Free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic Escherichia coli.

Abstract

The non-immunoglobulin component of human milk responsible for the inhibition of Escherichia coli cell adhesion (haemagglutination) mediated by colonisation factor antigen 1 (CFA1) was determined by chromatographic fractionation of human whey proteins with Sephadex G-200, DEAE cellulose and heparin-sepharose. Pure free secretory component (fSC) and pure lactoferrin (Lf) were isolated and both compounds inhibited the haemagglutination induced by E. coli CFA1+. The lowest concentrations of purified fSC and Lf able to inhibit the haemagglutination induced by E. coli strain TR50/3 CFA1+ were 0.06 mg/ml and 0.1 mg/ml respectively. Commercially available lactoferrin from human milk and transferrin from human serum, which has a great structural analogy to lactoferrin, also inhibited the haemagglutination. The lowest concentrations of the commercial lactoferrin and transferrin able to inhibit the haemagglutination induced by E. coli TR50/3 CFA1+ were 0.03 mg/ml and 0.4 mg/ml, respectively. These results indicate that fSC and Lf may be important non-specific defence factors against enterotoxigenic E. coli infections.

Full text

Page 1

J. Med. Microbiol. -
0 1995 The Pathological Society of Great Britain and Ireland
Vol. 42 (1995), 3-9
HOST DEFENCE MECHANISMS
Free secretory component and lactoferrin of human milk
inhibit the adhesion of enterotoxigenic Escherichia co/i
L. G. GIUGLIANO, S. T. G. RIBEIRO, M. H. VAINSTEIN and C. J. ULHOA"
Laboratorios de Microbiologia e * Enzimologia, Departamento de Biologia Celular, lnstituto de Biologia,
Universidade de Brasflia, 709 10- 900 Brasgia OF, Brazil
Summary. The non-immunoglobulin component of human milk responsible for the inhibition
of Escherichia coli cell adhesion (haemagglutination) mediated by colonisation factor antigen
1 (CFAl) was determined by chromatographic fractionation of human whey proteins with
Sephadex G-200, DEAE cellulose and heparin-sepharose. Pure free secretory component
(fSC) and pure lactoferrin (Lo were isolated and both compounds inhibited the haemag-
glutination induced by E. coli CFAl+. The lowest concentrations of purified fSC and Lf able
to inhibit the haemagglutination induced by E. coli strain TR50/3 CFA1' were 0-06 mg/ml
and 0.1 mg/ml respectively. Commercially available lactoferrin from human milk and
transferrin from human serum, which has a great structural analogy to lactoferrin, also
inhibited the haemagglutination. The lowest concentrations of the commercial lactoferrin
and transferrin able to inhibit the haemagglutination induced by E. coli TR50/3 CFA1+ were
0.03 mg/ml and 0-4 mg/ml, respectively. These results indicate that fSC and Lf may be
important non-specific defence factors against enterotoxigenic E. coli infections.
Introduction
Enterotoxigenic Escherichia coli (ETEC) is one of
the pathogens isolated most frequently from children
with diarrhoea in developing c~untries.l-~
of human ETEC strains to adhere to and colonise the
intestinal epithelium is associated with the presence of
colonisation factor antigens (CFA) in the bacterial
s~rface.~ Several CFAs have been characterised but
CFAl is the one found most frequently in ETEC
strains.5*
Many epidemiological studies of diarrhoea have
shown that breast feeding protects infants from in-
testinal infection^.^,^ The protective effect of human
milk has been attributed to its immunoglobulin
content, mainly to secretory immunoglobulin A
(~IgA),~-ll and to non-specific defence factors such
as lactoferrin (Lo, lysozyme, bifidus factor and
oligosaccharides .
Lf is an 80-kDa glycoprotein, found in high
concentrations in human milk,12?16 which has been
shown to inhibit bacterial growth in vitro. This
property is ascribed to its iron binding capacity that
leads to iron deprivation for the micro-organism.l29
It is also known that Lf binds to various microbial
and this binding seems to enhance its
The ability
'9 12-15
1 7 9 l8
Received 30 March 1994; accepted 17 June 1994.
antimicrobial capacity.23 Moreover, study of the
specific binding of Lf to enteropathogenic (EPEC),
enterotoxigenic (ETEC), enteroinvasive (EIEC) and
enterohaemorrhagic (EHEC) strains of E. coli showed
that ETEC strains bound more Lf than the other
groups of enter~pathogens.~~
The protective effect of human milk has also been
thought to be due to milk components that could act
as cell receptor analogues for bacterial adhesins and
enterotoxins. As the cell receptors are probably
glycoconjugates containing a receptor-specific oligo-
saccharide m ~ i e t y , ~ ~ - ~ ~ the receptor analogues should
be glycocompounds. Indeed, it has been shown that
fucosylated oligosaccharides are associated with the
inhibition of CFA 1 - and CFA2-mediated adhesion of
ETEC strains26 and inhibit localised adhesion of EPEC
strain^.^
Human milk contains many glycocompounds, some
of which are rich in fucosylated oligosaccharides, such
as Lf and free secretory component (fSC), which are
found in abundance.l2' l6,
ponent (SC) mediates the transport of sIgA into
external fluids on mucosal epithelial cells, and can be
found in secretions both complexed with sIgA (bound
SC) or as an uncomplexed 80-kDa glycoprotein called
free SC.31-33
This study investigated a possible role for glyco-
proteins from human milk in the inhibition of the
28-30 Th e secretory com-
3

