U.S. patent application number 10/028075 was filed with the patent office on 2003-06-19 for gene regulator.
Invention is credited to Benner, Robert, Khan, Nisar Asmed.
Application Number | 20030113733 10/028075 |
Document ID | / |
Family ID | 26077008 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113733 |
Kind Code |
A1 |
Khan, Nisar Asmed ; et
al. |
June 19, 2003 |
Gene regulator
Abstract
The invention relates to the modulation of gene expression in a
cell, also called gene control, in particular in relation to the
treatment of a variety of diseases. The invention provides a method
for modulating expression of a gene in a cell comprising providing
said cell with a signalling molecule comprising a peptide or
functional analogue thereof. Furthermore, the invention provides a
method for identifying or obtaining a signalling molecule
comprising a peptide or functional derivative or analogue thereof
capable of modulating expression of a gene in a cell comprising
providing said cell with a peptide or derivative or analogue
thereof and determining the activity and/or nuclear translocation
of a gene transcription factor.
Inventors: |
Khan, Nisar Asmed;
(Rotterdam, NL) ; Benner, Robert; (Barendrecht,
NL) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
26077008 |
Appl. No.: |
10/028075 |
Filed: |
December 21, 2001 |
Current U.S.
Class: |
435/6.16 ;
435/7.2; 506/14 |
Current CPC
Class: |
A61P 19/08 20180101;
C07K 7/06 20130101; A61P 11/06 20180101; C07K 14/59 20130101; C07K
5/101 20130101; C07K 5/0806 20130101; A61P 19/10 20180101; A61P
29/00 20180101; A61P 43/00 20180101; A61P 31/04 20180101; C07K
5/0808 20130101; C07K 5/06026 20130101; A61K 38/00 20130101; C07K
7/08 20130101; C07K 5/06008 20130101; C07K 5/1013 20130101; A61P
1/00 20180101; C07K 5/1008 20130101; C07K 5/081 20130101 |
Class at
Publication: |
435/6 ;
435/7.2 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 4, 2001 |
EP |
01203748.7 |
Claims
What is claimed is:
1. A method for obtaining information about the capacity or
tendency of an oligopeptide, or a modification or derivative
thereof, to regulate expression of a gene comprising the steps of:
a) contacting said oligopeptide, or a modification or derivative
thereof, with at leaste one cell; b) determining the presence of at
least one gene product in or derived from said cell.
2. The method according to claim 1 wherein said oligopeptide
comprises an amino acid sequence corresponding to a fragment of a
naturally occurring polypeptide.
3. The method according to claim 2 wherein said naturally occurring
polypeptide comprises human chorionic gonadotropic hormone
(hCG).
4. The method according to any one of claims 1 to 3 wherein said
cell comprises an eukaryotic cell.
5. The method according to any one of claims 1 to 4 further
comprising c) determining the presence of said gene product in or
derived from a cell which has not been contacted with said
oligopeptide, or a modification or derivative thereof, and
determining the ratio of gene product found in step b to gene
product found in step c.
6. A method for identifying or obtaining a signalling molecule
comprising a peptide or functional derivative or analogue thereof
capable of modulating expression of a gene in a cell comprising
providing said cell with a peptide or derivative or analogue
thereof and determining the activity and/or nuclear translocation
of a gene transcription factor and then synthesising the molecule
with the desired activity.
7. The method according to claim 6 further comprising determining
whether said signalling molecule is membrane-permeable.
8. The method according to claim 6 or 7 wherein said gene
transcription factor comprises a NF-kappaB/Rel protein.
9. A method for identifying or obtaining a signalling molecule
comprising a peptide or functional derivative or analogue thereof
capable of modulating expression of a gene in a cell comprising
providing said cell with a peptide or derivative or analogue
thereof and determining relative up-regulation and/or
down-regulation of at least one gene expressed in said cell and
then synthesising the molecule with the desired activity.
10. The method according to claim 6, 7, 8 or 9 further comprising
determining relative up-regulation and/or down-regulation of at
least one gene expressed in said cell.
11. The method according to claim 6 to 10 further comprising
determining relative up-regulation and/or down-regulation of a
multitude of genes expressed in said cell.
12. A method for identifying or obtaining a signalling molecule
comprising a peptide or functional derivative or analogue thereof
capable of modulating expression of a gene in a cell comprising
providing a peptide or derivative or analogue thereof and
determining binding of said peptide or derivative or analogue
thereof to a factor related to gene control and then synthesising
the molecule with the desired activity.
13 The method according to claim 12 further comprising providing a
multitude of peptides or derivatives or analogues thereof and
determining binding of at least one of said peptides or derivatives
or analogues thereof to a factor related to gene control.
14. The method according to claim 12 or 13 wherein said factor
related to gene control comprises a transcription factor.
15. The method according to claim 14 wherein said transcription
factor comprises a NF-kappaB-Rel protein.
16. The method according any one of claims 12 to 15 further
comprising providing a cell with said peptide or derivative or
analogue thereof and determining the activity and/or nuclear
translocation of a gene transcription factor in said cell.
17. The method according to any one of claims 12 to 16 further
comprising providing a cell with said peptide or derivative or
analogue thereof and determining relative up-regulation and/or
down-regulation of at least one gene expressed in said cell.
18. A signalling molecule useful in modulating expression of a gene
in a cell and identifiable or obtainable by employing a method
according to any one of claims 1 to 17.
19. A signalling molecule according to claim 18 selected from the
group of peptides LQG, AQG, LQGV, AQGV, LQGA, VLPALP, ALPALP,
VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLAALP, VLPALA,
VLPALPQ, VLAALPQ, VLPALPA, GVLPALP, LQGVLPALPQVVC, LPGCPRGVNPVVS,
LPGC, MTRV, MTR, VVC, and functional analogues or derivatives
thereof.
20. A signalling molecule capable of modulating expression of a
gene in a cell comprising a peptide of at most 30 amino acids or a
functional analogue or derivative thereof.
21. A signalling molecule according to claim 20 wherein said
peptide is an oligopeptide of from about 3 to at about 15 amino
acids long.
22. A modulator of NF-kappaB/Rel protein activation comprising a
signalling molecule according to anyone of claims 18 to 21.
23. Use of a signalling molecule according to anyone of claim 18 to
21 for the production of a pharmaceutical composition for the
modulation of gene expression.
24. Use according to claim 23 for the modulation of gene expression
by inhibiting NF-kappaB/Rel protein activation.
Description
[0001] Gene control is generally thought to occur at four levels:
1) transcription (either initiation or termination), 2) processing
of primary transcripts, 3) stabilization or destabilization of
mRNAs, and 4) mRNA translation. The primary function of gene
control in cells is to adjust the enzymatic machinery of the cell
to its nutritional, chemical and physical environment.
[0002] It is generally thought that gene expression is regulated at
both the level of transcription and translation. Modulation or
regulation of gene expression requires factors called
transcriptional factors. The term gene control or regulation also
refers to the formation and use of mRNA. Although control can be
exerted at a number of different molecular steps, differential
transcription probably most frequently underlies the differential
rate of protein synthesis in prokayotes as well as eukaryotes. It
is generally thought that activator proteins (also called
transcription factors or transcriptional activators) bind to DNA
and recruit the transcriptional machinery in a cell to a promotor,
thereby stimulating gene expression. Further, differential
processing of RNA transcripts in the cell nucleus, differential
stabilization of mRNA in the cytoplasm, and differential
translation of mRNA into protein are also important in eukaryotic
gene control. These steps define the regulatory decisions in a
transcriptional circuit and misregulation at any stage can result
in a variety of diseases.
[0003] Where in unicellular organisms, be it of prokaryotic or
eukaryotic origin, a cell's response to its environment is
influenced by many stimuli from the outside world, reflecting the
often widely variable environment of the single cell, most cells in
multicellular organisms experience a fairly constant environment.
Perhaps for this reason, genes that are devoted to responses to
environmental changes constitute a much smaller fraction of the
total number of genes in multicellular organisms than in
single-cell organisms.
[0004] As said above, cells react to environmental changes, which
they perceive through extracellular signals. These signals can be
either physical (e.g. light, temperature, pressure and electricity)
or chemical (e.g. food, hormones and neurotransmitters). Cells can
both sense and produce signals. This makes it possible that they
communicate with each other. In order to achieve this, there are
complex signal-sensing and -producing mechanisms in uni- and
multi-cellular organisms.
[0005] Two groups of chemical signals can be distinguished:
membrane permeable and membrane-impermeable signals. The
membrane-permeable signal molecules comprise the large family of
steroid hormones, such as estrogens, progesterone and androgens.
Steroids pass the plasma membrane and bind to specific receptors,
which are localized in the cytoplasm or nucleus of the cell. After
binding of the hormone, the receptor undergoes a conformational
change. The receptor is then able to bind to DNA itself or to
proteins which can in turn interact with DNA. In general, steroid
hormones can directly regulate gene expression by means of this
process. The membrane-impermeable signal molecules include
acetylcholine, growth factors, extracellular matrix components,
thrombin, lysophosphatidic acid, the yeast mating factors and, for
the social amoeba Dictyostellium discoideum, folic acid and cyclic
AMP. They are recognized by receptors, which are localized on the
plasma membrane of the cell. The receptors are specific for one
particular signal molecule or a family of closely related signal
molecules. Upon binding of their ligands, these receptors transduce
the signals by several mechanisms.
[0006] The most characteristic and exacting requirement of gene
control on multicellular organisms is the execution of precise
developmental decisions so that the right gene is activated in the
right cell at the right time. These developmental decisions include
not only those related to the development of an organism perse, as
for example can be seen during embryogenesis and organogenesis, or
in response to disease but also relate to the differentiation or
proliferation or apoptosis of those cells that merely carry out
their genetic program essentially without leaving progeny
behind.
[0007] Such cells, such as skin cells, precursors of red blood
cells, lens cells of the eye, antibody-producing cells, are also
often regulated by patterns of gene control that serve the need of
the whole organism and not the survival of an individual cell.
[0008] It is generally reasoned that there are at least three
components of gene control: molecular signals, levels and
mechanisms. Firstly, it is reasoned that specific signalling
molecules exist to which a specific gene can respond. Secondly,
control is exerted on one or more levels (i.e. the step or steps)
in the chain of events leading from the transcription of DNA to the
use of mRNA in protein synthesis. Thirdly, at each of those levels,
specific molecular mechanisms are employed to finally exert the
control over the gene to be expressed.
[0009] Many genes are regulated not by a signalling molecule that
enters the cells but by molecules that bind to specific receptors
on the surface of cells. Interaction between cell-surface receptors
and their ligands can be followed by a cascade of intracellular
events including variations in the intracellular levels of
so-called second messengers (diacylglycerol, Ca.sup.2+, cyclic
nucleotides). The second messengers in turn lead to changes in
protein phosphorylation through the action of cyclic AMP, cyclic
GMP, calcium-activated protein kinases, or protein kinase C, which
is activated by diaglycerol.
[0010] Many of the responses to binding of ligands to cell-surface
receptors are cytoplasmatic and do not involve immediate gene
activation in the nucleus. Some receptor-ligand interactions,
however, are known to cause prompt nuclear transcriptional
activation of a specific and limited set of genes. For example, one
proto-oncogene, c-fos, is known to be activated in some cell types
by elevation of almost every one of the known second messengers and
also by at least two growth factors, platelet-derived growth factor
and epidermal growth factor. However, progress has been slow in
determining exactly how such activation is achieved. In a few
cases, the transcriptional proteins that respond to cell-surface
signals have been characterized.
[0011] One of the clearest examples of activation of a pre-existing
inactive transcription factor following a cell-surface interaction
is the nuclear factor (NF)-kappaB, which was originally detected
because it stimulates the transcription of genes encoding
immunoglobulins of the kappa class in B-lymphocytes. The binding
site for NK-kappaB in the kappa gene is well defined (see for
example P. A. Baeuerle and D. Baltimore, 1988, Science 242:540),
providing an assay for the presence of the active factor. This
factor exists in the cytoplasm of lymphocytes complexed with an
inhibitor. Treatment of the isolated complex in vitro with mild
denaturing conditions dissociates the complex, thus freeing
NK-kappaB to bind to its DNA site. Release of active NF-kappaB in
cells is now known to occur after a variety of stimuli including
treating cells with bacterial lipopolysaccharide (LPS) and
extracellular polypeptides as well as chemical molecules (e.g.
phobol esters) that stimulate intracellular phosphokinases. Thus a
phosphorylation event triggered by many possible stimuli may
account for NF-kappaB conversion to the active state. The active
factor is then translocated to the cell nucleus to stimulate
transcription only of genes with a binding site for active
NF-kappaB.
[0012] The inflammatory response involves the sequential release of
mediators and the recruitment of circulating leukocytes, which
become activated at the inflammatory site and release further
mediators (Nat. Med. 7:1294;2001). This response is self-limiting
and resolves through the release of endogenous anti-inflammatory
mediators and the clearance of inflammatory cells. The persistent
accumulation and activation of leukocytes is a hallmark of chronic
inflammation. Current approaches to the treatment of inflammation
rely on the inhibition of pro-inflammatory mediator production and
of mechanisms that initiate the inflammatory response. However, the
mechanisms by which the inflammatory response resolves might
provide new targets in the treatment of chronic inflammation.
Studies in different experimental models of resolving inflammation
have identified several putative mechanisms and mediators of
inflammatory resolution. We have shown that cyclopentenone
prostaglandins (cyPGs) may be endogenous anti-inflammatory
mediators and promote the resolution of inflammation in vivo.
Others have shown a temporal shift to the production of
anti-inflammatory lipoxins during the resolution of inflammation.
In recent years, apoptosis has been identified as an important
mechanism for the resolution of inflammation in vivo. It has been
postulated that defects in leukocyte apoptosis are important in the
pathogenesis of inflammatory disease. In addition, the selective
induction of apoptosis in leukocytes may offer a new therapeutic
approach to inflammatory disease.
[0013] Considering that NF-kappaB is thought by many to be a
primary effector of disease (A. S. Baldwin, J. Clin. Invest., 2001,
107:3-6), numerous efforts are underway to develop safe inhibitors
of NF-kappaB to be used in treatment of both chronic and acute
disease situations. Specific inhibitors of NF-kappaB should reduce
side effects associated with drugs such as NSAIDS and
glucocorticoids and would offer significant potential for the
treatment of a variety of human and animal diseases. Specific
diseases or syndromes where patients would benefit from NF-kappaB
inhibition vary widely, and range from rheumatoid artrhritis,
athereosclerosis, multiple sclerosis, chronic inflammatory
demyelinating polyradiculoneuritis, asthma, inflammatory bowel
disease, to Helicobacter pylori-associated gastritis and other
inflammatory responses, and a variety of drugs that have effects on
NF-kappaB activity, such as corticosteroids, sulfasalazine,
5-aminosalicylic acid, aspirin, tepoxalin, leflunomide, curcumin,
antioxidants and proteasome inhibitors. These drugs are considered
to be non-specific and often only applicable in high concentrations
that may end up toxic for the individual treated.
[0014] Inactive cytoplasmatic forms of transcription factors can
thus be activated by removal of an inhibitor, as is the case with
NF-kapppaB, or alternatively, by association of two (or more)
proteins neither of which is active by itself as in the case of
interferon-alpha-stimulated factor (D. E. Levy et al., 1989, Genes
and Development 3:1362). After interferon-alpha attaches to its
cell-surface receptor, one of the proteins is changed within a
minute or less, and the two can combine. The active (combined)
factor is then translocated to the cell nucleus to stimulate
transcription only of genes with a binding site for the protein.
Considering that interferon-alpha is a mediator of responses of the
body directed at pathogens and self-antigens, modulating regulation
of genes that are under influence of the
interferon-alpha-stimulated factor would contribute to the
treatment of a variety of human and animal diseases.
[0015] Other typical examples of signalling molecules that affect
gene expression via cell-surface receptor interaction are
polypeptide hormones such as insulin, glucagon, various growth
factors such as EGF, VEGF, and so on.
[0016] The steroid hormones and their receptors represent one of
the best understood cases that affect transcription. Because
steroid hormones are soluble in lipid membranes, they can diffuse
into cells. They affect transcription by binding to specific
intracellular receptors that are site specific DNA-binding
molecules. Other examples of signalling molecules that enter the
cell and act intra-cellularly are thyroid hormone (T.sub.3),
vitamin D and retinoic acid, other small lipid-soluble signalling
molecules that enter cells and modulate gene expression. The
characteristic DNA-binding sites for the receptors for these
signalling molecules are also known as response elements.
[0017] Another example of a small molecule that is involved in
regulation of gene expression is ethylene, a gas that for example
induces the expression of genes involved in fruit ripening. Also,
small plant hormones, known as auxines and cytokinins regulate
plant growth and differentiation directly by regulating gene
expression.
[0018] Given the critical role of regulatory factors in gene
regulation, the development of artificial or synthetic counterparts
that could be used in methods to rectify errors in gene expression
has been a long standing goal at the interface of chemistry and
biology.
DISCLOSURE OF THE INVENTION
[0019] The invention also relates to the treatment of anthrax.
Anthrax, the disease caused by the spore-forming Bacillus anthracis
(B. anthracis), continues to be a worldwide problem, among
domesticated and wild herbivores in Asia and Africa and poses a
worldwide threat when being used as biological weapons for
biological warfare or bioterrorism. Human infections occur after
contact with infected animals or contaminated animal products.
Outbreaks or epidemics are a constant threat for endemic regions
because spores can persist in the soil for long periods of time.
Importation controls on certain animal products are necessary to
prevent the establishment of anthrax where the disease is not
endemic. Human anthrax is usually classified by the portal of entry
into the host. Cutaneous anthrax, which accounts for the vast
majority of human anthrax cases, is a localized infection with
generally mild systemic symptoms and characterized by a painless
papule that is surrounded by edema which can be quite extensive.
The papule ulcerates by day 5 or 6 and develops into the
characteristic black eschar of cutaneous anthrax. Inhalation
anthrax, which occurs after inhaling airborne spores,
gastrointestinal anthrax, resulting from ingestion of contaminated
food, and in some instances, untreated cutaneous anthrax, are
characterized by dissemination of the bacteria from the initial
site of infection with development of a massive septicemia and
toxemia. In inhalation anthrax, phagocytic cells transport the
spores from the lung alveoli to the regional lymph nodes, where the
spores germinate and bacteria multiply. The bacilli then spread
into the bloodstream, where they are temporarily removed by the
reticuloendothelial system. Prior to death, which occurs 2 to 5
days after infection, there is a sudden onset of acute symptoms
characterized by hypotension, edema, and fatal shock due to an
extensive septicemia and toxemia. Therapeutic intervention in
general must be initiated early, as septicemic infections are
nearly always fatal.
[0020] The invention relates to the modulation of gene expression
in a cell, also called gene control, in relation to the treatment
of a variety of diseases such as anthrax. As said, anthrax is a
disease of animals and humans, poses a significant threat as an
agent of biological warfare and terrorism. Inhalational anthrax,
inwhich spores of B. anthracis are inhaled, is almost always fatal,
as diagnosis is rarely possible before the disease has progressed
to a point where antibiotic treatment is ineffective. The major
virulence factors of B. anthracis are a poly-D-glutamic acid
capsule and anthrax toxin. Anthrax toxin consists of three distinct
proteins that act in concert: two enzymes, lethal factor (LF) and
edema factor (EF; an adenylate cyclase); and protective antigen
(PA). The PA is a four-domain protein that binds a host
cell-surface receptor by its carboxy-terminal domain; cleavage of
its N-terminal domain by a furin-like protease allows PA to form
heptamers that bind the toxic enzymes with high affinity through
homologous N-terminal domains. The complex is endocytosed;
acidification of the endosome leads to membrane insertion of the PA
heptamer by forming a 14-stranded beta-barrel, followed by
translocation of the toxic enzymes into the cytosol by an unknown
mechanism. The binary combination of PA and LF is sufficient to
induce rapid death in animals when given intravenously, and certain
metalloprotease inhibitors block the effects of the toxin in vitro.
Thus, LF is a potential target for therapeutic agents that would
inhibit its catalytic activity or block its association with PA. LF
is a protein (relative molecular mass 90,000) that is critical in
the pathogenesis of anthrax. It comprises four domains: domain I
binds the membrane-translocating component of anthrax toxin, the
PA; domains II, III and IV together create a long deep groove that
holds the 16-residue N-terminal tail of mitogen-activated protein
kinase kinase-2 (MAPKK-2) before cleavage. Domain II resembles the
ADP-ribosylating toxin from Bacillus cereus, but the active site
has been mutated and recruited to augment substrate recognition.
Domain III is inserted into domain II, and seems to have arisen
from a repeated duplication of a structural element of domain II.
Domain IV is distantly related to the zinc metalloprotease family,
and contains the catalytic centre; it also resembles domain I. The
structure thus reveals a protein that has evolved through a process
of gene duplication, mutation and fusion, into an enzyme with high
and unusual specificity.
[0021] The MAPKK family of proteins are the only known cellular
substrates of LF. Cleavage by LF near to their N termini removes
the docking sequence for the downstream cognate MAP kinase. The
effect of lethal toxin on tumour cells, for example, is to inhibit
tumour growth and angiogenesis, most probably by inhibiting the
MAPKK-1 and MAPKK-2 pathways. However, the primary cell type
affected in anthrax pathogenesis is the macrophage. LF has been
shown to cleave short N-terminal fragments from mitogen or
extracellular signal-regulated MAPKK-1, MAPKK-2, MAPKK-3, and
MAPPKK-6, the upstream activators of extracellular signal-regulated
kinase 1 (ERK1), ERK2, and p38. Recent data show that this results
in inhibiting release, but not production, of the pro-inflammatory
mediators, NO and tumor necrosis factor-alpha (TNF-alpha). In
addition, high levels of lethal toxin lead to lysis of macrophages
within a few hours, by an unknown mechanism. Recent data suggests
that this happens due to inhibition of growth-factor pathways
leading to macrophage death. These observations suggest that at an
early stage in infection, lethal toxin may reduce (or delay) the
immune response, whereas at a late stage in infection, high titres
of the bacterium in the bloodstream trigger macrophage lysis and
the sudden release of high levels of NO and TNF-alpha. This may
explain the symptoms before death which are characterized by the
hyperstimulation of host macrophage inflammatory pathways, leading
to dramatic hypotension and shock. These symptoms resemble those of
LPS induced septic shock, whereby it is of note LPS-nonresponder
mice such as C3H/HeJ are also quite resistant against anthrax
toxine.
[0022] The recognition sites for LF require the presence of the
proline (P) residue followed by a hydrophobic residue or a glycine
(G) residue, between which LF cleaves. The recognition sites
further require an uncharged amino acid following the hydrophobic
residue, and at least one positively charged amino acid (and no
negatively charged amino acid, such as Asp and Glu) within the 5
amino acids to the N-terminal side of the proline residue. Other
residues in the sequence provide appropriate spacing between the
critical residues or between the donor and acceptor, and thus their
composition is not critical, and can include any natural or
unnatural amino acid.
[0023] The invention provides a method for modulating expression of
a gene in a cell comprising providing the cell with a signalling
molecule comprising an oligopeptide or functional analogue or
derivative thereof. Such a molecule is herein also called NMPF and
referenced by number. Since peptides, and functional analogues and
derivatives of relatively short amino acid sequences, are easily
synthesised these days, the invention provides a method to modulate
gene expression with easily obtainable synthetic compounds such as
synthetic peptides or functional analogues or derivatives
thereof.
