U.S. patent application number 14/927333 was filed with the patent office on 2016-02-18 for probiotic strains for use in improving the enteric nervous system.
This patent application is currently assigned to COMPAGNE GERVAIS DANONE. The applicant listed for this patent is COMPAGNIE GERVAIS DANONE. Invention is credited to Sandrine Capronnier, Isabelle Chambaud, Marie-Christine Degivry, Gianfranco Grompone, Sophie Legrain-Raspaud, Biliana Lesic, Michel Neunlist, Tamara Smokvina.
Application Number | 20160045557 14/927333 |
Document ID | / |
Family ID | 43302705 |
Filed Date | 2016-02-18 |
United States Patent
Application |
20160045557 |
Kind Code |
A1 |
Legrain-Raspaud; Sophie ; et
al. |
February 18, 2016 |
PROBIOTIC STRAINS FOR USE IN IMPROVING THE ENTERIC NERVOUS
SYSTEM
Abstract
The invention relates to the use of lactic acid bacteria, for
use in modifying the enteric nervous system and more particularly
in treating and/or preventing intestinal disorders such as
constipation and/or irritable bowel disease.
Inventors: |
Legrain-Raspaud; Sophie;
(Limours, FR) ; Grompone; Gianfranco; (Paris,
FR) ; Capronnier; Sandrine; (Villemoisson Sur Orge,
FR) ; Chambaud; Isabelle; (Issy Les Moulineaux,
FR) ; Smokvina; Tamara; (Orsay, FR) ; Degivry;
Marie-Christine; (Le Plessis Robinson, FR) ; Lesic;
Biliana; (Palaiseau Cedex, FR) ; Neunlist;
Michel; (Nantes, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
COMPAGNIE GERVAIS DANONE |
Paris |
|
FR |
|
|
Assignee: |
COMPAGNE GERVAIS DANONE
Paris
FR
|
Family ID: |
43302705 |
Appl. No.: |
14/927333 |
Filed: |
October 29, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13700485 |
Mar 11, 2013 |
9198940 |
|
|
PCT/IB2011/052344 |
May 27, 2011 |
|
|
|
14927333 |
|
|
|
|
Current U.S.
Class: |
424/93.45 ;
424/93.4 |
Current CPC
Class: |
A61P 1/10 20180101; A23L
33/135 20160801; C12R 1/225 20130101; A61P 3/04 20180101; A23C
9/1234 20130101; A23V 2002/00 20130101; A61K 35/745 20130101; A61P
1/04 20180101; A61P 1/12 20180101; A61P 1/00 20180101; A61P 43/00
20180101; A61P 31/00 20180101; A23Y 2300/00 20130101; A61K 35/74
20130101; A61K 35/747 20130101; A23Y 2220/00 20130101; A61K 35/74
20130101; A61K 2300/00 20130101; A23Y 2220/00 20130101; A23Y
2300/00 20130101; A23V 2002/00 20130101; A23V 2200/3204
20130101 |
International
Class: |
A01N 63/00 20060101
A01N063/00; A23C 9/123 20060101 A23C009/123 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 2010 |
IB |
PCT/IB2010/001534 |
Claims
1. A method of: A. increasing vaso-active intestinal peptide (VIP)
levels of the enteric nervous system, or B. increasing Choline
AcetylTransferase ImmunoReactive neurones (ChAT) levels of the
enteric nervous system, or C. decreasing ChAT levels of the enteric
nervous system, comprising administering to a subject a composition
comprising at least one strain of bacteria selected from the group
consisting of lactobacilli and bifidobacteria, wherein the strain
of bacteria is selected from the group consisting of the following
strains: DN.sub.--173.sub.--010 deposited under Accession No.
1-2494 with Collection Nationale De Cultures De Micro-Organismes
(CNCM) on Jun. 20, 2000, DN.sub.--156.sub.--007 deposited under
Accession No. 1-2219 with CNCM on May 31, 1999, and
DN.sub.--119.sub.--0118 deposited under Accession No. 1-4279 with
CNCM on Feb. 25, 2010.
2. The method according to claim 1, wherein said composition:
increases VIP, provided that ChAT is not increased, or increases
ChAT, provided that VIP is not increased, decreases ChAT, provided
that VIP is not decreased, or decreases ChAT and decreases VIP.
3. The method according to claim 1, wherein said composition is
administered to the subject for: treatment and/or prevention of an
intestinal disorder, or treatment and/or prevention of a disorder
selected from the group consisting of constipation and irritable
bowel syndrome C (IBS-C), or treatment and/or prevention of a
disorder selected from the group consisting of diarrhoea,
intestinal infection, IBS-D, IBS-PI, and IBD, or treatment and/or
prevention of a disorder selected from the group consisting of IBS
and inflammatory bowel Disease (IBD), or treatment and/or
prevention of disorders found in elderly people, infants, and obese
people.
4. The method according to claim 1, wherein said composition: A.
increases VIP levels of the enteric nervous system, and is used in
treatment and/or prevention of an intestinal disorder, or B.
increases ChAT levels of the enteric nervous system, and is used in
treatment and/or prevention of a disorder selected form the group
consisting of constipation and IBS-C, or C. decreases ChAT levels
of the enteric nervous system, and is used in treatment and/or
prevention of a disorder selected from the group consisting of
diarrhoea, IBS-D, IBS-PI, IBD.
5. The method according to claim 1, wherein said composition: A3
increases VIP provided that ChAT is not increased, and is
administered to a subject for: treatment and/or prevention of a
disorder selected from the group consisting of IBS and IBD, or
treatment and/or prevention of disorders found in elderly people,
infants, or obese people.
6. The method according to claim 1, wherein said composition: B3.
increases ChAT, provided that VIP is not increased, and is
administered to a subject for treatment and/or prevention of a
disorder selected form the group consisting of constipation and
IBS-C.
7. The method according to claim 1, wherein said composition: C3.
decreases ChAT, provided that VIP is not decreased, and is
administered to a subject for treatment and/or prevention of a
disorder selected form the group consisting of constipation and
IBS-C.
8. The method according to claim 1, wherein said composition: C2.
decreases ChAT and decreases VIP, and is administered to a subject
for treatment and/or prevention of a disorder selected from the
group consisting of diarrhoea, intestinal infections, IBS-D,
IBS-PI, and IBD.
9-17. (canceled)
18. The method according to claim 3, wherein said composition is
administered to the subject for improving gastro-intestinal
motility, improving intestinal peristalsis and/or decreasing
intestinal permeability.
19-26. (canceled)
27. The method according to claim 1, wherein the composition is a
fermented milk product.
28. The method according to claim 27, wherein the fermented milk
product is a yogurt.
Description
FIELD
[0001] The present invention relates to compositions comprising
strains of lactic acid bacteria for use in modifying the enteric
nervous system. Such compositions are especially suitable to treat
and/or prevent intestinal disorders such as constipation and/or
irritable bowel disease.