Page 2

4
L. G. GIUGLIANO ET AL.
adhesion (haemagglutination) induced by ETEC
CFA1+ strains, assaying, step by step, every fraction
obtained in the chromatographic fractionation of the
milk.
Materials and methods
Breast milk samples
Human milk samples were obtained from the
Human Milk Bank of the Hospital Regional da Asa
Sul (Brasilia DF). Aliquots of individual frozen
samples were taken from at least 10 lactating mothers
up to 1 month after delivery and pooled.
Fractionation of human milk
The pooled human milk was diluted with an equal
volume of buffer (0.1 M Tris-HC1, pH 7.6, sup-
plemented with 0.5 M NaC1, 1 m M phenylmethane-
sulphonyl fluoride, NaN, 0.1 YO and 50 m M e-amino
n-caproic acid). The mixture was centrifuged at
15 000 g at 4°C for 1 h to remove lipids and cells. The
middle layer was collected, acidified to pH 4.2 with
acetic acid 2 % and centrifuged at 15 000 g for 40 min
to remove casein. The clear supernatant fluid was
removed and its pH was adjusted to neutrality with
0.1 M NaOH. Ammonium sulphate was added to 70 YO
final saturation and the mixture was kept overnight at
4°C. The precipitate which formed was collected by
centrifugation, dissolved in the buffer and dialysed
overnight at 4°C against the buffer. The sample was
then applied to a Sephadex G-200 column equilibrated
with the buffer.
Purification o f human milk fSC
Purification of fSC from the fractions eluted in the
second peak after Sephadex G-200 chromatography
was performed as described previo~sly.~~
fractions were pooled and dialysed against 0.0 1 M Tris-
HCl, pH 7.6, containing 0.05 M NaCl, and applied to a
DEAE cellulose column equilibrated with the same
buffer. Free SC is not retained and was eluted by
washing the column with the buffer. For further
purification, the eluted material was concentrated
by adding (NH,),SO, to 70% final saturation. The
precipitate which formed was collected by centri-
fugation and subsequently dissolved in and dialysed
overnight at 4°C against 0.05 M Tris-HCl, pH 8.0,
containing 0.2 M NaC1. This material was then applied
to a heparin-sepharose affinity column equilibrated
with the same buffer.
Briefly, the
Electrophoretic procedures
The protein content of the fractions was measured
by the method of Bradford.35
All fractions from the chromatographic procedures
were monitored before pooling by SDS-PAGE on
acrylamide 10% gels by the method of Laem~~lli.,~
Fractions from the heparin-sepharose chromato-
graphy were concentrated by precipitation with
trichloro-acetic acid 10 YO before electrophoresis. Gels
were stained with Coomassie Brilliant Blue G and also
with ~ilver.~'
Two-dimensional electrophoresis was performed3'
and the gels were stained with silver.
Purification of lactoferrin
Purified Lf was obtained either as a co-purification
product of the procedure used to obtain fSC, or by a
single chromatographic step on a heparin-sepharose
column equilibrated with 5 m M Veronal-HC1 buffer,
pH 7.4, containing 0.05 M NaC1.39 Lf binds more
strongly to heparin-sepharose than do the other whey
proteins, and it can be eluted from the resin in a pure
form with c. 0-6 M NaCl.
Inhibition assay
The test system used was agglutination of human
group A erythrocytes by ETEC CFAl+-mediated
a d h e s i ~ n . ~ ~ ' ~ ~ The ETEC CFA1+ strains used were
TR 50/3, kindly provided by Dr B. C. Guth, Escola
Paulista de Medicina, Sgo Paulo, Brazil and m452-C1
and CD79a provided by Dr J. Blanco, Universidade
de Santiago de Compostela, Lugo, Spain. The
erythrocytes were collected in sodium citrate and
washed three times in saline. The bacterial strains were
grown overnight in CFA medium,,, harvested, washed
two or three times in saline and resuspended in saline
to a concentration of 300 Klett units (measured in a
Klett-Summerson photo-electric colorirneter fitted
with a green filter), equivalent to 1.5 x lo9 cfu/ml.
The milk samples or fractions to be analysed were
serially diluted in 25pl of saline in plastic micro-
titration trays (Linbro Chemical Co., Inc., New
Haven, CT, USA). To each well, 25 pl of the bacterial
suspension was added and the trays were first
incubated for 15 min at room temperature and then
kept for 2 h at 4°C. Subsequently, 25 pl of the washed
erythrocytes 3 YO in saline containing D-mannose 4 %
were added and the trays were kept for 2 h at 4°C. The
haemagglutination pattern observed was graded from
- (no agglutination) to + + + + (complete aggluti-
nation) and the 50% agglutination reaction was
determined by interpolation when needed.
The highest dilution that inhibited 50% of the
haemagglutination reaction was considered to be the
inhibition titre of the fraction tested.
Reagents
Pharmacia, Uppsala.
The reagents were from Sigma and the resins from
Results
Our preliminary studies on adhesion inhibition
induced by human milk verified that both the dialysed

