U.S. patent application number 12/267602 was filed with the patent office on 2009-05-14 for immunomodulating compounds and related compositions and methods.
Invention is credited to DENNIS L. KASPER, SARKIS MAZMANIAN, RYAN MICHAEL O'CONNELL, JUNE L. ROUND.
Application Number | 20090124573 12/267602 |
Document ID | / |
Family ID | 40624345 |
Filed Date | 2009-05-14 |
United States Patent
Application |
20090124573 |
Kind Code |
A1 |
MAZMANIAN; SARKIS ; et
al. |
May 14, 2009 |
IMMUNOMODULATING COMPOUNDS AND RELATED COMPOSITIONS AND METHODS
Abstract
Provided herein are compounds, compositions and methods for
balancing a T-helper cell profile and in particular Th1, Th2, Th17
and Treg cell profiles, and related methods and compositions for
treating or preventing an inflammatory condition associated with an
imbalance of a T-helper cell profile.
Inventors: |
MAZMANIAN; SARKIS;
(PASADENA, CA) ; ROUND; JUNE L.; (PASADENA,
CA) ; O'CONNELL; RYAN MICHAEL; (PASADENA, CA)
; KASPER; DENNIS L.; (Brookline, MA) |
Correspondence
Address: |
Steinfl & Bruno
301 N Lake Ave Ste 810
Pasadena
CA
91101
US
|
Family ID: |
40624345 |
Appl. No.: |
12/267602 |
Filed: |
November 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61002705 |
Nov 9, 2007 |
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61008407 |
Dec 20, 2007 |
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61196046 |
Oct 14, 2008 |
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61P 37/02 20180101;
A61P 29/00 20180101; A61K 31/715 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/54 |
International
Class: |
A61K 31/715 20060101
A61K031/715; A61P 29/00 20060101 A61P029/00 |
Goverment Interests
STATEMENT OF GOVERNMENT GRANT
[0002] The U.S. Government has certain rights in this invention
pursuant to Grant No. AI039576 awarded by the National Institutes
of Health.
Claims
1. A method to balance a T-helper cell profile in an individual,
the method comprising administering to the individual an effective
amount of a zwitterionic polysaccharide.
2. The method of claim 1, wherein the T-helper cell is a subset of
T-helper cells, the subset consisting of at least one of Th1, Th2,
Th17, and Treg.
3. The method of claim 1, wherein the T-helper cell is a subset of
T-helper cells, the subset consisting of Th17 and at least one of
Th1, Th2, and Treg.
4. The method of claim 1, wherein the T-helper cell is Th17.
5. The method of claim 1, wherein the zwitterionic polysaccharide
is a naturally occurring bacterial capsular polysaccharide.
6. The method of claim 1, wherein the zwitterionic polysaccharide
is a B fragilis capsular polysaccharide A (PSA) or polysaccharide B
(PSB).
7. The method of claim 1, wherein the effective amount is in a
range from about 1 .mu.g to about 100 .mu.g of zwitterionic
polysaccharide per 0.025 kilograms of body weight.
8. The method of claim 1, wherein the effective amount is in a
range from about 0.001 .mu.g to about 1,000 .mu.g per 0.25
kilograms of body weight.
9. A method to control an inflammation associated with an imbalance
of a T-helper cell profile in an individual, the method comprising
administering to the individual an effective amount of a
zwitterionic polysaccharide.
10. The method of claim 9, wherein the T-helper cell is Th17.
11. The method of claim 9, wherein the zwitterionic polysaccharide
is a B fragilis capsular polysaccharide A (PSA) or polysaccharide B
(PSB).
12. The method of claim 9, wherein the method is a therapeutic
method for treating the cytokine mediated inflammation and the
effective amount of PSA is a therapeutically effective amount of a
zwitterionic polysaccharide.
13. A method to control cytokine production in an individual, the
cytokine being at least one of IL-1, IL-6, TNF-a, IL-17, IL21,
IL23, the method comprising administering to the individual an
effective amount of a zwitterionic polysaccharide.
14. The method of claim 13, wherein the cytokine is Il-17.
15. The method of claim 13, wherein the zwitterionic polysaccharide
is a B fragilis capsular polysaccharide A (PSA).
16. A method to control an inflammation associated with production
of at least one of IL-1, IL-6, TNF-a, IL-17, IL21, IL23 in an
individual, the method comprising administering to the individual
an effective amount of a zwitterionic polysaccharide.
17. The method of claim 16, wherein the method is a therapeutic
method for treating the inflammation and the effective amount of
zwitterionic polysaccharide is a therapeutically effective amount
of zwitterionic polysaccharide.
18. An anti-inflammatory composition comprising a zwitterionic
polysaccharide and a suitable vehicle, wherein the zwitterionic
polysaccharide is comprised in an amount of from about 1 .mu.g to
about 100 .mu.g.
19. The anti-inflammatory composition of claim 18, wherein the
zwitterionic polysaccharide is comprised in an amount of from about
0.01 .mu.g to about 1,000 .mu.g.
20. The anti-inflammatory composition of claim 18, wherein the
composition is a pharmaceutical composition and wherein the
suitable vehicle is a pharmaceutically acceptable vehicle.
21. The anti-inflammatory composition of claim 18, wherein the
zwitterionic polysaccharide is a B fragilis capsular polysaccharide
A (PSA) or polysaccharide B (PSB).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application entitled "Host-Bacterial Mutualism by a Microbial
Symbiosis Factor Prevents Inflammatory Disease" Ser. No.
61/002,705, filed on Nov. 9, 2007 Docket No. CIT5031-P, U.S.
Provisional Application entitled "Host-Bacterial Mutualism by a
Microbial Symbiosis Factor Prevents Inflammatory Disease" Ser. No.
61/008,407, filed on Dec. 20, 2007 Docket No. CIT5031-P2, and to
U.S. Provisional Application entitled "A Molecule from a Symbiotic
Gut Bacteria Controls Systemic Inflammation", Ser. No. 61/196,046,
filed on Oct. 13, 2008 Docket No. CIT5250-P, the disclosure of each
of which is incorporated herein by reference in its entirety.
FIELD
[0003] The present disclosure relates to the immune system, and, in
particular, to an immunomodulating compound able to control T cell
differentiation and/or cytokines production associated with an
immunitary response in an individual.
BACKGROUND
[0004] T cells belong to a group of white blood cells known as
lymphocytes, and play a central role in cell-mediated immunity. In
particular, T helper cells (also known as effector T cells or Th
cells) are a sub-group of lymphocytes (a type of white blood cell
or leukocyte) that plays an important role in establishing and
maximizing the capabilities of the immune system and in particular
in activating and directing other immune cells. More particularly,
Th cells are essential in determining B cell antibody class
switching, in the activation and growth of cytotoxic T cells, and
in maximizing bactericidal activity of phagocytes such as
macrophages.
[0005] Different types of Th cells have been identified that
originate in outcome of a differentiation process and are
associated with a specific phenotype. Following T cell development,
matured, naive (meaning they have never been exposed to the antigen
to which they can respond) T cells leave the thymus and begin to
spread throughout the body. Once the naive T cells encounter
antigens throughout the body, they can differentiate into a
T-helper 1 (Th1), T-helper 2 (Th2), T-helper 17 (Th17) or
regulatory T cell (Treg) phenotype.
[0006] Each of these Th cell types secretes cytokines, proteins or
peptides that stimulate or interact with other leukocytes,
including T.sub.h cells. However, each cell type has a peculiar
phenotype and activity that interferes and often conflict with the
other.
[0007] Th1, Th2, and Th17 (inflammatory T-helper or inflammatory
Th), promote inflammation responses trough secretion of
pro-inflammatory cytokines, such as IL-1, IL-6, TNF-a, IL-17, IL21,
IL23, and/or through activation and/or inhibition of other T cell
including other Th cells (for example Th1 cell suppresses Th2 and
Th17, Th2 suppresses Th1 and Th17). Tregs instead, are a component
of the immune system that suppresses biological activities of other
cells associated to an immune response. In particular, Tregs can
secrete immunosuppressive cytokines TGF-beta and Interleukin 10,
and are known to be able to limit or suppress inflammation.
[0008] An imbalance in the profile of any of the inflammatory
T-helper cells is usually associated with a condition in an
individual. For example, an increase profile for Th1 or Th17 leads
to autoimmunity, whereas an increased Th2 cell profile leads to
allergies and asthma. In particular, imbalance of Th17 cell profile
has been associated with several autoimmunitary conditions. Treg
cells suppress inflammation induced by all 3 other T cell lineages,
and thus are crucial for preventing uncontrolled inflammation,
which leads to disease. Therefore, a balanced T-helper profile is
critical for health in individuals.
SUMMARY
[0009] Provided herein, are immunomodulating compounds and related
methods and compositions that are suitable to balance a T-helper
cell profile, and in particular to balance the cell profile of at
least one of Th1, Th2, Th17 and Treg cells in an individual. More
particularly, provided herein are methods and compositions based on
the surprising immunomodulating properties of PSA polysaccharide A
(PSA) and other zwitterionic polysaccharides (ZPs) that make those
polysaccharides suitable for treatment, prevention and control of
inflammations and inflammatory conditions in an individual.
[0010] According to a first aspect, a method to balance a T-helper
cell profile in an individual is disclosed. The method comprises
administering to the individual an effective amount of a
zwitterionic polysaccharide.
[0011] According to a second aspect, a method to balance a cell
profile of at least one Th cell selected from the group consisting
of Th1, Th2, Th17 and Treg, in an individual is disclosed. The
method comprises administering to the individual an effective
amount of a zwitterionic polysaccharide.
[0012] According to a third aspect, a method to control cytokine
production in an individual, is disclosed, the cytokine being at
least one of IL-1, IL-6, TNF-a, IL-17, IL21, IL23. The method
comprises administering to the individual an effective amount of a
zwitterionic polysaccharide.
[0013] According to a fourth aspect, a method to control
inflammation associated with a Th-cell profile imbalance in an
individual is disclosed. The method comprises administering to the
individual an effective amount of a zwitterionic
polysaccharide.
[0014] According to a fifth aspect, a method to treat or prevent
conditions associated with an imbalanced cell profile of at least
one Th cell selected from the group consisting of Th1, Th2, Th17
and Treg in an individual is disclosed. The method comprises
administering to the individual an effective amount of a
zwitterionic polysaccharide.
[0015] According to a sixth aspect, a method to treat or prevent
conditions associated with production of at least one of IL-1,
IL-6, TNF-a, IL-17, IL21, IL23 cytokines in an individual, is
disclosed. The method comprises administering to the individual an
effective amount of a zwitterionic polysaccharide.
[0016] According to a seventh aspect, an anti-inflammatory
composition is disclosed. The anti-inflammatory composition
comprises a zwitterionic polysaccharide and a suitable vehicle,
wherein the zwitterionic polysaccharide is comprised in an amount
of from about 1 to about 100 .mu.g.
[0017] The compositions and methods herein disclosed can be used in
several embodiments to simultaneously control and balance the
profile of Th1, Th2, Th17 and Treg cells in an individual, thus
preventing or treating conditions associated with an imbalanced
profile for those cytokines in the individual.
[0018] The compositions and methods herein described can be used in
connection with medical, pharmaceutical, veterinary applications as
well as fundamental biological studies and various applications,
identifiable by a skilled person upon reading of the present
disclosure, wherein investigating the possible role of a
zwitterionic polysaccharide is desirable
[0019] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The accompanying drawings, which are incorporated into and
constitute a part of this specification, illustrate one or more
embodiments of the present disclosure and, together with the
detailed description, serve to explain the principles and
implementations of the disclosure.
[0021] FIG. 1 shows an exemplary ZP mediated protection from
experimental colitis in individuals according to some embodiments
herein disclosed. Panel (a) shows diagrams summarizing the results
of mono-association of germ-free mice with wild-type B. fragilis
and B. fragilis DPSA (mean percentages.+-.Standard Deviation (SD)
for 3 experiments: conventional, 38.4%.+-.2.2; germ-free,
26.7%.+-.1.3; B. fragilis, 40.8%.+-.3.1; B. fragilis DPSA,
28.8%.+-.2.6). All cells gated on CD4.sup.+ splenocytes. Panel (b)
shows a diagram illustrating the results of co-colonization
experiments of H. hepaticus with B. fragilis and B. fragilis DPSA
(two-tailed p value, 0.004; Mann-Whitney U test). Combined data
from 2 independent experiments are shown. Error bars show SD for
triplicate samples. Panel (c) shows a diagram illustrating the
results of ELISA test of colon organ cultures to detect TNFa levels
in animals co-colonized with H. hepaticus and wild-type B. fragilis
or B. fragilis DPSA. Panel (d) shows a diagram illustrating the
results of a Q-PCR for IL-23p19 performed on splenocytes,
normalized to L32 expression. Error bars show SD for triplicate
samples.