[0024] The invention also provides a method for the treatment of an
inflammatory condition comprising administering to a subject in
need of such treatment a molecule comprising an oligopeptide
peptide or functional analogue or derivative thereof the molecule
capable of reducing production of NO by a cell, in particular
wherein the molecule additionally is capable of modulating
translocation and/or activity of a gene transcription factor
present in a cell, especially wherein the gene transcription factor
comprises a NF-kappaB/Rel protein. Advantageously, the invention
provides a method wherein the modulating translocation and/or
activity of a gene transcription factor allows modulation of
TNF-alpha production by the cell, in particular wherein the
TNF-alpha production is reduced. Considering that TNF-alpha
production is central to almost all, if not all, inflammatory
conditions, reducing TNF-alpha production can greatly alleviate, or
mitigate, a great host of inflammatory conditions that are
described herein. In particular, the invention provides a method
wherein the inflammatory condition comprises an acute inflammatory
condition, and it is especially useful to treat anthrax related
disease, especially when considering that with anthrax, both NO and
TNF-alpha reduction will greatly mitigate the course of disease.
Table 6 lists oligopeptides according to the invention that have
such modulatory effect. In particular, the invention provides a
method of treatment wherein the treatment comprises administering
to the subject a pharmaceutical composition comprising an
oligopeptide or functional analogue or derivative thereof capable
of reducing production of NO by a cell, preferably wherein the
composition comprises at least two oligopeptides or functional
analogues or derivatives thereof capable of reducing production of
NO by a cell, example of such combinations can be selected under
guidance of table 6, whereby it suffices to select two, or more,
with a desired effect, such as wherein the at least two
oligopeptides are selected from the group LQGV, AQGV and
VLPALP.
[0025] The invention also provides an isolated, preferably
synthetic, oligopeptide or functional analogue or derivative
thereof or mixture of such oligopeptides or analogues or
derivatives capable of reducing production of NO by a cell. Such
cell is preferably of a macrophage or DC lineage, considering the
central role these cells play in the inflammatory process. The
invention also provides a pharmaceutical composition comprising an
oligopeptide or functional analogue or derivative according to the
invention or comprising at least two oligopeptides or functional
analogues or derivatives thereof capable of reducing production of
NO by a cell. Furthermore, the invention provides the use of an
oligopeptide or functional analogue or derivative thereof capable
of reducing production of NO by a cell for the production of a
pharmaceutical composition for the treatment of an inflammatory
condition by the reduction of NO production by macrophages or DC in
the subject to be treated.
[0026] A functional analogue or derivative of a peptide is defined
as an amino acid sequence, or other sequence monomers, which has
been altered such that the functional properties of the sequence
are essentially the same in kind, not necessarily in amount. An
analogue or derivative can be provided in many ways, for instance
through conservative amino acid substitution. Also peptidomimetic
compounds can be designed that functionally or structurally
resemble the original peptide taken as starting point but that are
for example composed of non-naturally occurring amino acids or
polyamides. With conservative amino acid substitution, one amino
acid residue is substituted with another residue with generally
similar properties (size, hydrophobicity, etc), such that the
overall functioning is likely not to be seriously affected.
However, it is often much more desirable to improve a specific
function. A derivative can also be provided by systematically
improving at least one desired property of an amino acid sequence.
This can for instance be done by an Ala-scan and/or replacement net
mapping method. With these methods, many different peptides are
generated, based on an original amino acid sequence but each
containing a substitution of at least one amino acid residue. The
amino acid residue may either be replaced by alanine (Ala-scan) or
by any other amino acid residue (replacement net mapping). This
way, many positional variants of the original amino acid sequence
are synthesized. Every positional variant is screened for a
specific activity. The generated data are used to design improved
peptide derivatives of a certain amino acid sequence.
[0027] A derivative or analogue can also for instance be generated
by substitution of an L-amino acid residue with a D-amino acid
residue. This substitution, leading to a peptide which does not
naturally occur in nature, can improve a property of an amino acid
sequence. It is for example useful to provide a peptide sequence of
known activity of all D-amino acids in retro inversion format,
thereby allowing for retained activity and increase half-life
values. By generating many positional variants of an original amino
acid sequence and screening for a specific activity, improved
peptide derivatives comprising such D-amino acids can be designed
with further improved characteristics.
[0028] A person skilled in the art is well able to generate
analogous compounds of an amino acid sequence. This can for
instance be done through screening of a peptide library. Such an
analogue has essentially the same functional properties of the
sequence in kind, not necessarily in amount. Also, peptides or
analogues can be circularised (for example by providing them with
(terminal) cysteines, dimerised or multimerised, for example by
linkage to lysine or cystein or other compounds with side-chains
that allow linkage or multimerisation, brought in tandem- or
repeat-configuration, conjugated or otherwise linked to carriers
known in the art, if only by a labile link that allows
dissociation.
[0029] The invention also provides a signalling molecule for
modulating expression of a gene in a cell comprising a small
peptide or functional analogue or derivative thereof. Surprisingly,
the inventors found that a small peptide acts as a signalling
molecule that can modulatesignal transduction pathways and gene
expression. A functional analogue or derivative of a small peptide
that acts as such a signalling molecule for modulating expression
of one or more genes in a cell can be identified or obtained by at
least one of various methods for finding such a signalling molecule
as provided herein.
[0030] For example, one method as provided herein for identifying
or obtaining a signalling molecule comprising a peptide or
functional derivative or analogue thereof capable of modulating
expression of a gene in a cell comprises providing the cell with a
peptide or derivative or analogue thereof and determining the
activity and/or nuclear translocation of one or more gene
transcription factors. Such activity can be determined in various
ways using means and/or methods honed to the specific transcription
factor(s) under study. In the detailed description, it is provided
to study NF-kappaB/Rel protein translocation and/or activity, but
it is of course also easily possible to study translocation and/or
activity of any other transcription factor for which such tools are
available or can be designed. One such other transcription factor
is for example the interferon-alpha-stimulated factor as discussed
above. Other useful transcription factors to study in this context
comprise c-Jun, ATF-2, Fos, and their complexes, ELK-1, EGR-1,
IRF-1, IRF-3/7, AP-1, NF-AT, C/EBPs, Sp1, CREB, PPARgamma, STAT
proteins to name a few. Considering that many proteins are subject
to proteolytic breakdown whereby oligopeptide fragments are
generated, many already before the full protein even has exerted a
function, it is hereby established that oligopeptide fragments of
such proteins (of which a non-extensive list is given in the
detailed description, but one can for example think of MAPKK-2 that
can give rise to a peptide MLARRKPVLPALTINP, and subsequently to a
peptide comprising MLARRKP or MLAR or VLPALT or VLPAL, but also of
nitric oxide synthase that can give rise to peptides FPGC or PGCP,
GVLPAVP, LPA, VLPAVP, or PAVP after proper proteolysis) are
involved in feedback mechanisms regulating gene expression, likely
by the effect of transcription factors on gene expression. In
addition, oligopeptide fragments of proteins (of which a
non-extensive list is given in the detailed description) can also
modulate the activity of extracellular components such as factor
XIII (example of oligopeptide fragments obtained from factor XIII
are LQGV, LQGVVPRGV, GVVP, VPRGV, PRG, PRGV) or activated protein C
(APC) and thereby eventually leading to the modulation of
intracellular signal transduction pathways and gene(s)
expression.
[0031] As said, the invention provides active oligopeptides acting
as a signalling molecule. To allow for improved bio-availability of
such a signalling molecule (which is useful as a pharmacon,
especially when produced artificially) the invention also provides
a method for determining whether a small peptide or derivative or
analogue thereof can act as a functional signalling molecule
according to the invention, the method further comprising
determining whether the signalling molecule is
membrane-permeable.
[0032] The invention for example provides a method a process or
method for obtaining information about the capacity or tendency of
an oligopeptide, or a modification or derivative thereof, to
regulate expression of a gene comprising the steps of:
[0033] a) contacting the oligopeptide, or a modification or
derivative thereof, with at least one cell;
[0034] b) determining the presence of at least one gene product in
or derived from the cell. It is preferred that the oligopeptide
comprises an amino acid sequence corresponding to a fragment of a
naturally occurring polypeptide, such as hCG, or MAPKK, or another
kinase, be it of plant or animal cell, or of eukaryotic or
prokaryotic origin, or a synthase of a regulatory protein in a
cell, such as wherein the regulatory protein is a (pro-)
inflammatory mediator, such as a cytokine. Several candidate
proteins and peptide fragments are listed in the detailed
description which are a first choice for such an analysis from the
inventors perspective, but the person skilled in the art, and
working in a specific field of interest in biotechnology shall
immediately understand which protein to select for such analyses
for his or her own purposes related to his or her field.
[0035] In particular it is provided to perform a process according
to the invention further comprising a step c) comprising
determining the presence of the gene product in or derived from a
cell which has not been contacted with the oligopeptide, or a
modification or derivative thereof, and determining the ratio of
gene product found in step b to gene product found in step c, as
can easily been done with the present day genechip technology (see
for example the detailed description herein) and related methods of
expression profiling known in the art.
[0036] Another method provided herein for identifying or obtaining
information on a signalling molecule, (or for that matter the
signalling molecule itself, considering that the next step of
synthesising the molecule, generally being a short peptide, is
whole within the art) comprising a peptide or functional derivative
or analogue thereof capable of modulating expression of a gene in a
cell comprises providing the cell with a peptide or derivative or
analogue thereof and determining relative up-regulation and/or
down-regulation of at least one gene expressed in the cell. The
up-regulation can classically be studied by determining via for
example Northern or Western blotting or nucleic acid detection by
PCR or immunological detection of proteins whether a cell or cells
make more (in the case of up-regulation) or less (in the case of
down-regulation) of a gene expression product such as mRNA or
protein after the cell or cells have been provided with the peptide
or derivative or analogue thereof. Of course, various methods of
the invention can be combined to better analyse the functional
analogue of the peptide or derivative or analogue under study.
Furthermore, relative up-regulation and/or down-regulation of a
multitude or clusters of genes expressed in the cell can be easily
studied as well, using libraries of positionally or spatially
addressable predetermined or known relevant nucleic acid sequences
or unique fragments thereof bound to an array or brought in an
array format, using for example a nucleic acid library or so-called
gene chip expression analysis systems. Lysates of cells or
preparations of cytoplasma and/or nuclei of cells that have been
provided with the peptide or derivative or analogue under study are
than contacted with the library and relative binding of for example
mRNA to individual nucleic acids of the library is than determined,
as further described herein in the detailed description.
[0037] A functional analogue or derivative of a small peptide that
can act as a signalling molecule for modulating expression of a
gene in a cell can also be identified or obtained by method for
identifying or obtaining a signalling molecule comprising an
oligopeptide or functional derivative or analogue thereof capable
of modulating expression of a gene in a cell comprising providing a
peptide or derivative or analogue thereof and determining binding
of the peptide or derivative or analogue thereof to a factor
related to gene control. Such a factor related to gene control can
be any factor related to transcription (either initiation or
termination), processing of primary transcripts, stabilization or
destabilization of mRNAs, and mRNA translation.
[0038] Binding of a peptide or derivative or analogue thereof to
such a factor can be determined by various methods known in the
art. Classically, peptides or derivatives or analogues can be
(radioactively) labelled and binding to the factor can be
determined by detection of a labelled peptide-factor complex, such
as by electrophoresis, or other separation methods known in the
art. However, for determining binding to such factors, array
techniques, such as used with peptide libraries, can also be
employed, comprising providing a multitude of peptides or
derivatives or analogues thereof and determining binding of at
least one of the peptides or derivatives or analogues thereof to a
factor related to gene control.
[0039] In a preferred embodiment, the factor related to gene
control comprises a transcription factor, such as a NF-kappaB-Rel
protein or another transcription factor desired to be studied. When
binding of a functional analogue according to the invention to such
factor has been established, it is of course possible to further
analyse the analogue by providing a cell with the peptide or
derivative or analogue thereof and determining the activity and/or
nuclear translocation of a gene transcription factor in the cell,
and/or by providing a cell with the peptide or derivative or
analogue thereof and determining relative up-regulation and/or
down-regulation of at least one gene expressed in the cell.
[0040] The invention thus provides a signalling molecule useful in
modulating expression of a gene in a cell and/or useful for
reducing NO production by a cell and identifiable or obtainable by
employing a method according to the invention. Useful examples of
such a signalling molecule can be selected from the group of
oligopeptides LQG, AQG, LQGV, AQGV, LQGA, VLPALPQVVC, VLPALP,
ALPALP, VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLAALP,
VLPALA, VLPALPQ, VLAALPQ, VLPALPA, GVLPALP, GVLPALPQ,
LQGVLPALPQVVC, VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL,
RPRCRPINATLAVEK, EGCPVCITVNTTICAGYCPT, SKAPPPSLPSPSRLPGPS,
SIRLPGCPRGVNPVVS, LPGCPRGVNPVVS, LPGC, MTRV, MTR, VVC, QVVC and
functional analogues or derivatives thereof.
[0041] A preferred size of a signalling molecule according to the
invention is at most 30-40 amino acids, although much smaller
molecules, in particular of oligopeptide size, have been shown to
be particularly effective. Surprisingly, the invention provides
here the insight that gene expression can be modulated or regulated
by small peptides, that are most likely breakdown products of
larger polypeptides such as chorionic gonadotrophin (CG) and growth
hormones or growth factors such as fibroblast growth factor, EGF,
VEGF, RNA 3'terminal phosphate cyclase and CAP18. In principle such
regulating peptide sequences can be derived from a part of any
protein of polypeptide molecule produced by prokaryotic and/or
eukaryotic cells, and the invention provides the insight that
breakdown products of polypeptides, preferably oligopeptides at
about the sizes as provided herein, are naturally involved as
signalling molecule in modulation of gene expression. In
particular, as signalling molecule a (synthetic) peptide is
provided obtainable or derivable from beta-human chorionic
gonadotrophin (beta-HCG), preferably from nicked beta-HCG. It was
thought before that breakdown products of nicked-beta hCG were
involved in immuno-modulation (PCT International Patent Application
WO99/59671) or in the treatment of wasting syndrome (PCT
International Patent Application WO97/49721) but a relationship
with modulation of gene expression was not forwarded in these
publications. Of course, such an oligopeptide, or functional
equivalent or derivative thereof is likely obtainable or derivable
from other proteins that are subject to breakdown or proteolysis
and that are close to a gene regulatory cascade. Preferably, the
peptide signalling molecule is obtained from a peptide having at
least 10 amino acids such as a peptide having an amino acid
sequence MTRVLQGVLPALPQVVC, SIRLPGCPRGVNPVVS,
VVCNYRDVRFESIRLPGCPRGVNPVVS- YAVALSCQCAL,
RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT, CALCRRSTTDCGGPKDHPLTC,
SKAPPPSLPSPSRLPGPS, CRRSTTDCGGPKDHPLTC,
TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTP- ILPQ or functional fragment
(e.g. a breakdown product) or functional analogue thereof.
Functional analogue herein relates to the signalling molecular
effect or activity as for example can be measured by measuring
nuclear translocation of a relevant transcription factor, such as
NF-kappaB in an NF-kappaB assay, or AP-1 in an AP-1 assay, or by
another method as provided herein. Fragments can be somewhat (i.e.
1 or 2 amino acids) smaller or larger on one or both sides, while
still providing functional activity.
[0042] Not wishing to be bound by theory, it is postulated herein
that an unexpected mode of gene regulation has been uncovered.
Polypeptides, such as endogenous CG, EGF etc, but also polypeptides
of pathogens such as viral, bacterial or protozoal polypeptides,
are subject to breakdown into distinct oligopeptides, for example
by intracellular proteolysis. Distinct proteolytic enzymes are
widely available in the cell, for example in eukaryotes in the
lysosomal or proteasomal system. Some of the resulting breakdown
products are oligopeptides of 3 to 15, preferably 4 to 9, most
preferably 4 to 6 amino acids long that are surprisingly not
without any function or effect to the cell, but as demonstrated
herein may be involved, possibly via a feedback mechanism in the
case of breakdown of endogenous polypeptides, as signalling
molecules in the regulation of gene expression, as demonstrated
herein by the regulation of the activity or translocation of a gene
transcription factor such as NF-kappaB by for example peptide LQGV,
VLPALPQVVC, LQGVLPALPQ, LQG, GVLPALPQ, VLPALP, VLPALPQ, GVLPALP,
VVC, MTRV, MTR. Synthetic versions of these oligopeptides as
described above, and functional analogues or derivatives of these
breakdown products are herein provided to modulate gene expression
in a cell and be used in methods to rectify errors in gene
expression or the treatment of disease. Oligopeptides such as LQG,
AQG, LQGV, AQGV, LQGA, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ,
VLPALAQ, LAGV, VLAALP, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA,
GVLPALP, GVLPALPQ, LQGVLPALPQVVC, SIRLPGCPRGVNPVVS,
SKAPPPSLPSPSRLPGPS, LPGCPRGVNPVVS, LPGC, MTRV, MTR, VVC, or
functional analogues or derivatives (including break down products)
of the longer sequences thereof, are particularly effective.
[0043] By using the insight as expressed herein, in a preferred
embodiment, the invention provides a method for modulating
expression of a gene in a cell comprising providing the cell with a
signalling molecule comprising an oligopeptide or functional
analogue or derivative thereof wherein the signalling molecule is
membrane-permeable in that it enters the cell. Most small peptides
as described herein have already an inherent propensity to become
intracellularly involved, but signalling molecules as provided
herein can also be provided with additional peptide sequences, such
as arginine or lysine rich stretches of amino acids, that allow for
improved internalisation across a lipid bilayer membrane, and may
possibly be cleaved off later by internal proteolytic activity.
[0044] In a preferred embodiment, the invention provides a method
for modulating expression of a gene in a cell comprising providing
the cell with a signalling molecule comprising a small peptide
(amino acid sequence) or functional analogue or derivative thereof,
wherein the signalling molecule modulates NF-kappaB/Rel protein
conversion or translocation. As said, NF-.kappa.B was originally
identified as a gene transcription factor that bound to an enhancer
element in the gene for the Ig.kappa. light chain and was believed
to be B cell-specific. However, subsequent studies revealed that
NF-kappaB/Rel proteins are ubiquitously expressed and play a
central role as transcription factor in regulating the expression
of many genes, particularly those involved in immune, inflammatory,
developmental and apoptotic processes. NF-.kappa.B related gene
transcription factors can be activated by different stimuli such as
microbial products, proinflammatory cytokines, T- and B-cell
mitogens, and physical and chemical stresses. NF-.kappa.B in turn
regulates the inducible expression of many cytokines, chemokines,
adhesion molecules, acute phase proteins, and antimicrobial
peptides.
[0045] NF-.kappa.B represents a group of structurally related and
evolutionarily conserved gene transcription factors. So far, five
mammalian NF-.kappa.B proteins named Rel (c-Rel), RelA (p65), RelB,
NF-.kappa.B (p50 and its precursor p105), and NF-.kappa.B (p52 and
it precursor p100) have been described. NF-.kappa.B proteins can
exist as homo- or heterodimers, and although most NF-.kappa.B
dimers are activators of transcription, the p50/p50 and p52/p52
homodimers often repress the transcription of their target genes.
In Drosophila, three NF-.kappa.B homologs named Dorsal, Dif, and
Relish have been identified and characterized. Structurally, all
NF-.kappa.B/Rel proteins share a highly conserved NH.sub.2-terminal
Rel homology domain (RHD) that is responsible for DNA binding,
dimerization, and association with inhibitory proteins known as
I.kappa.BS. In resting cells, NF-.kappa.B/Rel dimers are bound to
I.kappa.Bs and retained in an inactive form in the cytoplasm. Like
NF-.kappa.B, IkBs are also members of a multigene family containing
seven known mammalian members including I.kappa.B.alpha.,
I.kappa.B.beta., I.kappa.b.gamma., I.kappa.B.epsilon., Bcl-3, the
precursor Rel-proteins, p100, and p105, and one Drosophila
I.kappa.B named Cactus. The I.kappa.B family is characterized by
the presence of multiple copies of ankyrin repeats, which are
protein-protein interaction motifs that interact with NF-.kappa.B
via the RHD. Upon appropriate stimulation, I.kappa.B is
phosphorylated by I.kappa.B kinases (IKKs), polyubiquitinated by a
ubiquitin ligase complex, and degraded by the 26S proteosome.
Consequently, NF-.kappa.B is released and translocates into the
nucleus to initiate gene expression.
[0046] NF-.kappa.B related transcription factors regulate the
expression of a wide variety of genes that play critical roles in
innate immune responses. Such NF-.kappa.B target genes include
those encoding cytokines (e.g., IL-1, IL-2, IL-6, IL-12,
TNF-.alpha., LT.alpha., LT.beta., and GM-CSF), adhesion molecules
(e.g., ICAM, VCAM, endothelial leukocyte adhesion molecule [ELAM]),
acute phase proteins (e.g., SAA), and inducible enzymes (e.g., iNOS
and COX-2). In addition, it has been demonstrated recently that
several evolutionary conserved antimicrobial peptides, e.g.,
.beta.-defensins, are also regulated by NF-.kappa.B, a situation
similar to Drosophila. Besides regulating the expression of
molecules involved in innate immunity, NF-.kappa.B also plays a
role in the expression of molecules important for adaptive
immunity, such as MHC proteins, and the expression of critical
cytokines such as IL-2, IL-12 and IFN-.gamma.. Finally NF-.kappa.B
plays an important role in the overall immune response by affecting
the expression of genes that are critical for regulating the
apoptotic process, such as c-IAP-1 and c-IAP-2, Fas ligand, c-myc,
p53, and cyclin D1.
[0047] Under normal conditions, NF-kappaB is rapidly activated upon
microbial and viral invasion, and this activation usually
correlates with resistance of the host to infection. However,
persistent activation of NF-kappaB may lead to the production of
excessive amounts of pro-inflammatory mediators such as IL-12 and
TNF-alpha, resulting in tissue damage, as in insulin dependent
diabetes mellitus, atherosclerosis, Crohn's disease, organ failure,
and even death of the host, as in bacterial infection-induced
septic shock. It is interesting to note that in order to survive in
the host, certain pathogens, such as Bordetella, Yersinia,
Toxoplasma gondii and African Swine Fever Virus have evolved
mechanisms to counteract or escape the host system by inhibiting
NF-kappaB activation. On the other hand, some viruses, including
HIV-1, CMV and SV-40 take advantage of NF-kappaB as a host factor
that is activated at sites of infection.
[0048] Furthermore, the invention provides a method to explore
alterations in gene expression in antigen presenting cells such as
dendritic cells in response to microbial exposure by analysing a
gene-expression profile of dendritic cells in response to micro
organisms such as for example bacteria such as Escherichia coli or
other pathogenic bacteria, fungi or yeasts such as Candida
albicans, viruses such as influenza virus and the effect of
(simultameous) treatment of these diseases with a signalling
molecule according to the invention. For example, human
monocyte-derived dendritic cells are cultured with one or more
pathogens for 1-36 hours, and gene expression is analysed using an
oligonucleotide array representing a (be it large or small) set of
genes. When the pathogens regulate the expression of a core set of
a distinct number genes, these genes may be classified according to
their kinetics of expression and function. Generally, within 4
hours of pathogen exposure, genes associated with pathogen
recognition and phagocytosis will be downregulated, whereas genes
for antigen processing and presentation are upregulated-8 hours
post-exposure. Treatment of such dendritic cells with a signalling
molecule according to the invention (be it simultaneous or before
or after the treatment of the cells with the pathogen) allows
studying the effect of a signalling molecule according to the
invention has on the effect a pathogen has on an antigen presenting
cell.
[0049] In short, the invention surprisingly provides a signalling
molecule capable of modulating expression of a gene in a cell, the
molecule being a short peptide, preferably of at most 30 amino
acids long or a functional analogue or derivative thereof. In a
much preferred embodiment, the peptide is an oligopeptide of from
about 3 to at about 15 amino acids long, preferably 4 to 12, more
preferably 4 to 9, most preferably 4 to 6 amino acids long, or a
functional analogue or derivative thereof. Of course, such
signalling molecule can be longer, for example by extending it (N-
and/or C-terminally, with more amino acids or other side groups,
which can for example be (enzymatically) cleaved off when the
molecule enters the place of final destination. Such extension may
even be preferable to prevent the signalling molecule from becoming
active in an untimely fashion, however, the core or active fragment
of the molecule comprises the aforementioned oligopeptide or
analogue or derivative thereof.
