U.S. patent application number 10/946272 was filed with the patent office on 2005-02-17 for parasite-derived anti-inflammatory immunomodulatory protein.
This patent application is currently assigned to THE BOARD OF TRUSTEES OF THE UNIVERSITY OF ILLINOIS. Invention is credited to Kalyanasundaram, Ramaswamy, Salafsky, Bernard, Shibuya, Takeshi.
Application Number | 20050037970 10/946272 |
Document ID | / |
Family ID | 21759891 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050037970 |
Kind Code |
A1 |
Salafsky, Bernard ; et
al. |
February 17, 2005 |
Parasite-derived anti-inflammatory immunomodulatory protein
Abstract
A protein with anti-inflammatory, and immunomodulatory functions
was identified and isolated from the excretory/secretions of a
human parasite, Schistosoma mansoni. This protein, designated
herein as Sm 16.8, is released by schistosomes upon entry into
human skin. Under in vitro conditions Sm 16.8 is shown to stimulate
the production of anti-inflammatory cytokine Interleukin-1 receptor
antagonist (IL-1ra) in human keratinocytes and transcriptionally
down regulate the production of pro-inflamnmatory cytokines IL-1
and IL-1.
Inventors: |
Salafsky, Bernard;
(Rockford, IL) ; Kalyanasundaram, Ramaswamy;
(Rockford, IL) ; Shibuya, Takeshi; (Tokyo,
JP) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Assignee: |
THE BOARD OF TRUSTEES OF THE
UNIVERSITY OF ILLINOIS
Urbana
IL
|
Family ID: |
21759891 |
Appl. No.: |
10/946272 |
Filed: |
September 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10946272 |
Sep 20, 2004 |
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10114417 |
Apr 2, 2002 |
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10114417 |
Apr 2, 2002 |
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09180446 |
Oct 19, 1999 |
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6372219 |
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09180446 |
Oct 19, 1999 |
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PCT/US97/03953 |
Mar 14, 1997 |
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60013423 |
Mar 14, 1996 |
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Current U.S.
Class: |
424/184.1 ;
514/12.2; 514/18.7; 514/19.1; 530/350 |
Current CPC
Class: |
A61K 39/00 20130101;
A61K 38/00 20130101; Y02A 50/423 20180101; C07K 14/4354 20130101;
A61K 39/0003 20130101; Y02A 50/30 20180101 |
Class at
Publication: |
514/012 ;
530/350 |
International
Class: |
A61K 038/16; C07K
014/195 |
Claims
1-7. (Canceled)
8. A method for treating an inflammatory cutaneous diseases, the
method comprising administering to a subject a therapeutically
effective dose of an isolated Sm16.8 polypeptide comprising a
molecular weight of about 16.8 kilodaltons and a pI of 5.9, thereby
treating an inflammatory cutaneous disease.
9. The method of claim 8 wherein said inflammatory cutaneous
disease is selected from the group consisting of urticaria, atopic
dermatitis, contact sensitivity, and psoriasis.
10-11. (Canceled)
12. The method of claim 8 wherein the polypeptide is administered
by a route selected from the group consisting of intravenous
administration, intramuscular administration, subcutaneous
administration, oral administration and topical administration.
13. A method for modulating the expression of an interleukin
selected from the group consisting of interleukin-1.alpha.,
interleukin-1.beta. and interleukin-1ra, the method comprising
administering to a subject an effective dose of an isolated Sm16.8
polypeptide comprising a molecular weight of about 16.8 kilodaltons
and a pI of 5.9.
14. The method according to claim 13 wherein the interleukin is
selected from the group consisting of interleukin-1.alpha. and
interleukin-1.beta., further wherein the interleukin expression is
inhibited.
15. A method for inhibiting the expression of ICAM-1 in a cell, the
method comprising administering to a subject a therapeutically
effective dose of an isolated Sm16.8 polypeptide comprising a
molecular weight of about 16.8 kilodaltons and a pI of 5.9.
16. The method of claim 15 wherein the cell is selected from the
group consisting of an endothelial cell, a keratinocyte, a dermal
cell, and an epidermal cell.
17. A method for inhibiting infiltration of a cell to a site of
inflammation, wherein the cell is selected from the group
consisting of a lymphocyte and a neutrophil.
18. A method for modulating an immune response, the method
comprising administering to a subject an effective dose of an
isolated Sm16.8 polypeptide comprising a molecular weight of about
16.8 kilodaltons and a pI of 5.9.
Description
[0001] This application claims priority from U.S. provisional
application No. 60/013,423 filed on Mar. 14, 1996.
BACKGROUND OF THE INVENTION
[0002] In one of its life-stages the parasite Schistosoma mansoni
(S. mansoni), which causes the tropical disease schistosomiasis,
has the unique ability to penetrate intact, unabraded human skin.
After gaining entry into the host, the parasite spends over 48 hrs
in various layers of the skin without eliciting any marked tissue
response. This subdued inflammatory response is largely responsible
for the parasite's ability to pass through the skin virtually
undetected by its human host.
