New chemotactic dimeric peptides show high affinity and potency at the human formylpeptide receptor

Article abstract of DOI:10.1016/j.ejphar.2007.04.006

A number of analogues of the prototypical peptide for-Met-Leu-Phe-OMe (fMLP-OMe) have been studied in order to evaluate their ability to interact with formylpeptide receptors and to induce specific biological responses in human neutrophils. In vitro assay

Full text of DOI:10.1016/j.ejphar.2007.04.006

European Journal of Pharmacology 567 (2007) 171–176
www.elsevier.com/locate/ejphar
New chemotactic dimeric peptides show high affinity
and potency at the human formylpeptide receptor
Susanna Spisani a, Anna Fraulini c, Katia Varani b, Sofia Falzarano a, Giorgio Cavicchioni c,
⁎
a
Department of Biochemistry and Molecular Biology, University of Ferrara, 44100 Ferrara, Italy
b
Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, 44100 Ferrara, Italy
c
Department of Pharmaceutical Sciences, University of Ferrara, 44100 Ferrara, Italy
Received 7 November 2006; received in revised form 26 March 2007; accepted 3 April 2007
Available online 14 April 2007
Abstract
A number of analogues of the prototypical peptide for-Met-Leu-Phe-OMe (fMLP-OMe) have been studied in order to evaluate their ability to
interact with formylpeptide receptors and to induce specific biological responses in human neutrophils. In vitro assays were carried out and
receptor binding, chemotaxis, superoxide anion release and secretagogue activity were evaluated. The fMLP-OMe analogues synthesized, with the
general formula for-Met-Leu-Phe-Xaa-Lys(OMe)-Phe-Leu-Met-for (Xaa=Gly, β-Ala, γ-aminobutyric acid, 5-aminovaleric acid, and 6-
aminocaproic acid), were constituted by two fMLP units linked by a Lys residue, with an amino acid spacer between them. Competition binding
experiments revealed that the new compounds have much more affinity for formylpeptide receptors than the reference ligand, with good
correlation between receptor affinity and length of spacer. The EC50 values for the killing mechanisms of each analogue were similar to each other,
the affinity and potency, once again, being strictly dependent on the chain length. Furthermore the analogues proved to be more potent full
agonists than the prototype fMLP-OMe in these functions, while chemotaxis was poorly induced. The dimeric fMLP-OMe analogues are one of
the few examples of formylpeptides which exhibit a receptor affinity greater than the parent fMLP-OMe thereby rendering them suitable to be
used as carriers for various drugs.
© 2007 Elsevier B.V. All rights reserved.
Keywords: N-formylmethionyl peptide; Human neutrophil; Receptor binding; Chemotaxis; Superoxide anion generation; Lysozyme release
1. Introduction
to its receptor (Liu et al., 2004) leads to the activation, not only
of direction-specific movement [chemotaxis], but also a wide
range of functions required for host defence, including the
respiratory burst, phagocytosis and lipid mediator synthesis
(Ferretti et al., 2001; Niggli, 2003; Selvatici et al., 2006).
It has previously been demonstrated that the binding sites of
the fMLP receptor have two affinity states. fMLP binds the
formylpeptide receptor subtype, termed FPR, with high affinity,
and is activated by very low concentrations of the ligand, while
a second subtype (termed FPRL1) is activated by high concen-
trations of fMLP and is defined as a low-affinity receptor
(Durstin et al., 1994; Murphy, 1996; Prossnitz and Ye, 1997; Le
et al., 2002).
Neutrophils play a key role as primary phagocytic cells,
migrating along a concentration gradient of chemoattractant
molecules to infected and inflamed tissues (Kobayashi et al.,
2003), thereby constituting the first line of defence against
bacterial invasion contributing to inflammatory processes. A
variety of neutrophil cellular functions are stimulated by a
number of peptides such as formylpeptides, chemokines and
C5a. The N-formyltripeptide for-Met-Leu-Phe-OH (fMLP),
generated as a by-product or degradation product of bacterial
metabolism or deriving from disrupted mitochondria (Carp,
1982; Marasco et al., 1984), and its methylester derivative for-
Met-Leu-Phe-OMe (fMLP-OMe) (Schiffmann et al., 1975) are
reference chemotactic peptides. The binding of a formylpeptide
We decided to focus our attention on dimeric fMLP analogues
linked by a Lys residue and with amino acids of various lengths as
spacers because dimeric ligands of bioactive molecules, such as
dimeric enkephalins, neurokinins, and bradykinins, spaced with
methylene chains, show strong activity together with good
⁎
Corresponding author.