[0050] In particular, the invention provides a modulator of
NF-kappaB/Rel protein activation comprising a signalling molecule
according to the invention. Such modulators are widely searched
after these days. Furthermore, the invention provides use of a
signalling molecule according to the invention for the production
of a pharmaceutical composition for the modulation of gene
expression.
[0051] Also, the invention provides a method for the treatment of
bone disease such as osteoporosis comprising administering to a
subject in need of such treatment a molecule comprising an
oligopeptide peptide or functional analogue thereof the molecule
capable of modulating production of NO and/or TNF-alpha by a cell.
Such a method of treatment is particularly useful in
post-menopausal women that do not longer experience the benefits of
being provided with a natural source of several of the signalling
molecules as provided herein, hCG and its breakdown products.
Furthermore, the invention provides method for the treatment of an
inflammatory condition associated with TNF-alpha activity of
fibroblasts, such as seen with chronic arthritis or synovitis
comprising administering to a subject in need of such treatment a
molecule comprising an oligopeptide peptide or functional analogue
thereof wherein the molecule is capable of modulating translocation
and/or activity of a gene transcription factor present in a cell,
in particular of the NF-kappaB factor. Such a treatment can be
achieved by systemic administration of a signalling molecule
according to the invention, but local administration in joints,
bursae or tendon sheaths is provided as well. The molecule can be
selected from table 6 or identified in a method according to the
invention. It is preferred when the treatment comprises
administering to the subject a pharmaceutical composition
comprising an oligopeptide or functional analogue thereof also
capable of reducing production of NO by a ceel, for example wherein
the composition comprises at least two oligopeptides or functional
analogues thereof each capable of reducing production of NO and/or
TNF-alpha by a cell, in particular wherein the at least two
oligopeptides are selected from the group LQGV, AQGV and
VLPALP.
[0052] Furthermore, the invention provides use of an oligopeptide
or functional analogue thereof capable of reducing production of NO
and/or TNF-alpha by a cell for the production of a pharmaceutical
composition for the treatment of an inflammatory condition or a
post-meno-pausal condition, or a bone disease such as osteoporosis,
or for the induction of weight loss. The term pharmaceutical
composition as used herein is intended to cover both the active
signalling molecule alone or a composition containing the
signalling molecule together with a pharmaceutically acceptable
carrier, diluent or excipient. Acceptable diluents of an
oligopeptide as described herein in the detailed description are
for example physiological salt solutions or phosphate buffered salt
solutions. In one embodiment of the present invention, a signal
molecule is administered in an effective concentration to an animal
or human systemically, e.g. by intravenous, intra-muscular or
intraperitoneal administration. Another way of administration
comprises perfusion of organs or tissue, be it in vivo or ex vivo,
with a perfusion fluid comprising a signal molecule according to
the invention. Topical administration, e.g. in ointments or sprays,
may also apply, e.g. in inflammations of the skin or mucosal
surfaces of for example mouth, nose and/or genitals. Local
administration can occur in joints, bursae, tendon sheaths, in or
around the spinal cord at locations where nerve bundles branch off,
at the location of hernias, in or around infarcted areas in brain
or heart, etc. The administration may be done as a single dose, as
a discontinuous sequence of various doses, or continuously for a
period of time sufficient to permit substantial modulation of gene
expression In the case of a continuous administration, the duration
of the administration may vary depending upon a number of factors
which would readily be appreciated by those skilled in the art.
[0053] The administration dose of the active molecule may be varied
over a fairly broad range. The concentrations of an active molecule
which can be administered would be limited by efficacy at the lower
end and the solubility of the compound at the upper end. The
optimal dose or doses for a particular patient should and can be
determined by the physician or medical specialist involved, taking
into consideration well known relevant factors such as the
condition, weight and age of the patient, etc.
[0054] The active molecule may be administered directly in a
suitable vehicle, such as e.g. phosphate-buffered saline (PBS) or
as solutions in alcohol or DMSO. Pursuant to preferred embodiments
of the present invention, however, the active molecule is
administered through a single dose delivery using a drug-delivery
system, such as a sustained-release delivery system, which enables
the maintenance of the required concentrations of the active
molecule for a period of time sufficient for adequate modulation of
gene expression. A suitable drug-delivery system would be
pharmacologically inactive or at least tolerable. It should
preferably not be immunogenic nor cause inflammatory reactions, and
should permit release of the active molecule so as to maintain
effective levels thereof over the desired time period. A large
variety of alternatives are known in the art as suitable for
purposes of sustained release and are contemplated as within the
scope of the present invention. Suitable delivery vehicles include,
but are not limited to, the following: microcapsules or
microspheres; liposomes and other lipid-based release systems;
viscous instillates; absorbable and/or biodegradable mechanical
barriers and implants; and polymeric delivery materials, such as
polyethylene oxide/polypropylene oxide block copolymers,
polyesters, cross-linked polyvinylalcohols, polyanhydrides,
polymethacrylate and polymethacrylamide hydrogels, anionic
carbohydrate polymers, etc. Useful delivery systems are well known
in the art.
[0055] A highly suitable formulation to achieve the active molecule
release comprises injectable microcapsules or microspheres made
from a biodegradable polymer, such as poly(dl-lactide),
poly(dl-lactide-co-glyco- lide), polycaprolactone, polyglycolide,
polylactic acid-co-glycolide, poly(hydroxybutyric acid), polyesters
or polyacetals. Injectable systems comprising microcapsules or
microspheres having a diameter of about 50 to about 500 micrometers
offer advantages over other delivery systems. For example, they
generally use less active molecule and may be administered by
paramedical personnel. Moreover, such systems are inherently
flexible in the design of the duration and rate of separate drug
release by selection of microcapsule or microsphere size, drug
loading and dosage administered. Further, they can be successfully
sterilised by gamma irradiation.
[0056] The design, preparation and use of microcapsules and
microspheres are well within the reach of persons skilled in the
art and detailed information concerning these points is available
in the literature. Biodegradable polymers (such as lactide,
glycolide and caprolactone polymers) may also be used in
formulations other than microcapsules and microspheres; e.g.
pre-made films and spray-on films of these polymers containing the
active molecule would be suitable for use in accordance with the
present invention. Fibres or filaments comprising the active
molecule are also contemplated as within the scope of the present
invention.
[0057] Another highly suitable formulation for a single-dose
delivery of the active molecule in accordance with the present
invention involves liposomes. The encapsulation of an active
molecule in liposomes or multilamellar vesicles is a well known
technique for targeted drug delivery and prolonged drug residence.
The preparation and use of drug-loaded liposomes is well within the
reach of persons skilled in the art and well documented in the
literature.
[0058] Yet another suitable approach for single dose delivery of an
active molecule in accordance with the present invention involves
the use of viscous installates. In this technique, high molecular
weight carriers are used in admixture with the active molecule,
giving rise to a structure which produces a solution with high
viscosity. Suitable high molecular weight carriers include, but are
not limited to, the following: dextrans and cyclodextrans;
hydrogels; (cross-linked) viscous materials, including
(cross-linked) viscoelastics; carboxymethylcellulose; hyaluronic
acid; and chondroitin sulfate. The preparation and use of
drug-loaded viscous instillates is well known to persons skilled in
the art.
[0059] Pursuant to yet another approach, the active molecule may be
administered in combination with absorbable mechanical barriers
such oxidised regenerated cellulose. The active molecule may be
covalent or non-covalently (e.g. ionically) bound to such a
barrier, or it may simply be dispersed therein. A pharmaceutical
composition as provided herein is particularly useful for the
modulation of gene expression by inhibiting NF-kappaB/Rel protein
activation.
[0060] NF-kappaB/Rel proteins are a group of structurally related
and evolutionarily conserved proteins (Rel). Well known are c-Rel,
RelA (p65), RelB, NF-kappaB 1 (p50 and its precursor p105), and
NF-kappaB2 (p52 and its precursor p100). Most NF-kappaB dimers are
activators of transcription, p50/p50 and p52/p52 homodimers repress
the transcription of their target genes. All NF-kappaB/Rel proteins
share a highly conserved NH2-terminal Rel homology domain (RHD).
RHD is responsible for DNA binding, dimerization, association with
inhibitory proteins known as IkappaBs. In resting cells,
NF-kappaB/Rel dimers are bound to IkappaBs and retained in an
inactive form in the cytoplasm. IkappaBs are members of a multigene
family (IkappaBalpha, IkappaBbeta, IkappaBgamma, IkappaBepsilon,
Bcl-3, the precursor Rel-proteins, p100 and p105. Presence of
multiple copies of ankyrin repeats to interact with NF-kappaB via
the RHD (protein-protein interaction. Upon appropriate stimulation,
IkappaB is phosphorylated by IkappaB Kinase (IKKs),
polyubiquitinated by ubiquitin ligase complex, and degraded by the
26S proteosome. NF-kappaB is released and translocates into nucleus
to initiate gene expression.
[0061] NF-kappaB regulation of gene expression includes innate
immune responses: such as regulated by cytokines IL-1, IL-2, IL-6,
IL-12, TNF-alpha, LT-alpha, LT-beta, GM-CSF; expression of adhesion
molecules (ICAM, VCAM, endothelial leukocyte adhesion molecule
[ELAM]), acute phase proteins (SAA), inducible enzymes (iNOS and
COX-2) and antimicrobial peptides (beta-defensins). For adaptive
immunity MHC proteins, IL-2, IL-12 and IFN-alpha are regulated by
NF-kappaB. Regulation of overall immune response includes the
regulation of genes critical for regulation of apoptosis (c-IAP-1
and c-IAP-2, Fas Ligand, c-myc, p53 and cyclin D1.
[0062] Considering that NF-kappaB and related transcription factors
are cardinal pro-inflammatory transcription factors, and
considering that the invention provides a signalling molecule, such
as an oligopeptide and functional analogues or derivatives thereof
that are capable of inhibiting NF-kappaB and likely also other
pro-inflammatory transcription factors, herein also called
NF-kappaB inhibitors, the invention provides a method for
modulating NF-kappaB activated gene expression, in particular for
inhibiting the expression and thus inhibiting a central
pro-inflammatory pathway.
[0063] The consequence of this potency to inhibit this
pro-inflammatory pathway is wide and far reaching. The invention
for example provides a method to mitigate or treat inflammatory
airway disease such as asthma. Generally, asthma patients show
persistent activation of NF-kappaB of cells lining the respiratory
tract. Providing these patients, for example by aerosol
application, with a signalling molecule according to the invention,
such as LQGV or AQGV or MTRV or functional analogue or derivative
thereof, will alleviate the inflammatory airway response of these
individuals, by inhibiting NF-kappaB activation of the cells. Such
compositions can advantageously be made with signalling molecules
that are taken up in liposomes.
[0064] As said, inflammation involves the sequential activation of
signalling pathways leading to the production of both pro- and
anti-inflammatory mediators. Considering that much attention has
focused on pro-inflammatory pathways that initiate inflammation,
relatively little is known about the mechanisms that switch off
inflammation and resolve the inflammatory response. The
transcription factor NF-kB is thought to have a central role in the
induction of pro-inflammatory gene expression and has attracted
interest as a new target for the treatment of inflammatory disease.
However NF-kB activation of leukocytes recruited during the onset
of inflammation is also associated with pro-inflammatory gene
expression, whereas such activation during the resolution of
inflammation is associated with the expression of anti-inflammatory
genes and the induction of apoptosis. Inhibition of NF-kB during
the resolution of inflammation protracts the inflammatory response
and prevents apoptosis. This shows that NF-kB has an
anti-inflammatory role in vivo involving the regulation of
inflammatory resolution. The invention provides a tool to modulate
the inflammation at the end phase, a signalling molecule or
modulator as provided herein allows the modulation of the NF-kappaB
pathway at different stages of the inflammatory response in vivo,
in a particular embodiment, the invention provides a modulator of
NF-kappaB for use in the resolution of inflammation, for example
through the regulation of leukocyte apoptosis. Useful oligopeptides
can be found among those that accelerate shock.
[0065] The invention also provides a method to mitigate or treat
neonatal lung disease, also called chronic lung disease of
prematurity, a condition often seen with premature children who
develop a prolonged pulmonary inflammation or bronchopulmonary
dysplasia. Treating such premature children with an NF-kappaB
inhibitor, such as oligopeptide LQGV, or functional analogue or
derivative thereof, as provided herein allows such lung conditions
to be prevented or ameliorated as well.
[0066] Recent advances in bone biology provide insight into the
pathogenesis of bone diseases. The invention also provides a method
of treatment of a post-menopausal condition such as osteoporosis
comprising modulation and inhibition of osteoclast differentiation
and inhibiting TNF-alpha induced apoptosis of osteoblasts, thereby
limiting the dissolve of bone structures, otherwise so prominent in
post-menopausal women that have no longer a natural source of hCG
and thus lack the modulatory effect of the signal molecules that
are derived of hCG as shown herein. The invention thus also
provides a method of treatment of a bone disease, such as
osteoporosis (which is often, but not exclusively seen with
post-meno-pausal women) Furthermore, NO and TNF-alpha modulators as
provided herein inhibit the inflammatory response and bone loss in
periodontitis. Furthermore, considering that there is a correlation
between TNF-alpha activity, and severity of clinical manifestations
in ankylosing spondylitis, the invention provides the treatment of
spondylitis by use of a signalling molecule as provided herein.
[0067] Furthermore, considering that an important pathogenic
component in the development of insulin-dependent diabetes mellitus
(type 1) comprises over-activation of the NF-kappaB pathway as seen
in dendritic cells, treatment with an NF-kappaB inhibitor according
to the invention will lead to reduced symptoms of diabetes, or at
least to a prolonged time to onset of the disease. Particularly
effective oligopeptide signalling molecules according to the
invention in this context are GVLPALPQ, LQGV MTRV, VLPALPQVVC,
VLPALP, VLPALPQ, LPGCPRGVNPVVS, LPGC,
VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL, CPRGVNPVVS which were shown
herein to postpone onset of diabetes in an Non-obese Diabetic Mouse
(NOD). Another approach to treatment of diabetes, in particular
insulin-independent diabetes (type 2) comprises inhibition of the
PPARgamma cascade with an oligopeptide signalling molecule or
functional analogue or derivative thereof.
[0068] Other use that is provided relates to a method for combating
or treating auto-immune disease. A non-limiting list of an immune
diseases includes:
[0069] Hashimoto's thyroiditis, primary myxoedema thyrotoxicosis,
pernicious anaemia, autoimmune atrophic gastritis, Addison's
disease, premature menopause, insulin-dependent diabetes mellitus,
stiff-man syndrome, Goodpasture's syndrome, myasthenia gravis, male
infertility, pemphigus vulgaris, pemphigoid, sympathetic
ophthalmia, phacogenic uveitis, multiple sclerosis, autoimmune
haemolytic anaemia, idiopathic thrombocytopenic purpura, idiopathic
leucopenia, primary biliary cirrhosis, active chronic hepatitis,
cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome,
rheumatoid arthritis, dermatomyositis, polymyositis, scleroderma,
mixed connective tissue disease, discoid lupus erythematosus, and
systemic lupus erythematosus.
[0070] Other use that is provided relates to a method for combating
or treating infections caused by micro-organisms, in particular
those infections that are caused by micro-organisms that activate
the NF-kappaB pathway during infections.
[0071] Such micro-organisms are manifold, including bacteria,
viruses, fungi, protozoa, but other pathogens (e.g. worms) can have
the same effect. Activation of the NFkappaB pathway by a microbial
infection in general occurs via activation of the Toll-like
receptor pathway. The invention provides a method to modulate and
in particular to inhibit parts of gene expression that are related
to the inflammatory responses of an organism that are generally
activated through one of the Toll-like receptor pathways.
[0072] Toll-like receptor-mediated NF-kappa-B activation is central
in recognition of pathogens by a host. Such recognition of
pathogens generally occurs through germline-encoded molecules, the
so-called pattern recognition receptors (PRRs). These PRRs
recognize widespread pathogen associated molecular patterns
(PAMPs). The pattern recognition receptors are expressed as either
membrane-bound or soluble proteins. They include CD14,
beta2-integrins (CD11/CD18), C-type lectins, macrophage scavenger
receptors, complement receptors (CR1/CD35, CR2/CD21) and Toll-like
receptors (TLRs). TLRs are distinguished from other PRRs by their
ability to recognise and discriminate between different classes of
pathogens. TLRs represent a family of transmembrane proteins that
have an extracellular domain comprising multiple copies of leucine
rich repeats (LRRs) and a cytoplasmic: Toll/IL-IR (TIR) motif that
has significant homology to the intracellular signaling domain of
the type I IL-1 receptor (IL-1 RI). Therefore, TLRs are thought to
belong to the IL-IR superfamily.
[0073] Pathogen associated molecular patterns (PAMPS) are not
expressed by hosts but are components of the pathogenic
micro-organism. Such PAMPS comprise bacterial cell wall components
such as lipopolysaccharides (LPS), lipoproteins (BLP),
peptidoglycans (PGN), lipoarabinomannan (LAM), lipoteichoic acid
(LTA), DNA containing unmethylated CpG motifs, yeast and fungal
cell walls mannans and beta-glucans, double stranded RNA, several
unique glycosylated proteins and lipids of protozoa, and so on.
[0074] Recognition of these PAMPS foremost provides for
differential recognition of pathogens by TLRs. For example, TLR2 is
generally activated in response to BLPs, PGNs of gram-positive
bacteria, LAM of mycobacteria, and mannans of yeasts, whereas TLR4
is often activated by LPS of gram-negative bacteria and LTA of
gram-negative bacteria; also a secreted small molecule MD-2 can
account for TLR4 signalling.
[0075] Several oligopeptides capable of modulating gene expression
according to the invention have earlier been tested, both ex vivo
as in vivo, and in small animals but a relationship with modulation
of gene expression was not brought forward. A beneficial effect of
these oligopeptides on LPS induced sepsis in mice, namely the
inhibition of the effect of the sepsis, was observed.
Immunomodulatory effects with these oligopeptides have been
observed in vitro and in ex vivo such as in T-cell assays showing
the inhibition of pathological Th1 immune responses, suppression of
inflammatory cytokines (MIF), increase in production of
anti-inflammatory cytokines (IL-10, TGF-beta) and immunomodulatory
effects on antigen presenting cells (APC) like dendritic cells,
monocytes and macrophages.
[0076] Now that the insight has been provided that distinct
synthetic oligopeptides or functional analogues or derivatives
thereof, for example those that resemble breakdown products which
can be derived by proteolysis from endogenous proteins such as hCG,
can be used to modulate gene expression, for example by NF-kappaB
inhibition, such oligopeptides find much wider application. Release
of active NF-kappaB in cells is now known to occur after a variety
of stimuli including treating cells with bacterial
lipopolysaccharide (LPS) and the interaction with a Toll-like
receptor (see for example Guha and Mackman, Cell. Sign. 2001,
13:85-94). In particular LPS stimulation of dendritic cells,
monocytes and macrophages induces many genes that are under the
influence of activation by transcription factors such as NF-kappaB,
p50, EGR-1, IRF-1 and others that can be modulated by a signalling
molecule according to the invention. Considering that LPS induction
of EGR-1 is required for maximal induction of TNF-alpha, it is
foreseen that inhibition of EGR-1 considerably reduces the effects
of sepsis seen after LPS activation. Now knowing the gene
modulatory effect of the signalling molecules such as oligopeptides
as provided herein allows for rational design of signal molecule
mixtures that better alleviate the symptoms seen with sepsis. One
such mixture, a 1:1:1 mixture of LQGV, AQGV and VLPALP was
administered to primates in a gram-negative induced rhesus monkey
sepsis model for prevention of septic shock and found to be
effective in this primate model. Accordingly, the invention
provides a pharmaceutical composition for the treatment of sepsis
in a primate, and a method for the treatment of sepsis in a primate
comprising subjecting the primate to a signalling molecule
according to the invention, preferably to a mixture of such
signalling molecules. Administration of such a signalling molecule
or mixture preferably occurs systematically, e.g. by intravenous or
intraperitoneal administration. In a further embodiment, such
treatment also comprises the use of for example an antibiotic,
however, only when such use is not contra-indicated because of the
risk of generating further toxin loads because of lysis of the
bacteria subject to the action of those antibiotics in an
individual thus treated.
[0077] Other use that is contemplated relates to a method for
combating or treating viral infections, in particular those
infections that are caused by viruses that activate the NF-kappaB
pathway during infections. Such virus infections are manifold,
classical examples are hepatitis B virus induced cell
transformation by persistent activation of NF-kappaB. Use of a
signalling molecule according to the invention is herein provided
to counter or prevent this cell transformation.
[0078] Other disease where persistent NF-kappaB activation is
advantageously inhibited by a signalling molecule according to the
invention is a transplantation-related disease such as
transplantation-related immune responses,
graft-versus-host-disease, in particular with bone-marrow
transplants, acute or chronic xeno-transplant rejection, and
post-transfusion thrombocytopenia.
[0079] Other case where persistent NF-kappaB activation is
advantageously inhibited by a signalling molecule according to the
invention is found in the prevention or mitigation of
ischemia-related tissue damage seen after infarcts, seen for
example in vivo in brain or heart, or ex vivo in organs or tissue
that is being prepared or stored in preparation of further use as a
transplant. Ischemia related tissue damage can now be mitigated by
perfusing the (pre)ischemic area with a signalling molecule
according to the invention that inhibits NF-kappaB activation.
Example of conditions where ischemia (also called underperfusion)
plays a role are for example eclampsia which can be ascribed to
focal cerebral ischemia resulting from vasoconstriction, consistent
with the evidence of changes detected by new cerebral imaging
techniques. The liver dysfunction intrinsic to the HELLP
(hemolysis, elevated liver enzymes, and low platelet count)
syndrome could also be attributed to the effects of acute
underperfusion. Other conditions of ischemia are seen after
coronary occlusion leading to irreversible myocardial damage
produced by prolonged episodes of coronary artery occlusion and
reperfusion in vivo, which has already been discussed in
PCT/NL01/00259 as well.
[0080] Now that the insight has been provided that distinct
synthetic oligopeptides, for example those that resemble breakdown
products which can be derived by proteolysis from endogenous
proteins such as hCG, can be used to modulate gene expression, for
example by NF-kappaB inhibition, the oligopeptides find much wider
application. For example, the invention provides a method for
perfusing a transplant with a perfusing fluid comprising at least
one signalling molecule according to the invention; ischemic or
pre-implantation damage due to activation of NF-kappaB in the
transplant can than greatly be diminished, allowing a wider use of
the transplants.
[0081] The invention provides a signalling molecule useful in
modulating expression of a gene in a cell. Several examples of the
use of such a signalling molecule for the production of a
pharmaceutical composition for the treatment of medical or
veterinary conditions are herewith given. In one embodiment, the
invention provides such use in the treatment of an
immune-mediated-disorder, in particular of those cases whereby a
central role of NF-kappaB/Rel proteins in the immune response is
found. However as said, modulating gene expression via modulating
activity of other transcription factors, such as AP-1 or PPARgamma,
and others is also provided, now that the gene modulating role of
signalling molecules such as the oligopetides or analogues or
derivatives thereof is understood. As also said, now knowing that
oligopeptides, likely breakdown products, play such a central role
in modulation of gene expression, the invention provides
straightforward ways for identifying further gene expression
modulating oligopeptides, and provides synthetic versions of these,
and analogues and derivatives thereof for use in a wide variety of
disorders and for use in the preparation of a wide variety of
pharmaceutical compositions. Examples of such treatment and useful
pharmaceutical compositions are for example found in relation to
conditions wherein the immune-mediated disorder comprises chronic
inflammation, such as diabetes, multiple sclerosis or acute or
chronic transplant rejection, in particular in those cases whereby
antigen presenting cells (APC's) or dendritic cells (DCs) are
enhanced by (overactive) and persistent NF-kappaB expression or
wherein the immune-mediated disorder comprises acute inflammation,
such as septic or anaphylactic shock or acute transplant rejection.
