Clinical Science (1984) 66,217-224
217
The effect of leucocyte elastase on the immunoelectrophoretic
behaviour of al-antitrypsin
R. A. STOCKLEY
AND
S. C . AFFORD
The General Hospital, Bimingham, UK.
(Received 29 November 1982123 June 1983; accepted 2 August 1983)
summary
Introduction
1. Two-dimensionalimmunoelectrophoresis and
conventional sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed on
various mixtures of purified al-antitrypsin (alAT)
and leucocyte elastase (LE).
2. The results confirm that alAT inhibits LE
by the formation of enzyme-inhibitor complexes
demonstrable by both techniques.
3. The complexes break down with time and
are not affected by pH in the presence of excess
alAT. However, the breakdown is more rapid in
the presence of excess enzyme only at pH values
where LE remains active. The resultant products
of the complex breakdown include inactivated LE
and aIAT that has undergone limited proteolysis.
4. It is concluded that the presence or absence
of alAT-enzyme complexes as demonstrated by
two-dimensional immunoelectrophoresis must be
interpreted with caution when studying alAT
function in lung secretions. The absence of such
complexes does not mean that previous interaction with enzyme has not occurred, thereby
accounting for a reduction in alAT inhibitory
capacity.
al-Antitrypsin (alAT) is a major plasma and lung
inhibitor of serine proteinases. It is thought to
play a crucial role in protecting the lung from
enzyme-induced damage, which may result in
chronic diseases such as pulmonary emphysema
[l J . The mechanisms whereby proteolytic enzymes
can overcome the inhibitory capacity of alAT are
poorly understood; however, it is recognized that
a proportion of the alAT found in lung secretions
is no longer active as an inhibitor [Z-41.
al-Antitrypsin is known to inhibit proteolytic
enzymes, such as leucocyte elastase (the enzyme
implicated in emphysema), by the formation of
stable enzyme-inhibitor complexes, although some
dissociation can occur [S]. The presence of such
complexes is readily demonstrable by two-dimensional immunoelectrophoresis (2DIEP) and they
have been demonstrated in lung secretions [2].
This is an indication of prior interaction with
enzyme, thus accounting for some reduction in the
inhibitory capacity of the alAT.
However, later work [3,4] has shown a reduction in the inhibitory capacity of alAT in the
absence of such complexes seen on 2DIEP.
Furthermore, Stockley & Burnett [2] also demonstrated complete loss of inhibitory capacity of
&,AT in lung secretion samples that showed less
a,AT-enzyme complex than samples retaining
some inhibitory function. These results suggest
that alAT can be inactivated as an inhibitor in the
absence of alAT-enzyme complexes demonstrated
by 2DIEP.
The interpretation of these results depends
upon detailed knowledge of how alAT and
enzymes interact and what effect this has upon the
2DIEP behaviour of alAT. Firstly, the stability of
Key words: antibody, al-antitrypsin, immunoelectrophoresis, leucocyte elastase, polyacrylamide
gel electrophoresis.
Abbreviations: q A T , cY1-antitrypsin; 2DIEP, twodimensional electrophoresis; LE, leucocyte elastase;
SDS, sodium dodecyl sulphate.
Correspondence: Dr R. A. Stockley, The
General Hospital, Steelhouse Lane, Birmingham
B4 6NH, U.K.
218
R. A . Stockley and S. C Afford
alAT-enzyme complexes has not been evaluated
over long periods. This is of particular importance
since the lung secretion samples may have been
formed many hours before collection and analysis.
Secondly, it is of importance t o determine whether
a loss of inhibitory function of alAT and the production of decreasing proportions of the alAT
present as complexes is a natural consequence of
the interaction of alAT with increasing quantities
of enzyme that exceed the inhibitory capacity.
Finally, it is necessary to determine whether
enzyme-inhibitor complexes can be absent after
the interaction of a,AT with enzyme or whether
this absence means that no such interaction can
have taken place previously.
The present study was specifically designed to
clarify these points by investigating the interaction
of purified alAT and leucocyte elastase (LE) of
known concentration and function. In particular,
the 2DIEP pattern was studied to assess the effect
of time after the mixing of both proteins in various
proportions.
method has been described in detail elsewhere [8]
but slight modifications were introduced for the
present studies where 2DIEP assessment of the
alAT was performed.