BACKGROUND
[0002] Irritable bowel syndrome (IBS) or spastic colon is a
functional bowel disorder characterized by chronic abdominal pain,
discomfort, bloating, and alteration of bowel habits in the absence
of any detectable organic cause. In some cases, a low-grade gut
inflammation was reported. Diarrhoea or constipation may
predominate, or they may alternate (classified as IBS-D, IBS-C
respectively). IBS may begin after an infection (post-infectious,
IBS-PI), a stressful life event or onset of maturity without any
other medical indicators. In IBS, routine clinical tests yield no
abnormalities, though the bowels may be more sensitive to certain
stimuli, such as balloon insufflation testing.
[0003] IBS is a very common condition affecting approximately 15%
of the population at any one time. There are about twice as many
women as men with this condition. IBS is a source of chronic pain,
fatigue and other symptoms, and it increases a patient's medical
costs, and contributes to work absenteism. Researchers have
reported that the high prevalence of IBS, in conjunction with
increased costs produces a disease with a high societal cost. It is
also regarded as a chronic illness and can dramatically affect the
quality of a sufferer's life.
[0004] A leading theory about the cause of IBS relates to the
enteric nervous system (ENS). The enteric nervous system (ENS) is a
subdivision of the peripheral nervous system (PNS) that directly
controls gastro-intestinal (GI) functions and is embedded in the
lining of the gastrointestinal system. It includes efferent
neurons, afferent neurons, and interneurons. The structural and
physiological functioning of the ENS is performed by glial cells
(astrocytes). The ENS is organized into two major plexus with
functional specific roles.
[0005] The myenteric plexus, located between the longitudinal and
circular muscle, contains neurones mainly involved in the control
of intestinal motility. Through intestinal muscles, the efferent or
motor neurons control peristalsis and churning of intestinal
contents.
[0006] The motor neurons controlling motility are composed of two
major classes: [0007] excitatory myenteric neurons liberating
acetylcholine (referred to as Choline AcetylTransferase
ImmunoReactive neurons ("ChAT-IR" or "ChAT" or "ChAT neurones" or
"ChAT nerves") and/or substance P (SP) for contractions, and [0008]
inhibitory myenteric neurons liberating nitric oxide (identified as
Nitric Oxide Synthase neurons (NOS-IR)) and/or Vaso-active
Intestinal Peptide (VIP) for relaxation.
[0009] Choline acetyltransferase EC 2.3.1.6 is an enzyme that is
synthesized within the body of a neuron and transferred to the
nerve terminal. The role of ChAT is to join Acetyl-CoA to choline,
resulting in the formation of the neurotransmitter acetylcholine.
Experimentally, effect on acetylcholine production is extrapolated
from the determination of the number of ChAT neurons, typically an
increase in the number of ChAT nerves is indicative of an increase
of acetylcholine.
[0010] The submucosal plexus, located between the circular muscle
and the mucosa, contains neurons mainly involved in the control of
intestinal epithelial barrier (IEB) functions, such as paracellular
permeability. In particular, activation of enteric neurones in the
submucosal plexus decreases paracellular permeability, via the
liberation of VIP, whereas acetylcholine (Ach) increases
paracellular permeability, setting the basis of a fine `tuning` of
the IEBR permeability by the ENS. Thus, concerning the neuronal
control of paracellular permeability, the increase of VIP
liberation by submucosal neurons increases IEB integrity while the
increase of submucosal plexus ChAT neurons decreases IEB
resistance.
[0011] Although there is at current no cure for IBS, there are
treatments which attempt to relieve symptoms, including dietary
adjustments, medication and psychological interventions.
[0012] Probiotics, in particular strains of lactic acid bacteria,
are reported to be beneficial in the treatment and/or prevention of
IBS. Examples of such disclosures are WO 2007/036230, WO 03/010297,
and WO 2009/080800. However, the bacterial strains are selected for
their effect on the immune system, on intestinal permeability or on
the intestinal microbiota and not for their effect on improving the
function of the ENS. WO 2008/064489 discloses the use of probiotics
to block an intermediate conductance calcium dependent potassium
current resulting in an anti-inflammatory effect. WO 2007/132359
discloses the use of Lactobacillus and a cannabinoid receptor
agonist and/or an opioid receptor antagonist in relation to pain
perception. WO 2006/032542 discloses the use of Lactobacillus for
analgesic purposes. Kamm et al, 2004, Neurogastrointest. Motil 16:
53-60 disclosed effects of S. boulardii on decreasing calbindin-28
k (CALB) but not on other neuronal markers of the pig jejunum.
Metugriachuk et al, 2006, Rejuvenation Res. 9: 342-345 disclose
that a symbiotic preparation on motility of small and large
intestine in old Wistar rats significantly increased the
mnyoelectric activity of small intestine and colon, an increased
mRNA expression of VIP, but no significant effect on VIP
concentration.
[0013] Further research is therefore needed on individual strains
of probiotic bacteria with a beneficial effect on the ENS for use
in IBS, constipation and/or other disorders.
SUMMARY OF THE INVENTION
[0014] The inventors employed a new model system for screening and
selecting strains of lactic acid bacteria and bifidobacteria which
have an improved effect on the enteric nervous system (ENS). This
model contains a (mono)layer of intestinal epithelial cells from
human colon carcinoma and, on the basolateral side of the
monolayer, a mixture of a primary culture of enteric nervous system
cells including neurones from myenteric and submucosal plexus.
Using this model the effects of food grade components, in
particular strains of lactic acid bacteria and bifidobacteria, on
the ENS could be assessed by measuring the effects of apical or
luminal addition of these components on the expression of
vaso-intestinal peptide (VIP) and/or ChAT releasing nerves on the
basolateral side.
[0015] This model therefore allowed to screen and to select new
strains of lactic acid bacteria and bifidobacteria for use in
improving the function of the enteric nervous system. Such strains
improve intestinal motility and peristalsis. With some strains the
intestinal transit time can be reduced, which can address some
conditions such as constipation. With some strains the intestinal
transit time can be increased, which can address some conditions
such as diarrhoea. VIP increasing strains also improve intestinal
epithelial barrier integrity. Such strains are in particular useful
and more efficacious than existing strains in prevention and/or
treatment of IBS and/or constipation and other disorders associated
with a decreased function of the ENS.
[0016] Increasing cholinergic phenotype, in particular the
expression of ChAT neurons is of therapeutic interest in GI tract
pathologies associated with inhibition of colonic transit. Using
lactic acid bacteria or bifidobacteria selected to have an
increasing effect on enhancing cholinergic expression, i.e. ChAT,
in neurons are of therapeutical interest for constipated patients,
and patients suffering from IBS-C. Therefore, one group of lactic
acid bacteria or bifidobacteria strains of the present invention
advantageously increases the number of ChAT nerves, which is
indicative for an improved effect on intestinal motility, and
especially is beneficial for IBS patients, in particular IBS-C
patients, and patients suffering from constipation. This group is
referred to as group B). Good motility requires a high number of
ChAT nerves, which are responsible for contraction and have a
prokinetic effect. In some interesting embodiments for this group
the TEER levels are not decreased, since the IEB function and
ability to relax the muscles is preferably not impaired. The
subgroup according to these embodiments is referred to as group
B3).
