U.S. patent application number 11/478545 was filed with the patent office on 2007-06-21 for bifidobacterium in the treatment of inflammatory disease.
This patent application is currently assigned to University College Cork - National University of Ireland, Cork. Invention is credited to John Kevin Collins, Liam O'Mahony, Gerald Christopher O'Sullivan, Fergus Shanahan.
Application Number | 20070141039 11/478545 |
Document ID | / |
Family ID | 26320235 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141039 |
Kind Code |
A1 |
Collins; John Kevin ; et
al. |
June 21, 2007 |
Bifidobacterium in the treatment of inflammatory disease
Abstract
A strain of Bifidobacterium isolated from resected and washed
human gastrointestinal tract is significantly immunomodulatory
following oral consumption in humans. The strain is useful in the
prophylaxis and/or treatment of undesirable inflammatory activity,
especially gastrointestinal inflammatory activity such as
inflammatory bowel disease or irritable bowel syndrome. The
inflammatory activity may also be due to cancer.
Inventors: |
Collins; John Kevin;
(Doughcloyne, IE) ; O'Sullivan; Gerald Christopher;
(Ballinveltig, IE) ; O'Mahony; Liam;
(Ballintemple, IE) ; Shanahan; Fergus; (Kinsale,
IE) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
University College Cork - National
University of Ireland, Cork
Cork
IE
Enterprise Ireland (Trading as Bioresearch Ireland)
|
Family ID: |
26320235 |
Appl. No.: |
11/478545 |
Filed: |
June 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10975353 |
Oct 29, 2004 |
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11478545 |
Jun 29, 2006 |
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10388652 |
Mar 17, 2003 |
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10975353 |
Oct 29, 2004 |
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09903681 |
Jul 13, 2001 |
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10388652 |
Mar 17, 2003 |
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PCT/IE00/00008 |
Jan 17, 2000 |
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09903681 |
Jul 13, 2001 |
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Current U.S.
Class: |
424/93.45 ;
435/252.9 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
31/12 20180101; A23V 2002/00 20130101; A23C 9/1234 20130101; A61P
29/00 20180101; A23Y 2220/77 20130101; Y10S 435/853 20130101; A61P
37/04 20180101; Y10S 435/822 20130101; A61P 37/00 20180101; A61P
1/00 20180101; A61P 35/00 20180101; A61P 37/02 20180101; A61P 37/06
20180101; A61K 2039/542 20130101; A61P 1/12 20180101; A61P 19/02
20180101; A61P 31/14 20180101; C12N 1/20 20130101; A23L 33/135
20160801; A61K 2039/52 20130101; A61P 31/04 20180101; A61P 43/00
20180101; C12R 1/01 20130101; A61K 39/09 20130101; A61P 19/04
20180101; A23V 2002/00 20130101; A23V 2200/3204 20130101; A23V
2200/324 20130101 |
Class at
Publication: |
424/093.45 ;
435/252.9 |
International
Class: |
A61K 35/74 20060101
A61K035/74; C12N 1/20 20060101 C12N001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 15, 1999 |
IE |
990033 |
Sep 20, 1999 |
IE |
990782 |
Claims
1-54. (canceled)
55. An antimicrobial agent obtained from a strain of
Bifidobacterium isolated from a resected and washed human
gastrointestinal tract, which is significantly immunomodulatory
following oral consumption.
56. The antimicrobial agent of claim 55, wherein the antimicrobial
agent is significantly immunomodulatory in a human following oral
consumption by the human.
57. The antimicrobial agent of claim 56, wherein the
immunomodulatory effect is an increase in an anti-inflammatory
cytokine; a decrease in a pro-inflammatory cytokine; an increase in
an anti-inflammatory cytokine and a decrease in a pro-inflammatory
cytokine; or production of a high anti-inflammatory cytokine to
pro-inflammatory cytokine ratio in the human following oral
consumption.
58. The antimicrobial agent of claim wherein the anti-inflammatory
cytokine is IL-10 and the pro-inflammatory cytokine is IL-12 or
TNF-.alpha..
Description
INTRODUCTION
[0001] This invention relates to probiotic Bifidobacterium strains
which have various applications in foodstuffs and in medicine. More
particularly, the invention relates to probiotic strains of
bifidobacteria which are capable of beneficially modifying and
consequently alleviating observable symptoms in inflammatory
disease.
[0002] Consumers are becoming increasingly aware of matters which
may be necessary for maintenance of their environment, health and
nutrition. In response, scientific research has focussed upon the
roles that diet, stress, and modem medical practices (e.g.
antibiotics and radiotherapy) may play in threatening human health.
In particular, population dynamics shifting towards older societies
are increasing the incidence of illnesses which may be caused by
deficient or compromised microflora such as gastrointestinal tract
(GIT) infections, constipation, irritable bowel syndrome (IBS),
inflammatory bowel disease (IBD)--Crohn's disease and ulcerative
colitis, food allergies, antibiotic-induced diarrhoea,
cardiovascular disease, and certain cancers (e.g. colorectal
cancer).
[0003] Probiotics have been defined as live microbial food
supplements which beneficially affect the host by improving the
intestinal microbial balance, or more broadly, as living
micro-organisms, which upon ingestion in certain numbers, exert
health effects beyond inherent basic nutrition. Cocktails of
various micro-organisms, particularly species of Lactobacillus and
Streptococcus, have traditionally been used in fermented dairy
products to promote health.
[0004] In recent years the commercial manufacture and marketing of
functional foods (foods which affect functions of the body in a
targeted manner so as to bring about positive affects on physiology
and nutrition), particularly probiotic (Acidophilus-Bifidus)
yoghurts, has spread from the well-established Japanese niche
market place into the lucrative and expanding European Union. While
a number of probiotic bacteria of human origin are now being
exploited commercially (e.g., L. acidophilus LA-1), many consumers,
consumer organisations, and members of the scientific community are
sceptical of such products and their publicised probiotic claims.
The diary-food industry is therefore under considerable pressure to
scientifically validate these new probiotic food products.
[0005] Criteria which have been suggested for the selection of
potentially effective probiotic micro-organisms may be summarised
as follows: human origin, non-pathogenic behaviour, resistance to
technological processes (i.e., viability and activity in delivery
vehicles), resistance to gastric acidity and bile toxicity,
adhesion to gut epithelial tissue, ability to colonise the GIT,
production of antimicrobial substances, ability to modulate immune
responses, and the ability to influence metabolic activities (e.g.,
cholesterol assimilation, lactase activity, vitamin production)
(Huis in't Veld J, Shortt C. Selection criteria for probiotic
micro-organisms. In: Leeds, A. R., Rowland, I. R. eds. Gut Fora and
Health--Past, Present and Future. London: The Royal Society of
Medicine Press Ltd., 1996:19-26).
[0006] Bifidobacteria are one of several predominant culturable
bacteria present in the colonic microflora.
[0007] The functions of endogenous bifidobacteria in the colon have
not been completely elucidated. However it is recognised that
exclusively breast-fed infants have a reduced risk of diarrhoea
compared with formula-fed infants. The fact that these infants have
greater numbers of colonic bifidobacteria may in part explain this
observed health advantage as the occupation of available niches in
the GIT by large numbers of nonpathogenic bifidobacteria may help
prevent bacterial infection. The pathogenesis of Crohn's disease is
thought to be related to colonic bacterial microflora (Targan, S.
and Shanahan, F. Inflammatory bowel disease: From bench to bedside.
Williams and Wilkins 1994.) It has recently been found that
patients suffering from active Crohn's disease have significantly
less recoverable bifidobacteria in their faeces compared with
healthy individuals. This reduction in bifidobacteria numbers was
observed to be directly correlated with decreased levels of
.beta.-D galactosidase production and activity (Favier, C. et al;
Dig. Dis. Sci. 1997; 42:817-822). .beta.-D galactosidase is an
enzyme produced by bifidobacteria. These results support
suggestions proposed in other studies that strains of
bifidobacteria may play important roles in maintaining a balanced
healthy intestinal microflora.
[0008] Bifidobacteria are considered to be probiotics as they are
living organisms which exert healthy effects beyond basic nutrition
when ingested in sufficient numbers. Numerous ingested
bifidobacteria must reach the site of action in the gut in order to
exert a probiotic effect. A minimum level of approximately
10.sup.6-10.sup.7 viable bifidobacteria per gram intestinal
contents has been suggested (Bouhnik, Y., Lait 1993: 73:241-247).
