U.S. patent application number 11/584649 was filed with the patent office on 2007-02-15 for goodpasture's syndrome model mouse.
This patent application is currently assigned to Japan Science and Technology Agency. Invention is credited to Akira Nakamura, Toshihiro Nukiwa, Toshiyuki Takai.
Application Number | 20070039062 11/584649 |
Document ID | / |
Family ID | 16086102 |
Filed Date | 2007-02-15 |
United States Patent
Application |
20070039062 |
Kind Code |
A1 |
Nakamura; Akira ; et
al. |
February 15, 2007 |
Goodpasture's syndrome model mouse
Abstract
The present invention provides a non-human model animal of
Goodpasture's syndrome that contributes to the treatment of
Goodpasture's syndrome where the development of therapy had been
delayed due to the lack of adequate disease models, a method for
screening a remedy for Goodpasture's syndrome by using the model
animal, and a method for diagnosing Goodpasture's syndrome at the
early stage. A Goodpasture's syndrome model mouse is constructed by
immunizing immunoglobulin Fc.gamma. receptor IIB knockout mouse
with type IV collagen, thereby inducing Goodpasture's syndrome.
Moreover, a remedy for Goodpasture's syndrome is screened by
administration of test substances to the Goodpasture's syndrome
model mouse, followed by evaluating the severity of the expression
of Goodpasture's syndrome as an index, such as diffuse alveolar
hemorrhage, glomerulonephritis, the appearance of antikidney
glomerular basement membrane antibody, and the like.
Inventors: |
Nakamura; Akira;
(Sendai-shi, JP) ; Nukiwa; Toshihiro; (Sendai-shi,
JP) ; Takai; Toshiyuki; (Sendai-shi, JP) |
Correspondence
Address: |
VENABLE LLP
P.O. BOX 34385
WASHINGTON
DC
20043-9998
US
|
Assignee: |
Japan Science and Technology
Agency
Kawaguchi-shi
JP
|
Family ID: |
16086102 |
Appl. No.: |
11/584649 |
Filed: |
October 23, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10009950 |
Dec 14, 2001 |
7135607 |
|
|
11584649 |
Oct 23, 2006 |
|
|
|
Current U.S.
Class: |
800/9 ;
800/18 |
Current CPC
Class: |
A01K 2267/0325 20130101;
A01K 2227/105 20130101; A61P 11/00 20180101; A01K 2217/075
20130101; A61P 13/12 20180101; A01K 67/0276 20130101 |
Class at
Publication: |
800/009 ;
800/018 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 23, 2000 |
WO |
PCT/JP00/04132 |
Jun 25, 1999 |
JP |
H11-180600 |
Claims
1-7. (canceled)
8. A method for diagnosing Goodpasture's syndrome at the early
stage characterized in that a Fc.gamma. receptor IIB gene is
extracted from human test cells and is examined whether there is
any deficiency in the function of said gene.
Description
TECHNICAL FIELD
[0001] The present invention relates to a non-human model animal of
Goodpasture's syndrome, a method for screening a remedy for
Goodpasture's syndrome by using the model animal, and a method for
diagnosing Goodpasture's syndrome at the early stage.
PRIOR ART
[0002] Goodpasture's syndrome, which has the combination of diffuse
alveolar hemorrhage, glomerulonephritis, and the appearance of
antikidney glomerular basement membrane antibody, is similar as a
clinical feature with Wegener's granulomatosis, systemic
necrotizing vasculitis, systemic lupus erythematosus (SLE) and the
like, and is believed that antibodies common to the kidney
glomerular basement membrane and alveolar epithelium basement
membrane exist in the patient's serum, which bind to the target
tissues and induce lesion of inflammation based on type II
hypersensitivity reaction (J. Exp. Med. 126, 989-1004, 1987). Aside
from the characteristics of the above-mentioned clinical feature,
the diagnosis of Goodpasture's syndrome is made by proving the
deposition of immunoglobulin (hereinafter "Ig") on the kidney
glomerular basement membrane, where most of the anti basement
membrane antibodies belong to the IgG fraction, and in recent
years, it has been identified as an autoantibody to a part of the
.alpha..sub.3 chain of type IV collagen (Cell Mol. Biol. 5,
107-112, 1991).