[0017] Increasing VIP beneficially improves relaxation of the
muscles of the GI tract and improves IEB, which is beneficial for
patients suffering from IBS or inflammatory bowel disease (IBD) and
also for elderly people, infants, and obese people. For such
subjects a good IEB function is better if not essential. Although
patients suffering from IBS or IBD have an oversecretion of
neuropeptides such as VIP, this is supposed to be an adaptative
response of the ENS to control intestinal inflammation, to
re-establish intestinal barrier functions and to increase
neuroprotection. A group of lactic acid producing bacteria strains,
in particular bifidobacteria, of the present invention
advantageously increases VIP. This group is referred to as group
A). In one embodiment the ChAT level is not increased. The group
according to this embodiment is referred to as group A3).
Accordingly the ChAT level can remain substantially unchanged or
can decrease.
[0018] All the herein referred bacterial strains have been
deposited, according to the Budapest Treaty, before CNCM
("Collection Nationale de Cultures de Microorganismes", 25 rue du
Docteur Roux, Paris) as an International depositary authority.
[0019] Strains found with the screening method were belonging to
group B) are DN.sub.--154.sub.--0067 (CNCM I-4320 filed May 19,
2010), DN.sub.--116.sub.--0047 (CNCM I-4317 filed May 19, 2010) and
DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010). Strains
belonging to group A) are DN.sub.--173.sub.--010 (CNCM I-2494 filed
Jun. 20, 2000), DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19,
2010), DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999), and
DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010). Strain
DN.sub.--173.sub.--010 (CNCM I-2494 filed Jun. 20, 2000) has been
disclosed in International application WO 02/02800 and strain
DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999) has been
disclosed in International application WO 01/01785.
[0020] Compositions comprising at least one of these selected
strains are therefore part of the invention. A composition
comprising a mix of at least one strain belonging to group B) and
at least one strain belonging to group A) is preferred. Such a mix
will advantageously have an improved effect on motility by
improving both contractions and relaxations and additionally have
an advantageous effect on IEB function.
[0021] Thus, according to one aspect the invention concerns a
composition comprising at least one strain of bacteria, preferably
selected from the group consisting of lactobacilli and
bifidobacteria, for use in:
[0022] A) increasing vaso-active intestinal peptide (VIP) levels of
the enteric nervous system, or
[0023] B) increasing Choline AcetylTransferase ImmunoReactive
neurones (ChAT) levels of the enteric nervous system, or
[0024] C) decreasing ChAT levels of the enteric nervous system.
[0025] According to one aspect the invention concerns a composition
comprising at least one strain of bacteria selected from the group
consisting of the following strains:
[0026] DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010)
[A)-A3)],
[0027] DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999)
[A)-A3)],
[0028] DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010)
[A)-A3)],
[0029] DN.sub.--116.sub.--0047 (CNCM I-4317 filed May 19, 2010)
[B)-B3)],
[0030] DN.sub.--154.sub.--0067 (CNCM I-4320 filed May 19, 2010)
[B)-B3)], and
[0031] DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010)
[B)-B3],
[0032] for use in: [0033] treatment and/or prevention of an
intestinal disorder, preferably treatment and/or prevention of an
intestinal motility disorder, or [0034] treatment and/or prevention
of a disorder selected form the group consisting of constipation
and IBS-C, [0035] treatment and/or prevention of a disorder
selected from the group consisting of diarrhoea, intestinal
infection, IBS-D, IBS-PI, and IBD, or [0036] treatment and/or
prevention of a disorder selected from the group consisting of IBS
and IBD, or [0037] treatment and/or prevention of disorders found
in elderly people, infants, or obese people.
[0038] According to one aspect the invention concerns a composition
comprising at least one strain of bacteria selected from the group
consisting of the following strains:
[0039] DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010)
[A)-A3)],
[0040] DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999)
[A)-A3)],
[0041] DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010)
[A)-A3)],
[0042] DN.sub.--116.sub.--0047 (CNCM I-4317 filed May 19, 2010)
[B)-B3)],
[0043] DN.sub.--154.sub.--0067 (CNCM I-4320 filed May 19, 2010)
[B)-B3)], and
[0044] DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25,
2010)[B)-B3)],
[0045] for use in administration to subjects suffering from a
disorder selected from the group consisting of: [0046]
constipation, IBS-C, [0047] diarrhoea, intestinal infection, IBS-D,
IBS-PI, IBD, [0048] IBS, and [0049] disorders found in elderly
people, infants, or obese people.
[0050] According to one aspect the invention concerns a composition
as mentioned above for use in improving gastro-intestinal motility,
improving intestinal peristalsis and/or decreasing intestinal
permeability.
[0051] According to one aspect the invention concerns new strains
of bacteria selected from the group consisting of: [0052]
DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],
[0053] DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010)
[A)-A3)], [0054] DN.sub.--116.sub.--0047 (CNCM I-4317 filed May 19,
2010) [B)-B3)], and [0055] DN.sub.--154.sub.--0067 (CNCM I-4320
filed May 19, 2010) [B)-B3)].
[0056] According to one aspect the invention concerns compositions
comprising the new strains.
[0057] According to one aspect the invention concerns a composition
comprising: [0058] at least one strain of bacteria selected from
the group consisting of lactobacilli and bifidobacteria that B)
increases ChAT levels in the enteric nervous system, and [0059] at
least one strain of bacteria selected from the group consisting of
lactobacilli and bifidobacteria that A) increases vaso-active
intestinal peptide (VIP) levels in the enteric nervous system.
[0060] According to one aspect the invention concerns a method of
selecting strains of bacteria, said method comprising the steps
of:
[0061] a) Arranging a coculture of intestinal epithelial cells and
enteric neuronic cells, wherein said intestinal epithelial cell are
present as a monolayer and wherein said enteric neuronic cells are
present at the basoluteral side of the monolayer,
[0062] b) Adding strains of bacteria to the apical or luminal side
of the monolayer of intestinal epithelial cells, preferably in an
amount of about 4 to 400 bacterial cells per epithelial cell,
[0063] c) Incubating the coculture with the strain of lactic acid
bacteria,
[0064] d) Preferably isolating the neuronic cells,
[0065] e) Measuring the amount of VIP, ChAT, substance P, Nitrogen
Oxide nerves, ATP and/or pituitary adenylate cyclase activating
peptide (PACAP) produced by the neuronic cells and optionally
additionally the TransEpithelial Electrical Resistance (TEER) of
the intestinal epithelial cells layer.
[0066] In this method, the strain of bacteria preferably belongs to
the group consisting of lactobacilli and bifidobacteria.
DETAILED DESCRIPTION
Definitions
[0067] In the present application the use of a compound or a
composition is intended to cover the use itself, optionally with
the connected intention, but also any communication associated to
the compound or composition with commercial or legal consequences,
for example advertisement, instructions or recommendation on the
package of the compositions, instructions or recommendation on
commercial support such as leaflets, brochures, posters,
documentation filed in support to regulatory registrations for
safety purpose, efficacy purpose, or consumer protection, for
example at administrations such as EFSA in Europe.