There are reports in the literature which show that in vivo studies
completed in adults and in infants indicate that some strains of
bifidobacteria are capable of surviving passage through the
gastrointestinal tract. Significant differences have been observed
between the abilities of different bifidobacteria strains to
tolerate acid and bile salts, indicating that survival is an
important criterion for the selection of potential probiotic
strains.
[0009] Ingestion of bifidobacteria can improve gastrointestinal
transit.
[0010] Furthermore, indirect evidence in humans demonstrates that
consuming milk fermented by bifidobacteria can lead to reduced
levels of certain faecal enzymes such as .beta.-D galactosidase
implicated in the conversion of procarcinogens to carcinogens
(Bouhnik Y. et al; Eur. J. Clin. Nutr. 1996; 50:269-273).
Faecal-bome putrefaction metabolities such as p-cresol, indole and
ammonia were also reduced when subjects consumed milk fermented by
Bifidobacterium longum and S. thermophilus (Takiguchi, R. et al.
Bifidus--Flores, Fructus etSemina 1996;9:135-140).
[0011] Antimicrobial activity has been reported to be associated
with bifidobacteria. Also, bifidobacteria have been shown to
modulate various parameters of the immune system.
[0012] Mucosal inflammation in IL-10deficient mice has been
reported to be reduced by feeding the subject animals a preparation
of lactic acid bacteria (Madsen, K. et al. Gastroenterol. 1997;
112:A1030.). Further studies completed in rats have demonstrated
that ingestion of bifidobacteria can suppress aberrant crypt foci
(early preneoplastic lesions) formation in the colon (Kulkani, N.
and Reddy, B. Proc. Soc. Experim. Biol. Med. 1994; 207; 278-283.)
in addition to significant decreases in colon tumor incidence and
in the numbers of tumors present (Singh, J. et al Carcinogenesis
1997; 18:833-841).
[0013] There is an on-going search for probiotic strains with
particular beneficial effects on nutrition and therapy and on
health generally.
STATEMENTS OF INVENTION
[0014] The invention provides a strain of Bifidobacterium isolated
from resected and washed human gastrointestinal tract which is
significantly immunomodulatory following oral consumption in
humans.
[0015] The strain of Bifidobacterium preferably effects changes in
an immunological marker when introduced into a system comprising
cells which interact with the immune system and cells of the immune
system. Preferably the cells which interact with the immune system
are epithelial cells. Preferably the immunological marker is a
cytokine, especially TNF.alpha..
[0016] In a preferred embodiment the cells which interact with the
immune system and the immune system cells are of matched
origin.
[0017] The cells which interact with the immune system are of
gastrointestinal, respiratory or genitourinary origin.
[0018] The cells of the immune system are preferably of
gastrointestinal, respiratory or genitourinary origin.
[0019] The invention also provides a strain of Bifidobacterium
longum infantis isolated from resected and washed human
gastrointestinal tract which is significantly immunomodulatory
following oral consumption in humans.
[0020] The strain of Bifidobacterium which has significant
anti-inflammatory effect following oral consumption in humans.
[0021] The strain of Bifidobacterium is preferably isolated from
resected and washed human gastrointestinal tract which is capable
of combating the effects of inflammatory bowel disease, said
capability being maintained in the presence of physiological
concentrations of human bile and human gastric juice. The
capability of combating the effects of inflammatory bowel disease
is measured by measuring a reversal of a wasting disease induced in
severe combined immunodeficient recipient mice (SCID) which have
been administered purified CD4.sup.+, CD45RB.sup.high T cells.
[0022] The capability of the strain of Bifidobacterium longum
infantis to combat the effects of inflammatory bowel disease can
also be measured by measuring the reduction in colonic inflammation
in IL-10 deficient mice (IL-10.sup.+129 Svex strain) following
administration of one or more of the strains of Bifidobacterium
longum infantis according to the invention alone or in combination
with a strain of Lactobacillus salivarius as hereinafter
defined.
[0023] Interleukin 10 (IL-10) is an important regulatory cytokine
that supresses effector functions of macrophage/monocytes, T helper
1 (Th1) cells, and natural killer cells. In addition, IL-10
augments proliferation and differentiation of B cells. Murine
models lacking the IL-10 gene spontaneously develop inflammatory
bowel disease and gastrointestinal tumors. The gastrointestinal
flora have been implicated in the pathogenesis of these disease
states as germ free animals do not develop disease.
[0024] The strain of Bifidobacterium preferably has inhibitory
activity against a broad range of Gram positive and Gram negative
bacteria.
[0025] Preferably the strain of Bifidobacterium exhibits a
broad-spectrum of activity against bacteria including
Staphylococcus, Pseudomonas, Coliform and Bacillus species.
[0026] In a particular aspect the invention provides strain of
Bifidobacterium longum infantis UCC35624 or mutant or variant
thereof.
[0027] A deposit of Bifidobacterium longum infantis strain UCC
35624 was made at the National Collections of Industrial and Marine
Bacteria Limited (NCIMB) on Jan. 13, 1999 and accorded the
accession number NCIMB 41003.
[0028] In one embodiment the mutant is a genetically modified
mutant.
[0029] In one embodiment the variant is a naturally occurring
variant of Bifidobacterium longum infantis UCC35624.
[0030] The strain of Bifidobacterium may be in the form of viable
cells. Alternatively the strain of Bifidobacterium is in the form
of non-viable cells.
[0031] The invention also provides an antimicrobial agent obtained
from a strain of Bifidobacterium of the invention which is
antagonistic to the growth of other organisms.
[0032] In a further aspect the invention provides a formulation
which comprises a strain of Bifidobacterium of the invention.
[0033] The formulation may comprise two or more strains of
Bifidobacterium.
[0034] The formulation may include another probiotic material.
Alternatively or additionally the formulation includes a prebiotic
material.
[0035] The formulation may which include a strain of Lactobacillus
salivarius.
[0036] The strain of Lactobacillus salivarius may be in the form of
viable cells or in the form of non-viable cells.
[0037] The Lactobacillus salivarius is preferably isolated from
resected and washed human gastrointestinal tract, the Lactobacillus
salivarius being significantly immunomodulatory following oral
consumption in humans. Preferably the strain of Lactobacillus
salivarius is isolated from resected and washed human
gastrointestinal tract which inhibits a broad range of Gram
positive and Gram negative micro-organisms.
[0038] In a preferred embodiment the strain of Lactobacillus
salivarius secretes a product having antimicrobial activity into a
cell-free supernatant, said activity being produced only by growing
cells and being destroyed by proteinase K and pronase E, the
inhibitory properties of said strain and its secretory products
being maintained in the presence of physiological concentration of
human bile and human gastric juice.
[0039] Such strains of Lactobacillus salivarius are disclosed in WO
98/35014.
[0040] Ideally the strain of Lactobacillus salivarius is
Lactobacillus salivarius strain UCC 118 or a mutant or variant
thereof. The mutant is a genetically modified mutant. The variant
may be a naturally occurring variant of Lactobacillus
salivarius.
[0041] A deposit of Lactobacillus salivarius strain UCC 118 was
made at the NCIMB on Nov. 27, 1996 and accorded the accession
number NCIMB 40829.
[0042] Preferably the formulation includes an ingestable carrier.
The ingestable carrier may be a pharmaceutically acceptable carrier
such as a capsule, tablet or powder.
[0043] The ingestable carrier may be a food product such as
acidified milk, yoghurt, frozen yoghurt, milk powder, milk
concentrate, cheese spreads, dressings or beverages.
[0044] The formulation may comprise a protein and/or peptide, in
particular proteins and/or peptides that are rich in
glutamine/glutamate, a lipid, a carbohydrate, a vitamin, mineral
and/or trace element.
[0045] In one embodiment the Bifidobacterium is present at more
than 10.sup.6 cfu per gram of delivery system.
[0046] In another embodiment the formulation includes an
adjuvant.
[0047] The formulation may include a bacterial component. The
formulation may alternatively or additionally include a drug
entity. The formulation may also include a biological compound.
[0048] The formulation may be in a form for oral immunisation.
[0049] The invention further provides a strain of Bifidobacterium
or a formulation thereof for use in foodstuffs.