[0003] Other schistosomes such as Trichobilharzia ocellata (a bird
parasite) which also can penetrate intact human skin, causes a
severe inflammatory response in the skin (commonly called swimmer's
itch) which results in the parasite's elimination from the host.
Therefore, suppression of inflammatory responses in the skin
appears to be crucial for the survival of schistosomes in its human
host. Because Schistosoma mansoni does not elicit such a response,
it would be useful to determine the mechanisms by which the
infammatory response is reduced or suppressed and to exploit such
mechanisms as anti-inflammatory therapeutics for treating any
inflammatory condition.
SUMMARY OF THE INVENTION
[0004] The present invention is directed to an isolated purified
polypeptide designated Sm 16.8 or variants, fragments, derivatives,
homologs or analogs thereof. The variants, fragments, derivatives,
homologs or analogs may have a biological activity of Sm 16.8
and/or be immunologically active, that is, capable of inducing
antibody formation, the antibodies being directed to one or more
epitopes of the Sm 16.8 polypeptide.
[0005] Antibodies that specifically bind to polypeptides of the
present invention are also within the scope of the present
invention. Such antibodies may serve as therapeutics and/or
diagnostic products. The antibodies may be polyclonal or
monoclonal.
[0006] More particularly, the invention is directed to a
polypeptide having a molecular weight of 16.8 kDa on a non-reducing
SDS-polyacrylamide gel and has an isoelectric point of 5.9. The
polypeptide of the present invention is obtainable from the
parasite Schistosoma mansoni and may be a secretion/excretion
product of the schistosome.
[0007] Also, comprehended by the invention are DNAs encoding the
polypeptides of the present invention as well as vectors comprising
the DNAs of the invention, host cells transformed with the vector
and expression products derived therefrom. Host cells may be
procaryotic or eucaryotic host cells.
[0008] The invention is also directed to vaccines comprising one or
more polypeptides according to the present invention which may also
include suitable adjuvants, diluents or carrier substances, the
vaccines being useful for immunoprophylaxis of schistosomiasis.
[0009] Pharmaceutical compositions comprising Sm 16.8 variants,
fragments, derivatives, homologs or analogs of the polypeptide in
combination with pharmaceutically acceptable diluents, adjuvants
and carriers are also contemplated by the present invention.
[0010] The invention is further directed to methods of suppressing
or preventing inflammation by administering to a subject an
anti-inflammatory dose of a polypeptide or pharmaceutical
compositions according to the present invention.
[0011] Methods for treating inflammation associated diseases such
as cutaneous disease are also within the scope of the invention.
Such methods comprise administering to a subject a therapeutically
effective dose of a polypeptide or pharmaceutical composition
according to the present invention. The polypeptides may be
administered topically, for example, in a suitable cream, lotion or
salve that may include excipients useful in transporting the
biologically active polypeptides into the skin.
[0012] Methods for treating systemic diseases characterized by
inflammatory processes, using the polypeptides of the invention are
also contemplated by the present invention. Such diseases may be
autoimmune diseases.
DETAILED DESCRIPTION OF THE INVENTION
[0013] During penetration into the skin, parasites such as those of
the species Schistosoma continuously excrete/secrete substances
(ES) into their surroundings in order to aid their passage and/or
as part of their metabolism.
[0014] These ES products are known to contain different types of
proteins and lipids, many of whose function are not fully known. By
culturing these parasites (i.e., schistosomes) in vitro ES products
have been collected, purified and analyzed for their functional
capabilities. One of the major components of the ES products are
proteins. Given that S. mansoni and T. ocellata differ in their
ability to induce infammation in the skin, the proteins in the ES
products of the two parasites were analyzed in an attempt to
identify whether ES products of S. mansoni contain any factors that
have the capacity to modulate inflammatory responses in human skin
that are not contained in the ES products of T. ocellata. These
studies revealed the presence of a protein in the secretions of S.
mansoni but not T. ocellata, that has the ability to suppress
inflammation.
[0015] Cytokines, natural hormone like substances produced by many
cells in the skin, may play a central role in initiating, promoting
or suppressing inflammation. Cytokines such as Interleukin-1.alpha.
(IL-1.alpha.) and IL-1.beta. promote inflammation, whereas,
cytokines such as IL-1ra suppress inflammation. In the skin, cells
such as keratinocytes, Langerhans cells, lymphocytes or mast cells
can produce an array of pro-inflammatory cytokines upon activation.
Yet, penetration and migration of S. mansoni through the skin fails
to induce any inflammatory response.
[0016] Keratinocytes constitute over 95% of the cells in human
skin. It has been shown repeatedly that depending upon the
stimulus, human keratinocytes may produce the pro-inflammatory
cytokine IL-1.alpha. or the anti-inflammatory cytokine IL-1ra.
Production of large quantities of IL-1ra locally results in the
suppression of inflammatory responses.