Page 3

fSC AND Lf IN MILK INHIBIT E. COLI ADHESION
5
precipitate from human whey obtained with
ammonium sulphate at 50 YO saturation, which con-
tains most of the immunoglobulins of the milk,34 and
also the supernate, were capable of inhibiting
haemagglutination by the ETEC CFA1+ strains. By
increasing the ammonium sulphate to 70 % saturation,
the inhibition capacity was detected only in the
precipitate. Furthermore, treatment of the precipitate
obtained at 50 YO ammonium sulphate saturation and
its supernate with trypsin increased the inhibition titre
of both, whereas treatment with sodium metaperiodate
decreased or abolished their inhibitory activity (results
not shown). These preliminary results led us to the
hypothesis that the human milk non-immunoglobulin
component responsible for the inhibition was a com-
plexed glycoprotein, because it was precipitated with
ammonium sulphate, further activated by trypsin and
inactivated by sodium metaperiodate.
The inhibition titre of the whey proteins obtained by
precipitation at 70 YO ammonium sulphate was always
> 256 for all the ETEC CFAl+ strains tested, whereas
the supernate did not inhibit the reaction.
Every fraction obtained from the fractionation of
the human milk was tested for its capacity to inhibit
haemagglutination induced by E. coli CFA1+ TR50/3
and was also analysed by SDS-PAGE.
Chromatographic fractionation
A typical profile of human whey proteins eluted
from the Sephadex G-200 column is shown in fig. 1.
The fractions from the first peak (Pl-S), containing
mainly s I ~ A , ~ ~ inhibited the haemagglutination
induced by the ETEC CFAl+, as did the second peak
(P2-S), that contained the free secretory component.
This chromatographic step was repeated several times
and the highest inhibition titres induced by P1-S and
P2-S were always similar, varying between 64 and 128.
Fractions from P1-S analysed by SDS-PAGE
presented a typical profile of sIgA, showing three
protein bands corresponding to the SC, heavy chain
and light chain, with estimated mol. wts of 79, 57 and
21 kDa respectively (fig. 2, lane 2). The most inhibitory
fractions from P2-S showed two main proteins of
79 kDa (corresponding to fSC) and 60 kDa (fig. 2, lane
3). All active fractions with the same electrophoretic
pattern were pooled and applied to a DEAE cellulose
column. With this procedure, BC is not retained by
the resin and is eluted by washing the column with the
starting The proteins bound to the DEAE
were eluted by washing the column with the same
buffer supplemented with 1 M NaCl. Fig. 3 shows the
elution pattern of the pooled fractions from P2-2 in ion
exchange chromatography, and the titre of inhibition
of some representative fractions. Most of the activity
was found in the fractions containing the proteins not
retained by the column (Pl-D), but a six-fold lower
activity was also detected in the fractions containing
the proteins that were bound to the DEAE (P2-D).
Electrophoretic analyses of the active fractions of
P1-D showed one main protein of c. 79 kDa (fig. 2,
lane 4). However, when the active fractions were
pooled and concentrated with ammonium sulphate at
70 YO saturation, small amounts of two more proteins
of lower mol. wt were also found. This result indicated,
as expected, that the main compound of P1-D was a
79-kDa protein which corresponds to fSC.
Because the most prevalent contaminant in the
purification of fSC is la~toferrin,~~
of c. 80 kDa, and two-dimensional electrophoresis of
the concentrated P1-D fractions showed two proteins
of approximately the same mol. wt but with very
distinct iso-electric points to be present (fig. 5A), it was
thought that Lf was also present with fSC in P1-D.
Therefore, the pooled and concentrated fractions of
P1-D were applied to a heparin-sepharose column to
remove lactoferrin and any other whey proteins basic
enough to have affinity to the strongly negatively
charged resin. With the procedure used, fSC was not
adsorbed by the column and was eluted by washing the
which has a mol. wt
1
8
s"
0.5
0
- 512
- 128
- 32
' 8
- 2
0
10
20
30
40
50
60
70 80
Fraction number
Fig. 1. Chromatography of the human milk proteins on Sephadex
G-200 column (2.1 cmx 92 cm). The peaks were monitored at
280 nm (-*-)
and eluted with 0.1 M Tris-HC1, pH 7.6; the flow
rate was 20 ml/h. Fractions of 5 ml were collected and assayed for
inhibition of the haemagglutination (- - -0 - - -) induced by E. coli
CFA I+ TR50/3.
Fig. 2. SDS-PAGE of human milk fractions stained with Coomassie
Blue. Lane 1, whey proteins precipitated with ammonium sulphate
at 70% saturation; 2, PI-S; 3, P2-S; 4, P1-D; 5, fraction 7 from Pl-
H; 6, fraction 26 from P2-H; 7, lactoferrin isolated by one-step
chromatography; 8, transferrin (Sigma).

