U.S. patent application number 17/685993 was filed with the patent office on 2022-08-18 for use of dermcidin in sterile inflammatory conditions.
The applicant listed for this patent is THE FEINSTEIN INSTITUTES FOR MEDICAL RESEARCH. Invention is credited to Haichao Wang, Ping Wang.
Application Number | 20220257714 17/685993 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220257714 |
Kind Code |
A1 |
Wang; Ping ; et al. |
August 18, 2022 |
USE OF DERMCIDIN IN STERILE INFLAMMATORY CONDITIONS
Abstract
A method of treating a sterile inflammatory condition in a
subject using an isolated dermcidin peptide or an active fragment
thereof of or an active analog thereof is provided. Also provided
is a method of inhibiting organ transplantation-associated
ischemia/reperfusion and/or organ transplantation-associated
inflammation.
Inventors: |
Wang; Ping; (Roslyn, NY)
; Wang; Haichao; (Edison, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE FEINSTEIN INSTITUTES FOR MEDICAL RESEARCH |
Manhasset |
NY |
US |
|
|
Appl. No.: |
17/685993 |
Filed: |
March 3, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15769880 |
Apr 20, 2018 |
11266716 |
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PCT/US16/58027 |
Oct 21, 2016 |
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17685993 |
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62244779 |
Oct 22, 2015 |
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International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 38/16 20060101 A61K038/16; A61K 47/54 20060101
A61K047/54; A61K 47/60 20060101 A61K047/60; A61P 29/00 20060101
A61P029/00; A01N 1/02 20060101 A01N001/02; A61K 9/00 20060101
A61K009/00 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
nos. AT005076, GM063075, GM053008, and GM076179 awarded by the
National Institutes of Health. The government has certain rights in
the invention.
Claims
1. A method of treating a sterile inflammatory condition in a
subject comprising administering to the subject an amount of an
isolated dermcidin peptide, or an active fragment thereof, or an
active analog thereof, effective to treat a sterile inflammatory
condition.
2. The method of claim 1, wherein the sterile inflammatory
condition is caused by or associated with ischemia-reperfusion in
an organ in the subject.
3. The method of claim 1, wherein the sterile inflammatory
condition is caused by or associated with ischemia-reperfusion in a
gastrointestinal tract, liver, lung, kidney, heart, brain or
crushed limb of the subject.
4. The method of claim 1, wherein the active analog of dermcidin is
administered or used.
5. The method of claim 4, wherein the active analog of dermcidin
has a cysteine to serine substitution that prevents dimerization
via disulfide bonds between cysteine 34 of two dermcidin
peptides.
6. The method of claim 1, wherein the active fragment of dermcidin
is administered or used.
7. The method of claim 1, wherein the peptide, analog or fragment
is modified to improve its plasma half-life.
8. The method of claim 7, wherein the peptide, analog or fragment
is modified with PEGylation or mannosylation.
9. The method of claim 1, wherein the amount of an isolated
dermcidin peptide or an active fragment thereof or an active analog
thereof is administered by intra-arterial, intravenous,
intraventricular, or topical administration.
10. The method of claim 1, wherein the subject is a human
subject.
11. A dermcidin analog comprising the following sequence:
TABLE-US-00002 YDPEAASAPGSGNPSHEASAAQKENAGEDPGLARQAPKPRKQRSSLLEKG
LDGAKK.
12. The dermcidin analog of claim 11, wherein the peptide, analog
or fragment is modified with PEGylation or mannosylation.
13. A composition comprising the dermcidin analog of claim 11 in
monomer form.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/769,880, filed Apr. 20, 2018, which is a
U.S. national stage entry under 35 U.S.C. .sctn. 371 of PCT
International Patent Application No. PCT/US2016/058027, filed Oct.
21, 2016, which claims the benefit of U.S. Provisional Application
No. 62/244,779, filed Oct. 22, 2015, the contents of which are
incorporated herein by reference into the subject application.
BACKGROUND OF THE INVENTION
[0003] Throughout this application various patents and other
publications are referred to in parenthesis. Full citations for the
references may be found at the end of the specification. The
disclosures of these patents and publications are hereby
incorporated by reference in their entirety into the subject
application to more fully describe the art to which the subject
invention pertains.
[0004] Despite advances in medicine, conditions in which
inflammatory responses result in complications, injury or death
continue to be a problem area. This is true even in the case of
sterile inflammatory responses.
[0005] The present invention addresses the need for a new
anti-inflammatory treatments and compositions.