[0068] In the present application groups of strains refer to
strains that exhibit a specific property or set of properties. A
specific strain can thus pertain to several groups. In the present
application the term "or" is not exclusive.
[0069] In the present application a property such as VIP and/or
ChAT is considered as substantially unchanged compared to a control
if the variation does not exceed 10%, preferably 1% compared to the
control.
Preferred Embodiments
[0070] In preferred embodiments, the composition is for use in:
[0071] A3) increasing VIP, provided that ChAT is not increased,
or
[0072] B3) increasing ChAT, provided that VIP is not increased,
Electrical Resistance (TEER) of the intestinal epithelial cells
layer being not decreased or
[0073] C3) decreasing ChAT, provided that VIP is not decreased,
or
[0074] C2) decreasing ChAT and decreasing VIP.
For example the composition of the invention can be used in: [0075]
treatment and/or prevention of an intestinal disorder, preferably
treatment and/or prevention of an intestinal motility disorder, or
[0076] treatment and/or prevention of a disorder selected form the
group consisting of constipation and IBS-C, or [0077] treatment
and/or prevention of a disorder selected from the group consisting
of diarrhoea, intestinal infection, IBS-D, IBS-PI, and IBD, or
[0078] treatment and/or prevention of a disorder selected from the
group consisting of IBS and IBD, or [0079] treatment and/or
prevention of disorders found in elderly people, infants, and obese
people. In especially preferred embodiments, the compositions
can:
[0080] A) increase VIP levels of the enteric nervous system, and be
used in treatment and/or prevention of an intestinal disorder,
preferably treatment and/or prevention of an intestinal motility
disorder, or
[0081] B) increase ChAT levels of the enteric nervous system, and
be used in treatment and/or prevention of a disorder selected form
the group consisting of constipation and IBS-C, or
[0082] C) decrease ChAT levels of the enteric nervous system, and
be used in treatment and/or prevention of a disorder selected from
the group consisting of diarrhoea, IBS-D, IBS-PI, IBD.
The strain of bacteria can for example be selected from the group
consisting of the following strains: [0083] DN.sub.--173.sub.--010
(CNCM I-2494 filed Jun. 20, 2000) [A)-A3)], [0084]
DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],
[0085] DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999)
[A)-A3)], [0086] DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19,
2010) [A)-A3)], [0087] DN.sub.--116.sub.--0047 (CNCM I-4317 filed
May 19, 2010) [B)-B3)], [0088] DN.sub.--154.sub.--0067 (CNCM I-4320
filed May 19, 2010) [B)-B3)], and [0089] DN.sub.--119.sub.--0118
(CNCM I-4279 filed Feb. 25, 2010) [B)-B3)]. In one embodiment the
strain of bacteria is selected from the group consisting of the
following strains: [0090] DN.sub.--156.sub.--0032 (CNCM I-4321
filed May 19, 2010) [A)-A3)], [0091] DN.sub.--156.sub.--007 (CNCM
I-2219 filed May 31, 1999) [A)-A3)], and [0092]
DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010)
[A)-A3)],
[0093] and the composition is for use in: [0094] treatment and/or
prevention of a disorder selected from the group consisting of IRS
and IBD, or [0095] treatment and/or prevention of disorders found
in elderly people, infants, or obese people. In one embodiment the
strain of bacteria is selected from the group consisting of the
following strains: [0096] DN.sub.--116.sub.--0047 (CNCM I-4317
filed May 19, 2010) [B)-B3)], [0097] DN.sub.--154.sub.--0067 (CNCM
I-4320 filed May 19, 2010) [B)-B3)], and [0098]
DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010)
[B)-B3)],
[0099] and the composition for use in treatment and/or prevention
of a disorder selected from the group consisting of constipation
and IBS-C.
In one embodiment the strain of bacteria is selected from the group
consisting of the following strains: [0100] DN.sub.--156.sub.--0032
(CNCM I-4321 filed May 19, 2010) [A)-A3)], [0101]
DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999) [A)-A3)],
and [0102] DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010)
[A)-A3)],
[0103] and the composition is for use in:
[0104] A) increasing vaso-active intestinal peptide (VIP) levels of
the enteric nervous system, preferably for use in A3) increasing
VIP, provided that ChAT is not increased.
In a particular embodiment of this embodiment, the composition is
for use in: [0105] treatment and/or prevention of a disorder
selected from the group consisting of IBS and IBD, or [0106]
treatment and/or prevention of disorders found in elderly people,
infants, or obese people. In one embodiment the strain of bacteria
is selected from the group consisting of the following strains:
[0107] DN.sub.--116.sub.--0047 (CNCM I-4317 filed May 19, 2010)
[B)-B3)], [0108] DN.sub.--154.sub.--0067 (CNCM I-4320 filed May 19,
2010) [B)-B3)], and [0109] DN.sub.--119.sub.--0118 (CNCM I-4279
filed Feb. 25, 2010) [B)-B3)],
[0110] and the composition is for use in:
[0111] B) increasing ChAT levels of the enteric nervous system,
preferably 13) increasing ChAT, provided that VIP is not
increased.
In a particular embodiment of this embodiment, the composition is
for use in treatment and/or prevention of a disorder selected form
the group consisting of constipation and IBS-C.
Further Details of Strains of Bacteria
[0112] As mentioned above the composition comprises at least one or
two specific strains of bacteria, preferably lactic acid bacteria.
They are preferably selected from the group consisting of the genus
Lactobacillus and Bifidobacterium, Lactococcus and Streptococcus.
The said specific strain of bacteria were found to be capable to
affect VIP levels and/or to affect ChAT nerve levels in a coculture
model representing the interaction between the intestine and the
ENS.
[0113] A coculture model, described in more detail below, was used
to in vitro select strains of lactic acid bacteria or
bifidobacteria with these properties. 102 strains belonging to the
genera Lactobacillus, Streptococcus or Bifidobacterium were
screened.
[0114] Group 1) strains with increased effect on ChAT will
typically improve intestinal motility. Group B3) strains with
increased effect on ChAT, excluding strains wherein VIP is not
increased (i.e. VIP is substantially unchanged or VIP is decreased)
represent a specific preferred embodiment. Strains of these groups
are typically beneficial for treatment and/or prevention of a
disorder selected form the group consisting of constipation and
IBS-C. This might be of further particular interest for treatment
and/or prevention of disorders found in elderly people, which can
often suffer from constipation. Concerning increasing motility,
strains increasing ChAT expression would be favoured. This property
appears to be very rare. Interestingly, only three strains were
found to increase statistically ChAT. These are referred to group
B) or group 133) strains. Group B) or group B3) strains comprise
strains DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010),
DN.sub.--154.sub.--0067 (CNCM I-4320 filed May 19, 2010) and
DN.sub.--116.sub.--0047 (CNCM I-4317 filed May 19, 2010). Such
strains beneficially improve motility, especially contractions. It
is preferred that Electrical Resistance (TEER) of the intestinal
epithelial cells layer be not decreased. Such a property can be
indicative of a suitable barrier function. Strain
DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010)
significantly decreased VIP levels, whereas the other two strains
did not have a significant effect on VIP levels. Since a decrease
in VIP may have an adverse effect on IEB it was examined with an in
vitro model with a monolayer of intestinal epithelial cells whether
incubation of this strain resulted in a decrease transepithelial
electrical resistance (TEER). This turned out not to be the case,
indicating that the IEB function is not impaired.
