U.S. patent application number 14/217543 was filed with the patent office on 2014-11-06 for probiotic bifidobacterium strains.
This patent application is currently assigned to ALIMENTARY HEALTH LIMITED. The applicant listed for this patent is ALIMENTARY HEALTH LIMITED. Invention is credited to Barry Kiely, John MacSharry, Liam O'Mahony, David O'Sullivan.
Application Number | 20140328879 14/217543 |
Document ID | / |
Family ID | 39758481 |
Filed Date | 2014-11-06 |
United States Patent
Application |
20140328879 |
Kind Code |
A1 |
MacSharry; John ; et
al. |
November 6, 2014 |
PROBIOTIC BIFIDOBACTERIUM STRAINS
Abstract
Probiotic Bifidobacterium strain AH1205 or mutants or variants
thereof are immunomodulatory following oral consumption and are
useful in the prophylaxis and/or treatment of inflammatory activity
such as undesirable gastrointestinal inflammatory activity for
example inflammatory bowel disease.
Inventors: |
MacSharry; John; (Cork,
IE) ; O'Mahony; Liam; (County Cork, IE) ;
O'Sullivan; David; (County Cork, IE) ; Kiely;
Barry; (County Cork, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALIMENTARY HEALTH LIMITED |
Cork |
|
IE |
|
|
Assignee: |
ALIMENTARY HEALTH LIMITED
Cork
IE
|
Family ID: |
39758481 |
Appl. No.: |
14/217543 |
Filed: |
March 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12450451 |
Apr 26, 2010 |
8709398 |
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PCT/IE2008/000034 |
Mar 28, 2008 |
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14217543 |
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60907312 |
Mar 28, 2007 |
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Current U.S.
Class: |
424/234.1 ;
435/252.3 |
Current CPC
Class: |
A61P 9/04 20180101; A61P
1/00 20180101; A61P 9/00 20180101; A61P 17/02 20180101; A61P 29/00
20180101; A61P 1/16 20180101; A61P 17/00 20180101; A61P 37/04
20180101; A61P 3/12 20180101; A61P 43/00 20180101; A23V 2002/00
20130101; A61P 15/00 20180101; Y02A 50/30 20180101; A61P 37/00
20180101; A61P 3/04 20180101; A61P 11/06 20180101; A61P 31/10
20180101; A61P 17/06 20180101; A61P 25/28 20180101; A23L 33/135
20160801; A61P 17/10 20180101; A61P 1/10 20180101; A61P 7/06
20180101; A61P 31/12 20180101; A61P 37/08 20180101; A61P 19/10
20180101; A61P 1/02 20180101; A61P 1/04 20180101; A61P 3/00
20180101; A61P 7/02 20180101; A61P 19/02 20180101; C12R 1/01
20130101; A61P 9/10 20180101; A61P 25/00 20180101; A61P 37/02
20180101; A61P 7/00 20180101; A61P 35/04 20180101; A61P 31/04
20180101; A61K 35/745 20130101; A61P 1/14 20180101; A61P 3/10
20180101; Y02A 50/473 20180101; A61P 37/06 20180101; A61P 31/18
20180101; A61P 5/00 20180101; A61P 13/12 20180101; A61P 11/00
20180101; A61P 13/02 20180101; A61P 1/12 20180101; A61P 35/00
20180101 |
Class at
Publication: |
424/234.1 ;
435/252.3 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A23L 1/30 20060101 A23L001/30 |
Claims
1-39. (canceled)
40. A Bifidobacterium strain deposited at NCIMB with accession
number NCIMB 41387 or mutants or variants thereof.
41. The Bifidobacterium strain as claimed in claim 40 wherein the
mutant is a genetically modified mutant.
42. The Bifidobacterium strain as claimed in claim 40 wherein the
variant is a naturally occurring variant of Bifidobacterium.
43. A Bifidobacterium strain with sequence homology of 96% or more,
97% or more, 98% or more, or 99% or more with a Bifidobacterium
strain deposited at NCIMB with accession number NCIMB 41387.
44. A Bifidobacterium strain having at least 85%, at least 90%, or
at least 95% sequence homology to the 16s-23s intergenic spacer
polynucleotide sequence of a Bifidobacterium strain deposited at
NCIMB with accession number NCIMB 41387.
45. The Bifidobacterium strain as claimed in claim 40 which is a
probiotic.
46. The Bifidobacterium strain as claimed in claim 40 in the form
of viable cells.
47. The Bifidobacterium strain as claimed in claim 40 in the form
of non-viable cells.
48. The Bifidobacterium strain as claimed in claim 40 wherein the
Bifidobacterium is isolated from infant faeces.
49. The Bifidobacterium strain as claimed in claim 40 wherein the
strain is significantly immunomodulatory following oral consumption
in humans.
50. A formulation which comprises a Bifidobacterium strain as
claimed in claim 40.
51. The formulation as claimed in claim 50 which further comprises
a probiotic material.
52. The formulation as claimed in claim 50 which further comprises
a prebiotic material.
53. The formulation as claimed in claim 50 further comprising an
ingestible carrier.
54. The formulation as claimed in claim 50 wherein the
Bifidobacterium strain is present in an amount of more than
10.sup.6 cfu per gram of the formulation.
55. A food stuff comprising a Bifidobacterium strain as claimed in
claim 40.
56. A medicament comprising a Bifidobacterium strain as claimed in
claim 40.
57. A method for the prophylaxis and/or treatment of undesirable
inflammatory activity comprising the step of administering a
Bifidobacterium strain as claimed in claim 40.
Description
INTRODUCTION
[0001] The invention relates to a Bifidobacterium strain and its
use as a probiotic bacteria in particular as an immunomodulatory
biotherapeutic agent.