[0003] Moreover, the onset of Goodpasture's syndrome is found in a
wide range of age bracket, where without its diagnosis and therapy
at the early stage, 80% of the patients die within a year due to
the deterioration of nephrosis, and 30% of the patients die due to
pulmonary hemorrhage. Recently, therapy at the early stage has
enabled to increase the average of saving lives of Goodpasture's
syndrome to about 50%, but therapy only with the use of oral
steroid agent or oral immunosuppressive drug is insufficient, and
using high-dose of predonine pulse therapy is effective for
pulmonary hemorrhage. However, pulse therapy is not sufficient for
nephrosis, and is said that plasma exchange and high-dose of
predonine, with the use of cyclophosphamide at the same time is
effective, whereas to serious renal damage, artificial dialysis or
renal transplantation is considered.
[0004] On the other hand, receptors that recognize and bind to the
Fc portion of Ig (hereinafter "FcR") exist on the surface of cells
in such as the immune system and the like, and the Fc.gamma.
receptor (hereinafter "Fc.gamma.R") among them, which is a receptor
protein that binds specifically to the .gamma. chain of IgG in the
body fluid, is classified mainly into three types, type I (CD64
antigen), type II (CD32 antigen), and type III (CD16 antigen),
based on the similarity of gene structure. Among these,
Fc.gamma.RII differs from the other FcRs in that it has low
affinity to the IgG of the monomer, binds to the polyvalent IgG
that has become an immune complex, and is widely expressed in the
hemopoietic stem cells including monocytes, macrophages,
polymorphonuclear (PMN) leukocytes, mast cells, platelets, some of
the T cell lymphocytes and B cell lymphocytes. Moreover, three
types of receptors having different gene arrangements,
Fc.gamma.RIIA, Fc.gamma.RIIB, and Fc.gamma.RIIC, exist in the
Fc.gamma.RII, and it is also known that each of the chromosomes are
positioned in 1q23.
[0005] Unlike the other FcRs, the above-mentioned Fc.gamma.RIIB
does not associate with .gamma. chain, and has an amino acid
sequence (ITIM: Immunoreceptor Tyrosine-based Inhibition Motif)
which transmits supressive signal to the intracellular domain
(Immunol. Rev. 125, 49-76, 1992, Science 256, 1808-1812, 1992). In
order to elucidate these physiological functions of Fc.gamma.RIIB,
the inventors of the present invention had already constructed
Fc.gamma.RIIB-deficient mouse (Nature 379, 346-349, 1996), and
constructed arthritis model mouse which is generated by immunizing
Fc.gamma.RIIB-deficient mice with type II collagen (J. Exp. Med.
189, 187-194, 1999), and autoimmune disease model mouse (Japanese
Laid-Open Patent Publication No. 08-289699).
[0006] Model animals that are effective in the study on the
pathogenesis of Goodpasture's syndrome, where 80% of the patients
die within a year due to the deterioration of nephrosis and 30%
among the patients die due to pulmonary hemorrhage without its
diagnosis and therapy at the early stage, and in the development of
therapy for Goodpasture's syndrome, had not been known to the
present. An object of the present invention is to present a
non-human model animal of Goodpasture's syndrome contributing to
the treatment of Goodpasture's syndrome where the development of
therapy had been delayed due to the lack of adequate disease models
to elucidate its onset mechanism, a method for screening a remedy
for Goodpasture's syndrome by using the model animal, and a method
for diagnosing Goodpasture's syndrome at the early stage.
SUMMARY OF THE INVENTION
[0007] The inventors of the present invention have conducted
intensive study to elucidate the physiological functions of
Fc.gamma.RIIB, and have discovered that when a mouse whose function
of Fc.gamma.RIIB gene is deficient on its chromosome, namely, the
Fc.gamma.RIIB knockout mouse, is immunized with type IV collagen,
said Fc.gamma.RIIB knockout mouse indicates diagnostic sign of
Goodpasture's syndrome, and thus the present invention has been
completed.
[0008] The present invention relates to a non-human model animal of
Goodpasture's syndrome, especially Goodpasture's syndrome model
mouse, which is obtained by immunizing a non-human animal whose
function of immunoglobulin Fc.gamma. receptor IIB gene is deficient
on its chromosome with type IV collagen or peptide which includes a
part of its amino acid sequence.