[0017] The immunological basis for many cutaneous diseases such as
atopic dermatitis, urticaria, contact sensitivity, cutaneous
allergic conditions and psoriasis, is the accumulation of
inflammatory cells in the epidermis and dermis. Any drug that can
reduce or suppress accumulation of inflammatory cells in the skin
will alleviate the clinical symptoms associated with these
diseases.
[0018] Other inflammatory diseases are associated with the
inflammatory processes in other organs of the body and are likewise
susceptible to treatment with certain anti-inflammatory drugs.
[0019] A Schistosoma mansoni derived protein, Sm 16.8, that
selectively up regulates. IL-1ra production in human keratinocytes
was identified and characterized. When added to human keratinocyte
cultures at a concentration of 5 .mu.g/1.times.10.sup.5 cells, Sm
16.8 stimulated the production of IL-1ra within 4 hrs.
Intracellular message (mRNA) for IL-1ra in these cells increased
from 4 hrs after treatment and attained maximum values within 8
hrs. Statistically significant amounts of IL-1ra were also found
intracellularly and in the culture supernatants of human
keratinocytes after treatment with Sm 16.8. IL-1ra is a natural
antagonist of IL-1, in that it competes with IL-1.alpha. and
IL-1.beta. for the IL-1 receptor. Binding of IL-1.alpha. or
IL-1.beta. to IL-1 receptors expressed on many cells results in a
cascade of events leading to inflammation. However, binding of
IL-1ra to IL-1 receptors fails to induce any receptor mediated
responses. Therefore, occupancy of all available IL-1 receptors by
IL-1ra results in the blocking of all IL-1 mediated responses.
Since, IL-1ra binds to IL-1 receptors with affinity equal to or
higher than those of IL-1.alpha. or IL-1.beta. and, since the
dissociation rate constant of IL-1ra from IL-1 receptors is
several-fold lower than that for IL-1.alpha. and IL-1.beta., a
higher concentration of IL-1ra in the local microenvironment can
effectively block all the IL-1 mediated responses including
inflammation. Given evidence that Sm 16.8 can stimulate a 100-400
fold increase in IL-1ra production from human keratinocytes, which
are the major cell type in the skin, the use of Sm 16.8 as an
anti-inflammatory substance against inflammatory diseases including
inflammatory conditions in human skin is within the scope of the
present invention.
[0020] In addition to its effect in stimulating IL-1ra production,
Sm 16.8 suppresses IL-1.alpha. and IL-1.beta. synthesis in human
keratinocytes both at the transcriptional and translational levels.
When added to human keratinocyte cultures that were stimulated with
lipopolysaccharide--a bacterial envelope protein that induces
marked IL-1.alpha. production in human keratinocytes, the
parasite-derived protein suppressed IL-1.alpha. production by
keratinocytes in a dose dependent fashion. At a concentration of 5
.mu.g/ml per 1.times.10.sup.5 cells, Sm 16.8 completely inhibited
IL-1.alpha., and IL-1.beta. RNA production in human keratinocytes.
Thus, Sm 16.8 appears to act by providing a two-pronged approach
towards reducing inflammation, (i) by stimulating the production of
IL-1ra in human skin cells and (ii) by transcriptionally down
regulating the production of the pro-inflammatory cytokines
IL-1.alpha. and IL-1.beta..
[0021] Lymphocytes collected from infected animals often respond to
recall antigens (antigens to which an animal has been previously
exposed) by rapid multiplication (lymphoproliferation) and by
secreting cytokines, specifically IL-2. This immunological
phenomenon provides the basis of many vaccination protocols. It is
clearly shown herein that lymphocytes recovered from the spleen and
axiilary lymphnodes of S. mansoni infected mice exhibit recall
response to parasite Schistosoma antigens. However, when Sm 16.8 is
present in the antigen mixture, the lymphocytes are unable to
respond to the antigens. Once the antigen mixture is depleted of Sm
16.8 the recall response is regained. When Sm 16.8 is then added
back to the depleted antigen mixture, ability of the lymphocytes to
respond to the recall antigens is again lost. This demonstrates
that the anti-inflammatory protein Sm 16.8 also has
immunomodulatory functions.
[0022] Success of a vaccination protocol largely depends on the
initiation of an appropriate immune response at the site of
vaccination. In mice a radiation-attenuated vaccine [Richter et
al., Parasitology Today, 11:288-293 (1995)] has been shown to
confer protection against S. mansoni infection. Such vaccination
results in an initial interferon-.gamma. (IFN-.gamma.) response in
the skin and axillary lymph nodes. Whereas, a normal infection
results in an initial IL-4 and IL-10 but no IFN-.gamma. response.
Thus, development of an early IFN-.gamma. response in the skin and
its associated axillary lymph nodes is correlated with protection.
In vitro studies show that Sm 16.8 suppresses IFN-.gamma. response
in axillary lymph node cells stimulated with recall antigens.
Therefore, it is possible that Sm 16.8 may also interfere with
other aspects of the development of immune response against the
parasite.
