in Fig. 2A, collagen
type I and III were increased on the protein and mRNA levels on
day 14, whereas fibronectin expression levels remained low.
From day 70, there was a prominent fibronectin expression
documented on both protein and mRNA levels. The distribution pattern of
fibronectin was diffuse throughout fibrotic areas around endocrine and
remaining acinar cells on day 70 (Fig. 2C). Only
little fibronectin was present in normal mouse pancreas. The
distribution of collagen type I and III was similar in specific
immunofluorescence staining (data not shown). Although a further
increase of ECM was morphologically documented on day 330 (Fig. 1D), the collagen type I and III expression
remained stable. However, fibronectin expression levels continued to
increase from day 70 to day 330 (Fig. 2,
A and B). Collagen type IV was also enhancing
from day 14 to day 330 on the mRNA expression
level. However, on the protein level the expression remained similar throughout the time course (Fig. 2, A and B).
Laminin expression, known to be associated with basal membrane, was not
increased at the examined time points (data not shown).
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Growth factors in TGF-1 transgenic mouse pancreas.
The relative TGF-
1 transcript level was upregulated 9.8 ± 1.8-fold on day 14 and 15.8 ± 1.9-fold on day 70 compared with the wild-type level at the related time points as
determined by quantitative RT-PCR TaqMan analysis (Table
1). The RT-PCR TaqMan analysis revealed
no significant enhancement of TGF-
2, TGF-
3, CTGF, FGF-1, FGF-2,
IGF-I, PDGF A, and PDGF B on mRNA expression level on day
14.
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ECM-producing cells.
Immunofluorescence staining of pancreatic tissue of TGF-1 transgenic
mice showed an increased number of
-smooth muscle actin-positive cells in fibrotic areas (Fig.
6A). They were oval shaped and
grouped in areas where fibrosis was close to normal-appearing acinar
cells on day 14 (Fig. 6A, II) and in
fibrotic areas on day 70 (Fig. 6A,
III). They were not found in wild-type mice (Fig.
6A, I). The morphology and localization of these
cells corresponded to the cells identified in Fig. 5, which were
stained with CTGF or FGF-2, respectively.
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Effect of growth factors on PSC.
Primary cultures of PSC were immunohistochemically positive for
fibronectin, collagen type I, and collagen type III (Fig. 6B; similar staining for collagen type I). RNA analyses of
these cells revealed a low expression of fibronectin and collagen type I when cultured in DMEM only (Fig. 7).
However, incubation of PSC with 20 ng/ml TGF-1 in DMEM for 18 h
exhibited a strong increase of collagen type I but not of fibronectin
mRNA and protein expression levels. In contrast, incubation with 20 ng/ml FGF-2 in DMEM stimulated both fibronectin and collagen type I
mRNA and protein expression levels in PSC in vitro (Fig. 7).
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DISCUSSION |
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In the present study, we analyzed the development of pancreatic
fibrosis in a TGF-1 transgenic mouse model. Investigation of the
time course of ECM deposition in this model revealed additional insights into the regulation of ECM by TGF-
1 in vivo. Soon after birth on day 14, ECM was mainly composed of collagen type I
and type III in the transgenic mouse pancreas, whereas at the mRNA level, TGF-
1 was found to be overexpressed. However, in older mice
collagen and fibronectin were the principal components of ECM.
As a possible source of ECM, we could identify PSC in younger and older
mice. TGF-1 was shown to be a major factor in stimulating the
proliferation of PSC and their collagen synthesis capacity (2). Saotome et al. (31) also described
fibroblast-like cells in periacinar regions possessing characteristics
of myofibroblasts. In these cells,
-smooth muscle actin, the marker
of myofibroblast-like phenotype, as well as collagen synthesis could be
increased by TGF-
1 treatment.
In parallel to the increased fibronectin concentration in older
animals, we could demonstrate CTGF and FGF-2 expression in the TGF-1
transgenic mouse pancreas on day 70 by immunofluorescence staining. Both could be localized to oval-shaped cells in fibrotic areas. Staining of corresponding sections suggested myofibroblast-like cells as a source. TGF-
1 was shown to induce CTGF gene expression in
fibroblasts (14) and caused FGF-2 and FGF receptor mRNA
expression in human myofibroblastic liver cells, known as activated
hepatic stellate cells (29). Furthermore, analysis of mRNA
expression in the TGF-
1 transgenic mouse pancreas on day 70 revealed a significant increase of additional growth factors such
as CTGF, TGF-
2, FGF-1, and FGF-2.
CTGF is known as a downstream mediator of TGF- action on
fibroblasts. Wenger et al. (36) could identify slightly to
moderately elevated transcript levels for CTGF in 15 studied tissue
samples of chronic pancreatitis. Also, di Mola et al. (6)
recently showed an upregulation of CTGF mRNA expression levels in human chronic pancreatitis tissues. They revealed a positive correlation of
CTGF expression and degree of fibrosis in these samples.