[0115] Concerning the relaxation of the muscles and reinforcement
of IEB function, which is beneficial in IBS and IBD patients,
strains would be favoured that increase VIP expression (which would
have in addition anti-inflammatory effects). Such strains are
referred to as group A) strains. In a preferred embodiment group A)
strains do not increase ChAT expression (i.e. ChAT is substantially
unchanged or ChAT is decreased). Such strains are referred to as
group A3) strains. Strains of these groups are typically beneficial
to treatment and/or prevention of a disorder selected from the
group consisting of IBS and IBD, or to treatment and/or prevention
of disorders found in elderly people, infants, or obese people.
Group A) or group A3) strains comprise strains
DN.sub.--173.sub.--010 (CNCM I-2494 filed Jun. 20, 2000),
DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010),
DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999),
DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010). It is
interesting to note that all the strains having this property are
bifidobacteria except one: DN.sub.--121.sub.--0304 (CNCM I-4318
filed May 19, 2010). Furthermore, using another in vitro model with
a T84 monolayer and the transepithelial electric resistance (TEER)
especially strains DN.sub.--173.sub.--010 (CNCM I-2494 filed Jun.
20, 2000), DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999),
DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010) and
DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010) of group
A) or A3) were found to have a protective effect on the IEB in the
presence of LPS. Therefore, these particular strains are especially
preferred.
[0116] According to one embodiment the strains allow decreasing
ChAT levels of the enteric nervous system. The corresponding group
of strains is referred to as group C). In a particular embodiment
the strains allow decreasing ChAT, provided that VIP is not
decreased (i.e. VIP is substantially unchanged or VIP is
increased). This group is referred to as group C3). In a particular
embodiment the strains allow decreasing ChAT with decreasing VIP.
These strains are referred to as group C2). Strains of group C3)
are typically beneficial to treatment and/or prevention of a
disorder selected form the group consisting of diarrhoea, IBS-D.
This might be of further particular interest for treatment and/or
prevention of disorders found in elderly people, which can often
suffer from diarrhoea. Strains of group C2) are typically
beneficial to treatment and/or prevention of a disorder selected
from the group consisting of diarrhoea, IBS-D, IBS-PI, and IBD.
This might be of further particular interest for treatment and/or
prevention of disorders found in elderly people which can often
suffer from such conditions, especially diarrhoea.
[0117] The present invention also encompasses the use of the above
mentioned strains, but also mutant strains or genetically
transformed strains derived from any one of the parent strains
still having activity on VIP and effecting ChAT nerves. These
mutant or genetically transformed strains can be strains wherein
one or more endogenous gene(s) of the parent strain has (have) been
mutated, for instance to modify some of its metabolic properties
(e.g. its ability to ferment sugars, its resistance to acidity, its
survival to transport in the gastrointestinal tract, its
post-acidification or its metabolite production). They can also be
strains resulting from the genetic transformation of the parent
strain by one or more gene(s) of interest, for instance in order to
give to said strain additional physiological features, or to allow
it to express proteins of therapeutic or vaccinal interest that one
wishes to administer through said strains.
[0118] Preferably a mix of at least one strain belonging to group
B), preferably group B3), and at least one strain belonging to
group A), preferably group A3), is used. Such a mix will
advantageously have an improved effect on motility as well as on
IEB.
Co-Culture Model and Screening Assay
[0119] In one embodiment the present invention relates to a method
of selecting strains of lactic acid bacteria, said method
comprising the steps of:
[0120] a) Using a coculture of intestinal epithelial cells and
enteric neuronic cells, wherein the intestinal epithelial cells are
present as a monolayer and wherein the enteric neuronic cells are
present at the basolateral side of the monolayer,
[0121] b) Adding lactic acid bacteria or the apical or luminal side
of the monolayer, preferably in an amount of about 4 to 400
bacterial cells per epithelial cell,
[0122] c) Incubating the coculture with the lactic acid
bacteria,
[0123] d) Preferably isolating the neuronic cells, and
[0124] e) Measuring the amount of at least one neurotransmitter
selected from the group consisting of VIP, ChAT, substance P and
Nitrogen Oxide, ATP, PACAP produced by the neuronic cells, and
optionally additionally the TransEpithelial Electrical Resistance
(TEER) of the intestinal epithelial cells layer.
[0125] In agreement with the peristaltic reflex the ENS contains
hardwired circuits that consist of ascending excitatory motor
neurons that release acetylcholine and substance P, which contracts
smooth muscle through muscarinic receptors, and of descending
inhibitory neurons that release a cocktail of transmitters, like
NO, ATP, VIP and PACAP, all of which inhibit the circular
muscle.
Cell Culture
[0126] A suitable way to set up the coculture with a monolayer of
polarized intestinal epithelial cells is given in example 1 and is
also described in J. Chevalier et al, 2008, J. Physiol. 586
1963-1975.
[0127] All intestinal epithelial cell cultures forming monolayers
are suitable, such as Caco-2, T84, HT29, and TC7. Preferably T84
cells are used.
[0128] As primary enteric nerve system cells, preferably cells are
isolated from non-human mammalian foetuses, preferably rodents,
more preferably rats.
[0129] Preferably the bacteria strains tested are grown to late
exponential phase in a suitable growth medium and washed.
Preferably the bacteria are added to the apical side of the
coculture at an amount of 4 to 400 bacteria/epithelial cell, more
preferably 10 to 100 bacteria/epithelial cell, even more preferably
30 to 50 bacteria/epithelial cell. Preferably, as a control, no
bacteria are added. Preferably, as a positive control 1 mM butyrate
or 40 mM KCl is used.
[0130] Preferably the incubation step is performed at about
37.degree. C. Preferably the incubation step takes 1 to 72 h, more
preferably 2 to 36 h, even more preferably 4 to 12 h.
[0131] Preferably after co-incubation, the compartment containing
epithelial cells and bacteria is removed and primary neuronal cells
are incubated for 12 to 48 h, more preferably for 20 to 28 h in a
humidified incubator containing 5% CO.sub.2.
[0132] Preferably the amount of ChAT nerves versus total nerves is
measured using immunohistochemical staining, using anti-neurone
specific enolase (NSE) to count the total number of neurones and
anti-choline acetyl transferase to count the ChAT nerves.
[0133] Preferably VIP is determined by ELISA after collecting the
neuronal cells and extracting the proteins with the presence of a
protease inhibitor cocktail.