SUMMARY OF THE INVENTION
[0006] Provided is a method for treating a sterile inflammatory
condition in a subject comprising administering to the subject an
amount of an isolated dermcidin peptide, or an active fragment
thereof or an active analog thereof, effective to treat a sterile
inflammatory condition.
[0007] Also provided is a method of inhibiting organ
transplantation-associated ischemia/reperfusion and/or organ
transplantation-associated inflammation in a recipient subject
comprising storing and/or rinsing the organ to be transplanted in a
solution comprising an amount of an isolated dermcidin peptide, or
an active fragment thereof or an active analog thereof, effective
to inhibit organ transplantation-associated ischemia/reperfusion
and/or organ transplantation-associated inflammation in a recipient
subject.
[0008] Also provided is a method of treating an inflammatory
condition in a subject comprising administering to the subject an
amount of an isolated dermcidin peptide, or an active fragment
thereof or an active analog thereof, effective to treat an
inflammatory condition.
[0009] Additional objects of the invention will be apparent from
the description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A-1C. Expression and purification of recombinant
dermcidin. 1A). Amino acid sequence of dermcidin precursor and
various proteolytic peptides. (From top to bottom SEQ ID NOS:1, 7,
8, and 6, respectively). DCD-1L is a 48 amino acid peptide
corresponding to the C-terminal of the full length of dermcidin
precursor. 1B). Expression and purification of recombinant
histidine-tag dermcidin precursor (20-110) (DCD). Recombinant
dermcidin corresponding to residue 20-110 amino acid with an
N-terminal histidine tag was expressed in E. coli BL21 (DE3) pLysS
cells (Panel B, left gel), and purified by histidine-affinity
chromatography (Panel B, right gel) and Triton X-114 extraction to
remove contaminating endotoxins. Note that recombinant dermcidin
migrated on SDS-PAGE gel as a 12-14 kDa monomer (DCD M) in the
presence of a reducing agent (dithiothreitol, DTT), but migrated as
both a monomer and 24-28 kDa dimer (24-28 kDa) in the absence of
DTT, suggesting possible cross-linking between dimers through
disulfide bonds. 1C). Confirmation of the identify of recombinant
protein by Western blotting analysis using dermcidin-specific
antibodies.
[0011] FIG. 2. Dermcidin dose-dependently attenuated LPS- and
CIRP-induced NO release by murine macrophages. Murine macrophages
were stimulated with LPS or CIRP alone or in the presence of
recombinant dermcidin (DCD) for 16 hours, and extracellular levels
of nitric oxide (NO) were determined by the Griess Reagent. *,
P<0.05 versus "-control"; #, P<0.05 versus "+LPS", or "+CIRP"
alone.
[0012] FIG. 3A-3B. Dermcidin modulated LPS- and HMGB1-induced
chemokine release by human monocytes. Human peripheral mononuclear
cells (huPBMCs) were stimulated with LPS (0.8 .mu.g/ml) or HMGB1
(4.0 .mu.g/ml) alone, or in the presence of DCD (1.0 .mu.g/ml) for
16 hours, and extracellular levels of cytokines and chemokines were
determined by Cytokine Antibody Arrays (Panel A, B). 3A,
Representative cytokine antibody arrays. The name of the cytokines
and positive controls ("Pos", or "+Ctrl") were labeled in the table
below. 3B, Relative cytokine levels. The relative cytokine levels
were estimated by measuring the intensity of corresponding signal,
and expressed as mean.+-.SEM [% of positive controls ("+Ctrl") of
respective arrays] of two independent experiments. *, P<0.05
versus "untreated"; #, P<0.05 versus "+LPS" or "+HMGB1"
alone.
[0013] FIG. 4A-4B. Intravenous administration of dermcidin
conferred protection against hepatic ischemia/reperfusion (I/R)
injury. Male C57BL/6 mice (20-25 g) were subjected to hepatic
ischemia/reperfusion by temporal clamping the hepatic artery and
portal vein for 60 minutes, which typically produced ischemia in
70% of the liver. At the beginning of the reperfusion, 0.2 ml
saline or recombinant dermcidin solution ("DCD", 5.0 mg/kg BW) was
injected via the internal jugular vein. At 24 h after the onset of
ischemia, animals were euthanized to harvest blood to measure serum
levels of hepatic injury markers such as alanine aminotransferase
(ALT) (4A) and aspartate aminotransferase (AST) (4B) using
commercial kits. Note that dermcidin promoted significant
protection against I/R injury. *, P<0.05 versus sham control; #,
P<0.05 versus saline group ("I/R").