Page 4

6
L. G. GIUGLIANO ET AL.
0
p"
0
0.7 1
P2-D
0.6
0 -5
0.4
0.3
0.2
0 -1
0
0
5 10 15 20 25
30
35 40 45
51 2
256
128
64
32
16
8
4
2
Fraction number
Fig. 3. Chromatography of the pooled fractions from P2-S on DEAE cellulose column (2.1 cm x 25 cm). The peaks were monitored at 280 nm
(-0-). The unbound proteins (PI-D) were eluted in 0.01 M Tris-HC1, pH 7.6, with 0.05 M NaCl and the bound proteins (P2-D) were
recovered in the same buffer with 1 M NaCl; the flow rate was 36 ml/h. Fractions of 6 ml were c(
haemagglutination (---O
---) induced by E. coli CFAl+ TR50/3.
0 -5
0.4
0 *3
8
0
ni
0.2
0.1
0
0
3 6
9 12 15 18 21 24 27 30
Fraction number
lected and assayed for inhibition of the
1.0
0.8 -
I
W
.- s
H
c,
8
p
0-6
C
0.4
0.2
Fig. 4. Chromatography of the pooled and concentrated fractions of PI-D on heparin-sepharose (1.5 cm x 10 an). The peaks were monitored
at 280 nm ( - . - ) .
The unbound proteins (PI-H) were eluted in 0.05 M Tris-HC1, pH 8.0, with 0.2 M NaCl and the bound proteins were
recovered with an increasing NaCl gradient (0.2 M-1 M) (---) in the same buffer; the flow rate was 9.0 ml/h. Fractions of 2.5 ml were collected
and assayed for inhibition of haemagglutination induced by E. coli CFAl+ TR50/3 (-0-).
column with the starting
was retained but could be removed with c. 0.6 M
NaC1.34*39 Fig. 4 shows the chromatographic profile of
P1-D on heparin-sepharose and the inhibitory titres
induced by the fractions. Both the protein fractions
that did not bind to the column (Pl-H) and those that
did (P2-H) inhibited haemagglutination. Electro-
phoretic analyses of the active fractions 3-6 showed
the presence of a major protein of 79 kDa, corre-
sponding to fSC, and also, in very small amounts (only
revealed by silver stain) another protein of lower
mol. wt (not shown). However, fractions 7-10 showed
only the fSC even when developed with silver stain (fig.
However, lactoferrin
2, lane 5). Similarly, fractions 23-25 showed a major
protein of 79 kDa corresponding to lactoferrin and
two other smaller proteins that could be visualised
only by silver stain, but fractions 26-28 showed only
the lactoferrin band (fig. 2, lane 6). These results
showed that highly purified fSC and Lf were both able
to inhibit the haemagglutination induced by ETEC
CFAl+ strains.
The purity of the fSC (fraction 7) and Lf (fraction
26) preparations was examined by two-dimensional
gel electrophoresis and silver staining (fig. 5B and C).
Both fSC and Lf preparations showed the presence of
only one protein. The estimated PI ranges of fSC and

