Inflammatory Bowel Diseases威

8(2):71–80 © 2002 Crohn’s & Colitis Foundation of America, Inc.

Lactobacillus plantarum 299V in the Treatment and Prevention

of Spontaneous Colitis in Interleukin-10-Deficient Mice

*†Michael Schultz, *Claudia Veltkamp, *Levinus A. Dieleman, ‡Wetonia B. Grenther,

§Pricilla B. Wyrick, ‡Susan L. Tonkonogy, and *§R. Balfour Sartor
*Center for GI Biology and Disease, Department of Medicine, and §Department of Microbiology and Immunology, University of
North Carolina, Chapel Hill, North Carolina, U.S.A.; †Department of Internal Medicine I, University of Regensburg, Germany;
‡College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, U.S.A.

Summary: Interleukin (IL)-10-deficient (IL-10−/−) mice de-
velop colitis under specific pathogen-free (SPF) conditions and
remain disease free if kept sterile (germ free [GF]). We used
four different protocols that varied the time-points of oral ad-
ministration of Lactobacillus plantarum 299v (L. plantarum)
relative to colonization with SPF bacteria to determine whether
L. plantarum could prevent and treat colitis induced by SPF
bacteria in IL-10−/− mice and evaluated the effect of this pro-
biotic organism on mucosal immune activation. Assessment of
colitis included blinded histologic scores, measurements of se-
creted colonic immunoglobulin isotypes, IL-12 (p40 subunit),
and interferon (IFN)-␥ production by anti-CD3-stimulated
mesenteric lymph node cells. Treating SPF IL-10−/− mice with
L. plantarum attenuated previously established colonic inflam-
mation as manifested by decreased mucosal IL-12, IFN-␥, and
immunoglobulin G2a levels. Colonizing GF animals with L.
plantarum and SPF flora simultaneously had no protective ef-
fects. Gnotobiotic IL-10−/− mice monoassociated with L. plan-
tarum exhibited mild immune system activation but no colitis.
Pretreatment of GF mice by colonization with L. plantarum,
then exposure to SPF flora and continued probiotic therapy
significantly decreased histologic colitis scores. These results
demonstrate that L. plantarum can attenuate immune-mediated
colitis and suggest a potential therapeutic role for this agent in
clinical inflammatory bowel diseases. Key Words: Probiot-
ics—Experimental colitis—Interleukin-10 knockout mice.

INTRODUCTION
Chronic inflammatory bowel diseases (IBD) such as
Crohn’s disease, ulcerative colitis, and, to a certain ex-
tent, pouchitis, appear to be the result of an unrestrained
cell-mediated immune response to ubiquitous luminal
antigens, although the exact nature of the antigenic
stimuli remains unclear (1,2). Studies of experimental
colitis in various animal models demonstrated the impor-
tance of the resident luminal flora in initiation and per-
petuation of intestinal inflammation (2,3). The spontane-
ous colitis that develops in transgenic HLA-B27 rats (4),
in mice deficient for interleukin-2 (IL-2−/−) (5), IL-10
Received August 15, 2000; accepted September 6, 2001.
Address correspondence and reprint requests to R. Balfour Sartor,
M.D., Division of Digestive Diseases and Nutrition, School of Medi-
cine, CB#7038, Room 032 Glaxo Building, University of North Caro-
lina at Chapel Hill, Chapel Hill, NC 27599-7038, U.S.A. E-mail:
rbs@med.unc.edu
(6), T-cell receptors ␣/␤ (7), after bone marrow trans-
plantation in ␧26 mice transgenic for the human CD3␧
gene (8), or in the SAMP-1/Yit strain (9) requires the
presence of luminal bacteria. These genetically engi-
neered rodents develop no disease under germ-free (GF,
sterile) conditions with the exception of IL-2-deficient
mice, which develop mild, focal colitis, gastritis, and
hepatitis when raised sterilely but fed chow containing
dead bacteria (5). Furthermore, antibiotic therapy, di-
rected mainly against anaerobic bacteria, attenuates ex-
perimental colitis (10–13) and human chronic IBD
(14,15). Both IBD patients and mice with experimental
colitis have abnormally aggressive T lymphocyte re-
sponses to resident enteric bacteria (16,17). There is
growing evidence that the fecal flora, including the mu-
cosal-associated flora (18), differs in patients with active
IBD compared with quiescent disease or healthy volun-
teers. Multiple studies document decreased luminal lac-
tobacilli and bifidobacteria concentrations in patients