Further Details about Compositions
[0134] The invention encompasses compositions with strains of
bacteria which allow the above referenced uses or properties. The
invention also encompasses compositions comprising one or more of
the following strains (encompassing mutants or genetically
transformed strains derived thereof): [0135]
DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010) [A)-A3)],
[0136] DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999)
[A)-A3)], [0137] DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19,
2010) [A)-A3)], [0138] DN.sub.--116.sub.--0047 (CNCM I-4317 filed
May 19, 2010) [B)-B3)], [0139] DN.sub.--154.sub.--0067 (CNCM I-4320
filed May 19, 2010) [B)-B3)], and [0140] DN.sub.--119.sub.--0118
(CNCM I-4279 filed Feb. 25, 2010) [B)-B3)],
[0141] for use in: [0142] treatment and/or prevention of an
intestinal disorder, preferably treatment and/or prevention of an
intestinal motility disorder, or [0143] treatment and/or prevention
of a disorder selected form the group consisting of constipation
and IBS-C, or [0144] treatment and/or prevention of a disorder
selected from the group consisting of diarrhoea, intestinal
infection, IBS-D, IBS-PI, and IBD, or [0145] treatment and/or
prevention of a disorder selected from the group consisting of IBS
and IBD, or [0146] treatment and/or prevention of disorders found
in elderly people, infants, or obese people,
[0147] typically when administered in vivo to a subject.
[0148] In the compositions of the invention, said strains can be
used in the form of whole bacteria which may be living or not.
Alternatively, they can be used in the form of a bacterial lysate
or in the form of bacterial fractions; the bacterial fractions
suitable for this use can be chosen, for example, by testing their
properties of alleviating the effects on VIP levels and levels of
ChAT nerves of the coculture model described in the present
invention. Preferably the bacterial cells are present as living,
viable cells.
[0149] The compositions of the invention can be in any form
suitable for administration, in particular oral administration.
This includes for instance solids, semi-solids, liquids, and
powders. Liquid compositions are generally preferred for easier
administration, for instance as drinks.
[0150] The composition can for example comprise at least 10.sup.5,
preferably at least 1.times.10.sup.6, cfu per g dry weight, of at
least one strain of bacteria, preferably of strains of bacteria as
mentioned above. These are preferably selected from the group
consisting of lactobacilli and bifidobacteria.
[0151] When the bacteria are in the form of living bacteria, the
composition may typically comprise 10.sup.5 to 10.sup.13 colony
forming units (cfu), preferably at least 10.sup.6 cfu, more
preferably at least 10.sup.7 cfu, still more preferably at least
10.sup.8 cfu, and most preferably at least 10.sup.9 cfu per g dry
weight of the composition. In the case of a liquid composition,
this corresponds generally to 10.sup.4 to 10.sup.12 colony forming
units (cfu), preferably at least 10.sup.5 cfu, more preferably at
least 10.sup.6 cfu, still more preferably at least 10.sup.7 cfu,
and most preferably at least 10.sup.9 cfu/ml.
[0152] Examples of the compositions of the invention are
nutritional compositions, including food products and in particular
dairy products.
[0153] The composition can be for example a dairy product,
preferably a fermented dairy product. The administration in the
form of a fermented dairy product has the additional advantage of
low lactose levels, which is further beneficial for IBS.
Optionally, other strains of lactic acid bacteria may be present.
The fermented product can be present in the form of a liquid or
present in the form of a dry powder obtained by drying the
fermented liquid. Preferably the fermented product is a fresh
product. A fresh product, which has not undergone severe heat
treatment steps, has the advantage that bacterial strains present
are in the living form. Preferably the fermented product is a dairy
product, more preferably fermented milk and/or fermented whey.
Preferably the nutritional composition is yoghurt, or fermented
milk in set, stirred or drinkable form. Preferably the fermented
product is a cheese. Preferably the fermented product is a
fermented vegetable, such as fermented soy, cereals and/or fruits
in set, stirred or drinkable forms.
[0154] Preferably the present nutritional composition is a baby
food, an infant milk formula or an infant follow-on formula.
Preferably the present composition is a nutraceutical or a
pharmaceutical product, a nutritional supplement or medical
food.
[0155] Nutritional compositions of the invention also include food
supplements, and functional food. A "food supplement" designates a
product made from compounds usually used in foodstuffs, but which
is in the form of tablets, powder, capsules, potion or any other
form usually not associated with aliments, and which has beneficial
effects for one's health. A "functional food" is an aliment which
also has beneficial effects for one's health. In particular, food
supplements and functional food can have a physiological
effect--protective or curative--against a disease, for example
against a chronic disease.
[0156] A composition comprising a mix of at least one strain of
lactic acid bacterium or bifidobacterium increasing ChAT nerves and
at least one strain of lactic acid bacterium or bifidobacterium
increasing VIP levels is preferred. Such a mix will advantageously
have an improved effect on motility as well as on IEB.
[0157] A mix of at least one strain belonging to group B),
preferably B3), and at least one strain belonging to group A),
preferably A3), is preferred. Such a mix will advantageously have
an improved effect on motility as well as on IEB.
Therefore the present invention also relates to compositions
comprising: [0158] at least one strain of bacteria selected from
the group consisting of the following strains:
[0159] DN.sub.--116.sub.--0047 (CNCM I-4317 filed May 19, 2010)
[B)-B3)],
[0160] DN.sub.--154.sub.--0067 (CNCM I-4320 filed May 19, 2010)
[B)-B3)], and
[0161] DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25,
2010)[B)-B3)]; and [0162] at least one strain of bacteria selected
from the group consisting of the following strains:
[0163] DN.sub.--173.sub.--010 (CNCM I-2494 filed Jun. 20, 2000)
[A)-A3)],
[0164] DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010)
[A)-A3)],
[0165] DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999)
[A)-A3)], and
[0166] DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010)
[A)-A3)].
[0167] The compositions of the invention can also comprise one or
more other strain(s) of lactic acid bacteria, probiotic or not, for
instance one or more bacterial strain(s) selected from the genera
Lactobacillus, Lactococcus, Streptococcus, and Bifidobacteria. In
particular, this (these) other strain(s) can include one or more
strain(s) of Streptococcus thermophilus, and/or one or more
strain(s) of Lactobacillus bulgaricus.
Application
[0168] In one embodiment strains of the present invention were
found to increase the number of ChAT nerves. Choline
acetyltransfcrase EC 2.3.1.6 is an enzyme that is synthesized
within the body of a neuron and transferred to the nerve terminal.
The role of choline acetyltransferase is to join Acetyl-CoA to
choline, resulting in the formation of the neurotransmitter
acetylcholine. It is used as an immunohistochemical marker for
motor neurons. The effects on the ChAT nerve result in the improved
contractions resulting in improved peristalsis. Therefore, the
strains and compositions of the present invention able to increase
ChAT nerves are advantageously administered to improve the ENS, to
improve or enhance peristalsis, to improve intestinal motility
and/or to decrease the gastro-intestinal transit time. Increasing
cholinergic phenotype is of therapeutic interest in GI pathologies
associated with inhibition of colonic transit. In particular,
various studies have shown that slow transit could be associated
with a reduced expression of ChAT neurons. In particular, (i)
severely constipated patients generally have a lower amount of ChAT
nerves, (ii) the production of myenteric ACh significantly
decreased both during the course of infection and post infection
(PI), (iii) during aging a reduction of the proportion of
cholinergic neurons has been reported.