[0050] In another aspect the invention provides a strain of
Bifidobacterium or a formulation thereof for use as a
medicament.
[0051] The strain or formulation may be for use in the prophylaxis
and/or treatment of undesirable inflammatory activity.
[0052] The strain or formulation may be for use in the prophylaxis
and/or treatment of undesirable gastrointestinal inflammatory
activity such as inflammatory bowel disease eg. Crohns disease or
ulcerative colitis, irritable bowel syndrome, pouchitis or post
infection colitis.
[0053] The undesirable inflammatory activity may be due to
cancer.
[0054] In addition the strain or formulation may be for use in the
prophylaxis and/or treatment of gastrointestinal cancer(s).
[0055] The strain or formulation may be used for the prophylaxis of
cancer. Further, the strain or formulation may be for use in the
prophylaxis and/or treatment of systemic disease such as rheumatoid
arthritis.
[0056] The strain or formulation may be for use in the prophylaxis
and/or treatment of autoimmune disorders due to undesirable
inflammatory activity.
[0057] The strain or formulation may be for use in the prophylaxis
and/or treatment of cancer due to undesirable inflammatory
activity.
[0058] The strain or formulation may be for use in the prophylaxis
and/or treatment of diarrhoeal disease due undesirable inflammatory
activity, such as Clostridium difficile associated diarrhoea,
Rotavirus associated diarrhoea or post infective diarrhoea.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] In the accompanying drawings:
[0060] FIG. 1 is a graph of cfu/ml versus time for Bifidobacterium
longum infantis strain 35612 as described in Example 2;
[0061] FIG. 2 is a graph of cfu/ml versus time for Bifidobacterium
longum infantis strain 35624 as described in Example 2;
[0062] FIG. 3 is a graph of percentage weight change versus time
(days) for five SCID mice (1-5) administered strain UCC 35624 as
described in Example 5;
[0063] FIG. 4 is a graph of average percentage weight change versus
time (days) for the SCID mice (1-5) administered strain UCC 35624
as described in Example 5;
[0064] FIG. 5 is a graph of percentage weight change versus time
(days) for mice (6-10) administered a combination of strains
Lactobacillus salivarius UCC 118 and UCC 35624 as described in
Example 5;
[0065] FIG. 6 is a graph of average percentage weight change versus
time (days) for mice (6-10) administered a combination of strains
UCC 118 and UCC 35624 as described in Example 5;
[0066] FIG. 7 is a graph of percentage weight change versus time
(days) for mice (11-15) administered a combination of stains UCC
118 and UCC 35624 as described in Example 5;
[0067] FIG. 8 is a graph of average percentage weight change versus
time (days) for mice (11-15) administered a combination of strains
UCC 118 and UCC 35624 as described in Example 5;
[0068] FIG. 9 is a bar chart of TNF.alpha. levels in patient and
control samples in the presence of PBMCs and Bifidobacteria longum
infantis as described in Example 7;
[0069] FIG. 10 is a bar chart showing TNF.alpha. and IL-8 levels in
co-cultures of epithelial cells, PBMCs and Bifidobacterium longum
infantis as described in Example 7. Controls represent co-cultures
of epithelial cells and PBMCs alone;
[0070] FIG. 11 are bar charts of peripheral blood cytokine levels
following consumption of Bifidobacterium longum infantis by healthy
human volunteers (n=18) for three weeks as described in Example
8;
[0071] FIG. 12 are bar charts of serum levels of TNF.alpha. and
IL-IRA following consumption of Bifidobacterium longum infantis to
healthy human volunteers (n=18) as described in Example 8;
[0072] FIG. 13 is a bar chart of TNF.alpha. levels in cell-free
spent culture supernatant of Bifidobacterium longum infantis and an
MRS control as described in Example 9;
[0073] FIG. 14 is a diagrammatic representation of a SCID mouse
lower intestine after treatment with Bifidobacterium longum
infantis; and
[0074] FIG. 15 is a diagrammatic representation of the lower
intestine of an untreated SCID mouse.
DETAILED DESCRIPTION
[0075] We have isolated strains of probiotic bacteria which are
capable of beneficially modifying and consequently alleviating
observable symptoms in inflammatory disorders. These strains and
the formulations prepared may be used in a variety of foodstuffs
and medicaments to combat the effect of inflammatory disorders.
[0076] In vivo and in vitro studies were carried out using the
probiotic bacteria strains. It was found that humans fed with
yoghurt containing Bifidobacterium longum infantis UCC35624 show
marked decreases in their systemic levels of IL-8. This strain may
therefore have potential application in the treatment of a range of
inflammatory disorders, particularly if used in combination with
current anti-inflammatory therapies, such as non-steroid
anti-inflammatory drugs (NSAIDs) or Infliximab.
[0077] The consumption of Bifidobacterium longum infantis by SCID
mice was also examined. While this experiment significantly
attenuated inflammatory activity, mice consuming Bifidobacterium
longum infantis retained solid stools while control mice suffered
from diarrhoea. This anti-diarrhoeal effect could be related to the
anti-inflammatory activity of this invention, possibly mediated via
cAMP modulation.
[0078] It is unknown whether intact bacteria are required to exert
an anti-inflammatory effect or if individual active components of
the invention can be utilised alone. Proinflammatory components of
certain bacterial strains have been identified. The proinflammatory
effects of gram-negative bacteria are mediated by
lipopolysaccharide (LPS). LPS alone induces a proinflammatory
network, partially due to LPS binding to the CD14 receptor on
monocytes. It is assumed that components of probiotic bacteria
possess anti-inflammatory activity, due to the effects of the whole
cell. Upon isolation of these components, pharmaceutical grade
manipulation is anticipated.
[0079] The general use of Bifidobacterium longum infantis UCC35624
is in the form of viable cells. However, it can also be extended to
non-viable cells such as killed cultures or compositions containing
beneficial factors expressed by Bifidobacterium longum infantis
UCC35624. This could include thermally killed micro-organisms or
micro-organisms killed by exposure to altered pH or subjection to
pressure. With non-viable cells product preparation is simpler,
cells may be incorporated easily into pharmaceuticals and storage
requirements are much less limited than viable cells. Lactobacillus
casei YIT 9018 offers an example of the effective use of heat
killed cells as a method for the treatment and/or prevention of
tumour growth as described in U.S. Pat. No. 4,347,240.
[0080] The invention will be more clearly understood from the
following Examples.
EXAMPLE 1
Isolation of Probiotic Bacteria
[0081] Appendices and sections of the large and small intestine of
the human G.I.T., obtained during reconstructive surgery, were
screened for probiotic bacterial strains as shown in Table 1.
TABLE-US-00001 TABLE 1 Gastrointestinal tract tissue samples
screened for the presence of probiotic bacteria Sample Location A
Ileum B Colon C Ileal-caecal region D Appendix E Appendix F Ileum G
Ileal-caecal region
[0082] All samples were stored immediately after surgery at
-80.degree. C. in sterile containers. Frozen tissues were thawed,
weighed and placed in cysteinated (0.05%) one quarter strength
Ringers' solution. Each sample was gently shaken to remove loosely
adhering microorganisms (termed--wash `W`). Following transfer to a
second volume of Ringers' solution, the sample was vortexed for 7
min to remove tightly adhering bacteria (termed--Sample `S`). In
order to isolate tissue embedded bacteria, samples A, B and C were
also homogenised in a Braun blender (termed--homogenate `H`). The
solutions were serially diluted (dilution 10.sup.-1 from a wash
sample was labelled WI, dilution 10.sup.-2 was labelled W2 and the
same labelling system was used for the `S` and `H` samples) and
spread-plated (100 .mu.l) on to the following agar media: RCM
(reinforced clostridial media) and RCM adjusted to pH 5.5 using
acetic acid; TPY (trypticase, peptone and yeast extract),
Chevalier, P. et al. (1990) J Appl. Bacteriol 68, 619-624). MRS
(deMann, Rogosa and Sharpe); ROG (acetate medium (SL) of Rogosa);
LLA (Liver-lactose agar of Lapiere); BHI (brain heart infusion
agar); LBS (Lactobacillus selective agar) and TSAYE (tryptone soya
agar supplemented with 0.6% yeast extract). All agar media was
supplied by Oxoid Chemicals with the exception of TPY agar. Plates
were incubated in anaerobic jars (BBL, Oxoid) using CO.sub.2
generating kits (Anaerocult A, Merck) for 2-5 days at 37.degree.