In brief, a reversed inhibition study was performed. Increasing amounts of LE were added
to a fixed amount of alAT in 0.5 ml of buffer
(Tris/HCl; 0.2 mol/l, pH 8.8) and incubated at
37°C for 10min. An aliquot was then taken for
the 2DIEP and sodium dodecyl sulphate (SDS)polyacrylamide gel electrophoresis. A further
aliquot was incubated for 10 min with Succ Ala,
pNA (3 mg in 0.5 ml of buffer), the absorbance
read at 4 1 0 n m and compared with that of an
appropriate blank. In this way, the point of functional (molar) equivalence for LE and alAT was
determined (Fig. 1).
Further aliquots of the initial reaction mixtures
were removed at various time intervals for up to
48 h and reassessed as above to determine the
2DIEP, SDS-polyacrylamide gel electrophoresis
pattern and residual enzyme activity.
Methods
Effect of Pseudomonas broth
Proteins studied
Human leucocyte elastase (99% pure) was purified by the method of Ohlsson & Olsson [6] and
was a gift from Dr A. J. Barrett (Strangeways
Laboratories, Cambridge, U.K.). The enzyme was
shown to be 68% functional by active-site titration
by the method of Nakajima et al. [7], consistent
with some inactivation as a result of purification
procedures. The protein was stored in phosphate
buffer (0.2 mol/l, pH 8.0) at -70°C and fresh
aliquots were used for each experiment.
a,-Antitrypsin was purified from fresh citrated
human plasma as described elsewhere [8]. The
concentration was evaluated immunologically with
a high titre monospecific antiserum characterized
previously [8] and compared with a protein
standard of known &,AT concentration (Seward
Laboratories).
A Pseudomonas aeruginosa culture broth supernatant containing elastase activity was obtained
from Dr A. Jackson. The original organism was a
mucoid strain, obtained from the sputum of a
patient with cystic fibrosis, and grown in a synthetic chemically defined culture medium [9].
In a further series of experiments, a small
amount of Pseudomonas aeruginosa culture broth
supernatant was incubated for 30 min with &,AT
at 30'C. This broth had been previously shown to
contain proteolytic enzyme with elastolytic
properties [ l o ] . After the period of incubation,
1.5
1.0.
0
3
$
9
0.5
0
1
2
3
Molar ratio LE/a,AT
Leucocyte eIastase-a,-antitrypsinreaction mixtures
The inhibition of LE by alAT was first assessed
by using the chromogenic substrate succinyl trialanyl p-nitroanilide (Succ Ala,pNA) purchased
from the Peptide Institute (Osaka, Japan). This
FIG. 1. Results from one experiment of increasing
amounts of LE added to a constant amount of
alAT. The horizontal axis is the functional molar
ratio of LE/alAT and the vertical axis the absorbance determined at 410 nm (equivalent to LE
activity).
a,-Antittypsin-leucocyte elastase interaction
the 2DIEP and SDS-polyacrylamide gel electrophoresis characteristics of the alAT were determined, as well as its ability to inhibit LE, by using
the chromogenic substrate Succ AlaJpNA (which
is not affected by the Aeudomonas enzyme).
lbodimensional immunoelectrophoresis
The basic method has been described previously [2] and the technique is demonstrated in
Fig. 2. In brief, aliquots of the alAT-LE or alATAeudomonas broth mixtures were placed in a well
cut in strips of plain agarose gel. A current was
applied (15 V/cm) until a bromophenol blue dye
marker had reached the end of the strip. A second
agarose gel containing alAT antibody was then
poured on to a glass plate and electrophoresis
conducted overnight (3 V/cm) at right angles from
the plain agarose strip. The plates were then
pressed, dried and stained with 2% kenacid blue
(British Drug Houses Ltd).
The immunological proportion of alAT present
as an enzyme-inhibitor complex was determined
by planimetry, as shown in Fig. 2. The total alAT
precipitation arc was measured and the area of
complex measured separately, either in total or as
the hatched area and doubled. Both techniques
gave comparable results when expressed as a percentage of the whole alAT precipitation area.
A similar study of complexing on fresh whole
human plasma was also performed. Increasing
amounts of LE to enzyme excess were added to a
fixed volume of the plasma and aliquots assessed
by 2DIEP as above.