[0002] The defense mechanisms to protect the human gastrointestinal
tract from colonization by intestinal bacteria are highly complex
and involve both immunological and non-immunological aspects (1).
Innate defense mechanisms include the low pH of the stomach, bile
salts, peristalsis, mucin layers and anti-microbial compounds such
as lysozyme (2). Immunological mechanisms include specialized
lymphoid aggregates, underlying M cells, called peyers patches
which are distributed throughout the small intestine and colon (3).
Luminal antigens presented at these sites result in stimulation of
appropriate T and B cell subsets with establishment of cytokine
networks and secretion of antibodies into the gastrointestinal
tract (4). In addition, antigen presentation may occur via
epithelial cells to intraepithelial lymphocytes and to the
underlying lamina propria immune cells (5). Therefore, the host
invests substantially in immunological defense of the
gastrointestinal tract. However, as the gastrointestinal mucosa is
the largest surface at which the host interacts with the external
environment, specific control mechanisms must be in place to
regulate immune responsiveness to the 100 tons of food which is
handled by the gastrointestinal tract over an average lifetime.
Furthermore, the gut is colonized by over 500 species of bacteria
numbering 10.sup.11-10.sup.12/g in the colon. Thus, these control
mechanisms must be capable of distinguishing non-pathogenic
adherent bacteria from invasive pathogens, which would cause
significant damage to the host. In fact, the intestinal flora
contributes to defense of the host by competing with newly ingested
potentially pathogenic micro-organisms.
[0003] Bacteria present in the human gastrointestinal tract can
promote inflammation. Aberrant immune responses to the indigenous
microflora have been implicated in certain disease states, such as
inflammatory bowel disease. Antigens associated with the normal
flora usually lead to immunological tolerance and failure to
achieve this tolerance is a major mechanism of mucosal inflammation
(6). Evidence for this breakdown in tolerance includes an increase
in antibody levels directed against the gut flora in patients with
inflammatory bowel disease (IBD).
[0004] The present invention is directed towards a Bifidobacterium
strain which has been shown to have immunomodulatory effects, by
modulating cytokine levels or by antagonizing and excluding
pro-inflammatory micro-organisms from the gastrointestinal
tract.
STATEMENTS OF INVENTION
[0005] According to the invention there is provided Bifidobacterium
strain AH1205 (NCIMB41387) or mutants or variants thereof.
[0006] The mutant may be a genetically modified mutant. The variant
may be a naturally occurring variant of Bifidobacterium.
[0007] The strain may be a probiotic. It may be in the form of a
biologically pure culture.
[0008] The invention also provides an isolated strain of
Bifidobacterium NCIMB 41387 In one embodiment of the invention
Bifidobacterium strains are in the form of viable cells.
Alternatively Bifidobacterium strains are in the form of non-viable
cells.
[0009] In one embodiment of the invention the Bifidobacterium
strains are isolated from infant faeces, the Bifidobacterium
strains being significantly immunomodulatory following oral
consumption in humans.
[0010] The invention also provides a formulation which comprises
the Bifidobacterium strain of the invention.
[0011] In one embodiment of the invention the formulation includes
another probiotic material.
[0012] In one embodiment of the invention the formulation includes
a prebiotic material.
[0013] Preferably the formulation includes an ingestible carrier.
The ingestible carrier may be a pharmaceutically acceptable carrier
such as a capsule, tablet or powder. Preferably the ingestible
carrier is a food product such as acidified milk, yoghurt, frozen
yoghurt, milk powder, milk concentrate, cheese spreads, dressings
or beverages.
[0014] In one embodiment of the invention the formulation of the
invention further comprises 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.
[0015] In one embodiment of the invention the Bifidobacterium
strain is present in the formulation at more than 10.sup.6 cfu per
gram of delivery system. Preferably the formulation includes any
one or more of an adjuvant, a bacterial component, a drug entity or
a biological compound.
[0016] In one embodiment of the invention the formulation is for
immunisation and vaccination protocols.
[0017] The invention further provides a Bifidobacterium strain or a
formulation of the invention for use as foodstuffs, as a
medicament, for use in the prophylaxis and/or treatment of
undesirable inflammatory activity, for use in the prophylaxis
and/or treatment of undesirable respiratory inflammatory activity
such as asthma, 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, for use in the prophylaxis and/or treatment of
gastrointestinal cancer(s), for use in the prophylaxis and/or
treatment of systemic disease such as rheumatoid arthritis, for use
in the prophylaxis and/or treatment of autoimmune disorders due to
undesirable inflammatory activity, for use in the prophylaxis
and/or treatment of cancer due to undesirable inflammatory
activity, for use in the prophylaxis of cancer, for use in the
prophylaxis and/or treatment of diarrhoeal disease due to
undesirable inflammatory activity, such as Clostridium difficile
associated diarrhoea, Rotavirus associated diarrhoea or post
infective diarrhoea, for use in the prophylaxis and/or treatment of
diarrhoeal disease due to an infectious agent, such as E. coli.
[0018] The invention also provides a Bifidobacterium strain or a
formulation of the invention for use in the preparation of an
anti-inflammatory biotherapeutic agent for the prophylaxis and/or
treatment of undesirable inflammatory activity or for use in the
preparation of anti-inflammatory biotherapeutic agents for the
prophylaxis and/or treatment of undesirable inflammatory
activity.
[0019] In one embodiment of the invention the strain of the
invention act by antagonising and excluding proinflammatory
micro-organisms from the gastrointestinal tract.
[0020] The invention also provides a Bifidobacterium strain or a
formulation of the invention for use in the preparation of
anti-inflammatory biotherapeutic agents for reducing the levels of
pro-inflammatory cytokines.