C.
[0083] Gram positive, catalase negative rod-shaped or
bifurcated/pleomorphic bacteria isolates were streaked for purity
on to complex non-selective media (TPY). Isolates were routinely
cultivated in TPY medium unless otherwise stated at 37.degree. C.
under anaerobic conditions. Presumptive Bifidobacteria species were
stocked in 40% glycerol and stored at -20.degree. and -80.degree.
C.
Fermentation End-Product Analysis
[0084] Metabolism of the carbohydrate glucose and the subsequent
organic acid end-products were examined using an LKB Bromma, Aminex
HPX-87H High Performance Liquid Chromatography (HPLC) column. The
column was maintained at 60.degree. C. with a flow rate of 0.6
ml/min (constant pressure). The HPLC buffer used was 0.01 N
H.sub.2SO.sub.4. Prior to analysis, the column was calibrated using
10 mM citrate, 10 mM glucose, 20 mM lactate and 10 mM acetate as
standards. Cultures were propagated in modified MRS broth for 1-2
days at 37.degree. C. anaerobically. Following centrifugation for
10 min at 14,000 g, the supernatant was diluted 1:5 with HPLC
buffer and 200 .mu.l was analysed in the HPLC. All supernatants
were analysed in duplicate.
Biochemical and Physiological Characterisation
[0085] Biochemical and physiological traits of the bacterial
isolates were determined to aid identification. Nitrate reduction,
indole formation and expression of .beta.-galactosidase activity
were assayed. Growth at both 15.degree. C. and 45.degree. C. and
protease activity on gelatin were determined. Growth
characteristics of the strains in litmus milk were also
assessed.
Antibiotic Sensitivity Profiles
[0086] Antibiotic sensitivity profiles of the isolates were
determined using the `disc susceptibility` assay. Cultures were
grown up in the appropriate broth medium for 24-48 h, spread-plated
(100 .mu.l) onto agar media and discs containing known
concentrations of the antibiotics were placed onto the agar.
Strains were examined for antibiotic sensitivity after 1-2 days
incubation at 37.degree. C. under anaerobic conditions. Strains
were considered sensitive if zones of inhibition of 1 mm or greater
were seen.
Isolation of Bifidobacteria sp.
[0087] Seven tissue sections taken from the human G.I.T. were
screened for the presence of strains belonging to the
Bifidobacterium genus. There was some variation between tissue
samples as follows. Samples A (ileum) and E (appendix) had the
lowest counts with approximately 10.sup.2 cells isolated per gram
of tissue. In comparison, greater than 10.sup.3 cfu/g tissue were
recovered from the other samples. Similar numbers of bacteria were
isolated during the `wash` and `sample` steps with slightly higher
counts in the `sample` solutions of F (ileum) and G (ileal-caecal).
Of those screened for tightly-adhering bacteria (homogenised), C
(ileal-caecal) was the only tissue section that gave significant
counts.
[0088] During the screening of some tissue sections, for example C
and B, there was not a direct correlation between counts obtained
during a dilution series. This would indicate that some growth
factors, either blood or tissue derived were being provided for the
growth of the fastidious bacteria in the initial suspension which
was subsequently diluted out.
Strain Selection and Characterisation
[0089] Approximately fifteen hundred catalase negative bacterial
isolates from different samples were chosen and characterised in
terms of their Gram reaction, cell size and morphology, growth at
15.degree. C. and 45.degree. C. and fermentation end-products from
glucose. Greater than sixty percent of the isolates tested were
Gram positive, homofermentative cocci arranged either in tetrads,
chains or bunches. Eighteen percent of the isolates were Gram
negative rods and, heterofermentative coccobacilli.
[0090] The remaining isolates (twenty-two percent) were
predominantly homofermentative coccobacilli. Thirty eight strains
were characterised in more detail--13 isolates from G; 4 from F; 8
from D; 9 from C; 3 from B and 1 from E. All thirty eight isolates
tested negative both for nitrate reduction and production of indole
from tryptophan.
Antibiotic Sensitivity Profiles
[0091] Antibiotics of human clinical importance were used to
ascertain the sensitivity profiles of selected bifidobacteria. The
bifidobacteria tested were sensitive to ampicillin, amoxycillin
ceftaxime, ceftriaxone, ciprofloxacin, cephradine, rifampicin,
amikacin, gentamicin and chloramphenicol. They were also resistant
to netilmicin, trimethoprim, nalidixic acid, cefuroxime, vancomycin
and tetracycline.
EXAMPLE 2
Acid Resistance
[0092] The first line of host defence that a micro-organism reaches
following human consumption is gastric acid in the stomach. A key
factor influencing bacteria is survival in gastric juice. The
survival and growth of Bifidobacterium longum infantis strains
35612 and 35624 in a low pH environment were examined. The strains
were routinely cultured in trypticase-peptone-yeast extract (TPY)
medium at 37.degree. C. under strict anaerobic conditions (BBL Gas
jars using the Merck Anaerocult A gas pak system) for 12-24 h.
Human gastric juice was obtained from healthy subjects by
aspiration through a nasogastric tube (Mercy Hospital, Cork,
Ireland). It was immediately centrigued at 13,000 g for 30 min. to
remove all solid-particles, sterilised through 0.451 .mu.m filters
and 0.21 .mu.m filters and stored at 4.degree. C. The pH and pepsin
activity were measured prior to experimental use. Pepsin activity
was measured using the quantitative haemoglobin assay (Guantam, S.
and R. S. de la Motte. 1989. Proteolytic enzymes, a practical
approach. Chapter 3. R. J. Beynon and J. S. Bond(eds.), IRL Press,
Oxford University Press; Dawson, R. M. 1969. pH and buffers. In
Data for Biochemical Research p 138. R. M. Dawson, D. C. Elliot and
K. M. Jones(eds.), Clarendon Press, Oxford). Survival of the
strains at low pH in vitro was investigated using the following
assays:
[0093] (a) Cells were harvested from fresh overnight cultures,
washed twice in phosphate buffer (pH 6.5) and resuspended in MRS
broth adjusted to pH 3.5, 3.0, 2.5 and 2.0 (with 1 N HCl) to a
final concentration of approximately 10.sup.6 cfu/ml. Cells were
incubated at 37.degree. C. and survival measured at intervals of 5,
30, 60 and 120 min. using the plate count method.
[0094] The strains survived with no loss of viability at pH 3.5. At
pH 2.5 there was a 3 log reduction over the 60 min. incubation
period as depicted in FIGS. 1 and 2.
Survival of Bifidobacterium in Human Gastric Juice
[0095] Fresh overnight cultures were harvested, washed twice in
buffer (pH 6.5) and resuspended in human gastric juice to a final
concentration of 10.sup.6 cfu/ml. Survival was monitored over a
30-60 min incubation period at 37.degree. C. The experiment was
performed using gastric juice at pH 1.2 (unadjusted) and pH 2.0 and
2.5 (adjusted using 1N NaOH).
[0096] Survival of the strains was increased in gastric juice at pH
2.0, when compared with gastric juice at 1.2. After 30 min
incubation no viable cells were recovered at either pH as shown in
Table 2. TABLE-US-00002 TABLE 2 Survival of Bifidobacterium sp. in
human gastric juice* TIME (min) STRAIN pH 0 5 30 60 35612 1.2 7.56
0.00 0.00 0.00 2.0 6.27 6.31 2.88 0.00 35624 1.2 5.96 4.18 0.00
0.00 2.0 6.33 6.32 0.00 0.00 35652 1.2 6.16 3.78 0.00 0.00 2.0 8.45
8.40 3.45 0.00 35648 1.2 6.00 0.00 0.00 0.00 2.0 7.89 6.45 0.00
0.00 35687 1.2 6.68 0.00 0.00 0.00 2.0 8.75 8.77 3.34 0.00 BO 2.0
8.41 8.56 8.42 8.43 10 2.0 8.39 8.56 4.64 0.00 6.3 2.0 8.75 8.75
8.29 8.42 B. longum 6 2.0 8.15 8.02 0.00 0.00 *Survival expressed
as log.sub.10 cfu/ml
EXAMPLE 3
Bile Resistance
[0097] In the evaluation of the effectiveness of using lactic acid
bacteria as beneficial members of the gastrointestinal tract, it is
considered that resistance to bile acids is an important biological
strain characteristic required for survival in this hostile
environment and in addition they must not impinge on the health of
the host by producing toxic compounds such as deoxycholic (DCA) and
lithocholic acid (LCA) which have been implicated in a number of
cytotoxic phenomena.