219
Polyacry hmide gel electrophoresis
Aliquots of the LE-alAT mixtures were subjected to SDS-polyacrylamide gel electrophoresis
as described by Shapiro et el. [ l l ] . SDS/sucrose
was added to the aliquots to a final concentration
of 4%/6%respectively. Incubation was then continued for 2 h at 3 7 O C or the mixture was boiled
for 10 min only and a bromophenol blue marker
(0.25%; British Drug Houses Ltd) was added.
Each plate was run with a series of marker
proteins of known molecular weight. These included purified immunoglobulin G (Immunodiagnostic Research Laboratory, University of
Birmingham), transferrin (Hoeschst Pharmaceuticals), bovine serum albumin (Sigma), ovalbumin
(Hoeschst) and cytochrome C (British Drug Houses
Ltd).
Approximately 1 pg of each standard and each
reaction mixture was then subjected to electrophoresis into a 7% polyacrylamide gel (pH 8.6)
with a 5% stacking gel (pH 7.0) by using a vertical
slab system (Bio-Rad Laboratories, Hertfordshire,
U.K.). Electrophoresis was continued at 80 mV
until the bromophenol blue dye marker was 1 cm
from the anodal end of the gel. The gel was then
removed, measured and fixed in 10% trichloroacetic
acid before staining with 2% kenacid blue.
Effect of pH
The majority of experiments were performed at
pH 8.6 as described above. However, since the pH
of lung secretions studied previously [2] has ranged
FIG. 2. Stained two-dimensional electrophoresis plate of an a1AT-LE mixture. The
mixtures were applied to a well and electrophoresed from right to left. After this,
electrophoresis was performed from bottom to top into agarose containing alAT
antibody. Peak A, uncomplexed alAT; peak B, alAT-LE complex. The hatched area
wasmeasured, doubled and expressed as a percentage of the total area.
R. A. Stockley and S. C. Afford
220
from 6.7 to 8.8 (unpublished observation) we
carried out a further series of similar studies at pH
7.0, 6.6 and 6.0. The two last-named studies were
performed in 0.02 mol/l phosphate buffer.
iZ5E
50
h
Results
H
no-dimensional immunoelectrophoresis
6
The results of four experiments in which
samples of LE-alAT mixtures were incubated for
only 10 min are shown in Fig. 3. The addition of
increasing amounts of LE resulted in increasing
amounts of the alAT complex until functional
molar equivalence. At this point, 69.1%(SD f 6.9)
of the alAT was present as the electrophoretically
slow moving complex. The addition of greater
amounts of enzyme produced no further increase
in the complex and tended to result in a slight
decrease (e.g. 3 : 1 functional molar ratio LE/alAT
produced 55.9%complex; SD 3.6).
In contrast, similar studies with plasma demonstrated that at least 86.1%of the aIAT was capable
of forming electrophoretically distinct enzymeinhibitor complexes with LE (results not shown).
With time, there was a gradual disappearance
of this ‘complex’ seen on 2DIEP. The effect was
more rapid in the presence of functional excess of
LE. The results of preincubation of the mixtures
for 1 h is also shown in Fig. 3. At 1 h, no change
was seen in the proportion of ‘complex’ in the
presence of excess alAT up to molar equivalence,
whereas some disappearance was already seen in
the presence of excess enzyme ( 3 : l LE/a,AT
molar ratio = 24%).
*
loor
1
2
3
Molar ratio LE/(r,AT
FIG. 3. Degree of complexing between alAT and
LE obtained by two-dimensional immunoelectro-
phoresis. The horizontal axis is the functional
molar ratio of the proteins and the vertical axis
the percentage of alAT complex. 0-0, Mean results
from four experiments after 10 min incubation
the complex after 1 h
(bar lines f 1 SD);
incubation.
*-*,
h
E
0
1
2
3 4
Time (h)
24
48
FIG. 4. Effect of time (horizontal axis) on alAT
complex (vertical axis) for two mixtures: 0, 2:l
molar ratio alAT/LE; 0, 0.5 : 1 molar ratio aIAT/
LE .
The effect of longer periods upon the LE and
alAT mixtures is shown in Fig. 4. The complex
seen on 2DIEP rapidly disappears in the presence
of excess enzyme but a slow disappearance is also
seen in the presence of inhibitor excess.