[0022] FIG. 2 shows an exemplary ZP mediated cytokine control
according to some embodiments herein disclosed. In particular, FIG.
2 shows diagrams illustrating the results of ELISA tests for the
detection of the pro-inflammatory cytokines IL-12p40 (left) and
IL-1b (right) in animals co-colonized with H. hepaticus and
wild-type B. fragilis or B. fragilis DPSA over those in control
animals (C57BL/6). Results are from one trial of 2 independent
experiments. Error bars indicate SD values from studies of colons
recovered from 4 animals per group.
[0023] FIG. 3 shows an example of ZP mediated control of TNFa
expression by CD4.sup.+ T cells according to some embodiments
herein disclosed. In particular FIG. 3 CD4.sup.+ cells were
purified from pooled splenocytes from each group (4 mice per group)
and restimulated in vitro with PMA and ionomycin in the presence of
brefeldin A for 4 hours. Cells were stained for intracellular
TNF.alpha.. Cells within the lymphocyte gate were included in the
analysis, and numbers indicate the percentage of cells producing
TNF.alpha.. Purified cells were >90% CD4.sup.+. Animals
colonized with PSA-producing B. fragilis during protection
displayed lower TNFa levels than diseased animals.
[0024] FIG. 4 shows control experiments supporting various
embodiments herein described. Panel (a) shows an ethidium
bromide-stained gel electrophoresis of H. hepaticus-specific Q-PCR
performed following co-colonization with wild-type and various
mutants of B. fragilis after 8 weeks. M: Marker. 1: Rag2.sup.-/-
animals with CD4.sup.+ CD45Rb.sup.high T cell transfer colonized
with H. hepaticus alone. 2: Rag2.sup.-/- animals with CD4.sup.+
CD45Rb.sup.high T cell transfer colonized with H. hepaticus and B.
fragilis 9343 (wt). 3: Rag2.sup.-/- animals with CD4.sup.+
CD45Rb.sup.high T cell transfer colonized with H. hepaticus and B.
fragilis DPSA. 4: C57BL/6 mice colonized with H. hepaticus alone.
Note: H. hepaticus readily colonized animals but did not induce
disease (FIG. 1). Primers for H. hepaticus 16S rDNA: (HB-15)
5'-GAAACTGTTACTCTG-3' (SEQ ID NO: 1) and (HB-17)
5'-TCAAGCTCCCCGAAGGG-3' (SEQ ID NO: 2). Panel (b) shows ethidium
bromide-stained gel electrophoresis of B. fragilis-specific Q-PCR
performed following co-colonization with wild-type and various
mutants of B. fragilis after 8 weeks. A: Rag2.sup.-/- animals with
CD4.sup.+ CD45Rb.sup.high T cell transfer colonized with H.
hepaticus and B. fragilis 9343 (wt). B: Rag2.sup.-/- animals with
CD4.sup.+ CD45Rb.sup.high T cell transfer colonized with H.
hepaticus and B. fragilis DPSA. C: Rag2.sup.-/- animals with
CD4.sup.+ CD45Rb.sup.high T cell transfer colonized with H.
hepaticus alone. D: C57BL/6 mice colonized with H. hepaticus alone.
E: B. fragilis genomic DNA (positive control). M: Marker. Primers
for B. fragilis ssr3 (finB) gene: (ssr3-F) 5'-TATTTGCGAGAAGGTGAT-3'
(SEQ ID NO: 3) and (ssr3-r) 5'-TAAACGCTTTGCTGCTAT-3' (SEQ ID NO:
4).
[0025] FIG. 5 effects associated to a ZP-mediated protection
according to some embodiments herein disclosed. In particular, FIG.
5 shows a diagram illustrating the results of Q-PCR experiments
directed to quantitate H. hepaticus in animals co-colonized with H.
hepaticus and wild-type B. fragilis or B. fragilis DPSA. The
results was assessed according to Young et al., 2004.sup.1 as
log.sup.10 number of copies of a known gene (cytolethal distending
toxin). Animals contained equivalent levels of H. hepaticus at the
end of the experiment.
[0026] FIG. 6 shows a ZPs mediated protection according to some
embodiments herein disclosed. Panel (a) shows a diagram
illustrating the results of a colonization with H. hepaticus in
absence (second column) or in presence of purified PSA (third
column) (Kruskal-Wallis comparisons of all groups: p>0.05 for
dissimilar results, p<0.01 for similar results; Mann-Whitney U
test: two-tailed p value, 0.0002). Panel (b) shows a diagram
illustrating results of experiments directed to detect wasting
disease in Rag2.sup.-/- animals following transfer of CD4.sup.+
CD45Rb.sup.high T cells and colonization with H. hepaticus (PBS+Hh)
in presence or absence of PSA as indicated. ANOVA indicates that
comparisons between all indicated groups (asterisks) are
statistically significant. Panel (c) shows the architecture of
colonic sections from wild-type animals (left panel); following
transfer of CD4.sup.+ CD45Rb.sup.high T cell into
Helicobacter-colonized Rag2.sup.-/- mice (middle panel); oral PSA
treatment of Helicobacter-colonized animals (right panel). Images
in each row are the same magnification.
[0027] FIG. 7 shows a ZP modulated immune response according to
some embodiments herein disclosed. Panel (a) shows a diagram
illustrating the correlation between oral PSA administration and
body weight related to TNBS-treated PBS controls. ANOVA values for
all indicated groups (asterisks) are statistically significant.
Error bars show SD between 4 animals per group. Panel (b) shows
colon sections from TNBS+PBS-treated groups, from TNBS+PSA-treated
animals and from a control (representative sections from animals in
2 independent experiments). Panels (c, d) show diagram illustrating
the results of Q-PCR of purified CD4.sup.+ T cells from MLNs with
IL-17A (Panel c) and TNFa (Panel d) in presence or absence of PSA
during disease. Error bars are from duplicate runs of 3 independent
experiments. Panels (e, f) show diagrams illustrating
transcriptional expression of IL17A (Panel e) and TNF.alpha. (Panel
f) from homogenized colons of TNBS+PBS-treated groups, from
TNBS+PSA-treated animals and from a control. Error bars are from
duplicate runs of 3 independent experiments.
[0028] FIG. 8 shows a ZP mediated control of cytokine expression
according to some embodiments herein disclosed. Panel (a) shows a
diagram illustrating the results of Q-PCR assay of colons for IL-10
in wild type mice treated with ethanol (control), TNBS, or TNBS and
PSA. Error bars show SD for triplicate samples. Panel (b) shows a
diagram illustrating Q-PCR results for IL-10 expression in
CD4.sup.+ T cells purified from MLNs of TNBS-treated groups. Error
bars show SD for triplicate samples. Panel (c) shows a diagram
illustrating the effects of incubation of BMDC/T cell co-cultures
with purified PSA LPS and a-CD3/a-CD28 on IL-10 production. Error
bars show SD for triplicate samples. Panel (d) shows a diagram
illustrating the results of an infection of BMDC-T cell co-cultures
with increasing concentrations of H. hepaticus (multiplicity of
infection: 0.1, 1.0, and 10, as depicted by triangles) on TNFa
release in presence (middle three bars) or absence (left three
bars) of PSA and following the addition of aIL-10R right three
bars. Error bars show SD values of experiments run in
triplicate.
[0029] FIG. 9 shows a ZP mediated control of cytokine expression
according to some embodiments herein disclosed. In particular, FIG.
9 shows a diagram illustrating the results for an IL-10 ELISA of
supernatants of primary BMDC-T cell co-cultures incubated for 48
hours with H. hepaticus alone or with H. hepaticus and B. fragilis
(wild-type or .DELTA.PSA) at a multiplicity of infection of 5.
Error bars show SD values for samples run in duplicate and
represent 3 independent experiments.
[0030] FIG. 10 shows a ZP mediated control of cytokine expression
according to some embodiments herein disclosed. In particular, FIG.
10 shows a diagram illustrating the results of an infection of
BMDC-T cell co-cultures with increasing concentrations of live H.
hepaticus (multiplicity of infection: 0.1, 1.0, and 10, as depicted
by triangles) on release of the cytokine IL-1b in presence (middle
three bars) or absence (left three bars) of PSA and following the
addition of aIL-10R right three bars. Error bars show SD values for
experiments run in triplicate.
[0031] FIG. 11 shows a ZP mediated protection from inflammation
according to some embodiments herein disclosed. Panels (a, b) show
diagrams illustrating results of ELISA detection for
pro-inflammatory cytokines TNF.alpha. (Panel a) and IL-17A (Panel
b) in IL-10.sup.-/- mice left uncolonized (control) or colonized
with H. hepaticus (to induce inflammation) either alone or in
combination with B. fragilis (wild-type or .DELTA.PSA). Error bars
show SD for triplicate samples. Panel (c) shows a diagram
illustrating the colitis scores in Rag.sup.-/- animals with
CD4.sup.+ CD45Rb.sup.high T cell transfer colonized with H.
hepaticus with or without PSA and in presence of neutralizing
antibodies to IL-10 block (.alpha.-IL10R). Data represent 2
independent experiments. Panel (d) shows a diagram illustrating
colitis scores in Rag.sup.-/- animals with CD4.sup.+
CD45Rb.sup.high T cell transferred from IL-10.sup.-/- mice
colonized with H. hepaticus with PSA or PBS. Results are shown for
1 representative trial of 2 independent experiments. Panel (e)
shows histologic colon sections Rag.sup.-/- animals with CD4.sup.+
CD45Rb.sup.high T cell transferred from IL-10.sup.-/- mice
colonized with H. hepaticus with PSA or PBS. All images are the
same magnification. Panel (f) shows a diagram illustrating the mean
body weights for groups of Rag.sup.-/- animals (n=4) with CD4.sup.+
CD45Rb.sup.high T cell transferred from IL-10.sup.-/- mice
colonized with H. hepaticus with PSA or PBS.
[0032] FIG. 12 shows effect of a ZP administration supporting
embodiments herein disclosed. In particular, FIG. 12 shows a
diagram illustrating the variation on body weight in groups of 4
C57BL/6 mice treated with PSA (or PBS) and then subjected to rectal
administration of TNBS or vehicle (control). Mean body weights
(shown as percentages of initial weight) are shown for each group;
SD values indicate that, in the absence of IL-10, PSA cannot
restore TNBS-induced weight loss. ANOVA demonstrates that weight
loss in both TNBS-treated groups is statistically different from
that in control animals.
[0033] FIG. 13 shows effects of a ZP administration supporting some
embodiments herein disclosed. In particular, FIG. 13 shows results
of histologic analysis of H&E-stained sections from a
representative animal of groups of 4 C57BL/6 mice treated with PSA
(or PBS) and then subjected to rectal administration of TNBS or
vehicle (control). Results represent 2 independent experiments.
[0034] FIG. 14 shows inhibition of inflammation within
extra-intestinal immune compartments following oral administration
of ZPS according to some embodiments herein disclosed. In
particular, Panel (a) shows a diagram illustrating the colonic
histological score detected in untreated mice (control) and in mice
treated with TNBS or TNBS/PSA. Each dot represents an individual
mouse and the line indicates the average score of the group. Panel
(b) shows a diagram illustrating the percent of survival in time of
Balb/c mice undergoing TNBS induced colitis. n=16 mice in each
group. Panel (c) shows an image of the spleen of untreated mice
(control) and mice treated with TNBS or TNBS/PSA Panel (d) shows a
diagram illustrating the relative units of TNF-.alpha., IL-6,
IL-17A and IL-10 within CD4.sup.+ splenocytes in untreated mice
(control) and in mice treated with TNBS or TNBS/PSA. These data are
representative of three independent experiments.
[0035] FIG. 15 shows protection from TNBS induced intestinal
colitis following administration of ZPS to extra-intestinal sites
according to some embodiments herein disclosed. In particular,
Panel (a) shows a diagram illustrating the percent survival of mice
undergoing TNBS induced colitis. n=10 mice in each group. Panel (b)
shows a diagram illustrating variation of the spleen weight in
untreated mice (Etoh) and in mice treated with TNBS or TNBS/PSA
systemically administered. The weight of the spleen was used as an
indicator of size. Each diamond represents the weight of the spleen
from an individual animal. The bar indicates the average weight of
the group. P values were determined by students T test.