[0014] FIG. 5. DCD monomer protects against hepatic
ischemic/reperfusion injury. Representative liver histology at 24 h
post the onset of reperfusion. Note a normal liver parenchyma
architecture in the "Sham" control, but hepatic necrosis in the
"I/R". Unlike the DCD dimer-treated group ["I/R+DCD (d)"], the DCD
monomer-treated group ["I/R+DCD (m)"] exhibited a well-preserved
tissue structure. Liver injury was also assessed histologically
using the Suzuki liver injury scores, and expressed as
means.+-.S.E. of 3-6 animals per group. *P<0.05 vs. sham;
#P<0.05 vs. "I/R" group.
[0015] FIG. 6A-6B. DCD protects against hepatic
ischemic/reperfusion-induced lung injury. 6A). Histopathological
characteristics of lung injury after hepatic I/R. Representative
H&E histological images of lung sections at 24 h post the onset
of reperfusion. Note a normal lung architecture in the "Sham"
control, and extensive lung injury and neutrophil infiltration in
the "I/R" group. DCD treatment group ("I/R+DCD") exhibited a
well-preserved tissue structure. 6B). Histological Scores. Lung
injury was assessed histologically using American Thoracic Society
Documents' lung injury scores, and expressed as means.+-.S.E. of
3-6 animals per group. *P<0.05 vs. sham; #P<0.05 vs. "I/R"
group.
[0016] FIG. 7. DCD protects against lethal sepsis in mice. Balb/C
mice (7-10 weeks, 20-25 g, male) were subjected to sepsis by cecal
ligation and puncture as previously described. Briefly, Balb/c mice
were anesthetized with Ketamine (75 mg/kg, intramuscularly) and
xylazine (10 mg/kg, intramuscularly) before a 15 mm midline
incision was made to expose the cecum. A 4-0 Prolene suture
ligature was placed at a level 5.0 mm from the cecal tip away from
the ileocecal valve, and the ligated cecal stump was then punctured
once with a 22-gauge needle without direct extrusion of stool. The
cecum was then replaced back into its normal intra-abdominal
position, and the abdomen wound was closed with staples (wound
clips) to prevent leakage of fluid. All animals were resuscitated
with a normal saline solution (subcutaneously at 20 ml/kg of body
weight), and given a subcutaneous injection of imipenem (0.5
mg/mouse in 200 .mu.l sterile saline) (Primaxin, Merck & Co.,
Inc., West Point, Pa.) 30 minutes after the surgery. Saline or
recombinant DCD (0.2 mg/kg body weight) were given
intraperitoneally at +2 and +24 h post CLP surgery, and animal
survival rates were monitored for up to two weeks. The Kaplan-Meier
method was used to compare the differences in mortality rates
between groups. Shown in the figure was a summary of two
independent experiments with similar results. *, P<0.05 versus
saline control group.
DETAILED DESCRIPTION OF THE INVENTION
[0017] A method is provided for treating a sterile inflammatory
condition in a subject comprising administering to the subject an
amount of an isolated dermcidin peptide, or an active fragment
thereof of or an active analog thereof, effective to treat a
sterile inflammatory condition.
[0018] In an embodiment, the sterile inflammatory condition is
caused by or associated with ischemia-reperfusion in an organ in
the subject. In an embodiment, the sterile inflammatory condition
is caused by or associated with ischemia-reperfusion in a
gastrointestinal tract, liver, lung, kidney, heart, brain or
crushed limb of the subject.
[0019] Also provided is a method of inhibiting organ
transplantation-associated ischemia/reperfusion and/or organ
transplantation-associated inflammation in a recipient subject
comprising storing and/or rinsing the organ to be transplanted in a
solution comprising an amount of an isolated dermcidin peptide, or
an active fragment thereof or an active analog thereof, effective
to inhibit organ transplantation-associated ischemia/reperfusion
and/or organ transplantation-associated inflammation in a recipient
subject. In an embodiment, the isolated dermcidin peptide, or an
active fragment thereof or an active analog thereof, is used as an
adjuvant in an organ transplantation storage and/or organ
transplantation rinse solution. In an embodiment, the organ is a
kidney, liver, heart, or lung. The organ transplantation storage
and/or organ transplantation in an embodiment is a known and/or
clinically used organ transplantation storage and/or organ
transplantation.
[0020] In an embodiment of the methods, the isolated dermcidin
peptide is administered to the subject, or used in the storing
and/or rinsing, respectively. In an embodiment of the methods, the
isolated dermcidin peptide has the sequence of full-length human
dermcidin without its signal sequence.