E-mail address: g5z@unife.it (G. Cavicchioni).
0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2007.04.006

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S. Spisani et al. / European Journal of Pharmacology 567 (2007) 171–176
selectivity (Shimohigashi et al., 1982; Kondo et al., 1986; Kodama
et al., 1998a; Kodama et al., 1988b; Cheronis et al., 1992). From
these studies it appears that dimeric ligands may exhibit an
increased affinity, a function of the spacer length, as compared to
the corresponding monomeric analogues. Miyazaki et al. (1995)
synthesized dimeric fMLP analogues cross-linked with hydro-
philic mono-, di-, tri, and tetra-ethylene glycols. In chemotaxis
assays, all dimers showed higher activities in human neutrophils
thantheir monomeric prototype, and themagnitude of the response
was dependent on linker length. These dimeric fMLPs can also be
regarded as selective agonists for chemotaxis, as superoxide anion
production was not triggered with the same strength.
Based on the above reports, and as a continuation of our
previous studies in this field, we decided to study a new group
of dimeric fMLP-OMe analogues, with the general formula for-
Met-Leu-Phe-Xaa-Lys(OMe)-Phe-Leu-Met-for [Xaa=Gly 1,
β-Ala 2, γ-aminobutyric acid (GABA) 3, 5-aminovaleric acid
4, and 6-aminocaproic acid 5] (Fig. 1), in order to shed light on
the role of spacers, amino acids of various length, on the
properties and activity of chemotactic N-formylpeptides. The
agonist properties of these peptide analogues on human neutro-
phils were analysed by means of receptor binding assays, mea-
surements of chemotaxis, superoxide anion production and
lysosomal enzyme secretion.
Fig. 2. Scheme of synthesis.
were determined on a Reichert-Kofler block and remain
uncorrected.
Purification of all final products was achieved by reverse-
phase High Performance Liquid Chromatography (HPLC)
analysis on a Waters Delta Prep 3000. Products were revealed
with a Waters 484 UV spectrophotometer at 220 nm using, as a
stationary, less polar phase, a Delta Pack C 18-300 A column
(0.3×30 cm, particles 15 μm) with the correct eluting system.
Thin-layer chromatography was performed on pre-coated
silica gel F254 [Merck, Germany] with a dichloromethane:
toluene:methanol (17:1:2) solvent system.
Satisfactory microanalyses were obtained for all com-
pounds, analytical results being within 0.4% of the theoretical
values.
2. Materials and methods
Amino acid residues were purchased from Fluka (Milan,
Italy) as hydrochlorides. The t-Boc amino acids were synthe-
sized using di-tert-butyl dicarbonate as the di-tert-butyloxycar-
bonylating agent. Removal of the t-Boc group was performed by
treatment with a 1:1 mixture of trifluoroacetic acid [TFA]:
chloroform (CHCl3). Peptide coupling was achieved by the 1-
hydroxy-benzotriazole (HOBt)-N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide (EDC) hydrochloride method, while the
formyl group was introduced according to the N-ethoxycarbo-
nyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) method.
2.1. Chemistry
Molecular weight was determined with a MALDI-TOF
(Matrix Assisted Laser Desorption Ionization Time of Flight),
using a Hewlett Packard G2025A LD-TOF as mass spectromet-
ric system and α-cyano-4-hydroxycinnamic acid as a matrix.
Optical rotations were determined in MeOH at 20 °C
with a Perkin-Elmer Model 241 polarimeter. Melting points
2.1.1. Synthesis
The N-terminal fMLP-OH was coupled to H-Xaa-OMe, and
the resulting for-Met-Leu-Phe-Xaa-OMe was hydrolysed to
render it able to couple the carboxy-terminal fMLP. Synthesis
followed standard procedures in solution [Fig. 2].
for-Met-Leu-Phe-Gly-Lys[OMe]-Phe-Leu-Met-for 1 —
Colourless solid (mp. 115–118 °C; [α]D20 =−26.9° in MeOH,
c=1). Molecular ion mass spectr. plus Na [MS+Na]+ 1079.6.