[0021] The invention further provides a Bifidobacterium strain for
use in the preparation of anti-inflammatory biotherapeutic agents
for modifying the levels of IL-10.
[0022] The invention also provides for the use of a Bifidobacterium
strain as a anti-infective probiotic due to their ability to
antagonise the growth of pathogenic species.
[0023] We have found that particular strains of Bifidobacterium
elicit immunomodulatory effects in vitro.
[0024] The invention is therefore of major potential therapeutic
value in the prophylaxis or treatment of dysregulated immune
responses, such as undesirable inflammatory reactions for example
asthma.
[0025] Bifidobacterium are commensal microorganisms. They have been
isolated from the microbial flora within the human gastrointestinal
tract. The immune system within the gastrointestinal tract cannot
have a pronounced reaction to members of this flora, as the
resulting inflammatory activity would also destroy host cells and
tissue function. Therefore, some mechanism(s) exist whereby the
immune system can recognize commensal non-pathogenic members of the
gastrointestinal flora as being different to pathogenic organisms.
This ensures that damage to host tissues is restricted and a
defensive barrier is still maintained.
[0026] A deposit of Bifidobacterium longum strain AH1205 was made
at the NCIMB on May 11, 2006 and accorded the accession number
NCIMB 41387.
[0027] The Bifidobacterium longum may be a genetically modified
mutant or it may be a naturally occurring variant thereof.
[0028] Preferably the Bifidobacterium longum is in the form of
viable cells.
[0029] Alternatively the Bifidobacterium longum may be in the form
of non-viable cells.
[0030] It will be appreciated that the specific Bifidobacterium
strain of the invention may be administered to animals (including
humans) in an orally ingestible form in a conventional preparation
such as capsules, microcapsules, tablets, granules, powder,
troches, pills, suppositories, suspensions and syrups. Suitable
formulations may be prepared by methods commonly employed using
conventional organic and inorganic additives. The amount of active
ingredient in the medical composition may be at a level that will
exercise the desired therapeutic effect.
[0031] The formulation may also include a bacterial component, a
drug entity or a biological compound.
[0032] In addition a vaccine comprising the strains of the
invention may be prepared using any suitable known method and may
include a pharmaceutically acceptable carrier or adjuvant.
[0033] Throughout the specification the terms mutant, variant and
genetically modified mutant include a strain of Bifidobacteria
whose genetic and/or phenotypic properties are altered compared to
the parent strain. Naturally occurring variant of Bifidobacterium
longum includes the spontaneous alterations of targeted properties
selectively isolated. Deliberate alteration of parent strain
properties is accomplished by conventional (in vitro) genetic
manipulation technologies, such as gene disruption, conjugative
transfer, etc. Genetic modification includes introduction of
exogenous and/or endogenous DNA sequences into the genome of a
Bifidobacteria strain, for example by insertion into the genome of
the bacterial strain by vectors, including plasmid DNA, or
bacteriophages.
[0034] Natural or induced mutations include at least single base
alterations such as deletion, insertion, transversion or other DNA
modifications which may result in alteration of the amino acid
sequence encoded by the DNA sequence.
[0035] The terms mutant, variant and genetically modified mutant
also include a strain of Bifidobacteria that has undergone genetic
alterations that accumulate in a genome at a rate which is
consistent in nature for all micro-organisms and/or genetic
alterations which occur through spontaneous mutation and/or
acquisition of genes and/or loss of genes which is not achieved by
deliberate (in vitro) manipulation of the genome but is achieved
through the natural selection of variants and/or mutants that
provide a selective advantage to support the survival of the
bacterium when exposed to environmental pressures such as
antibiotics. A mutant can be created by the deliberate (in vitro)
insertion of specific genes into the genome which do not
fundamentally alter the biochemical functionality of the organism
but whose products can be used for identification or selection of
the bacterium, for example antibiotic resistance.
[0036] A person skilled in the art would appreciate that mutant or
variant strains of Bifidobacteria can be identified by DNA sequence
homology analysis with the parent strain. Strains of Bifidobacteria
having a close sequence identity with the parent strain are
considered to be mutant or variant strains. A Bifidobacteria strain
with a sequence identity (homology) of 96% or more, such as 97% or
more, or 98% or more, or 99% or more with the parent DNA sequence
may be considered to be a mutant or variant. Sequence homology may
be determined using on-line homology algorithm "BLAST" program,
publicly available at http://www.ncbi.nlm.nih,gov/BLAST/.
[0037] Mutants of the parent strain also include derived
Bifidobacteria strains having at least 85% sequence homology, such
as at least 90% sequence homology, or at least 95% sequence
homology to the 16s-23s intergenic spacer polynucleotide sequence
of the parent strain. These mutants may further comprise DNA
mutations in other DNA sequences in the bacterial genome.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a BOX PCR (bioanalyzer) barcode profile for B.
longum AH1205. Base pair sizes were determined using the Agilent
2100 software;
[0039] FIG. 2 is a graph illustrating the faecal recovery of B.