[0098] A number of Bifidobacterium longum infantis strains were
streaked onto TPY agar plates supplemented with porcine bile
(B-8631, Sigma Chemical Co. ltd., Poole) at concentrations of 0.3,
0.5, 1.0, 1.5, 5.0 and 7.5% (w/v) (Legrand-Defretin, R. et al.,
Lipids 1991.; 26 (8), 578-583). Porcine bile is the closest in
composition to human bile with respect to bile salt/cholesterol and
phospholipid/cholesterol ratios. Plates were incubated at
37.degree. C. under anaerobic conditions and growth was recorded
after 24-48 h. Strain 35624 was found to be strongly bile resistant
and grew to confluence at up to 55 porcine bile as shown in Table
3. TABLE-US-00003 TABLE 3 Growth of Bifidobacterium sp. isolates in
the presence of porcine bile % (w/v) PORCINE BILE STRAIN 0.0 0.3
0.5 1.0 1.5 5.0 7.5 34612 + - - - - - - 35624 + + + + + + - 35652 +
- - - - - - 35658 + + + + - - - 35687 + - - - - - - -, no growth;
+, confluent growth
[0099] Human bile was obtained from several human gall bladders and
sterilised at 80.degree. C. for 10 min. The bile acid composition
of human bile was determined using reverse phase High Performance
Liquid Chromatography (HPLC) in combination with a pulsed
amperometric detector according to the method of Dekcker, R. R. et
al., Chromatographia, 1991, 31 (11/12), 255-256: Human bile was
added at a concentration of 0.3% (v/v). Freshly streaked cultures
were examined for growth after 24 and 48 h.
[0100] Strain 35624 was capable of growth in the presence of
physiologically relevant human bile (0.3% (v/v)).
[0101] Growth of the strains was examined in the presence of
individual conjugated and deconjugated bile acids. Under
physiological conditions bile acids are often found as sodium
salts. The strains were screened for growth on TPY agar containing
the conjugated and deconjugated sodium salts of each of the
following bile acids.
[0102] (a) conjugated form: glycocholic acid (GCA);
glycodeoxycholic acid (GDCA); and glycochenodeoxycholic acid
(GCDCA);
[0103] (b) deconjugated form: lithocholic acid (LCA);
chenodeoxycholic acid (CDCA); deoxycholic acid (DCA) and cholic
acid (CA). For each bile acid concentrations of 1, 3 and 4 mM were
used. Growth was recorded after 24 and 48 h anaerobic
incubation.
[0104] The five strains studied grew on agar medium supplemented
with 5 mM GCA and GCDCA and on agar medium supplemented with 1 mM
GDCA as shown in Table 4. Strain 35624 was resistant to
concentrations of 5 mM LCA (data not shown) and strains 35612 and
35624 were capable of growth at concentrations of 5 mM CA as shown
in Table 5. No growth was observed in the presence of 1 mM CDCA
(data not shown). TABLE-US-00004 TABLE 4 Growth of Bifidobacterium
sp. isolates in the presence of glycine-conjugated bile acids BILE
ACIDS (mM) GCDCA GDCA GCA STRAIN 0 1 3 5 0 1 3 5 0 1 3 5 35612 + +
+ + + + + + + + + + 35624 + + + + + + + + + + + + 35652 + + + + + +
+ + + + + + 35658 + + + + + + + + + + + + 35687 + + + + + + + + + +
+ + -, no growth; +, confluent growth GCDCA, glycochenodeoxycholic
acid, GDCA, glycodeoxycholic acid; CGA, glycocholic acid.
[0105] TABLE-US-00005 TABLE 5 Growth of Bifidobacterium sp.
isolates in the presence of unconjugated cholic acid (CA) CHOLIC
ACID (mM) STRAIN 0 1 3 5 35612 + + + + 35624 + + + + 35652 + + - -
35658 + + - - 35687 + + - - -, no growth; +, confluent growth
EXAMPLE 4
Antimicrobial Activity
[0106] Bifidobacterium species exert inhibitory effects on other
bacteria by excluding long term colonisation by invasive pathogens.
Their antagonistic activity is due to the production of acetic and
lactic acid though fermentation (Scardovi, V. (1986)
Bifidobacterium in Bergey's Manual of systemic bacteriology, Vol.
2. Eds. Sheath, P. H., Main, N. S., Sharpe, M. and Holdt, J. G.,
Williams and Wilkins Publishers, Baltimore Md., p1418). Very few
reports exist on the production of antimicrobial compounds other
than acids (Anand, S. K. et al. Cult. Dairy Prods. 1985; J. 2,
21-23). Bacteriocins and other compounds may influence the survival
of a bacterium in an ecological niche and allow them to effectively
dominate fermenting ecosystems. Such a feature is a good trait for
a probiotic strain.
[0107] The inhibitory spectra of various bifidobacterial strains
was determined by the method of Tagg et al. (Tagg. J. R. et al.
Bacteriol. Rev. 1976; 40, 722-756). Cell free supernatant was
assayed for inhibitory activity against a wide range of Gram
positive and Gram negative micro-organisms. Overlays of each
indicator were prepared on agar plates and allowed to dry. Spots (5
ml) of cell free supernatant were placed on the seeded plates,
allowed to dry and the plates were incubated overnight.
[0108] It was observed that the strains were inhibitory to a wide
range of Staphylococcus, Pseudomonas, Coliform and Bacillus sp.
when tested on TPY medium. Zones of inhibition of up to 4.4 mm were
recorded against Pseudomonas and Staphylococcus and up to 7.0 mm
surrounding Bacillus sp. as shown in Tables 6 and 7. However, when
the deferred assays were performed on buffered TPY medium zones of
inhibition were not observed against any indicator strain.
Therefore, inhibition appeared to be, solely due to the presence of
acid produced by the bifidobacteria. TABLE-US-00006 TABLE 6
Inhibition of Staphylococcus strains by Bifidobacterium sp. on
unbuffered medium* B. longum 1 B. longum 9 B. longum 10 63 35612
35624 35652 35658 35675 35678 35687 S. aureus MHS 1.5 2 1.5 3.5 1.5
1 2 2 1 2.5 1.5 S. aureus HC 1.5 1.5 2 2.5 2 1.5 2.5 2 1.5 1.5 2 S.
aureus 771 1.5 3 1.5 3 2 2 2.5 2 3 2 3.5 S. aureus 949 2 3.5 2.5 2
3 3.5 3 2.5 3.5 3.5 2.5 S. aureus 1018 1 3.5 1.5 1.5 2 3.5 1 3 3.5
2.5 2 S. aureus 1502 1.5 3.5 1 2 2.5 2.5 1.5 3 4 2.5 1.5 S. aureus
1505 3 4 3 2.5 2.5 3 2.5 4.5 5.5 5 5.5 S. aureus 1511 1 3.5 2 1.5 2
2.5 3 3.5 4 2.5 3 S. aureus 1522 1.5 3 2.5 1 2.5 1.5 2.5 2.5 3.5
3.5 3 S. aureus 1499 1.5 3.5 1.5 1.5 2 2 3 2 3.5 3.5 1.5 S. aureus
1963 2 3 2 2.5 3.5 3.5 3.5 3.5 2.5 3 2.5 S. aureus PRMM 1 3.5 1 1.5
1 3.5 2 2 3 2 2.5 S. albus 1 2 1.5 1 2 2.5 2 1.5 2 1.5 1 S. camosus
1 1.5 2 2.5 2.5 2.5 2 2.5 2 1.5 1 *values given are radii of
inhibition zones in mm (distance from edge of producer colony to
the edge of zone of inhibition)
[0109] TABLE-US-00007 TABLE 7 Inhibition of Pseudomonas and
Bacillus strains by Bifidobacterium sp. on unbuffered medium* B.
longum 1 B. longum 9 B. longum 10 63 35612 35624 35652 35658 35675
35678 35687 P. fluorescens HC 1 2.5 1.5 1 1.5 2 3 2 1.5 2 2.5 P.
fluorescens MHP 1.5 4.5 3.5 2 2.5 3.5 2.5 2.5 3.5 2 4 P.