[0169] In this context, using strains of bacteria, preferably
lactic acid bacteria or bifidobacteria to enhance cholinergic
expression in neurons could be of future therapeutically interest
for severely constipated patient and IBS-C. Therefore, the strains
and compositions of the present invention are advantageously
administered to patients suffering from IBS-C, and/or constipation.
Strains that are most useful are the group B) or B3) strains
mentioned above.
[0170] In one particular embodiment the strains and compositions of
the present invention are used by or for elderly people. Elderly
people in the present invention are defined as human with an age
above 65 years, preferably above 70 years, preferably above 75
years, preferably above 80 years, preferably above 85 years.
Elderly people typically have decreased number of ChAT neurons in
the enteric nervous system located in the colon, most preferably in
the transversal colon. Therefore, the strains and compositions of
the present invention are advantageously administered to treat
and/or prevent IBS, preferably IBS-C, and/or constipation for
elderly people. Strains able to increase ChAT nerves are group B),
such as strain DN.sub.--154.sub.--0067 (CNCM I-4320 filed May 19,
2010), DN.sub.--116.sub.--0047 (CNCM I-4317 filed May 19, 2010) and
DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010). They
preferably do not decrease VIP, since VIP is necessary for a good
IEB function and for relaxation of the GI tract, another important
part of the GI tract motility such as peristalsis. The strains
meeting this criterion were DN.sub.--154.sub.--0067 (CNCM I-4320
filed May 19, 2010), and DN.sub.--116.sub.--0047 (CNCM I-4317 filed
May 19, 2010). However, also strain DN.sub.--119.sub.--0118 (CNCM
I-4279 filed Feb. 25, 2010) did not negatively affect IEB function
as determined by TEER experiments, as TEER did not decreased.
[0171] In one embodiment the strains of the present invention were
found to increase the levels of VIP. With respect to the digestive
system, VIP induces smooth muscle relaxation (lower oesophageal
sphincter, stomach, gallbladder), stimulates secretion of water
into pancreatic juice and bile, and causes inhibition of gastric
acid secretion and absorption from the intestinal lumen. Its role
in the intestine is to greatly stimulate secretion of water and
electrolytes, as well as dilating intestinal smooth muscle,
dilating peripheral blood vessels, stimulating pancreatic
bicarbonate secretion, and inhibiting gastrin-stimulated gastric
acid secretion. These effects work together to increase motility.
Therefore this finding is indicative for these strains to have an
improved effect on intestinal motility, in particular the
relaxation part of motility. VIP also beneficially increases IEB
function. Therefore, the strains and compositions of the present
invention are advantageously administered to improve the ENS, to
improve or enhance peristalsis, to decrease permeability, to
improve intestinal motility and/or to decrease the
gastro-intestinal transit time. Therefore, the strains and
compositions of the present invention are advantageously
administered for use in or to patients suffering from: [0172]
treatment and/or prevention of an intestinal disorder, preferably
treatment and/or prevention of an intestinal motility disorder, or
[0173] treatment and/or prevention of a disorder selected form the
group consisting of constipation and IBS-C, or [0174] treatment
and/or prevention of a disorder selected from the group consisting
of diarrhoea, intestinal infection, IBS-D, IBS-PI, and IBD, or
[0175] treatment and/or prevention of a disorder selected from the
group consisting of IBS and IBD, or [0176] treatment and/or
prevention of disorders found in elderly people, infants, and obese
people. Strains that are most useful are the group A) or A3)
strains mentioned above. Details or advantages of the present
invention can be found in the non limitative examples below.
EXAMPLES
Example 1
Screening of Probiotics in a Co-Culture Model Involving Epithelial
Cells and Enteric Neuronal Cells
Cell Culture
[0177] Pregnant Sprague-Dawley rats were purchased (CERJ, Le Genest
St Isle, France and Janvier-Breeding Center, Belgium) and killed by
an overdose of CO.sub.2 followed by severing the carotid arteries.
The embryos (35-45 per isolation from 3 pregnant rats) were removed
and killed by decapitation. The small intestines of embryos were
removed and finely diced in HBSS (Sigma, France). Tissue fragments
were collected in 5 mil of medium (DMEM-F.sub.12 1:1 medium) and
digested at 37.degree. C. for 15 min in 0.1% trypsin (Sigma). The
trypsin reaction was stopped by adding 10 ml of medium containing
10% foetal calf serum and then treated by DNAse 1 (0.01%, Sigma)
for 10 min at 37.degree. C. After triturating with a 10 ml pipette,
cells were centrifuged at 750 r.p.m. for 10 min. Cells were counted
and then seeded at a density of 2.4.times.10.sup.5 cells/cm.sup.2
on 24 well plates previously coated for 6 h with a solution of
gelatine (0.5%, Sigma) in sterile phosphate buffered saline (PBS).
After 24 h, the medium was replaced with a serum free medium
(DMEM-F12 1:1 containing 1% of N-2 supplement (Life technologies,
France). Cells were maintained in culture for 14 days to obtain
primary culture of enteric nervous system (ENS). Half of the medium
was replaced every other day. At 14 days the primary neuronal cells
were ready for the establishment of the co-culture model.
[0178] T84 cell line (EATCC) was cultured in DMEM-F12 (1:1, GIBCO)
supplemented with 10% heat inactivated FBS and 50 IU/ml penicillin
and 50 .mu.g/ml streptomycin. Cells were seeded in 12-well
Transwell.RTM. filters (Corning, N.Y. USA) at a density of
2.times.10.sup.5 cells/insert and cultured to obtain
confluence.
[0179] One day after epithelial cells arrived to confluence,
Transwell.RTM. filters were transferred in the 12-well plates
seeded at the bottom with enteric nervous cells. Epithelial and
neuronal cells were co-cultured in the medium for epithelial
cells.
Growing of Strains of Bacteria
[0180] Bacteria were grown for 16 hrs in TGYH for bifidobacteria
and lactobacilli, except for strain DN.sub.--173.sub.--010 (CNCM
I-2494 filed Jun. 20, 2000) which was grown on MRS+cysteine medium,
washed in PBS twice and adjusted to 410.sup.8 cfu/ml in order to
add consistently the same volume of bacterial suspension to the
filter. The strains were added in the filter compartment at a MOI
of 40 bacteria/epithelial cell. As a control, no bacteria were
added.
[0181] After 8 hrs of co-incubation, the filter compartment
containing epithelial cells and bacteria was removed and primary
neuronal cells were incubated for 24 h in a humidified incubator
containing 5% CO.sub.2. In the control wells neuronal cells where
stimulated with 1 mM butyrate and 40 mM KCl when ChAT and VIP
measurements were performed, respectively.