longum AH1205 over an 8 day feeding period which demonstrates the
ability of AH1205 to transit the murine gastrointestinal tract;
[0040] FIG. 3 is a bar graph showing the effect of B. longum AH1205
on IL-10 cytokine production by human PBMCs. Results are expressed
as mean+/-SE (n=6);
[0041] FIGS. 4A and B are graphs showing the effect of probiotic
bacterial strain AH1205 (A) and placebo (B) on total cell numbers
in bronchoalveolar lavage fluid following ovalbumin (OVA) challenge
in sensitised animals (n=10/group, *=p<0.05 compared to OVA
challenge alone);
[0042] FIGS. 5A and B are graphs showing the effect of probiotic
strain AH1205 treatment (A) and placebo (B) on airway
responsiveness to methacholine, as assessed by changes in enhanced
pause (Penh) in ovalbumin (OVA)-sensitised mice 24 hours after
intranaval challenge with OVA or saline. Each data paint represents
the mean.+-.SEM (n=10/groups * p=<0.05 compared to OVA
alone);
[0043] FIG. 6 A to E are graphs showing IL-10(A), IFN.gamma.(B),
TNF(C), IL-6(D) and CCL2(E) cytokine levels in bronchoalveolar
lavage (BAL) fluid from ovalbumin (OVA)-sensitised mice. Each
column represents the mean.+-.SEM (n=10, * p<0.05 compared to
OVA challenged, saline treated control);
[0044] FIG. 7 is a graph illustrating that CD4.sup.+ CD25.sup.+
Cells from AH1205 fed mice significantly reduced responder CD4 T
cell proliferation (n=7);
[0045] FIG. 8 is a graph showing the percentage of Payer's patch
cells in the CD4+ population that are also CD25+, as assessed by
flow cytometry;
[0046] FIGS. 9A and B are graphs showing the percentage of
CD4/CD25+ cells expressing the transcription factor Foxp3 is
significantly upregulated in germ free mice consuming AH1205.
(A)=spleen cells, (B)=MLNC cells (n=4/group for spleen assay, n=2/3
for MLNC assay);
[0047] FIG. 10 is a graph showing that the level of cytokines IL-6,
MCP-1 and IFN-.gamma. secreted by CD3/CD28 stimulated MLNC cultures
was reduced when germ-free mice consumed Bifidobacterium longum
AH1205. Results are expressed as the mean per group+/-standard
error. (n=4/group);
[0048] FIG. 11 is a graph showing that the level of cytokines IL-6
and TNF-.alpha. secreted by CD3/CD28 stimulated splenocyte cultures
was reduced when germ-free mice consumed Bifidobacterium longum
AH1205. Results are expressed as the mean per group+/-standard
error (n=4/group); and
[0049] FIG. 12 is a graph illustrating the stability of probiotic
strain AH1205 over 3 months compared to Lactobacillus GG.
DETAILED DESCRIPTION
[0050] We have found that Bifidobacterium longum strain AH1205 is
not only acid and bile tolerant and transits the gastrointestinal
tracts but also, surprisingly has immunomodulatory effects, by
modulating cytokine levels or by antagonising and excluding
pro-inflammatory or immunomodulatory micro-organisms from the
gastrointestinal tract.
[0051] The general use of probiotic bacteria 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 the probiotic bacteria. 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.
[0052] It is unknown whether intact bacteria are required to exert
an immunomodulatory effect or if individual active components of
the invention can be utilized 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 CD 14 receptor on
monocytes. It is assumed that components of probiotic bacteria
possess immunomodulatory activity, due to the effects of the whole
cell. Upon isolation of these components, pharmaceutical grade
manipulation is anticipated.
[0053] IL-10 is produced by T cells, B cells, monocytes and
macrophages. This cytokine augments the proliferation and
differentiation of B cells into antibody secreting cells. IL-10
exhibits mostly anti-inflammatory activities. It up-regulates
IL-IRA expression by monocytes and suppresses the majority of
monocyte inflammatory activities. IL-10 inhibits monocyte
production of cytokines, reactive oxygen and nitrogen
intermediates, MHC class II expression, parasite killing and IL-10
production via a feed back mechanism (7). This cytokine has also
been shown to block monocyte production of intestinal collagenase
and type IV collagenase by interfering with a PGE.sub.2-cAMP
dependant pathway and therefore may be an important regulator of
the connective tissue destruction seen in chronic inflammatory
diseases.
[0054] The host response to infection is characterised by innate
and acquired cellular and humoral immune reactions, designed to
limit spread of the offending organism and to restore organ
homeostasis. However, to limit the aggressiveness of collateral
damage to host tissues, a range of regulatory constraints may be
activated. Regulatory T cells (Tregs) serve one such mechanism.
These are derived from the thymus but may also be induced in
peripheral organs, including the gut mucosa. Deliberate
administration of Treg cells suppresses inflammatory disease in a
wide range of murine models including experimental autoimmune
encephalomyelitis, inflammatory bowel disease, bacterial-induced
colitis, collagen-induced arthritis, type I diabetes, airway
osinophilic inflammation, graft-vs-host disease and organ
transplantation. The forkhead transcription factor Foxp3 (forkhead
box P3) is selectively expressed in Treg cells, is required for
Treg development and function, and is sufficient to induce a Treg
phenotype in conventional CD4 cells (19). Mutations in Foxp3 cause
severe, multi-organ autoimmunity in both human and mouse. We have
described a Bifidobacterium strain that generates CD25
positive/Foxp3 positive T regulatory cells in vivo.
[0055] The invention will be more clearly understood from the
following examples.
Example 1
Characterisation of Bacteria Isolated from Infant Faeces
Demonstration of Probiotic Traits
Isolation of Probiotic Bacteria
[0056] Fresh faeces was obtained from a 3 day old male breast fed
infant and serially dilutions were plated on TPY (trypticase,
peptone and yeast extract) and MRS (deMann, Rogosa and Sharpe)
media supplemented with 0.05% cysteine and mupirocin. 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. Gram
positive, catalase negative rod-shaped or bifurcated/pleomorphic
bacteria isolates were streaked for purity on to complex
non-selective media (MRS and TPY). Isolates were routinely
cultivated in MRS or TPY medium unless otherwise stated at
37.degree. C. under anaerobic conditions. Presumptive
Bifidobacterium were stocked in 40% glycerol and stored at
-20.degree. C. and -80.degree. C.