[0023] In an important aspect of the invention, antagonists of Sm
16.8 activity such as peptides (the preparation of which are
discussed below) and antibodies are produced which, upon challenge
with S. mansoni, will block the anti-inflammatory activity/or
immunomodulatory effects of Sm 16.8. Antibodies are produced by
immunization of a host animal with polypeptides according to the
present invention using methods well known in the art.
[0024] Another anti-inflammatory protein designated Transthyretin
(TTR) [Borish et al., Inflammation, 16(5):471-484 (1992)], has a
molecular mass close to the parasite-derived protein Sm 16.8 of the
present invention. TTR has a molecular mass of 17 kDa and is
produced by human liver cells. However, studies show that TTR is
different from Sm 16.8 both in its physical property and function.
TTR was originally purified from human serum by an anion exchange
chromatography, molecular sieve HPLC and hydroxyl apatite
chromatography suggesting that TTR is a basic protein. Whereas Sm
16.8 is an acidic protein with a pI of 5.8. [See, e.g., Borish et
al., Inflammation, 16(5):471-484 (1992); and Zocchi et al.,
Immunol. Letters, 13(5):245-253 (1986).]
[0025] The anti-inflammatory activity of TTR depends on its ability
to inhibit secretion of IL-1.alpha. and IL-1.beta. from endothelial
cells and monocytes similar to Sm 16.8. However, TTR does not
inhibit the intracellular synthesis of IL-1 as does Sm 16.8, rather
TTR increases the level of IL-1 mRNA and IL-1 protein concentration
intracellularly. This means the TTR treated cells are continually
synthesizing the pro-inflammatory cytokine IL-1 in their cytoplasm
but are unable to secrete the cytokine, whereas, in Sm 16.8 treated
cells the synthesis of pro-inflammatory cytokines IL-1.alpha. and
IL-1.beta. is completely inhibited. In addition, unlike TTR, Sm
16.8 is shown to stimulate the production and secretion of
significant quantities of the anti-inflammatory cytokine IL-1ra.
Thus overall, the function of Sm 16.8 provides advantages in
reducing an inflammatory response in the skin not obtainable with
TTR because it reduces or eliminates the pro-inflammatory cytokines
present in the microenvironment of the tissue.
[0026] Having isolated and purified Sm 16.8 its amino acid
composition and its N-terminal amino acid sequence are obtainable
using routine methodologies well known to those of ordinary skill
in the art. Knowledge of the N-terminal amino acid sequence of Sm
16.8 enables a person of ordinary skill in the art to obtain the
DNA encoding the protein by synthesizing appropriate polynucleotide
probes or primers based on the determined amino acid sequence and
by using the polynucleotides or probes to screen genomic DNA
libraries or cDNA libraries obtainable from S. mansoni by
hybridization methods, or by use of methods such as the polymerase
chain reaction (PCR). Upon isolating and determining the sequence
of a DNA encoding Sm 16.8, a person of ordinary skill in the art
having knowledge of the genetic code would be readily able to
deduce the complete amino acid sequence of Sm 16.8. Vectors
comprising a DNA according to the present invention (e.g.,
plasmids, viruses, bacteriophage, cosmids and others) are useful
for expressing the Sm 16.8 DNA in host cells. Host cells may be
eucaryotic or procaryotic cells. Such cells include yeast cells and
bacterial cells such as E. coli and others. Host cells expressing
DNAs of the present invention provide an abundant reproducible
source of Sm 16.8 for use in the practice of the invention.
[0027] DNA of the present invention are also useful for preparing
muteins, variants or analogs of Sm 16.8, having amino acid
substitutions at specified sites in the protein and which retain a
biological activity of Sm 16.8. Methods for selecting candidate
amino acids for substitution are well known in the art. [See, e.g.,
Kyte et al., J. Mol. Biol., 157:105-132 (1982).] The DNA may also
be used to prepare truncations or fragments of Sm 16.8 and to
prepare derivatives of Sm 16.8 such as Sm 16.8 fusion proteins.
Exemplary proteins for fusion to Sm 16.8 include
.beta.galactosidase, glutathione-S-transferase, (His-tags) and
others well known in the art. The fusion may be made at the
N-terminus, carboxyl terminus of the polypeptide or may be inserted
at an internal site of the protein. Such fusion proteins may be
useful in preparing vaccines for enhancing the immune response
directed to Sm 16.8 and thereby serving as an immunoprophylactic
against infection with S. mansoni. The vaccine may also comprise
suitable diluents, adjuvants, and carriers.
[0028] Useful fragments of Sm 16.8 may also be prepared by the
proteolytic digestion of the purified protein with one or more of a
variety of well known proteolytic reagents such as cyanogen bromide
and/or proteolytic enzymes such as the carboxypeptidases,
asparaginases and others well known in the art.
[0029] DNAs, according to the present invention, may also be used
to isolate DNA homologous from other species by hybridization at
high stringency or by PCR under stringent conditions. Molecular
techniques for accomplishing the foregoing are well known and
described in detail in numerous publications including Ausubel et
al., Current Protocols in Molecular Biology, John Wiley and Sons
(1996), which is incorporated herein by reference.