Immunohistochemical Staining. Measuring ChAT Nerves
[0182] After the incubation, immunohistochemistry was performed to
detect neuronal cell populations. After cells fixation (in 0.1 M
PBS containing 4% paraformaldehyde for 1 h at room temperature),
cells were washed 3 times in PBS, then permeabilized for 30 min in
PBS/NaN.sub.3 containing 0.5% Triton X-100 and 4% horse serum.
Primary antibody: rabbit anti-neurone specific enolase (NSE)
(1:2000; Biovalley, France) and rabbit anti-choline acetyl
transferase was diluted in PBS/NaN.sub.3, 0.5% Triton X-100 and 4%
horse serum and incubated overnight at room temperature. After
incubation with primary antiserum, cells were washed 3 times with
PBS and incubated for 3 h with donkey anti-rabbit IgG conjugated to
fluoresceine isothiocyanate (FITC) (1:200 Immunotech, France) and
7-amino-4-methyl-coumarin-3-acetate respectively. Specimens were
viewed under an Olympus IXSO fluorescence microscope fitted with
white video camera (Mod. 4910, Cohu Inc, Germany) connected to
macintosh computer through a frame grabber card (Scion Image, SL
Microtest).
VIP Measurements:
[0183] For VIP determination, neuronal cells were collected from
the 12-well plates, the proteins were extracted using RIPA lysis
buffer (Millipore, France) containing protease inhibitor cocktail
(Roche Diagnostics, France) and VIP levels were measured by ELISA
(Bachem, Germany).
Results
[0184] Differential response of primary enteric neurones on VIP and
ChAT markers following interaction of some of 102 probiotic strains
including lactic acid bacteria and Bifidobacteria are shown in
Table 1. Only some strains belonging to group A3) or B3) or C3) are
shown. Additionally it is mentioned that 26 strains were shown to
have no significant effect on VIP and ChAT (including strains
Bifidobacterium longum NCC 2705 (CNCM I-2618), Lactobacillus
rhamnosus GG (ATCC 53103) and Lactobacillus casei Shirota), 11
strains belonging to group C2) were shown to decrease both VIP and
ChAT (including strain Bifidobacterium longum W11 of Alfa-Wass (LMG
P-21586)), 10 strains decreased VIP and had no effect on ChAT
(including bench mark strains Bifidobacterium infantis UCC 3564,
Bifidobacterium longum Bb536, Bifidobacterium animalis spp lactis
Bb12 (DSM 15954), and Bifidobacterium animalis spp lactis Bi-07
(ATCC SD5220) and 41 strains belonging to group C3) decreased ChAT
and had no effect on VIP including strains Lactobacillus johnsonii
La1 (CNCM I-1225), Lactobacillus plantarum 299v (DSM 9843)
Lactobacillus reuteri SD 2122 (ATCC 55730)).
TABLE-US-00001 TABLE 1 Effect of incubation with lactic acid
bacteria and bifidobacteria on VIP and ChAT levels in a coculture
model with epithelial cell monolayer and primary ENS cells. VIP
ChAT Estimated Estimated DN Number difference* p Empiric difference
p Empiric Group species (CNCM number) vs control value mean vs
control value Mean 1 DN_154_0067 (CNCM I- -0.0097 0.9 0.0790 0.2709
0.09 0.1796 4320 filed May 19 2010) Bifidobacterium bifidum 1
DN_116_0047 (CNCM I- -0.0389 0.7 0.0397 0.3151 0.10 0.2535 4317
filed May 19 2010) Lactobacillus rhamnosus 1 DN_119_118 (CNCM I-
-0.1329 0.09 -0.2221 0.2847 0.02 0.2796 4279 filed Feb. 25 2010)
Lactobacillus acidophillus 2 DN_173_010 (CNCM I- 0.2345 0.01 0.2001
-0.2615 0.05 -0.0825 2494 filed Jun. 20 2000) Bifidobacterium
lactis 2 DN_156_0032 (CNCM I- 0.2248 0.01 0.2020 -0.5450 0.00
-0.5723 4321 filed May 19 2010) Bifidobacterium breve 2 DN_156_007
(CNCM I- 0.2715 0.02 0.3552 -0.3632 0.01 -0.1281 2219 filed May 31
1999) Bifidobacterium breve 2 DN_121_0304 (CNCM I- 0.5976 0.00
0.6813 -0.6269 0.00 -0.3918 4318 filed May 19 2010) Lactobacillus
plantarum *Values are given as a difference compared to the
control, where no bacterial strains were added.
[0185] Although strain DN.sub.--119.sub.--0118 (CNCM I-4279 filed
Feb. 25, 2010) decreases VIP levels, it turned out with a TEER
model (Hirotani et al, 2008, Yakugaku Zasshi September;
128(9):1363-8) that incubation with the strain for 4 or 6 h did not
significantly reduce TEER values, even in presence of damage vs.
control. In short, bacteria were cultured in TGYH. The culture
suspensions were washed with PBS. Subsequently, the bacteria (100
cfu/cell) were added to the apical side of the T84 cell monolayers.
After 2 h incubation, LPS (L4516,--EPEC--0127: B8) was added on the
apical side at 40 ng/ml or not added. Then, after 2 h and 4 h
incubation, the TEER value was measured to assess epithelial
barrier function. All experiments were performed three times
independently and in triplicate in presence and in absence of LPS.
The value of the T84 at t=0 was set at 100%. In the absence of LPS
TEER at T4 was 98.7% and at T6 100.2% with strain
DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010); for T84
alone this was still 100%. In the presence of LPS the control T84
at T4 was 56.2% compared to t=0 and with strain
DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010) 47.9%; At
T6 the T84 control was 46.7% and with strain
DN.sub.--119.sub.--0118 (CNCM I-4279 filed Feb. 25, 2010)
52.2%.
[0186] Using this same TEER model especially strain
DN.sub.--173.sub.--010 (CNCM I-2494 filed Jun. 20, 2000),
DN.sub.--156.sub.--007 (CNCM I-2219 filed May 31, 1999),
DN.sub.--121.sub.--0304 (CNCM I-4318 filed May 19, 2010) and
DN.sub.--156.sub.--0032 (CNCM I-4321 filed May 19, 2010), all
belonging to group A3), showed good results on the intestinal
barrier function as assessed by TEER in presence of LPS. See Table
2.
TABLE-US-00002 TABLE 2 TEER results in presence of LPS of selected
bacteria showing the best results TEER T4/ TEER T6/ TEER T0 (%)
TEER T0 (%) Signif- Empiric Signif- Empiric Strain icance mean
icance mean T84 control 56.20 46.76 DN_173_010 B. lactis *** 71.27
*** 51.03 (CNCM I-2494 filed Jun. 20 2000) DN_156_007 B. breve ***
70.70 *** 55.87 (CNCM I-2219 filed May 31 1999) DN_121_0304 L.
plantarum *** 65.84 *** 64.67 (CNCM I-4318 filed May 19 2010)
DN_156_0032 B. breve *** 84.44 *** 80.38 (CNCM I-432 filled May 19
2010) ***p value < 0.05
* * * * *