Title: Selection and use of
lactic acid bacteria for reducing inflammation caused by Helicobacter
United States Patent: 7,105,336
Issued: September 12, 2006
Inventors: Connolly; Eamonn
(Lidingo, SE), Mollstam; Bo (Lerum, SE)
Assignee: Biogaia AB
Filed: October 7, 2002
Master of Science in Law
Strains of Lactobacillus which have been
selected for their capability of reducing gastrointestinal inflammation,
such as that due to Helicobacter pylori, and products derived from these
strains, including agents for treatment or prophylaxis of inflammation
associated with Helicobacter pylori for administration to humans and
include conditioned media in which the selected strains have grown and
protein-containing extracts of the conditioned media.
SUMMARY OF THE
The invention herein comprises certain
Lactobacillus strains which have been selected for their capability of
reducing gastrointestinal inflammation, such as that due to Helicobacter
pylori, and products derived from said strains, including agents for
treatment or prophylaxis of inflammation associated with Helicobacter
pylori for administration to humans, and include conditioned media in
which said strains have grown and protein-containing extracts of the
OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF
The present invention herein comprises
strains of Lactobacillus which have been selected for their capability of
reducing gastrointestinal inflammation, such as that due to Helicobacter
pylori. Such strains include Lactobacillus coryniformis MM7, ATCC PTA4660
and Lactobacillus reuteri MM2 3, ATCC PTA-4659 deposited under the
Budapest Treaty at the American Type Culture Collection University Blvd.,
Manassas, VA 20110-2209, on Sept. 11,2002. Products such as foods,
nutritional additives and formulations, pharmaceuticals or medical devices
containing whole cells or components derived from these strains may be
formulated as is known in the art, and generally include an ingestible
support as known plus the Lactobacillus-strain, or its derived component.
Previously known strains, now identified to have good TNF.alpha. reducing
capacity, such as L. rhamnosus GG ATCC 53103, L. reuteri ATCC 55730 and
others, can also be used in the above formulations. These products are
agents for treatment or prophylaxis of inflammation associated with
Helicobacter pylori for administration to mammals.
Model systems using the appropriate cytokines are used to determine
factors that reduce or increase inflammation. In the examples provided
herein, a mouse macrophage assay, using the RAW 264.7 macrophage cells (ATCC,
Rockville, Md., ATCC # TIB-71), is used to screen strains of bacteria,
primarily lactobacilli, for their effect on the inflammatory pathway.
IL-10 is used in this assay as a positive control, with treatments with
IL-10 showing inhibition of pro-inflammatory cytokines such as TNF.alpha.
(tumor necrosis factor alpha). After individual growth of the
Lactobacillus strains to be screened in laboratory media, the live
bacterial cells are removed by filtration and the supernatant fluid (also
called the "conditioned-medium" herein) is tested in the macrophage assay.
The macrophages are first stimulated with the pro-inflammatory antigen for
example, purified LPS (E. coli derived lipopolysaccharide), S. aureus
derived lipoteichoic acid (LTA) or cell free E. coli or Helicobacter
conditioned media, to produce the pro-inflammatory cytokines including
TNF.alpha.. The conditioned medium from the Lactobacillus strain,
containing the putative immunomodulating substances derived from the
bacteria to be screened, is co-incubated with the antigen-activated
macrophages. The capacity of the conditioned medium to modulate the immune
response of the macrophages is monitored by the change in TNF.alpha.
production by the cells. The TNF.alpha. profile from the assay enables a
selection of the strains most effective in reducing the production of
TNF.alpha. by the macrophages. Control experiments with pH adjustment in
the assay system eliminates the possibility that a changed pH could cause
the observed effect.
Surprisingly, apparently similar bacterial isolates and strains of
Lactobacillus, even coming from very similar human sources show varying
and widely different abilities to influence the production of TNF.alpha.
by macrophages in response to a pro-inflammatory antigen. These strains
cannot be identified even by genetic fingerprinting since they can be up
to 98% similar genetically but still show very different effects on the
immune cells. The strains thus screened and found to have a strong
inhibitory effect against stimulated, proinflammatory cytokine production
by macrophages are especially effective in the treatment of inflammation
in the gastrointestinal tract of man, including H. pylori caused
inflammation in the stomach.
The features of the present invention will be more clearly understood by
reference to the following examples, which are not to be construed as
limiting the invention.
Selection of Anti-Inflammatory Strains.
Lactobacillus spp. (including for example L. rhamnosus GG ATCC 53103, L.
johnsonii ATCC 33200, L. reuteri MM2 3 ATCC PTA-4659, L. coryniformis,
MM7, ATCC PTA-4660) and E. coli Nissle were grown in de Man, Rogosa,
Sharpe (MRS) and Luria-Bertani (LB) media (Difco, Sparks, Md.),
respectively. Overnight cultures of lactobacilli were diluted to an
OD.sub.600 of 1.0 (representing approximately 10.sup.9 cells/ml) and
further diluted 1:10 and grown for an additional 4, 8 and 24 h.