[0036] FIG. 16 shows inhibition of inflammatory cytokines at both
intestinal and systemic immune compartments following systemic
administration of ZPS during TNBS induced colitis according to some
embodiments herein disclosed. Panel (a) shows a diagram
illustrating TNF-.alpha. production in CD4.sup.+ T lymphocytes
residing within the mesenteric lymph nodes (MLN) splenocytes in
untreated mice (control) and in mice treated with TNBS or TNBS/PSA.
Cells were collected from the MLN and stained with antibodies
recognizing CD4 or TNF-a. Numbers within quadrants represent the
percentage of cells. Panel (b) shows a diagram illustrating
analysis of the expression of IL-12, IL-23, and IL-17 in colon of
untreated mice (control) and mice treated with TNBS or TNBS/PSA.
Panel (c) show a diagram illustrating TNF-.alpha. production in
CD4.sup.+ T lymphocytes residing within the spleen of untreated
mice (control) and of mice treated with TNBS or TNBS/PSA. Numbers
within quadrants represent the percentage of cells. Panel (d) a
diagram illustrating analysis of the expression of IL-12, IL-6, and
IL-17 in spleen of untreated mice (control) and mice treated with
TNBS or TNBS/PSA.
[0037] FIG. 17 shows inhibition of inflammation and death
associated with systemic septic shock following administration of
ZPS according to some embodiments herein disclosed. Panel (a):
shows a diagram illustrating TNF-a serum levels in mice 1 and 4
hours post-administration of 100 .mu.g of LPS alone. Mice were
either pre-treated with PBS or 50 ug of PSA three times every other
day before LPS administration. * indicates statistical significance
as determined by a students t test. SD was determined from the
serum of individual mice. These data are representative of three
independent experiments. Panel (b): shows a diagram illustrating
IL-6 serum levels in mice 1 and 4 hours post-administration of 100
.mu.g of LPS. Pre-treatment as in panel a * indicates statistical
significance as determined by a students t test. SD was determined
from the serum of individual mice. These data are representative of
three independent experiments. Panel (c): shows a diagram
illustrating variation of the spleen weight in untreated mice (con)
and in mice administered LPS within the intraperitoneal cavity
(LPS) and pre-treated with PBS or PSA as in panel a. Each dot
represents the weight of the spleen from an individual mouse. P
values were determined by a students T test. Panel (d): shows a
diagram illustrating the survival rate of animals undergoing septic
shock induced by high dose (500 .mu.g) administration of LPS and
pre-treated with PSA or PBS. N=12 mice in each group. Panel (e):
shows a diagram illustrating the serum concentrations of TNF-a in
mice post-administration of 500 .mu.g of LPS alone or pre-treated
with PSA or PBS. p values were determined by students T test. Each
dot represents an individual mouse. Panel (f) shows a diagram
illustrating the serum concentrations of IL-6 in mice
post-administration of 500 g of LPS alone and pre-treated with PSA
or PBS. p values were determined by students T test. Each dot
represents an individual mouse.
[0038] FIG. 18 shows inhibition of inflammation and death
associated with systemic septic shock following administration of
ZPS according to some embodiments herein disclosed. Panel (a):
shows a diagram illustrating TNF-a serum levels in mice pre-treated
with PBS or PSA and administered LPS. Serum was collected 1 and 4
hours post-administration of LPS in IL-10.sup.-/- mice. * indicates
statistical significance as determined by a students t test. SD was
determined from the serum of individual mice. Panel (b): shows a
diagram illustrating IL-6 serum level in mice pre-treated with PSA
or PBS. Serum was collected 1 and 4 hours post-administration of
LPS in IL-10.sup.-/- mice. * indicates statistical significance as
determined by a students t test. SD was determined from the serum
of individual mice Panel (c): shows a diagram illustrating percent
survival in mice post-administration of LPS alone or together with
PSA in IL-10.sup.31/ - mice. N=8 mice in each group.
[0039] FIG. 19 shows a diagram illustrating additional effects of
ZPS administration on inflamed tissues according to some
embodiments herein disclosed.
DETAILED DESCRIPTION
[0040] Methods and compositions are herein disclosed that allow
balancing a T-helper cell profile in an individual, based on the
use of PSA or another zwitterionic polysaccharide.
[0041] The term "T-helper" as used herein with reference to cells
indicates a sub-group of lymphocytes (a type of white blood cell or
leukocyte) including different cell types identifiable by a skilled
person. In particular, T-helper cell according to the present
disclosure include effector T.sub.h cells (such as Th1, Th2 and
Th17)-i.e. Th cells that secrete cytokines, proteins or peptides
that stimulate or interact with other leukocytes, including T.sub.h
cells--and suppressor Th cells (such as Treg) i.e. Th cells that
suppress activation of the immune system and thereby maintain
immune system homeostasis and tolerance to self-antigens. Mature
T.sub.h cells are believed to always express the surface protein
CD4. T cells expressing CD4 are also known as CD4.sup.+ T cells.
CD4.sup.+ T cells are generally treated as having a pre-defined
role as helper T cells within the immune system, although there are
known rare exceptions. For example, there are sub-groups of
suppressor T cells, natural killer T cells, and cytotoxic T cells
that are known to express CD4 (although cytotoxic examples have
been observed in extremely low numbers in specific disease states,
they are usually considered non-existent).
[0042] The term "cell profile as used herein indicates a detectable
set of data portraying the characterizing features of a cell that
distinguish the characterized cell from another. In particular,
when referred to a T helper cell, the wording "cell profile"
indicates a detectable set of data related to a marker cytokine
that is produced by the Th cell and characterizes the Th cell with
respect to another. For example, marker cytokine for Th1 cell is
Interferon-g; marker cytokine for Th2 is IL-4, marker cytokine for
Th7 is 11-17 and marker cytokine for Treg is IL-10. Accordingly,
the wording "Th17 cell profile" as used herein indicates the
detectable set of data, such as presence and amount, related to
production of IL-17 in a certain organ or tissue of the individual
wherein the presence and/or activity of Th1 cell is investigated.
Similar definitions apply to the other Th cell types. On the other
hand, when the wording "cell profile" is referred to a subset of Th
cell including more then one Th cell type, the wording "T-helper
cell profile" indicates a detectable set of data related to each
marker cytokine that is produced by and characterizes each, of the
T-helper cells of the subset.
[0043] The term "balance" as used herein with reference to a "Th
cell profile" as used herein indicates the activity of bringing the
cell profile to a status associated with absence of an inflammatory
response. Similarly the term "balanced Th profile" indicates the Th
cell profile status associated with absence of an inflammatory
response and in particular to the detectable set of data related to
a marker cytokine that is produced by the T helper cell and
characterizes the T helper cell with respect to another in absence
of an inflammatory response. When the term "T-helper cell" profile
refers to a subset of Th cell including more then one Th cell type,
the term "balanced Th profile" refers instead to the relative ratio
between the detectable set of data related to each marker cytokine
that is produced by and characterizes each, of the T-helper cells.
For example, a "balanced Th cell profile" referred to a Th cells
subset comprising Th1, Th2 and Th17 indicates the relative ratio of
data related to Interferon-gamma, IL-4 and IL17 associated with
absence of an inflammatory response.
[0044] The term "zwitterionic polysaccharide" as used herein
indicates synthetic or natural polymers comprising one or more
monosaccharides joined together by glicosidic bonds, and including
at least one positively charged moiety and at least one negatively
charged moiety. Zwitterionic polysaccharides include but are not
limited to polymers of any length, from a mono- or di-saccharide
polymer to polymers including hundreds or thousands of
monosaccharides. In some embodiments, a zwitterionic polysaccharide
can include repeating units wherein each repeating unit includes
from two to ten monosaccharides, a positively charged moiety (e.g.
an free positively charged amino moiety) and a negatively charged
moiety (such as sulfonate, sulfate, phosphate and phosphonate). In
some embodiment ZPs can have a molecular weight comprised between
500 Da and 2,000,000 Da. In some embodiments, the ZPs can have a
molecular weight comprised between 200 and 2500. Exemplary ZPS
include but are not limited to PSA and PSB from Bacteroides
Fragilis, CP5/CD8 from Staphylococcus aureus, and Sp1/CP1 from
Streptococcus pneumonia. Zwitterionic polysaccharides can be
isolated from natural sources, and in particular from bacterial
sources, e.g. by purification. Zwitterionic polysaccharides can
also be produced by chemical or biochemical methods, as well as by
recombinant microorganism technologies all identifiable by a
skilled person. Thus, those methods and technologies will not be
further described herein in detail.
[0045] The wording "polysaccharide A" as used herein indicates a
molecule produced by the PSA locus of Bacteroides Fragilis and
derivatives thereof which include but are not limited to polymers
of the repeating unit
{.fwdarw.3).alpha.-d-AATGalp(1.fwdarw.4)[.beta.-d-Galf(1.fwdarw.3)]-d-Gal-
pNAc(1 3).beta.-d-Galp(1.fwdarw.}, where AATGal is
acetamido-amino-2,4,6-trideoxygalactose, and the galactopyranosyl
residue is modified by a pyruvate substituent spanning O-4 and O-6.
The term "derivative" as used herein with reference to a first
polysaccharide (e.g., PSA), indicates a second polysaccharide that
is structurally related to the first polysaccharide and is
derivable from the first polysaccharide by a modification that
introduces a feature that is not present in the first
polysaccharide while retaining functional properties of the first
polysaccharide. Accordingly, a derivative polysaccharide of PSA,
usually differs from the original polysaccharide by modification of
the repeating units or of the saccharidic component of one or more
of the repeating units that might or might not be associated with
an additional function not present in the original polysaccharide.
A derivative polysaccharide of PSA retains however one or more
functional activities that are herein described in connection with
PSA in association with the anti-inflammatory activity of PSA.
[0046] In some embodiments, the zwitterionic polysaccharide can be
PSA and/or PSB. In some embodiments, the effective amount of ZP and
in particular PSA and/or PSB is from about 1-100 micrograms to
about 25 grams of body weight and the T-helper cell profile is
balanced by balancing at least one of Th1, Th2, Th17 and Treg, in
particular at least one of Th1, Th 2 and Treg and Th17. More
particularly, in some embodiments, balance Th cell profile can be
performed by balancing the Th17 cell profile
[0047] In some embodiments, a ZP can be used to control cytokine
production associated with inflammation in an individual. In
particular, in some embodiments, ZPs can be administered to inhibit
production of pro-inflammatory cytokine molecules such as TNF-a,
IL1 or IL-6, IL21, IL23 and IL17.
[0048] The term "control" as used herein indicates the activity of
affecting and in particular inhibiting a biological reaction or
process, which include but are not limited to biological and in
particular biochemical events occurring in a biological system,
such as an organism (e.g. animal, plant, fungus, or micro-organism)
or a portion thereof (e.g. a cell, a tissue, an organ, an
apparatus).
[0049] The terms "inhibiting" and "inhibit", as used herein
indicate the activity of decreasing the biological reaction or
process. Accordingly, a substance "inhibits" a certain biological
reaction or process if it is capable of decreasing that biological
reaction or process by interfering with said reaction or process.
For example, a substance can inhibit a certain biological reaction
or process by reducing or suppressing the activity of another
substance (e.g. an enzyme) associated to the biological reaction or
process, e.g. by binding, (in some cases specifically), said other
substance. Inhibition of the biological reaction or process can be
detected by detection of an analyte associated with the biological
reaction or process. The term "detect" or "detection" as used
herein indicates the determination of the existence, presence or
fact of an analyte or related signal in a limited portion of space,
including but not limited to a sample, a reaction mixture, a
molecular complex and a substrate. A detection is "quantitative"
when it refers, relates to, or involves the measurement of quantity
or amount of the analyte or related signal (also referred as
quantitation), which includes but is not limited to any analysis
designed to determine the amounts or proportions of the analyte or
related signal. A detection is "qualitative" when it refers,
relates to, or involves identification of a quality or kind of the
analyte or related signal in terms of relative abundance to another
analyte or related signal, which is not quantified.