Other immune-mediated disorders that can be treated with a
pharmaceutical composition comprising a signalling molecule
according to the invention comprise auto-immune disease, such as
systemic lupus erythematosus or rheumatoid arthritis (in particular
by inhibiting IL-8 and/or IL-15 production by inhibiting NF-kappaB
activity on the expression of these genes), allergy, such as asthma
or parasitic disease, overly strong immune responses directed
against an infectious agent, such as a virus or bacterium (in
particular responses that include rapid haemorrhagic disease caused
by infection with organisms such as Yersinia pestis, Ebola-virus,
Staphylococcus aureus (e.g. in cases of tampon-disease), bacterial
(such as meningococcal) or viral meningitis and/or encephalitis,
and other life-threatening conditions). Such overly strong
responses are seen with for example pre-eclampsia, recurrent
spontaneous abortions (RSA) or preterm parturition or other
pregnancy related disorders. Especially with forms of
eclampsia/pre-eclampsia that are associated with genetically
programmed increased production of tumour-growth factor beta-1,
treatment according to the invention is recommended. Also, in
situations where RSA is likely attributable to increased IL-10
levels during pregnancy, or to increased TNF-alpha activity, for
example due to the presence of an unfavourable allele, in
particular of a G to A polymorphism in the promotor of the gene
encoding TNF-alpha, treatment with a pharmaceutical composition as
provided herein is recommended. Treatment directed at such
pregnancy-related immune disorders is herein also provided by
inhibiting NF-kappaB activity directed at activating natural killer
(NK) cell activity. Also, LPS induced reduced fertility, or
abortions, seen in pregnant sows, can be reduced by applying a
signalling molecule or method as provided herein.
[0082] Such use in treatment of an immune-mediated disorder
preferably comprises regulating relative ratios and/or cytokine
activity of lymphocyte, dendritic or antigen presenting cell
subset-populations in a treated individual, in particular wherein
the subset populations comprise Th1 or Th2, or DC1 or DC2 cells.
Other embodiments of the invention comprise use of a signalling
molecule according to the invention for the manufacture of a
medicament for modulating a cardiovascular or circulatory disorder,
such as coronary arterial occlusion and also in a pregnancy related
cardiovascular or circulatory disorder.
[0083] Furthermore, the invention provides a pharmaceutical
composition for modulating a cardiovascular or circulatory
disorder, in particular a pregnancy related cardiovascular or
circulatory disorder, comprising a signalling molecule according to
the invention or mixtures thereof. Such a composition finds it use
in a method for modulating a cardiovascular or circulatory
disorder, in particular a pregnancy related cardiovascular or
circulatory disorder, comprising subjecting an animal (in
particular a mammal) to treatment with at least one signalling
molecule according to the invention. Non-pregnancy related
disorders, that are for example related to hypercholesterolaemia,
are susceptible to treatment with a signalling molecule according
to the invention as well. For example, apolipoprotein E (apo E)
deficiency is associated with a series of pathological conditions
including dyslipidemia, atherosclerosis, Alzheimer's disease,
increase body weight and shorter life span. Inheritance of
different alleles of the POLYMORPHIC apoE gene is responsible for
10% of the variation in plasma cholesterol in most populations.
Individuals HOMOZYGOUS for one variant. apoE2, can develop type III
dysbetalipoproteinaemia if an additional genetic or environmental
factor is present. Some much rarer alleles of apoE produce dominant
expression of this disorder in heterozygous individuals. ApoE, is a
ligand for the LDL receptor and its effects on plasma cholesterol
are mediated by differences in the affinity of the LDL receptor for
lipoproteins carrying variant apoE proteins. The factors that
regulate apoE gene transcription have been investigated extensively
by the expression of gene constructs in transgenic mice and involve
complex interactions between factors that bind elements in the 5'
promoter region, in the first intron and in 3' regions many
kilobases distant from the structural gene. Deletion of the apo E
gene is associated with changes in lipoprotein metabolism [plasma
total cholesterol), HDL cholesterol, HDL/TC, and HDL/LDL ratios,
esterification rate in apo B-depleted plasma, plasma triglyceride,
hepatic HMG-CoA reductase activity, hepatic cholesterol content,
decreased plasma homocyst(e)ine and glucose levels, and severe
atherosclerosis and cutaneous xanthomatosis. The invention provides
a method and a signalling molecule for the treatment of conditions
that are associated with dysfunctional LDL receptors such as ApoE
and other members of the apolipoprotein family. In particular, use
of a signalling molecule comprising GVLPALPQ and/or VLPALP or a
functional analogue or derivative thereof is preferred.
[0084] The invention also provides use of a signalling molecule for
the preparation of a pharmaceutical composition or medicament and
methods of treatment for various medical conditions that are other
than use in the preparation of a pharmaceutical composition for the
treatment of an immune-mediated-disorder or a method of treatment
of an immune-mediated-disorder. For example, the invention provides
topical application, for example in an ointment or spray comprising
a signal molecule according to the invention, for the prevention or
mitigation of skin afflictions, such as eczemas, psoriasis, but
also of skin damage related to over-exposure to UV-light.
[0085] Also, use is contemplated in palliative control, whereby a
gene related to prostaglandin synthesis is modulated such that COX2
pathways are effected.
[0086] Furthermore, the invention also provides use of a signalling
molecule for the preparation of a pharmaceutical composition or
medicament and methods of treatment for various medical conditions
that are other than use in the preparation of a pharmaceutical
composition for the treatment of wasting syndrome, such as the
treatment of particular individuals that are suffering from
infection with HIV or a method of treatment of wasting syndrome of
such individuals.
[0087] In one embodiment, the invention provides the use of a
signalling molecule according to the invention for the preparation
of a pharmaceutical composition or medicament for modulating
angiogenesis or vascularisation, in particular during embryonal
development, or after transplantation to stimulate vascularisation
into the transplanted organ or inhibit it in a later phase.
Signalling molecules that effect angiogenesis are disclosed herein
in the detailed description.
[0088] Use as provided herein also comprises regulating TNF-alpha
receptor (e.g. CD27) expression on cells, thereby modulating the
relative ratios and/or cytokine activity of lymphocyte, dendritic
or antigen presenting cell subset-populations in a treated
individual. As for example described in the detailed description,
particular oligopeptide according to the invention is capable of
down-regulating CD27 expression on cells of the T-cell lineage.
[0089] Down-regulating TNF-alpha itself is also particularly useful
in septic-shock-like conditions that not only display increased
TNF-alpha activity but display further release of other
inflammatory compounds, such as NO. NO production is a central
mediator of the vascular and inflammatory response. Our results
show that inflammatory cells like macrophages stimulated with an
inflammatory active compound such as LPS produce large amounts of
NO. However, these cells co-stimulated with most of the NMPF
peptides (NMPF peptide 1 to 14, 43 to 66 and 69), even in a very
low dose (1 pg/ml), inhibited production of NO. Typical
septic-shock-like conditions that can preferably be treated by
down-regulating TNF-alpha and NO production comprise disease
conditions such as those caused by Bacillus anthracis (anthrax) and
Yersinia pestis toxins or infections with these micro-organisms
likely involved in bio-terrorism. Anthrax toxin is produced by
Bacillus anthracis, the causative agent of anthrax, and is
responsible for the major symptoms of the disease. Clinical anthrax
is rare, but there is growing concern over the potential use of B.
anthracis in biological warfare and terrorism. Although a vaccine
against anthrax exists, various factors make mass vaccination
impractical. The bacteria can be eradicated from the host by
treatment with antibiotics, but because of the continuing action of
the toxin, such therapy is of little value once symptoms have
become evident. Thus, a specific inhibitor of the toxin's action
will prove a valuable adjunct to antibiotic therapy. The toxin
consists of a single receptor-binding moiety, termed protective
antigen (PA), and two enzymatic moieties, termed edema factor (EF)
and lethal factor (LF). After release from the bacteria as nontoxic
monomers, these three proteins diffuse to the surface of mammalian
cells and assemble into toxic, cell-bound complexes.
[0090] Cleavage of PA into two fragments by a cell-surface protease
enables the fragment that remains bound to the cell, PA63, to
heptamerize and bind EF and LF with high affinity. After
internalization by receptor-mediated endocytosis, the complexes are
trafficked to the endosome. There, at low pH, the PA moiety inserts
into the membrane and mediates translocation of EF and LF to the
cytosol. EF is an adenylate cyclase that has an inhibitory effect
on professional phagocytes, and LF is a protease that acts
specifically on macrophages, causing their death and the death of
the host.
[0091] Down-regulating TNF-alpha itself, and/or a receptor for
TNF-alpha, as is herein also provided, is also beneficial in
individuals with Chagas cardiomyopathy.
[0092] Also, use of a signalling molecule according to the
invention for the preparation of a pharmaceutical composition for
modulation of vascularisation or angiogenesis in wound repair, in
particular of burns, is herein provided. Also, use of a
pharmaceutical composition as provided herein is provided in cases
of post-operative physiological stress, whereby not only
vascularisation will benefit from treatment, but the general
well-being of the patient is improved as well.
[0093] Another use of a signalling molecule according to the
invention comprises its use for the preparation of another
pharmaceutical composition for the treatment of cancer. Such a
pharmaceutical composition preferably acts viamodulating and
up-regulating apoptotic responses that are classically
down-regulated by NF-kappaB activity. Inhibiting the activity with
a signalling molecule according to the invention allows for
increased cell death of tumorous cells. Another anti-cancerous
activity of a signalling molecule as provided herein comprises
down-regulation of c-myb, in particular in the case of
hematopoietic tumors in humans. In this context, down-regulation of
14.3.3 protein is also provided.
[0094] A further use of a signalling molecule according to the
invention comprises its use for the preparation of a further
pharmaceutical composition for the treatment of cancer. Such a
pharmaceutical composition preferably acts via modulating and
down-regulating transferrine receptor availability, in particular
on tumorous cells. Transferrine receptors are classically
up-regulated by NF-kappaB activity. Inhibiting the activity with a
signalling molecule according to the invention allows for reduced
iron up-take and increased cell death of tumorous cells. In
particular erythroid and thromboid cells are susceptible to the
treatment.
[0095] Yet a further use of a signalling molecule according to the
invention comprises its use for the preparation of yet another
pharmaceutical composition for the treatment of cancer, in
particular of cancers that are caused by viruses, such as is the
case with retroviral-induced malignancies and other viral-induuced
malignancies. Such a pharmaceutical composition preferably acts via
modulating and down-regulating cell-proliferative responses that
are classically up-regulated by virus-induced transcriptional or
NF-kappaB activity. Inhibiting the activity with a signalling
molecule according to the invention allows for decreased
proliferation and increased cell death of tumorous cells. Such a
pharmaceutial composition may also act via modulating angiogenic
responses induced by IL-8, whereby for example inhibition of IL-8
expression via inhibition of transcription factor AP-1 or NF-kappaB
expression results in the inhibition of vascularisation dependent
tumor growth.
[0096] Furthermore, the invention provides the use of a signalling
molecule for the preparation of a pharmaceutical composition for
optimising human or animal fertility and embryo survival, and a
method for optimising fertility and embryo survival. In particular,
the invention provided for a method and composition allowing the
down-regulation of TNF-alpha in the fertilized individual,
optimally in combination with a composition and method for
up-regulating IL-10 in the individual. Such a composition and
method finds immediate use in both human and veterinary
medicine.
[0097] Also, the invention provides the use of a signalling
molecule for the preparation of a pharmaceutical composition for
modulating the body weight of an individual, in particular by
modulating gene expression of a gene under influence of peroxisome
proliferator-activated receptor gamma (PPARgamma) activation and
lipid metabolism by applying a signalling molecule according to the
invention, and a method for modulating bodyweight comprising
providing an individual with a signalling molecule according to the
invention.
[0098] A further use of a signalling molecule as provided herein
lies in the modulation of expression of a gene in a cultured cell.
Such a method as provided herein comprises subjecting a signalling
molecule according to the invention to the cultured cell.
Proliferation and/or differentiation of cultured cells (cells
having been or being under conditions of in vitro cell culture
known in the art) can be modulated by subjecting the cultured cell
to a signalling molecule according to the invention. It is
contemplated that for example research into proliferation or
differentiation of cells, such as stem-cell research will benefit
greatly from understanding that a third major way of effecting gene
modulation exists, and considering the ease of application of
synthetic peptides, and analogues or derivatives thereof.
[0099] Furthermore, it is contemplated that a signalling molecule
as provided herein finds an advantageous use as a co-stimulatory
substance in a vaccine, accompanying modern day adjuvants or
replacing the classically used mycobacterial adjuvants, especially
considering that certain mycobacteria express hCG-like proteins, of
which it is now postulated that these bacteria have already made
use of the this third pathway found in gene modulation as provided
herein by providing the host with breakdown products mimicking the
signalling molecules identified herein. Treatment and use of the
compositions as provided herein is not restricted to animals only,
plants and other organisms are also subject to this third pathway
as provided herein. Furthermore, now that the existence of such a
pathway has been demonstrated, it is herein provided to make it
subject of diagnosis as well, for example to determine the gene
modulatory state of a cell in a method comprising determining the
presence or absence of a signalling molecule as provided herein or
determining the presence or absence of a protease capable of
generating such a signalling molecule from a (preferable
endogenous) protein.
BRIEF DESCRIPTION OF THE FIGURES
[0100] FIGS. 1-2. Bone marrow (BM) cell yield of treated BALB/c
mice (n=6). BM cells were isolated from treated mice and cultured
in vitro in the presence of rmGM-CSF for nine days. These figures
show cell yield after nine days of culture of BM cells isolated
from mice treated with PBS, LPS or LPS in combination with NMPF
peptides 4, 46, 7 and 60. In these figures cell yield is expressed
in relative percentage of cells compared to PBS. Each condition
consists of 6 Petri dishes and results shown in these figures are
representative of 6 dishes. Differences of .gtoreq.20% were
considered significant and line bars represent significant data as
compared to LPS control group. A representative experiment is
shown. Findings involving all experimental conditions were entirely
reproduced in 3 additional experiments.
[0101] FIG. 3. Effect of in vivo treatment on MHC-II expression on
CD11c.sup.+ cells. Bone marrow (BM) cells were isolated from
treated BALB/c mice (n=6) and cultured in vitro in the presence of
rmGM-CSF for nine days. This figure shows MHC-II expression
expressed in mean fluorescence intensity (MFI) after nine days of
culturing of BM cells isolated from PBS, LPS or LPS in combination
with NMPF. Each condition consists of 6 Petri dishes and results
shown in these figures are representative of 6 dishes. Differences
of .gtoreq.20% were considered significant and line bars represent
significant data as compared to LPS control group. A representative
experiment is shown. Findings involving all experimental conditions
were entirely reproduced in 3 additional experiments.
[0102] FIGS. 4-7. Bone marrow (BM) cell yield of in vitro treated
BM cultures. BM cells from BALB/c mice (n=3) were cultured in vitro
and treated with either PBS, LPS (t=6), NMPF 4, 7, 46, 60 (t=0 or
t=6) or combination of NMPF with LPS (t=6), in the presence of
rmGM-CSF for nine days. These figures show cell yield expressed in
relative percentage of cells compared to PBS after nine days of
culture of BM cells. Each condition consists of 6 Petri dishes and
results shown in these figures are representative of 6 dishes.
Differences of .gtoreq.20% were considered significant. Line bars
represent significant data as compared to LPS control group and
dotted bars represent significant data as compared to PBS group. A
representative experiment is shown. Findings involving all
experimental conditions were entirely reproduced in 3 additional
experiments.
[0103] FIGS. 8-11. Effect of in vitro treatment on MHC-II
expression on CD11c.sup.+ cells. BM cells from BALB/c mice (n=3)
were cultured in vitro and treated with either PBS, LPS (t=6), NMPF
4, 7, 46, 60 (t=0 or t=6 days) or combination of NMPF with LPS (t=6
days), in the presence of rmGM-CSF for nine days. These figures
show MHC-11 expression expressed in mean fluorescence intensity
(MFI) of CD11c positive cells after nine days of culturing of BM
cells. Each condition consists of 6 Petri dishes, and results shown
in these figures are representative of 6 dishes. Differences of
.gtoreq.20% were considered significant. Line bars represent
significant data as compared to LPS control group and dotted bars
represent significant data as compared to PBS group. A
representative experiment is shown. Findings involving all
experimental conditions were entirely reproduced in 3 additional
experiments.
[0104] FIGS. 12-15. Bone marrow (BM) cell yield of treated BALB/c
mice (n=6). BM cells were isolated from treated mice and cultured
in vitro in the presence of rmGM-CSF for nine days. These figures
show cell yield after nine days of culture of BM cells in
suspension (unattached) and attached to Petri dish (attached). BM
cells were isolated from mice treated with PBS, LPS or LPS in
combination with different NMPF peptides. In these figures cell
yield is expressed in relative percentage of cells compared to PBS.
Each condition consists of 6 Petri dishes and results shown here
are representative of 6 dishes. Differences of .gtoreq.20% were
considered significant and line bars represent significant data as
compared to LPS control group. A representative experiment is
shown. Findings involving all experimental conditions were entirely
reproduced in 3 additional experiments.
[0105] FIGS. 16-17. Bone marrow (BM) cell yield of in vitro treated
BM cultures from NOD mice. BM cells from 15 weeks old female NOD
mice (n=3) were cultured in vitro and treated with either PBS or
NMPF in the presence of rmGM-CSF for nine days. These figures show
cell yield after nine days of culture of BM cells in suspension
(unattached) and attached to Petri dishes (attached). In these
figures cell yield is expressed in relative percentage of cells
compared to PBS. Each condition consists of 6 Petri dishes and
results shown here are representative of 6 dishes. Differences of
.gtoreq.20% were considered significant and dotted bars represent
significant data as compared to PBS control group. A representative
experiment is shown. Findings involving all experimental conditions
were entirely reproduced in 3 additional experiments.
[0106] FIGS. 20-32. In vivo treatment of fertilised chicken eggs
with NMPF and the effect of NMPF on angiogenesis. Fertile chicken
eggs (day 0) were treated with either PBS, NMPF, VEGF or VEGF in
combination with NMPF. Ten eggs were injected for every condition.
On day 8 of incubation, the embryos were removed from the eggs and
were placed in a 100-mm Petri dish. The embryo and the blood
vessels were photographed in vivo with the use of a microscope. Of
each egg one overview picture was taken and 4 detail pictures of
the blood vessels were taken. Quantification of angiogenesis
(vessels branches) was accomplished by counting the number of blood
vessels branches. Quantification of this vasculogenesis was
accomplished by measuring the blood vessel thickness. The number of
blood vessel branches and vessel thichness was measured in the
pictures and were correlated to a raster (in the pictures) of 10
mm.sup.2 for comparison. The mean number of branches and the mean
blood vessel thickness of each condition (N=10) were calculated and
compared to either the PBS or VEGF controls using a Student's
T-test. Line bars represent significant (p<0.05) data as
compared to PBS control group and dotted bars represent significant
(p<0.05) data as compared to VEGF group. FIGS. 20-30 show the
effect of NMPF on vessel branches. FIGS. 31-32 show the effect of
NMPF on vessel thickness.
[0107] FIG. 33. Detection of NF-kB via EMSA. This figure shows the
presence of NF-kB in the nuclear extracts of RAW264.7 cells treated
with LPS or NMPF in combination with LPS for 4 hours. Numbers 1-13
correspond to nuclear extracts from cells treated with NMPF and
LPS. CTL corresponds to nuclear extracts from cells treated with
LPS only. Specificity of the radioactively labeled NF-kB probe is
shown by competition with the unlabeld oligonucleotide (u1, u2, u3)
in three different concentrations (1.times., 10.times., 100.times.)
with nuclear extracts of CTL and olg corresponds to samples
containing only labeled oligonucleotide (without nuclear extract).
Description: (NMPF-1)VLPALPQVVC, (NMPF-2)LQGVLPALPQ, (NMPF-3)LQG,
(NMPF-4)LQGV, (NMPF-5)GVLPALPQ, (NMPF-6)VLPALP, (NF-7)VLPALPQ,
(NMPF-8)GVLPALP, (NMPF-9)VVC, (NMPF-1)MTRV, (NMPF-12)MTR
[0108] FIG. 34. HPLC chromatograph (wave length 206) in which data
profile obtained from the nuclear protein extracts of LPS and LPS
in combination with NMPF stimulated RAW264.7 cells are
overlayed.
[0109] FIG. 35. MSn analysis of NMPF-4 peptide.
[0110] FIG. 36. MSn analysis of fraction from nuclear extract of
LPS stimulated RAW264.7 cells. Upper panel shows full spectrum of
the fraction and lower panel shows the MS/MS spectrum of mass
413.13.
[0111] FIG. 37. MSn analysis of fraction from nuclear extract of
LPS in combination with NMPF-4 stimulated RAW264.7 cells. Upper
panel shows full spectrum of the fraction and lower panel shows the
MS/MS spectrum of mass 416.07.
[0112] FIGS. 38-49 Effect of NMPF on septic shock syndrome in
Rhesus monkeys. On the time point 70 minutes E.coli was infused and
at the end of E.coli infusion (time point 190 minutes) the
antibiotic Baytril was injected. The control monkey (monkey 429)
was treated with 0.9% NaCl at the time point of 100 minutes,
whereas the NMPF treated monkeys (monkey 459 and 427) received the
NMPF treatment at the same time point as the control monkey. Heart
rate (beats per minute), blood pressure (mmHg), difference between
systolic and diastolic blood pressure and blood oxygen
concentration (saturation in %) of the control monkey 429 (FIGS.
38-41), NMPF treated monkey 459 (FIGS. 42-45) and 427 (FIGS. 46-49)
in the time (minutes) during the experiment are shown.
[0113] FIG. 50 These figures show the NO production of LPS (10
.mu.g/ml) stimulated RAW 264.7 macrophages co-stimulated with
different NMPF peptides (1 pg/ml).
[0114] FIG. 51 These figures show the NO production of LPS (10
.mu.g/ml) stimulated RAW 264.7 macrophages co-stimulated with
different NMPF peptides with three different concentrations.
[0115] FIG. 52 This figure shows the percentage of diabetic NOD
mice treated for 2 weeks with the various NMPF peptides
[0116] FIG. 53 This figure shows the performed glucose tolerance
test (GTT) in NOD mice treated with NMPF peptides (A), and fasting
blood glucose levels (B).
DETAILED DESCRIPTION OF THE INVENTION
[0117] Cells react to environmental and intrinsic changes, which
they perceive through extracellular and inter- as well as
intracellular signals. The nature of these signals can be either
for example physical or chemical. Moreover, different classes of
molecules present in blood react to each other and induce cascade
of reaction that have direct effect on other molecules and/or
eventually lead to cellular responses, for example complement
system and blood coagulation proteins.
[0118] Many genes are regulated not by a signalling molecule that
enters the cells but by molecules that bind to specific receptors
on the surface of cells for example receptors with enzymatic
activity (receptor tyrosine kinases, receptor-like protein tyrosine
phosphatases, receptor serine/threonine kinases, histidine kinases,
guanylyl cyclases) and receptors without enzymatic activity
(cytokine receptors, integrins, G-protein-coupled receptors).
Interaction between cell-surface receptors and their ligands can be
followed by a cascade of intracellular events that modulate one or
more intracellular transducing proteins, including variations in
the intracellular levels of so-called second messengers
(diacylglycerol, Ca.sup.2+, cyclic nucleotides, inositol(1,4,5)
trisphosphate, phosphatidylinositol(3,4,5) trisphosphate,
phosphatidylinositol transfer protein (PITP)). This leads to the
activation or inhibition of a so-called `effector protein`. The
second messengers in turn lead to changes in protein for example
protein phosphorylation through the action of cyclic AMP, cyclic
GMP, calcium-activated protein kinases, or protein kinases (for
example AGC group serine/threonine protein kinases, CAMK group
serine/threonine protein kinases, CMGC group serine/threonine
kinases, protein tyrosine kinase group, or others like MEK/Ste7p).