[0030] Polypeptides of the present invention may also be covalently
modified by the addition of chemical moieties. Exemplary chemical
moieties include polyethylene glycol.
[0031] Once the primary amino acid composition of the protein is
obtained the three-dimensional structure of the protein is
determined, e.g., by crystallization and x-ray diffraction, and the
active binding sites could be identified for specific therapeutic
applications or immunomodulation. [See generally, e.g.,
PCT/US93/05548 published 23 Dec. 1993.] Such therapeutics may be
small peptides or polypeptide fragments that bind to the active
site thereby blocking its activity or may be other small molecules
which interfere with the active site of the polypeptide.
EXAMPLE 1
Isolation and Purification of Sm 16.8
[0032] Biomphalaria glabrata species of snails infected with S.
mansoni were obtained from Dr. Yung-san Liang, University of
Massachusetts Lowell, Mass. as part of a sub-contract from National
Institute of Allergy and Infectious Diseases (AI # 052590).
Cercarial stages of the parasite were collected from these infected
snails (suspended in warm distilled water) by exposing them to a
bright light source for 1 hour. Emerged cercariae were than
concentrated by passing through a wire mesh sieve (38 .mu./m;
Newark Wire Cloth Co., Newark, N.J.) and washed several times with
phosphate buffered saline containing 10 .mu.g/ml gentamicin.
Cercariae suspended in phosphate buffered saline (at a
concentration of 10,000 cercariae/ml) and were then transformed
into schistosomulae by incubating then in linolenic acid coated
culture flasks (coated with 2 mg/ml linoleic acid) for 30 min at
37.degree. C. Following incubation, the transformed schistosomulae
were separated from the tails and resuspended in sterile RPMI 1640
media containing 10 .mu.g/ml gentamicin. ES products from the
transformed schistosomulae were then collected by incubating the
schistosomulae in a culture flask for 16-24 hrs at 37.degree.
C.
[0033] Following incubation, the culture supernatant containing the
ES products were sterile filtered (0.2 .mu.m, Costar, Cambridge,
Mass.), concentrated up to 15-fold using a Centriprep concentrator
(3000 dalton cut off; Amicon, Beverly, Mass.) and the proteins were
separated in a 12-15% sodium dodecyl sulfate-polyacrylamide gel by
electrophoresis (SDS-PAGE) under non-reducing conditions (Bio-Rad,
Cambridge, Mass.). Molecular weight standards purchased from Sigma
Chemicals (St. Louis, Mo.) were run simultaneously in the same gel
to estimate the molecular mass of the proteins in the ES products.
Following separation, the protein bands in the SDS-PAGE gel were
stained with copper stain (Bio-Rad) and protein bands corresponding
to 211, 155.8, 67.8, 53.6, 38.2, 28.5, 21.2 and 16.8 kDa were cut
out using a sharp blade. The proteins were then electroeluted
(Bio-Rad) from the gel slices, and dialyzed against sterile
phosphate buffered saline. The eluted proteins were then tested for
anti-inflammatory activity as described below. Purity of the 16.8
kDa protein preparation was tested by running in an SDS-PAGE gel.
The 16.8 kDa is designated herein as Sm 16.8.
[0034] Isoelectric focusing and separation of the purified protein
in a two-dimensional SDS-PAGE showed that Sm 16.8 contains only a
single species of protein with a pI of 5.9. Treatment of Sm 16.8
with 2-mercaptoethanol (reducing conditions) had no effect on the
size of the protein suggesting that there are no disulfide bonds in
its structure. In addition Sm 16.8 has been shown to be trypsin
sensitive.
EXAMPLE 2
Effects of Sm 16.8 on Cytokine Expression
[0035] In order to characterize the mechanisms by which ES products
and particularly Sm 16.8 inhibits inflammation, approximately 5
.mu.g of purified Sm 16.8 purified was added to human keratinocyte
cultures (Clone C1, purchased from Clonetics Corporation, San
Diego, Calif.) grown in 6 well plates (1.times.10.sup.5 cells/well)
in keratinocyte growth medium KGM (Clonetics Corp., San Diego,
Calif.). At different time intervals after the start of the culture
(i.e. 4 hrs, 8 hrs, 16 hrs, 24 hrs, 48 hrs and 72 hrs) samples of
cells and their supernatants were collected for measuring
intracellular and secreted levels of the cytokines IL-1, IL-1,
IL-1ra, IL-2, IL-6, IL-10, and IFN-.gamma. by sandwich
enzyme-linked immunosorbent assay (ELISA), metabolic labelling and
immunoprecipitation studies, Northern blot analysis or polymerase
chain reaction (PCR). [See, e.g., Ramaswamy et al., Parasite
Immunology, 16:435-445 (1994); and Ausubel et al., Current
Protocols in Molecular Biology, eds. J. Wiley and Sons, (1997),
both of which are incorporated herein by reference.]