Page 5

f s c AND Lf IN MILK INHIBIT E. coLr ADHESION
7
Fig. 5. Two-dimensional electrophoresis of human milk fractions. A,
pooled and concentrated fractions of PI-D, arrow indicates two
proteins of an estimated 79 kDa with distinct iso-electric points; B,
fraction 26 from P2-H, purified lactoferrin; C, fraction 7 from PI-H,
purified free secretory component.
Lf were 6-9-7-5 and 7.7-9.3 respectively, which is in
good agreement with published
The protein concentration of fraction 7 (fSC) was
1*4mg/ml, and its inhibitory titre was 24. The in-
hibitory titre of fraction 26 (Lf) was 12, and its protein
concentration was 1.2 mg/ml. Therefore, the highest
dilutions of fSC and Lf able to inhibit the haemag-
glutination induced by strain TR50/3 were 0.06 mg/ml
and 0.1 mg/ml, respectively.
Lf in pure form and in high yield was also obtained
by a single chromatographic step on heparin-
sepharose (fig. 2, lane 7). Although this Lf preparation
was at least seven times more concentrated than the Lf
preparation obtained by multiple chromatographic
fractionation, it induced an inhibition titre at least six-
fold lower. This may be explained by the differences in
the procedures used for the isolation of Lf. In the
single chromatographic method, Veronal-HC1 buffer
was used throughout the procedure, whereas in the
multiple chromatographic method Tris-HC1 buffer
44
was used. When the Lf isolated in Verona1 buffer was
dialysed against Tris-HCl, the inhibition titre of the
preparation increased four-fold.
Commercial lactoferrin and transferrin
Commercially available lactoferrin from human
milk and transferrin from human serum (which has a
great structural analogy to lactoferrin) at 2 mg/ml and
10 mg/ml, respectively, in 0.05 M Tris-HCl, pH 7.6,
were also assayed and shown to inhibit haemag-
glutination by ETEC CFAl+ strains. The lowest
concentrations of lactoferrin and transferrin able to
inhibit haemagglutination by strain TR50/3 were
0-03 mg/ml and 0.4 mg/ml, respectively. These results
corroborate our finding that purified preparations of
Lf had inhibitory activity and showed that transferrin
also inhibited the haemagglutination, but at a higher
concentration.
The minimal inhibitory concentration of lactoferrin
varied with the CFAl+ strain used. The lowest con-
centration of lactoferrin that inhibited the haemag-
glutination induced by strain CD79a in our standard
assay was 0.062 mg/ml, whereas the highest concen-
tration of lactoferrin used in the assay (1 mg/ml) did
not inhibit the haemagglutination induced by strain
m452-C1. However, when the inhibition assay was
performed with bacterial suspensions adjusted to
150 pKlett, strains CD79a and m452-Cl were in-
hibited by 0.02 mg/ml and 0.5 mg/ml, respectively.
These results may reflect the differing ability of each
strain to express CFA1.
Discussion
This study demonstrated that both fSC and Lf
in human milk inhibited adhesion to erythrocytes
(haemagglutination) by ETEC CFAl+ strains. Human
serum transferrin, which has a great structural analogy
to Lf,28 also inhibited haemagglutination but at higher
concentrations than Lf.
Lf is considered to be an important non-specific
defence factor against gastrointestinal infections by its
iron-scavenging ability and consequent iron depri-
vation of micro-organisms.12*
specific Lf-binding receptors have been described in
several bacterial path~gens.l~-~~
terial surface may damage the outer cell membrane
and enhance the antimicrobial activity of Lf.239
Studying the specific binding of Lf to E. coli isolated
from human intestinal infection, Naidu et a1.24 showed
that ETEC strains bound significantly more Lf than
other pathogenic groups. As we found that Lf also
inhibits ETEC adhesion, it may indicate that Lf binds
to CFAs which are found only in ETEC strains.
Previous studiesg*
ation between the presence of fucosylated oligo-
saccharides in human milk and adhesion inhibition of
ETEC and EPEC strains to eukaryotic cells. Our
findings may support these reports, as the sugar chain
13*
' ' 7
l8 M ore recently,
Lf binding to a bac-
45
1 5 9 26 have established the associ-