Phosphorylation by protein kinases is one of the regulatory
mechanisms in signal transmission that modulate different cellular
pathways such as Ras/MAPK pathway, MAP kinase pathway, JAK-STAT
pathway, wnt-pathway. In many instances this all results in altered
gene expression (for example genes for the regulation of other
genes, cell survival, growth, differentiation, maturation,
functional activity).
[0119] Many of the responses to binding of ligands to cell-surface
receptors are cytoplasmatic and do not involve immediate gene
activation in the nucleus. In some instances a pre-existing
inactive transcription factor following a cell-surface interaction
is activated that lead to immediate gene activation. For example
the protein NF-kappaB, which can be activated within minutes by a
variety of stimuli, including membrane receptors (for example
pattern recognition receptors like Toll-like receptor binding to
pathogen-associated molecular patterns), inflammatory cytokines
such as TNF-.alpha., IL-1, T-cell activation signals, growth
factors and stress inducers.
[0120] Our genomic experiment with NMPF peptide LQGV showed very
immediate effects on signal transduction and gene regulation since
the cells were treated with the peptide for only four hours. In
this short period of time LQGV down-regulated at least 120 genes
and up-regulatedat least 6 genes in the presence of a strong
stimulator (PHA/IL-2 stimulated T-cell line (PM1)), demonstrating
the profound effect on signalling molecule according to the
invention and modulatory effect on gene expression. The genes
affected by LQGV include onco-genes, genes for transcription
factors, intracellular enzymes, membrane receptors, intracellular
receptors, signal transducing proteins (for example kinases) and
some genes for unkown molecules. This shows that LQGV as an example
of the synthetic signalling molecule (oligopeptide or functional
analogue or derivative thereof) as described here, has broad
spectrum of effects at different extracellular and intracellular
levels. In addition, our HPLC/MS data have shown the presence of
LQGV in the nucleus of a macrophage cell line (RAW267.4) within
half hour and also indicates the direct effects on DNA level as
well as at intracellular level which is further supported by
NF-kappaB experiments. The ultimate modulatory effect of LQGV is
dependent on, for example, type of the cell, differentiation and
maturation status of the cell, the functional status and the
presence of other regulatory molecules. This was evident by shock
experiment in which different NMPF peptides had similar or
different effects on the disease. Same results were obtained with
DC, fertilized chicken egg experiments, and CAO experiments; NMPF
effects were dependent on type of co-stimulation (GM-CSF alone or
in combination with LPS, or VEGF) and time of the treatment. Due to
this NMPF have the ability to modulate cellular responses at
different levels.
[0121] The invention is further explained with the aid of the
following illustrative examples.
EXAMPLES
[0122] Material and Methods
[0123] Peptide Synthesis
[0124] The peptides as mentioned in this document such as LQG, AQG,
LQGV, AQGV, LQGA, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ,
VLPALAQ, LAGV, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA, GVLPALP,
VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL,
RPRCRPINATLAVEKEGCPVCITVNTTICAGYC- PT, SKAPPPSLPSPSRLPGPS,
LQGVLPALPQVVC, SIRLPGCPRGVNPVVS, LPGCPRGVNPVVS, LPGC, MTRV, MTR,
VVC were prepared by solid-phase synthesis (Merrifield, 1963) using
the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology
(Atherton, 1985) with 2-chlorotrityl chloride resin (Barlos, 1991)
as the solid support. The side-chain of glutamine was protected
with a trityl function. The peptides were synthesised manually.
Each coupling consisted of the following steps: (i) removal of the
alpha-amino Fmoc-protection by piperidine in dimethylformamide
(DMF) (ii) coupling of the Fmoc amino acid (3 eq) with
diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) in
DMF/N-methylformamide (NMP) (iii) capping of the remaining amino
functions with acetic anhydride/diisopropylethylamine (DIEA) in
DMF/NMP. Upon completion of the synthesis, the peptide resin was
treated with a mixture of trifluoroacetic acid
(TFA)/H.sub.2O/triisopropylsilane (TIS) 95:2.5:2.5. After 30
minutes TIS was added until decouloration. The solution was
evaporated in vacuo and the peptide precipitated with diethylether.
The crude peptides were dissolved in water (50-100 mg/ml) and
purified by reverse-phase high-performance liquid chromatography
(RP-HPLC). HPLC conditions were: column: Vydac TP21810C18
(10.times.250 mm); elution system: gradient system of 0.1% TFA in
water v/v (A) and 0.1% TFA in acetonitrile (ACN) v/v (B); flow rate
6 ml/min; absorbance was detected from 190-370 nm. There were
different gradient systems used. For example for peptides LQG and
LQGV: 10 minutes 100% A followed by linear gradient 0-10% B in 50
minutes. For example for peptides VLPALP and VLPALPQ: 5 minutes 5%
B followed by linear gradient 1% B/minute. The collected fractions
were concentrated to about 5 ml by rotation film evaporation under
reduced pressure at 40.degree. C. The remaining TFA was exchanged
against acetate by eluting two times over a column with anion
exchange resin (Merck II) in acetate form. The elute was
concentrated and lyophilised in 28 hours. Peptides later were
prepared for use by dissolving them in PBS.
[0125] Transcription Factor Experiment
[0126] Macrophage cell line. The RAW 264.7 macrophages, obtained
from American Type Culture Collection (Manassas, Va.), were
cultured at 37.degree. C. in 5% CO.sub.2 using DMEM containing 10%
FBS and antibiotics (100 U/ml of penicillin, and 100 .mu.g/ml
streptomycin). Cells (1.times.10.sup.6/ml) were incubated with
peptide (10 .mu.g/ml) in a volume of 2 ml. After 8 h of cultures
cells were washed and prepared for nuclear extracts.
[0127] Nuclear extracts. Nuclear extracts and EMSA were prepared
according to Schreiber et al. Methods (Schriber et al. 1989,
Nucleic Acids Research 17). Briefly, nuclear extracts from peptide
stimulated or nonstimulated macrophages were prepared by cell lysis
followed by nuclear lysis. Cells were then suspended in 400 .mu.l
of buffer (10 mM HEPES pH 7.9), 10 mM KCl, 0.1 mM KCL, 0.1 mM EDTA,
0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitors),
vigorously vortexed for 15 s, left standing at 4.degree. C. for 15
min, and centrifuged at 15,000 rpm for 2 min. The pelleted nuclei
were resuspended in buffer (20 mM HEPES (pH 7.9), 10% glycerol, 400
mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and protease
inhibitors) for 30 min on ice, then the lysates were centrifuged at
15,000 rpm for 2 min. The supernatants containing the solubilized
nuclear proteins were stored at -70.degree. C. until used for the
Electrophoretic Mobility Shift Assays (EMSA).
[0128] EMSA. Electrophoretic mobility shift assays were performed
by incubating nuclear extracts prepared from control (RAW 264.7)
and peptide treated RAW 264.7 cells with a 32P-labeled
double-stranded probe (5' AGCTCAGAGGGGGACTTTCCGAGAG 3') synthesized
to represent the NF-kappaB binding sequence. Shortly, the probe was
end-labeled with T4 polynucleotide kinase according to
manufacturer's instructions (Promega, Madison, Wis.). The annealed
probe was incubated with nuclear extract as follows: in EMSA,
binding reaction mixtures (20 .mu.l) contained 0.25 .mu.g of
poly(dI-dC) (Amersham Pharmacia Biotech) and 20,000 rpm of
32P-labeled DNA probe in binding buffer consisting of 5 mM EDTA,
20% Ficoll, 5 mM DTT, 300 mM KCl and 50 mM HEPES. The binding
reaction was started by the addition of cell extracts (10 .mu.g)
and was continued for 30 min at room temperature. The DNA-protein
complex was resolved from free oligonucleotide by electrophoresis
in a 6% polyacrylamide gel. The gels were dried and exposed to
x-ray films.
[0129] Apo E Experiments
[0130] Apolipoprotein E (apo E) deficiency is associated with a
series of pathological conditions including dyslipidemia,
atherosclerosis, Alzheimer's disease, increase body weight and
shorter life span. Inheritance of different alleles of the
POLYMORPHIC apoE gene is responsible for 10% of the variation in
plasma cholesterol in most populations. Individuals HOMOZYGOUS for
one variant. apoE2, can develop type III dysbetalipoproteinaemia if
an additional genetic or environmental factor is present. Some much
rarer alleles of apoE produce dominant expression of this disorder
in heterozygous individuals. ApoE, is a ligand for the LDL receptor
and its effects on plasma cholesterol are mediated by differences
in the affinity of the LDL receptor for lipoproteins carrying
variant apoE proteins. The factors that regulate apoE gene
transcription have been investigated extensively by the expression
of gene constructs in transgenic mice and involve complex
interactions between factors that bind elements in the 5' promoter
region, in the first intron and in 3' regions many kilobases
distant from the structural gene. Deletion of the apo E gene is
associated with changes in lipoprotein metabolism [plasma total
cholesterol), HDL cholesterol, HDL/TC, and HDL/LDL ratios,
esterification rate in apo B-depleted plasma, plasma triglyceride,
hepatic HMG-CoA reductase activity, hepatic cholesterol content,
decreased plasma homocyst(e)ine and glucose levels, and severe
atherosclerosis and cutaneous xanthomatosis.
[0131] Results
[0132] NF-kB Experiments
[0133] The transcription factor NF-kB participates in the
transcriptional regulation of a variety of genes. Nuclear protein
extracts were prepared from LPS and peptide treated RAW264.7 cells
or from LPS treated RAW264.7 cells. In order to determine whether
the peptide modulates the translocation of NF-kB into the nucleus,
on these extracts EMSA was performed. FIG. 33 shows the amount of
NF-kB present in the nuclear extracts of RAW264.7 cells treated
with LPS or LPS in combination with peptide for 4 hours. Here we
see that indeed some peptides are able to modulate the
translocation of NF-kB since the amount of labeled oligonucleotide
for NF-kB is reduced. In this experiment peptides that show the
modulation of translocation of NF-kB are: (NMPF-1)VLPALPQVVC,
(NMPF-2)LQGVLPALPQ, (NMPF-3)LQG, (NMPF-4)LQGV, (NMPF-5)GVLPALPQ,
(NMPF-6)VLPALP, (NMPF-7)VLPALPQ, (NMPF-8)GVLPALP, (NMPF-9)VVC,
(NMPF-11)MTRV, (NMPF-12)MTR.
[0134] Nuclear Location of Peptide Experiment
[0135] Reverse-phase high-performance liquid chromatography
(RP-HPLC) method was used to prove the presence of synthetic
oligo-peptide in the nuclear extracts. We used a Shimadzu HPLC
system equipped with Vydac monomeric C18 column (column218MS54,
LC/MS C18 reversed phase, 300A, 5 .quadrature.m, 4.6 mm
ID.times.250 mm L); elution system: gradient system of 0.01% TFA
and 5% acetonitrile (CAN) in water v/v (A) and 0.01% TFA in 80%
acetonitrile (ACN) v/v (B); flow rate 0.5 ml/min; absorbance was
detected from 190-370 nm. The gradient time programe was as
follows:
1 Time (min) Buffer B concentration 0.01 0 5.0 0 30.0 80 40.0 100
60.0 100 65.0 0 70.0 0
[0136] The elution time of peptide LQGV was determined by injecting
2 .quadrature.g of the peptide in a separate run. Mass spectrometry
(MS) analysis of fraction which contained possible NMPF-4 (LQGV)
(elution time was determined by injecting the peptide in the same
or separate run) was performed on LCQ Deca XP (Thermo
Finnigan).
[0137] Results
[0138] Nuclear Location of Peptide Experiment
[0139] The nuclear protein extracts used in EMSA experiments were
also checked for the presence of LQGV by means of HPLC and MS. FIG.
34 shows HPLC chromatograph (wave length 206) in which data profile
obtained from the nuclear protein extracts of LPS and LPS in
combination with NMPF-4 (LQGV) stimulated RAW264.7 cells are
overlayed. This figure also show the presence or absence of number
of molecule signals in the nuclear extracts of oligopeptide+LPS
treated cells as compared to nuclear extracts of LPS treated cells.
Since HPLC profile of LQGV showed that the peptide elutes at around
12 minutes (data not shown), fraction corresponding to region 10-15
minutes was collected and analysed for the presence of this peptide
in MS.
[0140] The peptide's molecular weight is around 416 Daltons.
Besides 416 mass FIG. 35 shows some other molecular weights. This
is to be explained by the high concentration of the peptide which
induces the formation of dimers and sodium-adducts (m/z 416-[M+H]+,
438-[M+Na]+, 831-[2M+H]+, 853-[2M+Na]+, 1245-[3M+H]+,
1257-[3M+Na]+). FIG. 36 shows the MS results of 10-15 min. fraction
of nuclear extract obtained from LPS stimulated cells. These
results show the absence of 416 dalton mass, while FIG. 37 shows
the presence of 416 dalton mass of which the MSn data (FIG. 37) and
MS-sequence confirm the presence of LQGV peptide in the nuclear
protein extract obtained from LQGV+LPS stimulated RAW264.7
cells.
[0141] Endotoxin Shock Model (Sepsis)
[0142] Sepsis. For the endotoxin model, BALB/c mice were injected
i.p. with 8-9 mg/kg LPS (E. coli 026:B6; Difco Lab., Detroit,
Mich., USA). Control groups (PBS) were treated with PBS i.p. only.
To test the effect of NMPF from different sources (synthetic,
commercial hCG preparation [c-hCG]), we treated BALB/c with a dose
of 300-700 IU of different hCG preparations (PG23; Pregnyl batch
no. 235863, PG25; Pregnyl batch no. 255957) and with synthetic
peptides (5 mg/kg) after two hours of LPS injection. In other
experiments BALB/c mice were injected i.p. either with 10 mg/kg or
with 11 mg/kg LPS (E. coli 026:B6; Difco Lab., Detroit, Mich.,
USA). Subsequently mice were treated after 2 hours and 24 hours of
LPS treatment with NMPF peptides.
[0143] Semi-quantitative sickness measurements. Mice were scored
for sickness severity using the following measurement scheme:
[0144] 1 Percolated fur, but no detectable behaviour differences
compared to normal mice.
[0145] 2 Percolated fur, huddle reflex, responds to stimuli (such
as tap on cage), just as active during handling as healthy
mouse.
[0146] 3 Slower response to tap on cage, passive or docile when
handled, but still curious when alone in a new setting.
[0147] 4 Lack of curiosity, little or no response to stimuli, quite
immobile.
[0148] 5 Laboured breathing, inability or slow to self-right after
being rolled onto back (moribund)
[0149] 6 Sacrificed
[0150] Results
[0151] Endotoxin Shock Model (Sepsis)
[0152] Sepsis experiments. To determine the effect of synthetic
peptides (NMPF) in high-dose LPS shock model, BALB/c mice were
injected intraperitoneally with different doses of LPS and survival
was assessed daily for 5 days. In this experiment (for the LPS
endotoxin model) BALB/c mice were injected i.p. with 8-9 mg/kg LPS
(E. coli 026:B6; Difco Lab., Detroit, Mich., USA). Control groups
(PBS) were treated with PBS i.p. only. We treated BALB/c mice with
a dose of 300-700 IU of different hCG preparations (PG23; Pregnyl
batch no. 235863, PG25; Pregnyl batch no. 255957) or with peptides
(5 mg/kg) after two hours of LPS injection.
[0153] These experiments showed (table 1.) that NMPF peptides 4, 6,
66 and PG23 inhibited shock completely (all mice had in first 24
hours sickness scores not higher than 2; shortly thereafter they
recovered completely and had sickness scores of 0), while peptides
2, 3 and 7 accelerated shock (all mice had in first 24 hours
sickness scores of 5 and most of them died, while the control mice
treated with LPS+PBS had sickness scores of 3-4 in first 24 hours
and they most of them died after 48 hours with sickness scores of 5
(17% survival rate at 72 hours). In addition, peptides 1, 5, 8, 9,
11, 12, 13, 14 and 64 showed in number of different experiments
variability in effectiveness as well as in the kind (inhibitory vs
accelerating) of activity. This variability is likely attributable
to the rate of breakdown of the various peptides, and the different
effects the various peptides and their breakdown products have in
vivo. In addition, these experiments also showed the variability in
anti-shock activity in c-hCG preparations that is likely
attributable to the variation in the presence of anti-shock and
shock accelerating NMPF. Visible signs of sickness were apparent in
all of the experimental animals, but the kinetics and obviously the
severity of this sickness were significantly different. These data
are representative of at least 10 separate experiments.
[0154] In Table 2 we see the effect of ALA-replacement (PEPSCAN) in
peptide LQG, LQGV, VLPALP, VLPALPQ in septic shock experiments. We
conclude, that the change in even one amino acid by a neutral amino
acid can lead to different activity. So, genomic differences as
well as polymorphism in these peptides can regulate the immune
response very precise. Derivatives of these peptides, for example
(but not limited to) by addition of classical and non-classical
amino acids or derivatives that are differentially modified during
or after synthesis, for example benzylation, amidation,
glycosylation, proteolytic cleavage, linkage to an antibody
molecule or other cellular ligand etc. could also lead to a better
effectiveness of the activity.
[0155] To determine whether treatment of BALB/c mice with NMPF
inhibit septic shock at different stages of disease, synthetic
peptides (NMPF) were injected i.p. at 2 and 24 hours after the
induction of septic shock with high dose LPS (10 mg/kg).
[0156] As shown in Tables 3 and 4, control mice treated PBS after
the shock induction, reached a sickness score of 5 at 14 and 24
hours, and remained so after the second injection with PBS. The
survival rate in control group mice was 0% at 48 hours. In contrast
to control mice, mice treated with NMPF 9, 11, 12, 43, 46, 50 and
60 reached a maximum sickness score of 2-3 at 24 hours after the
induction of septic shock and further reached a maximum sickness
score of 1-2 at 48 hours after the second injection of NMPF. In
addition, mice treated with NMPF 5, 7, 8, 45, 53 and 58 reached a
sickness score of 5 and after the second injection with NMPF all
mice returned to a sickness score of 1-2 and survival rates in NMPF
groups were 100%. Mice treated with NMPF 3 reached sickness scores
of 3-4 and the second NMPF injection did save these mice. These
experiments show that NMPF peptides have anti-shock activity at
different stages of the disease and NMPF have anti-shock activity
even at disease stage when otherwise irreversible damage had been
done. This indicates that NMPF have effects on different cellular
levels and also have repairing and regenerating capacity.
[0157] Dendritic Cells Experiments
[0158] Mice. The mouse strain used in this study was BALB/c
(Harlan, Bicester, Oxon, GB). All mice used in experiments were
females between 8 and 12 weeks of age.
[0159] Mice were housed in a specific-pathogen-free facility. The
Animal Use Committee at the Erasmus University Rotterdam, The
Netherlands approved all studies.
[0160] In vivo treatment. At least six mice per group were injected
intraperitonally (i.p) with LPS (10 mg/kg; Sigma). After 2 and 24
hrs of LPS induction, mice were injected i.p. with either NMPF (5
mg/kg) or Phosphate Buffered Saline (PBS), in a volume of 100
.mu.l. LPS induced shock in this model had more than 90% mortality
at 48 hrs.
[0161] Bone marrow cell culture. From treated mice, bone-marrow
cells were isolated and cultured as follows. BALB/c mice were
killed by suffocation with CO.sub.2. The femurs and tibiae were
removed and freed of muscles and tendons under aseptic conditions.
The bones were placed in R10 medium (RPMI 1640, supplemented with
50 U/ml penicillin, 50 .mu.g/ml streptomycin, 0.2 M Na-pyruvate, 2
mM glutamine, 50 .mu.M 2-mercaptoethanol and 10% fetal calf serum
(Bio Whittaker, Europe)).
[0162] The bones were then cleaned more thoroughly by using an
aseptic tissue and were transferred to an ice cold mortier with 2
ml of R10 medium. The bones were crushed with a mortel to get the
cells out of the bones. Cells were filtered through a sterile 100
.mu.M filter (Beckton Dickinson Labware) and collected in a 50 ml
tube (FALCON). This procedure was repeated until bone parts
appeared translucent.
[0163] The isolated cells were resuspended in 10 ml of R10 and 30
ml of Geys medium was added. The cell suspension was kept on ice
for 30 minutes, to lyse the red blood cells. Thereafter, the cells
were washed twice in R10 medium. Upon initiation of the culture,
the cell concentration was adjusted to 2.times.1 cells per ml in
R10 medium supplemented with 20 ng/ml recombinant mouse Granulocyte
Monocyte-Colony Stimulating Factor (rmGM-CSF; BioSource
International, Inc., USA) and seeded in 100 mm non-adherent
bacteriological Petri dishes (Falcon). For each condition six Petri
dishes were used and for further analysis, cells were pooled and
analysed as described ahead. The cultures were placed in a 5%
CO.sub.2-incubator at 37.degree. C. Every three days after culture
initiation, 10 ml fresh R10 medium supplemented with rmGM-CSF at 20
ng/ml was added to each dish.
[0164] Nine days after culture initiation, non-adherent cells were
collected and counted with a Coulter Counter (Coulter).
[0165] Alternatively, BM cells from untreated mice were isolated
and cultured as described above and were in vitro treated with the
following conditions: NMPF 4, NMPF 46, NMPF 7, NMPF 60 (20
.mu.g/ml) were added to the culture either at day 0 or day 6 after
culture initiation. Or LPS (1 .mu.g/ml) was added to the culture at
day 6 with or without the NMPF.
[0166] Immunofluorescence staining. Cells (2.times.10.sup.5) were
washed with FACS-buffer (PBS with 1% BSA and 0.02% sodium azide),
and transferred to a round-bottomed 96-well plate (NUNC). The
antibodies used for staining were against MHC-II (I-A/I-E) PE and
CD11c/CD18 FITC (PharMingen/Becton Dickinson, Franklin Lakes, N.J.,
US).
[0167] Cells were resuspended in 200 .mu.l FACS-buffer containing
both of the antibodies at a concentration of 2.5 ng/.mu.l per
antibody. Cells were then incubated for 30 min at 4.degree. C.
Thereafter, cells were washed 3 times and finally resuspended in
200 .mu.l FACS-buffer for flow-cytometric analysis in a FACSCalibur
flow cytometer (Becton Dickinson, Heidelberg, Germany). All
FACS-data were analyzed with CellQuest software (Becton Dickinson,
Heidelberg, Germany).
[0168] Statistical analysis All differences greater than 20% are
considered to be significant.
[0169] Results
[0170] Dendritic Cells Experiments
[0171] Cell yield of ex vivo bone-marrow cell cultures. To
determine the in vivo effect of LPS and NMPF treatment on the cell
yield obtained from a nine-day culture of bone-marrow with
rmGM-CSF, cells were isolated from the BM of treated mice and
cultured, harvested and counted as described. As shown in FIGS. 1
and 2, the cell yield of the bone-marrow cultures of LPS (10 mg/kg)
treated mice is significantly decreased compared to PBS treated
mice. Mice treated with NMPF 4, NMPF 7, NMPF 46 and NMPF 60 after
LPS shock induction, had a significantly increased cell yield
compared to LPS in the presence of rmGM-CSF. In addition, BM
cultures from NMPF 46 treated mice gave a significantly increased
cell yield even compared to the PBS group.
[0172] Immunofluorescence staining of in vivo treated bone-marrow
derived DC. Culture of BM cells in the presence of rmGM-CSF gave
rise to an increased population of cells that are positive for
CD11c and MHC-II. Cells positive for these cell membrane markers
are bone-marrow derived dendritic cells (DC). DC are potent antigen
presenting cells (APC) and modulate immune responses. In order to
determine the maturation state of myeloid derived DC, cells were
stained with CD11c and MHC-II.
[0173] As shown in FIG. 3, the expression of the MHC-II molecule
was significantly decreased on CD11c-positive cells from LPS
treated mice as compared to the PBS group. This decrease in MHC-II
expression was further potentiated by the in vivo treatment with
NMPF 4 and NMPF 46. However, treatment of mice with NMPF 7 and NMPF
60 significantly increased the expression of the MHC-II molecule
even as compared to the PBS group.
