U.S. patent application number 16/463826 was filed with the patent office on 2020-03-05 for prophylactic agent, onset-suppressing agent or therapeutic agent for progressive immune demyelinating diseases.
The applicant listed for this patent is NATIONAL CENTER OF NEUROLOGY AND PSYCHIATRY. Invention is credited to Shinji OKI, Takashi YAMAMURA.
Application Number | 20200071408 16/463826 |
Document ID | / |
Family ID | 62242207 |
Filed Date | 2020-03-05 |
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United States Patent
Application |
20200071408 |
Kind Code |
A1 |
OKI; Shinji ; et
al. |
March 5, 2020 |
PROPHYLACTIC AGENT, ONSET-SUPPRESSING AGENT OR THERAPEUTIC AGENT
FOR PROGRESSIVE IMMUNE DEMYELINATING DISEASES
Abstract
The present invention provides a prophylactic agent,
onset-suppressing agent, or therapeutic agent for progressive
immune demyelinating diseases comprising, as an active ingredient,
a substance capable of suppressing or inhibiting production of
prolactin.
Inventors: |
OKI; Shinji; (Kodaira-shi,
Tokyo, JP) ; YAMAMURA; Takashi; (Kodaira-shi, Tokyo,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL CENTER OF NEUROLOGY AND PSYCHIATRY |
Kodaira-shi, Tokyo |
|
JP |
|
|
Family ID: |
62242207 |
Appl. No.: |
16/463826 |
Filed: |
November 28, 2017 |
PCT Filed: |
November 28, 2017 |
PCT NO: |
PCT/JP2017/042629 |
371 Date: |
May 23, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/02 20130101; A61P
25/00 20180101; A61P 37/02 20180101; A61K 45/06 20130101; A61K
39/395 20130101; C07K 16/2866 20130101; A61K 45/00 20130101; A61P
43/00 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C12Q 1/02 20060101 C12Q001/02; A61P 25/00 20060101
A61P025/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 29, 2016 |
JP |
2016-231364 |
Claims
1. A prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases comprising, as
an active ingredient, a substance capable of suppressing or
inhibiting production of prolactin.
2. The prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases according to
claim 1, wherein the substance comprises a substance capable of
suppressing or inhibiting generation of Zbtb20.
3. The prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases according to
claim 1, wherein the substance comprises a dopamine receptor
agonist or a dopamine receptor partial agonist.
4. The prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases according to
claim 1, wherein the progressive immune demyelinating disease is
secondary progressive multiple sclerosis.
5. A method for preventing or suppressing a progression into
progressive immune demyelinating diseases, comprising
administering, to a subject, a substance capable of suppressing or
inhibiting production of prolactin.
6. The method according to claim 5, wherein the substance comprises
a substance capable of suppressing or inhibiting generation of
Zbtb20.
7. The method according to claim 5, wherein the substance comprises
a dopamine receptor agonist or a dopamine receptor partial
agonist.
8. The method according to claim 5, wherein the progressive immune
demyelinating disease is secondary progressive multiple
sclerosis.
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. A prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases comprising, as
an active ingredient, a substance capable of suppressing or
inhibiting activation of CX3CR1 receptor.
14. The prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases according to
claim 13, wherein the substance is an anti-CX3CR1 antibody or an
antigen-binding fragment thereof.
15. A method of collecting data for diagnosing a progression into
progressive immune demyelinating diseases comprising: collecting
microglia from a human subject; and measuring an expression level
of at least one of IL-9, IFN-.alpha., and IFN-.beta.1.
16. The prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases according to
claim 2, wherein the progressive immune demyelinating disease is
secondary progressive multiple sclerosis.
17. The prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases according to
claim 3, wherein the progressive immune demyelinating disease is
secondary progressive multiple sclerosis.
18. The method according to claim 6, wherein the progressive immune
demyelinating disease is secondary progressive multiple
sclerosis.
19. The method according to claim 7, wherein the progressive immune
demyelinating disease is secondary progressive multiple sclerosis.
Description
TECHNICAL FIELD
[0001] The present invention relates to a prophylactic agent,
onset-suppressing agent, or therapeutic agent for progressive
immune demyelinating diseases.
BACKGROUND ART
[0002] Multiple sclerosis (MS) is one of autoimmune diseases and a
disease in which multiple inflammations targeting myelin sheaths
and nerve axons are triggered, resulting in nerve conduction
disorders caused by widespread demyelination. When multiple
sclerosis pathology is advanced, severe neurological symptoms such
as motor impairment and visual disturbance appear.
[0003] Multiple sclerosis includes: relapsing-remitting MS (RR-MS)
having repeated acute exacerbation and remission; and progressive
MS. It has been known that the progressive MS includes: primary
progressive MS (PP-MS); secondary progressive MS (SP-MS) in which
RR-MS pathology continues for a certain period and then progresses
into advanced pathology; and progressive relapsing MS (PR-MS) in
which the pathology progresses with repeated relapse (Non Patent
Literatures 1 to 3).
[0004] Type-1 interferons, anti-inflammatory agents, and
immunosuppressive agents have been known as disease-modifying drugs
(DMDs) for RR-MS. Unfortunately, use of DMDs for RR-MS cannot exert
effects on progressive MS and effective DMDs for progressive MS are
not currently known. As a treatment strategy for progressive MS,
symptomatic treatment such as intraspinal injection of baclofen or
dosing of persistent 4-aminopyridine preparation occupies an
important position.
[0005] To date, the mechanism of producing progressive MS pathology
has not been revealed, including whether the mechanism is identical
to or different from the mechanism of producing RR-MS pathology.
Meanwhile, Non Patent Literature 4 reports that in the central
nervous system (CNS) of each SP-MS patient, a plurality of cells
and tissues are damaged and the damage spreads not only into white
matter but also into gray matter. In addition, Non Patent
Literature 4 reports that in the brain of patients with multiple
sclerosis, the expression level of PAR2 receptor, which belongs to
the PAR (Protease-activated receptors) receptor family, is changed,
indicating that the PAR2 receptors participate in neuroinflammatory
symptoms.
[0006] The present inventors have found that in NR4A2-deficient
mice, in which monophasic experimental autoimmune encephalomyelitis
(EAE) is induced, EAE pathology usually accompanied by quadriplegia
is not observed at the early stage of the induction; but at the
late stage of the induction (about 28 days after the induction),
EAE pathology (hereinafter, sometimes referred to as "late-stage
EAE pathology") is observed; and this late-stage EAE pathology can
be a model for progressive MS pathology (Patent Literature 1). In
addition, the present inventors have considered that the late-stage
EAE pathology including neurodegeneration results from persistent
neuronopathy caused by stimulation-dependent release of granzyme B
and have found that inhibition of PAR1 receptor by using, for
instance, a PAR1 receptor antagonist causes the late-stage EAE
pathology to improve (Patent Literature 2).
CITATION LIST
Patent Literature
[0007] Patent Literature 1: WO 2016/002827 [0008] Patent Literature
2: WO 2016/114386
Non Patent Literature
[0008] [0009] Non Patent Literature 1: Nature Reviews Neurology
2012, 8, 647-656. [0010] Non Patent Literature 2: Nature Reviews
Neurology 2013, 9, 496-503. [0011] Non Patent Literature 3:
Multiple Sclerosis Journal 2013, 19: 1428-1436. [0012] Non Patent
Literature 4: Biochimica et Biophysica Acta 1802 (2010), 66-79.
SUMMARY OF INVENTION
Technical Problem
[0013] The present invention has been made in light of these
situations and the main purpose thereof is to provide a
prophylactic agent, onset-suppressing agent, or therapeutic agent
for progressive immune demyelinating diseases. Another purpose of
the present invention is to provide a method for preventing or
suppressing a progression into progressive immune demyelinating
diseases.
Solution to Problem
[0014] The present inventors have discovered that in the late-stage
EAE pathology of NR4A2-deficient mice, expression of Eomes molecule
in Th cells is induced by stimulation from CNS-derived
antigen-presenting cells and prolactin produced from the
antigen-presenting cells can promote induction of the Eomes
molecule expression. The present invention is based on these new
findings. Note that it has been known that prolactin can be
produced from not only the pituitary but also brain tissues other
than the pituitary, the mammary gland, mammary papilla tissues,
placenta, uterus, immune tissues (e.g., lymphocytes, the thymus,
the spleen), etc. The above tissue-derived prolactin is distinct
from the pituitary-derived prolactin and is also called ectopic
prolactin.
[0015] Specifically, the present invention provides the following
(1) to (15).
(1) A prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases comprising, as
an active ingredient, a substance capable of suppressing or
inhibiting production of prolactin. (2) The prophylactic agent,
onset-suppressing agent, or therapeutic agent for progressive
immune demyelinating diseases according to (1), wherein the
substance comprises a substance capable of suppressing or
inhibiting generation of Zbtb20. (3) The prophylactic agent,
onset-suppressing agent, or therapeutic agent for progressive
immune demyelinating diseases according to (1), wherein the
substance comprises a dopamine receptor agonist or a dopamine
receptor partial agonist. (4) The prophylactic agent,
onset-suppressing agent, or therapeutic agent for progressive
immune demyelinating diseases according to any one of (1) to (3),
wherein the progressive immune demyelinating disease is secondary
progressive multiple sclerosis. (5) A method for preventing or
suppressing a progression into progressive immune demyelinating
diseases, comprising administering, to a subject, a substance
capable of suppressing or inhibiting production of prolactin. (6)
The method according to (5), wherein the substance comprises a
substance capable of suppressing or inhibiting generation of
Zbtb20. (7) The method according to (5), wherein the substance
comprises a dopamine receptor agonist or a dopamine receptor
partial agonist. (8) The method according to any one of (5) to (7),
wherein the progressive immune demyelinating disease is secondary
progressive multiple sclerosis. (9) Use of a substance capable of
suppressing or inhibiting production of prolactin in the
manufacture of a prophylactic agent, onset-suppressing agent, or
therapeutic agent for progressive immune demyelinating diseases.