Page 6

8
L. G. GIUGLIANO ET AL.
patterns of fSC and Lf are similar and rich in
fucosylated oligosaccharide
oligosaccharides found to inhibit bacterial adherence
might even be the degradation products of glyco-
compounds of the milk, as it is known that human
milk contains specific hydrolytic enzymes46 and fSC is
very sensitive to prote~lysis.~~*
containing glycopeptides from Lf were shown to
inhibit the adherence of Shigella JEexneri to intestinal
cells .47
There is little information about the role of fSC in
secretions but some workers suggest that it may have
a protective role against diarrhoea.48*
showed that fSC inhibited the adherence of ETEC
strains in vitro, indicating that this compound may be
an important non-specific defence factor because it is
also found in intestinal secretion~,~~
protection for the mucous membranes which are the
initial target in most infections.
We, as well as other author^,^'^^ have verified that
the immunoglobulin fraction (P1 -S) also inhibited
bacterial adherence. As the main immunoglobulin
in human milk is sIgA and its molecule contains
covalently bound SC, the inhibitory effect of sIgA may
be partially due to the bound SC. In this context, Lf is
a strongly basic protein which has been shown to
30 Some of the
34 Furthermore, fucose-
49 This report
hence providing
References
1. Black RE, Lopez de Romana G, Brown KH, Bravo N, Bazalar
OG, Kanashiro HC. Incidence and etiology of infantile
diarrhea and major routes of transmission in Huascar,
Peru. Am J Epidemioll989; 129: 785-799.
2. Black RE, Merson MH, Rahman ASMM et al. A two-year
study of bacterial, viral, and parasitic agents associated
with diarrhea in Bangladesh. J Infect Dis 1980; 142:
660-664.
3. Guerrant RL, Kirchhoff LV, Shields DS et al. Prospective study
of diarrheal illnesses in Northeastern Brazil : patterns of
disease, nutritional impact, etiologies, and risk factor. J
Infect Dis 1983; 148: 986-997.
4. Levine MM.
Escherichia coli that cause diarrhea:
enterotoxigenic, enteropathogenic, enteroinvasive, entero-
hemorrhagic, and enteroadherent. J Infect Dis 1987; 155:
377-3 89.
5. Changchawalit S, Echeverria P, Taylor DN et al. Colonization
factors associated with enterotoxigenic Escherichia coli
isolated in Thailand. Infect Immun 1984; 45: 525-527.
6. Lopez-Vidal Y, Calva, JJ, Trujillo A et al. Enterotoxins and
adhesins of enterotoxigenic Escherichia coli: are they risk
factors for acute diarrhea in the community? J Infect Dis
1990; 162: 442-447.
7. Kovar MG, Serdula MK, Marks JS, Fraser DW. Review of the
epidemiologic evidence for an association between infant
feeding and infant health. Pediatrics 1984; 74: 61 5-638.
8. Young HB, Buckley AE, Hamza My Mandarano C. Milk and
lactation : some social and developmental correlates among
1000 infants. Pediatrics 1982; 69: 169-175.
9. Cravioto A, Tello A, Villafan H, Ruiz J, Vedovo S, Neeser JR.
Inhibition of localized adhesion of enteropathogenic
Escherichia coli to HEp-2 cells by immunoglobulin and
oligosaccharide fractiolis of human colostrum and breast
milk. JInfect Dis 1991; 163: 1247-1255.
10. Cruz JR, Gil L, Can0 F, Caceres P, Pareja G. Breast milk anti-
Escherichia coli heat-labile toxin IgA antibodies protect
against toxin-induced infantile diarrhea. Actu Puediatr
Scand 1988; 77: 658-662.
1 1. Glass RI, Svennerholm A-M, Stoll BJ et al. Protection against
cholera in breast-fed children by antibodies in breast milk.
N Engl JMed 1983; 308: 1389-1392.
12. Brock JH. Lactoferrin in human milk: its role in iron absorption