[0021] In an embodiment of the methods, the active analog of
dermcidin is administered to the subject, or used in the storing
and/or rinsing, respectively. In an embodiment of the methods, the
active analog of dermcidin has a Cysteine.fwdarw.Serine
substitution that prevents dimerization via disulfide bonds between
cysteine 34 of two dermcidin peptides. In an embodiment of the
methods, the active fragment of dermcidin is administered to the
subject, or used in the storing and/or rinsing, respectively.
[0022] In an embodiment, a pharmaceutically acceptable salt of
dermcidin peptide or of a dermcidin fragment or analog is used.
[0023] In an embodiment, the dermcidin peptide has the sequence:
YDPEAASAPGSGNPCHEASAAQKENAGEDPGLARQAPKPRKQRSSLLEKGLDGAKK
AVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL (SEQ ID NO:1). In an
embodiment, the "cysteine 34" as referred to herein is the
underlined C in SEQ ID NO:1. In an embodiment, the dermcidin active
fragment has one of the following sequences:
TABLE-US-00001 (SEQ ID NO: 2) ESVGKGAVHDVKDVLDS; (SEQ ID NO: 3)
LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; (SEQ ID NO: 4)
LEKGLDGAKKAVGGLGKLGKDAVE; (SEQ ID NO: 5)
SSLLEKGLDGAKKAVGGLGKLGKDAVEDL; or (SEQ ID NO: 6)
SSLLEKGLDGAKKAVGGLGKLGKDA.
[0024] In an embodiment, the dermcidin fragment is DCD-1L, DCD-1 or
SSL25 as shown in FIG. 1A.
[0025] In an embodiment of the methods, the peptide, analog or
fragment is modified to improve its plasma half-life. In an
embodiment of the methods, the peptide, analog or fragment is
modified by being PEGylated or mannosylated.
[0026] In an embodiment of the methods wherein administration is
employed, the amount of an isolated dermcidin peptide or an active
fragment thereof or an active analog thereof is administered by
intra-arterial, intravenous, intraventricular, or topical
administration. Other routes of medicament administration known in
the art may also be used.
[0027] Also provided is a method of treating an inflammatory
condition in a subject comprising administering to the subject an
amount of an isolated dermcidin peptide, or an active fragment
thereof or an active analog thereof, effective to treat an
inflammatory condition.
[0028] In an embodiment, the inflammatory condition is sepsis,
septicemia or endotoxemia.
[0029] In an embodiment, the inflammatory condition is sepsis. In
an embodiment, the isolated dermcidin peptide, or an active
fragment thereof or an active analog thereof is administered
intraperitoneally.
[0030] In an embodiment, the inflammatory condition is septicemia.
In an embodiment, the isolated dermcidin peptide, or an active
fragment thereof or an active analog thereof is administered
intravascularly.
[0031] In an embodiment, the inflammatory condition is
endotoxemia.
[0032] In an embodiment of the methods, the active fragment of
dermcidin is administered or used. In an embodiment of the methods,
the peptide, analog or fragment is modified to improve its plasma
half-life. In an embodiment of the methods, the peptide, analog or
fragment is modified with PEGylation or mannosylation. In an
embodiment of the methods, the active analog of dermcidin is
administered or used. In an embodiment of the methods, the isolated
dermcidin peptide is administered or used. In an embodiment of the
methods, the isolated dermcidin peptide has the sequence of
full-length human dermcidin without its signal sequence. In an
embodiment of the methods, the active analog of dermcidin has a
Cysteine.fwdarw.Serine substitution that prevents dimerization via
disulfide bonds between cysteine 34 of two dermcidin peptides.
[0033] In an embodiment of the methods, the subject is a human
subject.
[0034] All combinations of the various elements described herein
are within the scope of the invention unless otherwise indicated
herein or otherwise clearly contradicted by context.
[0035] This invention will be better understood from the
Experimental Details, which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims that follow thereafter.
Experimental Details
Introduction
[0036] Cohabitating with various microbes over millions of years,
animals have developed multiple strategies to deal with microbial
infections. The epidermal barriers of the skin serve as the first
layer of defense by limiting the physical access of many pathogens
to the blood circulation. In addition, sweat glands secrete a wide
array of antimicrobial peptides, which restrain the growth of
various microbes on the skin. For instance, during rigorous
physical exercise, an antimicrobial peptide, called dermcidin, is
secreted by the sweat glands onto the epidermal surface of the skin
(1). It has been proposed that dermcidin can be activated in salty
and slightly acidic sweat to form channels that can possibly
perforate microbe membranes, allowing water and Zn.sup.+2 ions in
sweat to gush across the cell membrane, killing the microbe (2, 3).