(10) The use according to (9), wherein the substance comprises a
substance capable of suppressing or inhibiting generation of
Zbtb20. (11) The use according to (9), wherein the substance
comprises a dopamine receptor agonist or a dopamine receptor
partial agonist. (12) The use according to any one of (9) to (11),
wherein the progressive immune demyelinating disease is secondary
progressive multiple sclerosis. (13) A prophylactic agent,
onset-suppressing agent, or therapeutic agent for progressive
immune demyelinating diseases comprising, as an active ingredient,
a substance capable of suppressing or inhibiting activation of
CX3CR1 receptor. (14) The prophylactic agent, onset-suppressing
agent, or therapeutic agent for progressive immune demyelinating
diseases according to (13), wherein the substance is an anti-CX3CR1
antibody or an antigen-binding fragment thereof. (15) A method of
collecting data for diagnosing a progression into progressive
immune demyelinating diseases comprising: collecting microglia from
a human subject; and measuring an expression level of at least one
of IL-9, IFN-.alpha., and IFN-.beta.1.
Advantageous Effects of Invention
[0016] The present invention makes it possible to provide a
prophylactic agent, onset-suppressing agent, or therapeutic agent
for progressive immune demyelinating diseases and to provide a
method for preventing or suppressing a progression into progressive
immune demyelinating diseases.
BRIEF DESCRIPTION OF DRAWINGS
[0017] FIG. 1 is cytograms showing expressions of CD107a and Eomes
on and in Th cells co-cultured with antigen-presenting cells
(CD19.sup.+ B cells or non-B/class II.sup.+ cells) infiltrated into
the CNS of each NR4A2-deficient mouse in which monophasic EAE was
induced.
[0018] FIG. 2 is graphs showing the Eomes expression levels in Th
cells co-cultured with antigen-presenting cells (CD19.sup.+ B cells
or non-B/class II.sup.+ cells) infiltrated into the CNS of each
NR4A2-deficient mouse in which monophasic EAE was induced (summary
data of FIG. 1).
[0019] FIG. 3(a) is a graph showing when the brain and the spinal
cord in each progression stage of EAE pathology were collected and
the three dashed lines indicate the collection time points
corresponding to the respective early-stage EAE pathology,
mid-stage EAE pathology, and late-stage EAE pathology. FIG. 3(b) is
cytograms of T cells in each progression stage of EAE pathology and
a graph indicating the percentage of Eomes.sup.+ CD4.sup.+ T cells
in each progression stage of EAE pathology.
[0020] FIG. 4 is cytograms of CD19.sup.+ B cells or CD19.sup.+
class II.sup.+ cells in each progression stage of EAE pathology and
graphs indicating the percentages regarding each cell type.
[0021] FIG. 5 is graphs showing the expression level of prolactin
or growth hormone in antigen-presenting cells (CD19.sup.+ B cells
or non-B/class II.sup.+ cells) infiltrated into the CNS of each
NR4A2-deficient mouse in which monophasic EAE was induced.
[0022] FIG. 6 is graphs showing the expression level of prolactin
or growth hormone in the CSF in each progression stage of EAE
pathology.
[0023] FIG. 7 is graphs showing the effects of culturing in the
co-presence of prolactin on Eomes expression.
[0024] FIG. 8 is graphs showing the effects of culturing in the
co-presence of prolactin on Eomes expression.
[0025] FIG. 9 is graphs showing the gene expression levels of each
of prolactin and Zbtb20 in antigen-presenting cells infiltrated
into the CNS of each NR4A2-deficient mouse in which monophasic EAE
was induced.
[0026] FIG. 10 is cytograms showing the Zbtb20 protein expression
in antigen-presenting cells infiltrated into the CNS of each
NR4A2-deficient mouse in which monophasic EAE was induced.
[0027] FIG. 11 is graphs showing Zbtb20 or prolactin protein
expression in antigen-presenting cells infiltrated into the CNS of
each NR4A2-deficient mouse in which monophasic EAE was induced.
[0028] FIG. 12 is cytograms and a graph showing the IL-10 and
Zbtb20 gene expression levels in B cells collected from the CNS of
each mouse with the late-stage EAE pathology.
[0029] FIG. 13 is cytograms and a graph showing the Zbtb20 gene
expression levels in non-B antigen-presenting cells collected from
the CNS of each mouse with the late-stage EAE pathology.
[0030] FIG. 14 is graphs showing the expression level of prolactin
or growth hormone in the pituitary.
[0031] FIG. 15 is a graph showing the Eomes expression levels in
spleen-derived Th cells cultured in the presence or absence of
prolactin.
[0032] FIG. 16 is cytograms showing Eomes expression in
spleen-derived CD226.sup.+ Th cells cultured in the presence or
absence of prolactin.
[0033] FIG. 17 is a graph showing the EAE scores when bromocriptine
was administered to each NR4A2-deficient mouse in which monophasic
EAE had been induced.
[0034] FIG. 18 is graphs showing Eomes expression in Th cells
infiltrated into the CNS after bromocriptine was administered to
each NR4A2-deficient mouse in which monophasic EAE had been
induced.
[0035] FIG. 19 is a graph showing the percentage of Eomes.sup.+
CD4.sup.+ T cells after bromocriptine dosing.
[0036] FIG. 20 is cytograms showing expression of CD107a and Eomes
in Th cells infiltrated into the CNS when bromocriptine was
administered to each NR4A2-deficient mouse in which monophasic EAE
had been induced.
[0037] FIG. 21 is graphs showing the percentages of Eomes.sup.+
CD4.sup.+ T cells and the percentages of CD107a.sup.+ CD4.sup.+ T
cells after bromocriptine dosing.
[0038] FIG. 22 is graphs showing Eomes gene expression after
dopamine dosing.
[0039] FIG. 23 is graphs showing the clinical scores in each EAE
pathology mouse after L-dopa dosing.
[0040] FIG. 24 is graphs showing expression of Eomes protein or
Zbtb20 protein after dopamine dosing.
[0041] FIG. 25 is graphs showing the prolactin gene expression
levels after L-dopa dosing.
[0042] FIG. 26 is a graph showing the EAE scores when an
anti-CX3CR1 antibody was administered to each NR4A2-deficient mouse
in which monophasic EAE had been induced.
[0043] FIG. 27 is graphs showing the percentage of Eomes.sup.+
CD4.sup.+ Th cells with respect to CD4.sup.+ Th cells when an
anti-CX3CR1 antibody was administered to each NR4A2-deficient mouse
in which monophasic EAE had been induced.
[0044] FIG. 28 is graphs showing the clinical scores in each EAE
pathology mouse after Zbtb20-specific siRNA dosing.
[0045] FIG. 29 is graphs showing expression of Eomes protein or
Zbtb20 protein after Zbtb20-specific siRNA dosing.
[0046] FIG. 30 is graphs showing expression of prolactin gene or
Zbtb20 gene after Zbtb20-specific siRNA dosing.
[0047] FIGS. 31(a) and (b) are graphs showing the clinical scores
in each EAE pathology mouse after anti-CD20 antibody dosing. FIG.
31(c) is graphs showing Zbtb20 protein expression after anti-CD20
antibody dosing.
[0048] FIG. 32 is graphs showing expression of Eomes protein or
Zbtb20 protein after anti-CD20 antibody dosing.
[0049] FIG. 33 is graphs showing Zbtb20 protein expression under
culturing in the presence of each different cytokine.
[0050] FIG. 34 is graphs showing expression of Zbtb20 gene or
prolactin gene under culturing in the presence of each different
cytokine.
[0051] FIG. 35 is graphs showing the changes in expression of each
different cytokine during the course of progression of EAE
pathology.
[0052] FIG. 36 is graphs showing the changes in expression of each
different cytokine during the course of progression of EAE
pathology.
[0053] FIG. 37 is cytograms and graphs showing expression of
prolactin gene or Zbtb20 gene under culturing in the presence of
microglia collected from each mouse having the late-stage EAE
pathology.
[0054] FIG. 38 is graphs showing the changes in expression of each
different cytokine during the course of progression of EAE
pathology.
[0055] FIG. 39 is graphs showing the changes in expression of each
different cytokine during the course of progression of EAE
pathology.
[0056] FIG. 40 is graphs showing the changes in expression of each
different chemokine during the course of progression of EAE
pathology.
DESCRIPTION OF EMBODIMENTS
Definitions
[0057] As used herein, the "progressive immune demyelinating
diseases" means diseases caused by myelin sheath impairment
resulting from an immune reaction, namely a persistently
progressive diseases without remission. The progressive immune
demyelinating disease is preferably CNS progressive immune
demyelinating diseases. Examples of the progressive immune
demyelinating diseases include progressive multiple sclerosis such
as PP-MS, SP-MS, and PR-MS.
[0058] NR4A2 gene is also referred to as Nurr1 gene, NOT gene, or
RNR1 gene and is a kind of orphan nuclear receptor. The major
expression site of NR4A2 gene lies in the central nervous system
and the NR4A2 gene is strongly expressed, in particular, in the
ventral mesencephalon, brain stem, and spinal cord. Meanwhile,
expression of NR4A2 can be induced in response to prostaglandins,
growth factors, inflammatory cytokines, and/or T-cell receptor
cross-linking, and NR4A2 can directly bind to DNA, in a
ligand-dependent or -independent manner, to regulate transcription.
The accession number of human NR4A2 gene transcript in the NCBI
Reference Sequences is NM_006186.3.
[0059] Eomes gene is also called Eomesodermin or Tbr2, is a kind of
T-box transcription factor, and is a protein participating in
development and differentiation of vertebrates. It has been known
that Eomes gene is expressed in CD8.sup.+ T cells (cytotoxic T
lymphocytes; CTL) and NK cells. In addition, it is also known that
Eomes gene can directly induce expression of perforin and granzyme
B. The accession numbers of human Eomes gene transcripts in the
NCBI Reference Sequences are NM_001278182.1 (variant 1),
NM_005442.3 (variant 2), and NM_001278183.1 (variant 3).