interact electrostatically with acidic molecules includ-
ing s I ~ A , ~ ~ ~
also be involved in the inhibition of adhesion by some
sIgA preparations.
The protection provided by human milk against
intestinal infections is well documented7? and has
been attributed traditionally to its high content of Lf
and sIgA.9-13,17,18 It may be that both sIgA and Lf are
degraded by gastrointestinal digestive enzymes freeing
SC from sIgA and eventually oligosaccharide residues
from fSC and Lf, which could act as important defence
factors.
Despite all the evidence indicating a relevant pro-
tective role for the isolated compounds, caution is
needed in extrapolating the results obtained in vitro
to explain the protection conferred by human milk
in vivo, since it may not reflect the real conditions
to which these compounds are submitted in the
gastrointestinal tract. Therefore, in-vivo studies are
required to establish the role of Lf and fSC against the
adhesive capacity of enteric pathogens.
51 very often forming complexes, and could
We are grateful to Drs R. Giugliano, B. D. de Lima and C.
Martins de Sa for many helpful discussions and suggestions, H. P.
Coelho and A. C. A. Lob0 for technical assistance and the Human
Milk Bank of Hospital Regional da Asa Sul for providing milk
samples.
and protection against enteric infection in the newborn
infant. Arch Dis Child 1980; 55: 417-421.
13. Bullen JJ, Rogers HJ, Leigh L. Iron-binding proteins in milk
and resistance to Escherichia coli infection in infants. B M J
1972; 1: 69-75.
14. Jelliffe DB, Jelliffe EPF. Protection and hazards. In : Jelliffe DB,
Jelliffe EPF (eds) Human milk in the modern world.
Psychosocial, nutritional and economic significance.
Oxford, Oxford University Press. 1978 : 84-1 12.
15. Newburg DS, Picketing LK, McCluer RH, Cleary TG.
Fucosylated oligosaccharides of human milk protect suck-
ling mice from heat-stable enterotoxin of Escherichia coli. J
Infect Dis 1990; 162: 1075-1080.
16. Reiter B, Oram JD. Bacterial inhibitors in milk and other
biological fluids. Nature 1967; 216: 328-330.
17. Griffiths E, Humphreys J. Bacteriostatic effect of human milk
and bovine colostrum on Escherichia coli: importance of
bicarbonate. Infect Immun 1977 ; 15 : 39640 1.
18. Oram JD, Reiter B. Inhibition of bacteria by lactoferrin and
other iron-chelating agents. Biochim Biophys Acta 1968 ;
170: 351-365.
19. Lee BC, Schryvers AB. Specificity of the lactoferrin and
transferrin receptors in Neisseria gonorrhoeae. Mol
Microbioll988; 2: 827-829.
20. Naidu AS, Andersson M, Forsgren A. Identification of a
human lactoferrin-binding protein in Staphylococcus
aureus. J Med Microbioll992; 36: 177-183.
21. Schryvers AB. Identification of the transferrin- and lactoferrin-
binding proteins in Haemophilus inJuenzae. J Med
Microbiol 1989; 29: 121-130.
22. Tigyi Z, Kishore AR, Maland JAY Forsgren A, Naidu AS.
Lactoferrin-binding proteins in Shigella Jexneri. Infect
Immun 1992 ; 60 : 26 19-2626.
23. Dalamastri C, Valenti P, Visca P, Orsi N. Enhanced
antimicrobial activity of lactoferrin by binding to the
bacterial surface. Miccobiologica 1978 ; 11 : 225-230.
24. Naidu SS, Erdei J, Czir6k E et al. Specific binding of lactoferrin
to Escherichia coli isolated from human intestinal
infections. APMIS 1991 ; 99: 1142-1 150.
25. Andersson By Porras 0, Hanson LA, LagergHrd TS, Svanborg-
Ed& C. Inhibition of attachment of Streptococcus
pneumoniae and Haemophilus inJuenzue by human milk
and receptor oligosaccharides. J Infect Dis 1986; 153:
232-237.