71
72 M. SCHULTZ ET AL.
with active Crohn’s disease or ulcerative colitis (19–21).
Together, these results indicate an important role of com-
mensal luminal bacteria in the induction and mainte-
nance of chronic immune-mediated colitis and suggest
an altered equilibrium of aggressive and protective bac-
terial species in patients with IBD.
A subset of the autochthonous (resident) intestinal mi-
croflora, including various lactic-acid bacteria (Lactoba-
cillus and bifidobacteria species), confer benefits to the
host. These probiotic bacteria have therapeutic activity,
particularly in the treatment and prevention of relapsing
Clostridium difficile diarrhea (22,23), traveler’s diarrhea
(24,25), and rotavirus infection in children (26,27). More
recently, probiotics have been used in the treatment of
IBD. Gionchetti et al. presented evidence that a cocktail
of eight probiotic bacteria prevented relapse of chronic
pouchitis (28). This group also reported similar effects of
this probiotic mixture in patients with ulcerative colitis
(29). The nonpathogenic Escherichia coli strain Nissle
was shown to be effective in maintaining remission of
ulcerative colitis (30–32). However, as recently reviewed
(33–35), the effects of probiotics in IBD and infectious
colitis remain inconclusive because of inconsistent re-
sults, the relatively small number of patients treated, and
the difficulty in comparing results with different probi-
otic preparations.
Moreover, the therapeutic mechanisms of probiotic
bacteria are still largely unknown. Most lactobacilli spe-
cies are able to adhere to colonic epithelial cells, thereby
interfering with the adherence of other potentially patho-
genic bacteria (36) and possibly competing for nutrients
while they temporarily colonize the intestine. Recent in
vitro data suggest that this inhibition of the adherence of
attaching and effacing E. coli to intestinal epithelial cells
by lactobacilli is mediated through increased expression
of intestinal mucin genes (37). Some probiotic bacterial
species are able to restore damaged intestinal permeabil-
ity (38–40), which is deranged in IBD and experimental
colitis. An additional mechanism of protection is direct
inhibition of the growth of potential pathogenic bacterial
species. For example, Lactobacillus GG produces a bac-
teriocin (41), and both lactobacilli and bifidobacteria
lower the luminal pH (29,42). Finally, probiotic organ-
isms can directly or indirectly influence mucosal and
systemic immune function by altering cytokine, immu-
noglobulin profiles, and cellular immune responses (43–
49). However, literature reports are still inconclusive,
and data from in vitro experiments cannot be extrapo-
lated to humans and animal models.
The role of bacteria in the pathogenesis of experimen-
tal colitis has been particularly well studied in IL-10
gene–deficient mice (IL-10−/−). These mice spontane-
Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002
ously develop a TH1-mediated chronic colonic inflam-
mation predominantly in the rectum if raised under spe-
cific pathogen-free (SPF) conditions (50,51) but remain
healthy with no evidence of immune activation if kept
GF (6). Transfer of GF IL-10−/− mice into SPF condi-
tions leads to the development of colitis within 1 week,
with progressive inflammation leading to severe cecal
inflammation (typhlitis) within 4–5 weeks (6). Madsen et
al. (40) demonstrated an intestinal mucosal permeability
defect in IL-10−/− mice, which occurred before the onset
of colitis. Furthermore, these investigators documented
decreased fecal lactobacilli concentrations in IL-10−/−
mice with active inflammation and showed that admin-
istration of endogenous Lactobacillus spp. or broad-
spectrum antibiotics could prevent the onset of colitis
(11,40).
The aims of the current study are to determine whether
oral administration of a well-characterized probiotic spe-
cies of human origin, Lactobacillus plantarum 299v (52),
could treat established immune-mediated colitis in SPF
IL-10−/− mice, prevent the onset of disease in GF IL-
10−/− mice simultaneously or subsequently colonized
with SPF bacteria, and alter pathologic immune re-
sponses in this model. In addition, we sought to deter-
mine whether a probiotic bacterial strain could have ben-
eficial effects in a host species different from their origin.
To achieve these goals, different protocols for adminis-
tering this strain of Lactobacillus were evaluated in SPF
and gnotobiotic IL-10−/− mice.
MATERIALS AND METHODS
Mice
Germ-free IL-10−/− (C57BL6 × 129/Ola) outbred mice
were derived at the University of Wisconsin by cesarean
section as previously described (6). A GF breeding
colony (IL-10−/− × IL-10−/− mice) was maintained in the
Gnotobiotic Facility of the Center of GI Biology and
Disease at the North Carolina State University, College
of Veterinary Medicine, Raleigh, NC. Mice were geno-
typed by amplification of the IL-10 gene by PCR using
murine IL-10 specific primers (Nucleic Acid Core Facil-
ity, Lineberger Comprehensive Cancer Center, Univer-
sity of North Carolina, Chapel Hill, NC, U.S.A.) as de-
scribed elsewhere (51). The GF colonies were housed in
Trexler flexible film isolators with autoclaved food and
water ad libitum. Sterility was tested regularly by fecal
pellet Gram stain and by culturing stool and bedding in
thioglycollate medium (Becton Dickinson Microbiology
Systems, Cockeysville, MD, U.S.A.) and on blood agar
 L. PLANTARUM ATTENUATES COLITIS 73

plates for aerobic and anaerobic growth. PCR analysis of colonized with SPF bacteria. Mice were killed after 1 or
feces from SPF IL-10−/− mice excluded contamination 4 weeks (n ⳱ 3–5/group). Persistent colonization with L.
with intestinal Helicobacter species, including H. hepati- plantarum was confirmed by culture of serially diluted
cus (6,53). SPF IL-10−/− (C57BL6 × 129/Ola) mice were cecal contents at necropsy and morphologic comparison
bred in the Laboratory Animal Resources facilities at of the grown cultures from fecal pellets to L. plantarum
North Carolina State University, College of Veterinary colonies grown from the stock provided by the company.
Medicine. 4) In a precolonization prevention protocol, GF IL-10−/−
mice (n ⳱ 8/group) were given L. plantarum as de-
scribed for protocol 3. After 2 weeks, the mice were
Oral Therapy with Lactobacillus plantarum 299v in transferred to an SPF environment with continuous ad-
Drinking Water ministration of L. plantarum (1 × 109 CFU/ml) in drink-
ing water for another 4 weeks. This group was compared
Lactobacillus plantarum 299v (ConAgra, Inc.,
with GF IL-10−/− mice of the same age, which were
Omaha, NE, U.S.A.) (52) was expanded from a lyophi-
transferred without L. plantarum treatment to the same
lized stock in deMan-Rogosa-Sharpe medium (MRS,
SPF environment and received water only (no L. plan-
Difco, Detroit, MI, U.S.A.) at 37°C overnight under re-
tarum) after colonization with SPF bacteria. Successful
stricted aerobic conditions. Culture on MRS and blood
colonization with SPF flora and/or with L. plantarum
agar plates showed minor contamination. The suspension
299v in all groups was regularly confirmed by fecal cul-
was washed once in sterile phosphate-buffered-saline
tures on blood agar and MRS plates, with cultures con-
(PBS, pH 7.5). Concentration was determined by photo-
firmed by Gram staining. Quantification of L. plantarum
metric comparison with a previously established growth
was performed after serial dilutions of homogenized fe-
curve. Mice were given 1 × 109 colony-forming units
cal pellets or cecal luminal contents and anaerobic
(CFU)/ml in autoclaved drinking water. The mice drank
growth on MRS agar plates. L. plantarum could not be
an average of 5 ml water/d, leading to a daily consump-
detected in the stool before administration of the study
tion of approximately 5 × 109 CFU L. plantarum. After
medication. One day after administration, stool pellets
an expansion of viable L. plantarum within the first 24
contained 4.6 × 107 CFU Lactobacillus/g; this concen-
hours in drinking water, counts reached a plateau of 1 ×
tration increased to 1 × 108 CFU/g by 6 days of admin-
1010 CFU/ml and stayed stable for at least 72 hours.
istration and remained stable at this plateau during the
Therefore, water bottles in studies performed in the SPF
study.
environment were changed every 2–3 days to keep the
dosage stable.
Clinical Assessment
Therapeutic Protocols
Clinical evidence of intestinal inflammation including
Four different protocols were studied: 1) In the treat-
diarrhea (absence of formed fecal pellets), progressive
ment arm of the study, SPF IL-10−/− mice of 10–12
wasting, and rectal prolapse were assessed twice weekly.
weeks of age with established colonic disease received
Mice were weighed before necropsy and then were eu-
either 1 × 109 CFU/ml L. plantarum in drinking water for
thanized by CO2 asphyxiation. Segments of the rectum,
4 weeks or water only (n ⳱ 6/group). The water bottles
colon, cecum, small intestine, and the mesenteric lymph
were changed every 2–3 days so that drinking water
nodes were processed for histology, immunoglobulin,
contained freshly prepared viable organisms. 2) In a si-
and cytokine measurements.
multaneous prevention protocol, 12–14-week-old GF IL-
10−/− disease-free mice (n ⳱ 8/group) were colonized
with SPF mouse flora (free of Helicobacter species), as
previously described (6) and treated with 1 × 109 Histological Assessment of Intestinal Inflammation
CFU/ml L. plantarum or water only for 4 weeks, begin-
ning on the first day of transfer from GF housing con- Intestinal tissues were fixed for 24 hours in 10% saline
ditions. 3) In a selective colonization protocol, GF IL- buffered formalin. Tissues were embedded in paraffin
10−/− mice were given L. plantarum (1 × 109 CFU/ml) in and stained with hematoxylin and eosin. A gastrointes-
drinking water. Water bottles provided to GF mice were tinal histologic inflammatory score ranging from 0 to 4,
not changed to avoid frequent manipulations of the iso- previously validated in IL-10−/− mice (6), was applied in
lators. Negative littermate controls remained GF or were a blinded fashion to quantify intestinal inflammation.

Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002

74
The histologic scores are either given for the cecum
(maximum score 4) or as a total colonic score that was
calculated as a summation of the individual scores for the
proximal colon, transverse colon, and rectum (maximum
score 12).
Colon Fragment Cultures
Sections of the transverse colon of individual mice
were prepared as previously described (6). Briefly, colon
sections were washed with PBS to remove fecal contents,
shaken at 280 rpm at room temperature for 30 minutes in
RPMI 1640 plus 50 ␮g/ml gentamicin (both from the
Tissue Culture Facility, Lineberger Comprehensive Can-
cer Center, University of North Carolina, Chapel Hill,
NC, U.S.A.), and cut into small fragments. Tissue frag-
ments (100 mg wet weight) were incubated in 1.0 ml of
RPMI 1640 supplemented with 50 ␮g/ml gentamicin,
100 U/ml penicillin, 100 ␮g/ml streptomycin, 0.25
␮g/ml fungizone (Gibco Life Technologies, Grand Is-
land, NY, U.S.A.), and 5% heat-inactivated fetal calf
serum (Irvine Scientific, Santa Ana, CA, U.S.A.) at 37°C
for 18 hours. Supernatants were collected and stored at
−20°C until further processing.
Measurement of IL-12 (p40) from Cultured
Colonic Fragments
IL-12 secretion in colon fragment culture supernatants
was measured by enzyme-linked immunosorbent assay
(ELISA) using commercially available anti-IL-12 anti-
bodies C15.6 and biotinylated C17.8 (PharMingen, San
Diego, CA, U.S.A.) for capture and detection, respec-
tively, of the inducible IL-12 p40 subunit (6). The
amount of binding of biotinylated antibody to IL-12 from
culture supernatants was detected using horseradish per-
oxidase–labeled streptavidin (Southern Biotechnology
Associates, Birmingham, AL, U.S.A.). Concentrations of
IL-12 were established by comparison of values against
a standard curve generated for each assay using recom-
binant murine IL-12 p70 (PharMingen).
Measurement of Interferon-␥ from Anti-CD3
Stimulated Mesenteric Lymph Node Cells
Mesenteric lymph nodes were removed and single-cell
suspensions were prepared by gentle teasing. Lymphoid
cells were stimulated with immobilized anti-CD3 mono-
clonal antibody as previously described (6). For inter-
feron (IFN)-␥ detection, supernatants were collected on
day 3 and stored at −20°C until being assayed by ELISA
as described (54) using the following antibody pairs:
Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002
M. SCHULTZ ET AL.
unlabeled rat anti-mouse IFN-␥ designated R46A2 for
capture and biotinylated rat anti-mouse designated
XMG-1.2 for detection, both prepared by S. Tonkonogy
from hybridoma culture supernatants.
Isotype-Specific ELISA to Measure Immunoglobulin
Detection of immunoglobulin production in colon cul-
ture supernatants was performed as described in detail
previously (5,54). The following affinity-purified poly-
clonal antibodies were used for capture: goat anti-mouse
IgA (KPL), goat anti-mouse IgG1, and goat anti-mouse
IgG2a (Southern Biotechnology Associates). Horseradish
peroxidase-labeled goat anti-mouse IgA (KPL), and goat
anti-mouse IgG1, and IgG2a (Southern Biotechnology
Associates) were used to detect the different isotypes.
The concentration of antibody was calculated by com-
parison with a standard curve of purified mouse IgA,
IgG1 and IgG2a (PharMingen).
Statistical Analysis
Values are expressed as mean ± SE. For each mea-
surement, one-way analysis of variance was carried out
to compare groups. A p value < 0.05 was regarded as
significant.
RESULTS
Administration of L. plantarum 299v led to less diar-
rhea in all groups; however, there was no significant
change in body weight or stool pH. In accordance with
previous observations (6,50), the colitis that spontane-
ously developed in SPF IL-10−/− mice was not associated
with inflammation in other parts of the gastrointestinal
tract such as the stomach, duodenum, small intestine, and
liver but is most prominent in the rectum when colonized
at birth and in the cecum when GF mice were colonized
as adults.
Treatment of Established Colitis in SPF IL-10−/−
Mice with L. plantarum
L. plantarum at a concentration of 1 × 109 CFU/ml
was given daily in drinking water for 4 weeks to IL-10−/−
mice that had been raised to the age of 10–12 weeks in
an SPF environment. Colonic inflammation is well es-
tablished in SPF IL-10−/− mice at this age. Results were
compared with untreated (water only) SPF IL-10−/−
mice. The histologic score varied among the different
sections of the large intestine; however, the total colonic,
rectal, and cecal histologic scores in the IL-10−/− mice
 L. PLANTARUM ATTENUATES COLITIS 75