[0036] Cytokine values in cells and supernatants collected from
control cultures that were grown simultaneously in media alone
(i.e., not stimulated with the protein) was used as the baseline
value.
[0037] Results of the assay show that treatment of the cells with
Sm 16.8 resulted in 30-33 fold increase in secreted and
intracellular levels of IL-1ra as measured by ELISA. Metabolic
labelling studies showed that the increased intracellular synthesis
of IL-1ra occurred as early as 4 hrs after treatment with the
protein. Northern blot analysis confirmed this finding by showing
that message (mRNA) levels for IL-1ra increased in human
keratinocytes within 4 hrs after treatment with the protein.
[0038] Analysis of intracellular and secreted levels of IL-1.alpha.
and IL-1.beta. in human keratinocytes cultures treated with Sm 16.8
at 5 .mu.g/ml per 1.times.10.sup.5 cells showed that these two
pro-inflammatory cytokines are absent or are present only below the
base line value. This finding was confirmed by both Northern blot
analysis and metabolic labelling studies. Message (mRNA) levels for
both IL-1.alpha. and IL-1.beta. were absent in human keratinocytes
4 hrs after stimulation with the protein.
[0039] Thus, the parasite-derived protein Sm 16.8, suppressed
IL-1.alpha. and IL-1.beta. production in human keratinocytes within
4 hrs after exposure.
EXAMPLE 3
Effects of Sm 16.8 on Cytokine Production in Human Skin Organ
Culture
[0040] Parasitic stages of the S. mansoni that secrete Sm 16.8 were
applied to human skin organ cultures and cytokines released into
the culture medium were analyzed. Skin was cultured according to
the method of Buchshaun et al., J. Cut. Path., 20:21-27 (1993)
incorporated herein by reference. Approximately 3 cm.sup.3 neonatal
normal human foreskin obtained from Swedish American Hospital,
Rockford Ill. were exposed to 250 parasites (this translates to
approximately 50 .mu.g of the anti-inflammatory protein) and grown
in air/liquid biphasic cultures containing RPMI 1640, 5% fetal
bovine serum (FBS), 5% human AB serum (Sigma), 10 .mu.g/ml
gentamicin and 25 .mu.g/ml fungizone (Gibco, Grand Island, N.Y.) at
37.degree. C. and 5% CO.sub.2. At the end of 72 hrs of culture,
concentration of cytokines (IL-1, IL-1ra, IL-10 and IFN-.gamma.) in
the culture supernatant was measured as described above as were the
concentrations in an epidermal cell lysate prepared from the
culture. Epidermal cell lysates were prepared by freeze-thawing in
liquid nitrogen and cell debris was removed by centrifugation.
[0041] Control skin samples grown in media without exposure to the
parasite were used for obtaining baseline cytokine values.
[0042] Results of the ELISA assay indicate that exposure to the
parasite resulted in 15-20 fold increase in secreted levels of
IL-1ra and greater than 110-fold increase in epidermal cell lysate
of human skin organ cultures. The levels of IL-1.alpha. on the
other hand was increased only by 0.5 to 1 fold over the base line.
These data demonstrate that migration of schistosomes through human
skin stimulate the production of substantial quantities of the
anti-inflammatory cytokine IL-1ra, as predicted from the cell
culture system.
EXAMPLE 4
Immunomodulatory Effects of Sm 16.8
[0043] Single cell suspension of spleen and axiliary lymph node
cells (lymphocytes) were prepared from wht CD1 strain of mice
(Charles River, Wilmington, Mass.) that were infected 10 weeks
earlier with S. mansoni using methods well known in the art. See,
e.g., Mosca et al., Immunol. Lett., 15; 13(5):245-253 (1986) and
Ramaswamy et al., Parasite Immunol., 16:435-445 (1994). Cells were
placed in RPMI 1640 medium containing 2 mM glutamine, 0.04% sodium
bicarbonate (Sigma), 1 mM sodium pyruvate (Gibco), 25 mM HEPES
(Sigma), 50 mM 2-mercaptoethanol and 10 .mu.g/ml gentamicin.
[0044] During the process of proliferation, lymphocytes take up and
incorporate thymidine. By incubating lymphocytes in media
containing .sup.3H-labelled thymidine for a defuiite period of
time, and then measuring the amount of .sup.3H-thymidine that is
incorporated into the cells, it is possible to calculate the
proportion of dividing cells in the cultures. Addition of ES
products depleted of Sm 16.8 resulted in 1.8-fold increase in
.sup.3H-thymidine uptake by spleen cells and 9-fold increase in
.sup.3H-thymidine up take by axiliary lymph node cells indicating
that ES depleted of Sm 16.8 was capable of stimulating
proliferation of lymphocytes from infected mice..sup.1 Prior
incubation of spleen and axillary lymph node cells with Sm 16.8 for
6 hrs before addition of the ES products depleted of Sm 16.8,
resulted in near baseline values of .sup.3H-thymidine up take by
the cells indicating that Sm 16.8 is capable of suppressing antigen
specific lymphoproliferation. .sup.1 ES products were depleted of
Sm 16.8 by running ES products on non-reducing gel SDS-PAGE gel as
described in Example 1. The band corresponding to Sm 16.8 was
excised nd set aside. The remaining bands were electroeluted,
pooled and dialyzed against PBS. The dialyzing membrane had a
molecular weight cutoff of 3 kDa.