Despite its capacity in binding to various bacterial strains,
dermcidin has not yet been reproducibly shown to permeabilize
bacterial membranes (4), calling for further investigation in this
arena. Nevertheless, at body sites in frequent contact with
pathogenic microbes, a higher amount of dermcidin peptide is
detected in sweat (5), supporting the essential role of sweat in
the regulation of skin microbial flora.
[0037] Prior to the disclosure herein, however, to applicants'
knowledge it was not known that dermcidin also has mammalian immune
system-modulating properties and anti-inflammatory properties.
[0038] As the first line of defense against microbial infection,
monocytes continuously patrol the body in search of invading
pathogens or damaged tissues, and can immediately infiltrate the
infected/injured tissue upon the detection of microbial products or
host-derived chemotactic factors. Once reaching extravascular
tissues, these monocytes are differentiated into tissue-specific
resident macrophages, which ingest and eliminate invading pathogens
in conjunction with other phagocytes (e.g., neutrophils).
Additionally, macrophages/monocytes are equipped with pattern
recognition receptors [such as the Toll-like receptors (TLRs) TLR2,
TLR3, TLR4, and TLR9] (7) for various pathogen-associated molecular
patterns (PAMPs, such as bacterial peptidoglycan, double-stranded
RNA, endotoxin, and CpG-DNA) (8). The engagement of various PAMPs
with respective receptors triggers release of various
proinflammatory mediators such as high mobility group box 1 (HMGB1)
(9), cold-inducible RNA-binding protein (CIRP) (10, 11) as well as
nitric oxide (NO) (12). In addition to active secretion, HMGB1 can
also be passively released from damaged cells (13) following
ischemia/reperfusion (14), trauma (15), or toxemia (16), thereby
serving as a damage-associated molecular pattern molecule (DAMP).
Thus, infection and injury converge on a common process,
inflammation (17), which is orchestrated by HMGB1 and other
proinflammatory mediators (e.g., CIRP) derived from activated
immune cells and damaged tissues (10). If dysregulated, the
excessive production of these proinflammatory mediators (e.g.,
HMGB1, NO, and CIRP) (9, 10, 12, 18), individually or in
combination, contribute to the pathogenesis of inflammatory
diseases.
[0039] Herein is provided evidence that dermcidin exhibits
immune-modulating properties in response to PAMP or DAMP.
[0040] Dermcidin is expressed in sweat glands, and in the absence
of an inflammatory stimulus, is constitutively secreted as a
full-length protein (1). This full length precursor can be further
processed by unknown proteases in human sweat, to form several
shorter peptides that exhibit anti-oxidant and antimicrobial
activities (FIG. 1A). For instance, the N-terminal peptide (residue
20-62) has been shown to protect various types of cells against
oxidative or hypoxic stresses (25-27). On the other hand, many
C-terminal peptides exhibit anti-microbial properties against S.
aureus, E. coli, E. faecalis, and C. albicans (1). In addition to
sweat glands, innate immune cells (e.g., monocytes) also express
dermcidin in response to viral infection (28). Furthermore, the
full-length dermcidin precursor (residue 22-110) also exhibits
bacterial killing activities towards S. aureus, E. coli, and P.
acnes (29). A C-terminal peptide, DCD-1L, has been shown to
activate keratinocytes to produce cytokines (e.g., TNF) and
chemokines (e.g., IL-8/CXCL8, CXCL10, and CCL20) (30). Herein it is
disclosed that dermcidin precursor divergently modulated PAMP- and
DAMP-induced production of TNF, NO, and chemokines by innate immune
cells.
[0041] Recombinant human dermcidin protein was generated in E.