[0057] Following isolation of a pure bifidobacteria strain,
assigned the designation AH1205, microbiological characteristics
were assessed and are summarized in Table 1 below. AH1205 is a gram
positive, catalase negative pleomorphic shaped bacterium which is
Fructose-6-Phoshate Phosphoketolase positive confirming its
identity as a bifidobacterium. Using minimal media in which a
single carbon source was inserted, AH1205 was able to grow on all
carbon sources tested (Glucose, Lactose, Ribose, Arabinose,
Galactose, Raffinose, Fructose, Malt Extract, Mannose, Maltose,
Sucrose).
TABLE-US-00001 TABLE 1 Physiochemical characteristics of B. longum
AH1205 B. longum AH1205 Strain Characteristics Gram Stain +
Catalase - Motility - F6PPK* + Milk coagulation + 45.degree. C.
anaerobic culture - 45.degree. C. aerobic culture - CHO
Fermentation: Glucose + Lactose + Ribose + Arabinose + Galactose +
Raffinose + Fructose + Malt Extract + Mannose + Maltose + Sucrose +
*signifies Fructose-6-Phoshate Phosphoketolase Assay
Species Identification
[0058] 16s intergenic spacer (IGS) sequencing was performed to
identify the species of bifidobacteria isolated. Briefly, DNA was
isolated from AH1205 using 100 .mu.l of Extraction Solution and 25
.mu.l of Tissue Preparation solution (Sigma, XNAT2 Kit). The
samples were incubated for 5 minutes at 95.degree. C. and then 100
.mu.l of Neutralization Solution (XNAT2 kit) was added. Genomic DNA
solution was quantified using a Nanodrop spectrophotometer and
stored at 4.degree. C. PCR was performed using the IGS primers, IGS
L: 5'-GCTGGATCACCTCCTTTC-3' (SEQ ID NO. 3) which is based on SEQ ID
NO. 1 and IGS R: 5'-CTGGTGCCAAGGCATCCA-3' (SEQ ID NO. 4) which is
based on SEQ ID NO. 2. The cycling conditions were 94.degree. C.
for 3 min (1 cycle), 94.degree. C. for 30 sec, 53.degree. C. for 30
sec, 72.degree. C. for 30 sec (28 cycles). The PCR reaction
contained 4 .mu.l (50 ng) of DNA, PCR mix (XNAT2 kit), 0.4 .mu.M
IGS L and R primer (MWG Biotech, Germany). The PCR reactions were
performed on an Eppendorf thermocycler. The PCR products (10 .mu.l)
were ran alongside a molecular weight marker (100 bp Ladder, Roche)
on a 2% agarose EtBr stained gel in TAE, to determine the IGS
profile. PCR products of Bifidobacterium (single band) were
purified using the Promega Wizard PCR purification kit. The
purified PCR products were sequenced using the primer sequences
(above) for the intergenic spacer region. Sequence data was then
searched against the NCBI nucleotide database to determine the
identity of the strain by nucleotide homology. The resultant DNA
sequence data was subjected to the NCBI standard
nucleotide-to-nucleotide homology BLAST search engine
(http://www.ncbi.nlm.nih.gov/BLAST/). The nearest match to the
sequence was identified and then the sequences were aligned for
comparison using DNASTAR MegAlign software. The sequences (SEQ ID
NO. 1 [IGS forward sequence] and SEQ ID NO. 2 [IGS reverse
sequence]) obtained can be viewed in the sequence listing.
Searching the NCIMB database revealed that AH1205 has a unique IGS
(SEQ ID NO. 1 [forward sequence] and SEQ ID NO. 2 [reverse
sequence]) sequence with its closest sequence homology to a
Bifidobacterium longum.
[0059] In order to develop a barcode PCR profile for AH1205, PCR
was performed using BOX primers (8). The cycling conditions were
94.degree. C. for 7 min (1 cycle); 94.degree. C. for 1 minute,
65.degree. C. for 8 minutes, (30 cycles) and 65.degree. C. for 16
minutes. The PCR reaction contained 50 ng of DNA, PCR mix (XNAT2
kit) and 0.3 .mu.M BOXA1R primer (5'-CTACGGCAAGGCGACGCTGACG-3')
(SEQ ID NO. 5) (MWG Biotech, Germany). The PCR reactions were
performed on an Eppendorf thermocycler. The PCR products (1 .mu.l)
were ran alongside a molecular weight marker (DNA 7500 ladder,
Agilent, Germany) using the DNA 7500 LabChip.RTM. on the Agilent
2100 Bioanalyzer (Agilent, Germany). The barcode (PCR product
profile) was determined using the Agilent Bioanalyzer software
where peak number (PCR products) and size were identified (FIG.
1).
Antibiotic Sensitivity Profiles
[0060] Antibiotic sensitivity profiles of the B. longum strain was
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. The minimum inhibitory concentration (MIC) for each
antibiotic was independently assessed. The MIC for clindamycin,
vancomycin and metronidazole were 0.032, 0.75 and >256
respectively.
Intestinal Transit
[0061] To determine whether Bifidobacterium longum could survive at
low pH values equivalent to those found in the stomach, bacterial
cells were harvested from fresh overnight cultures, washed twice in
phosphate buffer (pH 6.5) and resuspended in TPY broth adjusted to
pH 2.5 (with 1M HCl). Cells were incubated at 37.degree. C. and
survival measured at intervals of 5, 30, 60 and 120 minutes using
the plate count method. AH1205 survived well for 5 minutes at pH
2.5 while no viable cells were recovered after 30 minutes.