[0050] The term "cytokine" as used herein indicates a category of
signaling proteins and glycoproteins extensively used in cellular
communication that are produced by a wide variety of hematopoietic
and non-hematopoietic cell types and can have autocrine, paracrine
and endocrine effects, sometimes strongly dependent on the presence
of other chemicals. The cytokine family consists mainly of smaller,
water-soluble proteins and glycoproteins with a mass between 8 and
30 kDa. Cytokines are critical to the development and functioning
of both the innate and adaptive immune response. They are often
secreted by immune cells that have encountered a pathogen, thereby
activating and recruiting further immune cells to increase the
system's response to the pathogen.
[0051] Detection of inhibition of cytokine production can be
performed by methods known to a skilled person including but not
limited to ELISA, Q-PCR and intracellular cytokine staining
detected by FACs and any other methods identifiable by a skilled
person upon reading of the present disclosure.
[0052] In some embodiments, a ZP can be administered to inhibit
production of at least one of TNF-a, IL-6, IL-17, IL-21 and IL-23.
In particular, in some of those embodiments ZP can be administered
systemically and in particular, orally, sub cutaneously, intra
peritoneally, and intravenously. In some embodiments ZP can be
administered in an amount between about 1 and about 100
micrograms/25 grams of body weight.
[0053] Methods and compositions are herein disclosed that allow
control of an inflammation associated with an imbalanced Th cell
profile and or to production of at least one of the
pro-inflammatory cytokines IL-1, IL-6, TNF-a, IL-17, IL21, IL23,
and TGF-.beta. in an individual.
[0054] The term "inflammation" and "inflammatory response as used
herein indicate the complex biological response of vascular tissues
of an individual to harmful stimuli, such as pathogens, damaged
cells, or irritants, and includes secretion of cytokines and more
particularly of pro-inflammatory cytokine, i.e. cytokines which are
produced predominantly by activated immune cells such as microglia
and are involved in the amplification of inflammatory reactions.
Exemplary pro-inflammatory cytokines include but are not limited to
IL-1, IL-6, TNF-a, IL-17, IL21, IL23, and TGF-.beta.. Exemplary
inflammations include acute inflammation and chronic inflammation.
The wording "acute inflammation" as used herein indicates a
short-term process characterized by the classic signs of
inflammation (swelling, redness, pain, heat, and loss of function)
due to the infiltration of the tissues by plasma and leukocytes. An
acute inflammation typically occurs as long as the injurious
stimulus is present and ceases once the stimulus has been removed,
broken down, or walled off by scarring (fibrosis). The wording
"chronic inflammation" as used herein indicates a condition
characterized by concurrent active inflammation, tissue
destruction, and attempts at repair. Chronic inflammation is not
characterized by the classic signs of acute inflammation listed
above. Instead, chronically inflamed tissue is characterized by the
infiltration of mononuclear immune cells (monocytes, macrophages,
lymphocytes, and plasma cells), tissue destruction, and attempts at
healing, which include angiogenesis and fibrosis. An inflammation
can be controlled in the sense of the present disclosure by
affecting and in particular inhibiting anyone of the events that
form the complex biological response associated with an
inflammation in an individual. In particular, in some embodiments,
an inflammation can be controlled by affecting and in particular
inhibiting cytokine production, and more particularly production of
pro-inflammatory cytokines, following administration of a
zwitterionic polysaccharide.
[0055] More particularly, in some embodiments, a ZP can be used to
control an inflammation associated with IL-1, IL-6, TNF-a, IL-17,
IL21, IL23, and/or TGF-.beta. mediated inflammation in an
individual. The wording "cytokine mediated inflammation" as used
herein indicates an inflammation wherein the complex biological
response to a harmful stimulus is controlled by cytokine molecules,
such as pro-inflammatory cytokine molecules (e.g. TNF-a, IL1 and/or
IL-6) and anti-inflammatory cytokine molecules (e.g. IL-10).
Exemplary cytokine mediated inflammation include but are not
limited to conditions mediated by IL-1, IL-6, TNF-.alpha.,
IL-12p35, IL-17A, IL-21, IL-22, IFN-.gamma. and/or IL-23p19.
[0056] In some embodiments, the cytokine is at least one of TNF-a,
IL-17, IL-21, and IL-23 and the cytokine mediated inflammation is a
IBD, asthma, type I diabetes, multiple sclerosis, obesity, type 2
diabetes, hay fever, food allergies, skin allergies, or rheumatoid
arthritis. Reference is also made to Mazmanian et al 2008.sup.43,
in particular the figures and related portion of the paper herein
incorporated by reference in its entirety.
[0057] In some embodiments, the inflammation is a systemic
inflammation. Systemic inflammations include but are not limited to
an inflammatory response in the circulatory system, an inflammatory
response which is not confined in a specific organ, and an
inflammatory response that extends to a plurality (up to all)
tissues and organs in an individual.
[0058] In some embodiments, a ZP can be used to control an
inflammation associated with an imbalance of T-helper cell profile
and in particular to a Th17 cell profile, including but not limited
to rheumatoid arthritis, respiratory diseases, allograft rejection,
systemic lupus erythematosis, tumorgenesis, multiple sclerosis,
systemic sclerosis and chronic inflammatory bowel disease.
[0059] In some embodiments, PSA can be administered systemically to
the individual. The wording "systemic administration" as used
herein indicates a route of administration by which PSA is brought
in contact with the body of the individual, so that the desired
effect is systemic (i.e. non limited to the specific tissue where
the inflammation occurs). Systemic administration includes enteral
and parenteral administration. Enteral administration is a systemic
route of administration where the substance is given via the
digestive tract, and includes but is not limited to oral
administration, administration by gastric feeding tube,
administration by duodenal feeding tube, gastrostomy, enteral
nutrition, and rectal administration. Parenteral administration is
a systemic route of administration where the substance is given by
route other than the digestive tract and includes but is not
limited to intravenous administration, intra-arterial
administration, intramuscular administration, subcutaneous
administration, intradermal, administration, intraperitoneal
administration, and intravesical infusion.
[0060] In some embodiments, administration is performed
intravenously by introducing a liquid formulation including a ZP in
a vein of an individual using intravenous access methods
identifiable by a skilled person, including access through the skin
into a peripheral vein. In some embodiments, administration of a ZP
is performed intraperitoneally, by injecting a ZP in the peritoneum
of an individual, and in particular of animals or humans.
Intraperitoneal administration is generally preferred when large
amounts of blood replacement fluids are needed, or when low blood
pressure or other problems prevent the use of a suitable blood
vessel for intravenous injection. In some embodiments
administration is performed intragastrically, including
administration through a feeding tube. In some embodiments,
administration of a ZP is performed intracranially. In some
embodiments a ZP can be administered topically by applying the ZP
usually included in an appropriate formulation directly where its
action is desired. Topical administration include but is not
limited to epicutaneous administration, inhalational administration
(e.g. in asthma medications), enema, eye drops (E.G. onto the
conjunctiva), ear drops, intranasal route (e.g. decongestant nasal
sprays), and vaginal administration.
[0061] In some embodiments, the inflammation is an inflammation of
in a tissue and in particular in pancreas, lungs, joints, skin,
brains and central nervous system, and eyes. In some embodiments,
PSA is used in a method of treating or preventing a condition
associated with inflammation in an individual. The method comprises
administering to the individual a therapeutically effective amount
of the PSA. The term "individual" as used herein includes a single
biological organism wherein inflammation can occur including but
not limited to animals and in particular higher animals and in
particular vertebrates such as mammals and in particular human
beings.
[0062] The term "condition" as used herein indicates a usually the
physical status of the body of an individual, as a whole or of one
or more of its parts, that does not conform to a physical status of
the individual, as a whole or of one or more of its parts, that is
associated with a state of complete physical, mental and possibly
social well-being. Conditions herein described include but are not
limited disorders and diseases wherein the term "disorder"
indicates a condition of the living individual that is associated
to a functional abnormality of the body or of any of its parts, and
the term "disease" indicates a condition of the living individual
that impairs normal functioning of the body or of any of its parts
and is typically manifested by distinguishing signs and symptoms.
Exemplary conditions include but are not limited to injuries,
disabilities, disorders (including mental and physical disorders),
syndromes, infections, deviant behaviors of the individual and
atypical variations of structure and functions of the body of an
individual or parts thereof.
[0063] The wording "associated to" as used herein with reference to
two items indicates a relation between the two items such that the
occurrence of a first item is accompanied by the occurrence of the
second item, which includes but is not limited to a cause-effect
relation and sign/symptoms-disease relation.
[0064] Conditions associated with an inflammation include but are
not limited to inflammatory bowel disease, including but not
limited to Chron's disease and ulcerative colitis, asthma,
dermatitis, arthritis, myasthenia gravis, Grave's disease,
sclerosis, psoriasis.
[0065] The term "treatment" as used herein indicates any activity
that is part of a medical care for or deals with a condition
medically or surgically.
[0066] The term "prevention" as used herein indicates any activity,
which reduces the burden of mortality or morbidity from a condition
in an individual. This takes place at primary, secondary and
tertiary prevention levels, wherein: a) primary prevention avoids
the development of a disease; b) secondary prevention activities
are aimed at early disease treatment, thereby increasing
opportunities for interventions to prevent progression of the
disease and emergence of symptoms; and c) tertiary prevention
reduces the negative impact of an already established disease by
restoring function and reducing disease-related complications.
[0067] An effective amount and in particular a therapeutically
effective amount of PSA is for example in the range of between
about 1 .mu.g to about 100 .mu.g of PSA per 0.025 kilograms of body
weight. In some embodiments, the effective amount is in a range
from about 001 to about 1,000 .mu.g per 25 grams of body
weight.
[0068] In some embodiments, PSA is comprised in a composition
together with a suitable vehicle. The term "vehicle" as used herein
indicates any of various media acting usually as solvents,
carriers, binders or diluents for PSA comprised in the composition
as an active ingredient.
[0069] In some embodiments, where the composition is to be
administered to an individual the composition can be a
pharmaceutical anti-inflammatory composition, and comprises PSA and
a pharmaceutically acceptable vehicle.
[0070] In some embodiments, PSA can be included in pharmaceutical
compositions together with an excipient or diluent. In particular,
in some embodiments, pharmaceutical compositions are disclosed
which contain PSA, in combination with one or more compatible and
pharmaceutically acceptable vehicle, and in particular with
pharmaceutically acceptable diluents or excipients.
[0071] The term "excipient" as used herein indicates an inactive
substance used as a carrier for the active ingredients of a
medication. Suitable excipients for the pharmaceutical compositions
herein disclosed include any substance that enhances the ability of
the body of an individual to absorb PSA. Suitable excipients also
include any substance that can be used to bulk up formulations with
PSA to allow for convenient and accurate dosage. In addition to
their use in the single-dosage quantity, excipients can be used in
the manufacturing process to aid in the handling of PSA. Depending
on the route of administration, and form of medication, different
excipients may be used. Exemplary excipients include but are not
limited to antiadherents, binders, coatings disintegrants, fillers,
flavors (such as sweeteners) and colors, glidants, lubricants,
preservatives, sorbents.
[0072] The term "diluent" as used herein indicates a diluting agent
which is issued to dilute or carry an active ingredient of a
composition. Suitable diluent include any substance that can
decrease the viscosity of a medicinal preparation.
[0073] In certain embodiments, compositions and, in particular,
pharmaceutical compositions can be formulated for systemic
administration, which includes enteral and parenteral
administration.
[0074] Exemplary compositions for parenteral administration include
but are not limited to sterile aqueous solutions, injectable
solutions or suspensions including PSA. In some embodiments, a
composition for parenteral administration can be prepared at the
time of use by dissolving a powdered composition, previously
prepared in lyophilized form, in a biologically compatible aqueous
liquid (distilled water, physiological solution or other aqueous
solution).
[0075] Exemplary compositions for enteral administration include
but are not limited to a tablet, a capsule, drops, and
suppositories.
[0076] The Examples section of the present disclosure illustrates
examples of the compositions and methods herein described as well
as the studies carried out by applicants in order to investigate
the functional and physical interactions of PSA
[0077] Further advantages and characteristics of the present
disclosure will become more apparent hereinafter from the following
detailed disclosure in the Examples given by way or illustration
only with reference to an experimental section.
EXAMPLES
[0078] The methods and system herein disclosed are further
illustrated in the following examples, which are provided by way of
illustration and are not intended to be limiting.
[0079] In particular, in the following examples, the following
materials and methods were used.