treated with L. plantarum slightly improved, compared

with the untreated mice (L. plantarum treated versus un-
treated mice: total colonic histologic score 3.3 ± 0.5 ver-
sus 4.8 ± 0.8; p < 0.07, rectal score 2.1 ± 0.4 versus 2.5
± 0.2; p < 0.2, cecal score 1.0 ± 0.2 versus 1.6 ± 0.3; p
< 0.08). However, spontaneous colonic IL-12 production
(an indicator of intestinal macrophage activation) as well
as IFN-␥ production by anti-CD3 stimulated MLN cells
(an indicator of the presence of functional TH1 lympho-
cytes) was significantly lower in mice treated with L.
plantarum (Fig. 1). In addition, secretion of IgG2a was
significantly lower in colonic fragment culture superna-
tants from SPF mice treated with L. plantarum compared
with untreated controls (Fig. 2A). Significantly lower
amounts of colonic IgG1 were also detected in L. plan-
tarum treated SPF mice (L. plantarum treated versus un-
treated mice: 1.5 ± 0.3 versus 2.9 ± 0.6 ␮g/ml; p < 0.02).
IgA levels were variable and not different between
groups (Fig. 2B).

Prevention of Colitis in IL-10−/− Mice

Simultaneously Colonized with L. plantarum and
SPF Bacteria

To determine the ability of L. plantarum to prevent the

onset of experimental colitis, we administered a daily
dose of 1 × 109 CFU/ml in drinking water to IL-10−/−
mice beginning on the day of transfer from the GF en-
vironment to SPF conditions. The results were compared
with littermates identically colonized with SPF bacteria,
but which received water only. The administration of L.
plantarum did not prevent the development of colitis
(total colonic histologic score of IL-10−/− mice receiving
L. plantarum versus untreated mice: 5.3 ± 0.4 versus 4.5
± 0.3; p < 0.08). There was a trend toward reduced
colonic IL-12 levels after the administration of L. plan-
tarum; however, amounts were not significantly differ-
ent, and no differences in MLN cell IFN-␥ secretion
were noted (Fig. 1). Consistent with the histologic and
cytokine results, no significant differences were seen in
mucosal immunoglobulin levels in the prevention proto-
col (data not shown).
Colonization of GF IL-10−/− Mice with L.
plantarum 299v
To determine immune system activation and the pro-
inflammatory potential of L. plantarum, GF IL-10−/−
mice were colonized selectively with L. plantarum. L.
plantarum induced no clinical or histologic evidence of
colitis and led to only minimal immune system activation
(Table 1). For all parameters measured, gnotobiotic IL-
FIG. 1. Cytokine secretion in specific pathogen-free (SPF) inter-
leukin (IL)-10−/− mice receiving Lactobacillus plantarum in pre-
vention and treatment protocols. L. plantarum (1 × 109 colony-
forming units [CFU]/ml in drinking water) was administered daily
to 10–12-week-old SPF IL-10−/− mice for 4 weeks (treatment) or
daily for 4 weeks beginning at the time of colonizing germ-free
IL-10−/− mice with SPF bacteria (prevention). Control IL-10−/−
mice received water only. A: Spontaneous IL-12 (p40) produc-
tion, measured by enzyme-linked immunosorbent assay in cul-
tured unstimulated colonic mucosal fragments from treated and
control mice. Values represent ± SE of IL-12 pg/ml of culture
containing 100 mg of colonic tissue. *p < 0.05 versus untreated
control mice. B: Interferon (IFN)-␥ production by mesenteric
lymph node cells from the same mice stimulated in vitro with
anti-CD3 antibody for 3 days. Values represent mean ± SE of
IFN-␥ units per ml of culture. *p < 0.05 versus control mice. N =
6/group in treatment protocol, n = 8/group in prevention protocol.
10−/− mice colonized with L. plantarum for 1 or 4 weeks
had significantly lower cytokine, immunoglobulin, and
histology values than mice colonized with SPF bacteria
for 4 weeks (p < 0.02 to p < 0.0002).

Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002

76 M. SCHULTZ ET AL.

TABLE 1. Intestinal inflammatory parameters after colonization of germ-free interleukin-10−/− mice with Lactobacillus
plantarum 299v
L. plantarum L. plantarum SPF IL-10−/−
GF IL-10−/− 1 week 4 weeks 4 weeks
Number of mice 5 3 3 4
Total colonic histology score 0 0 0 4.8 ± 0.8c
IL-12 (pg/ml/0.1 g colonic tissue) 79 ± 3a 351 ± 125 198 ± 34b 1525 ± 238c
IgG2a (␮g/ml/0.1 g colonic tissue) 0.05 ± 0.01 0.08 ± 0.01 0.06 ± 0.01 2.5 ± 0.4c
IgA (␮g/ml/0.1 g colonic tissue) 1.2 ± 0.2 0.5 ± 0.09 2.9 ± 0.7 53.2 ± 5.4c
a
Values represent mean ± SE.
b
p < 0.05 vs. GF IL-10−/−.
c
p < 0.05 vs. L. plantarum 4 weeks.
GF, germ free; SPF, specific pathogen free; IL, interleukin.

Pretreatment of GF IL-10−/− Mice with L. and/or prevent colitis in IL-10−/− mice. IL-10−/− mice
plantarum 299v, Followed by Colonization with SPF develop immune-mediated colitis if raised under or
Flora and Continued L. plantarum transferred into SPF conditions and remain disease-free
299v Administration if kept sterile (6). The resulting colitis is characterized by

Based on the results outlined above, we colonized GF

IL-10−/− mice with L. plantarum alone for 2 weeks be-
fore their transfer into a SPF environment. The probiotic
therapy was continued after SPF bacterial colonization
with the daily administration of 1 × 10 9 CFU L.
plantarum/ml in drinking water for 4 weeks. Treated
mice were compared with mice that were transferred to a
SPF environment but were not given L. plantarum. There
was histologic improvement in all segments of the large
intestine of L. plantarum-treated mice, with significantly
decreased histologic scores in the cecum and colon (Fig.
3). Colonic inflammation in untreated IL-10−/− mice was
characterized by mucosal hyperplasia and infiltration of
the lamina propria with mononuclear cells, loss of goblet
cells, crypt abscesses, and occasional deep ulcerations
(Fig. 4A). After Lactobacillus treatment, the mucosa was
still somewhat hyperplastic and contained some infiltrat-
ing cells, but there were no ulcerations and crypt ab-
scesses and substantially more goblet cells were visible
(Fig. 4B). In contrast to the significant histologic im-
provement, there was only a trend toward decreased lev-
els of colonic IL-12 production and IFN-␥ secretion by
anti-CD3 stimulated MLN cells precolonized with L.
plantarum. Cytokine production in pretreated versus un-
treated mice was as follows: IL-12: L. plantarum 1,905 ±
158 pg/ml/0.1g colonic tissue versus water controls
2,290 ± 356 pg/ml/0.1 g colonic tissue; NS; IFN-␥: L.
plantarum 3,616 ± 350 U/ml versus water controls 4,349
± 436 U/ml; NS.
FIG. 2. IgG2a (A) and IgA (B) production on the treatment pro-
DISCUSSION tocol studied in Figure 1, measured in cultured unstimulated co-
lonic mucosal fragments by enzyme-linked immunosorbent as-
say. Values represent mean ± SE of Ig per ml of culture contain-
The aim of the current study was to determine whether ing 100 mg of colonic tissue. *p < 0.0001 versus control,
oral administration of L. plantarum 299v could treat untreated animals.

Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002

 L. PLANTARUM ATTENUATES COLITIS
FIG. 3. Blinded histological scores following colonization of gno-
tobiotic interleukin (IL)-10−/− mice with Lactobacillus plantarum
299v for 2 weeks before transfer into a specific pathogen-free
(SPF) environment with daily administration of L. plantarum 299v
(1 × 109 colony-free units [CFU] in drinking water) for 4 weeks
compared with germ-free IL-10−/− mice colonized with SPF bac-
teria and not receiving L. plantarum (control). Control (n = 8);
treatment with L. plantarum 299v (n = 8). *p < 0.003; **p < 0.001
versus no pretreatment.
mucosal hyperplasia caused by infiltrating cells and
stimulation of the mucosal immune system with elevated
colonic IL-12 levels and increased IFN-␥ secretion by
anti-CD3-stimulated MLN cells. In the current study, we
demonstrate that selective colonization of GF IL-10−/−
mice with L. plantarum for 2 weeks, followed by colo-
nization with SPF flora and continued probiotic therapy
attenuated the development of colitis. Furthermore, se-
lective colonization of GF IL-10−/− mice with L. planta-
rum for up to 4 weeks induced only mild mucosal im-
mune activation but no colitis. Finally, L. plantarum
treatment of SPF IL-10−/− mice with previously estab-
lished colonic inflammation attenuated colitis but was
not able to prevent disease from developing in GF adult
mice simultaneously colonized with L. plantarum and
SPF bacteria.
Successful prevention of colitis in young SPF IL-10−/−
mice by native Lactobacillus species was previously
demonstrated by Madsen et al. (40). However, there are
several differences that make the current study unique
with respect to a proposed clinical use of lactobacilli in
the treatment of IBD. To test the ability of a probiotic
agent to both prevent and treat experimental colitis, we
used a well-defined Lactobacillus strain at a defined con-
centration for daily oral administration in drinking water
(55) in contrast to the protocol of a rectal enema and
daily anal swabbing with endogenous murine L. reuteri
77
described in the study by Madsen et al. (40). Because L.
plantarum is not a natural component of the intestinal
flora in IL-10−/− mice and was not found in fecal cultures
before the study, our observations can therefore be fully
attributed to the therapeutic administration of L. planta-
rum. A preventive effect of L. plantarum was also shown
in a rat model with methotrexate-induced enterocolitis
(56) and is therefore not species specific, fulfilling a
critical requirement for extrapolating results from rodent
models to humans. Additional differences between the
current study and the report by Madsen et al. (40) include
the genetic background of IL-10−/− mice. Our SPF and
GF colonies of IL-10−/− mice were outbred populations
on a C57BL/6 × 129/Ola background, whereas the pre-
vious study used mice on an inbred 129Sv/Ev back-
ground (40). In preliminary studies using our optimal
protocol (i.e., 2-week colonization of GF mice with lac-
tobacilli, then daily administration in drinking water after
SPF colonization), we have demonstrated a more active
role for L. plantarum than for L. rhamnosus subsp. GG in
preventing the onset of colitis in gnotobiotic IL-10−/−
mice on an inbred 129Sv/Ev background colonized with
SPF bacteria (57). The importance of the host genetic
background has been investigated by Berg et al., who
showed a difference in the development of colitis in in-
bred and crossbred IL-10−/− mice (51). In addition, the
current study investigated the immunologic and histo-
logic consequences of colonizing GF IL-10−/− mice with
L. plantarum. Finally, and most importantly, we demon-
strated that lactobacilli more effectively treated estab-
lished colitis than prevented it, as opposed to the pre-
vention of onset of disease in mice treated beginning at
1 week of age described by Madsen et al. (40). Clinical
efficacy of experimental agents in human IBD has cor-
related better with therapeutic activity in animal models
than with the ability to prevent onset of experimental
inflammation. One possible explanation for the lack of
prevention is that in the protocol where L. plantarum and
SPF bacteria are used to simultaneously colonize GF
mice, the mucosal immune system was suddenly con-
fronted with up to 400 different bacterial species. The
positive effects of lactobacilli were possibly over-
whelmed by the more aggressive inflammatory effects of
other luminal constituents. This hypothesis is supported
by our observation that initial colonization of sterile IL-
10−/− mice with lactobacilli, followed 2 weeks later by
exposure to SPF flora had a more pronounced effect in
attenuating the developing colitis. By the time of colo-
nization with SPF bacteria, lactobacilli have presumably
occupied their niche within the intestinal ecosystem and
perhaps induced protective mucosal barrier function and
immune responses. This concept is supported by the ob-
Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002
78 M. SCHULTZ ET AL.
servation that Lactobacillus species rapidly colonize the
colon of neonatal suckling mice before the development
of a more complex microenvironment (11). To extrapo-
late these findings into a clinical situation, one can pos-
tulate that decreasing the concentration of luminal bac-
teria by antibiotic treatment before the initiation of pro-
biotic administration might potentiate the beneficial
effects of probiotics. This combination protocol was suc-
cessfully used by Gionchetti et al., who started VSL #3
probiotic treatment of patients with chronic pouchitis af-
ter a 4-week administration of ciprofloxacin and rifaxi-
min (28) and by Dieleman et al., who showed protection
with broad-spectrum antibiotics and L. rhamnosus subsp.
GG in HLA-B27 transgenic rats (58).