Molecular ion mass spectr. plus K [MS+K]+ 1095.8.
for-Met-Leu-Phe-βAla-Lys[OMe]-Phe-Leu-Met-for 2 —
Colourless solid (mp. 111–113 °C; [α]D20 =−25.2° in MeOH,
c=1). Molecular ion mass spectr. plus H [M+H]+ 1070.9.
for-Met-Leu-Phe-GABA-Lys[OMe]-Phe-Leu-Met-for 3 —
Colourless solid (mp. 138–140 °C; [α]D20 =−7.3° in MeOH,
c=1). Molecular ion mass spectr. plus H [M+H]+ 1085.8. Mole-
cular ion mass spectr. plus Na [MS+Na]+ 1107.6. Molecular ion
mass spectr. plus K [MS+K]+ 1123.6.
for-Met-Leu-Phe-5-aminovaleric acid-Lys[OMe]-Phe-Leu-
Met-for 4 — Colourless solid (mp. 120–123 °C; [α]D20 =−15.3°
in MeOH, c=1). Molecular ion mass spectr. plus Na [MS+Na]+
1122.8. Molecular ion mass spectr. plus K [MS+K]+ 1138.5.
Fig. 1. Chemical structure of analogues.

S. Spisani et al. / European Journal of Pharmacology 567 (2007) 171–176
173
for-Met-Leu-Phe-6-aminocaproic acid-Lys[OMe]-Phe-Leu-
Met-for 5 — Colourless solid (mp. 132–135 °C; [α]D20 =−20.8°
in MeOH, c=1). Molecular ion mass spectr. plus Na [MS+Na]+
1135.3. Molecular ion mass spectr. plus K [MS+K]+ 1151.7.
Italy), and migration into the filter was evaluated by the leading-
front method according to Zigmond and Hirsch (Zigmond
and Hirsch, 1973). The random movement used as control was
32 μm 3 SE of ten separate experiments performed in
duplicate. Chemotaxis was studied by adding each peptide to
the lower compartment of the chemotaxis chamber. Peptides
were diluted from a stock solution (10−2 M in DMSO) with
KRPG containing 1 mg/ml of bovine serum albumin and used at
concentrations ranging from 10−13 to 10−5 M. Data were
expressed in terms of the chemotactic index (C.I.), being the
ratio: (migration toward test attractant minus migration toward
the buffer)/(migration toward the buffer).
2.2. Biological assays
2.2.1. Peptides
10−2 M stock solutions of fMLP-OMe (Sigma Chemical Co.
St. Louis MO, U.S.A.) and the alkyl spacer analogues were
prepared in dimethyl-sulfoxide (DMSO, Sigma) and diluted in
Krebs–Ringer-phosphate containing 0.1% w/v glucose [KRPG,
pH 7.4], before use. KRPG was made up as a stock solution
with the following composition: NaCl, 40 g/l; KCl, 1.875 g/l;
Na2HPO4. 2H2O, 0.6 g/l; KH2PO4, 0.125 g/l; NaHCO3, 1.25 g/l;
glucose, 10 g/l. This solution was five times working strength.
1 mM MgCl2 and CaCl2 supplemented the buffer before
biological tests. All reagents were of the purest commercially
available grade.
2.2.5. Superoxide anion production
Superoxide anion production was measured by the superox-
ide dismutase-inhibitable reduction of ferricytochrome C modi-
fied for microplate-based assays (Cavicchioni et al., 2003).