[0045] When IL-2 levels were measured (by ELISA) in the
supernatants of spleen and axillary lymph node cell cultures that
were set up as described above, a significant reduction in the
ability of the lymphocytes to secrete IL-2 was observed in the
presence of Sm 16.8. This suggests that the Sm 16.8 also has an
immunomodulatory function.
EXAMPLE 5
Effects of Sm 16.8 LPS Induction of IL-1.alpha.
[0046] Based on its properties described in Examples 2 & 3, Sm
16.8 appears to specifically suppress the pro-inflammatory cytokine
IL-1.alpha. in cutaneous cells. To test the extent to which Sm 16.8
can suppress IL-1.alpha. production in human keratinocytes, human
keratinocytes were stimulated in vitro with lipopolysaccharide
(LPS), a potent IL-1.alpha. inducing molecule, and tested the
ability of Sm 16.8 to reverse or prevent the IL-1.alpha. inducing
effect of LPS on human keratinocytes. In these studies a titration
curve for LPS was plotted (0.00001-10 .mu.g/ml) to identify the
minimum concentration of LPS needed to induce maximum IL-1.alpha.
secretion from keratinocytes. Tissue culture methods and cytokine
analysis used in these experiments were as described in Example 2.
These experiments showed that LPS at a concentration of 0.1
.mu.g/ml or above had maximal effect on IL-1.alpha. production by
human keratinocytes (19-21 ng of IL-1.alpha. per 1.times.10.sup.6
cells). To test the modulatory effect of Sm 16.8 on IL-1.alpha.
secretion in keratinocytes, we added Sm 16.8 in varying
concentration (0.6125 to 20 .mu.g/ml) simultaneously to LPS treated
(1 mg/ml) human keratinocytes in vitro. The results showed a
concentration dependent decrease in LPS induced IL-1.alpha.
secretion by Sm 16.8 treated human keratinocytes. A concentration
of 2.5 .mu.g/ml of Sm 16.8 in the culture media was highly
effective in suppressing LPS induced IL-1.alpha. secretion by human
keratinocytes.
EXAMPLE 6
Effects of Sm 16.8 on Neutrophil Infiltration
[0047] IL-1 plays a major role in acute cutaneous inflammation.
Since the preceding in vitro studies showed that Sm 16.8 can
suppress LPS induced IL-1.alpha. in keratinocytes, Sm 16.8 was
tested for its ability to suppress lymphocyte (neutrophil)
infiltration (a hallmark of acute inflammation) in vivo in the
skin. In these studies 50 .mu.g of a 1 mg/ml suspension of LPS in
PBS was injected subcutaneously into the skin of CD1 strain of mice
(Charles River Laboratories, Wilmington, Mass.) to induce
neutrophil infiltration. Preliminary titration experiments showed
that this concentration of LPS induced maximum neutrophil
infiltration into the skin of mice within 24 hrs. To determine the
modulatory effect of Sm 16.8 on neutrophil infiltration, we added
Sm 16.8 in varying concentrations (0.6125 to 20 .mu.g/ml) to 1
mg/ml of LPS in PBS and injected subcutaneously into the skin of
mice.
[0048] The number of neutrophils infiltrated in the skin 24 hrs
after injection were counted after removing the treated area of the
skin, fixing it in buffered formalin, processing the tissue in
paraffin, cutting five .mu.m serial sections of the entire skin
piece and staining with hematoxylin/eosin. Neutrophils were
identified morphologically in these sections and their total
numbers (in 30-60 fields) were counted using a 3 mm.sup.2 grid
(100.times.). The values were then expressed as mean cell .+-.SD
per 3 mm.sup.2 area of skin. These studies showed that LPS induced
significant neutrophil infiltration (51.+-.3 per 3 mm.sup.2) into
the skin.
[0049] Sm 16.8 was shown to completely suppress the LPS induced
neutrophil infiltration into the skin in a dose dependent manner.
Maximum suppression was evident at a dose of 2.5 .mu.g/ml and
above. These data thus suggested that Sm 16.8 prevented neutrophil
infiltration into the skin in an in vivo model.
EXAMPLE 7
Effects of Sm 16.8 on Expression of ICAM-1
[0050] Infiltration and entry of neutrophils from systemic
circulation into the skin is facilitated by cytokine induced
expression of intercellular adhesion molecules, for example ICAM-1,
on the surface of endothelial cells. Besides the endothelial cells,
keratinocytes, dermal dendritic cells and possibly other dermal
cells may also express ICAM-1 in response to pro-inflammatory
cytokines such as IL-1.alpha.. Since the foregoing Examples show
that Sm 16.8 suppresses IL-1.alpha. expression and prevents
neutrophil infiltration into the skin, studies were conducted to
determine whether Sm 16.8 interferes with expression of ICAM-1 in
the skin.