Recent studies have confirmed the important role of
normal luminal bacteria in the pathogenesis of chronic,
immune-mediated colitis in multiple rodent models (4–
8,59). However, not all endogenous bacteria possess
equal abilities to induce inflammation. Rath et al.
showed that Bacteroides species could initiate the in-
flammatory process in HLA-B27 transgenic rats (4). In
contrast, monoassociation with E. coli did not lead to
colitis (59). Kim et al. recently demonstrated that IL-
10−/− mice selectively colonized with Enterococcus fae-
calis developed active distal colitis (60). In our present
study, colonization of GF IL-10−/− mice with L. planta-
rum did not lead to colitis and caused only minimally
elevated levels of colonic IL-12 and of IFN-␥ secreted by
anti-CD3 stimulated MLN cells, indicating very slight
immune system activation that was significantly less
than that after colonization with SPF enteric bacteria.
Likewise, Dieleman et al. reported that monoassociation
with Helicobacter hepaticus did not induce colitis in
gnotobiotic IL-10−/− mice (61). Moreover, the ability of
Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002
FIG. 4. A: Photomicrograph of the
transverse colon of a control interleu-
kin (IL)-10−/− mouse 4 weeks after
colonization with specific pathogen-
free (SPF) bacteria, not colonized or
treated with Lactobacillus plantarum.
The inflammation is characterized by
severe mucosal hyperplasia with infil-
trating lymphocytes, elongation of the
crypts, and crypt abscesses (×100).
B: Transverse colon of a previously
germ-free IL-10−/− mouse following 2
weeks colonization with L. plantarum
before transfer to SPF conditions and
continued administration of 1 × 109
colony-free units/ml L. plantarum in
the drinking water for 4 weeks. The
number of infiltrating cells is reduced,
mucosal hyperplasia is less evident,
and goblet cells are numerous. There
are no ulcerations or crypt abscesses
(×100).
L. plantarum to reverse established colitis indicates that
although some resident bacteria have the capacity to in-
duce immune-mediated colitis in genetically susceptible
hosts, other components of the luminal flora have pro-
tective qualities.
The possible mechanism(s) by which probiotic bacte-
ria modulate the inflammatory process is still unclear.
One obvious possibility is a local effect caused by inter-
actions between lactobacilli species and other constitu-
ents of the luminal flora or intestinal epithelial cells.
Some nonpathogenic bacterial species can exert a regu-
latory effect of epithelial immune responses by inhibition
of I␬B-␣ ubiquitination (62), but this mechanism has not
been proven for probiotic microorganisms. Mack et al.
provided in vitro evidence that probiotics inhibit entero-
pathogenic E. coli adherence to epithelial cells by induc-
ing intestinal mucin gene expression (37). Our data dem-
onstrate that colonization with L. plantarum did not di-
minish concentrations of resident luminal bacteria. The
role of probiotic species in systemic immune modulation
remains uncertain. In vitro, the production of tumor ne-
crosis factor-␣, IL-6, and IL-10 by human peripheral
blood mononuclear cells is induced by lactic acid bacte-
ria (48), and recent observations suggest a possible shift
from a Th1-mediated immune response toward a Th2
profile after the administration of a probiotic agent (45).
In preliminary observations, Helwig et al. documented
an increased IL-10 concentration in the ileal pouches of
patients treated with a cocktail of eight different probi-
otic bacteria, including four Lactobacillus spp. (43). A
potentially clinically relevant way of enhancing luminal
IL-10 levels is by colonizing the colon with genetically
engineered bacteria (63). IL-10−/− mice colonized with
SPF bacteria exhibit increased amounts of Th1 cyto-
 L. PLANTARUM ATTENUATES COLITIS
kines, including IFN-␥ by stimulated MLN cells and the
inducible p40 subunit of IL-12 in culture supernatants of
inflamed colon fragments, presumably reflecting in-
creases of the biologically active IL-12 p70 heterodimer.
Live bacteria, bacterial products such as lipopolysaccha-
rides and lipoteichoic acid, as well as prokaryotic DNA
have all been demonstrated to increase production of
IL-12 by macrophages and monocytes in vitro (64)
through stimulation of Toll-like receptors (65). De-
creased levels of IL-12 and IFN-␥, although not statisti-
cally significant in all protocols, in our mice treated with
L. plantarum are consistent with the observed attenuation
of histologic inflammation, and could therefore be ex-
plained as a phenomenon secondary to the diminished
inflammation. Our results demonstrated variability of cy-
tokine results, which were significantly decreased in the
treatment protocol but not significantly inhibited despite
a 16–17% decrease in IFN-␥ and IL-12 concentrations in
the precolonization/prevention protocol, probably be-
cause of the small sample size. Stimulation of IgA se-
cretion by Lactobacilli, as shown in human studies by
other investigators (46,66), was not demonstrated in our
SPF mice.
In summary, the present data provide evidence that L.
plantarum 299v can be of therapeutic benefit in experi-
mental intestinal inflammation and merits consideration
for the treatment of clinical IBD. However, the timing of
probiotic administration might be crucial for successful
clinical outcomes. More in depth studies in experimental
models are needed to further explore dosage, duration of
treatment, comparison of various probiotic species, and
possible adjunctive use of antibiotics to optimize clinical
protocols, as well as to define the mechanisms of pro-
tection.
Acknowledgment: The authors thank Susie May for secre-
tarial support and Julie Kwon for expert technical assistance.
REFERENCES
1. Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis.