[0060] Zbtb20 is a protein belonging to one of subfamilies of C2H2
Kruppel-like zinc finger proteins and BTB/POZ domain-containing
zinc finger proteins. In addition, Zbtb20 can bind to a promoter of
prolactin gene and can thus promote transcriptional activation of
prolactin. Zbtb20 is highly expressed in all mature endocrine cell
types in the anterior pituitary gland. When Zbtb20 is deficient,
expression and secretion of prolactin decrease markedly.
[0061] [Prophylactic Agent, Onset-Suppressing Agent, or Therapeutic
Agent for Progressive Immune Demyelinating Diseases]
[0062] A prophylactic agent, onset-suppressing agent, or
therapeutic agent for progressive immune demyelinating diseases
according to a first embodiment of the present invention comprises,
as an active ingredient, a substance capable of suppressing or
inhibiting production of prolactin.
[0063] The progressive immune demyelinating disease is preferably
CNS progressive immune demyelinating diseases and more preferably
secondary progressive multiple sclerosis (SP-MS).
[0064] Examples of the substance capable of suppressing or
inhibiting production of prolactin include: expression suppressors
capable of suppressing expression of prolactin gene; dopamine
receptor agonists; dopamine receptor partial agonists; dopamine
reuptake inhibitors; dopamine degrading enzyme inhibitors; dopamine
analogs; and substances capable of suppressing or inhibiting
generation of Zbtb20. It is preferable that each dopamine receptor
agonist or partial agonist is a dopamine D2 receptor agonist or
partial agonist.
[0065] Each expression suppressor capable of suppressing expression
of prolactin gene may be a substance capable of suppressing a
prolactin from functioning as a protein. Examples of the expression
suppressor include: substances capable of suppressing expression of
prolactin gene at a transcription or translation level; and
substances capable of suppressing functional expression while
binding to a functional site of prolactin.
[0066] Examples of the substances capable of suppressing expression
of prolactin gene at a transcription or translation level include
nucleic acids, peptides, sugars or glycoproteins, and
low-molecular-weight compounds with a molecular weight of 1000 or
less, that can suppress gene expression of prolactin. Examples of
the nucleic acids capable of suppressing gene expression of
prolactin include at least one kind selected from the group
consisting of anti-sense oligonucleotides, siRNAs, shRNAs, miRNAs,
and ribozymes for prolactin gene.
[0067] Examples of the substances capable of suppressing functional
expression while binding to a functional site of prolactin include
anti-prolactin antibodies (e.g., neutralizing antibodies) or
antigen-binding fragments thereof.
[0068] Each expression suppressor capable of suppressing expression
of prolactin may be designed and produced, by known procedures in
the art, based on information on the genome sequence and mRNA
sequence of prolactin gene, the sequence and conformation of
prolactin protein, etc.
[0069] Examples of the dopamine receptor agonists include dopamine,
apomorphine, bromocriptine, cabergoline, ciladopa, dihydrexidine,
dinapsoline, doxanthrine, epicriptine, lisuride, pergolide,
piribedil, pramipexole, propylapomorphine, quinagolide, talipexole,
ropinirole, rotigotine, roxindole, and sumanirole.
[0070] Examples of the dopamine receptor partial agonists include
aripiprazole, brexpiprazole, phencyclidine, salvinorin A, and
quinpirole.
[0071] Examples of the dopamine reuptake inhibitors include
altropane, amfonelic acid, amineptine, BTCP, DBL-583, difluoropin,
GBR-12783, GBR-12935, GBR-13069, GBR-13098, GYKI-52895, lometopane,
methylphenidate, RTI-229, and vanoxerine.
[0072] Examples of the dopamine degrading enzyme inhibitors
include: monoamine oxidase B inhibitors such as selegiline,
zonisamide; and catechol-o-methyltransferase (COMT) inhibitors such
as entacapone.
[0073] Examples of the dopamine analogs include L-dopa (levodopa,
L-3,4-dihydroxyphenylalanine) and droxidopa
(L-threo-dihydroxyphenylserine).
[0074] Each expression suppressor capable of suppressing expression
of Zbtb20 gene may be a substance capable of suppressing the
functioning of ZBtb20 as a protein. Examples of the expression
suppressor include: substances capable of suppressing expression of
Zbtb20 gene at a transcription or translation level; and substances
capable of suppressing functional expression while binding to a
functional site of Zbtb20. Examples of the substances capable of
suppressing expression of Zbtb20 gene at a transcription or
translation level include nucleic acids, peptides, sugars or
glycoproteins, and low-molecular-weight compounds with a molecular
weight of 1000 or less, that can suppress gene expression of
Zbtb20. Examples of the nucleic acids capable of suppressing gene
expression of Zbtb20 include at least one kind selected from the
group consisting of anti-sense oligonucleotides, siRNAs, shRNAs,
miRNAs, and ribozymes for Zbtb20 gene. Examples of the substances
capable of suppressing functional expression while binding to a
functional site of Zbtb20 include nucleic acids, peptides, sugars
or glycoproteins, low-molecular-weight compounds with a molecular
weight of 1000 or less, and anti-Zbtb20 antibodies (e.g.,
neutralizing antibodies) or antigen-binding fragments thereof that
can suppress functional expression while binding to a functional
site of Zbtb20.
[0075] Each expression suppressor capable of suppressing expression
of Zbtb20 may be designed and produced, by known procedures in the
art, based on information on the genome sequence and mRNA sequence
of Zbtb20 gene, the sequence and conformation of Zbtb20 protein,
etc. The expression suppressors capable of suppressing expression
of Zbtb20 may be, for instance, substances capable of inhibiting or
suppressing a microRNA122/CUX1/microRNA214/ZBTB20 pathway. It has
been known that forced expression of microRNA214 or microRNA214
causes expression of Zbtb20 gene to be suppressed (Kojima et al.
Nature communications 2011, 2: 338, 1-10). In addition, the
expression suppressors capable of suppressing expression of Zbtb20
may be, for instance, substances capable of suppressing expression
of LMP2A (Latent membrane protein 2A) and/or IRF4 (Interferon
regulatory factor 4). It has been known that in LMP2A-expressing B
cells, expression of Irf4 and Zbtb20 genes is enhanced; and IRF4
protein encoded by Irf4 binds to a Zbtb20 promoter, thereby
enhancing expression of Zbtb20 gene (Minamitani et al. Proc. Natl.
Acad. Sci. 2015, 112, 37, 11612-11617).
[0076] The content of the above active ingredient (substance
capable of suppressing or inhibiting production of prolactin) in
the prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases according to
this embodiment is not particularly limited; and for instance, the
content may be from 0.001 to 100 mass % based on the total amount
of the prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases.
[0077] The prophylactic agent, onset-suppressing agent, or
therapeutic agent for progressive immune demyelinating diseases may
be constituted only of the above active ingredient or may contain,
in addition to the above active ingredient, an additional drug
usable for multiple sclerosis prophylactic, onset-suppressing, or
therapeutic purposes; and/or an additive(s) usually used in the
technical field of formulation, such as an excipient, a buffering
agent, a stabilizer, an antioxidant, a binder, a disintegrating
agent, a filler, an emulsifier, and/or a flow modifying additive.
In addition, it is preferable that the above additional drug exerts
the therapeutic efficacy by a mechanism different from the one for
suppressing or inhibiting signal transduction starting from the
prolactin receptor.
[0078] Examples of the additional drug include voltage-dependent
sodium channel inhibitors (e.g., lamotrigine), potassium channel
blockers (e.g., fampridine), voltage-dependent calcium channel
suppressors (e.g., gabapentin), siponimod (BAF312), HMG-CoA
inhibitors (e.g., statins such as simbastatin), SIP receptor
antagonists (e.g., fingolimod (FTY720), c-kit receptor inhibitors
(e.g., masitinib), MIS416, toeluna, potassium-sparing diuretics
(e.g., amiloride), riluzole, phosphodiesterase inhibitors (e.g.,
ibudilast (MN-166)), cyclophosphamide, steroids (e.g.,
methylprednisolone, prednisone), topoisomerase II inhibitors (e.g.,
mitoxantrone), ELND002, MD1003, ritalin, quetiapine fumarate, NMDA
receptor inhibitors (e.g., memantine), tacrolimus, teriflunomide
(HMR1726), BHT-3009-01, sunphenon EGCG (sunphenon epigallocatechin
gallate), CS-0777, glatiramer acetate (Copoxone (registered
trademark)), ONO-4641, purine metabolism antagonists (e.g.,
cladribine), lipoic acid, inosine, cannabis, nabiximols (Sativex
(registered trademark)), erythropoietin, interferon .beta.-1b,
adrenocorticotropic hormone (ACTH), estriol, synthetic partial
myelin basic protein peptides (e.g., dirucotide (MBP8298)),
monoclonal anti-human CD20 antibodies (e.g., rituximab), humanized
monoclonal anti-.alpha.4 integrin antibodies (e.g., natalizumab
(BG00002)), monoclonal anti-CD25 antibodies (e.g., daclizumab),
humanized monoclonal anti-IL-12 antibodies, monoclonal
anti-IL-12/23 antibodies (e.g., ABT-874 (briakinumab)), and
humanized monoclonal Nogo-A antibodies (e.g., ozanezumab,
GSK1223249).
[0079] Examples of a dosage form of the prophylactic agent,
onset-suppressing agent, or therapeutic agent for progressive
immune demyelinating diseases according to the first embodiment
include any dosage forms such as powder, pills, granules, tablets,
syrups, pastilles, capsules, and injections.