[0009] Moreover, the present invention relates to a method for
screening a remedy for Goodpasture's syndrome characterized in
that: test substances are administered to a non-human animal whose
function of immunoglobulin Fc.gamma. receptor IIB gene is deficient
on its chromosome before, after or at the same time it is immunized
with type IV collagen; and the severity of the expression of
Goodpasture's syndrome as an index is evaluated, a method for
screening a remedy for Goodpasture's syndrome characterized in
that: test substances are administered to a non-human model animal
of Goodpasture's syndrome; and the severity of the expression of
Goodpasture's syndrome as an index is evaluated, the
above-mentioned method for screening a remedy for Goodpasture's
syndrome characterized in that: a comparative evaluation with the
wild-type non-human animal used as a control is made when the
severity of the expression of Goodpasture's syndrome as an index is
evaluated, the above-mentioned method for screening a remedy for
Goodpasture's syndrome characterized in that: the expression of
Goodpasture's syndrome is at least one among diffuse alveolar
hemorrhage, glomerulonephritis, and the appearance of antikidney
glomerular basement membrane antibody, and the above-mentioned
method for screening a remedy for Goodpasture's syndrome
characterized in that: the non-human animal is a mouse.
[0010] In addition, the present invention relates to a method for
diagnosing Goodpasture's syndrome at the early stage, wherein a
Fc.gamma. receptor IIB gene is extracted from human test cells, and
is examined whether there is any deficiency in the function of said
gene.
BRIEF EXPLANATION OF DRAWINGS
[0011] FIG. 1 is a view showing Goodpasture's syndrome-like
alveolar hemorrhage by immunization with type IV collagen.
[0012] FIG. 2 is a view showing Goodpasture's syndrome-like
glomerulonephritis by immunization with type IV collagen.
[0013] FIG. 3 is a graph showing the level of antibody titer of
immunization with type IV collagen.
BEST MODE FOR CARRYING OUT THE INVENTION
[0014] In the present invention, a "non-human animal whose function
of Fc.gamma.RIIB gene is deficient on its chromosome" means a
non-human animal whose function of expressing Fc.gamma.RIIB is
impaired by inactivation of endogenous genes of the non-human
animal encoding Fc.gamma.RIIB caused by genetic mutation such as
destruction, deficiency, substitution or the like. Specific
examples of "non-human animal" in the present invention include a
rodent such as a mouse or a rat, but are not limited to these
examples.
[0015] For example, mouse whose function of Fc.gamma.RIIB gene is
deficient on its chromosome, namely, the Fc.gamma.RIIB knockout
mouse, can be constructed by using the method as previously
described (Nature 379, 346-349, 1996) by the present inventors, or
the like. Specifically, a gene fragment obtained from a mouse
genomic library using methods such as PCR or the like is used to
screen the Fc.gamma.RIIB gene, and the Fc.gamma.RIIB gene which had
been screened is subcloned by using a viral vector or the like, and
is determined by DNA sequencing. A fragment that includes S.sub.2
exon and EC.sub.1 exon of the said clone was substituted with pMC1
neo gene cassette or the like, and a target vector was
generated.
[0016] The linearized vector was introduced into ES cells by
methods such as electroporation or the like, followed by homologous
recombination, and ES cells indicating resistance to G418 and the
like were selected from said homologous recombinants, and then the
clone of said cells were microinjected into the blastocysts of the
mice, said blastocysts were returned to the tentative parent mice,
and chimeric mice were generated. These chimeric mice were
intercrossed with wild-type mice to obtain heterozygous mice, and
these heterozygous mice were intercrossed to obtain Fc.gamma.RIIB
knockout mice.
[0017] In the present invention, type IV collagen is a specific
exemplification of an immunogen used to induce Goodpasture's
syndrome to a non-human animal whose function of Fc.gamma.RIIB gene
is deficient on its chromosome, however, anything such as a peptide
which includes a part of the amino acid sequence of type IV
collagen or the like can be used, as long as Goodpasture's syndrome
can be induced to the non-human animal whose function of
Fc.gamma.RIIB gene is deficient on its chromosome.
[0018] In the present invention, a "non-human model animal of
Goodpasture's syndrome" can be any kind of non-human animal, as
long as it is a non-human animal such as a mouse or the like, which
has the combination of three diagnostic signs of diffuse alveolar
hemorrhage, glomerulonephritis, and the appearance of antikidney
glomerular basement membrane antibody. For example, it can be
obtained by immunizing a non-human animal whose function of
Fc.gamma.RIIB gene is deficient on its chromosome with type IV
collagen.