Gastroenterology 1998;115:182–205.
2. Sartor RB. Microbial factors in the pathogenesis of Crohn’s dis-
ease, ulcerative colitis and experimental intestinal inflammation.
In: Kirsner JB, ed. Inflammatory Bowel Disease. 5th Ed. Philadel-
phia: WB Saunders, 1999:153–78.
3. Sartor RB. Intestinal microflora in human and experimental in-
flammatory bowel disease. Curr Opin Gastroenterol 2001;17:
324–30.
4. Rath HC, Herfarth HH, Ikeda JS, et al. Normal luminal bacteria,
especially Bacteroides species, mediate chronic colitis, gastritis,
and arthritis in HLA-B27/human beta2 microglobulin transgenic
rats. J Clin Invest 1996;98:945–53.
5. Schultz M, Tonkonogy SL, Sellon RK, et al. IL-2-deficient mice
raised under germfree conditions develop delayed mild focal in-
testinal inflammation. Am J Physiol 1999;276:G1461–72.
79
6. Sellon RK, Tonkonogy SL, Schultz M, et al. Resident enteric
bacteria are necessary for development of spontaneous colitis and
immune system activation in interleukin-10-deficient mice. Infect
Immun 1998;66:5224–31.
7. Dianda L, Hanby AM, Wright NA, et al. T cell receptor-alpha
beta-deficient mice fail to develop colitis in the absence of a mi-
crobial environment. Am J Pathol 1997;150:91–7.
8. Veltkamp C, Tonkongy SL, De Jong YP, et al. Continuous stimu-
lation by normal luminal bacteria is essential for the development
and perpetuation of colitis in Tg␧26 mice. Gastroenterology 2001;
120:900–13.
9. Matsumoto S, Okabe Y, Setoyama H, et al. Inflammatory bowel
disease-like enteritis and caecitis in a senescence accelerated
mouse P1/Yit strain. Gut 1998;43:71–8.
10. Rath HC, Schultz M, Freitag R, et al. Different subsets of enteric
bacteria induce and perpetuate experimental colitis in rats and
mice. Infect Immun 2001;69:2277–85.
11. Madsen K, Doyle JS, Tavernini MM, et al. Antibiotic therapy
attenuates colitis in interleukin 10 gene-deficient mice. Gastroen-
terology 2000;118:1094–105.
12. Panwala CM, Jones JC, Viney JL. A novel model of inflammatory
bowel disease: mice deficient for the multiple drug resistance gene,
mdr1a, spontaneously develop colitis. J Immunol
1998;161:5733–44.
13. Videla S, Vilaseca J, Guarner F, et al. Role of intestinal microflora
in chronic inflammation and ulceration of the rat colon. Gut 1994;
35:1090–7.
14. Sutherland L, Singleton J, Sessions J, et al. Double blind, placebo
controlled trial of metronidazole in Crohn’s disease. Gut 1991;32:
1071–5.
15. Turunen U, Farkkila M, Hakala K, et al. Long-term treatment of
ulcerative colitis with ciprofloxacin: a prospective, double-blind,
placebo-controlled study. Gastroenterology 1998;115:1072–8.
16. Duchmann R, May E, Heike M, et al. T cell specificity and cross
reactivity towards Enterobacteria, Bacteroides, Bifidobacterium,
and antigens from resident intestinal flora in humans. Gut 1999;
44:812–8.
17. Cong Y, Brandwein SL, McCabe RP, et al. CD4+ T cells reactive
to enteric bacterial antigens in spontaneously colitic C3H/HeJBir
mice: increased T helper cell type 1 response and ability to transfer
disease. J Exp Med 1998;187:855–64.
18. Schultsz C, Van Den Berg FM, Ten Kate FW, et al. The intestinal
mucus layer from patients with inflammatory bowel disease har-
bors high numbers of bacteria compared with controls. Gastroen-
terology 1999;117:1089–97.
19. Giaffer MH, Holdsworth CD, Duerden BI. The assessment of fecal
flora in patients with inflammatory bowel disease by a simplified
bacteriological technique. J Med Microbiol 1991;35:238–43.
20. Fabia R, Ar’Rajab A, Johansson ML, et al. Impairment of bacterial
flora in human ulcerative colitis and experimental colitis in the rat.
Digestion 1993;54:248–55.
21. Favier C, Neut C, Mizon C, et al. Fecal beta-D-galactosidase pro-
duction and Bifidobacteria are decreased in Crohn’s disease. Dig
Dis Sci 1997;42:817–22.
22. Gorbach SL, Chang TW, Goldin BR. Successful treatment of re-
lapsing Clostridium difficile colitis with Lactobacillus GG. Lancet
1987;26:1519.
23. Pochapin M. The effect of probiotics on Clostridium difficile di-
arrhea. Am J Gastroenterol 2000;95:S11–3.
24. Hilton E, Kolakowski P, Smith M, et al. Efficacy of Lactobacillus
GG as a diarrheal preventative in travelers. J Travel Med 1997;4:
41–3.
25. Oksanen P, Salminen S, Saxelin M, et al. Prevention of traveler’s
diarrhea by Lactobacillus GG. Ann Med 1990;22:53–6.
26. Isolauri E, Juntunen M, Rautanen T, et al. A human Lactobacillus
strain (Lactobacillus casei sp strain GG) promotes recovery from
acute diarrhea in children. Pediatrics 1991;88:90–7.
27. Saavedra J. Probiotics and infectious diarrhea. Am J Gastroenterol
2000;95:S16–8.
Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002
80 M. SCHULTZ ET AL.
28. Gionchetti P, Rizzello F, Venturi A, et al. Oral bacteriotherapy as
maintenance treatment in patients with chronic pouchitis: a double-
blind, placebo-controlled trial. Gastroenterology 2000;119:305–9.
29. Venturi A, Gionchetti P, Rizzello F, et al. Impact on the compo-
sition of the faecal flora by a new probiotic preparation: prelimi-
nary data on maintenance treatment of patients with ulcerative
colitis. Aliment Pharmacol Ther 1999;13:1103–8.
30. Kruis W, Schutz E, Fric P, et al. Double-blind comparison of an
oral Escherichia coli preparation and mesalazine in maintaining
remission of ulcerative colitis. Aliment Pharmacol Ther 1997;11:
853–8.
31. Rembacken BJ, Snelling AM, Hawkey PM, et al. Non-pathogenic
Escherichia coli versus mesalazine for the treatment of ulcerative
colitis: a randomised trial. Lancet 1999;354:635–9.
32. Kruis W, Fric P, Stolte M, The Mutaflor Study Group. Mainte-
nance of remission in ulcerative colitis is equally effective with
Escherichia coli Nissle 1917 and with standard mesalamine [Ab-
stract]. Gastroenterology 2001;120:680.
33. Schultz M, Sartor RB. Probiotics and inflammatory bowel dis-