[0174] Cell yields of in vitro bone-marrow cell cultures. To
determine the effect of LPS and NMPF in vitro on the cell yield of
a nine-day culture of bone-marrow cells, we isolated the BM cells
from untreated BALB/c mice and cultured in the presence of
rmGM-CSF. In addition to rmGM-CSF, cultures were supplemented with
NMPF at either day 0 or day 6 with or without the addition of LPS
at day 6.
[0175] As shown in FIGS. 4-7, there is a significant decrease in
cell yield in LPS treated BM cells as compared to PBS. BM cells
treated with NMPF 4, 7, 46 or 60 at time point t=0 or t=6 without
LPS, showed a significant increase in cell yield as compared to the
PBS group. However, BM cell cultures treated with NMPF 4 at time
point t=6 showed significant decrease in cell yield as compared to
the PBS group and this effect is comparable with the effect of LPS
(FIG. 4). In addition, BM cells treated with NMPF 4, 7, 46 or 60 at
time point t=6 in combination with LPS showed a significant
increase in cell yield as compared to the LPS group and even in the
group of NMPF 7 the cell yield was significantly increased as
compared to the PBS group. Immunofluorescence staining of in vitro
treated bone-marrow derived DC. To determine the maturation state
of DC, CD11c positive cells were stained for MHC-II antibody. FIGS.
7-11 show that there is an opposite effect of LPS on MHC-II
expression as compared to in vivo experiments, namely, MHC-11
expression is significantly increased with LPS treatment in vitro
as compared to PBS. NMPF 4 with LPS further potentiated the effect
of LPS, while NMPF 7 with or without LPS (t=6), significantly
inhibited the expression of MHC-11 as compared to LPS and PBS,
respectively. However, cells treated with NMPF 46 without LPS (t=0)
showed significantly increased expression of MHC-II on CD11c
positive cells. Furthermore, no significant differences were found
in the group NMPF 60 with or without LPS on MHC-II expression as
compared to LPS and PBS treated cells.
[0176] To determine the in vivo effect of LPS and NMPF treatment on
the cell yield obtained from a nine-day culture of bone-marrow with
rmGM-CSF, cells were isolated from the BM of treated mice and
cultured, harvested and counted as described. The cell yield of
`attached` cells was significant increased with NMPF 4, 7, 9, 11,
43, 46, 47, 50, 53, 58 60 and even in the group of NMPF 7, 46 and
60 the cell yield was significant increased as compared to the PBS
group (FIGS. 14-15). In addition, cell yield of `un-attached` cells
was significant increased with NMPF 4, 7, 9, 11, 46, 50, 53, 58 60
and agin in the group of NMPF 46 the cell yield was significant
increased as compared to the PBS group (FIGS. 12-13).
[0177] To determine the effect of LPS and NMPF in vitro on the cell
yield of a nine-day culture of bone-marrow cells of female NOD
mice, we isolated the BM cells from untreated NOD mice and cultured
in the presence of rmGM-CSF. In addition to rmGM-CSF, cultures were
supplemented with NMPF. In these experiments the bone-marrow cell
yield of `un-attached` cells was significant increased with NMPF 1,
2, 3, 4, 5, 6, 7, 8, 9, 12 and 13 as compared to PBS group and no
effect was observed with NMPF 11 (FIG. 16). The `attached`
bone-marrow cells of these experiments showed different yield than
the `un-attached` cells, namely there was a significant increased
in cell yield in cultures treated with NMPF 3 and 13, while
cultures treated with NMPF 2 and 6 showed significant decrease in
the cell yield as compared to PBS (FIG. 17) (more additional
results are summarised in table 5).
[0178] Coronary Artery Occlusion (CAO) Experiments
[0179] CAO induction and treatment. NMPF have immunoregulatory
effects in chronic inflammatory as well as acute inflammatory mice
models. Since certain cytokines like TGF-beta1, TNF-alpha, IL-1 and
ROS (reactive oxygen species) have been implicated in irreversible
myocardial damage produced by prolonged episodes of coronary artery
occlusion and reperfusion in vivo that leads to ischaemia and
myocardial infarct, we tested the cardio-protective properties of
peptides in ad libitum fed male Wistar rats (300 g). The
experiments were performed in accordance with the Guiding
principles in the Care and Use of Animals as approved by the
Council of the American Physiological Society and under the
regulations of the Animal Care Committee of the Erasmus University
Rotterdam. Shortly, rats (n--3) were stabilised for 30 minutes
followed by i.v. 1 ml of peptide treatment (0.5 mg/ml) in 10
minutes. Five minutes after completion of treatment, rats were
subjected to a 60-min coronary artery occlusion (CAO). In the last
5 minutes of CAO, rats were again treated over 10 minutes i.v. with
1 ml of peptide (0.5 mg/ml) followed by 120 minutes of reperfusion
(IP). Experimental and surgical procedures are described in detail
in Cardiovascular Research 37(1998) 76-81. At the end of each
experiment, the coronary artery was re-occluded and was perfused
with 10 ml Trypan Blue (0.4%, Sigma Chemical Co.) to stain the
normally perfused myocardium dark blue and delineate the nonstained
area at risk (AR). The heart was then quickly excised and cut into
slices of 1 mm from apex to base. From each slice, the right
ventricle was removed and the left ventricle was divided into the
AR and the remaining left ventricle, using micro-surgical scissors.
The AR was then incubated for 10 min in 37.degree. C.
Nitro-Blue-Tetrazolium (Sigma Chemical Co.; 1 mg per 1 ml Sorensen
buffer, pH 7.4), which stains vital tissue purple but leaves
infarcted tissue unstained. After the infarcted area (IA) was
isolated from the noninfarcted area, the different areas of the LV
were dried and weighed separately. Infarct size was expressed as
percentage of the AR. Control rats were treated with PBS.
[0180] Results
[0181] Coronary Artery Occlusion (CAO) Experiments
[0182] Our CAO data showed that 15 rats in control group treated
with only PBS had infarcted area of 70.+-.2% (average.+-.standard
error) after 60-minutes of CAO followed by 2 hours of reperfusion.
While rats treated with peptide VLPALP, LQGV, VLPALPQVVC,
LQGVLPALPQ, LAGV, LQAV and MTRV showed infarcted area of 62.+-.6%,
55.+-.6%, 55.+-.5%, 67.+-.2%, 51.+-.4%, 62.+-.6% and 68.+-.2%,
respectively. Here, we see that certain peptides (such as VLPALP,
LQGV, VLPALPQVVC, LAGV) have a protective effect on the area at
risk for infarction. In addition, peptide LQAV showed smaller
infarcted area but in some instances the area was haemorhagic
infarcted. In addition NMPF-64 (LPGCPRGVNPVVS) had also protective
effect (35%) in CAO experiment. It is important to note that mice
treated with certain above mentioned peptides showed less
viscousity of blood. Apart from immunological effect, these
peptides may have also effect on blood coagulation system directly
or indirectly since there is certain homology with blood
coagulation factors (for additional results of NMPF peptides see
table 5.) So, in both models the circulatory system plays an
important role in the pathogenesis of the disease.
[0183] Chicken Eggs Experiments
[0184] In vivo treatment of fertilised chicken eggs with NMPF.
Fertile chicken eggs (Drost Loosdrecht BV, the Netherlands) were
incubated in a diagonal position in an incubator (Pas Reform BV,
the Netherlands) at 37.degree. C. and 32% relative humidity.
[0185] Solutions of NMPF peptides (1 mg/ml) and VEGF were made in
PBS. At least ten eggs were injected for every condition. The
treatment was performed as follows: on day 0 of incubation, a hole
was drilled into the eggshell to open the air cell. A second hole
was drilled 10 mm lower and right from the first hole for
injection. The holes in the eggshell were disinfected with jodium.
The NMPF peptides (100 .quadrature.g/egg) and/or VEGF (100 ng/ml)
were injected in volume of 100 .mu.l. The holes in the eggshell
were sealed with tape (Scotch Magic.TM. Tape, 3M) and the eggs were
placed into the incubator.
[0186] Quantification of angiogenesis. On day 7 of incubation, the
eggs were viewed under an UV lamp to check if the embryos were
developing in a normal way and the dead embryos were counted. On
day 8 of incubation, the embryos were removed from the eggs by
opening the shell at the bottom of the eggs. The shell membrane was
carefully dissected and removed. The embryos were placed in a
100-mm Petri dish. The embryo and the blood vessels were
photographed (Nikon E990, Japan) in vivo with the use of a
microscope (Zeiss Stemi SV6, Germany). One overview picture was
taken and 4 detail pictures of the blood vessels were taken. Only
eggs with vital embryos were evaluated.
[0187] Data analysis. Quantification of angiogenesis was
accomplished by counting the number of blood vessels branches.
Quantification of vasculogenesis was accomplished by measuring the
blood vessel thickness. The number of blood vessel branches and the
blood vessel thickness were counted in the pictures (4
pictures/egg) using Corel Draw 7. Thereafter, the number of blood
vessel branches and the thickness of the blood vessels were
correlated to a raster of microscope (10 mm.sup.2) for comparison.
The mean number of branches and the mean blood vessel thickness of
each condition (n=10) were calculated and compared to the PBS
control eggs using a Student's T-test.
[0188] Results
[0189] Chicken Eggs Experiments
[0190] In order to determine the effect of NMPF on angiogenesis and
vasculogenesis we treated fertilized chicken eggs with NMPF or NMPF
in combination with VEGF as described in materials and methods
section. FIGS. 20-30 show that NMPF 3, 4, 9 and 11 promoted
angiogenesis (p<0.05), while NMPF VEGF, 7, 43, 44, 45, 46, 51
and 56 inhibited angiogenesis (p<0.05). NMPF 6, 7, 12, 45, 46
and 66 were able to inhibit angiogenesis induced by VEGF. Moreover,
NMPF 6 itself did not show any effect on angiogensis, but it
inhibited (p<0.05) NMPF 3 induced angiogenesis.
[0191] FIGS. 31-32 show that NMPF 1, 2, 3, 4, 6, 7, 8, 12, 50, 51,
and 52 had vasculogenesis inhibiting (p<0.05) effect, while only
NMPF 44 promoted (p<0.05) vasculogenesis.
[0192] NOD Experiment
[0193] Mice. Female NOD mice at the age of 13-14 weeks were treated
i.p. with PBS (n=6) or NMPF peptides (VLPALPQVVC, LQGV, GVLPALPQ,
VLPALP, VLPALPQ, MTRV, LPGCPRGVNPVVS, CPRGVNPVVS, LPGC,
MTRVLQGVLPALPQVVC, VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL) (5
mg/kg, n=6) three times a week for 2 weeks. Every four days urine
was checked for the presence of glucose (Gluketur Test; Boehringer
Mannheim, Mannheim, Germany). All mice used in these studies were
maintained in a pathogen-free facility. They were given free access
to food and water. The experiments were approved by the Animal
Experiments Committee of the Erasmus University Rotterdam. Diabetes
was assessed by measurement of the venous blood glucose level using
an Abbott Medisense Precision glucometer. Mice were considered
diabetic after two consecutive glucose measurements .gtoreq.11
mmol/l (200 mg/dl). Onset of diabetes was dated from the first
consecutive reading.
[0194] Glucose tolerance test (GTT) test was performed at 28 weeks
of age in fasted mice (n=5) by injecting 1 g/kg D-glucose
intraperitoneally (i.p.). At 0 (fasting), 5, 30 and 60 minutes
blood samples were collected from the tail and tested for glucose
content.
[0195] NO Experiment
[0196] Cell culture. The RAW 264.7 murine macrophage cell line,
obtained from American Type Culture Collection (Manassas, Va.,
USA), were cultured at 37.degree. C. in 5% CO.sub.2 using DMEM
containing 10% fetal calf serum (FCS), 50 U/ml penicillin, 50
.mu.g/ml streptomycin, 0.2 M Na-pyruvate, 2 mM glutamine and 50
.mu.M 2-mercaptoethanol (Bio Whittaker, Europe). The medium was
changed every 2 days.
[0197] Nitrite measurements. Nitrite production was measured in the
RAW 264.7 macrophage supernatants. The cells
(7.5.times.10.sup.5/ml) were cultured in 48-well plates in 500
.quadrature.l of culture medium. The cells were stimulated with LPS
(10 microg/ml) and/or NMPF (1 pg/ml, 1 ng/ml, 1 .quadrature.g/ml)
for 24 hours, then the culture media were collected. Nitrite was
measured by adding 100 microl of Griess reagent (Sigma) to 100
microl samples of culture medium. The OD.sub.540 was measured using
a microplate reader, and the nitrite concentration was calculated
by comparison with the OD.sub.540 produced using standard solutions
of sodium nitrite in the culture medium.
[0198] Results
[0199] NOD Experiment
[0200] In order to determine whether NMPF has effect on the disease
development in NOD mice, we tested NMPF on pre-diabetic female NOD
mice at the age of 13-14 weeks. After only two weeks of treatment
(injection of NMPF (5 mg/kg) every other day) glucosuria data of
all NOD mice was analysed at the of 17 weeks. Profound
anti-diabetic effect (mice negative for glucosuria) was observed in
different NMPF groups as compared to PBS group, especially in NMPF
groups treated with peptide VLPALPQVVC, VLPALP, MTRV, LPGCPRGVNPVVS
and LPGC. In addition, impairment of the glucose tolerance test was
positively correlated to insulitis, but negatively correlated to
the number of functional beta cells, also this test showed that NOD
mice successfully treated with NMPF were tolerant for glucose as
compared to PBS group. Our results show that PBS treated NOD mice
were all diabetic at the age of 23 weeks. Whereas, NOD mice treated
with three times a week for two weeks with NMPF showed profound
inhibition of diabetes development. The strongest anti-diabetic
effects were seen with NMPF-1, 4, 5, 6, 7, 65, 66 and commercial
hCG preparation (Pregnyl, Organon, Oss, The Netherlands, batch no.
235863). These mice had a low fasting blood glucose level and were
tolerant for glucose (data partially shown). However, NMPF-71
showed no effect on the incidence of diabetes, while NMPF-64 and
NMPF-11 had a moderate anti-diabetic effect.
[0201] NO Experiment
[0202] NO production is a central mediator of the vascular and
inflammatory response. Our results show that macrophages (RAW
264.7) stimulated with LPS produce large amount of NO. However,
these cells co-stimulated with most of the NMPF peptides (NMPF
peptide 1 to 14, 43 to 66 and 69) even in a very low dose (1 pg/ml)
inhibited the production of NO.
[0203] Results
[0204] ApoE Experiment
[0205] The invention provides a method and a signalling molecule
for the treatment of conditions that are associated with
dysfunctional LDL receptors such as ApoE and other members of the
apolipoprotein family. In particular, use of a signalling molecule
comprising GVLPALPQ (NMPF-5) and/or VLPALP (NMPF-6) or a functional
analogue or derivative thereof is preferred. Groups of ApoE
deficient mice (n=6 per group) were fed a high cholesterol food and
given PBS or NMPF every other day intraperitonealy. After 2.5 weeks
body weight was determined as shown in the Table below.
2 Average Weight (g) SD (g) p-value ApoE -/- PBS 31.667 1.007 ApoE
-/- NMPF-4 31.256 1.496 0.536 ApoE -/- NMPF-5 29.743 1.160 0.019
Background/PBS 26.760 1.582 10.sup.-6 ApoE -/- NMPF-6 29.614 1.064
0.004
[0206] Analysis of Different Peptides in Data Bases
[0207] Examples of different data bases in which peptides were
analysed are:
[0208] Proteomics tools: Similarity searches
[0209] BLAST data base (ExPasy, NCBI)
[0210] SMART (EMBL)
[0211] PATTINPROT (PBIL)
[0212] Post-translational modification prediction
[0213] SignalP (CBS)
[0214] Primary structure analysis
[0215] HLA Peptide Binding Predictions (BIMAS)
[0216] Prediction of MHC type I and II peptide binding
[0217] (SYFPEITHI)
[0218] Amino acid scale representation (Hydrophobicity, other
conformational parameters, etc.) (PROTSCALE)
[0219] Representations of a protein fragment as a helical
wheel(HelixWheel/HelixDraw)
[0220] Results
[0221] A non-extensive l,ist of relevant oligopeptides useful for
application in a method to identify signalling molecules according
to the invention derivable from protein data bases
[0222] pdb.vertline.1DE7.vertline.1DE7-A INTERACTION OF FACTOR XIII
ACTIVATION PEPTIDE WITH ALPHA-THROMBIN
[0223] LQGV, LQGVV, LQGVVP
[0224] pdb.vertline.1DL6.vertline.1DL6-A SOLUTION STRUCTURE OF
HUMAN TFIIB N-TERMINAL DOMAIN
[0225] LDALP
[0226] pdb.vertline.1QMH.vertline.1QMH-A CRYSTAL STRUCTURE OF RNA
3'-TERMINAL PHOSPHATE CYCLASE, AN UBIQUITOUS ENZYME
[0227] LQTV, VLPAL, LVLQTVLPAL
[0228] pdb.vertline.1LYP.vertline.1LYP CAP18 (RESIDUES 106-137)
[0229] IQG, IQGL, LPKL, LLPKL
[0230] pdb.vertline.1B9O.vertline.1B9O-A HUMAN
ALPHA-LACTALBUMIN
[0231] LPEL
[0232] pdb.vertline.1GLU.vertline.1GLU-A GLUCOCORTICOID RECEPTOR
(DNA-BINDING DOMAIN)
[0233] PARP
[0234] pdb.vertline.2KIN.vertline.2KIN-B KINESIN (MONOMERIC) FROM
RATTUS NORVEGICUS
[0235] MTR1
[0236] pdb.vertline.1SMP.vertline.1SMP-I MOL_ID: 1; MOLECULE:
SERRATIA METALLO PROTEINASE; CHAIN: A
[0237] LQKL, LQKLL, PEAP, LQKLLPEAP
[0238] pdb.vertline.1ES7.vertline.1ES7-B COMPLEX BETWEEN BMP-2 AND
TWO BMP RECEPTOR IA ECTODOMAINS
[0239] LPQ, PTLP, LQPTL
[0240] pdb.vertline.1BHX.vertline.1BHX-F X-RAY STRUCTURE OF THE
COMPLEX OF HUMAN ALPHA THROMBIN WITH THE INHIBITOR SDZ 229-357
[0241] LQV, LQVV
[0242] pdb.vertline.1VCB.vertline.1VCB-A THE VHL-ELONGINC-ELONGINB
STRUCTURE
[0243] PELP
[0244] pdb.vertline.1CQK.vertline.1CQK-A CRYSTAL STRUCTURE OF THE
CH3 DOMAIN FROM THE MAK33 ANTIBODY
[0245] PAAP, PAAPQ, PAAPQV
[0246] pdb.vertline.1FCB.vertline.1FCB-A FLAVOCYTOCHROME
[0247] LQG,
[0248] pdb.vertline.1LDC.vertline.1LDC-A L-LACTATE DEHYDROGENASE:
CYTOCHROME C OXIDOREDUCTASE (FLAVOCYTOCHROME B=2=) (E.C.1.1.2.3)
MUTANT WITH TYR 143 REPLACED BY PHE (Y143F) COMPLEXED WITH
PYRUVATE
[0249] LQG
[0250] pdb.vertline.1BFB.vertline.1BFB BASIC FIBROBLAST GROWTH
FACTOR COMPLEXED WITH HEPARIN TETRAMER FRAGMENT
[0251] LPAL, PALP, PALPE
[0252] pdb.vertline.1MBF.vertline.1MBF MOUSE C-MYB DNA-BINDING
DOMAIN REPEAT 1
[0253] LPN
[0254] pdb.vertline.1R2A.vertline.1R2A-A THE MOLECULAR BASIS FOR
PROTEIN KINASE A
[0255] LQG, LTELL
[0256] pdb.vertline.1CKA.vertline.1CKA-B C-CRK (N-TERMINAL SH3
DOMAIN) (C-CRKSH3-N) COMPLEXED WITH C3G PEPTIDE
(PRO-PRO-PRO-ALA-LEU-PRO-PRO-LYS-- LYS-ARG)
[0257] PALP
[0258] pdb.vertline.1RLQ.vertline.1RLQ-R C-SRC(SH3 DOMAIN)
COMPLEXED WITH THE PROLINE-RICH LIGAND RLP2 (RALPPLPRY) (NMR,
MINIMIZED AVERAGE STRUCTURE)
[0259] LPPL, PPLP
[0260] pdb.vertline.1TNT.vertline.1TNT MU TRANSPOSASE (DNA-BINDING
DOMAIN) (NMR, 33 STRUCTURES)
[0261] LPG, LPGL, LPK
[0262] pdb.vertline.1GJS.vertline.1GJS-A SOLUTION STRUCTURE OF THE
ALBUMIN BINDING DOMAIN OF STREPTOCOCCAL PROTEIN G
[0263] LAAL, LAALP
[0264] pdb.vertline.1GBR.vertline.1GBR-B GROWTH FACTOR
RECEPTOR-BOUND PROTEIN 2 (GRB2, N-TERMINAL SH3 DOMAIN) COMPLEXED