Growth factors such as FGF-1 and FGF-2 were also found to be
overexpressed in human chronic pancreatitis, and a role in pancreatic fibrosis was implicated (9). Here we could demonstrate the upregulation of FGF-1 and FGF-2 in the TGF-1 transgenic mouse pancreas on day 70. Little is known about the potential of
FGF-1 in fibrosis development. A role of FGF-1 in the proliferation of
interstitial fibroblasts in human kidney diseases was suggested (20). FGF-2 is predominantly recognized for its mitogenic
effect on fibroblasts (29). The effects of FGF-2 on
synthesis of ECM components by various cell types were contradictory
(7, 15, 32). However, in fibroblasts derived from the
pancreas, FGF-2 stimulated mRNA concentrations of collagen type I and
III and fibronectin (26).
Because CTGF and FGF-2 were not increased at early stages, this
suggests that TGF-1 alone is sufficient to induce pancreatic fibrosis by the induction of PSC and the associated collagen expression in this transgenic mouse model. However, CTGF and FGF-2 appear to be
cofactors at later stages. The concomitant effect of TGF-
1, FGF-2,
and CTGF in older animals was combined with a change in ECM composition
when fibronectin expression was getting prominent and collagen
deposition remained constant.
This was confirmed by our in vitro analysis. Thereby, TGF-1
stimulated collagen type I but not fibronectin expression in cells that
grew out of the TGF-
1 transgenic mouse pancreas in cell culture
medium. In vitro, these cells were
-smooth muscle actin- and
desmin-positive, identifying them as mouse PSC. However, TGF-
1 was
described to stimulate collagen type I and III and fibronectin
expression in fibroblasts and human PSC in vitro (3, 18).
Furthermore, TGF-
1 was shown to regulate fibronectin gene transcription in different cell lines, including fibroblasts. The
regulation occurred rapidly and did not require protein synthesis, indicating a direct mechanism (5). However, it is well
known that TGF-
exhibits differential effects on the same cell type under different experimental conditions. There is evidence that early
cultured PSC show different ECM expression and responsiveness to growth
factor stimulation compared with fibroblast cell lines (18,
19). In contrast, it was also shown that protein synthesis was
required for TGF-
to induce ECM protein gene expression, indicating
that some other intermediate factor or factors may be involved in the
signaling pathways (5, 28). One of these factors was shown
to be CTGF, stimulating fibronectin and collagen type I synthesis in
cultured fibroblasts after being induced by TGF-
(14).
In the present study, the difference that TGF-
did not directly
influence fibronectin expression in mouse PSC underscores the idea that
TGF-
effects were partially mediated by induction of additional
peptide growth factors and emphasizes the complexity in the control of
ECM composition in vivo. Our in vitro data were in agreement with the
in vivo situation, in which TGF-
1 stimulated the activation and
proliferation of PSC on day 14, which produced and deposited
predominantly collagen matrix.
The complexity of ECM control by TGF- is also accentuated by the
findings of collagen type IV regulation in this in vivo model. In vitro
data in hepatic stellate cells showed the induction of collagen type IV
by TGF-
1 (18). However, despite the induction of
collagen type IV on the mRNA level, it remained constant on the protein
expression level throughout the examined stages of life of TGF-
transgenic mice compared with the wild type.
Furthermore, we could demonstrate that FGF-2 stimulated collagen type I
and fibronectin expression and synthesis in mouse PSC in vitro. As
outlined above, FGF-2 was shown to induce collagen and fibronectin
expression in human PSC and fibroblasts derived from pancreatic tissue
(3, 26). The mouse PSC could also be demonstrated in vivo
in the TGF-1 transgenic mouse pancreas on day 70 after
birth, when fibronectin and FGF-2 expression was increased. The in
vitro effect of FGF-2 on fibronectin expression in mouse PSC suggests a
concomitant effect of TGF-
1 and FGF-2 on fibronectin and collagen
production in this transgenic mouse model at a later time point.
In conclusion, transgenic mice selectively overexpressing TGF-1 in
the pancreas revealed an increased production and deposition of ECM in
pancreatic tissue. PSC were identified as a source of this ECM
assembly. At early time points, when TGF-
1 was the predominant fibrotic factor, the matrix was mainly composed of collagen type I and
III. Later, when fibronectin was also a prominent part of ECM, growth
factors such as CTGF and FGF-2 were also found to be increased. In
vitro, FGF-2 is able to stimulate fibronectin and collagen expression,
whereas TGF-
1 mainly increased collagen expression in mouse PSC.
This points to an indirect mechanism by which TGF-
regulates
pancreatic ECM assembly by induction of additional growth factors.
Future studies will be necessary to look further into the cross-talk of
the different growth factors to understand the complexity of fibrosis
development in inflammatory pancreatic diseases.
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ACKNOWLEDGEMENTS |
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We would like to thank L. Orci (Dept. of Morphology, University of
Geneva, Switzerland) for kindly providing the TGF- transgenic mouse
model. Furthermore, we thank S. Braeg for excellent technical assistance and C. Wenger for kindly providing the CTGF cDNA.
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FOOTNOTES |
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This work was supported by grant KN 200 DFG Forschergruppe obtained from the Deutsche Forschungsgemeinschaft.
Address for reprint requests and other correspondence: R. Vogelmann, Dept. of Internal Medicine I, Univ. of Ulm, Robert-Koch-Str. 8, 89081 Ulm, Germany (E-mail: roger.vogelmann{at}medizin.uni-ulm.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 January 2000; accepted in final form 20 July 2000.
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