[0062] Upon exiting the stomach, putative probiotics are exposed to
bile salts in the small intestine. In order to determine the
ability of B. longum to survive exposure to bile, cultures were
streaked on TPY agar plates supplemented with 0.3% (w/v), 0.5%, 1%,
2%, 5%, 7.5% or 10% porcine bile. B. longum AH1205 growth was
observed on plates containing up to 0.5% bile.
[0063] In a murine model, the ability of B. longum AH1205 to
transit the gastrointestinal tract was assessed. Mice consumed
1.times.10.sup.9 AH1205 daily and faecal pellets were examined for
the presence of the fed micro-organism. Detection of AH1205 was
facilitated by isolating a spontaneous rifampicin resistant variant
of the bifidobacteria--incorporation of rifampicin in the TPY
plates used to assess transit ensured that only the fed rifampicin
resistant bifidobacteria was cultured. Faecal samples were
collected daily and B. longum transit through the gastrointestinal
tract was confirmed (FIG. 2).
Anti-Microbial Activity
[0064] The indicator pathogenic micro-organisms used in this study
were propagated in the following medium under the following growth
conditions: Salmonella typhimurium (37.degree. C., aerobic) in
Tryptone Soya broth/agar supplemented with 0.6% yeast extract
(TSAYE, Oxoid), Campylobacter jejuni (37.degree. C., anaerobic) and
E. coli O157:H7 (37.degree. C., anaerobic) on Blood agar medium,
Clostridium difficile (37.degree. C., anaerobic) in reinforced
Clostridial medium (RCM, Oxoid). All strains were inoculated into
fresh growth medium and grown overnight before being used in
experiments.
[0065] Antimicrobial activity was detected using the deferred
method (9). Briefly, B. longum AH1205 was incubated for 36-48 h.
Ten-fold serial dilutions were spread-plated (100 .mu.l) onto TPY
agar medium. After overnight incubation, plates with distinct
colonies were overlayed with the indicator bacterium. The indicator
lawn was prepared by inoculating a molten overlay with 2% (v/v) of
an overnight indicator culture which was poured over the surface of
the inoculated TPY plates. The plates were re-incubated overnight
under conditions suitable for growth of the indicator bacterium.
Indicator cultures with inhibition zones greater than 1 mm in
radius were considered sensitive to the test bacterium. B. longum
AH1205 inhibited the growth of all pathogenic organisms tested,
with zones of clearing measuring 8.67, >80, 4.33 and 11.67 mm
for Salmonella typhimurium, Campylobacter jejuni, E. coli O157:H7
and Clostridium difficile respectively.
Example 2
Cytokine Production by PBMCs in Response to B. longum
[0066] Peripheral blood mononuclear cells (PBMCs) were isolated
from healthy donors by density gradient centrifugation. PBMCs were
stimulated with the probiotic bacterial strain for a 72 hour period
at 37.degree. C. At this time culture supernatants were collected,
centrifuged, aliquoted and stored at -70.degree. C. until being
assessed for IL-10 levels using cytometric bead arrays (BD
BioSciences). AH1205 induced significant secretion of IL-10 by
human PBMCs suggesting this probiotic strain would induce a
anti-inflammatory response in vivo (FIG. 3).
Example 3
B. Longum AH1205 Attenuates Respiratory Disease in a Murine Model
of Asthma
[0067] This study investigated whether the probiotic bacteria
Bifidobacterium longum AH1205, suppresses allergic responses in an
ovalbumin (OVA) sensitized mouse model of allergic airway
inflammation. Briefly, adult male BALB/c mice were sensitized by
i.p. injection of OVA day 0 and day 6. On days 12 and 14, mice were
challenged intranasally with OVA. Twenty-four hours after the last
challenge (day 15), mice were subjected to measurements of airway
responsiveness followed by BAL procedure. OVA/alum-sensitized,
saline-challenged mice served as controls. Animals received
probiotic or placebo throughout the trial. Airway inflammation
(cytokine and cell counts) was assessed by inflammatory cell counts
in bronchoalveolar lavage (BAL) fluid. Airway responsiveness was
also measured using the Buxco whole-body plethysmograph.
Splenocytes were also isolated from OVA sensitized mice and were
incubated in the presence of anti-CD3 and anti-CD28 antibodies
after which cytokine levels were measured in the supernatants by
flow cytometry.
[0068] B. longum AH1205 caused no significant reduction in cells
recovered from BAL fluid following OVA challenge, when compared to
broth fed animals (FIG. 4). Airway responsiveness was measured and
challenge of sensitized mice with OVA resulted in an enhancement of
AHR to methacholine when compared with saline-challenged mice.
AH1205 did not modulate this enhanced airway responsiveness to
methacholine, as assessed by changes in enhanced pause (FIG.
5).
[0069] BAL cytokine levels were measured by cytometric bead array
and demonstrated that animals fed AH1205 had significantly reduced
TNF-.alpha. levels compared to OVA control (FIG. 6C). No
significant differences were noted for IL-10, IFN-.gamma., IL-6 and
CCL2 levels. (FIG. 6)
Example 4
Treg Effector Model
[0070] This study investigated the effect of probiotic consumption
on regulatory T cell number and activity in healthy mice. BALB/c
mice (10/group) were fed Bifidobacterium longum AH1205 or placebo
for three weeks. Following probiotic/placebo consumption, CD4+CD25+
T-regulatory cells were isolated and their in vitro suppressive
activity was determined by measuring proliferation of anti-CD3/CD28
stimulated CFSE-labelled CD4+ responder T cells using flow
cytometry. CD4+ responder T cells were co-incubated with CD4+CD25-
T cells as a control. The percentage of CD4+CD25+ cells (Regulatory
T cells) in murine splenocytes that are also FoxP3 positive was
determined in the spleens of probiotic or placebo-fed mice.