[0080] Bacterial strains and animals. B. fragilis NCTC9343 and H.
hepaticus ATCC51149 were obtained from the American Type Culture
Collection. Conventionally reared SPF mice of strains C57BL/6NTac,
C57BL/6NTac IL-10.sup.-/-, and B6.129S6-Rag2.sup.tm/Fwa N12
(Rag2.sup.-/-) were purchased from Taconic Farms (Germantown, N.Y.)
and screened negative for B. fragilis and H. hepaticus.
Swiss-Webster germ-free (SWGF) mice were purchased from Taconic
Farms. Upon delivery in sterile shipping containers, the mice were
transferred to sterile isolators (Class Biologically Clean,
Madison, Wis.) in our animal facility. Animals were screened weekly
for bacterial, viral, and fungal contamination as previously
described.sup.40. All animals were cared for under established
protocols and the IACUC guidelines of Harvard Medical School and
the California Institute of Technology.
[0081] Model of inflammation: Three models of intestinal
inflammation were used: 1) CD4.sup.+ CD45Rb.sup.high T cells were
purified from the spleens of wild-type or IL-10.sup.-/- donor mice
by flow cytometry and transferred into Rag.sup.-/- (C57B1/6)
recipients as described. 2) TNBS colitis was induced by
pre-sensitization of wild-type (C57B1/6) mice on the skin with a
TNBS/acetone mix. Seven days after sensitization, 2.5% TNBS in
ethanol was administered rectally; mice were sacrificed 3-6 days
later. 3) IL10.sup.-/- mice were colonized (by oral gavage) with H.
hepaticus alone or in combination with wild-type B. fragilis or B.
fragilis .DELTA.PSA.
[0082] Assays and scoring systems: Cytokines from the spleen,
colons, or MLNs were assayed by ELISA, Q-PCR, or flow cytometry.
Colitis was assessed with tissue sections (fixed, paraffin
embedded, sectioned onto a slide, and stained with hematoxylin and
eosin) and was scored by a blinded pathologist (Dr. R. T. Bronson,
Harvard Medical School) according to a standard scoring system: 0,
no thickening of colonic tissues and no inflammation (infiltration
of lymphocytes); 1, mild thickening of tissues but no inflammation;
2, mild thickening of tissues and mild inflammation; 3, severe
thickening and severe inflammation. BMDCs were purified from femurs
of mice after extraction and washing in PBS. Cells were cultured
for 8 days in C--RPMI-10 in the presence of GM-CSF (20 ng/mL;
Biosource, Camarillo, Calif.). CD4.sup.+ T cells were purified by
negative selection over a magnetic column (Miltenyi or R& D
Systems).
[0083] Flow cytometry, fluorescence-activated cell sorting (FACS),
and staining. Lymphocytes were isolated from mouse spleens that
were mechanically disrupted into single-cell preparations. Red
blood cells were lysed, and splenocytes (1.times.10.sup.6) were
incubated with various combinations of antibodies (BD Pharmingen,
San Diego, Calif.) at 2 mg/mL for 30 min at 4.degree. C. Cells were
then washed and either fixed or used directly. For intracellular
cytokine flow cytometry, samples were analyzed on a model FC500
cytometer (Beckman Coulter, Fullerton, Calif.) or a FacsCalibur
(Becton Dickson), and data were analyzed with RXP Analysis Software
(Beckman Coulter) or FlowJO. FACS was performed on a BD FACSAria,
and cell purity was always >99%.
[0084] In vitro cytokine assays. For colon organ cultures,
procedures were followed as previously reported.sup.41. For
co-culture, CD4.sup.+ T cells were purified from splenic
lymphocytes (prepared as described above) with a CD4.sup.+ T Cell
Subset Kit (R&D Systems, Minneapolis, Minn.) used as instructed
by the manufacturer. Cell purity was always >95%. BMDCs were
purified from femurs of mice after extraction and washing in PBS.
Cells were cultured for 8 days in C--RPMI-10 in the presence of
GM-CSF (20 ng/mL; Biosource, Camarillo, Calif.). Medium was
replaced after 4 days, and adherent cells were cultured for an
additional 4 days, at which point nonadherent cells were recovered,
washed, and used directly. Cells were >95% CD11c.sup.+ at the
time of use. Purified CD4.sup.+ T cells (1.times.10.sup.6) were
mixed with purified CD11c.sup.+ BMDCs (1.times.10.sup.6) in a
48-well plate and were incubated at 37.degree. C. in an atmosphere
containing 5% CO.sub.2. Various stimuli were used, as described in
Results. ELISA was performed with pre-coated plate kits (BD
Pharmingen) according to the manufacturer's guidelines. In some
assays, H. hepaticus, with or without wild-type B. fragilis or B.
fragilis .DELTA.PSA, was added at various concentrations.
[0085] Induction of experimental colitis. As assessed by PCR,
Rag2.sup.-/- and control C57Bl/6 mice were negative for H.
hepaticus colonization at the time of delivery. Splenic lymphocytes
were harvested from wild-type donor mice, and CD4.sup.+
CD45Rb.sup.high cells were purified from lymphocyte populations by
FACS as described above. Cells were washed with PBS, and
3.times.10.sup.5 cells were injected intraperitoneally in a volume
of 0.2 mL into recipient H. hepaticus-colonized Rag2.sup.-/-
animals. For colonization experiments, both H. hepaticus
(1.times.10.sup.8 organisms) and B. fragilis (1.times.10.sup.8
organisms) were introduced at the time of cell transfer. Throughout
PSA treatment studies, animals received 50 .mu.g of PSA by gavage 3
times per week. Animals were weighed throughout the experiment
until sacrifice at 8 weeks.
[0086] Induction of intestinal inflammation-TNBS colitis. The backs
of wild-type (C57BL/6) male mice were shaved, and pre-sensitization
solution (150 .mu.L; acetone with olive oil in a 4:1 ratio mixed
with 5% TNBS in a 4:1 ratio) was slowly applied. Seven days after
sensitization, mice were anesthetized with isofluorene and TNBS
solution (100 .mu.L; 1:1 5% TNBS with absolute ethanol)
administered rectally through a 3.5 F catheter (Instech Solomon;
SIL-C35). Mice were analyzed 4-6 days after TNBS
administration.
[0087] Histologic tissue analysis. Mouse tissues in Bouin's
fixative (VWR, West Chester, Pa.) were embedded in paraffin,
sectioned (6-.mu.m slices), mounted onto slides, and stained with
hematoxylin and eosin. Sections were evaluated in blinded fashion
by a single pathologist (Dr. R. T. Bronson, Harvard Medical
School).
[0088] Quantitative real-time PCR. RNA was extracted with Trizol
per the manufacturer's instructions (Invitrogen). RNA (1 .mu.g) was
reverse transcribed into cDNA with an iScript cDNA synthesis kit
(Bio-Rad). cDNA was diluted by addition of 60 .mu.L of water, and a
2-.mu.L volume of this solution was used for Q-PCR. Q-PCR was
performed using IQ SYBR Green supermix (Bio-Rad) and primers were
used at 0.2 .mu.m. Q-PCR was performed on a Bio-Rad iCycler IQ5.
Sequences of Q-PCR primers were as follows 5'-3': IL-23 (p19) F:
AGC TAT GAA TCT ACT AAG AGA GGG ACA (SEQ ID NO: 5) R: GTC CTA GTA
GGG AGG TGT GAA GTT G (SEQ ID NO: 6). IL-17A F: TTA AGG TTC TCT CCT
CTG AA (SEQ ID NO: 7) R: TAG GGA GCT AAA TTA TCC AA. (SEQ ID NO: 8)
TNF.alpha. F: ACG GCA TGG ATC TCA AAG AC (SEQ ID NO: 9) R: GTG GGT
GAG GAG CAC GTA GT (SEQ ID NO: 10). IL-10 F: CTG GAC AAC ATA CTG
CTA ACC G (SEQ ID NO: 11) R: GGG CAT CAC TTC TAC CAG GTA A (SEQ ID
NO: 12) RORyT F: CCG CTG AGA GGG CTT CAC (SEQ ID NO: 13) R: TGC AGG
AGT AGG CCA CAT TAC A (SEQ ID NO: 14) IL-21 F: ATC CTG AAC TTC TAT
CAG CTC CAC (SEQ ID NO: 15) R: GCA TTT AGC TAT GTG CTT CTG TTT C
(SEQ ID NO: 16) IL-27 F: CTG TTG CTG CTA CCC TTG CTT (SEQ ID NO:
17) R: CAC TCC TGG CAA TCG AGA TTC (SEQ ID NO: 18).
Example 1
PSA Balances the Th1/Th2 Profile of the Mammalian Immune System
[0089] The two subtypes of effector CD4.sup.+ T cells, T.sub.H1 and
T.sub.H2, are defined by expression of the cytokines interferon g
(IFNg) and interleukin 4 (IL-4), respectively (Janeway et al.,
2001). As shown above, PSA induces CD4.sup.+ T cell expansion in B.
fragilis-colonized mice and in vitro. To further characterize the
effects of PSA-mediated T cell activation, we assessed cytokine
profiles using purified cellular components. Co-culture of DCs and
CD4.sup.+ T cells in the presence of PSA yields dose-dependent
up-expression of the T.sub.H1 cytokine IFNg. The level of IFNg
production associated with PSA is comparable to that associated
with several known potent IFNg inducers (a-CD3, LPS, and
staphylococcal enterotoxin A [SEA]) and requires both DCs and T
cells. Specificity is evidenced by the lack of T.sub.H1 cytokine
production after NAc-PSA treatment.
[0090] T.sub.H1 cytokine production suppresses T.sub.H2 responses;
conversely, T.sub.H2 cytokine expression inhibits T.sub.H1
responses. Normal immune responses require a controlled balance of
these opposing signals. Examination of IL-4 expression in response
to PSA treatment reveals no cytokine production by purified
CD4.sup.+ T cells. a-CD3 and the superantigen SEA are potent
stimulators of both classes of cytokine. As T.sub.H2 cytokine
production is a "default pathway" in many systems (Kidd, 2003;
Amsen et al. 2004)) and T.sub.H1 cytokine production is
antagonistic to T.sub.H2 expression, the specific stimulation of
IFNg by PSA in vitro may provide a mechanism for establishing
commensal-mediated homeostasis of the host immune system by
balancing T.sub.H1/T.sub.H2 responses.
Example 2
PSA is Required for Appropriate CD4.sup.+ T-Helper Cytokine
Production During Colonization
[0091] A proper T.sub.H1/T.sub.H2 balance is critical for human and
animal health; over- or underproduction of either response is
associated with immunologic disorders. We investigated the effects
of PSA on T.sub.H1/T.sub.H2 cytokine responses in colonized
animals, again using germ-free mice. CD4.sup.+ T cells from mouse
spleens were purified and tested by ELISA for cytokine production.
Overproduction of the T.sub.H2 cytokine IL-4 in spleens of
germ-free mice compared with levels in conventional mice. This
result is consistent with previous reports of the appreciably
T.sub.H2-skewed profile of mice devoid of bacterial contamination
and reflects the human neonatal (precolonization) cytokine profile
(Kirjavainen and Gibson, 1999; Prescott et al., 1998; Adkins, 2000;
Kidd, 2003). This "default" T.sub.H2-bias in the absence of
bacterial colonization again highlights the profound contributions
of the microflora to immune development and provides a model to
test the effects of symbiotic bacteria on the establishment of
appropriate host cytokine production.
[0092] Mice colonized with wild-type B. fragilis alone display a
level of IL-4 production similar to that in conventional mice with
a complex microflora; this similarity shows the organism's
sufficiency to correct systemic immune defects. Moreover, mice
colonized with B. fragilis DPSA produce T.sub.H2 cytokines at
elevated levels similar to those in germ-free mice. Thus the
expression of a single bacterial antigen allows B. fragilis to
correct the IL-4 cytokine imbalance found in uncolonized
animals.
[0093] Examination of IFNg production by purified splenic CD4.sup.+
T cells reveals that germ-free mice, which are T.sub.H2-skewed, are
deficient in production of this prototypical T.sub.H1 marker when
compared to conventional mice. Colonization with wild-type B.
fragilis alone is sufficient to correct the defect in IFNg
expression in germ-free mice, with levels nearly as high as those
in conventional mice. Lack of PSA production by the B. fragilis
mutant during colonization of germ-free mice results in low-level
production of T.sub.H1 cytokines. These results were corroborated
by intracellular cytokine staining of splenic lymphocytes from each
group, which confirms that IFNg production is attributable to
CD4.sup.+ T cells. The production of IL-2, another T.sub.H1
cytokine, by CD4.sup.+ T cells in gnotobiotic mice also requires
PSA production data not shown) Together, these results demonstrate
that intestinal colonization of germ-free mice by B. fragilis alone
is sufficient to establish a proper systemic T.sub.H1/T.sub.H2
balance within the host a fundamental aspect of the mammalian
immune response required for health.