fluorescens DW 1.5 4 4 3.5 2.5 3.5 2.5 4.5 5.5 3.5 5 B. cereus 3 3
5 3 4 4 3.5 5 6 4.5 5.5 B. subtilis 2 2.5 5 2 3 6 3 6 7 3 6 B.
circulans 1 2 4 1.5 2.5 1.5 5 3.5 4.5 2 4.5 B. thuringensis 2.5 3.5
5 3 3.5 4.5 4 5.5 6.5 4.5 5.5 *values given are radii of inhibition
zones in mm (distance from edge of producer colony to the edge of
zone of inhibition)
EXAMPLE 5
Murine Feeding Trial to Investigate the Ability of Lactobacillus
salivarius Subsp. Salivarius UCC 118 and Bifidobacterium longum
infantis 35624 to Alleviate the Symptoms of Inflammatory Bowel
Disease (IBD)
Background
[0110] A number of mouse models have recently been generated by
either genetic or immunological means to study the mechanisms of
IBD. One of these models involves the transfer of spleen or lymph
node-derived CD4.sup.+T lymphocytes from normal mice into severe
combined immunodeficient recipient mice (SCID). It has been
demonstrated that mice who receive purified CD4.sup.+,
CD45RB.sup.high T cells develop a wasting disease characterised by
chronic intestinal inflammation which is more severe in the colon.
In this study a control group of SCID mice was injected with
CD4.sup.+CD45RB.sup.high and the mice developed a progressive
wasting disease including hunched over appearance, piloerection of
the coat, diarrhoea, weight loss and macro and microscopic colon
damage. A feeding trail was set up administering UCC 118 and strain
35624 (also referred to herein as UCC 35624) to determine if the
symptoms of IBD could be modified in this model.
Bacterial Strains
[0111] Lactobacillus salivarius subsp. Salivarius UCC 118 and
Bifidobacterium longum infantis UCC 35624 were isolated from the
ileal-caecal region of an adult human as described in Example 1. In
this example, spontaneous rifampicin and streptomycin resistant
derivatives of the strains were generated by plating cells,
previously grown overnight and subsequently washed in quarter
strength Ringer's solution on MRS and TPY agar containing 50
.mu.g/ml rifampicin (Sigma) respectively and MRS containing 400
.mu.g/ml streptomycin (Sigma). Plates were incubated for 2 days at
37.degree. C. anaerobically. The resulting antibiotic resistant
derivatives were determined to be otherwise phenotypically similar
to the parent strain. This selectable trait enabled the strains to
be readily enumerated following gut transit.
Animals and Maintenance
[0112] Donor mice (C57BL/6.times.BALB/c) F1 were purchased from
Simosen Laboratories (Gilroy, Calif.) and maintained at the
University of California--Los Angeles vivarium in ventilated cage
racks (Thoren caging systems, Hazelton, Pa.) under specific
pathogen free (SPF) conditions. CB-17 SCID mice were bred in
ventilated cage racks originally obtained from the University of
California--Los Angeles SCID core facility. The mice were reduced
flora (RF) mice rather than germ free and acting as the recipient
mice (Aranda R. et al. J. of Immunol. 1997; 158(7), 3464-3473).
[0113] Eight week old, female CB17 (SCID) mice were housed in pairs
in filter top cages in ventilated racks. The mice were divided into
four groups Group A: consumed 10% skim milk, control; Group B:
consumed Lactobacillus salivarius UCC 118, Group C: consumed
Lactobacillus salivarius UCC 118 and Bifidobacterium longum UCC
35624 9 (1:1 ratio); Group D: consumed Bifidobacterium longum UCC
35624. UCC 118 and UCC 35624 which were grown overnight in MRS
broth and MRS broth supplemented with 0.05% cysteine (Sigma)
respectively, were washed in PBS, resuspended in skim milk (10%
(v/v)) and administered in the otherwise sterile drinking water
(PBS). The mice in each respective group received
2.55.times.10.sup.8 cfu/ml of UCC 118 and 2.35.times.10.sup.8
cfu/ml of UCC 35624 daily for the duration of the feeding period.
Control mice received sterile milk diluted in sterile phosphate
buffered saline (PBS) and were maintained under identical
conditions as the test group.
Experimental Design
[0114] All CB 17 mice were administered their respective feed
according to their grouping for 2 days prior to injection with the
CD4.sup.+CD45RB.sup.high cells. The sorted donor lymphocytes
(3-4.times.10.sup.5) were represented in 200 .mu.l of sterile PBS
and injected i.p. into the recipient CB-17 SCID mice. All mice were
weighed initially, then twice weekly thereafter. They were observed
for clinical signs of illness: hunched over appearance,
piloerection of the coat and diarrhoea.
Evaluation of the Effects of the Administered Probiotics on the
Numbers of Indigenous Bacteria Culturable from Mouse Faeces.
[0115] The influence exerted by the administered UCC 118 and UCC
35624 when either administered alone or in combination with each
other, on the microflora of the CB-17 SCID murine gut was
investigated. Faecal samples were collected from each mouse weekly,
weighed and resuspended in 10 ml PBS. The samples were then
serially diluted in PBS and either pour plated or spread plated in
appropriate dilutions on appropriate media in duplicate. The
following bacterial groups were enumerated: lactobacilli;
bifidobacteria; enterococci; bacteroides and coliforms. The
selective media used were; de Mann Rogosa & Sharpe (MRS) agar,
MRS agar supplemented with 0.2% lithium chloride (BDH), 0.3% sodium
propionate (Fluke chemie), 0.5% cysteine hydrochloride (Sigma), and
5% sheep's blood; Slanetz and Bartley agar; Wilkins and Chalgren
agar supplemented with anaerobic supplement SR 108 and 5% horse
blood; and Violet Red Bile Agar. (All Oxoid unless otherwise
stated). VRBA and Slanetz ald Barrley plates were incubated
aerobically for 24 and 45 h respectively. All other plates were
incubated anaerobically for 48 h at 37.degree. C.
Enumeration of Culturable Indigenous Flora from Specific Segments
of the CB.17 SCID Murine G.I.T.
[0116] After the feeding period all mice were sacrificed and
dissected. Segments of the ileal-caecal region, small intestine,
and the large intestine were removed. A peripheral lymph node
(PLN), mesenteric lymph node (MLN) and a piece of the spleen were
also taken. All tissues were weighed before being resuspended in 10
ml of PBS. Samples were then homogenised and serially diluted in
PBS and either spread plated or pour plated in appropriate
dilutions on appropriate media in duplicate. The bacterial groups
were enumerated the same as those enumerated in the faecal analysis
and samples were incubated as described previously.
Preparation of Intraepithelial and Lamniinapropria Lymphocytes
[0117] The isolation of the mucosal lymphocytes was carried out
according to the method of Aranda, R. et al ((1997) supra).
Flow Cytometic Analysis of Lymphocyte Populations.
[0118] The analysis was conducted as described by Aranda, R. et al.
((1997) supra)
Preparation of Tissue for Histopathological Analysis
[0119] Tissue samples were taken from the small intestine, large
intestine, and ileal caecal region and fixed in 10% formalin. The
procedure was as described in Aranda, R. et al. ((1997) supra).
[0120] It was observed from the experiment carried out that,
consistent with previous results, the SCID mice reconstituted with
CD4.sup.+CD45RB.sup.high T lymphocytes and consuming skim milk
alone (control) developed a progressive wasting disease, identified
by their significant weight loss. Disease became apparent at about
2 and a half to three weeks and the sick mice characteristically
manifested a hunched over appearance, piloerection of their coat,
and loose stool. One of the mice in the control group (mouse 4)
died after 25 days and mice 1, 2, 3 and 5 showed a -20%, 25%, 21%
and -35% percentage weight change respectively as depicted in FIGS.
3 and 4.
[0121] CB-17 SCID mice consuming UCC 118 alone gave a similar
result as the controls with the characteristic weight loss. Mouse 3
died after 14 days, and mice 4, 5 and 6 showed a -15%,-25% and -28%
percentage weight change respectively (data not shown). The mice
consuming a combination of UCC 118 and UCC 35624 were found to have
a marked improvement on the control mice. These mice did not lose
as much weight as the control mice over the feeding period. Even
after 35 days three of the mice in this group showed little
percentage weight change. (FIGS. 5 and 6). Two of the mice in this
group showed a weight loss only after about 30 days whereas control
mice showed weight loss at 14 days (FIGS. 3 and 4).