The addition of small amounts of the Pseudomonas broth supernatant produced complete
inactivation of the alAT as an enzyme inhibitor.
The 2DIEP pattern of this inactivated alAT showed
no electrophoretically slow moving complex.
Furthermore, no such complex was produced by
the subsequent addition of LE to the inactivated
a1AT.
SDS-polyacrylamide gel electrophoresis
The SDS-polyacrylamide gel electrophoresis
results for our pure alAT preparation showed that
it was present in three forms. The major part consisted of two bands of molecular weight 53000
and 55000, consistent with the active inhibitor,
and the remainder gave a band of molecular weight
49 000, consistent with a degree of limited proteolysis. This is thought t o be a result of the purification procedure and accounts for the partial
inactivity of our alAT preparation and its failure
to produce as much enzyme-inhibitor complex
as the whole plasma tested (see above). Also, the
two bands of molecular weight 53 000 and 55 000
are thought to be due to minor differences in sialic
acid residues of the active alAT. Subsequent
neuraminidase treatment of our aIAT produced
only one band on SDS-polyacrylamide gel electrophoresis at 53 000 and the 49 000 band remained
unchanged .
The addition of LE t o alAT produced two new
bands seen on SDS-polyacrylamide gel electrophoresis of molecular weights approximately
82 000 and 66 000. This was associated with disap-
a1-Antitrypsin-leucocyte edastase interaction
221
FIG. 5 . SDS-polyacrylamide gel electrophoresis of alAT-LE mixtures. A, SDS-polyacrylamide gel electrophoresis for 2 h. The a1AT bands are indicated for the purified
protein alone (1 and 2, active alAT; 3, proteolysed alAT) and the full complex (C)
and intermediate complex (IC). Channels: a, LE; b, a l A T alone; c, 2 : l alAT/LE;
d, functional equivalence; e, 1: 2 alAT/LE; f, 2 : 1 aIAT/LE at 24 h; g, 1: 2 atAT/LE
at 24 h; h, a1AT alone. High molecular weight proteins run towards the top of the gel.
pearance of the alAT preparation bands at molecular weights 53 000 and 55 000. The complex band
was predominantly that of 82 000 molecular weight
for the samples boiled in SDS for 10 min alone
whereas intermediate complex (molecular weight
66 000) was clearly present in all samples incubated
with SDS for 2 h. With time and particularly in the
presence of excess LE these higher molecular
weight bands disappeared. All the alAT was concentrated in the 49 000 molecular weight band in
the samples exposed to excess LE whereas those
for excess inhibitor retained three bands at moleculat weights 55 000,53 000 and 49 000.
Inactivation by the Pseudomonas broth was
associated with a loss of the higher molecular
weight bands, all the alAT being present as a small
molecular weight form (molecular weight '49 000).
These experiments are summarized in Fig. 5.
a,AT (d
FIG. 6. Inhibitory capacity of alAT that had been
incubated with LE for 24 h at a molar ratio of 2 :1
alAT/LE (x, x = 107 pl), compared with fresh
alAT (0, x = 46 pl) and alAT incubated with
buffer only for 24 h (0,x = 53 111). The inhibition
lines are shown for each batch tested and the intercepts (amount of alAT solution required to cause
complete inhibition of 5 pg of LE) are indicated
Functional reassessment o f samples
above ( x ) .
In view of the demonstrable breakdown of
complex, the samples were reassessed for their
enzyme activity as well as the inhibitory capacity
of the alAT.
Samples which consisted of excess LE retained
some enzyme activity dependent upon the original
functional molar ratios. Similarly, samples with
excess inhibitor showed not only absence of
enzyme activity when reassessed, but also retained
the expected degree of functional alAT (Fig. 6).
Effect o f pH
The slow breakdown of alAT-LE complexes
seen on 2DIEP in the presence of excess alAT
was unaffected by alteration of the pH at which
the studies were conducted. The results of four
such studies at pH 6.0 and 8.6 are summarized in
Fig. 7.
However, differences in the rate of breakdown
of the complex at different pH values were seen in
R. A . Stockley and S. C Afford
222
5Or
I
"
0
1
2
4
Time (h)
4-'
FIG. 7. Rate of disappearance of alAT complex in
the presence of excess a1AT at pH 8.6 ( 0 ) and
6.0 (0). The vertical axis is the proportion of alAT
present as slow moving complex seen on twodimensional immunoelectrophoresis. The mean
value is shown at each time interval. The bar lines
are ?(: SD (n = 4).