Example 3
PSA Suppresses Th-17 Induced Inflammation
[0094] Experimental colitis and human IBD result from an initial
inflammatory response that--lacking repression--advances in an
uncontrolled fashion and ultimately leads to intestinal pathology
and disease. To elucidate how PSA affects these primary
inflammatory responses, Applicants employed an animal model of
chemically induced colonic inflammation. Rectal administration of
trinitrobenzene sulphonic acid (TNBS) to wild-type mice mimics the
initiation of colitis by eliciting inflammatory T cell responses.
Disease was induced by administration of TNBS (or vehicle, as a
negative control), and oral treatment of PSA was evaluated.
[0095] The results illustrated in FIG. 7 show that the intestinal
immune response are beneficially modulated by PSA. In particular,
the results illustrated in FIG. 7a show that TNBS-treated animals
display weight loss that is statistically significant relative to
figures for vehicle-treated and PSA-treated animals, although
partial weight loss is observed in the PSA group (FIG. 7a).
Histological analysis confirmed PSA protection of colonic tissues
against the massive epithelial hyperplasia and loss of colonocyte
organization seen after TNBS treatment (FIG. 7b). Studies have
shown that pathogenic T.sub.H17 cells, which produce IL-17, mediate
the induction of experimental colitis.sup.30. Indeed, IL-17 levels
are increased among purified CD4.sup.+ T cells from mesenteric
lymph nodes (MLNs; FIG. 7c) of diseased animals but not from those
of PSA-treated animals. The increased level of TNFa among CD4.sup.+
T cells from MLNs of TNBS-treated animals is also reduced in
PSA-treated groups (FIG. 7d). Transcriptional analysis of
TNBS-treated colons revealed that expression of both IL-17 and TNFa
is highly elevated in diseased but not in PSA-protected animals
(FIGS. 7e and 7f).
[0096] Therefore, the above results show that PSA inhibits
intestinal pathology and inflammation in a chemically induced model
of experimental colitis.
Example 4
PSA Induces the Differentiation of IL-10 Producing Treg to Suppress
Inflammation
[0097] Protection from experimental colitis is engendered through
anti-inflammatory processes that prevent undesirable reactions
against the intestinal microbiota.sup.23. Interleukin-10-deficient
(IL-10-/-) animals develop colitis.sup.31. IL-10, one of the most
potent anti-inflammatory cytokines, is required for protection in
many animal models of inflammation.sup.21,27,32.
[0098] The results of a series of experiments directed to test the
effect of PSA on IL-10 production are illustrated in FIG. 8, and
show that PSA induces IL-10 expression in TNBS-treated animals and
inhibits pro-inflammatory cytokine production in primary cultured
cells through IL-10 production. In particular, as assayed by
real-time PCR, transcriptional levels of IL-10 within colons of
PSA-treated mice are significantly higher than those in control and
TNBS-treated mice (FIG. 8a). IL-10 is produced by many cell types.
However, since CD4.sup.+ T cells that express IL-10 display
immunosuppressive activities that inhibit inflammation during
experimental colitis.sup.33, Applicants tested the IL-10 production
in CD4.sup.+ T. When fresh CD4.sup.+ T cells were purified from
MLNs of PSA-treated mice (in which inflammation is reduced), highly
elevated levels of the IL-10 transcript were observed (FIG. 8b).
Applicants then assessed whether PSA is sufficient to induce IL-10
in vitro; when bone marrow-derived dendritic cells (BMDCs) and
naive CD4.sup.+ T cells were treated with purified PSA, a specific
increase in IL-10 production was observed (FIG. 8c).
[0099] A further series of experiments illustrated in FIG. 9, shows
that PSA from B. fragilis induces expression of IL-10 in vitro. In
particular, BMDCs and naive CD4.sup.+ T cells were infected with H.
hepaticus co-cultured with B. fragilis, and a specific expression
of IL-10 from culture supernatants was observed; co-culture with B.
fragilis DPSA induces significantly lower levels of IL-10 (FIG. 9).
Since PSA induces expression of IL-10 in vitro, to test whether
this molecule is required for inhibition of inflammatory responses
to H. hepaticus, BMDC-T cell co-cultures were infected with live H.
hepaticus and measured expression of the critical pro-inflammatory
cytokine TNFa. Addition of increasing concentrations of the
pathogenic commensal causes a dose-dependent increase in TNFa
production, as measured by ELISA of culture supernatants (FIG. 8d;
left three bars). Treatment of cells with purified PSA markedly
decreases TNFa production in response to H. hepaticus (FIG. 8d;
middle three bars). Most importantly, co-incubation of cell
cultures with H. hepaticus and PSA in the presence of a
neutralizing IL-10 receptor antibody (aIL-10R) completely reverses
this phenotypic effect and increases expression of TNFa (FIG. 8d;
right three bars).
[0100] The results are similar for the related pro-inflammatory
cytokine IL-1b, as shown by the results of experiments illustrated
in FIG. 10. In particular, infection of BMDC-T cell co-cultures
with increasing concentrations of live H. hepaticus (see FIG. 10
multiplicity of infection: 0.1, 1.0, and 10, as depicted by
triangles) results in release of the cytokine IL-1b Treatment of
infected cells with PSA reduces IL-1b levels, as shown in the
middle three bars. Neutralization of IL-10 signaling by addition of
an IL-10 receptor antibody (aIL-10R) alleviates suppression of in
vitro inflammatory responses, resulting in increased levels of
IL-1b FIG. 10 left three bars.
[0101] Thus, the results illustrated in the present example support
the conclusion that IL-10 produced in response to PSA is required
for inhibition of inflammatory reactions in cell cultures.
Example 5
PSA Administration Results in Differentiation of Treg, Inhibition
of TNF-a and IL-17 Cytokine Production and in Colitis
Suppression
[0102] Applicants investigated the requirement for IL-10 in
suppression of intestinal inflammation. Initially, IL-10.sup.-/-
animals were colonized with H. hepaticus alone or in combination
with B. fragilis (wild-type or DPSA). Applicants subsequently
harvested MLNs and re-stimulated cells in culture with soluble
Helicobacter antigens in an assay previously developed to measure
antigen-specific responses to H. hepaticus.sup.27. In particular,
IL-10.sup.-/- mice were left uncolonized (control) or were
colonized with H. hepaticus (to induce inflammation) either alone
or in combination with B. fragilis (wild-type or .rarw.PSA). MLNs
from experimental groups were pooled and re-stimulated with soluble
Helicobacter antigen (5 .mu.g/ml) for 48 hours. Secretion of
pro-inflammatory cytokines TNF.alpha. (a) and IL-17A (b) was
analyzed by ELISA
[0103] The results of these experiments, illustrated in FIG.
11a-11c, show that Helicobacter-colonized animals display increased
production of TNFa and IL-17; however, in the absence of IL-10
production in colonized animals, B. fragilis co-colonization dos
not reduce levels of these pro-inflammatory molecules (FIGS. 11a
and b, respectively). As expected, the absence of PSA has no
effect. Using the cell transfer model of colitis (see Examples 6 to
8 below, Applicants transferred CD4.sup.+ CD45Rb.sup.high T cells
to Helicobacter-colonized Rag.sup.-/- animals. Administration of
aIL-10R to mice (to block IL-10 signaling) during oral treatment
with PSA abrogates protection from colitis (FIG. 11c). In
particular, colitis scores show that PSA protection requires aIL-10
signaling, as neutralizing antibodies to IL-10 block PSA's
suppressive activity. Treatment with IL-10 OR abrogates
PSA-mediated protection. (FIG. 11c).
[0104] Additionally, when IL-10.sup.-/- animals were treated with
TNBS in the presence or absence of PSA, weight and histology data
illustrated in FIGS. 12 and 13, indicated that IL-10 production is
required for PSA-elicited reduction of intestinal immune responses.
In particular, in a first series of experiments, groups of 4
C57BL/6 mice were treated with PSA (or PBS) and then subjected to
rectal administration of TNBS or vehicle (control). SD values
illustrated in FIG. 12, indicate that, in the absence of IL-10, PSA
cannot restore TNBS-induced weight loss. ANOVA demonstrates that
weight loss in both TNBS-treated groups is statistically different
from that in control animals and that PSA does not prevent weight
loss in TNBS-treated IL-10.sup.-/- animals (FIG. 12).
[0105] In a second series of experiments, groups of 4 C57BL/6 mice
were treated with PSA (or PBS) and then subjected to rectal
administration of TNBS or vehicle (control). Histologic analysis of
H&E-stained sections from a representative animal from each
group is shown in FIG. 13. Thickening of the colon and epithelial
hyperplasia are noted in both TNBS-treated groups of IL-10.sup.-/-
animals, regardless of PSA treatment. Thus, the results illustrated
in FIG. 13 show that in the absence of IL-10, PSA does not reduce
intestinal injury in TNBS-treated IL-10.sup.-/- mice.
[0106] The above data suggest that PSA-mediated protection entails
the generation and/or expansion of IL-10-producing CD4.sup.+ T
cells. To determine whether IL-10 production by CD4.sup.+ T cells
is required for protection, Applicants transferred CD4.sup.+
CD45Rb.sup.high T cells from IL-10.sup.-/- donor mice into
Rag.sup.-/- recipients and then colonized the recipients with H.
hepaticus.
[0107] The results illustrated in FIGS. 11d-11f show that, as
expected, groups of mice receiving IL-10.sup.-/- T cells along with
H. hepaticus develop severe colitis (FIG. 11d; left bar) and are
not protected by PSA (FIG. 11d; middle bar). This result, supported
by histological findings in colons, indicates that PSA induces
protection from "previously pathogenic" CD4.sup.+ CD45Rb.sup.high T
cells in an IL-10-dependent manner (FIG. 11e). Weight analysis at
sacrifice shows that colitic PBS- and PSA-treated animals receiving
IL-10.sup.-/- CD4.sup.+ CD45Rb.sup.high T cells (unlike control
animals receiving no transferred cells) develop wasting disease
(FIG. 11 f). Thus, IL-10 production by CD4.sup.+ T cells is
required for PSA-mediated protection from experimental colitis.
These results constitute the first reported evidence of a symbiotic
bacterial molecule that networks with the immune system to
coordinate anti-inflammatory responses required for mammalian
health.
Example 6
PSA Balances the CD4.sup.+ CD45Rb.sup.high/CD4.sup.+ CD45Rb.sup.low
T Cells Ratio
[0108] CD4.sup.+ T cells of the mammalian immune system can be
generally divided into a naive (`uneducated`) CD4.sup.+
CD45Rb.sup.high population and an antigen-experienced (`educated`)
CD4 CD45Rb.sup.low population.sup.16.
[0109] In a first series of experiments, mono-association of
germ-free mice with wild-type B. fragilis was performed to analyze
the effect on the CD4.sup.+ CD45Rb.sup.low T cells v. CD4.sup.+
CD45Rb.sup.high proportion. In particular, the ability of B.
fragilis to correct deficiencies in the CD4.sup.+ CD45Rb.sup.low T
cell population in spleen.
[0110] The results illustrated in FIG. 1a show that association of
B. fragilis expands the proportions of CD4.sup.+ CD45Rb.sup.low T
cells in a PSA-dependent manner Remarkably, Applicants found that
splenic cells from germ-free animals include a smaller proportion
of CD4.sup.+ CD45Rb.sup.low T cells than do those from age-matched
conventional mice with a complete bacterial microbiota (FIG. 1a).
Additionally, it appears that mono-colonization of germ-free mice
with wild-type B. fragilis alone restores the CD4.sup.+ CD45Rb
profile in animals with a complete bacterial microbiota (FIG. 1a;
left panels). Most notably, colonization with a mutant strain
defective in the ability to produce PSA (B. fragilis DPSA) does not
generate an expansion of the CD4.sup.+ CD45Rb.sup.low T cell
population (FIG. 1a; lower right). It is well established that the
latter population possesses potent anti-inflammatory properties and
confers protection in animal models of inflammation.sup.17. These
results suggested that PSA mediate protection from
inflammation.