coli, and purified to homogeneity in the absence or presence of a
reducing agent (DTT, FIG. 1B). Although migrating at a slightly
lower rate than the predicted molecular weight, the identity of
this recombinant dermcidin was confirmed by Western blotting
analysis using a commercially available antibody (FIG. 1C). This is
consistent with a recent report that recombinant histidine tag-DCD
migrated as a 15-16 kDa band on SDS-PAGE gel, even though its
molecular weight was determined to be .about.9.25 kDa by mass
spectrometry (29). Using purified dermcidin, we then tested its
immune-modulating properties using macrophage and monocyte
cultures. In response to PAMPs (e.g., bacterial endotoxin, LPS) or
endogenous cytokines (e.g., CIRP), macrophages released large
amounts of nitric oxide (NO, FIG. 2). However, dermcidin (DCD)
dose-dependently and significantly attenuated both LPS- and
CIRP-induced NO release (FIG. 2). Although activated monocytes
cannot produce NO, they do produce various proinflammatory
cytokines or chemokines. Human monocytes were stimulated with PAMPs
(e.g., LPS) or DAMPs (e.g., HMGB1), in the absence or presence of
DCD. The, relative levels of various cytokines/chemokines in the
monocyte-conditioned culture medium were measured using Cytokine
Antibody Arrays (FIG. 3). As shown in FIG. 3, both LPS and HMGB1
elevated the relative levels of several chemokines such as
GRO-.alpha. and MCP-3. Similarly, dermcidin effectively inhibited
LPS- and HMGB1-induced release of GRO-.alpha. and MCP-3 from human
monocyte cultures (FIG. 3). Despite the inhibitory effects on the
above chemokines, dermcidin slightly stimulated TNF secretion, an
early proinflammatory cytokine that propagates protective innate
immune response against microbial infection. In agreement with the
stability of dermcidin's anti-bacterial properties over a broad pH
range and salt concentrations (1), it was found that dermcidin's
immune-modulating properties were also relatively stable. Although
dermcidin tended to form dimers in the absence of reducing agents
(FIG. 1B), its immune modulating properties remained unaltered
(data not shown), indicating the relative stability of dermcidin's
biological activities in vitro.
[0042] In animal models of peritoneal microbial infection induced
by surgical perforation of the cecum, a technique known as cecal
ligation and puncture (CLP) (31), neutralizing antibodies against
TNF worsens the outcome (32), supporting a beneficial role of TNF
in the innate immunity against bacterial infection. Although
appropriate inflammatory responses might be needed for the innate
immunity against microbial infection, excessive recruitment of
leukocyte to infection or injury sites might be harmful to the
host. As a critical element of the innate immune response,
leukocyte recruitment is governed by chemotactic functions of
bacterial products and chemokines (such as GRO-.alpha. and MCP-3)
(33). The dermcidin-mediated suppression of both PAMP- and
DAMP-induced chemokines (such as GRO-.alpha. and MCP-3) might
attenuate leukocyte recruitment to the infection and injury site,
and likely prevent excessive inflammatory responses to infection or
injury.
[0043] Although many anti-inflammatory agents have failed to
improve outcomes of many inflammatory diseases (such as sepsis),
the investigation of pathogenic cytokines in animal models of
diseases has led to the development of successful
cytokine-targeting therapeutic strategies (e.g., anti-TNF antibody,
infliximab) for autoimmune diseases such as rheumatoid arthritis
(34, 35). The dual anti-bacterial (29) and anti-inflammatory (FIG.
2 and FIG. 3) properties may distinguish dermcidin from previously
tested anti-inflammatory agents, positioning it as a unique
experimental agent for preclinical testing using various animal
models of inflammatory diseases. Given the complex and redundant
roles of various cytokines and chemokines in various inflammatory
diseases, it is now particularly important to test the hypothesis
that dermcidin may occupy an important role in the regulation of
local or systemic inflammation in preclinical animal models. For
instance, injection of bacterial endotoxin directly into the skin
(e.g., footpad, subcutaneously) provides a murine model of local
inflammation and edema. It is interesting to determine whether
local co-administration of dermcidin attenuates paw edema at
various time points after endotoxin challenge. Additionally,
systemic inflammation can be induced in animals by infusion of
bacterial endotoxin such as LPS (31), or surgical perforation of
the cecum, a technique aforementioned as CLP (31). Systemic
administration (intraperitoneally or intravenously) of dermcidin
may confer a dose-dependent protection against lethal endotoxemia
or CLP-induced bacteremia.
[0044] In further experiments, intravenous administration of
dermcidin conferred protection against hepatic ischemia/reperfusion
(I/R) injury. Male C57BL/6 mice (20-25 g) were subjected to hepatic
ischemia/reperfusion by temporal clamping the hepatic artery and
portal vein for 60 minutes, which typically produced ischemia in
70% of the liver. At the beginning of the reperfusion, 0.2 ml
saline or recombinant dermcidin solution ("DCD", 5.0 mg/kg BW) was
injected via the internal jugular vein. At 24 h after the onset of
ischemia, animals were euthanized to harvest blood to measure serum
levels of hepatic injury markers such as alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) using commercial kits.