[0080] The prophylactic agent, onset-suppressing agent, or
therapeutic agent for progressive immune demyelinating diseases
according to the first embodiment may be administered orally or
parenterally. When the agent is administered to, for instance,
adult human males (with a body weight of 60 kg), the daily dose of
the prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases, as a specific
example of the dose, is usually from 0.0001 .mu.g to 10000
mg/day/person in terms of the active ingredient content.
[0081] The above first embodiment may also provide a method for
preventing or suppressing a progression into progressive immune
demyelinating diseases comprising a step of administering, to a
human subject of need, a substance capable of suppressing or
inhibiting production of prolactin. For instance, the above method
according to the first embodiment can prevent or suppress a
progression from relapsing-remitting immune demyelinating disease
to progressive immune demyelinating diseases.
[0082] A prophylactic agent, onset-suppressing agent, or
therapeutic agent for progressive immune demyelinating diseases
according to a second embodiment of the present invention
comprises, as an active ingredient, an anti-CX3CR1 antibody or an
antigen-binding fragment thereof.
[0083] The progressive immune demyelinating disease is preferably
CNS progressive immune demyelinating diseases and more preferably
secondary progressive multiple sclerosis (SP-MS).
[0084] The anti-CX3CR1 antibody or antigen-binding fragment thereof
may be a monoclonal or polyclonal antibody. The anti-CX3CR1
antibody may be any of a mouse antibody, a rat antibody, a guinea
pig antibody, a hamster antibody, a rabbit antibody, a monkey
antibody, a dog antibody, a chimeric antibody, a humanized
antibody, or a human antibody. The anti-CX3CR1 antibody may be
chemically modified in order to improve physical properties such as
blood retention. In addition, the anti-CX3CR1 antibody may be
conjugated to any radionuclide or toxin, etc., in order to increase
therapeutic efficacy.
[0085] The anti-CX3CR1 antibody may be a monoclonal or polyclonal
antibody. In addition, the anti-CX3CR1 antibody may be any of a
mouse antibody, a rat antibody, a guinea pig antibody, a hamster
antibody, a rabbit antibody, a monkey antibody, a dog antibody, a
chimeric antibody, a humanized antibody, or a human antibody. The
anti-CX3CR1 antibody may be chemically modified in order to improve
physical properties such as blood retention. In addition, the
anti-CX3CR1 antibody may be conjugated to any radionuclide or
toxin, etc., in order to increase therapeutic efficacy.
[0086] The antigen-binding fragment may be an antibody fragment
containing an antigen-binding site of the antibody; and examples
include Fab, Fab', F(ab').sub.2, scFv, and diabody.
[0087] The content of the above active ingredient (anti-CX3CR1
antibody or antigen-binding fragment thereof) in the prophylactic
agent, onset-suppressing agent, or therapeutic agent for
progressive immune demyelinating diseases according to this
embodiment is not particularly limited; and for instance, the
content may be from 0.001 to 100 mass % based on the total amount
of the prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases.
[0088] The prophylactic agent, onset-suppressing agent, or
therapeutic agent for progressive immune demyelinating diseases may
be constituted only of the above active ingredient or may contain,
in addition to the above active ingredient, an additional drug
usable for multiple sclerosis prophylactic, onset-suppressing, or
therapeutic purposes; and/or an additive(s) usually used in the
technical field of formulation, such as an excipient, a buffering
agent, a stabilizer, an antioxidant, a binder, a disintegrating
agent, a filler, an emulsifier, and/or a flow modifying additive.
In addition, it is preferable that the above additional drug exerts
the therapeutic efficacy by a mechanism different from the one for
suppressing or inhibiting signal transduction starting from the
CX3CR1 receptor. The additional drug is identical to the drug
designated in the first embodiment.
[0089] Examples of a dosage form of the prophylactic agent,
onset-suppressing agent, or therapeutic agent for progressive
immune demyelinating diseases according to the second embodiment
include any dosage forms such as powder, pills, granules, tablets,
syrups, pastilles, capsules, and injections.
[0090] The prophylactic agent, onset-suppressing agent, or
therapeutic agent for progressive immune demyelinating diseases
according to the second embodiment may be administered orally or
parenterally. When the agent is administered to, for instance,
adult human males (with a body weight of 60 kg), the daily dose of
the prophylactic agent, onset-suppressing agent, or therapeutic
agent for progressive immune demyelinating diseases, as a specific
example of the dose, is usually from 0.0001 .mu.g to 10000
mg/day/person in terms of the active ingredient content.
[0091] The above second embodiment may also provide a method for
treating progressive immune demyelinating diseases or suppressing
its pathology progression, the method comprising a step of
administering, to a human subject of need, an anti-CX3CR1 antibody
or an antigen-binding fragment thereof.
[0092] In the above first or second embodiment, the effects of
preventing progressive immune demyelinating diseases, suppressing
its onset, and suppressing its pathology progression may be
determined by analyzing an increase in the Eomes expression levels
on the Th cell surface. Examples of the procedure for analyzing an
increase in the Eomes expression levels on the Th cell surface
include a method for detecting Eomes.sup.+ CD4.sup.+ T cells in
lymphocyte-containing body fluid. The body fluid collected from
each human subject may be lymphocyte-containing body fluid.
Examples of the body fluid collected from each human subject
include blood and cerebrospinal fluid. The blood may be peripheral
blood. The Eomes.sup.+ CD4.sup.+ T cells may be detected in
accordance with conventional methods in the art.
[0093] The Eomes.sup.+ CD4.sup.+ T cell detection is not limited to
the above and may be performed by a step of separating, in
accordance with a conventional method, PBMCs from a
lymphocyte-containing body fluid sample collected from a human
subject and a step of causing the PBMCs to react with a labeled
anti-CD3 antibody or antigen-binding fragment thereof, a labeled
anti-CD4 antibody or antigen-binding fragment thereof, and a
labeled anti-Eomes antibody or antigen-binding fragment thereof so
as to detect Eomes.sup.+ CD4.sup.+ T cells by using a flow
cytometer.
[0094] The present invention also provides a method of collecting
data for diagnosing a progression into progressive immune
demyelinating diseases comprising: collecting microglia from a
human subject; and measuring an expression level of at least one of
IL-9, IFN-.alpha., and IFN-.beta.1.
[0095] As shown in FIG. 36, in the course of progression of EAE
pathology, the expression level of IFN-.alpha., IFN-.beta.1, and
IL-9 in microglia at the time of mid-EAE pathology increase
markedly. That is, the marked increase in the gene expression level
of each of these cytokines in microglia of each human subject
allows for collection of data for determining progression into
progressive immune demyelinating diseases.
[0096] This means that each cytokine increase is larger than a
threshold set based on the gene expression level of each cytokine
in microglia of each healthy adult or human subject who
undoubtfully has no progression into progressive immune
demyelinating diseases.
Examples
[0097] Hereinafter, the present invention will be more specifically
described based on Examples. However, the present invention is not
limited to the following Examples.
[0098] 1. Analysis of EAE in NR4A2c KO Mice
(1) Animals
[0099] Mice used were all 6 to 8 weeks old and reared under
specific pathogen-free conditions. A targeting vector having NR4A2
gene flanked by loxp sequences was used to establish
NR4A2.sup.fl/fl mice. Specifically, the NR4A2 gene flanked by loxp
sequences to be introduced was injected into a C57BL/6 embryonic
stem cell by microinjection. The established strain was crossed
with C57BL/6 FLPe mice (Riken BioResource Research Center) and the
resulting strains in which a neomycin cassette was deleted were
crossed with each other to generate a homozygous NR4A2.sup.fl/fl
C57BL/6 mouse. The resulting mouse was crossed with C57BL/6 CD4-Cre
mice (Taconic Farms, Inc.) to establish CD4-specific NR4A2c KO
C57BL/6 mice (C57BL/6 Cre-CD4/NR4A2.sup.fl\fl mice).
(2) EAE Induction (Monophasic EAE)
[0100] Equal volumes of 100 .mu.g of a peptide corresponding to
MOG.sub.35-55 residues (synthesized in Toray Research Center, Inc.,
Tokyo, Japan; hereinafter, sometimes referred to as a "MOG
peptide") and 1 mg of dead M. tuberculosis H37Ra (Difco, Kansas,
USA) emulsified using Complete Freund's adjuvant were mixed and
emulsified with a homogenizer to prepare MOG emulsion. The
resulting MOG emulsion was injected subcutaneously in 1 to 2 sites
of the back of each CD4-specific NR4A2c KO C57BL/6 mouse
(Cre-CD4/NR4A2.sup.fl/fl C57BL/6 mouse; NR4A2c KO) and, as a
control, each NR4A2.sup.fl/fl C57BL/6 mouse (Control) to immunize
them. Further, day 0 and day 2 after the immunization, 200 .mu.L of
PBS solution containing 200 ng per mouse of pertussis toxin (List
Biological Laboratories, USA) was injected intraperitoneally into
each mouse.
(3) Infiltration ofT Cells into Central Nervous System
[0101] From each C57BL/6 mouse (Control) and each NR4A2-deficient
mouse (NR4A2c KO) in which monophasic EAE had been induced in a
similar manner to the above 1.(2), the brain and the spinal cord
were collected at day 10 after the induction (corresponding to the
early-stage EAE pathology), day 14 after the induction
(corresponding to the mid-stage EAE pathology), and day 18 after
the induction (corresponding to the late-stage EAE pathology).
Then, a flow cytometer was used to separate CD19.sup.+ B cells and
non-B/class II.sup.+ cells infiltrated into the CNS. Specifically,
each tissue was cut into small pieces, which were then further
dissociated at 37.degree. C. for 40 min in RPMI 1640 medium
(manufactured by Invitrogen, Inc.) containing 1.4 mg/mL collagenase
H and 100 .mu.g/mL DNase I (manufactured by Roche Inc.). The
resulting tissue homogenate was made to pass through a 70-.mu.m
cell strainer (manufactured by GE Healthcare, Inc.), and was
centrifuged on a Percoll discontinuous density gradient (37%/80%)
to enrich leukocytes. Next, CD19.sup.+ B cells or non-B/class
II.sup.+ cells infiltrated into the CNS were sorted by FACS using a
FACS ARIA II (manufactured by BD Cytometry Systems, Inc.). The
respective sorted CD19.sup.+ B cells or non-B/class II.sup.+ cells
were co-cultured with spleen-derived CD226.sup.+ Th cells for 8 h.