Tests were carried out in a final volume of 200 μl containing
4×105 neutrophils, 100 nmol cytochrome C (Sigma) and
KRPG. Neutrophils were pre-incubated with 5 μg/ml cytocha-
lasin B [Sigma] for 5 min prior to activation by peptides. At zero
time, different amounts (10−11–10−5 M) of each peptide were
added, and the plates were incubated in a microplate reader
[Ceres 900, Bio-Tek instruments, INC] with the compartment T
set at 37 °C. Absorbance was recorded at wavelengths of 550
and 468 nm. Differences in absorbance at the two wavelengths
were used to calculate nmol of O−2 produced, using a molar
2.2.2. Preparation of human neutrophils
A sample of cells were obtained from the peripheral blood of
healthy subjects, and neutrophils were extracted employing the
standard techniques of dextran sedimentation (Pharmacia,
Uppsala, Sweden), centrifugation on Ficoll-Paque (Pharmacia)
and hypotonic lysis of contaminating red blood cells. The cells
were washed twice and resuspended in KRPG at a final
concentration of 50×106 cells/ml and used immediately. The
neutrophil purity was 98–100% and viability ≥99%, as deter-
mined by the Trypan blue exclusion test. All donors were non-
smokers and none had received any medication during the
3 days preceding donation. The study was approved by the local
Ethics Committee, and informed consent was obtained from all
participants.
extinction coefficient for cytochrome C of 18.5 mM−1 cm−1
.
2.2.6. Granule enzyme assay
Release of neutrophil granule enzymes was evaluated by
determining lysozyme activity modified for microplate-based
assays (Cavicchioni et al., 2003). 3×106 cells were incubated in
microplate wells in the presence of each peptide, with a final
concentration of 10−11–10−5 M, for 15 min at 37 °C. The plates
were then centrifuged for 5 min at 400 g, and the lysozyme was
quantified nephelometrically by the rate of lysis of cell wall
suspension of Micrococcus lysodeikticus [Sigma]. Neutrophils
were pre-incubated with 5 μg/ml cytochalasin B for 15 min at
37 °C, prior to activation by the peptides. Reaction rate was
2.2.3. Receptor binding assays
Binding experiments were carried out according to Spisani
et al. (2003). In saturation assays, human neutrophils were
incubated with 10–14 different concentrations of [3H]fMLP,
ranging from 1 to 300 nM, in a total volume of 250 μl
containing KRPG supplemented with 1 mM MgCl2 and 1 mM
CaCl2 at pH 7.4. The incubation time was 15 min at 37 °C, as
suggested by previous time course experiments. In competition
experiments, carried out to determine Ki values, 10 nM of [3H]
fMLP was incubated with 100 μl of human neutrophils (5×106)
and at least 6–8 different concentrations of the examined
compounds at 37 °C for 15 min. Non-specific binding was
measured in the presence of 10 μM fMLP, and constituted
approximately 20% of total binding. Bound and free radioac-
tivity was separated by rapid filtration through Whatman GF/C
glass-filters, which were washed with ice-cold buffer. The filter-
bound radioactivity was measured by a scintillation spectrom-
eter with an efficiency of 57%.
Fig. 3. Saturation of [3H]fMLP binding to human neutrophils. The Kd value was
32.5 3.6 nM and the Bmax was 23 3 fmol/106 cells. Values are expressed as the
mean, and vertical lines are the standard error of the mean (S.E.M.) of three
independent experiments. The inset is a Scatchard plot of the same data.
2.2.4. Random locomotion and chemotaxis
Random locomotion and chemotaxis studies were performed
with a 48-well microchemotaxis chamber (BioProbe, Milan,

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S. Spisani et al. / European Journal of Pharmacology 567 (2007) 171–176
Fig. 4. Competition curves of specific [3H]fMLP binding to human neutrophils
by the test compounds. The curves represent the mean of a series of five
independent experiments performed in duplicate. Non-specific binding was
determined in the presence of 10 μM fMLP-OMe.
Fig. 5. Chemotactic activity of fMLP-OMe and its synthetic derivatives towards
human neutrophils. The data expressed are the means of five separate experi-
ments performed in duplicate. S.E.M.s are in 0.02–0.09 chemotactic index
range.
measured with a microplate reader at 465 nm. Enzyme release was
expressed as the net percentage of total enzyme content released
by 0.1% Triton X-100. Spontaneous release was less than 10%
and total enzyme activity was 85 1 μg/1×107 cells/min.
The Scatchard plot was essentially linear, indicating that only one
affinity-binding site was present under our experimental condi-
tions. The dissociation binding constant (Kd) was 32.5 3.6 nM,
and the receptor density (Bmax) was 23 3 fmol/106 cells.