Dermcidin promoted significant protection against I/R injury. (see
FIG. 4)
[0045] The DCD monomer protects against hepatic
ischemic/reperfusion injury, as shown in FIG. 5. A representative
liver histology at 24 h post the onset of reperfusion is shown,
note a normal liver parenchyma architecture in the "Sham" control,
and hepatic necrosis in the "I/R". Unlike the DCD dimer-treated
group ["I/R+DCD (d)"], the DCD monomer-treated group ["I/R+DCD
(m)"] exhibited a well-preserved tissue structure. Liver injury was
also assessed histologically using the Suzuki liver injury scores,
and expressed as means.+-.S.E. of 3-6 animals per group. *P<0.05
vs. sham; #P<0.05 vs. "I/R" group.
[0046] DCD also protects against hepatic
ischemic/reperfusion-induced lung injury. FIG. 6 shows
histopathological characteristics of lung injury after hepatic I/R.
In FIG. 6A are shown representative H&E histological images of
lung sections at 24 h post the onset of reperfusion. Note a normal
lung architecture in the "Sham" control, and extensive lung injury
and neutrophil infiltration in the "I/R" group. DCD treatment group
("I/R+DCD") exhibited a well-preserved tissue structure. FIG. 6B
shows histological Scores. Lung injury was assessed histologically
using American Thoracic Society Documents' lung injury scores, and
expressed as means.+-.S.E. of 3-6 animals per group. *P<0.05 vs.
sham; #P<0.05 vs. "I/R" group.
[0047] DCD was also found to protect against lethal sepsis in mice.
Balb/C mice (7-10 weeks, 20-25 g, male) were subjected to sepsis by
cecal ligation and puncture as previously described. Briefly,
Balb/c mice were anesthetized with Ketamine (75 mg/kg,
intramuscularly) and xylazine (10 mg/kg, intramuscularly) before a
15 mm midline incision was made to expose the cecum. A 4-0 Prolene
suture ligature was placed at a level 5.0 mm from the cecal tip
away from the ileocecal valve, and the ligated cecal stump was then
punctured once with a 22-gauge needle without direct extrusion of
stool. The cecum was then replaced back into its normal
intra-abdominal position, and the abdomen wound was closed with
staples (wound clips) to prevent leakage of fluid. All animals were
resuscitated with a normal saline solution (subcutaneously at 20
ml/kg of body weight), and given a subcutaneous injection of
imipenem (0.5 mg/mouse in 200 .mu.l sterile saline) (Primaxin,
Merck & Co., Inc., West Point, PA) 30 minutes after the
surgery. Saline or recombinant DCD (0.2 mg/kg body weight) were
given intraperitoneally at +2 and +24 h post CLP surgery, and
animal survival rates were monitored for up to two weeks. The
Kaplan-Meier method was used to compare the differences in
mortality rates between groups. Shown in FIG. 7 is a summary of two
independent experiments with similar results. *, P<0.05 versus
saline control group.
Materials and Methods
[0048] Materials: Bacterial endotoxin (lipopolysaccharide, LPS, E.
coli 0111:B4, Cat. No. L4130) was obtained from Sigma-Aldrich (St.
Louis, Mo.). Dulbecco's Modified Eagle's Medium (DMEM, Cat. No.
11995-065), penicillin/streptomycin (Cat. No. 15140-122) and fetal
bovine serum (FBS, Cat. No. 26140079) were from Invitrogen (Grand
Island, N.Y.). Recombinant HMGB1 and CIRP were expressed in E.
coli, and purified to remove contaminating endotoxin by Triton
X-114 extraction as previously described (10, 19). To express
recombinant dermcidin, the cDNA encoding for the mature form of
dermcidin (DCD, NM_053283.2) (corresponding to residues 20-110,
without the N-terminal signal peptide, amino acid 1-19) was cloned
onto a pReceiver-B01 (CS-T3198-B01-01, GeneCopoeia) vector, and the
recombinant DCD was expressed in E. coli BL21 (DE3) pLysS cells.
Recombinant DCD containing an N-terminal histidine tag (His-DCD)
was isolated and purified to remove contaminating endotoxin by
Triton X-114 extraction.
[0049] Cell culture: Murine macrophage-like RAW 264.7 were obtained
from the American Type Culture Collection (ATCC, Rockville, Md.),
and were cultured in DMEM supplemented with 1%
penicillin/streptomycin and 10% FBS. Human blood was purchased from
the Long Island Blood Bank (Melville, N.Y.), and human peripheral
blood mononuclear cells (HuPBMCs) were isolated by density gradient
centrifugation through Ficoll (Ficoll-Paque PLUS, Pharmacia,
Piscataway, N.J.) as previously described (20-22). Adherent
macrophages or HuPBMCs were gently washed with, and cultured in,
DMEM before stimulation with LPS (0.4 .mu.g/ml), CIRP (2.0
.mu.g/ml), or HMGB1 (1.0 .mu.g/ml) in the absence or presence of
recombinant dermcidin for 16 h. Subsequently, the cell-conditioned
culture media were analyzed respectively for levels of nitric
oxide, and other cytokines by the Griess Reaction and Cytokine
Antibodies Arrays as previously described (19, 23).