After the culturing, a flow cytometer was used to analyze a change
in the expression level of each of Eomes and CD107a in and on the
Th cells recovered. As antibodies used at the sorting were an
anti-CD3 antibody (manufactured by Biolegend, Inc.), an anti-Eomes
antibody (manufactured by eBioscience, Inc.), and an anti-CD107a
antibody (manufactured by Biolegend, Inc.).
[0102] The results are shown in FIGS. 1 and 2. FIG. 1 demonstrates
that in each mouse with EAE at day 18 after the induction
(corresponding to the late-stage EAE pathology), the CNS-derived
antigen-presenting cells have an ability of inducing marked Eomes
expression in the Th cells.
(4) Eomes Expression at Each Progression Stage of EAE Pathology
[0103] From each wild-type C57BL/6 mouse in which monophasic EAE
had been induced in a similar manner to the above 1.(2), the brain
and the spinal cord were collected at day 10 after the induction
(the early-stage EAE pathology), day 14 (the mid-stage EAE
pathology), and day 18 (the late-stage EAE pathology). Then, a flow
cytometer was used to separate CD4.sup.+ T cells infiltrated into
the CNS. Note that the collection timings correspond to the Early,
Mid, and Late in the graph of FIG. 3(a). Specifically, each tissue
was cut into small pieces, which were then further dissociated at
37.degree. C. for 40 min in RPMI 1640 medium (manufactured by
Invitrogen, Inc.) containing 1.4 mg/mL collagenase H and 100
.mu.g/mL DNase I (manufactured by Roche Inc.). The resulting tissue
homogenate was made to pass through a 70-.mu.m cell strainer
(manufactured by GE Healthcare, Inc.), and was centrifuged on a
Percoll discontinuous density gradient (37%/80%) to enrich
leukocytes. Next, CD4.sup.+ T cells infiltrated into the CNS were
sorted by FACS using a FACS ARIA II (manufactured by BD Cytometry
Systems, Inc.).
[0104] Then, a flow cytometer was used to analyze a change in the
Eomes expression levels in the CD4.sup.+ T cells separated. As
antibodies used at the detection were an anti-CD4 antibody
(manufactured by Biolegend, Inc.) and an anti-Eomes antibody
(manufactured by eBioscience, Inc.).
[0105] Next, each CD4.sup.+ T cell was subjected to intracellular
staining to measure each Eomes expression level. The results are
shown in FIG. 3(b). The graph of FIG. 3(b) indicates the percentage
(%) of Eomes.sup.+ T cells with respect to the CD4.sup.+ T cells in
each pathology progression stage. It was observed that the
percentage of Eomes.sup.+ CD4.sup.+ T cells increased markedly in
the late-stage EAE pathology.
[0106] In addition, a flow cytometer was used to separate, from the
brain and the spinal cord collected, CD45.sup.hi cells, CD19.sup.+
B cells, and CD19.sup.- B cells infiltrated into the CNS. The
separated CD45.sup.hi cells, CD19.sup.+ B cells, or CD19.sup.- B
cells were sorted by FACS. As antibodies used at the detection were
an anti-CD45 antibody (manufactured by Biolegend, Inc.), an
anti-CD19 antibody (manufactured by Biolegend, Inc.), an
anti-TCR.beta. antibody (manufactured by Biolegend, Inc.), and an
anti-MHC class II antibody (manufactured by Biolegend, Inc.).
[0107] The results are shown in FIG. 4. It was observed that in the
mid-stage EAE pathology, infiltration of the CD19.sup.+ B cells or
CD19.sup.- non-B/class II.sup.+ antigen-presenting cells into the
CNS increased markedly; and in the late-stage EAE pathology, the
numbers of these types of cells decreased when compared with those
in the mid-stage EAE pathology.
(5) Gene Expression Level of Each of Prolactin and Growth Hormone
in Each EAE Pathology
[0108] In a similar manner to the above 1.(3), CD19.sup.+ B cells
or non-B/class II.sup.+ cells were isolated over time from the CNS
of each mouse with EAE. Then, the gene expression levels of each of
prolactin (Prl) and growth hormone (Gh) were analyzed by a
quantitative PCR assay. As primers for prolactin and growth hormone
were used those in Mm_Prl_1_SG QuantiTect Primer Assay and
Mm_Gh_1_SG QuantiTect Primer Assay (both from QIAGEN Inc.),
respectively.
[0109] The results are shown in FIG. 5. In FIG. 5, the "EB" means
CD19.sup.+ B cells isolated from the early-stage EAE conditions;
the "ED" means non-B/class II.sup.+ cells isolated from the
early-stage EAE conditions; the "MB" means CD19.sup.+ B cells
isolated from the mid-stage EAE conditions; the "MD" means
non-B/class II.sup.+ cells isolated from the mid-stage EAE
conditions; the "LB" means CD19.sup.+ B cells isolated from the
late-stage EAE conditions; and the "LD" means non-B/class II.sup.+
cells isolated from the late-stage EAE conditions. According to
FIG. 5, it was observed that the expression level of each of
prolactin and growth hormone increased markedly in the
antigen-presenting cells derived from the CNS in the late-stage EAE
conditions.
(6) Expression of Prolactin or Growth Hormone in CSF from Each
Progression Stage of EAE Pathology
[0110] A triple anesthesia (medetomidine, midazolam, and
butorphanol) was intraperitoneally (ip) administered to anesthetize
each mouse. The mice used included intact mice, mice with the
early-stage EAE pathology, mice with the mid-stage EAE pathology,
and mice with the late-stage EAE pathology. The skin of the
occipital region in each mouse was cut like an arrowhead; the
subcutaneous tissue and muscles were removed carefully; and a
surface of dura mater covering cisterna magna was exposed. A glass
capillary was put into the exposed dura mater for paracentesis and
CSF was collected by utilizing a capillary phenomenon. Each CSF
obtained was stored in a freezer at -80.degree. C.
[0111] Then, a Luminex system (manufactured by Luminex Corporation)
was used to measure protein expression levels of prolactin (PRL) or
growth hormone (GH) in each CSF collected.
[0112] The results are shown in FIG. 6. It was observed that in the
mid-stage EAE pathology, the protein expression levels of growth
hormone increased; and in the late-stage EAE pathology, the protein
expression levels of each of prolactin and growth hormone
increased.
(7) Effects of Culturing in Co-Presence of Prolactin on Eomes
Expression
[0113] Spleen-derived CD226.sup.+ CD4.sup.+ T cells collected from
each intact wild-type B6 mouse were cultured for 4 h or 8 h in the
absence of prolactin or in the presence of each specific amount of
prolactin. The Eomes expression levels in the respective cells
after the culturing were measured by using a flow cytometer or a
quantitative real-time PCR assay. As antibodies used at the
detection were an anti-CD4 antibody (manufactured by Biolegend,
Inc.) and an anti-Eomes antibody (manufactured by eBioscience,
Inc.).
[0114] The results are shown in FIG. 7. It was observed that when
cultured in the presence of prolactin at 30 or 100 ng/mL, the
percentage of CD4.sup.+ Eomes.sup.+ T cells increased markedly
regardless of the culturing time. FIG. 8(a) collectively provides,
as a graph, each percentage of Eomes.sup.+ cells on the basis of
each flow cytogram shown in FIG. 7. FIG. 8(b) shows, as a graph,
the relative expression level of prolactin protein in the
respective cells. In FIG. 8, It was also observed that when the
cells were cultured in the presence of prolactin at 30 ng/mL, the
gene expression levels of prolactin protein increased.
(8) Change in Gene Expression Levels of Each of Prolactin and
Zbtb20 During Course of Progression of EAE Pathology
[0115] In a similar manner to the above 1.(3), CD19.sup.+ B cells
or non-B/class II.sup.+ cells were isolated over time from the CNS
of each mouse with EAE. Then, the gene expression levels of each of
prolactin and Zbtb20 were analyzed by a quantitative PCR assay. As
primers for prolactin and Zbtb20 were used those in Mm_Prl_1_SG
QuantiTect Primer Assay and Mm_Zbtb20_1_SG QuantiTect Primer Assay
(both from QIAGEN Inc.), respectively.
[0116] The results are shown in FIG. 9. The "EB", "MB", "LB", "ED",
"MD", and "LD" in FIG. 9 are each defined in the same manner as in
FIG. 5. According to FIG. 9, it was observed that the gene
expression levels of each of prolactin and Zbtb20 increased in the
antigen-presenting cells derived from the CNS in the late-stage EAE
conditions.
(9) Change in Protein Expression Levels of Each of Prolactin and
Zbtb20 During Course of Progression of EAE Pathology
[0117] From each C57BL/6 mouse (Control) and each NR4A2-deficient
mouse (NR4A2c KO) in which monophasic EAE had been induced in a
similar manner to the above 1.(2), the brain and the spinal cord
were collected at day 9 after the induction (corresponding to the
early-stage EAE pathology) and day 19 after the induction
(corresponding to the late-stage EAE pathology). Then, a flow
cytometer was used to separate CD19.sup.+ B cells and non-B/class
II.sup.+ cells infiltrated into the CNS. Specifically, each tissue
was cut into small pieces, which were then further dissociated at
37.degree. C. for 40 min in RPMI 1640 medium (manufactured by
Invitrogen, Inc.) containing 1.4 mg/mL collagenase H and 100
.mu.g/mL DNase I (manufactured by Roche Inc.). The resulting tissue
homogenate was made to pass through a 70 .mu.m cell strainer
(manufactured by GE Healthcare, Inc.), and was centrifuged on a
Percoll discontinuous density gradient (37%/80%) to enrich
leukocytes. Next, CD19.sup.+ B cells or non-B/class II.sup.+ cells
infiltrated into the CNS were analyzed with a FACS CANTO II
(manufactured by BD Cytometry Systems, Inc.). The flow cytometer
was used to analyze a change in the expression level of Zbtb20 in
the CD19.sup.+ T cells or non-B/class II.sup.+ cells separated. As
an antibody used at the detection was an anti-Zbtb20 antibody
(manufactured by BD Bioscience, Inc.).