Page 7

fSC AND Lf IN MILK INHIBIT E. COLI ADHESION
9
26. Ashkenazi S, Mirelman D. Nonimmunoglobulin fraction of
human milk inhibits the adherence of certain entero-
toxigenic Escherichia coli strains to guinea pig intestinal
tract. Pediatr Res 1987; 22: 130-134.
27. Holmgren J, Svennerholm A-M, Lindblad M. Receptor-like
glycocompounds in human milk that inhibit classical and
El Tor Vibrio cholerae cell adherence (hemagglutination).
Infect Immun 1983; 39: 147-154.
28. Aisen P, Listowsky I. Iron transport and storage proteins. Annu
Rev Biochem 1980; 49: 357-393.
29. Brandtzaeg P. Human secretory immunoglobulins. 4.
Quantitation of free secretory piece. Acta Path Microbiol
Scand 1971 ; Section B 79: 189-203.
30. Mizoguchi A, Mizuochi T, Kobata A. Structures of the
carbohydrate moieties of secretory component purified
from human milk. J Biol Chem 1982; 257: 9612-9621.
31. Chintalacharuvu KR, Piskurich JF, Lamm ME, Kaetzel CS.
Cell polarity regulates the release of secretory component,
the epithelial receptor for polymeric immunoglobulins,
from the surface of HT-29 colon carcinoma cells. J Cell
Physioll991; 148: 35-47.
32. Mestecky J, McGhee JR. Immunoglobulin A (IgA): molecular
and cellular interactions involved in IgA biosynthesis and
immune response. Adv Immunol 1987; 40: 153-245.
33. Mostov KE, Deitcher DL. Polymeric immunoglobulin receptor
expressed in MDCK cells transcytoses IgA. Cell 1986; 46:
61 3-62 1.
34. Mestecky J, Kilian M. Immunoglobulin A (IgA). Methods
Enzymoll985; 116: 37-75.
35. Bradford MM. A rapid and sensitive method for quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 1976; 72 : 248-254.
36. Laemmli UK. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 1970;
227: 68M85.
37. Blum H, Beier H, Gross HJ. Improved silver staining of plant
proteins, RNA and DNA in polyacrylamide gels. Electro-
phoresis 1987; 8: 93-99.
38. O’Farrell PZ, Goodman H, O’Farrell PH. High resolution
two-dimensional electrophoresis of basic as well as acidic
proteins. Cell 1977; 12: 1133-1141.
39. Blackberg L, Hernell 0. Isolation of lactoferrin from human
whey by a single chromatographic step. FEBS Lett 1980;
109: 180-183.
40. Evans DG, Evans DJ, Tjoa W. Hemagglutination of human
group A erythrocytes by enterotoxigenic Escherichia coli
isolated from adults with diarrhea: correlation with
colonization factor. Infect Immun 1977; 18: 330-337.
41. Holmgren J, Svennerholm AM, Ahren C. Nonimmunoglobulin
fraction of human milk inhibits bacterial adhesion
(hemagglutination) and enterotoxin binding of Escherichia
coli and Vibrio cholerae. Infect Immun 1981; 33: 136-141.
42. Evans DG, Evans DJ. New surface-associated heat-labile
colonization factor antigen (CFA/II) produced by
enterotoxigenic Escherichia coli of serogroups 0 6 and 08.
Infect Immun 1978; 21: 638-647.
43. Malamud D, Drysdale J W .
Isoelectric points of proteins: a
table. Anal Biochem 1978; 86: 620-647.
44. Roberts TK, Masson PL, Heremans JF. Heterogeneity of
human lactoferrin. In: Bratanov K, Edwards RG,
Vulchanov VH, Dikov V, Somlev B (eds) Immunology of
reproduction. Sofia, Bulgarian Academy of Sciences Press.
1973: 706.
45. Ellison RT, Giehl TJ, LaForce FM. Damage of the outer
membrane of enteric gram-negative bacteria by lactoferrin
and transferrin. Infect Immun 1988 ; 56 : 2774-278 1.
46. Coppa GV, Catassi C, Felici L, Gabrielli 0, Giorgi PL. Acid
glycohydrolases in human colostrum. In : Proceedings of
XX Annual Meeting of European Society for Paediatric
Gastroenterology and Nutrition (ESPGAN), Lisbon,
Portugal. 1987: 90.
47. Izhar M, Nuchamowitz Y, Mirelman D. Adherence of Shigella
Jexneri to guinea-pig intestinal cells is mediated by a
mucosal adhesin. Infect Immun 1982; 35: 11 10-1 118.
48. Buts JP, Bernasconi P, Vaerman JP, Dive C. Stimulation of
secretory IgA and secretory component of immuno-
globulins in small intestine of rats treated with Saccharo-
myces boulardii. Dig Dis Sci 1990; 35: 251-256.
49. Nussinson E, Lahav M, Berebi A, Estrov Z, Zur S, Resnitzky P.
Secretory piece and IgA deficiency in a patient with
Waldenstrom’s macroglobulinemia. Am J Gastroenterol
1986; 81: 995-998.
50. Ahnen DA, Brown WR, Kloppel TM. Secretory component:
the polymeric immunoglobulin receptor. What’s in it for
the gastroenterologist and hematologist? Gastroenterology
1985; 89: 667-682.
5 1. Hekman A. Association of lactoferrin with other proteins, as
demonstrated by changes in electrophoretic mobility.
Biochim Biophys Acta 1971 ; 251 : 380-387.