[0122] Mice consuming UCC 35624 alone appeared in good health and
again weight loss when compared to the controls was considerably
less (FIGS. 7 and 8). It can be concluded therefore that
consumption of UCC 35624 either alone or in combination with UCC
118 alleviates the symptoms of inflammatory bowel disease.
[0123] Table 8 is a summary of experimental data for the study on
the treatment of CD45RB colitis induced CB 17 and SCID mice with a
cocktail of UCC 118 and UCC 35624.
[0124] It was found in the studies that the mice were successfully
reconstituted with lymphocytes and lymphocytes having been derived
from the donor model (data not shown). TABLE-US-00008 TABLE 8
Treatment of CD45RB colitis induced CB 17 SCID mice with a cocktail
of Lactobacillus salivarius UCC 118 and Bifidobacteria. Mouse 1
Untreated Mouse 2 Untreated Mouse 3 Mouse 4 Mouse 5 Mouse 6 (RB hi
cells + skimmed (RB hi cells + skimmed Cocktail Cocktail Cocktail
Cocktail Organ milk) milk) Treated Treated Treated Treated % weight
loss 31.25 27.74 14.50 14.05 21.88 11.18 Final looks ill very ill
very healthy slightly ill healthy healthy Appearance Stool very
mushy very mushy mushy solid semi solid semi solid Appearance Colon
thickened very slightly slightproximal slightly slightproximal
Appearance thickened thickened thickening thickened thickening No.
SIEL 100,000 200,000 0 0 512,000 28,000 No. LIEL 25,000 72,000
100,000 50,000 384,000 96,000 No. SLPL 200,000 100,000 264,000
200,000 640,000 104,000 No. LLPL 96,000 256,000 160,000 160,000
256,000 160,000 No. MLN 0 N/A 81,900 N/A 28,800 N/A No. PLN 0
192,000 0 120,000 64,000 0 Spleen # 960,000 512,000 640,000 640,000
512,000 6,400,000 Lymphos. CD3+/H-2Kb+ Flow correction % No. SIEL
62,000 114,000 0 0 450,560 17,920 No. LIEL 21,250 48,960 74,800
38,000 345,600 65,280 No. SLPL 74,000 42,000 158,400 136,000
384,000 66,460 No. LLPL 67,200 161,280 115,200 108,000 184,320
108,800 No. MLN 0 N/A 130,00 N/A 64,000 N/A No. PLN 0 126,720 0
87,600 54,400 0 Spleen 518,400 102,400 211,200 307,200 230,400
4,480,000 UCC 118 bacterial counts (per biopsy) post mortem SI 0 0
1,200 0 0 0 LI 0 0 >30,000 >30,000 100 11,600 Caecum 0 0
>30,000 >30,000 >30,000 >30,000 Spleen 0 0 0 1,350 0 0
Colon Pathological Scoring A(0-3) -- 1.0 1.0 2.0 -- -- B(0-2) --
1.5 1.0 1.0 -- -- C(0-3) -- 2.5 1.0 2.0 -- -- D(0-3) -- 2.0 3.0 3.0
-- -- E(1-3) -- 1.0 1.0 2.0 -- Remarks Total Score -- 8.0 7.0 10.0
-- -- A: Degree of inflammatory infiltrate; B: Mucin depletion; C:
Epithelia hyperplasia; D: No. of TEL in the crypts; E: No. of
inflammatory foci per high power fields
EXAMPLE 7
In vitro Studies to Examine the Immune Perception of
Bifidobacterium longum infantis
[0125] Overnight washed cultures of Bifidobacteria were incubated
with human peripheral blood mononuclear cells (PBMCs) from both
healthy volunteers (n=9) and patients suffering from inflammatory
bowel disease (n=5). Production of the proinflammatory cytokine
tumour necrosis factor a (TNF.alpha.) was measured by ELISA in
seventy two hour culture supernatants. Co-incubation of
Bifidobacterium longum infantis with human PBMCs did not result in
the stimulation of TNF.alpha. production (FIG. 9). Thus, exposure
of the systemic immune system to this bacterium does induce an
inflammatory response.
[0126] In order to assess the immune perception of Bifidobacterium
longum infantis at mucosal surfaces, co-culturing of epithelial
cells and PBMCs was performed in transwell chambers. Briefly, an
epithelial cell monolayer air was grown in the upper chamber and
PBMCs were incubated in the lower compartment. These were seperated
from each other by a porous membrane which allowed the passage of
soluble mediators between the two compartments but did not allow
cell-cell contact. Using this model, the production of TNF.alpha.
and interleukin-8 (IL-8) was measured in the presence and absence
of Bifidobacterium longum infantis in the PBMC compartment.
Co-culture of epithelial cells, PBMCs and Bifidobacterium longum
infantis resulted in significant suppression of TNF.alpha. and IL-8
production (FIG. 10). Thus, a tri-cellular network involving
epithelial cells, PBMCs and Bifidobacterium longum infantis results
in suppression of proinflammatory cytokine production.
EXAMPLE 8
In vivo Anti-Inflammatory Activity of Bifidobacterium longum
infantis
[0127] Bifidobacterium longum infantis (1.times.10.sup.9 cells per
day) was consumed by 18 healthy humans in a fermented milk
(yoghurt) product for three weeks. Serum was collected for cytokine
analysis pre and post consumption of this probiotic strain. Faecal
samples were obtained for microbiological analysis.
[0128] Considerable modification of peripheral blood cytokine
levels were observed in this feeding study. Serum soluble
Interleukin-6 receptor (sIL-6R, p=0.007), Interferon-.gamma.
(IFN.gamma., p=0.041) and IL-8 (p=0.004) levels were significantly
reduced following consumption of this probiotic strain (FIG. 11).
No alteration in serum TNF.alpha. and Interleukin-1 receptor
antagonist (IL-1RA) levels were observed (FIG. 12). Bifidobacterium
longum infantis was detected at approximately 1.times.10.sup.5
colony forming units per gram of faecal matter over the course of
this feeding study.
[0129] Targeted in vitro selection criteria reflecting the complex
interactions of the GI environment allow for the identification of
probiotic strains capable of functioning effectively when
reintroduced into that environment. Using the selection criteria
outlined above, the probiotic bacteria Bifidobacterium longum
infantis has demonstrable immunomodulating properties in vitro.
Following consumption by SCID mice and human volunteers,
significant modification of systemic immune parameters was noted.
Thus, the use of Bifidobacterium longum infantis as a
biotherapeutic agent in the treatment of immune mediated diseases
is warranted.
EXAMPLE 9
Measurement of TNF.alpha. in Bifidobacterium longum infantis UCC
35624 Cell Free Supernatant
[0130] Overnight cultures of Bifidobacterium longum infantis were
centrifuged and the cell-free culture supernatant was examined for
the presence of cytokine inhibitors. Cell free supernatants were
incubated with human TNF.alpha. for 20 minutes at 37.degree. C.
TNF.alpha. levels were quantified thereafter by ELISA. Following
exposure to the Bifidobacteria supernatant, TNF.alpha. levels were
significantly reduced (FIG. 13). Thus, Bifidobacterium longum
infantis UCC35624 secretes a factor that antagonises TNF.alpha.
activity. Production of this factor by Bifidobacterium longum
infantis at the surface of the gastrointestinal tract, in vivo,
would significantly restrict the host inflammatory response.
[0131] This indicates that the antagonism of TNF.alpha. also occurs
at a molecular level due to a soluble factor released by UCC
35624
Inflammation
[0132] Inflammation is the term used to describe the local
accumulation of fluid, plasma proteins and white blood cells at a
site that has sustained physical damage, infection or where there
is an ongoing immune response. Control of the inflammatory response
is exerted on a number of levels (for review see Henderson B., and
Wilson M. 1998. In "Bacteria-Cytokine interactions in health and
disease. Portland Press, 79-130). The controlling factors include
cytokines, hormones (e.g. hydrocortisone), prostaglandins, reactive
intermediates and leukotrienes. Cytokines are low molecular weight
biologically active proteins that are involved in the generation
and control of immunological and inflammatory responses, while also
regulating development, tissue repair and haematopoiesis. They
provide a means of communication between leukocytes themselves and
also with other cell types. Most cytokines are pleiotrophic and
express multiple biologically overlapping activities. Cytokine
cascades and networks control the inflammatory response rather than
the action of a particular cytokine on a particular cell type (Arai
K I, et al., Annu Rev Biochem 1990; 59:783-836). Waning of the
inflammatory response results in lower concentrations of the
appropriate activating signals and other inflammatory mediators
leading to the cessation of the inflammatory response. TNF.alpha.
is a pivotal proinflammatory cytokine as it initiates a cascade of
cytokines and biological effects resulting in the inflammatory
state. Therefore, agents which inhibit TNF.alpha. are currently
being used for the treatment of inflammatory diseases, e.g.
infliximab.