[0118] The results are shown in FIGS. 10 and 11. In FIG. 10, it was
observed that the gene expression levels of Zbtb20 increased
significantly in the late-stage EAE pathology. Meanwhile, FIG.
11(a) shows, as a graph, each percentage of Zbtb20.sup.+ cells; and
it was observed that the Zbtb20 expression increased markedly in
the late-stage EAE pathology. In addition, as shown in FIG. 11(b),
the gene expression levels of prolactin gene (Prl) were measured in
the B cells or the non-B antigen-presenting cells; and it was
observed that the relative expression level of prolactin gene (Prl)
increased markedly in the late-stage EAE pathology.
[0119] In addition, B cells collected from the CNS of each mouse
with the late-stage EAE pathology were cultured for 6 h in the
presence of 10 ng/mL lipopolysaccharide (LPS) and BD GolgiPlug. The
cultured B cells were sorted, based on the staining conditions of
CD1d and CD5 genes, into 4 different fractions (Fr1, Fr2, Fr3, and
Fr4) as shown in FIG. 12(a). The IL-10 and Zbtb20 gene expression
levels in each fraction were measured in FACS plots. The results
are shown in FIG. 12(b).
[0120] Further, non-B antigen-presenting cells collected from the
CNS of each mouse with the late-stage EAE pathology were stained
and analyzed by FACS. The non-B antigen-presenting cells were
sorted, based on the staining conditions of CD11c and PDCA-1 genes,
into 3 different fractions (Fr1, Fr2, and Fr3) as shown in FIG.
13(a). The Zbtb20 gene expression levels in each fraction were
measured in FACS plots. As antibodies used at the detection were an
anti-CD11c antibody (manufactured by Biolegend, Inc.), an
anti-PDCA-1 antibody (manufactured by eBioscience, Inc.), and an
anti-zbtb20 antibody (manufactured by Becton Dickinson, Inc.).
[0121] The results are shown in FIG. 13(a). In addition, FIG. 13(b)
shows the percentage of Zbtb20.sup.+ cells in each fraction.
(10) Expression of Prolactin or Growth Hormone in Pituitary
[0122] From each intact mouse or each mouse with the early-, mid-,
or late-stage EAE pathology, the pituitary was excised, total RNA
was isolated, and the expression levels of prolactin gene (Prl)
were then measured by using a quantitative real-time PCR assay. The
results are shown in FIG. 14(a).
[0123] In addition, serum of each mouse was collected and a Luminex
system was used to measure the protein levels of each of prolactin
(PRL) and growth hormone (GH) in each serum. The results are shown
in FIG. 14(b). In any of the progression stages of EAE pathology,
it was observed that the serum prolactin (PRL) levels were markedly
lower than those of each intact mouse.
[0124] Further, from the CNS of each intact mouse or each mouse
with the early-, mid-, or late-stage EAE pathology, a FACS ARIA was
used to purify CD19.sup.+ B cells, PDCA-1.sup.+ CD11c.sup.+ plasma
cell-like dendritic cells (pDCs), and CD45.sup.int CD11b.sup.+
microglial cells. Then, the prolactin gene (Prl) and zbtb20 gene
expression levels were measured by using a quantitative real-time
PCR assay. As antibodies used at the detection were an anti-CD19
antibody (manufactured by Biolegend, Inc.), an anti-PDCA-1 antibody
(manufactured by eBioscience, Inc.), an anti-CD45 antibody
(manufactured by Biolegend, Inc.), an anti-CD11c antibody
(manufactured by Biolegend, Inc.), and an anti-zbtb20 antibody
(manufactured by Becton Dickinson, Inc.). To quantify Prl and GH, a
Mouse pituitary magnetic bead panel (manufactured by Millipore,
Inc.) was used.
[0125] The results are shown in FIG. 14(c). It was observed that
the expression levels of prolactin gene increased markedly in
CD19.sup.+ B cells and PDCA-1.sup.+ B220.sup.+ pDCs in the
late-stage EAE pathology. In addition, it was observed that the
expression levels of zbtb20 gene increased in CD19.sup.+ B cells
and CD45.sup.int CD11b.sup.+ microglial cells as the EAE pathology
progressed.
[0126] 2. Change in Late-Stage EAE Pathology by Prolactin
(1) Prolactin Addition and Enhanced Expression of Eomes Gene-1
[0127] Spleen-derived Th cells of each intact mouse were cultured
for 8 h in the absence or presence of recombinant prolactin. After
the culturing, total RNA was extracted from the Th cells. A first
strand cDNA synthesis kit (manufactured by Takara Bio Inc.) was
used to synthesize cDNA from the total RNA obtained. A Light Cycler
instrument was used under conditions using a Light Cycler-Fast
Start DNA Master SYBR Green I kit (manufactured by Roche
Diagnostics, Inc.) or an ABI 7300 real-time PCR instrument was used
under conditions using a Power SYBR Green Master mix (manufactured
by Applied Biosystems, Inc.) to perform a quantitative real-time
PCR assay by using commercially available primers (QuantiTect
Primer Assay, QT01074332; manufactured by Qiagen, Inc.). The Eomes
gene expression levels were corrected based on the expression level
of a housekeeping gene GAPDH.
[0128] The results are shown in FIG. 15. In FIG. 15, a reference
numeral "1" denotes the level obtained by culturing without
addition of prolactin; a reference numeral "2" denotes the level
obtained by culturing in the presence of prolactin at a low
concentration; and a reference numeral "3" denotes the level
obtained by culturing in the presence of prolactin at a high
concentration. In FIG. 15, the ordinate represents the relative
expression level of Eomes relative to that of a housekeeping gene
(.beta.2-microglobulin); and the Eomes expression level increased
in a prolactin concentration-dependent fashion.
(2) Prolactin Addition and Enhanced Expression of Eomes
Protein-2
[0129] Spleen-derived CD226.sup.+ Th cells were cultured for 8 h or
48 h in the absence or presence of prolactin. After the culturing,
the CD226.sup.+ Th cells were stained with an anti-CD226 antibody
and an anti-Eomes antibody, and a FACS CANTO II (manufactured by BD
Cytometry Systems, Inc.) was used to analyze CD226.sup.+
Eomes.sup.+ T cells, CD226.sup.+ Eomes.sup.- T cells, and
CD226.sup.- T cells. As the antibodies used during the analysis
were an anti-CD226 antibody (manufactured by Biolegend, Inc.) and
an anti-Eomes antibody (manufactured by Biolegend, Inc.).
[0130] The results are shown in FIG. 16. The Eomes expression
levels in the cells after cultured for 48 h in the presence of
prolactin increased more markedly than in the cells after cultured
in the absence of prolactin.
(3) Suppression of Late-Stage EAE Pathology by D2 Receptor
Agonist
[0131] Into each control mouse (Control) or each
CD4-Cre/NR4A2.sup.fl/fl mouse in which monophasic EAE had been
induced in a similar manner to the above 1.(2), bromocriptine was
intraperitoneally injected every other day from day 4 after the
induction. After the injection, the EAE pathology of each mouse was
daily evaluated in accordance with the EAE criteria as indicated
below.
<EAE Criteria>
[0132] 0: No clinical symptoms; 1: partial tail paralysis; 2: limp
tail; 3: partial hindlimb paralysis; 4: complete hindlimb paralysis
5: hindlimb and forelimb paralysis.
[0133] The results are shown in FIG. 17. Each arrow in FIG. 17
denotes the day when bromocriptine was administered. The
bromocriptine dosing caused the late-stage EAE pathology to be
suppressed significantly.
(4) Suppression of Expression of Eomes Protein by D2 Receptor
Agonist (Bromocriptine) Dosing-1
[0134] Regarding each CD4-Cre/NR4A2.sup.fl/fl mouse in which
monophasic EAE had been induced in a similar manner to the above
1.(2), the brain and the spinal cord were collected from each
non-injected mouse and each mouse into which bromocriptine was
intravenously injected every other day from day 4 after the
induction. Then, a flow cytometer was used to separate Th cells
infiltrated into the CNS. Specifically, each tissue was cut into
small pieces, which were then further dissociated at 37.degree. C.
for 40 min in RPMI 1640 medium (manufactured by Invitrogen, Inc.)
containing 1.4 mg/mL collagenase H and 100 .mu.g/mL DNase I
(manufactured by Roche Inc.). The resulting tissue homogenate was
made to pass through a 70-.mu.m cell strainer (manufactured by GE
Healthcare, Inc.), and was centrifuged on a Percoll discontinuous
density gradient (37%/80%) to enrich leukocytes. Next, Th cells
infiltrated into the CNS were sorted by FACS using a FACS ARIA II
(manufactured by BD Cytometry Systems, Inc.). Then, a flow
cytometer was used to analyze a change in the expression level of
each of Eomes and CD4 in the Th cells recovered. As antibodies used
at the sorting were an anti-CD4 antibody (manufactured by
Biolegend, Inc.) and an anti-Eomes antibody (manufactured by
eBioscience, Inc.).
[0135] The results are shown in FIGS. 18 and 19. As shown in FIG.
18, the bromocriptine dosing caused the gene expression levels of
Eomes protein in the CNS-infiltrated Th cells to be suppressed
significantly. FIG. 19 is graphs showing changes in the percentage
and the actual number of Eomes.sup.+ CD4.sup.+ T cells.