[0050] Nitric oxide (NO) assay: The levels of NO in the culture
medium were determined indirectly by measuring the NO.sup.2-
production with a colorimetric assay based on the Griess reaction
(21, 24). NO.sup.2- concentrations were determined with reference
to a standard curve generated with sodium nitrite at various
dilutions.
[0051] Cytokine antibody array: Human Cytokine Antibody Array C3
(Cat. No. AAH-CYT-3-4, RayBiotech Inc., Norcross, Ga., USA), which
respectively detect 42 cytokines on one membrane, were used to
determine cytokine levels in human monocyte-conditioned culture
medium as previously described (21, 24). Briefly, the membranes
were sequentially incubated with equal volumes of cell culture
medium (200 .mu.l), primary biotin-conjugated antibodies, and
horseradish peroxidase-conjugated streptavidin. After exposing to
X-ray film, the relative signal intensity was determined using the
Scion Image software.
[0052] Statistical analysis: Data are expressed as mean.+-.SEM of
two independent experiments in triplicates. One-way analyses of
variance (ANOVA) followed by the Tukey's test for multiple
comparisons were used to compare between different groups. A P
value less than 0.05 was considered statistically significant.
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Sequence CWU 1
1
8191PRTHomo sapiens 1Tyr Asp Pro Glu Ala Ala Ser Ala Pro Gly Ser
Gly Asn Pro Cys His1 5 10 15Glu Ala Ser Ala Ala Gln Lys Glu Asn Ala
Gly Glu Asp Pro Gly Leu 20 25 30Ala Arg Gln Ala Pro Lys Pro Arg Lys
Gln Arg Ser Ser Leu Leu Glu 35 40 45Lys Gly Leu Asp Gly Ala Lys Lys
Ala Val Gly Gly Leu Gly Lys Leu 50 55 60Gly Lys Asp Ala Val Glu Asp
Leu Glu Ser Val Gly Lys Gly Ala Val65 70 75 80His Asp Val Lys Asp
Val Leu Asp Ser Val Leu 85 90217PRTHomo sapiens 2Glu Ser Val Gly
Lys Gly Ala Val His Asp Val Lys Asp Val Leu Asp1 5 10
15Ser337PRTHomo sapiens 3Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys
Glu Lys Ile Gly Lys Glu1 5 10 15Phe Lys Arg Ile Val Gln Arg Ile Lys
Asp Phe Leu Arg Asn Leu Val 20 25 30Pro Arg Thr Glu Ser
35424PRTHomo sapiens 4Leu Glu Lys Gly Leu Asp Gly Ala Lys Lys Ala
Val Gly Gly Leu Gly1 5 10 15Lys Leu Gly Lys Asp Ala Val Glu
20529PRTHomo sapiens 5Ser Ser Leu Leu Glu Lys Gly Leu Asp Gly Ala
Lys Lys Ala Val Gly1 5 10 15Gly Leu Gly Lys Leu Gly Lys Asp Ala Val
Glu Asp Leu 20 25625PRTHomo sapiens 6Ser Ser Leu Leu Glu Lys Gly
Leu Asp Gly Ala Lys Lys Ala Val Gly1 5 10 15Gly Leu Gly Lys Leu Gly
Lys Asp Ala 20 25748PRTHOMO SAPIENS 7Ser Ser Leu Leu Glu Lys Gly
Leu Asp Gly Ala Lys Lys Ala Val Gly1 5 10 15Gly Leu Gly Lys Leu Gly
Lys Asp Ala Val Glu Asp Leu Glu Ser Val 20 25 30Gly Lys Gly Ala Val
His Asp Val Lys Asp Val Leu Asp Ser Val Leu 35 40 45847PRTHOMO
SAPIENS 8Ser Ser Leu Leu Glu Lys Gly Leu Asp Gly Ala Lys Lys Ala
Val Gly1 5 10 15Gly Leu Gly Lys Leu Gly Lys Asp Ala Val Glu Asp Leu
Glu Ser Val 20 25 30Gly Lys Gly Ala Val His Asp Val Lys Asp Val Leu
Asp Ser Val 35 40 45
* * * * *