[0071] The % of CD4+ cells that proliferated when co-incubated with
CD4+CD25+ cells from the probiotic/placebo fed mice was compared to
the % of CD4+ cells that proliferated when co-incubated with
CD4+CD25- cells from the same trial mouse. In each case, T cell
proliferation was less in cultures containing CD4+CD25+ cells
compared in cultures containing CD4 cells alone and depleted of the
CD25+ cells (FIG. 7).
[0072] The % of cells in the CD4+ population that were also CD25+
was determined (FIG. 8). The Bifidobacterium longum AH1205 fed
group had significantly more CD4+ T cells that were CD25+ (i.e.
T-Regulatory cells) than their placebo-fed counterparts. This
suggests that the % of T-Regulatory cells within the CD4+
population was increased significantly by feeding with AH1205.
[0073] The number of CD4+CD25+FoxP3+ cells in the whole splenocyte
populations of probiotic or placebo-fed mice was also determined.
The number of CD4+CD25+ T-Regulatory cells expressing FoxP3 was
unchanged in the spleens of probiotic fed mice relative to placebo
or unfed mice.
Example 5
Germ Free Model
[0074] Germ free mice were purchased at 6 weeks of age and
maintained in the germ-free unit at the biological services unit in
UCC. Animals consumed the probiotic strain Bifidobacterium longum
AH1205 for 9 days or remained germ free. Induction of T regulatory
cells was assessed by flow cytometry and cytokine levels were
quantified by CBA.
[0075] AH1205 transit was assessed by measuring bifidobacterial
counts on selective agar over the course of the study. AH1205 did
not transit the gut of germ free mice in measurable numbers even
though approximately 1.times.10.sup.9 organisms were administered
daily. The results suggests that additional microbial or host
factors are required for bifidobacterial survival within the
gut.
[0076] While AH1205 did not transit the gut in detectable numbers,
the bacteria did interact with the host immune system. The numbers
of CD4+CD25+Foxp3+ cells in the mesenteric lymph node and spleen of
AH1205 fed germ-free animals was significantly increased following
9 days of feeding (FIG. 9). Total CD3/CD4 or CD3/CD8 counts
remained unaltered.
[0077] Isolated mesenteric lymph node cells (MLNC) and splenocytes
were stimulated in vitro with anti-CD3/CD28 antibodies or LPS or
remained un-stimulated as negative controls. MLNC secretion of IL-6
and IFN-.gamma., following CD3/CD28 stimulation, was substantially
decreased in culture supernatants while MCP-1 levels were
significantly suppressed when mice were pre-fed AH1205 (FIG. 10).
IL-10 levels remained similar between the groups. Splenocyte
release of IL-6 and TNF-.alpha. was substantially, but not
significantly, decreased when pre-fed AH1205 (FIG. 11). No
significant differences were noted for the un-stimulated or LPS
stimulated cultures but overall we observed less pro-inflammatory
cytokine production from the Bifidobacteria AH1205-fed animals.
Example 6
Stability Results
[0078] The stability of the probiotic strain AH1205, varied over 3
months at 30.degree. C. (FIG. 12)
[0079] Lactobacillus GG was a poor performer over the test period
with a 2 log drop over the 3 month period whereas strain AH 1205
declined in viability by up to approximately 1 log over the same
test period.
Immunomodulation
[0080] The human immune system plays a significant role in the
aetiology and pathology of a vast range of human diseases. Hyper
and hypo-immune responsiveness results in, or is a component of,
the majority of disease states. One family of biological entities,
termed cytokines, are particularly important to the control of
immune processes. Pertubances of these delicate cytokine networks
are being increasingly associated with many diseases. These
diseases include but are not limited to inflammatory disorders,
immunodeficiency, inflammatory bowel disease, irritable bowel
syndrome, cancer (particularly those of the gastrointestinal and
immune systems), diarrhoeal disease, antibiotic associated
diarrhoea, paediatric diarrhoea, appendicitis, autoimmune
disorders, multiple sclerosis, Alzheimer's disease, rheumatoid
arthritis, coeliac disease, diabetes mellitus, organ
transplantation, bacterial infections, viral infections, fungal
infections, periodontal disease, urogenital disease, sexually
transmitted disease, HIV infection, HIV replication, HIV associated
diarrhoea, surgical associated trauma, surgical-induced metastatic
disease, sepsis, weight loss, anorexia, fever control, cachexia,
wound healing, ulcers, gut barrier function, allergy, asthma,
respiratory disorders, circulatory disorders, coronary heart
disease, anaemia, disorders of the blood coagulation system, renal
disease, disorders of the central nervous system, hepatic disease,
ischaemia, nutritional disorders, osteoporosis, endocrine
disorders, epidermal disorders, psoriasis and acne vulgaris. The
effects on cytokine production are specific for each of the
probiotic strains examined. Thus specific probiotic strains may be
selected for normalising an exclusive cytokine imbalance particular
for a specific disease type. Customisation of disease specific
therapies can be accomplished using either a single strain of
AH1205 or mutants or variants thereof or a selection of these
strains.
Immune Education
[0081] The enteric flora is important to the development and proper
function of the intestinal immune system. In the absence of an
enteric flora, the intestinal immune system is underdeveloped, as
demonstrated in germ free animal models, and certain functional
parameters are diminished, such as macrophage phagocytic ability
and immunoglobulin production (10). The importance of the gut flora
in stimulating non-damaging immune responses is becoming more
evident. The increase in incidence and severity of allergies in the
western world has been linked with an increase in hygiene and
sanitation, concomitant with a decrease in the number and range of
infectious challenges encountered by the host. This lack of immune
stimulation may allow the host to react to non-pathogenic, but
antigenic, agents resulting in allergy or autoimmunity. Deliberate
consumption of a series of non-pathogenic immunomodulatory bacteria
would provide the host with the necessary and appropriate
educational stimuli for proper development and control of immune
function.