[0051] In vivo experiments were designed in CD1 mice similar to
those described in Example 6. The mice were injected subcutaneously
with same concentration of LPS (1 mg/ml) and varying concentrations
of Sm 16.8 (0.6125 to 20 .mu.g/ml). Skin samples from the injected
sites were snap frozen in OCT compound (Miles Inc. Elkhart, Ind.)
at -70.degree. C. and were processed for cryostat sectioning.
Sections of 7-8.mu. thickness were prepared on APES coated slides
(Sigma, St. Louis, Mo.), fixed in acetone and used in an
immunohistochemical assay to detect ICAM-1.
[0052] To detect ICAM-1 antigens in the skin, sections were
incubated (for 60 min at room temperature) with biotinylated
hamster anti-mouse CD54 antibodies (anti-ICAM-1 antibodies)
(Pharminigen, San Diego, Calif.) after blocking with Superblock
(Pierce Chemicals, Rockford, Ill.). Aline phosphatase labeled
streptavidin (Pierce Chemicals) was used to detect the biotin
associated with the primary antibody and the color was developed
using Fast Red (Pierce Chemicals).
[0053] Endogenous alkaline phosphatase activity was blocked using a
blocking buffer (Pierce Chemicals) for 15 min before the addition
of Fast Red. Appropriate controls without the primary and secondary
antibodies were processed simultaneously. These studies showed that
ICAM-1 expression is significantly increased in the endothelial
cells lining the capillaries of dermis and hypodermis, and on
several epidermal and dermal cells after treatment with LPS.
However, treatment with Sm 16.8 completely suppressed the LPS
induced ICAM-1 expression in the skin in a dose dependent manner.
Maximum ICAM-1 suppression was evident in mice treated with 10
.mu.g/ml or more of Sm 16.8. Thus, these studies demonstrate that
one of the mechanisms underlying the anti-inflammatory and
immunomodulatory activities of Sm 16.8 is its ability to down
regulate or to suppress ICAM-1 expression in the skin.
EXAMPLE 8
Therapeutic and Prophylactic Uses of Sm 16.8
[0054] The anti-inflammatory immunomodulatory properties of Sm 16.8
and related polypeptides described in detail above may be exploited
in therapeutic methods for the treatment of diseases characterized
by inflammatory process and with production of pro-inflammatory
cytokines. Exemplary diseases include cutaneous diseases such as
urticaria, atopic dermatitis, cutaneous allergic conditions such as
contact sensitivity, and psoriasis. See, e.g., Cecil, Textbook of
Medicine, Wyngaarden et al. eds., W.B. Sanders, Philadelphia, Pa.
(1985).
[0055] Therapeutic applications of Sm 16.8 and Sm 16.8-related
polypeptides are not limited to cutaneous diseases. Many other
diseases (systemic) are associated with inflammatory processes and
are amenable to treatment according to the present invention.
Exemplary diseases include but are not limited to Hashimoto's
Thyroiditis, inflammatory polyneuropathy, chronic granulomatous
disease, rheumatoid arthritis and others.
[0056] Methods for treating diseases such as those exemplified
above involve the administration of the polypeptides or antibodies
of the present invention via an appropriate route of
administration. For example, treatment of cutaneous diseases may be
accomplished by the topical application of the polypeptides of the
invention preferably in a suitable carrier such as pharmaceutically
acceptable lotions, creams, salves or other vehicles or by
subcutaneous, intradermal, or hypodermal injection in an
appropriate vehicle. The carrier may also include excipients which
facilitate the penetration of the polypeptides into the skin. The
determination of the appropriate dosages and dosage schedules for
optimal therapeutic effect is readily determined using routine
methods such as those described in the forgoing examples.
[0057] The polypeptides and pharmaceutical compositions of the
present invention are also useful in treating systemic inflammatory
diseases such as those described above. In such methods, the
polypeptides or pharmaceutical compositions of the present
invention may be administered via intravenous, intramuscular,
subcutaneous, oral or other routes of administration.
Therapeutically effective doses are readily determined by methods
well known in the art.
[0058] Polypeptides according to the present invention are also
useful for producing vaccines for immunoprophylaxis of
schistosomiasis. Useful vaccines induce the production of
antibodies directed to one or more epitopes of Sm16.8 and serve to
neutralize the anti-inflammatory effects of the protein, thereby
allowing the host to mount an effective immune response against the
invading parasite. Vaccines comprise one or more polypeptides
according to the present invention and optionally, a suitable
diluent, adjuvant or carrier.
[0059] The foregoing Examples are presented by way of illustration
and are not intended to in any way limit the scope of the present
invention as set out in the appended claims. All references set out
herein are incorporated by reference.
* * * * *