Example 7
PSA Controls IL23, IL1b and TNF-a Production in Inflamed Tissues,
Thus Controlling Th17 and Th1-Mediated Cytokine Production
[0111] The well-established CD4.sup.+CD45Rb transfer model of
experimental colitis.sup.18 was employed to investigate whether B.
fragilis colonization protects animals from inflammatory disease.
In this model, pathogenic CD4.sup.+ CD45Rb.sup.high T cells are
separated from protective CD4.sup.+ CD45Rb.sup.low cells and
transferred into specific pathogen-free (SPF) Rag.sup.-/- mice.
Upon cell transfer, mice are colonized with Helicobacter
hepaticus.sup.8,19, a pathobiont that is a benign commensal in
wild-type animals but an opportunistic pathogen causing colitis in
immuncompromised mice. After 8 weeks, animals are sacrificed and
colitis is assessed with a standard scoring system.sup.20.
[0112] The pathology scores illustrated in FIG. 1b, show that H.
hepaticus colonization and CD4.sup.+ CD45Rb.sup.high T cell
transfer are sufficient to induce severe colitis in Rag.sup.-/-
mice (FIG. 1b; first column), as previously reported.sup.19,21.
Co-colonization with wild-type B. fragilis results in significant
protection from disease (FIG. 1b; second column), whereas
co-colonization with B. fragilis DPSA does not (FIG. 1b; third
column).
[0113] Tissue damage in colitis is widely believed to result from
production of inflammatory cytokines in response to commensal
bacteria.sup.22. The pro-inflammatory cytokines tumor necrosis
factor a (TNFa, interleukin-1b (IL-1b and IL-23 are central to
disease initiation and progression in this experimental model of
colitis.sup.23. Furthermore, levels of these cytokines are elevated
in patients with IBD.sup.24, and therapies neutralizing TNFa have
yielded promising results in clinical trials in patients with
Crohn's disease.sup.25. Accordingly, Applicants decided to test the
inflammatory cytokine levels during disease by directly culturing
intestinal tissues of T cell recipient colonized animals.sup.26.
The results illustrated in FIGS. 1c, 1d, 2 and 3 show that PSA
alters cytokine levels in affected tissue.
[0114] In particular, the results of ELISA experiments of colon
organ cultures illustrated in FIG. 1c show an increased expression
of pro-inflammatory cytokine TNFa in diseased colons, with
significant reductions in animals co-colonized with wild-type B.
fragilis but not with B. fragilis DPSA.
[0115] The results of Q-PCR for IL-23p19 performed on splenocytes,
normalized to L32 expression illustrated in FIG. 1d show that
increases in IL-23 production by splenocytes following disease
induction are completely suppressed by intestinal colonization with
PSA-producing B. fragilis.
[0116] ELISA results for the pro-inflammatory cytokines IL-12p40
and IL-1b in colon and small intestines shown in FIG. 2 show a
specific increase in pro-inflammatory cytokines in diseased colons
but not in small intestines. This increase is significantly reduced
in animals co-colonized with PSA-producing B. fragilis. Conversely,
animals colonized with B. fragilis DPSA express greatly increased
pro-inflammatory cytokine levels over those in control animals
(C57BL/6) (FIG. 2).
[0117] The results of experiments illustrated in FIG. 3 show that
the expression of the TNFa by CD4.sup.+ T cells is reduced by
wild-type B. fragilis colonization during experimental colitis.
CD4.sup.+ cells were purified from pooled splenocytes from each
group (4 mice per group) and restimulated in vitro with PMA and
ionomycin in the presence of brefeldin A for 4 hours. Cells were
stained for intracellular TNF.alpha.. Cells within the lymphocyte
gate were included in the analysis, and numbers indicate the
percentage of cells producing TNF.alpha.. Purified cells were
>90% CD4.sup.+. Animals colonized with PSA-producing B. fragilis
during protection displayed lower TNFa levels than diseased
animals.
[0118] Overall these above results show that PSA performs its
effect by altering cytokine levels in affected tissues. In
particular, levels of the pro-inflammatory cytokines TNFa (FIG.
1c), IL-12p40, and IL-1b (FIG. 2) are elevated in the colons of
Rag.sup.-/- recipient mice colonized with H. hepaticus but not in
sections of small intestine (a site not affected in this model).
Consistent with the protection observed by pathophysiologic
analysis of experimental colitis, TNFa levels are not elevated when
these animals are co-colonized with wild-type B. fragilis. T cell
transfer plus co-colonization with H. hepaticus and B. fragilis
DPSA results in increased colonic cytokine production similar to
that seen in Rag.sup.-/- animals colonized with H. hepaticus alone.
Moreover, purified splenic CD4.sup.+ T cells from H.
hepaticus-colonized animals, display increased TNFa production;
this condition is corrected by colonization with wild-type B.
fragilis but not with the PSA deletion strain (FIG. 3). Expression
of IL-23 is critical in the cascade of events leading to
experimental colitis.sup.27,28. Applicants found that increases in
IL-23 production by splenocytes following disease induction are
completely suppressed by intestinal colonization with PSA-producing
B. fragilis (FIG. 1d).
[0119] Experiments directed to rule out bacterial clearance were
performed to show whether, over the course of the experiments,
levels of H. hepaticus and B. fragilis colonization did differ
between groups. The results illustrated in FIGS. 4 and 5 show that
protection is not the result of bacterial clearance.
[0120] In particular, the results shown in FIG. 4 show that
experimental animals remain colonized with H. hepaticus and B.
fragilis throughout the course of disease. More particularly, the
ethidium bromide-stained gel electrophoresis of H.
hepaticus-specific Q-PCR of FIG. 4a shows that co-colonization with
B. fragilis does not induce clearance of bacteria after 8 weeks.
The primers used for H. hepaticus 16S rDNA were: (HB-15)
5'-GAAACTGTTACTCTG-3' (SEQ ID NO: 1) and (HB-17)
5'-TCAAGCTCCCCGAAGGG-3' (SEQ ID NO: 2). Ethidium bromide-stained
gel electrophoresis of B. fragilis-specific Q-PCR of FIG. 4b show
stable bacterial colonization after 8 weeks; the primers used for
B. fragilis ssr3 (finB) gene were: (ssr3-F)
5'-TATTTGCGAGAAGGTGAT-3' (SEQ ID NO: 3) and (ssr3-r)
5'-TAAACGCTTTGCTGCTAT-3' (SEQ ID NO: 4).
[0121] In an additional series of experiments, quantitation of H.
hepaticus was performed to verify whether PSA administration
affected the presence of the organism. The results of quantitation
of H. hepaticus colonization experiments of FIG. 5 demonstrate that
the organism is present in equal numbers regardless of PSA-mediated
protection. In particular, in the experiments of FIG. 5 fecal
samples were collected from each experimental group, and total DNA
was extracted (Qiagen DNAeasy tissue kit). Equal amounts of DNA (50
ng) were used in Q-PCR (Bio-rad) with H. hepaticus-specific
primers. Q-PCR for H. hepaticus colonization was assessed according
to Young et al., 2004.sup.1 as log.sup.10 number of copies of a
known gene (cytolethal distending toxin). Animals contained
equivalent levels of H. hepaticus at the end of the experiment.
[0122] The results illustrated in this example support the
conclusion that PSA is a specific immunomodulatory molecule that
orchestrates beneficial immune responses to prevent B. Fragilis
host from developing experimental colitis.
Example 8
PSA Suppresses Inflammation Associated with CD4.sup.+
CD45Rb.sup.high T Cells
[0123] To determine whether PSA is sufficient for protection in the
absence of the intact B. fragilis organism, Applicants purified PSA
to homogeneity.sup.29 and administered it by gavage (orally) to
Rag.sup.-/- mice. Disease progression was then measured by various
pathologic and histologic criteria.
[0124] The results of related experiments illustrated in FIG. 6,
show that purified PSA orally administered protects against
experimental colitis.
[0125] In particular, in a first series of experiments illustrated
in FIG. 6a, colitis scores after CD4.sup.+ CD45Rb.sup.high T cell
transfer in the absence of H. hepaticus colonization indicated the
development of very mild colitis due to inflammation elicited by
the animals' SPF microbiota (FIG. 6a; first column). However,
Helicobacter-colonized Rag.sup.-/- animals that receive CD4.sup.+
CD45Rb.sup.high T cell transfers develop severe colitis (FIG. 6a;
second column). Oral PSA administration almost completely protects
animals against H. hepaticus-induced colitis (FIG. 6a; third
column), reducing disease to levels of control animals without T
cell transfer, that known not to develop colitis (FIG. 6a; fourth
column).
[0126] A second set of experiments was then performed to test the
inability to gain weight, a hallmark of colitis in this
experimental setting.sup.4. In particular, transfer of CD4.sup.+
CD45Rb.sup.high T cells and colonization with H. hepaticus (PBS+Hh)
in Rag2.sup.-/- animals was performed and the animals were
subsequently tested for wasting disease. The results illustrated in
FIG. 6b show that wasting disease in Rag.sup.-/- animals follows
transfer of CD4.sup.+ CD45Rb.sup.high cells and colonization with
H. hepaticus (FIG. 6b; PBS+Hh). These animals also develop
intestinal pathology and express pro-inflammatory cytokines (as
described above). Oral administration of PSA from the outset
completely protects animals against H. hepaticus-mediated wasting
disease (PSA+Hh). H. hepaticus provides the necessary antigens for
inflammation induction; no pathology is observed in uncolonized
animals (PBS-Hh) or in animals without cell transfer. Therefore
these experiments show that oral administration of PSA protects
animals against wasting (PSA+Hh).
[0127] In a further set of experiments, histologic sections of
colons of wild-type animals and animals subjected to transfer of
CD4.sup.+ CD45Rb.sup.high T cells and colonization with H.
hepaticus (PBS+Hh) were examined to verify the presence of
inflammation resulting in experimental colitis. The results
illustrated in FIG. 6c show that transfer of CD4.sup.+
CD45Rb.sup.high T cells into Helicobacter-colonized Rag.sup.-/-
mice results in onset of severe colitis, as evidenced by massive
epithelial cell hyperplasia and gross thickening of the gut wall
(FIG. 6c; second panel). Furthermore, consistent with previous
studies, the combination of CD4.sup.+ CD45Rb.sup.high T cell
transfer plus H. hepaticus colonization results in infiltration of
affected tissues by leukocytes--a hallmark of inflammation and
disease (FIG. 6c second panel, bottom).sup.19,21. Additionally,
oral administration of PSA to H. hepaticus-colonized cell transfer
recipients confers complete protection against experimentally
induced colonic hyperplasia (FIG. 6c; third panel); furthermore,
PSA-treated animals display no leukocyte infiltration in colonic
tissues (FIG. 6c third panel, bottom)--a result indicating
protection against inflammation.
[0128] Taken together, these results indicate that oral
administration of PSA prevents colitis and protects mice against
the associated weight loss and inflammatory cell infiltration
observed in diseased animals.
Example 9
PSA is Effective in Systemic Immune Compartments Suppressing
Cytokine Production by Th1 and Th17 Cells
[0129] In further series of experiments, mice were treated with
TNBS or TNB/PSA, orally administered to the mice. The relevant
colonic sections were subsequently analyzed by a blinded
pathologist who provided a histological score. The results
illustrated in FIG. 14a provide further evidence that oral PSA
administration reduces colitis.
[0130] While oral treatment with purified PSA protects from
experimental colitis (FIG. 14a), colonization by a B. fragilis
mutant that does not make PSA (B. fragilis .DELTA.PSA) is unable to
protect. During the course of the experiments exemplified in
Examples 1 to 8, Applicants noted strong effects of PSA in systemic
immune compartments. To further understand these systemic responses
Applicants utilized the TNBS induced model in the susceptible
Balb/c mouse strain. As this model allows for disease induction in
an immune-competent animal, it permits analysis of all immune cells
involved in both disease induction and protection.
[0131] Balb/c mice were orally administered purified PSA before
induction of colitis. Indeed, oral treatment of PSA protected from
weight loss associated with experimental colitis and inflammation
within the intestine (not shown).