[0019] Examples of methods for screening a remedy for Goodpasture's
syndrome in the present invention are: a method in which test
substances that are expectants for the remedy for Goodpasture's
syndrome are administered to the non-human animal whose function of
Fc.gamma.RIIB gene is deficient on its chromosome before or after
Goodpasture's syndrome is induced to the non-human animal by
immunization with type IV collagen; or at the same time
Goodpasture's syndrome is induced to the non-human animal by
immunization with type IV collagen; and the severity of the
expression of Goodpasture's syndrome (appearance of diagnostic
signs) as an index is evaluated, and a method in which test
substances that are expectants for the remedy for Goodpasture's
syndrome are administered to the Goodpasture's syndrome non-human
model animal; and the severity of the expression of Goodpasture's
syndrome as an index is evaluated.
[0020] In addition, when evaluating the severity of the expression
of Goodpasture's syndrome as an index, a wild-type non-human animal
of the same species as the non-human model animal of Goodpasture's
syndrome can be used as a control, and a comparative evaluation of
the severity of the expression of Goodpasture's syndrome between
the non-human model animal of Goodpasture's syndrome and the
wild-type non-human animal of the same species used as a control
can be made.
[0021] As an index of the severity of expression (appearance of
diagnostic signs) of Goodpasture's syndrome, at least one among
diffuse alveolar hemorrhage in the pulmonary tissues,
glomerulonephritis in the renal tissues, and the appearance of
antikidney glomerular basement membrane antibody can be favorably
exemplificated, including other examples such as the expression of
anti basement membrane antibody depositing in the pulmonary
alveoli, serum creatinine level, the glomus filtration value, or
the like. Evaluating at least one of these indexes can screen the
remedy for Goodpasture's syndrome.
[0022] In the present invention, a specific method for diagnosing
Goodpasture's syndrome at the early stage is to extract
Fc.gamma.RIIB gene from human test cells, and examine whether there
is any deficiency in the function of said gene. Examples of human
test cells used as Fc.gamma.RIIB gene source are macrophages, mast
cells, B cells, dendritic cells and the like, and an example of a
method for examining whether there is any deficiency in the
function of Fc.gamma.RIIB gene is to express the cloned
Fc.gamma.RIIB gene in the human cell line by ordinary method, and
examine the function of Fc.gamma.RIIB of the expression product,
such as the binding to IgG immune complex. Onset of Goodpasture's
syndrome is possible when there is a deficiency in the function of
Fc.gamma.RIIB in the expression product, and as mentioned above, a
method for diagnosing Goodpasture's syndrome at the early stage
becomes possible by examining whether there is any deficiency in
the function of Fc.gamma.RIIB gene.
[0023] The present invention will now be explained more
specifically with the following examples, however, the technical
scope of the invention is not limited to these examples.
Reference (Generation of Fc.gamma.RIIB-Deficient Mice)
[0024] A genomic DNA clone for Fc.gamma.RIIB gene was isolated by
screening a 129/Sv/J-lineage-derived mouse genomic DNA library. A
targeting vector was constructed by replacing a 2.65 Kb fragment
which includes two separate exons of S.sub.2 and EC.sub.1 of said
clone to a pMC1 neo gene cassette (Toyobo Co., Ltd.). This
linearized vector was introduced into ES cells (J1) by
electroporation, and was homologously recombined.
[0025] The ES clone was isolated from the ES cells that were
homologously recombined as mentioned above, a neomycin-resistant ES
clone was screened to G418 and GANC (ganciclovir), and homologous
recombinants were identified by Southern blot. Genomic DNA isolated
from the identified homologous recombinants was digested with Hind
III, and the existence of targeting allele containing pMC1 neo gene
cassette was confirmed. The said identified ES clone was
microinjected into the blastocysts to generate chimeric mice, and
the generated mice were intercrossed with wild-type C57BL/6J mice
to obtain heterozygous mice, then these heterozygous mice were
intercrossed to obtain homozygous mice, and mice whose
Fc.gamma.RIIB gene is deficient on its chromosome and its wild-type
mice were generated.
EXAMPLE 1
Construction of Goodpasuture's Syndrome Model Mice
[0026] A Cellmatrix IV (Nitta Gellatin, Inc.) prepared from bovine
crystalline lens at 3 mg/ml protein concentration in 1 mM HCL
solution (pH 3.0), was added NaOH to a final concentration of 1 mM,
to generate type IV collagen (pH 8.0). Two types of emulsions were
generated by mixing 3 mg/ml of said type IV collagen (pH 8.0) and 3
mg/ml of complete Freund's adjuvant (CFA) comprised of liquid
paraffin, surface-active agent, and dead Mycobacterium tuberculosis
in a connected syringe, and by mixing 3 mg/ml type IV collagen (pH
8.0) and 3 mg/ml incomplete Freund's adjuvant (IFA) comprised of
liquid paraffin and surface-active agent in a connected
syringe.