,
0
1
4
2
4- '
Time (h)
FIG. 8. Rate of disappearance of a1AT complex in
the presence of excess LE is shown at pH 8 . 6 (o),
6.6 ( 0 ) and 6.0 (m). The bar lines are f 1 SD ( n = 4).
the presence of excess enzyme. Some of the results
are summarized in Fig. 8; The breakdown of complex was slow and similar in rate to that seen in
the presence of excess alAT when samples were
left at pH 6.0 (when LE is almost totally inactivated), but more rapid at pH values associated
with increasing activity of the free enzyme (pH
6.6, 7.0 and 8.6).
Discussion
The present study confirms that alAT inhibits LE
by the formation of enzyme-inhibitor complexes
which are readily demonstrable by 2DIEP and
SDS-polyacrylamide gel electrophoresis. Previous
workers have suggested that this interaction occurs
on a molar basis [5] and several aspects of the
current study would support the concept. Having
previously determined our enzyme activity from
active-site titrations, we have shown that only
about 70% of our alAT preparation was functional
as an inhibitor [8]. With interaction mixtures that
are prepared at functional molar equivalence, we
were able to demonstrate that about 69% of the
alAT was present as the electrophoretically slow
moving form consistent with enzyme-inhibitor
complex. Furthermore, this was associated with
disappearance of all the alAT visible in the 55 000
and 53 000 molecular weight bands on SDS-polyacrylamide gel electrophoresis (Fig. 5).
Although this proportion capable of forming
complex is lower than that of fresh plasma, we
believe this merely reflects some inactivation of
alAT during the purification procedure associated
with the presence of a partially proteolysed form
of a l A T (molecular weight 49000) and suggests
that all the remaining functional alAT is capable
of forming enzyme-inhibitor complexes.
The complexes formed on SDS-polyacrylamide
gel electrophoresis seem to be of two forms (molecular weights 82000 and 66000). The exact
explanation and nature of these complexes seems
uncertain at present, although similar full and
intermediate size complexes have been shown for
other enzymes inactivated by alAT [12,13]. The
intermediate complexes were only clearly seen in
samples left incubating with SDS for 2 h and may
represent a partial breakdown product of the full
complex. However, in the samples boiled with
SDS, intermediate complexes were absent even in
the sample with alAT excess over LE that had
been left for 24 h (see channel f of plates A and
B, Fig. 5). Subsequent studies with samples incubated with SDS for 2 h and then boiled for 10 min
have produced polyacrylamide gel electrophoresis
plates with only the 82 000 molecular weight complex. This suggests that the intermediate complex
may be an artifact due to failure to coat all the
protein with SDS adequately over 2 h, although
heat instability of the intermediate complex
remains a possibility. Further studies will be necessary to clarify this. Nevertheless, these complexes
were indistinguishable on 2DIEP.
The enzyme-inhibitor complexes showed dissociation with time. The effect on the complexes
seen on 2DIEP was rapid in the presence of excess
enzyme and associated with a change of the alAT
from the large molecular weight (82000) of the
enzyme-inhibitor complex to a small molecular
weight (49 000) protein, suggesting partial proteolysis. In view of the physical treatment and time
involved in coating the proteins with SDS and
running the gels, it was not thought appropriate
to analyse the SDS results in depth. However, the
a ,-Antitiypsin-leucocy te elastase interaction
results do suggest that breakdown of the enzymeinhibitor complexes is associated with the presence
of alAT that has undergone partial or limited
proteolysis (track f, Fig. 5). Similar changes have
been demonstrated when alAT interacts with pig
pancreatic elastase [14]. The alAT can either be
cleaved at the C-terminal end, inactivating it and
preventing complexing with enzyme, or be cleaved
at the N-terminal end, resulting in the formation
of enzyme-inhibitor complexes.
The rapid breakdown of the complexes seen on
2DIEP in the present study appears to be dependent upon the presence of an excess of active
enzyme, since lowering the pH, and hence enzyme
activity, reduces the rate of breakdown of the
complex. The exact nature of the process remains
uncertain without studies of the amino acid
sequences of the protein products generated.