WITH SOS-A PEPTIDE (NMR, 29 STRUCTURES)
[0265] LPKL, PKLP
[0266] pdb.vertline.1A78.vertline.1A78-A COMPLEX OF TOAD OVARY
GALECTIN WITH THIO-DIGALACTOSE
[0267] VLPSIP
[0268] pdb.vertline.1ISA.vertline.1ISA-A IRON(II) SUPEROXIDE
DISMUTASE (E.C.1.15.1.1)
[0269] LPAL, PALP
[0270] pdb.vertline.1FZV.vertline.1FZV-A THE CRYSTAL STRUCTURE OF
HUMAN PLACENTA GROWTH FACTOR-1 (PLGF-1), AN ANGIOGENIC PROTEIN AT
2.0A RESOLUTION
[0271] PAVP, MLPAVP
[0272] pdb.vertline.1JLI.vertline.1JLI HUMAN INTERLEUKIN 3 (IL-3)
MUTANT WITH TRUNCATION AT BOTH N- AND C-TERMINI AND 14 RESIDUE
CHANGES, NMR, MINIMIZED AVERAGE
[0273] LPC, LPCL, PCLP
[0274] pdb.vertline.1HSS.vertline.1HSS-A 0.19 ALPHA-AMYLASE
INHIBITOR FROM WHEAT
[0275] VPALP
[0276] pdb.vertline.3CRX.vertline.3CRX-A CRE RECOMBINASE/DNA
COMPLEX INTERMEDIATE I
[0277] LPA, LPAL, PALP
[0278] pdb.vertline.1PRX.vertline.1PRX-A HORF6 A NOVEL HUMAN
PEROXIDASE ENZYME
[0279] PTIP, VLPTIP
[0280] pdb.vertline.1RCY.vertline.1RCY RUSTICYANIN (RC) FROM
THIOBACILLUS FERROOXIDANS
[0281] VLPGFP
[0282] pdb.vertline.1A3Z.vertline.1A3Z REDUCED RUSTICYANIN AT 1.9
ANGSTROMS
[0283] PGFP, VLPGFP
[0284] pdb.vertline.1GER.vertline.1GER-A GLUTATHIONE REDUCTASE
(E.C.1.6.4.2) COMPLEXED WITH FAD
[0285] LPALP, PALP
[0286] pdb.vertline.1PBW.vertline.1PBW-A STRUCTURE OF BCR-HOMOLOGY
(BH) DOMAIN
[0287] PALP
[0288] pdb.vertline.1BBS.vertline.1BBS RENIN (E.C.3.4.23.15)
[0289] MPALP
[0290] AI188872 11.3 366 327 18 382 [Homo sapiens]qd27c01.x1
Soares_placenta.sub.--8 to 9weeks.sub.--2NbHP8 to 9W
[0291] Homo sapiens cDNA clone IMAGE:1724928 3' similar to
gb:J00117 CHORIOGONADOTROPIN BETA CHAIN PRECURSOR (HUMAN);, mRNA
sequence.; minus strand; translated
[0292] MXRVLQGVLPALPQVVC, MXRV, MXR,
[0293] AI126906 19.8 418 343 1 418 [Homo sapiens]qb95f01.x1
Soares_fetal_heart_NbHH19W
[0294] Homo sapiens cDNA clone IMAGE:1707865 3' similar to
gb:J00117 CHORIOGONADOTROPIN BETA CHAIN PRECURSOR (HUMAN);, mRNA
sequence.; minus strand; translated
[0295] ITRVMQGVIPALPQVVC
[0296] AI221581 29.1 456 341 23 510 [Homo sapiens]qg20a03.x1
Soares_placenta.sub.--8 to 9 weeks 2 NbHP8 to 9W Homo sapiens cDNA
clone IMAGE:1760044 3' similar to gb:J00117 CHORIOGONADOTROPIN BETA
CHAIN PRECURSOR (HUMAN);, mRNA sequence.; minus strand;
translated
[0297] MTRVLQVVLLALPQLV
[0298] Mm.42246.3 Mm.42246 101.3 837 304 28 768 GENE=Pck1
PROTSIM=pir:T24168 phosphoenolpyruvate carboxykinase 1, cytosolic;
translated
[0299] KVIQGSLDSLPQAV, LDSL, LPQ
[0300] Mm.22430.1 Mm.22430 209.4 1275 157 75 1535 GENE=Ask-pending
PROTSIM=pir:T02633 activator of S phase kinase; translated
[0301] VLQAILPSAPQ, LQA, LQAIL, PSAP, LPS
[0302] Hs.63758.4 Hs.63758 93.8 3092 1210 51 2719 GENE=TFR2
PROTSIM=pir:T30154 transferrin receptor 2; translated
KVLQGRLPAVAQAV, LQG, LPA, LPAV
[0303] Mm.129320.2 Mm.129320 173.0 3220 571 55 2769
GENE=PROTSIM=pir:T16409 Sequence 8 from Patent WO9950284;
translated
[0304] LVQKVVPMLPRLLC, LVQ, LPRL, PMLP
[0305] Mm.22430.1 Mm.22430 209.4 1275 157 75 1535 GENE=Ask-pending
PROTSIM=pir:T02633 activator of S phase kinase; translated
[0306] VLQAILPSAPQ, LQA, LQAIL, PSAP, PSAPQ
[0307] P20155 IAC2_HUMAN Acrosin-trypsin inhibitor II precursor
(HUSI-II) [SPINK2] [Homo sapiens]
[0308] LPGCPRHFNPV, LPG, LPGC
[0309] Rn.2337.1 Rn.2337 113.0 322 104 1 327
GENE=PROTSIM=PRF:1402234A Rat pancreatic secretory trypsin
inhibitor type II (PSTI-II) mRNA, complete cds; minus strand;
translated
[0310] LVGCPRDYDPV, LVG, LVGC
[0311] Hs.297775.1 Hs.297775 43.8 1167 753 31 1291
GENE=PROTSIM=sp:000268 ESTs, Weakly similar to T2D3_HUMAN
TRANSCRIPTION INITIATION FACTOR TFIID 135 KDA SUBUNIT [H.sapiens];
minus strand; translated
[0312] PGCPRG, PGCP
[0313] Mm.1359.1 Mm.1359 PROTSTM=pir.A39743 urokinase plasmiogen
activator receptor
[0314] LPGCP, PGCP, LPG, LPGC
[0315] sptremb1.vertline.O56177.vertline.O56177 ENVELOPE
GLYCOPROTEIN
[0316] VLPAAP, PAAP
[0317] sptrembl.vertline.Q9W234.vertline.Q9W234 CG 13509
PROTEIN.//:trembl.vertline.AE003458.vertline.AE003458.sub.--7 gene:
"CG13509"; Drosophila melanogaster genomic scaffold
[0318] LAGTIPATP, LAG, PATP
[0319] swiss.vertline.P81272.vertline.NS2B HUMAN NITRIC-OXIDE
SYNTHASE IIB (EC 1.14.13.39) (NOS, TYPE II B) (NOSIIB)
(FRAGMENTS)
[0320] GVLPAVP, LPA, VLPAVP, PAVP
[0321] sptrembl.vertline.O30137.vertline.O30137 HYPOTHETICAL 17.2
KDA
[0322] GVLPALP, PALP, LPAL
[0323] sptrembl.vertline.Q91YZ3.vertline.Q91YZ3 DNA POLYMERASE
[0324] GLLPCLP, LPC, LPCL, PCLP
[0325] sptrembl.vertline.Q9PVW5.vertline.Q9PVW5 NUCLEAR PROTEIN
NP220
[0326] PGAP, LPQRPRGPNP, LPQ, PRGP, PNP
[0327] Hs.303116.2 PROTSIM=pir; T33097 stromal cell-derived factor
2-like1; translated
[0328] GCPR
[0329] pdb.vertline.1DU3.vertline.1DU3-A CRYSTAL STRUCTURE OF
TRAIL-SDR5
[0330] GCPRGM
[0331] pdb.vertline.1D0G.vertline.1D0G-R CRYSTAL STRUCTURE OF DEATH
RECEPTOR 5 (DR5) BOUND TO APO2L/TRAIL
[0332] GCPRGM
[0333] pdb.vertline.1BIO.vertline.1BIO HUMAN COMPLEMENT FACTOR D IN
COMPLEX WITH ISATOIC ANHYDRIDE INHIBITOR
[0334] LQHV
[0335] pdb.vertline.4NOS.vertline.4NOS-A HUMAN INDUCIBLE NITRIC
OXIDE SYNTHASE WITH INHIBITOR
[0336] FPGC, PGCP
[0337] pdb.vertline.1FL7.vertline.1FL7-B HUMAN FOLLICLE STIMULATING
HORMONE
[0338] PARP, VPGC
[0339] pdb.vertline.1HR6.vertline.1HR6-A YEAST MITOCHONDRIAL
PROCESSING PEPTIDASE
[0340] CPRG, LKGC
[0341] pdb.vertline.1BFA.vertline.1BFA RECOMBINANT BIFUNCTIONAL
HAGEMAN FACTOR/AMYLASE INHIBITOR FROM
[0342] PPGP, LPGCPREV, LPGC, PGCP, CPRE
[0343] swissnew.vertline.P01229.vertline.LSHB HUMAN Lutropin beta
chain precursor
[0344] MMRVLQAVLPPLPQVVC, MMR, MMRV, LQA, LQAV, VLPPLP, PPLP, QVVC,
VVC, VLPPLPQ, AVLPPLP, AVLPPLPQ
[0345] swissnew.vertline.P07434.vertline.CGHB PAPAN
Choriogonadotropin beta chain precursor
[0346] MMRVLQAVLPPVPQVVC, MMR, MMRV, LQA, LQAG, VLPPVP, VLPPVPQ,
QVVC, VVC, AVLPPVP, AVLPPVPQ
[0347] swissnew.vertline.Q28376.vertline.TSHB HORSE Thyrotropin
beta chain precursor
[0348] MTRD, LPK, QDVC, DVC, IPGC, PGCP
[0349] swissnew.vertline.P95180.vertline.NUOB MYCTU NADH
dehydrogenase I chain B
[0350] LPGC, PGCP
[0351] sptrembl.vertline.Q9Z284.vertline.Q9Z284 NEUTROPHIL
ELASTASE
[0352] PALP, PALPS
[0353] sptrembl.vertline.Q9UCG8.vertline.Q9UCG8 URINARY
GONADOTROPHIN PEPTIDE (FRAGMENT).
[0354] LPGGPR, LPG, LPGG, GGPR
[0355] XP.sub.--028754 growth hormone releasing hormone [Homo
sapiens]
[0356] LQRG, LQRGV, LGQL
SignalP (CBS)
[0357] SignalP predictions: (for example)
3 SignalP (CBS) SignalP pre- (for example) dictions: MTRVLQGVLPALP
QVVC HLA Peptide Binding Predictions (BIMAS) (For example) Half
time of dissociation HLA molecule VLQGVLPAL (84) type I (A_
GVLPALPQV (51) 0201): VLPALPQVV (48) RLPGCPRGV (14) TMTRVLQGV (115)
scores MHC II (H2-Ak CPTMTRVLQGVLPAL 14 15-mers) PGCPRGVNPVVSYAV 14
HLA-DRB1*0101 PRGVNPVVSYAVALS 29 15-mers TRVLQGVLPALPQVV 28
LQGVLPALPQVVCNY 22 HLA-DRB1*0301 CPTMTRVLQGVLPAL 26 (DR17) 15-mers
MTRVLQGVLPALPQV 21 SIRLPGCPRGVNPVV 17
[0358]
4TABLE 1 Results of shock experiments in mice % SURVIVAL IN TEST
SUBSTANCE TIME (HRS) 0 16 40 72 PBS 100 100 67 17 PG23 100 100 100
100 PG25 100 83 83 83 PEPTIDE NMPF SEQUENCE 1 VLPALPQVVC 100 100 50
17 2 LQGVLPALPQ 100 67 0 0 3 LQG 100 83 20 17 4 LQGV 100 100 100
100 5 GVLPALPQ 100 100 80 17 6 VLPALP 100 100 100 100 7 VLPALPQ 100
83 0 0 8 GVLPALP 100 100 83 67 9 VVC 100 100 50 50 11 MTRV 100 100
67 50 12 MTR 100 100 67 50 13 LQGVLPALPQVVC 100 100 100 100 14
(CYCLIC) LQGVLPALPQVVC 100 83 83 83 64 LPGCPRGVNPVVS 100 100 100
100 66 LPGC 100 100 100 100
[0359]
5TABLE 2 Additional results of shock experiments NMPF SEQUENCE ID:
ANTI-SHOCK EFFECT LQGV +++ AQGV +++ LQGA +++ VLPALP +++ ALPALP ++
VAPALP ++ ALPALPQ ++ VLPAAPQ ++ VLPALAQ +++ SHOCK ACCELERATING
EFFECT LAGV +++ LQAV +++ VLAALP +++ VLPAAP +++ VLPALA +++ VLPALPQ
+++ VLAALPQ +++ VLPALPA +++
[0360]
6TABLE 3 Further additional results of shock experiments NMPF
PEPTIDES % SURVIVAL IN TIME (IRS) Tx Tx 0 14 24 48 PBS 100 100 100
0 NMPF-3 100 100 100 0 NMPF-5 100 100 100 100 NMPF-7 100 100 100 67
NMPF-8 100 100 100 100 NMPF-9 100 100 100 100 NMPF-11 100 100 100
100 NMPF-12 100 100 100 100 NMPF-43 100 100 100 100 NMPF-45 100 100
100 100 NMPF-46 100 100 100 100 NMPF-50 100 100 100 100 NMPF-53 100
100 100 100 NMPF-58 100 100 100 100 NMPF-60 100 100 100 100
[0361]
7TABLE 4 Further additional results NMPF PEPTIDES SICKNESS SCORES
Tx Tx 0 14 24 48 PBS 0,0,0,0,0,0 5,5,5,5,4,4 5,5,5,5,5,5
.dagger..dagger..dagger..dagger.- .dagger..dagger. NMPF-3
0,0,0,0,0,0 3,3,3,3,3,4 4,4,4,4,4,4
.dagger..dagger..dagger..dagger..dagger..dagger. NMPF-5 0,0,0,0,0,0
5,5,5,5,5,5 5,5,5,5,5,5 2,2,2,2,2,2 NMPF-7 0,0,0,0,0,0 1,1,4,4,4,4
5,5,5,5,5,5 2,2,2,2,.dagger..dagger. NMPF-8 0,0,0,0,0,0 3,3,5,5,5,5
5,5,5,5,5,5 2,2,4,4,4,5 NMPF-9 0,0,0,0,0,0 3,3,4,4,5,5 2,2,2,2,2,2
1,1,2,2,2,2 NMPF-11 0,0,0,0,0,0 1,1,3,3,4,4, 2,2,2,2,4,4
1,1,1,1,1,1 NMPF-12 0,0,0,0,0,0 1,1,1,1,3,3 1,1,1,1,1,1 1,1,1,1,1,1
NMPF-43 0,0,0,0,0,0 1,1,4,4,4,4 1,1,1,1,3,3 2,2,2,2,2,2 NMPF-45
0,0,0,0,0,0 5,5,5,5,4,4 3,3,4,4,5,5 2,2,4,4,5,5 NFPF-46 0,0,0,0,0,0
1,1,2,2,3,3 1,1,2,2,2,2 1,1,1,1,1,1 NMPF-50 0,0,0,0,0,0 1,1,1,1,3,3
2,2,2,2,3,3 1,1,1,1,1,1 NMPF-53 0,0,0,0,0,0 5,5,5,5,5,5 5,5,5,5,5,5
1,1,2,2,2,2 NMPF-58 0,0,0,0,0,0 5,5,5,5,3,3 5,5,5,5,3,3 1,1,1,1,1,1
NMPF-60 0,0,0,0,0,0 1,1,4,4,2,2 2,2,2,2,4,4 1,1,1,1,1,1
[0362]
8TABLE 5 Summary of results of the various peptides in the various
experiments. ID SEQUENCE SEPSIS ANGIOGENSIS CAO DC NOD NMPF-1
VLPALPQVVC -+ + + NMPF-2 LQGVLPALPQ -+ + NMPF-3 LQG -+ + + + NMPF-4
LQGV + + + + NMPF-5 GVLPALPQ -+ + NMPF-6 VLPALP + + + + NMPF-7
VLPALPQ + + + NMPF-8 GVLPALP -+ + NMPF-9 VVC + + + NMPF-11 MTRV + +
+ + NMPF-12 MTR -+ + + NMPF-13 LQGVLPALPQVVC + + NMPF-14
cyclic-LQGVLPALPQVVC + NMPF-43 AQG + + + NMPF-44 LAG + NMPF-45 LQA
+ + NMPF-46 AQGV + + + NMPF-47 LAGV -+ + + NMPF-48 LQAV NMPF-49
LQGA + NMPF-50 ALPALP + + NMPF-51 VAPALP + + NMPF-52 VLAALP NMPF-53
VLPAAP + + NMPF-54 VLPALA NMPF-55 ALPALPQ + NMPF-56 VAPALPQ +
NMPF-57 VLAALPQ NMPF-58 VLPAAPQ + + NMPF-59 VLPALAQ + + NMPF-60
VLPALPA + + NMPF-61 VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL -+ +
NMPF-62 VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSGQ NMPF-63 LPGCPRGVNPVVS +
NMPF-64 LPGCPRGVNPVVS + NMPF-65 CPRGVNPVVS NMPF-66 LPGC + + +
NMPF-67 CPRGVNP NMPF-68 PGCP -+ NMPF-69
RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT NMPF-70 MTRVLQGVLPALPQ -+
NMPF-71 MTRVLPGVLPALPQVVC -+ NMPF-74 CALCRRSTTDCGGPKDHPLTC NMPF-75
SKAPPPSLPSPSRLPGPC NMPF-76 TCGGPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ +
= effects; -+ = variable effect; no entry is no effect or not yet
tested when table was assembled
[0363]
9TABLE 6 MODULATION OF NO AND/OR TNF-.alpha. ID SEQUENCE TNF-A NO
TNF-A and NO NMPF-1 VLPALPQVVC ++ ++++ ++++ NMPF-2 LQGVLPALPQ -+
++++ ++++ NMPF-3 LQG + ++++ ++++ NMPF-4 LQGV ++++ ++++ +++++++
NMPF-5 GVLPALPQ ++++ ++++ +++++++ NMPF-6 VLPALP ++++ ++++ +++++++
NMPF-7 VLPALPQ ++++ ++++ +++++++ NMPF-8 GVLPALP ++++ ++++ +++++++
NMPF-9 VVC ++++ ++++ +++++++ NMPF-10 QVVC ++++ +++ ++++ NMPF-11
MTRV ++++ ++++ ++++ NMPF-12 MTR ++++ ++++ ++++ NMPF-13
LQGVLPALPQVVC ++ ++++ ++++ NMPF-14 cyclic-LQGVLPALPQVVC ++ ++++
++++ NMPF-43 SQG ++++ ++++ +++++++ NMPF-44 LAG -+ ++++ ++++ NMPF-45
LQA ++++ ++++ +++++++ NMPF-46 AQGV ++++ ++++ +++++++ NMPF-47 LAGV
++ ++++ ++++ NMPF-48 LQAV ++ ++++ ++++ NMPF-49 LQGA ++ ++++ ++++
NMPF-50 ALPALP ++++ ++++ +++++++ NMPF-51 VAPALP + +++ ++++ NMPF-52
VLAALP ++ ++++ ++++ NMPF-53 VLPAAP ++++ ++++ +++++++ NMPF-54 VLPALA
+ ++++ ++++ NMPF-55 APALPQ + ++++ ++++ NMPF-56 VAPALPQ -+ ++++ ++++
NMPF-57 VLAALPQ + ++++ ++++ NMPF-58 VLPAAPQ ++++ ++++ +++++++
NMPF-59 VLPALAQ ++ ++++ ++++ NMPF-60 VLPALPA ++++ ++++ +++++++
VVCNYRDVRIFESIRLPGCPRGVNPVVSY NMPF-61 AVALSCQCAL -+ ++++ ++++
VVCNYRDVRFESIRLPGCPRGVNPVVSY NMPF-62 AVALSCQ -+ +++ ++++ NMPF-63
SIRLPGCPRGVNPVVS -+ ++ ++ NMPF-64 LPGCPRGVNPVVS ++ ++++ ++++
NMPF-65 CPRGVNPVVS ++ +++ +++ NMPF-66 LPGC +++ ++ +++ NMPF-67
CPRGVNP -+ + + NMPF-68 LGCP + + +++ RCRPINATLAVEK NMPF-69
EGCPVCITVNTTICAGYCPT -+ ++ ++ NMPF-70 MTRVLQGVLPALPQ -+ + + NMPF-71
MTRVLPGVLPALPQVVC -+ -+ -+ NMPF-74 CALCRRSTTDCGGPKDHPLTC -+ ++ +
NMPF-75 SKAPPPSLPSPSRLPGPS + ++ ++ TCDDPRFQDSSSSKAPPPSLPSPSRLPGPS
NMPF-76 DTPILPQ + + + NMPF-78 CRRSTTDCGGPKDHPLTC + + + from -+ to
+++++++ indicates from barely active to very active in
modulating
[0364] Monkey Experiment
[0365] Efficacy of NMPF, here a mixture 1:1:1 of LQGV, AQGV and
VLPALP, administered in a gram-negative induced rhesus monkey
sepsis model for prevention of septic shock. Overwhelming
inflammatory and immune responses are essential features of septic
shock and play a central part in the pathogenesis of tissue damage,
multiple organ failure, and death induced by sepsis. Cytokines,
especially tumour necrosis factor (TNF)-.quadrature. interleukin
(IL)-1.quadrature., and macrophage migration inhibitory factor
(MIF) have been shown to be critical mediators of septic shock.
Yet, traditional anti-TNF and anti-IL-1 therapies have not
demonstrated much benefit for patients with severe sepsis. We have
designed peptides that block completely LPS induced septic shock in
mice, even when treatment with these peptides is started up to 24
hours after LPS injection. These peptides are also able to inhibit
the production of MIF. This finding provides the possibility of
therapeutic use of these peptides for the treatment of patients
suffering from septic shock. Since primates are evolutionary more
closer to humans, we tested these peptides for their safety and
effectiveness in a primate system.
[0366] Experimental Design
10 EXPERIMENTAL TREATMENT (independent variable, e.g. placebo
treated GROUP) control group) BIOTECHNIQUES NUMBER animal I i.v.
infusion of a lethal Live E.coli infusion N = 1 dose of live Blood
sampling Escherichia.coli (10E10 No CFU/kg) recovery + antibiotics
(section) + placebo treated animal II i.v. infusion of a lethal
Live E.coli infusion N = 1 dose of live Blood sampling
Escherichia.coli (10E10 No CFU/kg) recovery + antibiotics (section)
+ oligopeptide (5 mg/kg of each of 3 peptides)
[0367] Only naive monkeys were used in this preclinical study to
exclude any interaction with previous treatments. The animals were
sedated with ketamine hydrochloride. Animals were intubated orally
and are allowed to breathe freely. The animals were kept
anesthesized with O.sub.2/N.sub.2O/isoflurane. The animals received
atropin as pre-medication for O.sub.2/N.sub.2O/isoflurane
anesthesia. A level of surgical anesthesia was maintained during
the 2 h infusion of E.coli and for 6 h following E.coli challenge
after which the endothracheal tubes were removed and the animals
were euthanized. Before bacteria were induced, a 1 hour
pre-infusion monitoring of heart-rate and blood pressure was
performed.
[0368] Two rhesus monkeys were infused with a 10 .sup.10CFU per kg
of the Gram negative bacterium E.coli to induce a fatal septic
shock. One monkey received placebo-treatment and was sacrificed
within 7 hours after infusion of the bacteria without recovery from
the anesthesia. The second monkey received treatment with test
compound and was sacrificed at the same time point.
[0369] In a limited dose-titration experiment performed with the
same bacterium strain in 1991 the used dose proved to induce fatal
shock within 8 hours. In recent experiments a 3-fold lower dose was
used inducing clear clinical and pathomorphological signs of septic
shock without fatal outcome.
[0370] The monkeys were kept anaesthetized throughout the
observation period and sacrificed 7 hours after the start of the
bacterium infusion for pathological examination. The animals
underwent a gross necropsy in which the abdominal and throrac
cavities were opened and internal organs examined in situ.
[0371] Full Description of the Experiment With Three Rhesus
Monkeys
[0372] The study was conducted in rhesus monkeys (Maccaca mulatta).
Only experimentally naive monkeys were used in study to exclude any
interaction with previous treatments. Prior to the experiment the
state of health of the animals was assessed physically by a
veterinarian. All animals had been declared to be in good health
and were free of pathogenic ecto- and endoparasites and common
bacteriological infections: Yersinia pestis, Yersinia
enterocolitica, Yersinia pseudotuberculosis, Shigella, Aeromonas
hydrophilia, pathogenic Campylobacter species and Salmonella.
[0373] Reagents. The Escherichia coli strain was purchased from
ATCC (E-coli; 086a: K61 serotype, ATCC 33985). In a control
experiment the strain proved equally susceptible to bactericidal
factors in human and rhesus monkey serum.
[0374] Prior to the experiment a fresh culture was set-up; the
E.coli strain was cultured for one day, harvested and washed
thoroughly to remove free endotoxine. Prior to infusion into the
animal the number and viability of the bacteria was assessed.
Serial dilutions of the E.coli stock were plated on BHI agar and
cultured overnight at 37oC. The colonies on each plate were counted
and the number of colony forming units per ml were calculated. The
body weight measurement of the day of the experiment was used to
calculate the E.coli dose and E.coli stock was suspended in
isotonic saline (N.P.B.I., Emmer-Compascuum, The Netherlands) at
the concentration needed for infusion (total dose volume for
infusion approximately 10 ml/kg. The E.coli suspension was kept on
ice until infusion.
[0375] Antibiotic was used to synchronise the shock induction in
the monkeys. Baytril (Baytril 2.5%, Bayer, Germany) was used
instead of gentamycin, as the strain proved only marginally
susceptible to the latter antibiotic.
[0376] Individual animals were identified by a number or letter
combination tattooed on the chest.
[0377] Experimental Design.
11 EXPERIMENTAL GROUP TREATMENT (number/ (independent variable,
letter or other e.g. placebo treated identification control group)
NUMBER SEX Animal I i.v. infusion of a lethal Live E. N = 1 F dose
of live coli Escherichia.coli (10E10 infusion CFU/kg) Blood +
antibiotic sampling placebo treated No recovery Animal II i.v.
infusion of a lethal Live E. N = 1 F dose of live coli
Escherichia.coli (10E10 infusion CFU/kg) Blood antibiotic sampling
NMPF-4, 6, 46; each No 5 mg/kg recovery (section) Animal III i.v.
infusion of a lethal Live E. N = 1 F dose of live coli
Escherichia.coli (10E10 infusion CFU/kg) Blood antibioti sampling +
NMPF-4, 6, 46; each Recovery 5 mg/kg and survival
[0378] Anesthesia. All animals were fasted overnight prior to the
experiment. On the morning of the experiment the animals were
sedated with ketamine hydrochloride (Tesink, The Netherlands) and
transported to the surgery. The animal was placed on its side on a
temperature controlled heating pad to support body temperature.