[0133] Pro-inflammatory cytokines are thought to play a major role
in the pathogenesis of many inflammatory diseases, including
inflammatory bowel disease (IBD). Current therapies for treating
IBD are aimed at reducing the levels of these pro-inflammatory
cytokines, including IL-8 and TNF.alpha.. It has been suggested
that such therapies may also play a significant role in the
treatment of systemic inflammatory diseases such as rheumatoid
arthritis. Humans fed with yoghurt containing Bifidobacterium
longum infantis UCC35624 have shown marked decreases in their
systemic levels of IL-8. This strain may therefore have potential
application in the treatment of a range of inflammatory diseases,
particularly if used in combination with current anti-inflammatory
therapies, such as non-steroid anti-inflammatory drugs (NSAIDs) or
Infliximab.
Diarrhoeal Disease.
[0134] The barrier function of the intestinal epithelium can be
diminished during nervous (acetylcholine) and immune (histamine)
mediated secretion. Certain bacterial toxins may also induce Ca2+
and PKC dependent secretion and thereby can disturb the epithelial
barrier (Ganguly N K and Kaur T. Indian J Med Res 1996; 104:2837,
Groot J A. Vet Q 1998; 20(S3):45-9). Several studies have examined
the prevention and treatment of diarrhoea using probiotic bacteria.
Prospective studies have demonstrated the efficacy of lactic acid
bacteria administration for both prophylactic and therapeutic use
against diarrhoea in pre-mature infants, new borns, children
(Isolauri E, et al., Dig Dis Sci 1994 December; 39(12):2595-600)
and in the treatment of antibiotic related diarrhoea (Siitonen S,
et al, Ann Med 1990 February; 22(1):57-9) and traveller's diarrhoea
(Oksanen P J, et al., Ann Med 1990 February; 22(1):53-6).
[0135] We have examined consumption of Bifidobacterium longum
infantis UCC 35624 by SCID mice. It was found that inflammatory
activity was significantly attenuated and mice consuming
Bifidobacterium longum infantis UCC 35624 retained solid stools
while control mice suffered from diarrhoea. FIGS. 14 and 15
illustrate the lower intestine of treated and untreated SCID mice.
The lower intestine shown includes the caecum 2, intestine 3 and
anus 5. In FIG. 14 the mice were treated with Bifidobacterium
longum infantis UCC 35624 and it is apparent that solid stools 4
have been retained in the intestine. In comparison FIG. 15 shows
the untreated mouse intestine 3, characteristically inflamed. No
water absorption has occurred so that no solid stools are retained
resulting in diarrhoea.
[0136] The anti-diarrhoeal effect observed may be related to the
anti-inflammatory activity, possibly mediated via cAMP modulation.
Cyclic AMP-dependent CI-secretion is the major secretory pathway in
the human intestine (Brzuszczak I M, et al., J. Gastroenterol.
Hepatol. 1996; 11(9):804-10). It can be inferred that the
anti-diarrhoeal effect of Bifidobacterium longum infantis UCC 35624
is not restricted just to diarrhoea resulting from gastrointestinal
inflammation, but can be applied to the general treatment of
diarrhoeal disease.
Autoimmune Disease
[0137] The immune system has a large repertoire of specificities
expressed by B and T cells. Some of these specificities will be
directed to self-components. Self-recognition is normally
controlled by clonal deletion and inactivation of self-reactive
lymphocytes. However, there is a constant background of
autoimmunity with antibodies to many proteins being found in serum.
A breakdown in the self-nonself recognition system results in
autoimmunity. When autoimmune disease does occur, the resulting
immune response damages the tissue bearing the offending antigen.
Immune complex deposition, type II hypersensitivity and
cell-mediated reactions are the most important mechanisms by which
immunopathological damage occurs. Examples of autoimmune diseases
include, but are not limited to, systemic lupus erythematosus,
rheumatoid arthritis, insulin dependent diabetes mellitus,
myasthenia gravis and pernicious anaemia. Bifidobacterium longum
infantis and Lactobacillus salivarius subsp. salivarius are
immunomodulatory bacteria. Thus, consumption either as single
components or in combination of these bacteria by patients
suffering from autoimmune disease may restrict organ damage and
help restore normal body homeostasis.
Inflammation and Cancer
[0138] The production of multifunctional cytokines across a wide
spectrum of tumour types suggests that significant inflammatory
responses are ongoing in patients with cancer. It is currently
unclear what protective effect this response has against the growth
and development of tumour cells in vivo. However, these
inflammatory responses could adversely affect the tumour bearing
host. Complex cytokine interactions are involved in the regulation
of cytokine production and cell proliferation within tumour and
normal tissues (McGee, D W, et al., Immunology 1995 September;
86(1):6-11, Wu S, et al., Gynecol Oncol 1994 April; 53(1):59-63).
It has long been recognised that weight loss (cachexia) is the
single most common cause of death in patients with cancer (Inagaki
J, et al., Cancer 1974 February; 33(2):568-73) and initial
malnutrition indicates a poor prognosis (Van Eys J. Nutr Rev 1982
December; 40(12):353-9). For a tumour to grow and spread it must
induce the formation of new blood vessels and degrade the
extracellular matrix. The inflammatory response may have
significant roles to play in the above mechanisms, thus
contributing to the decline of the host and progression of the
tumour. Due to the anti-inflammatory nature of these bacterial
strains they may reduce the rate of malignant cell transformation.
Furthermore, intestinal bacteria can produce, from dietary
compounds, substances with genotoxic, carcinogenic and
tumour-promoting activity and gut bacteria can activate
pro-carcinogens to DNA reactive agents (Rowland I. R. (1995).
Toxicology of the colon: role of the intestinal microflora. In:
Gibson G. R. (ed). Human colonic bacteria: role in nutrition,
physiology and pathology, pp 155-174. Boca Raton CRC Press). In
general, species of Bifidobacteria and Lactobacillus have low
activities of xenobiotic metabolising enzymes compared to other
populations within the gut such as bacteroides, eubacteria and
clostridia (Saito Y., et al., Microb. Ecol. Health Dis., 1992; 5,
105-110). Therefore, increasing the number of lactic acid bacteria
in the gut could beneficially modify the levels of these
enzymes.
Prebiotics
[0139] The introduction of probiotic organisms is accomplished by
the ingestion of the microorganism in a suitable carrier. It would
be advantageous to provide a medium that would promote the growth
of these probiotic strains in the large bowel. The addition of one
or more oligosaccharides, polysaccharides, or other prebiotics
enhances the growth of lactic acid bacteria in the gastrointestinal
tract (Gibson, G R. Br. J. Nutr. 1998; 80 (4):S209-12). Prebiotics
refers to any non-viable food component that is specifically
fermented in the colon by indigenous bacteria thought to be of
positive value, e.g. bifidobacteria, lactobacilli. Types of
prebiotics may include those which contain fructose, xylose, soya,
galactose, glucose and mannose. The combined administration of a
probiotic strain with one or more prebiotic compounds may enhance
the growth of the administered probiotic in vivo resulting in a
more pronounced health benefit, and is termed synbiotic. Other
Active Ingredients It will be appreciated that the Bifidobacterium
may be administered prophylactically or as a method of treatment
either on its own or with other probiotic and/or prebiotic
materials as described above. In addition, the bacteria may be used
as part of a prophylactic or treatment regime using other active
materials such as those used for treating inflammation or other
disorders, especially those of the gastrointestinal tract. Such
combinations may be administered in a single formulation or as
separate formulations administered at the same or different times
and using the same or different routes of administration.
[0140] The invention is not limited to the embodiments hereinbefore
described which may be varied in detail.
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