[0132] Additionally, pre-treatment of Balb/c mice undergoing TNBS
induced colitis, with PSA dramatically increases the survival of
animals with disease from 40% to 90%, (see FIG. 14b), further
attesting to the powerful anti-inflammatory effects of PSA. Since
splenomegaly is commonly seen in this model of IBD and demonstrates
the systemic nature of this disease, the Applicants analyzed the
spleen of mice treated with TNBS, and TNBS/PSA. The results
illustrated in FIG. 14c show that oral administration of ZPS is
protects from the splenomegaly. Furthermore, analysis of cytokine
expression showed that animals undergoing TNBS induced colitis have
severe splenomegaly with increases in the expression of
inflammatory cytokines from CD4+ T lymphocytes residing within the
spleen, as illustrated in FIG. 14d. Orally administered PSA
significantly reduces splenomegaly and the expression of
TNF-.alpha., IL-17 and IL-21 in CD4+ T lymphocytes from the spleen
during mucosal disease (FIG. 14d). The experiments outlined in
Example 5 demonstrate that PSA is able to protect from colitis
through induction of IL-10 from CD4+ T cells residing within the
intestinal compartments. Consistent with previous data, Applicants
find that IL-10 levels are elevated within the CD4+ T lymphocytes
in spleen (FIG. 14d). Taken together, these data suggest that PSA
residing within the intestine is capable of effecting systemic
immunity. In particular these results show that oral administration
of ZPS can not only protect from intestinal disease but also
suppresses inflammation within extra-intestinal immune
compartments, such as the spleen.
Example 10
Parenteral Administration of PSA Protects from Inflammation and
Controls TNF-a, IL-17 and IL23 Production in Intestine and
Spleen
[0133] Distinct subsets of cells reside within the intestinal
compartment, including CD8.alpha..alpha. T cells, mucosal
.gamma..delta. T cells and CD103+ dendritic cells. Recent studies
have demonstrated that these various cell types have distinct
functions from their systemic immune counterparts. To determine
whether PSA acts specifically within the intestine, purified PSA
was administered intravenously and mucosal inflammation was
induced. In a first series of experiments illustrated in FIG. 15,
PSA was administered before inflammation was induced. In a second
series of experiments, illustrated in FIG. 16, PSA was administered
after inflammation was induced.
[0134] The results illustrated in FIG. 15, show that delivery of
ZPS to extra-intestinal sites is able to protect from induced
intestinal colitis. In particular, systemic administration of PSA
enhances the survival of diseased animals and protects from
splenomegaly (60% survival vs. 90%) (FIGS. 15a and 15b).
Additionally, it is expected that colons of animals that treated
with PSA systemically, have significantly less hyperplasia and
inflammatory infiltrate.
[0135] The results illustrated in FIG. 16 show that while disease
is exacerbated by the increased production of inflammatory
cytokines at the site of induction, systemic administration of PSA
during TNBS induced colitis suppresses inflammatory cytokines at
both intestinal and systemic immune compartments. In particular,
TNF-.alpha. from Mesenteric Lymph Nodes (MLN) CD4+ T cells is
increased in expression during TNBS induced colitis, but is reduced
by PSA systemically administered (FIG. 16a). Additionally,
inflammatory cytokines IL12p35 IL-23p19 and IL-17 are elevated in
the colons of diseased mice, as shown by analysis of transcripts
from RNA extracted from colons of mice undergoing TNBS induced
colitis, but are reduced by administration of PSA (FIG. 16b). Also
in spleen, systemic administration of ZPS reduces the production of
TNF-.alpha. from CD4+ T lymphocytes within the spleen as shown by
the results illustrated in FIG. 16c. Furthermore, systemic
administration of ZPS reduces expression of the transcripts IL-17,
and IL-6 within the spleen as shown by the results illustrated in
FIG. 16d.
[0136] Additional experiments also demonstrated that while PSA
decreases expression of inflammatory cytokines, intravenous
treatment with PSA leads to an elevation in the production of IL-10
within the intestine (supplementary data). These data indicate that
systemically administered PSA is capable of extending to mucosal
sites and protecting from inflammatory bowel disease.
[0137] The data illustrated in this example also show that systemic
administration of PSA during TNBS induced colitis suppresses
inflammatory cytokines at both intestinal and systemic immune
compartments.
Example 11
Parenteral Administration of PSA Modulates Cytokine Expression and
Protects from Systemic Inflammation Caused by Th1 and Th17
Cells
[0138] Endotoxic shock occurs during severe gram-negative bacterial
infections and is characterized by hypotension, multi-organ failure
and potentially death. This syndrome results from the production of
multiple inflammatory cytokines, including TNF-a and IL-6, in
response to the lipopolysaccharides (LPS) found in the cell wall of
gram negative bacteria. IL-10 has been demonstrated to be a central
regulator of the inflammatory response to LPS, indeed a single dose
of IL-10 prevents death in murine models of endotoxic shock.sup.42.
The dramatic effects of PSA within the systemic immune compartments
lead us to investigate whether PSA could ameliorate systemic
inflammation.
[0139] To determine whether PSA was capable of suppressing
inflammation associated with endotoxic shock Applicants injected
Balb/c mice with a low dose (100) ug) of LPS and monitored serum
levels of the cytokines TNF-.alpha. and IL-6. In particular, serum
was collected from mice 1 and 4 hours post-administration of 100
.mu.g or 500 .mu.g of LPS and TNF-.alpha. and IL-6 protein levels
in the serum were determined by ELISA.
[0140] The results illustrated in FIG. 17a to c, show that
untreated mice had undetectable levels of serum TNF-.alpha. and
IL-6 at both time points collected. In particular, consistent with
previous studies, in absence of PSA administration, LPS treated
mice experienced an over 300 fold increase in serum TNF-.alpha.
levels that peaked at one hour post injection and decreased to
basal levels by four hours post injection (FIG. 17a). In absence of
PSA treatment, also IL-6 levels in the serum of LPS injected mice
was detectable as early as 1 hour and continues to increase in
expression by 4 hours, (FIG. 17b). Remarkably, mice that had been
pre-treated with PSA had a significant reduction in serum levels of
TNF-.alpha. and IL-6 at both time points (FIGS. 17a and 17b),
indicating that PSA is able to prevent the early induction of
inflammatory cytokines in response to LPS.). Additionally, in
absence of PSA treatment, splenomegaly occurs within three days of
LPS injection and results from the recruitment of inflammatory cell
types. Animals pre-treated with PSA, have smaller spleens and
express lower levels of inflammatory cytokines at this site (data
not shown and FIG. 17c).
[0141] This data demonstrates that PSA is capable of suppressing
systemic inflammatory responses induced by a low dose
administration of LPS.
Example 12
Parenteral Administration of PSA Results in TNF-a Modulation and
Treatment Systemic Inflammation
[0142] Death occurring during endotoxic shock is a result of the
elevated levels of inflammatory cytokines that occur within hours
of the response to LPS. Indeed, blockage of the inflammatory
mediator TNF-.alpha. completely rescues animals from LPS induced
mortality. That PSA had such a dramatic effect on the levels of the
cytokines expressed during low dose administration of LPS,
suggested that PSA might prevent death associated with endotoxic
shock. Applicants therefore administered high dose levels of LPS
(500 that cause death with 24-96 hours and accessed both cytokine
levels within the serum and monitored survival.
[0143] The results illustrated in FIGS. 17d and 17e, show that
while animals that were administered PBS all die within 60 hours of
administration of LPS, those animals that received PSA treatment
have a significantly increased survival rate (FIG. 17d).
Remarkably, while PBS animals have an over 3000 fold induction of
TNF-.alpha. when administered LPS, those mice receiving PSA have
very little TNF-a induction (FIG. 17e). These data demonstrate that
PSA is able to suppress the systemic inflammatory response that
ensues in response to LPS and is able to protect from septic
shock.
[0144] As shown in the exemplary experiments of Example 7 PSA
mediated protection from IBD is reliant on IL-10 production from a
CD4.sup.+ T lymphocyte. To determine whether IL-10 is required for
protection from LPS induced death Applicants pretreated IL10
deficient animals with PBS or purified PSA and administer levels of
LPS that would result in septic shock. The cytokine level and
percentage survival were monitored.
[0145] The results illustrated in FIG. 18, show that consistent
with previous data IL10-deficient animals were more sensitive to
lower doses of LPS and TNF alpha levels continue increase (FIG.
18a). Interestingly, PSA treated animals have a drastic decrease in
the levels of serum TNF-a in response to LPS that drops to
negligible levels by 4 hours post LPS administration (FIG. 18a),
indicating that decreased TNF-a levels by PSA is not dependent on
the ability of PSA to induce IL-10. Strikingly, decreased IL-6
production by PSA is IL-10 dependent as levels are similar to PBS
treated animals, indicating multiple mechanisms are employed by PSA
to alleviate endotoxic shock (FIG. 18b). Finally, IL10 deficient
mice receiving PSA are completely protected from LPS induced death
(FIG. 18c).
[0146] Additional experiments were performed to detect additional
effects of PSA administration in connection with low dose LPS
administration in mice. The results illustrated in FIG. 19 show
that other effects of ZPS administration during low dose LPS
administration include a reduction in CD11b and GR1 expression on
the surface of neutrophils as well as reduced neutrophil
recruitment in the blood.
[0147] Taken together, the data of this example and of example
indicate that PSA is capable of blocking extra-intestinal disease
and is expected to be a novel therapeutic agent to reduce systemic
inflammation.
[0148] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the compounds compositions
and methods of the disclosure, and are not intended to limit the
scope of what the inventors regard as their disclosure.
Modifications of the above-described modes for carrying out the
disclosure that are obvious to persons of skill in the art are
intended to be within the scope of the following claims. All
patents and publications mentioned in the specification are
indicative of the levels of skill of those skilled in the art to
which the disclosure pertains. All references cited in this
disclosure are incorporated by reference to the same extent as if
each reference had been incorporated by reference in its entirety
individually.
[0149] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background,
Summary, Detailed Description, and Examples is hereby incorporated
herein by reference.
[0150] Further, the hard copy of the sequence listing submitted
herewith and the corresponding computer readable form are both
incorporated herein by reference in their entireties.
[0151] It is to be understood that the disclosures are not limited
to particular compositions or biological systems, which can, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting. As used in this
specification and the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the content clearly
dictates otherwise. The term "plurality" includes two or more
referents unless the content clearly dictates otherwise. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the disclosure pertains.
[0152] Although any methods and materials similar or equivalent to
those described herein can be used in the practice for testing of
the specific examples of appropriate materials and methods are
described herein.
[0153] A number of embodiments of the disclosure have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the present disclosure. Accordingly, other embodiments are
within the scope of the following claims.
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Sequence CWU 1
1
18115DNAArtificial SequenceSynthetic Polynucleotide 1gaaactgtta
ctctg 15217DNAArtificial SequenceSynthetic Polynucleotide
2tcaagctccc cgaaggg 17318DNAArtificial SequenceSynthetic
Polynucleotide 3tatttgcgag aaggtgat 18418DNAArtificial
SequenceSynthetic Polynucleotide 4taaacgcttt gctgctat
18527DNAArtificial SequenceSynthetic Polynucleotide 5agctatgaat
ctactaagag agggaca 27625DNAArtificial SequenceSynthetic
Polynucleotide 6gtcctagtag ggaggtgtga agttg 25720DNAArtificial
SequenceSynthetic Polynucleotide 7ttaaggttct ctcctctgaa
20820DNAArtificial SequenceSynthetic Polynucleotide 8tagggagcta
aattatccaa 20920DNAArtificial SequenceSynthetic Polynucleotide
9acggcatgga tctcaaagac 201020DNAArtificial SequenceSynthetic
Polynucleotide 10gtgggtgagg agcacgtagt 201122DNAArtificial
SequenceSynthetic Polynucleotide 11ctggacaaca tactgctaac cg
221222DNAArtificial SequenceSynthetic Polynucleotide 12gggcatcact
tctaccaggt aa 221318DNAArtificial SequenceSynthetic Polynucleotide
13ccgctgagag ggcttcac 181422DNAArtificial SequenceSynthetic
Polynucleotide 14tgcaggagta ggccacatta ca 221524DNAArtificial
SequenceSynthetic Polynucleotide 15atcctgaact tctatcagct ccac
241625DNAArtificial SequenceSynthetic Polynucleotide 16gcatttagct
atgtgcttct gtttc 251721DNAArtificial SequenceSynthetic
Polynucleotide 17ctgttgctgc tacccttgct t 211821DNAArtificial
SequenceSynthetic Polynucleotide 18cactcctggc aatcgagatt c 21
* * * * *