(5) Suppression of Expression of Eomes Gene by D2 Receptor Agonist
(Bromocriptine) Dosing-2
[0136] Regarding each CD4-Cre/NR4A2.sup.fl/fl mouse in which
monophasic EAE had been induced in a similar manner to the above
1.(3), the brain and the spinal cord were collected, at day 27
after the induction, from each non-injected mouse and each mouse
into which bromocriptine was intravenously injected, every other
day from day 4 after the induction. Next, CD19.sup.+ B cells or
non-B/class II.sup.+ cells infiltrated into the CNS were sorted by
FACS using a FACS ARIA II (manufactured by BD Cytometry Systems,
Inc.). The respective sorted CD19.sup.+ B cells or non-B/class
II.sup.+ cells were co-cultured with spleen-derived CD4.sup.+ Th
cells for 8 h. After the culturing, a flow cytometer was used to
analyze a change in the expression level of each of Eomes and
CD107a in and on the Th cells recovered. As antibodies used at the
detection were an anti-Eomes antibody (manufactured by eBioscience,
Inc.) and an anti-CD107a antibody (manufactured by Biolegend,
Inc.).
[0137] The results are shown in FIG. 20. According to FIG. 20, the
bromocriptine dosing significantly suppressed the abilities of the
CNS-derived antigen-presenting cells to induce CD107a expression
and Eomes protein expression on and in the Th cells.
(6) Expression of Eomes Gene and CD107a Gene by D2 Receptor Agonist
(Bromocriptine) Dosing
[0138] Into each CD4-Cre/NR4A2.sup.fl/fl mouse in which monophasic
EAE had been induced in a similar manner to the above 1.(3),
bromocriptine or a placebo (DMSO and PBS) was intraperitoneally
injected every other day from day 4 after the induction. Next, the
brain and the spinal cord were collected at day 32 after the
induction; and CD19.sup.+ B cells or CD19.sup.- CD45.sup.hi
non-B/class II.sup.+ antigen-presenting cells infiltrated into the
CNS were sorted by FACS using a FACS ARIA II (manufactured by BD
Cytometry Systems, Inc.). The sorted cells were co-cultured, in the
presence of an FITC-conjugated anti-CD107a antibody, with CD4.sup.+
T cells sorted by FACS from the spleen derived from each intact
mouse of similar genetic strain. The respective cells were
co-cultured for 8 h, stained, and analyzed regarding the Eomes
expression. As antibodies used at the detection were an anti-CD4
antibody (manufactured by Biolegend, Inc.), an anti-Eomes antibody
(manufactured by eBioscience, Inc.), an anti-CD45 antibody
(manufactured by Biolegend, Inc.), and an FITC-conjugated CD107a
antibody (manufactured by Biolegend, Inc.).
[0139] The results are shown in FIG. 21. It was observed that the
Eomes gene and CD107a gene expression levels on the cell surface of
the co-cultured CD4.sup.+ T cells even when co-cultured with any of
the CD19.sup.+ B cells and CD19.sup.- CD45.sup.hi non-B/class
II.sup.+ antigen-presenting cells decreased by the bromocriptine
dosing.
(7) Suppression of Expression of Eomes Gene by D2 Receptor Agonist
(Dopamine) Dosing
[0140] CD19.sup.+ B cells or CD19.sup.- CD45.sup.hi non-B/class
II.sup.+ antigen-presenting cells were purified from each mouse
with the late-stage EAE pathology by sorting by FACS. The
respective purified cells were cultured for 24, 48, or 96 h in the
absence of dopamine or in the presence of a specific amount of
dopamine. Next, the cultured cells were recovered and the prolactin
gene (Prl) and Zbtb20 gene expression levels were analyzed by using
a quantitative real-time PCR assay.
[0141] The results are shown in FIG. 22. FIG. 22(a) shows the gene
expression levels in CD19.sup.+ B cells; and FIG. 22(b) shows the
gene expression levels in CD19.sup.- CD45.sup.hi non-B class
II.sup.+ cells. It was observed that in either case, the prolactin
and Zbtb20 gene expression levels decreased markedly after
culturing in the co-presence of dopamine.
(8) Suppression of Expression of Eomes Gene by Dopamine Precursor
Substance (L-Dopa) Dosing
[0142] Into each CD4-Cre/NR4A2.sup.fl/fl mouse in which monophasic
EAE had been induced in a similar manner to the above 1.(3), L-dopa
or a placebo (DMSO and PBS) was intraperitoneally injected every
other day from day 4 after the induction.
[0143] The EAE scores of each mouse are shown in FIG. 23. It was
observed that repeated dosing of L-dopa caused the EAE scores to
improve markedly. In the right graph of FIG. 23, the dashed lines
each indicate 95% confidence interval. In comparison with the
cumulative EAE scores, an improvement in the EAE scores by the
repeated dosing of L-dopa was confirmed.
[0144] Next, the brain and the spinal cord were collected from each
mouse at day 32 after the induction; and CD4.sup.+ T cells,
CD19.sup.+ B cells, or CD19.sup.- CD45.sup.hi non-B/class II.sup.+
antigen-presenting cells infiltrated into the CNS were sorted by
FACS using a FACS ARIA II (manufactured by BD Cytometry Systems,
Inc.). The respective sorted cells were stained and analyzed
regarding the Eomes and Zbtb20 gene expressions. As antibodies used
at the detection were an anti-CD4 antibody (manufactured by
Biolegend, Inc.), an anti-Eomes antibody (manufactured by
eBioscience, Inc.), an anti-CD19 antibody (manufactured by
Biolegend, Inc.), and an anti-Zbtb20 antibody (manufactured by
Becton Dickinson, Inc.).
[0145] The results are shown in FIG. 24. As shown in FIG. 24(a), it
was observed that the repeated dosing of L-dopa caused the
percentage of CD4.sup.+ Eomes.sup.+ T cells to decrease markedly in
the CD4.sup.+ T cells. In addition, as shown in FIGS. 24(b) and
(c), it was observed that the repeated dosing of L-dopa caused the
percentage of Zbtb20.sup.+ cells to decrease in the CD19.sup.+ B
cells and the CD19.sup.- CD45.sup.hi non-B/class II.sup.+
antigen-presenting cells.
[0146] Next, CD19.sup.+ B cells and CD19.sup.- CD45.sup.hi
non-B/class II.sup.+ antigen-presenting cells were separated from
the CNS or the spleen of each L-dopa-administered mouse or each
intact mouse; and the expression levels of prolactin gene (Prl) in
these types of cells were analyzed by using a quantitative
real-time PCR assay. As antibodies used at the detection were an
anti-CD19 antibody (manufactured by Biolegend, Inc.) and an
anti-CD45 antibody (manufactured by Biolegend, Inc.).
[0147] The results are shown in FIG. 25. It was observed that the
expression levels of prolactin gene were markedly high in the
CNS-derived B cells and non-B/class II.sup.+ antigen-presenting
cells; and the repeated dosing of L-dopa caused the expression
levels to decrease.
[0148] 3. Suppression of Expression of Eomes Gene by Anti-CX3CR1
Antibody Dosing
[0149] An anti-CX3CR1 antibody (manufactured by Biolegend, Inc.) or
an isotype thereof (manufactured by Biolegend, Inc.) was
administered to each NR4A2-deficient mouse, in which monophasic EAE
had been induced in a similar manner to the above 1.(3), at day 10
after the induction. Then, the EAE pathology of each mouse was
daily evaluated in accordance with the above-described EAE
criteria.
[0150] The results are shown in FIG. 26. FIG. 26 shows that when
the anti-CX3CR1 antibody was administered (CX3CR1), the late-stage
EAE pathology improved more significantly than when the isotype
(Isotype) was administered.
[0151] In addition, at day 22 and day 28 after the induction, the
CNS of each mouse to which the anti-CX3CR1 antibody or the isotype
thereof was administered was collected and CD4.sup.+ cells were
separated. Regarding the CD4.sup.+ cells obtained, the percentages
and the absolute numbers of Eomes.sup.+ CD4.sup.+ cells or
Eomes.sup.++ CD4.sup.+ cells (Eomes-strong-positive, CD4-positive
Th cells) with respect to the CD4.sup.+ cells were measured.
[0152] The results are shown in FIG. 27. As shown in FIG. 27, in
any of the Eomes.sup.+ CD4.sup.+ cells and the Eomes.sup.++
CD4.sup.+ cells, the percentages and the absolute numbers of the
Eomes-positive Th cells with respect to the CD4.sup.+ cells in the
late-stage EAE pathology decreased significantly by the anti-CX3CR1
antibody dosing.
[0153] 4. Suppression of Expression of Eomes Gene by
Zbtb20-Specific siRNA Dosing
(1) Change in Clinical Scores of EAE Pathology
[0154] Into each NR4A2-deficient mouse in which monophasic EAE had
been induced in a similar manner to the above 1.(3), atelocollagen
matrix-stabilized Zbtb20-specific siRNA (manufactured by KOKEN Co.,
LTD.) or control scrambled siRNA (manufactured by KOKEN Co., LTD.)
was intravenously injected at day 7 after the induction
(corresponding to the early-stage EAE pathology). Then, the EAE
pathology of each mouse was daily evaluated in accordance with the
above-described EAE criteria.
[0155] The results are shown in FIG. 28. In the right graph of FIG.
28, the ordinate represents a cumulative clinical score. In each
control scrambled siRNA-administered mouse, the clinical scores
were substantially the same as in each intact mouse; by contrast,
in each Zbtb20-specific siRNA-administered mouse, a marked decrease
in the clinical scores was recorded. In the right graph of FIG. 28,
the right graph shows, as a graph, the cumulative clinical scores;
and the dashed lines each indicate 95% confidence interval.