Fig. 4 displays the competition curves of compounds 1–5 in
human neutrophils. These compounds showed affinity values in
the nanomolar range, with higher affinity towards formylpep-
tide receptors than fMLP-OMe. The order of potency in [3H]-
fMLP displacement assays for the tested peptide was: 1N2N3N
4N5, indicating that affinity is linked to spacer length. Indeed
the peptide carrying the smallest spacer had the highest affinity
(Table 1). In particular, the affinity values of the fMLP-OMe-
derived compounds are in a similarly narrow range, 3.5 0.4≤
IC50 ≤8.2 0.8 nM, and are one order of magnitude lower than
fMLP-OMe (52 6 nM), thereby suggesting that the new com-
pounds have a much higher affinity with respect to the reference
ligand.
2.3. Statistical analysis
Saturation and competition binding experiments were analysed
with the program LIGAND (Munson and Rodbard, 1980), which
performs a weighted, non-linear, least-square curve-fitting pro-
gram. Inhibitory binding constant, Ki, values were also calculated
from the IC50 values according to the Cheng and Prusoff equation
(Cheng and Prusoff, 1973). EC50 values obtained in biological
assays were computed by non-linear regression analysis using the
equation for a sigmoid concentration–response curve (GraphPAD
Prism, San Diego, CA, USA). All experimental data were ex-
pressed as the arithmetic mean standard error of the mean (SEM)
of five independent experiments performed in duplicate. Analysis
of difference on EC50 values between new examined compounds
and FMLP data was done with Student's t test. Differences were
considered significant at a value of pb0.05.
3.2. Biological activity
The biological activity of the synthetic peptides was deter-
mined on human neutrophils. Migration activity and killing
mechanisms, i.e. superoxide anion production and granular
lysozyme release are shown in Figs. 5, 6 and 7, respectively.
3. Results
3.1. Saturation and competition binding experiments
Fig. 3 shows a saturation curve of [3H]fMLP, with the
corresponding Scatchard plot in the inset, on human neutrophils.
Table 1
fMLP-OMe receptor binding and biological activity for dimeric analogues
Peptide
Receptor binding Chemotaxis O−2 production Lysozyme
Ki (nM)
EC50 (pM)
EC50 (nM)
EC50 (nM)
fMLP-OMe 41.5 4.7
6.1 0.5
190 25
160 22
120 15
27.2 3.4
2.4 0.3
4.2 0.5
8.2 0.9
7.2 0.8
5.7 0.6
11.5 1.3
1.2 0.2
3.2 0.4
4.5 0.5
5.8 0.6
6.1 0.7
1
2
3
4
5
2.7 0.3
3.3 0.4
4.2 0.5
4.9 0.6
6.4 0.7
60
8
90 10
Data are mean S.E.M. from five independent experiments. Ki values represent
the concentration of drug able to displace 50% of the radioligand. EC50 is the
concentration of tested compounds that inhibits the 50% of the maximal effect.
Fig. 6. Superoxide anion production of fMLP-OMe and its synthetic derivatives
towards human neutrophils. The data reported are the means of five separate
experiments performed in duplicate. S.E.M.s are in 0.1–4 nmol O−2 range.

S. Spisani et al. / European Journal of Pharmacology 567 (2007) 171–176
175
These analogues showed a selective ability in stimulating
human neutrophils. In fact, as a general feature, they are all able
to trigger killing mechanisms, although chemotaxis is poorly
induced.
These data are not surprising as it has long been known that
the transduction pathway underlying the chemotactic response
is different from those responsible for superoxide anion produc-
tion and lysozyme release (Selvatici et al., 2003), as distinct
mechanisms are involved in each of these neutrophil responses.
In fact, for each stimulus capable of a unique set of cellular
responses, a distinctive imprint of signal protein activation may
exist. Moreover, it has been demonstrated that each physiolog-
ical function shows different requirements for receptor
occupancy. Superoxide anion production requires continuous
occupancy of almost 100% of the receptors to achieve and
maintain an optimal response, whereas chemotaxis does not
(Prossnitz and Ye, 1997).
Fig. 7. Release of neutrophil granule enzymes evaluated by determining
lysozyme activity induced by fMLP-OMe and its synthetic derivatives. The data
expressed are the means of five separate experiments performed in duplicate.
S.E.M.s are in 1–6% range.
Table 1 summarizes the EC50 values of the synthetic com-
pounds in each assay.