Inflammation
[0082] 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 (11). 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 (12). 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.
[0083] 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.. Such therapies may also
play a significant role in the treatment of systemic inflammatory
diseases such as rheumatoid arthritis.
[0084] The strains of the present invention may have potential
application in the treatment of a range of inflammatory diseases,
particularly if used in combination with other anti-inflammatory
therapies, such as non-steroid anti-inflammatory drugs (NSAIDs) or
Infliximab.
Cytokines and Cancer
[0085] 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 (13,14). It has long been recognized that weight
loss (cachexia) is the single most common cause of death in
patients with cancer and initial malnutrition indicates a poor
prognosis. 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 properties of Bifidobacterium longum infantis
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 (15). In general, species of
Bifidobacterium have low activities of xenobiotic metabolizing
enzymes compared to other populations within the gut such as
bacteroides, eubacteria and clostridia. Therefore, increasing the
number of Bifidobacterium bacteria in the gut could beneficially
modify the levels of these enzymes.
Vaccine/Drug Delivery
[0086] The majority of pathogenic organisms gain entry via mucosal
surfaces. Efficient vaccination of these sites protects against
invasion by a particular infectious agent. Oral vaccination
strategies have concentrated, to date, on the use of attenuated
live pathogenic organisms or purified encapsulated antigens (16).
Probiotic bacteria, engineered to produce antigens from an
infectious agent, in vivo, may provide an attractive alternative as
these bacteria are considered to be safe for human consumption
(GRAS status).
[0087] Murine studies have demonstrated that consumption of
probiotic bacteria expressing foreign antigens can elicit
protective immune responses. The gene encoding tetanus toxin
fragment C (TTFC) was expressed in Lactococcus lactis and mice were
immunized via the oral route. This system was able to induce
antibody titers significantly high enough to protect the mice from
lethal toxin challenge. In addition to antigen presentation, live
bacterial vectors can produce bioactive compounds, such as
immunostimulatory cytokines, in vivo. L. lactis secreting bioactive
human IL-2 or IL-6 and TTFC induced 10-15 fold higher serum IgG
titres in mice immunized intranasally (17). However, with this
particular bacterial strain, the total IgA level was not increased
by coexpression with these cytokines. Other bacterial strains, such
as Streptococcus gordonii, are also being examined for their
usefulness as mucosal vaccines. Recombinant S. gordonii colonizing
the murine oral and vaginal cavities induced both mucosal and
systemic antibody responses to antigens expressed by this bacterial
(18). Thus oral immunization using probiotic bacteria as vectors
would not only protect the host from infection, but may replace the
immunological stimuli that the pathogen would normally elicit thus
contributing to the immunological education of the host.
Prebiotics
[0088] The introduction of probiotic organisms is accomplished by
the ingestion of the micro-organism 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. 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 that 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 symbiotic.
Other Active Ingredients
[0089] It will be appreciated that the probiotic strains 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 with an immunological involvement. 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.
[0090] The invention is not limited to the embodiments herein
before described which may be varied in detail.
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Sequence CWU 1
1
51494DNABifidobacterium longummisc_featuren can be any of a,c,g or
t 1gcctagnctt ncngncacac gtcaccacac ggtgtcgcat ggccccgntn
ggcatccttc 60ctagcaaatt cccaggacga caaatcatca cactaaaatg atcacaaaac
gatcgaaaca 120aacactaaaa atagagttng attngaaatt aacagcaaga
acgaggaatn aaaggnaacc 180ccgtnttgnt tgngtccact atncagtttt
naagccacca cgcaccacca cgccgtncgg 240acgggaccag cccgccatna
ggnacgatgg gcatngaatc gcgccaggnc aaancctggg 300gtggcgatnc
gggagcccaa aagcgcatnc acaccactnc cgcggaacat nccacgacgg
360acgcaccgna agnccatgat tttttncaca ccancagccc caagncgccg
cgactgncgc 420gacgccnggg ctcgcaccgc cngacgaaca tncggncgtn
ttntncgtan aaaggaggtt 480cccancnann ncng 4942479DNABifidobacterium
longummisc_featuren can be any of a,c,g or t 2aananaaacg ccgcngttct
ccgcggtgcg tgccccgtcg tcncggcagt cgcggcggcc 60tggggctgct ggtgtggaag
agatcatggg ctttcggtgc gtccgtcntg ggatgttccg 120cgggagtggt
gtgnatgcgc ttttggnctc ccggatcgcc accncaggct ttggcctggc
180gcgattcgat gcccatcgtg cctgatggcg ggctggtccc gtccggacgg
cntggtggtg 240cgtggtggct tgagaactgg atagtggacg cgagcaagac
ngggtttcct ttgattcctc 300ttcttgctgt tgatttcgaa tcgaactcta
tttttantgt ttgnttccat cgttttgtga 360ncattttaat gtgangantt
gtcctctggg aatttgctan gaangancct tgnngccang 420cncaccntgn
ngnncctgtt gcctgcaang gcgnanggng gaagcccttg canccagaa
479318DNAArtificial SequencePrimer 3gctggatcac ctcctttc
18418DNAArtificial SequencePrimer 4ctggtgccaa ggcatcca
18522DNAArtificial SequencePrimer 5ctacggcaag gcgacgctga cg 22
* * * * *
References