(2) Suppression of Expression of Eomes or Zbtb20 Gene by
Zbtb20-Specific siRNA Dosing
[0156] The brain and the spinal cord were collected from each
intact mouse, each Zbtb20-specific siRNA-administered mouse, or
each control scrambled siRNA-administered mouse treated in a
similar manner to the above 4.(1) were collected and CD4.sup.+ T
cells and B cells were separated. The expression of Eomes or Zbtb20
gene in the CD4.sup.+ T cells or B cells, respectively, from each
mouse group obtained was analyzed with a flow cytometer. As
antibodies used at the detection were an anti-CD4 antibody
(manufactured by Biolegend, Inc.), an anti-Eomes antibody
(manufactured by eBioscience, Inc.), an anti-CD19 antibody
(manufactured by Biolegend, Inc.), and an anti-Zbtb20 antibody
(manufactured by Becton Dickinson, Inc.).
[0157] The results are shown in FIG. 29. As shown in FIG. 29(a), it
was observed that the expression of Eomes gene in the CD4.sup.+ T
cells and the expression of Zbtb20 gene in the B cells in each
Zbtb20-specific siRNA-administered mouse were both markedly
suppressed. FIG. 29(b) shows, as a graph for comparison, the
percentage of Eomes- or Zbtb20-expressing cells in each mouse
group.
(3) Suppression of Expression of Prolactin Gene by Zbtb20-Specific
siRNA Dosing
[0158] By using each intact mouse, each Zbtb20-specific
siRNA-administered mouse, or each control scrambled
siRNA-administered mouse treated in a similar manner to the above
4.(1), antigen-presenting cells were separated from the CNS of each
mouse with the late-stage EAE pathology. Next, CD19.sup.-
CD45.sup.hi non-B/class II.sup.+ antigen-presenting cells were
purified from the obtained antigen-presenting cells by sorting by
FACS using a FACS ARIA II (manufactured by BD Cytometry Systems,
Inc.). The purified cells were transfected with the Zbtb20-specific
siRNA or the control scrambled siRNA and cultured for 24 h in the
presence of LPS; and the Zbtb20 or prolactin (Prl) expression
levels were then measured by using a quantitative real-time PCR
assay. As antibodies used at the detection were an anti-CD4
antibody (manufactured by Biolegend, Inc.), an anti-Eomes antibody
(manufactured by eBioscience, Inc.), an anti-CD19 antibody
(manufactured by Biolegend, Inc.), and an anti-CD45 antibody
(manufactured by Biolegend, Inc.).
[0159] The results are shown in FIG. 30. It was observed that the
dosing of the Zbtb20-specific siRNA caused the Zbtb20 gene and Prl
gene expression levels to decrease markedly.
[0160] 5. Suppression of Expression of Eomes Gene by Anti-CD20
Antibody Dosing
(1) Change in Clinical Scores of EAE Pathology
[0161] Into each NR4A2-deficient mouse in which monophasic EAE had
been induced in a similar manner to the above 1.(3), an anti-CD20
antibody or control IgG was intravenously injected 7 days before or
at day 13 after the induction; and the EAE pathology of each mouse
was daily evaluated in accordance with the above-described EAE
criteria.
[0162] The results are shown in FIGS. 31(a) and (b). Each arrow in
FIGS. 31(a) and (b) denotes the day when the antibody was
administered. FIG. 31(a) shows the results obtained when the
antibody was administered 7 days before the induction; and FIG.
31(b) shows the results obtained when the antibody was administered
at day 13 after the induction. As shown in FIG. 31(b), the clinical
scores improved markedly by dosing the anti-CD20 antibody at day 13
after the induction. The right graph of FIG. 31(b) shows, as a
graph, the cumulative clinical scores; and the dashed lines each
indicate 95% confidence interval.
(2) Suppression of Expression of Eomes or Zbtb20 Gene by Anti-CD20
Antibody
[0163] The brain and the spinal cord were collected from each
intact mouse, each anti-CD20 antibody-administered mouse, or each
control IgG-administered mouse treated in a similar manner to the
above 5.(1), and CD4.sup.+ T cells and B cells were separated. The
expression of Eomes or Zbtb20 gene in the CD4.sup.+ T cells or B
cells, respectively, from each mouse group obtained was analyzed
with a flow cytometer. As antibodies used at the detection were an
anti-CD4 antibody (manufactured by Biolegend, Inc.), an anti-Eomes
antibody (manufactured by eBioscience, Inc.), an anti-CD20 antibody
(manufactured by Biolegend, Inc.), an anti-CD45 antibody
(manufactured by Biolegend, Inc.), and an anti-Zbtb20 antibody
(manufactured by Becton Dickinson, Inc.).
[0164] The results are shown in FIGS. 31(c) and 32. As shown in
FIG. 31(c), it was observed that the expression of Eomes gene in
the CD4.sup.+ T cells and the expression of Zbtb20 gene in the B
cells in each anti-CD20 antibody-administered mouse were both
suppressed markedly. FIG. 32 shows, as a graph for comparison, the
percentage of Eomes- or Zbtb20-expressing cells in each mouse
group.
[0165] 6. Changes in Zbtb20 Gene Expression by Various
Cytokines
(1) Changes in Zbtb20 Gene Expression by Various Cytokines
[0166] Spleen-derived CD19.sup.+ B cells were isolated and purified
by sorting by FACS using a FACS ARIA II (manufactured by BD
Cytometry Systems, Inc.). The purified B cells were cultured for 24
h in the presence of LPS and in the co-presence or absence of each
cytokine designated in FIG. 33(a). After the culturing, the
expression levels of Zbtb20 gene in the respective cells were
measured. As antibodies used at the detection were an anti-CD19
antibody (manufactured by Biolegend, Inc.) and an anti-Zbtb20
antibody (manufactured by Becton Dickinson, Inc.).
[0167] The results are shown in FIGS. 33 and 34. In FIG. 33(a), the
gray graphs each indicate the expression level of Zbtb20 gene in
the cells cultured in the absence of each cytokine; and the black
line graphs each indicate the expression level in the case of
culturing in the co-presence of each different cytokine. After the
culturing in the co-presence of each cytokine designated, it was
observed that the expression of Zbtb20 gene increased; and an
increase in the expression of Zbtb20 gene by culturing in the
co-presence of IFN.alpha. or IFN.beta.1 was the most remarkable.
FIG. 33(b) shows, as a graph, each percentage of Zbtb20.sup.+ cells
under each condition. FIG. 34 shows how many folds each percentage
of the Zbtb20.sup.+ cells or Prl.sup.+ cells increased in the case
of culturing in the co-presence of each different cytokine compared
to the case of culturing in the absence of the cytokine.
(2) Changes in Each Different Cytokine Expression During Course of
Progression of EAE Pathology-1
[0168] The CSF and plasma were collected from each mouse at each
progression stage (early-stage EAE pathology, mid-stage EAE
pathology, or late-stage EAE pathology) and the expression level of
each different cytokine was measured by using a Luminex system. In
addition, the expression levels of each of type 1 interferons
(IFN.alpha.2, IFN.beta.1), IL-6, IL-9, and Cxc113 in CNS-derived B
cells, pDCs, or microglia were likewise measured by using a
quantitative real-time PCR assay. To detect IFN.alpha.2 and
IFN.beta.1, ProcartaPlex Mouse IFNa/b (manufactured by ThermoFisher
Scientific, Inc.) was used. To detect other cytokines, BioPlex Pro
Cytokine GI 23-plex panel and BioPlex Pr Cytokine GIII TH17 8-plex
B panel (manufactured by Bio-Rad, Inc.) were used.
[0169] The results are shown in FIGS. 35 and 36. FIG. 35 shows
IL-1.alpha., IL-1.beta., IL-6, IL-9, IL-12 (p40), IL-12 (p70), and
IFN-.gamma. expression levels. FIG. 35(a) shows the IFN-.alpha. or
IFN-.beta. expression levels. FIG. 35(b) shows an amount of the
IFN.alpha.2 or IFN.beta.1 expression in the CNS-derived B cells,
pDCs, or microglia. FIG. 35(c) shows an amount of the IL-6, IL-9,
or Cxc113 expression in the CNS-derived B cells, pDCs, or
microglia. As shown in FIGS. 35(b) and (c), it was observed that
the amounts of expression of IFN-.alpha.2, IFN-.beta.1, and IL-9 in
microglia at the time of mid-EAE pathology increased markedly.
(3) Effects of Microglia Derived from Each Mouse with Late-Stage
EAE Pathology on Zbtb20 Expression
[0170] CD19.sup.+ B cells were sorted from the spleen of each
intact mouse of similar genetic strain by FACS and were co-cultured
for 24 h with microglial cells derived from each intact mouse or
each mouse with the late-stage EAE pathology. The amounts of
expression of Zbtb20 gene in the cultured B cells were measured
with a flow cytometer. In addition, the B cells were purified by
sorting by FACS, and Zbtb20 and Prl RNA levels were detected by
using a real-time PCR assay. As antibodies used at the detection
were an anti-CD19 antibody (manufactured by Biolegend, Inc.) and an
anti-Zbtb20 antibody (manufactured by eBioscience, Inc.).
[0171] The results are shown in FIG. 37. As shown in FIG. 37(a), it
was observed that the expression of Zbtb20 gene was enhanced
markedly when the CD19.sup.+ B cells were co-cultured with
microglia of each mouse with the late-stage EAE pathology. In
addition, as shown in FIG. 37(b), it was observed that the Zbtb20
and Prl RNA levels also increased relatively.
(4) Changes in Each Different Cytokine Expression During Course of
Progression of EAE Pathology
[0172] The CSF and plasma were collected from each intact mouse or
each mouse with each progression stage of EAE pathology, and a
Luminex system was used to measure the protein levels of various
cytokines (IL-2, IL-4, IL-3, IL-5, IL-10, IL-13, IL-17, G-CSF,
GM-CSF, TNF-.alpha., Eotaxin, KC, MCP-1, MIP-1b, RANTES, and
MIP-1a). For detection, BioPlex Pro Cytokine GI 23-plex panel and
BioPlex Pr Cytokine GIII TH17 8-plex B panel (manufactured by
Bio-Rad, Inc.) were used.
[0173] The results are shown in FIGS. 38 to 40.
* * * * *