Helicobacter pylori, (Sydney strain SS1) and Helicobacter hepaticus
3B1(ATCC 51449) were cultured for 48 h in Brucella broth (Difco)
supplemented with 10% fetal bovine serum (FBS). Cultures were diluted 1:10
and grown for another 24 and 48 h. Bacterial cell-free conditioned medium
was collected by centrifugation at 8500 rpm for 10 min at 4.degree. C.
Conditioned medium was separated from the cell pellet and then filtered
through a 0.22 .mu.m pore filter unit (Millipore, Bedford, Mass.).
Mouse monocyte/macrophage cell lines, RAW 264.7 (ATCC TIB-71) and RAW
264.7 gamma NO(-) (ATCC CRL-2278), were used as a reporter cells for
studying the inflammatory response pathway. RAW 264.7 cells were grown in
either Dulbecco's Modified Eagle Medium (wild-type) or RPMI Medium 1640
(gamma NO--) (Gibco-Invitrogen, Carlsbad, Calif.) supplemented with 10%
FBS and 2% antibiotic (5000 units/ml Penicillin and 5 mg/ml Streptomycin,
Sigma) at 5% CO.sub.2 37.degree. C. until 80 90% confluent. Approximately
5.times.104 cells were seeded into 96-well cell culture clusters and
allowed to adhere for 2 h prior to lipopolysaccharide (LPS) activation and
addition of conditioned medium. Naive RAW 264.7 cells were exposed to
purified LPS from E. coli serotype O127:B8 (Sigma). Activation medium was
made by adding 2 ng LPS to 20 .mu.l conditioned medium per well.
Macrophages were either pre-incubated or co-incubated with cell-free
Lactobacillus conditioned medium. Recombinant mIL-10 (R&D Systems,
Minneapolis, Minn.) was used as a positive control. Cell viability was
assessed by Trypan-blue (Invitrogen) exclusion. The presence of TNF-.alpha.
in cell culture supernatant was measured with a sandwich enzyme
immunoassay, Quantikine M.RTM. Mouse TNF-.alpha. Immunoassay (R & D
The effect of Lactobacillus-conditioned media on TNF.alpha. production by
LPS-activated macrophages is shown in FIG. 1, which shows that of the 45
strains tested, several different strains are capable of decreasing
TNF.alpha. production by the activated macrophages. FIG. 2 shows the fold
change in TNF.alpha. expression with various Lactobacillus strains
compared to LPS alone. The results of these studies are then used to
select the most efficient strains. The strains mentioned in the figures
but not specifically mentioned in the text are various strains of
Lactobacillus, primarily L. reuteri that were tested.
Strain MM2 3 is thus shown (FIG. 2) to be extremely effective in
decreasing TNF.alpha. production by activated macrophages with a reduction
to approximately 10 pg/10e5 cells as compared to over 700 pg/10e5 cells in
the control and as compared to generally near or over 300 pg/10e5 cells in
other strains. In fact this reduction was over 35-fold change in TNF.alpha.
(FIG. 1). Further, other data show that when strains are screened for
inhibition of IL-8 production in Toxin A-stimulated cultured HT29 human
intestinal epithelial cells, strain MM2 3 consistently shows greater than
a 50% partial inhibition of this proinflammatory cytokine. This strain is
very acid tolerant, and shows a much greater survival in pH 2.0 gastric
juice than other strains, having complete survival for a 30-minute period,
while the other tested strains are reduced by 1 6 logs in their numbers.
Not all Lactobacillus strains show strong aggregation, nor do they all
adhere to mucus, but strain MM2 3 has strong adhesion to mucus with and
without pre-incubation of the cells with mucins to induce binding
activity. Thus, this strain is the optimal choice for anti-inflammation,
especially in an acid environment like the stomach.
In this example, L. coryniformis MM7, ATCC PTA-4660, was selected by using
the method above, for addition to a standard yogurt. The L. coryniformis
strain was grown and lyophilized, using standard methods for growing
Lactobacillus in the dairy industry. This culture was then added to
previously fermented milk, using traditional yogurt cultures, at a level
of 10E+7 CFU/gram of yogurt, and the yogurt was used by humans as a
prevention of gastritis caused by H. pylor.
Claim 1 of 1 Claim
1. A biologically pure culture
of Lactobacillus reuteri strain MM2 3, ATCC PTA-4659 which strain exhibits
all of the identifying data and parameters as shown in FIGS. 1 and 2 having
the properties with respect to TNF.alpha..
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