[0027] The Fc.gamma.RIIB gene deficient mice (eight weeks of age:
gender at randomly chosen) generated from the method described in
the above-mentioned reference were anesthetized by ether and its
tail base were shaved, and 100 .mu.l emulsion containing 150 .mu.g
each of type IV collagen and CFA were injected to the mice's skin
for primary immunization. After the primary immunization, on day
14, day 28, and day 42, 100 .mu.l emulsion containing 150 .mu.g
each of type IV collagen and IFA were injected to their skin, the
mice were killed on day 56, and the pulmonary tissues and renal
tissues were extracted. In addition, wild-type mice were used as a
control.
[0028] As shown in FIG. 1, Fc.gamma.RIIB gene deficient mice
immunized with type IV collagen (Fc.gamma.RIIB.sup.-/.sup.-),
compared to control wild-type mice (WT), showed remarkable signs of
alveolar hemorrhage in the pulmonary tissues at a wide range
including the infiltration of inflammatory cells such as
macrophages, neutrophils, and the like. In addition, as shown in
FIG. 2, degeneration of glomus and proximal renal tubule in the
renal tissues were displayed and renal lesion mainly as
glomerulonephritis occurred. From these results, it can be
understood that Goodpasuture's syndrome model mice can be obtained
by immunizing Fc.gamma.RIIB gene deficient mice with type IV
collagen.
EXAMPLE 2
Examination of Antibody Titer to Type IV Collagen
[0029] After each of Fc.gamma.RIIB knockout mice, FcR.gamma.
knockout mice, and wild-type mice were immunized with type IV
collagen, blood was extracted from the orbit after a set period of
time, and the antibody titer to type IV collagen was tested by the
following method, where improvement had been added to the ELISA
analysis previously described (Cell. Immunol. 145, 299-310,
1992).
[0030] 20 .mu.g type IV collagen was lysed in 1 ml phosphate
buffered solution (PBS), and this lysate solution was used at 50
.mu.l/well, and after coating a 96-well microplate (Falcon; Becton
Dickinson Labware) at 4 C. .degree. for overnight, was washed three
times with PBS containing 0.05% Tween 20 and 0.1% BSA, and then
blocked with PBS containing 0.2% BSA at 250 .mu.l/well at 4 C.
.degree. overnight.
[0031] The serum obtained from the blood mentioned above was then
diluted to 400 to 20000 times, and the diluted serum was added to
the aforementioned 96-well microplate at 50 .mu.l/well, and allowed
to react at 4 C. .degree. overnight. After the reaction, the
96-well microplate was washed three times with PBS containing 0.05%
Tween 20, added 50 .mu.l of horseradish peroxidase (Sigma Chemical
Co.)-conjugated goat anti-mouse IgG1, IgG2a, or IgG2b diluted to
200 times, and was then incubated at 4 C. .degree. for 2 hours.
After incubation, it was washed again three times with PBS
containing 0.05% Tween 20, and developed enzyme reaction at room
temperature for 30 minutes with 0.1 ml of True Blue Peroxidase
Substrate (Kirkegaard & Perry Labs). The OD 450 was then read
by using a Microplate Reader (Biolumin960; Molecular Dynamics
Japan, Inc.). The results are shown in FIG. 3.
[0032] From these results, increase in antibody titer to type IV
collagen (IgG1, IgG2a, IgG2b or IgM) can be seen in Fc.gamma.RIIB
knockout mice (IIB-KO), compared to FcR.gamma. knockout mice
(.gamma.-KO) and wild-type mice (Wild), and since this is not
inconsistent with the observations to Goodpasture's syndrome, it
was found that Goodpasture's syndrome model mouse was
generated.
INDUSTRIAL APPLICABILITY
[0033] According to the present invention, a non-human model animal
of Goodpasture's syndrome, a method for screening a remedy for
Goodpasture's syndrome using the model animal, and a method for
diagnosing Goodpasture's syndrome at the early age can be provided,
which leads to the therapy of Goodpasture's syndrome, where the
development of therapy had been deleyed due to the lack of adequate
disease models for elucidating its onset mechanism.
* * * * *