The results in samples containing excess inhibitor are less clear since the assessment of the SDSpolyacrylamide gel electrophoresis results cannot
be comparable or quantitative. There is little
doubt that the enzyme-inhibitor complexes disappear slowly with time on SDS-polyacrylamide gel
electrophoresis as well as on 2DIEP and the rate
appears similar to that seen in enzyme excess after
the LE has been virtually inactivated by a pH
change (to 6.0). However, direct evidence of the
nature of the &,AT is not possible from this study.
Some indirect evidence can be obtained by using
our previous results on the immunological assessment of &,AT [8]. The immunological properties
of alAT are dependent upon the structural integrity
of the protein and we have shown that partial
proteolysis of alAT results in overestimation of
the protein quantitatively when compared with a
standard of native q A T alone and by using
polyclonal antiserum. Thus if breakdown of the
alAT-enzyme complex in alAT excess is associated
with the release of alAT that has undergone partial
proteolysis, we should be able to detect a change
in the immunological quantification in the sample
leading to an overestimation of the protein, which
increases as the complex disappears. This is shown
in Fig. 9, confuming an immunological change
suggesting that the release of partially proteolysed
alAT is also a feature of the breakdown of alATLE complexes in the presence of excess a1AT.
Nevertheless, this process results in the inactivation
of the LE, since no enzyme activity can be detected at 24 h and the remaining alAT retains its
expected inhibitory function (Fig. 6).
With the results obtained here, it is possible to
add further interpretation to previous studies of
lung alAT employing 2DIEP. In our previous
studies, we have found less alAT complex in lung
secretions containing free elastase activity, and the
223
180r
a,AT complex (% of total)
FIG. 9. Relationship between spontaneous breakdown of complex, at a 0.5:1 molar ratio of LE/
alAT, and the rise in immunological value for
alAT (determined by rocket immunoelectrophoresis) over 24 h. The horizontal axis is the
percentage of a1AT complex and the vertical axis
the alAT value (% of control, alAT alone). r =
-0.94.
non-complexed alAT was no longer functional as
an inhibitor. This contrasts with samples containing no active elastase where the alAT retained some
of its inhibitory function [2, 15, 161. We hypothesized that the alAT had been inactivated in
elastase excess, preventing the formation of complexes [ 2 ] , and the present study would suggest
this interpretation to be correct and arising from
the more rapid dissociation of enzyme-inhibitor
complexes in enzyme excess. Furthermore, subtle
electrophoretic changes suggested that the lung
alAT had undergone limited proteolysis [15,16]
and the present data confirm this as the probable
explanation.
With time, the enzyme-inhibitor complexes
break down completely and can no longer be
detected by 2DIEP. This effect is rapid in enzyme
excess where the enzyme remains active and would
produce alAT, which is proteolysed and no longer
functional. This may well explain the results of
previous workers [4,17,18] although only the
two last-named authors [17,18] commented on
the presence of excess enzyme in the samples.
However, interpretation of samples which
retain some a,AT inhibitory function is less clear.
Firstly, the breakdown of enzyme-inhibitor complexes is less rapid in a1AT excess though it can be
complete by 24-48 h. Obviously, the time between
formation and collection of lung secretions is critical to the interpretation of results and no such
data are currently available. Nevertheless, the
absence of alAT-enzyme complexes on 2DIEP
does not necessarily mean that interaction with
enzyme has not occurred. Furthermore, bacterial
enzymes, such as those studied here, and macrophage enzymes [19] can inactivate &,AT without
224
R. A . Stockley and S. C Afford
the formation of enzyme-inhibitor complexes and
may be responsible for some of the reduced alAT
function found in the secretions of smokers (31.
Clearly, more direct studies including assessment of the presence of alAT that has undergone
limited proteolysis in lung secretions are necessary
before firm conclusions can be reached.
In conclusion, the presence or absence of alAT
complexes in lung secretions provides little evidence
of whether such a change has occurred before collection without information concerning the nature
of the remaining alAT. Caution must therefore be
observed in the interpretation of mechanisms which
inactivate alAT in lung secretions.
Acknowledgments
We thank Dr D. Burnett for his comments on the
manuscript and Mrs C. Seymour for her typing.
The study was supported by grants from the West
Midlands Regional Health Authority, the Endowment Fund and Boehringer Ingelheim.
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