In particular, it was found that:
3.2.1. Chemotaxis
1. Compounds 1–5 exhibited a lower chemotactic efficacy with
respect to the prototype fMLP-OMe; a good correlation
between potency and spacer length emerges, thereby sug-
gesting that the peptide carrying the smallest spacer has the
lowest potency.
Chemotactic responses of human neutrophils to fMLP-OMe
and 1–5 analogues are shown in Fig. 5. All peptides, both the
reference fMLP-OMe and dimeric analogues, evidence optimum
concentration at 10−9 M, even though compounds 1–5 exhibited
a lower efficacy (pb0.01, versus EC50 values) with respect to the
prototype. In particular, the potency of compounds 4 and 5 was
slightly higher (60 pMbEC50b90 pM) than compounds 3, 2 and
1 (120 pMbEC50b190 pM), with an order of potency of 4N5N
3N2N1. All of the new compounds show a less potent chemo-
tactic response than fMLP-OMe (EC50=6.1 pM).
2. Both superoxide anion production and lysosomal enzyme
release can be efficaciously triggered by peptides; the
maximal activity was seen for peptides bearing the shorter
chains. These analogues are some of the few examples of
formylpeptides which exhibit a greater receptor affinity than
the parent fMLP-OMe. Indeed, their ability to bind FPR is
approximately ten times greater than that of the prototype.
3. The results of the above reported binding studies are in
accordance with high activity in relation to O2− production
and lysosomal enzyme release, but not with the weak activity
found in chemotaxis. This different behaviour can be
rationalized on the basis of the existence of at least two
different functional receptor subtypes or isoforms (Dalpiaz
et al., 2003). Low doses of a full agonist (or a “pure” chemo-
attractant) are required to interact with a high-affinity
receptor subtype which activates the transduction pathway
responsible for chemotactic response, while an increase in
agonist concentration allows binding with the low-affinity
subtype, able to activate the transduction pathways respon-
sible for O2− production and lysozyme release. As a conse-
quence, a peptide selective for killing mechanisms which
preferentially interacts with the low-affinity subtypes is
efficient in effectively displacing the full agonist [3H]-fMLP
(Cavicchioni et al., 2005).
3.2.2. Superoxide anion production
In superoxide anion production tests (Fig. 6), all dimers
exhibited higher activity than the monomeric fMLP-OMe
(pb0.01, versus EC50 values). EC50 values were in the high
nanomolar range, 2.4 nMbEC50b8.2 nM, with respect to the
prototype (EC50=27 nM). The maximal activity was seen for
peptides 1 and 2 [1N2]. In particular, peptide 1 [-Gly-Lys(OMe)-]
was 10 times more potent than the prototype. The order of potency
of the other peptides was: 5N4N3.
3.2.3. Lysozyme enzyme release
Fig. 7 shows the dose–response curves relative to the
capability of dimeric derivatives to stimulate lysosomal enzyme
release with respect to the reference compound. Once again,
peptide 1 was 10 times more potent than the prototype (pb0.01,
versus EC50 values). The EC50 values of O2− production and
lysozyme release were very similar.
The results have been quantitatively reported in Table 1 for a
comprehensive picture. Comparisons were made between each
peptide and the parent fMLP-OMe.
The availability of ligands capable of differentiating between
killing mechanisms and chemotactic activity should provide
useful tools in further investigation of the structural requirements
of FPR, and should aid research into how the overall fit into the
receptor pocket can be influenced by structural modifications by
adding amino acid spacers in dimeric fMLP-OMe and to design
new potential peptide drugs in inflammatory diseases.
Additional studies are planned to further explore these
topics.
4. Discussion
A series of dimeric formyltripeptides were synthesized in
order to specifically investigate their ability to better stimulate
and/or selectively trigger neutrophil functions.

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S. Spisani et al. / European Journal of Pharmacology 567 (2007) 171–176
COOH-terminal heptapeptide fragments enhanced the selectivity for
Acknowledgments
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Le, Y., Murphy, P.M., Wang, J.M., 2002. Formyl-peptide receptors revised.
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This work was supported by MURST [Research Funds ex
60%] and the Fondazione Cassa di Risparmio di Ferrara, Italy.
We are grateful to the Banca del Sangue of Ferrara for providing
fresh blood and Anna Forster for the English revision of the
text.
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