eases. Am J Gastroenterol 2000;95:S19–21.
34. Shanahan F. Probiotics in inlfammatory bowel disease. Gut 2001;
48:609.
35. Rolfe RD. The role of probiotic cultures in the control of gastro-
intestinal health. J Nutr 2000;130:396–402S.
36. Chauviére G, Coconnier M-H, Kernéis S, et al. Adhesion of Lac-
tobacillus acidophilus strain LB to human enterocyte-like Caco-2
cells. J Gen Microbiol 1992;138:1689–96.
37. Mack DR, Michail S, Wei S, et al. Probiotics inhibit enteropatho-
genic E. coli adherence in vitro by inducing intestinal mucin gene
expression. Am J Physiol 1999;276:G941–50.
38. Isolauri E, Majamaa H, Arvola T, et al. Lactobacillus casei strain
GG reverses increased permeability induced by cow milk in suck-
ling rats. Gastroenterology 1993;105:1643–50.
39. Salminen S, Isolauri E, Salminen E. Clinical uses of probiotics for
stabilizing the gut mucosal barrier: successful strains and future
challenges. Antonie van Leeuwenhoek 1996;70:347–58.
40. Madsen KL, Doyle JS, Jewell LD, et al. Lactobacillus species
prevents colitis in interleukin-10 gene-deficient mice. Gastroen-
terology 1999;116:1107–14.
41. Silva M, Jacobus NV, Deneke C, et al. Antimicrobial substance
from a human Lactobacillus strain. Antimicrobial Agents Che-
mother 1987;31:1231–3.
42. Rowland IR, Rumney CJ, Coutts JT, et al. Effect of Bifidobacte-
rium longum and inulin on gut bacterial metabolism and carcino-
gen-induced aberrant crypt foci in rats. Carcinogenesis 1998;19:
281–5.
43. Helwig U, Rizzello F, Cifone G, et al. Elevated IL-10 levels in
pouch-tissue after probiotic therapy [Abstract]. Immunol Lett 1999;
69:159.
44. Cunningham-Rundles S, Ahrne S, Bengmark S, et al. Probiotics
and immune response. Am J Gastroenterol 2000;95:S22–5.
45. Schultz M, Linde HJ, Stebbing JF, et al. Oral administration of
Lactobacillus GG (L. GG) induces an antiinflammatory, TH-2 me-
diated systemic immune response towards intestinal organisms
[Abstract]. Gastroenterology 2000;118:A781.
46. Kim P-H, Ko E-J. Bifidobacterium is a strong stimulant for IgA
antibody production by mucosal B lymphocytes [Abstract]. FASEB
J 1998;12:A5278.
47. Ulisse S, Gionchetti P, D’Alò S, et al. Expression of cytokines,
inducible nitric oxide synthetase and matrix metalloproteinases in
Inflammatory Bowel Diseases威, Vol. 8, No. 2, March 2002
pouchitis: effects of probiotic treatment. Am J Gastroenterol
2001;96:2691–9.
48. Miettinen M, Vuopio-Varkila J, Varkila K. Production of human
tumor necrosis factor alpha, interleukin-6, and interleukin-10 is
induced by lactic acid bacteria. Infect Immun 1996;64:5403–5.
49. Haller D, Blum S, Bode C, et al. Activation of human peripheral
blood mononuclear cells by nonpathogenic bacteria in vitro: evi-
dence of NK cells as primary targets. Infect Immun 2000;68:752–9.
50. Kuhn R, Lohler J, Rennick D, et al. Interleukin-10-deficient mice
develop chronic enterocolitis. Cell 1993;75:263–74.
51. Berg DJ, Davidson N, Kuhn R, et al. Enterocolitis and colon cancer
in interleukin-10-deficient mice are associated with aberrant cyto-
kine production and CD4(+) TH1-like responses. J Clin Invest
1996;98:1010–20.
52. Johansson M-L, Nobaek S, Berggren A, et al. Survival of Lacto-
bacillus plantarum DSM 9843 (299v), and effect on the short-
chain fatty acid content of faeces after ingestion of a rose-hip drink
with fermented oats. Int J Food Microbial 1998;42:1010–20.
53. Beckwith CS, Franklin CL, Hook RR, et al. Fecal PCR assay for
diagnosis of Helicobacter infection in laboratory rodents. J Clin
Microbiol 1997;35:1620–3.
54. Tonkonogy SL, Sartor RB. Immune system activation in C3H/HeJ
Bir mice exhibiting spontaneous perianal ulceration. Inflammatory
Bowel Diseases 1997;3:10–9.
55. Johansson ML, Molin G, Jeppsson B, et al. Administration of
different Lactobacillus strains in fermented oatmeal soup: in vivo
colonization of human intestinal mucosa and effect on the indig-
enous flora. Appl Environ Microbiol 1993;59:15–20.
56. Mao Y, Nobaek S, Kasravi B, et al. The effects of Lactobacillus
strains and oat fiber on methotrexate-induced enterocolitis in rats.
Gastroenterology 1996;111:334–44.
57. Veltkamp C, Tonkongy SL, Schultz M, et al. Lactobacillus plan-
tarum is superior to Lactobacillus GG in preventing colitis in IL-10
deficient mice [Abstract]. Gastroenterology 1999;116:A838.
58. Dieleman LA, Goerres M, Arends A, et al. Lactobacillus GG pre-
vents recurrence of colitis in HLA B27 transgenic rats after anti-
biotic treatment [Abstract]. Gastroenterology 2000;118:A814.
59. Rath HC, Wilson KH, Sartor RB. Differential induction of colitis
and gastritis in HLA-B27 transgenic rats selectively colonized with
Bacteroides vulgatus or Escherichia coli. Infect Immun 1999;67:
2969–74.
60. Kim SC, Tonkonogy SL, Balish E, et al. IL-10 deficient mice
monoassociated with non-pathogenic Enterococcus faecalis de-
velop chronic colitis [Abstract]. Gastroenterology 2001;120:A441.
61. Dieleman LA, Arends A, Tonkonogy SL, et al. Helicobacter he-
paticus does not induce or perpetuate colitis in interleukin-10-
deficient mice. Infect Immun 2000;68:5107–13.
62. Neish AS, Gewirtz AT, Zeng H, et al. Prokaryotic regulation of
epithelial responses by inhibition of I␬B-␣ ubiquitination. Science
2000;289:1560–63.
63. Steidler L, Hans W, Schotte L, et al. Treatment of murine colitis by
Lactococcus lactis secreting interleukin-10. Science 2000;289:
1352–5.
64. Halpern MD, Kurlander RJ, Pisetsky DS. Bacterial DNA induced
murine interferon-␥ production by stimulation of interleukin-12
and tumor necrosis factor-␣. Cell Immunol 1996;167:72–8.
65. Kimbrell DA, Beutler B. The evolution and genetics of innate
immunity. Nat Rev Genet 2001;2:256–67.
66. Kaila M, Isolauri E, Soppi E, et al. Enhancement of the circulating
antibody secreting cell response in human diarrhea by a human
Lactobacillus strain. Pediatr Res 1992;32:141–4.