Rectal temperature was monitored using a Vet-OX 5700. The animals
were intubated orally and were allowed to breathe freely. The
animals were kept anesthesized using O.sub.2/N.sub.2O/isoflurane
inhalation anaesthesia during the E.coli infusion and the 7 hour
observation period following E.coli challenge after which the
endothracheal tubes were removed and the animals were euthanized or
allowed to recover from anesthesia. The femoral or the cephalic
vein was cannulated and used for infusing isotonic saline, live
E.coli and antibiotic administration. Insensible fluid loss was
compensated for by infusing isotonic saline containing 2.5% glucose
(Fresenius, 's Hertogenbosch, The Netherlands) at a rate of 3.3
ml/kg/hr.
[0379] Preparative actions. During anesthesia the animals were
instrumented for measurement of blood pressure (with an automatic
cuff), heart rate and body temperature. Isotonic saline was infused
at 3.3 ml/kg/hr to compensate for fluid loss. Femoral vessels were
cannulated for infusion of E.coli and antibiotics. Temperature
controlled heating pads were used to support body temperature. The
monkeys were continuously monitored during E.coli challenge and for
the 6 hr period following E.coli administration. After 7 hrs 2
animals (the control animal and one treated with NMPF) were
sacrificed to compare the direct effect of the compound at the
level of histology. The 3.sup.rd animal, treated with NMPF, was
allowed to recover from anaesthesia and was intensively observed
during the first 12 hours after recovery followed by frequent daily
observation. The decision to allow 3.sup.rd animal to recover was
made after consulting with the veterinarian.
[0380] Induction of septic shock. Before the infusion of E.coli, a
1 hr pre-infusion monitoring of heart-rate and blood pressure was
performed. All three animals received an i.v. injection of E.coli
086 (k61 serotype; ATCC 33985) at a lethal dose of 10.times.109
CFU/kg bodyweight. In a dose titration study with this batch
performed in 1991, this bacterial dose induced lethal shock within
8 hrs after the start of the infusion. The infusion period was 2
hrs.
[0381] Antibiotics. Baytril was administered intravenously
immediately after completion of the 2 h. E.coli infusion (i.v.;
dose 9 mg/kg).
[0382] Treatment with NMPF. 30 minutes post-onset of E.coli
infusion the animals were administered a single intravenous bolus
injection a mixer of NMPF oligopeptide. The oligopeptide mixer
contained the following NMPF peptides: LQGV (5 mg/kg), AQGV (5
mg/kg) and VLPALP (5 mg/kg). These NMPF peptides were dissolved in
0.9% sodium chloride for injection (N.P.B.I., Emmer Compascuum, The
Netherlands).
[0383] Results
[0384] Preliminary Monkey Results
[0385] An anti-shock effect of the test compound on sepsis in the
monkey treated with the oligopeptide mixture, namely the inhibition
of the effect of the sepsis in this early 7-hour trajectory of this
primate model was observed. Immunomodulatory effects with these
peptides have been observed in vitrolex vivo such as in T-cell
assays the inhibition of pathological Th1 immune responses,
suppression of inflammatory cytokines (MIF), increase in production
of anti-inflammatory cytokines (IL-10, TGF-beta) and
immunomodulatory effects on antigen presenting cells (APC) like
dendritic cells and macrophages.
[0386] The following organs were weighed and a bacterial count were
performed:
[0387] kidneys
[0388] liver
[0389] lungs
[0390] lymph nodes
[0391] gross lesions
[0392] Tissues of all organs were preserved in neutral aqueous
phosphate buffered 4% solution of formaldehyde. Lymphoid organs
were be cryopreserved. All tissues will be processed for
histopathological examination.
[0393] Further Results Obtained in the Three-Monkey Experiment
[0394] Monkey 429(control). Female monkey (5.66 kg) received an
i.v. injection of E.coli 086 (10E10 CFU/kg). In a dose titration
study with this batch performed in 1991, this bacterial dose
induced lethal shock within 8 hrs after the start of the infusion.
The infusion period was 2 hrs. Baytril was administered
intravenously immediately after completion of the 2 h. E.coli
infusion (i.v.; dose 9 mg/kg). After the E.coli injection the
monkey was observed by the authorized veterinarian without knowing
which of the monkey received NMPF treatment. The clinical
observations were as follows: vomiting, undetectable pulse, heart
arythmia, abnormalities in ECG: signs of ventricle dilatation/heart
decompensation (prolonged QRS complex, extra systoles), decreased
blood clotting and forced respiration. In addition, there was big
fluctuation in heart rate (30-150 beats per minute), collapse of
both systolic and diastolic blood pressure (35/20 mmHg) and
decrease in blood oxygen concentration (80-70%). Seven hours after
the start of the E.coli infusion, monkey began to vomit blood and
faeces, and have convulsions. After final examination, the
veterinarian did not gave permission to let this monkey awake. At
this time point control monkey was euthanised. Hereafter, post
mortem examination was conducted and internal organs were examined
in situ. Number of internal bleedings were found by the
pathologist.
[0395] Monkey 459(NMPF). Female monkey (5.44 kg) received an i.v.
injection of E.coli 086 (10E10 CFU/kg). In a dose titration study
with this batch performed in 1991, this bacterial dose induced
lethal shock within 8 hrs after the start of the infusion. The
infusion period was 2 hrs. 30 minutes after the initiation of
E.coli infusion, NMPF was i.v. injected in a single bolus
injection. Baytril was administered intravenously immediately after
completion of the 2 h. E.coli infusion (i.v.; dose 9 mg/kg). After
the E.coli injection this monkey was also observed by the
authorized veterinarian without knowing which of the monkey
received NMPF treatment. The clinical observations were as follows:
normal pulse, heart sounds normal, normal ECG, higher heart-rate
but otherwise stable (180 beats per minute), no hypotension (75/30
mmHg), normal blood oxygen concentration (95-85%), lungs sound
normal, normal turgor. Seven hours after the start of the E.coli
infusion, the clinical condition of the monkey was stable. After
final examination, the veterinarian did give permission to let this
monkey awake due to her stable condition. In order to compare the
hematological and immunological parameters between the control and
NMPF treated monkey, at this time point NMPF treated monkey 459 was
euthanised. Hereafter, post mortem examination was conducted and
internal organs were examined in situ. No macroscopic internal
bleedings were found by the pathologist.
[0396] Monkey 427(NMPF). Female monkey (4.84 kg) received an i.v.
injection of E.coli 086 (10E10 CFU/kg). In a dose titration study
with this batch performed in 1991, this bacterial dose induced
lethal shock within 8 hrs after the start of the infusion. The
infusion period was 2 hrs. 30 minutes after the initiation of
E.coli infusion, NMPF was i.v. injected. Baytril was administered
intravenously immediately after completion of the 2 h. E.coli
infusion (i.v.; dose 9 mg/kg). After the E.coli injection this
monkey was also observed by the authorised veterinarian doctor
without knowing which of the monkey received NMPF treatment. The
clinical observations were as follows: normal pulse, heart sounds
normal, normal ECG, moderately higher heart-rate but otherwise
stable (160 beats per minute), no hypotension (70/30 mmHg), normal
blood oxygen concentration (95-90%), lungs sound normal, normal
turgor. Seven hours after the start of the E.coli infusion, the
clinical condition of the monkey was stable. After final
examination, the veterinarian did give permission to let this
monkey wake up due to her stable condition. Monkey woke up quickly,
she was alert and there was a slow disappearance of oedema.
[0397] Genomic Experiment
[0398] PM1 T-cell line was obtained from American Type Culture
Collection (Manassas, Va.) and was cultured at 37.degree. C. in 5%
CO.sub.2. These cells were maintained and cultured in RPMI 1640,
10% fetal bovine serum, 2 mM L-glutamine, and antibiotics
penicillin and streptomycin. For genomic experiments cells
(2.times.10.sup.6/ml) were incubated with phytohemagglutinin (PHA,
10 .quadrature.g/ml) and IL-2 (200 IU/ml) or PHA, IL-2 and peptide
LQGV (10 mg/ml) in a volume of 2 ml in 6-wells plates. After 4 h of
cultures 10.times.10.sup.6 cells were washed and prepared for
genechip probe arrays experiment. The genechip expression analysis
was performed according to the manufacturer's instructions
(Expression Analysis, Technical Manual, Affymetrix Genechip). The
following major steps outline Genechip Expression Analysis: 1)
Target preparation 2) Target hybridization 3) Experiment and
fluidics station setup 4) Probe Array washing and staining 5) Probe
array scan and 6) Data analysis.
[0399] Results
[0400] Genomic Experiment
[0401] The gene chip expression analysis revealed that due to LQGV
treatment of PM1 (T-cell line) cells for 4 hours in the presence of
PHA/IL-2 down-regulated at least 120 genes more than 2 fold as
compared to control PM1 cells (stimulated with PHA/IL-2) only.
Moreover, at least 6 genes were up-regulated more than 2 fold in
peptide treated cells as compared to control cells.
[0402] Down regulated genes due to treatment with LQGV in genomics
experiment
12 Fold Change/Descriptions 21.2 M11507 Human transferrin receptor
mRNA, complete cds (_5, _M, _3 represent transcript regions 5
prime, Middle, and 3 prime respectively) 10.1 Human (c-myb) gene,
complete primary cds, and five complete alternatively spliced cds
(U22376 /FEATURE = cds#5 /DEFTNITION = HSU22376) 9.7 Cluster Incl.
X68836:H.sapiens mRNA for S-adenosylmethionine synthetase (cds =
(65,1252) /gb = X68836 /gi = 36326 /ug = Hs.77502 /len = 1283) 9.3
M97935 Homo sapiens transcription factor ISGF-3 mRNA, complete cds
(_5, _MA, _MB, _3 represent transcript regions 5 prime, MiddleA,
MiddleB, and 3 prime respectively) 8.7 Human mRNA for
phosphatidylinositol transfer protein (PJ-TPbeta), complete cds
(D30037 /FEATURE = /DEF1NTTION = HUMPITPB) 7.5 Cluster Incl.
U28964:Homo sapiens 14-3-3 protein mRNA, complete cds (cds =
(126,863) /gb = U28964 /gi = 899458 /ug = Hs.75103 /len = 1030) 6.7
Human CDK tyrosine 15-kinase WEE1Hu (Wee1Hu) mRNA, complete cds
(U10564 /FEATURE = /DEFINITION = HSU10564) 6.7 Homo sapiens
E2F-related transcription factor (DP-1) mRNA, complete cds (L23959
/FEATURE = /DEFINITION = HUMDP1A) 6.5 Cluster Incl. W29030:55c4
Homo sapiens cDNA (gb = W29030 /gi = 1308987 /ug = Hs.4963 /len =
758) 6.1 Cluster Incl. U08997:Human glutamate dehydrogenase gene,
complete cds (cds = (0,1676) /gb = U08997 /gi = 478987 /ug =
Hs.239377 /len = 1677) 5.7 M97935 Homo sapiens transcription factor
ISGF-3 mRNA, complete cds (_5, _MA, _MB, _3 represent transcript
regions 5 prime, MiddleA, MiddleB, and 3 prime respectively) 5.6
Cluster Incl. Y00638:Human mRNA for leukocyte common antigen (T200)
(cds = (86,4000) /gb = Y00638 /gi = 34280 /ug = Hs.170121 /len =
4315) 5.3 Ras-Like Protein Tc21 5.3 H.sapiens mRNA for Fas/Apo-1
(clone pCRTM11-Fasdelta(4,7))(X83492 / FEATURE = exons #1-2
/DEFINITION = HSFAS47) 4.8 Cluster Incl. AJ002428:Homo sapiens
VDAC1 pseudogene (cds = (0,853) / gb = AJ002428 /gi = 3183956 /ug =
Hs.201553 /len = 854) 4.7 Ras-Related Protein Rap1b 4.6 Cluster
Incl. AL080119:Homo sapiens mRNA; cDNA DKFZp564M2423 (from clone
DKFZp564M2423) (cds = (85,1248) /gb = AL080119 /gi = 5262550 / ug =
Hs.165998 /len = 2183) 4.5 Cluster Incl. AF047448:Homo sapiens
TLS-associated protein TASR mRNA, complete cds (cds = (29,580) /gb
= AF047448 /gi = 2961148 /ug = Hs.239041 / len = 620) 4.5 Cluster
Incl. D14710:Human mRNA for ATP synthase alpha subunit, complete
cds (cds = (63,1724) /gb = 14710 /gi = 559324 /ug = Hs.155101 /len
= 1857) 4.5 Cluster Incl. X59618:H.sapiens RR2 mRNA for small
subunit ribonucleotide reductase (cds = (194,1363) /gb = X59618 /gi
= 36154 /ug = Hs.75319 / len = 2475) 4.5 Human mRNA for annexin II,
5 UTR (sequence from the 5 cap to the start codon) (D28364/FEATURE
= /DEFINITION = HUMAI23) 4.5 Cluster Incl. AA477898:zu34f08.rl Homo
sapiens cDNA, 5 end / clone = IMAGE-739911 /clone_end = 5 (gb =
AA477898 /gi = 2206532 /ug = Hs.239414 / len = 449) 4.4 Cluster
Incl. L19161:Human translation initiation factor eIF-2 gamma
subunit mRNA, complete cds (cds = (0,1418) /gb = L19161 /gi =
306899 /ug = Hs.211539 / len = 1440gb = AA477898 / gi = 2206532 /ug
= Hs.239414 /len = 449) 4.4 Human serine/threonine-protein kinase
PRP4h (PRP4h) mRNA, complete cds (U48736 /FEATURE = /DEFINITION =
HSU48736) 4.4 Cluster Incl. L43821:Homo sapiens enhancer of
filamentation (HEF1) mRNA, complete cds (cds = (163,2667) /gb =
L43821 /gi = 1294780 / ug = Hs.80261 /len = 3817) 4.4 Ras-Like
Protein Tc21 4.4 Human (c-myb) gene, complete primary cds, and five
complete alternatively spliced cds (U22376 /FEATURE = cds #3
/DEFINITION = HSU22376) 4.3 Cluster Incl. U18271 :Human
thymopoietin (TMPO) gene (cds = (313,2397) /gb = U18271 /gi =
2182141 /ug = Hs.170225 /len = 2796) 4.2 Fk506-Binding Protein,
Alt. Splice 2 4.2 Human proliferating cell nuclear antigen (PCNA)
gene, promoter region (J05614 /FEATURE = mRNA /DEFINITION =
HUMPCNAPRM) 4.1 Human insulin-stimulated protein kinase 1 (ISPK-1)
mRNA, complete cds (U08316 /FEATURE = /DEFINITION = HSU08316) 4.1
Cluster Incl. W28732:50h7 Homo sapiens cDNA (gb = W28732 /gi =
1308680 /ug = Hs.177496 /len = 818) 4.1 Cluster Incl. Y00638:Human
mRNA for leukocyte common antigen (T200) (cds = (86,4000) /gb =
Y00638 /gi = 34280 /ug = Hs.170121 /len = 4315) 4 Homo sapiens
putative purinergic receptor P2Y10 gene, complete cds (AF000545
/FEATURE = cds /DEFINITION = HSAF000545) 3.8 Cluster Incl.
U08997:Human glutamate dehydrogenase gene, complete cds (cds =
(0,1676) /gb = U08997 /gi = 478987 /ug = Hs.239377 /len = 1677) 3.6
Human mRNA for raf oncogene (X03484 /FEATURE = cds /DEFINITION =
HSRAFR) 3.6 Cluster Incl. M32886:Human sorcin CP-22 mRNA, complete
cds (cds = (12,608) /gb = M32886 /gi = 338481 /ug = Hs.117816 /len
= 952) 3.6 Homo sapiens GTP-binding protein (RAB1) mRNA, complete
cds (M28209 /FEATURE = /DEFINITION = HUMRAB1A) 3.5 Human
FKBP-rapamycin associated protein (FRAP) mRNA, complete cds (L34075
/FEATURE = /DEFINITION = HUMFRAPX) 3.5 Human DNA topoisomerase II
(top2) mRNA, complete cds (J04088 /FEATURE = /DEFINITION =
HUMTOPII) 3.4 Human translation initiation factor eIF-2 gamma
subunit mRNA, complete cds (L19161 /FEATURE = /DEFINITION =
HUMIEF2G) 3.4 Human mRNA for pre-mRNA splicing factor SRp20, 5 UTR
(sequence from the 5 cap to the start codon) (D28423 /FEATUIRE =
/DEFINITION = HUMLPSF82) 3.4 Cluster Incl. AA442560:zv75g07.r1 Homo
sapiens cDNA, 5 end / clone = IMAGE-759516 /clone_end = 5 (gb =
AA4425 60 /gi = 2154438 /ug = Hs.135198 / len = 566) 3.4 Cluster
Incl. X98248:H.sapiens mRNA for sortilin /cds = (21,2522) (gb =
X98248 /gi = 1834494 /ug = Hs.104247 /len = 3723) 3.3 Cluster Incl.
AB020670:Homo sapiens mRNA for KIAA0863 protein, complete cds (cds
= (185,3580) /gb = AB020670 /gi = 4240214 /ug = Hs.131915 /len =
4313) 3.3 Cluster Incl. W28869:53h2 Homo sapiens cDNA (gb = W28869
/ gi = 1308880 /ug = Hs.74637 /len = 975) 3.3 Cluster Incl.
Z12830:H.sapiens mRNA for SSR alpha subunit /cds = (29,889) (gb =
Z12830 /gi = 551637 /ug = Hs.76152 /len = 974). 3.3 Cluster Incl.
AL021546:Human DNA sequence from BAC 15E1 on chromosome 12.
Contains Cytochrome C Oxidase Polypeptide VIa-liver precursor gene,
60S ribosomal protein L31 pseudogene, pre-mRNA splicing factor
SRp30c gene, two putative genes, ESTs, STSs and putative CpG
islands (cds = (0,230) /gb = AL021546 /gi = 2826890 / ug =
Hs.234768 /len = 547) 3.2 Cluster Incl. U78082:Human RNA polymerase
transcriptional regulation mediator (h-MED6) mRNA, complete cds
(cds = (50,523) /gb = U78082 /gi = 2618737 /ug = Hs.167738 /len =
885) 3.2 H.sapiens RbAp48 mRNA encoding retinoblastoma binding
protein (X74262 /FEATURE = cds /DEFINITION = HSRBAP48) 3.1 Cluster
Incl. M64174:Human protein-tyrosine kinase (JAK1) mRNA, complete
cds (cds = (75,3503) /gb = M64174 /gi = 190734 /ug = Hs.50651 /len
= 3541) 3.1 Cluster Incl. A1862521:wj15a06.x1 Homo sapiens cDNA, 3
end / clone = IMAGE-2402866 /clone_end = 3 (gb = A1862521 /gi =
5526628 /ug = Hs.146861 /len = 606) 3.1 Cluster Incl. W27517:31h6
Homo sapiens cDNA (gW = W27517 /gi = 1307321 /ug = Hs.13662 /len =
732) 3 Human rab GDI mRNA, complete cds (D13988 /FEATURE =
/DEFINITION = HUMRABGDI) 3 Cluster Incl. AL080119:Homo sapiens
mRNA; cDNA DKFZp564M2423 (from clone DKFZp564M2423) (cds =
(85,1248) /gb = AL080119 /gi = 5262550 /ug = Hs.165998 /len = 2183)
3 Human cAMP-dependent protein kinase type I-alpha subunit
(PRKAR1A) mRNA, complete cds (M33336 /FEATURE = /DEFINITION =
HUMCAMPPK) 3 Cluster Incl. L75847:Human zinc finger protein 45
(ZNF45) mRLNA, complete cds (cds = (103,2151) /gb = L75847 /gi =
1480436 /ug = Hs.41728 / len = 2409) 3 Cluster Incl. M21154:Human
S-adenosylmethionine decarboxylase mRNA, complete cds (cds =
(248,1252) /gb = M21154 /gi = 178517 /ug = Hs.75744 /len = 1805) 3
Cluster Incl. AA675900:g02504r Homo sapiens cDNA, 5 end / clone =
g02504 /clone_end = 5 (gb = AA675900 /gi = 2775247 /ug = Hs.119325
/len = 647) 3 Cluster Incl. M97936:Human transcription factor
ISGF-3 mRNA sequence (cds = UNKNOWN /gb = M97936 /gi = 475254 /ug =
Hs.21486 /len = 2607) 2 M33336 /DEFINITION = HUMCAMPPK Human
cAMP-dependent protein kinase type I-alpha subunit (PRKAR1A) mRNA,
complete cds 2 U16720 /FEATURE = mRNA /DEFINITION = HSU16720 Human
interleukin 10 (IL10) gene, complete cds 2 M33336 HUMCAMPPK Human
cAMP-dependent protein kinase type I-alpha subunit (PRKAR1A) mRNA 2
U50079 /FEATURE = /DEFINITION = HSU50079 Human histone deacetylase
HD1 mRNA, complete cds 2 U16720 /FEATURE = mRNA /DEFINITION =
HSU16720 Human interleukin 10 (IL10) gene, complete cds 2 X87212
/FEATURE = cds /DEFINITION = HSCATHCGE H.sapiens mRNA for cathepsin
C 2 Cluster Incl. AI740522:wg16b07.x1 Homo sapiens cDNA, 3 end
/clone = IMAGE- 2365237 /clone_end = 3 /gb = A1740522 2 M21154
/FEATURE = mRNA /DEFINITION = HUMAMD Human S adenosylmethionine
decarboxylase mRNA, complete cds 2 X00737 /FEATURE = cds
/DEFINITION = HSPNP Human mRNA for purine nucleoside phosphorylase
(PNP; EC 2.4.2.1) 2.1 Cluster Incl. AF034956:Homo sapiens RAD51D
mRNA, complete cds / cds = (124,993) /gb = AF034956 /gi = 2920581
2.1 Ras Inhibitor Inf 2.1 Cluster Incl. M27749:Human
immunoglobulin-related 14.1 protein mRNA, complete cds /cds =
(118,759) /gb = M27749 2.1 Ras-Like Protein Tc4 2.1 X92106 /FEATURE
= cds /DEFINITION = HSBLEO H.sapiens mRNA for bleomycin hydrolase
2.1 D88674 /FEATURE = /DEFINITION = D88674 Homo sapiens mRNA for
antizyme inhibitor, complete cds 2.1 Cluster Incl.
H15872:ym22b12.r1 Homo sapiens cDNA, 5 end /clone = IMAGE- 48838
/clone_end = 5 /gb = H15872 2.1 Cluster Incl. L07541:Human
replication factor C, 38-kDa subunit mRNA, complete cds /cds =
(9,1079) /gb = L07541 2.1 V01512 /FEATURE = mRNA #1 /DEFINITION =
HSCFOS Human cellular oncogene c-fos (complete sequence) 2.1
Cluster Incl. L23959:Homo sapiens E2F-related transcription factor
(DP-1) mRNA, complete cds /cds = (37,1269) 2.1 Stimulatory Gdp/Gtp
Exchange Protein For C-Ki-Ras P21 And Sing P21 2.1 Cluster Incl.
L13943:Human glycerol kinase (GK) mRNA exons 1-4, complete cds /
cds = (66,1640) /gb = L13943 /gi = 348166 2.1 Cluster Incl.
X78925:H.sapiens HZF2 mRNA for zinc finger protein /cds = (0,2198)
/ gb = X78925 /gi = 498722 /ug = Hs.2480 2.1 X74794 /FEATURE = cds
/DEFINITION = HSP1CDC21 H.sapiens P1-Cdc21 mRNA 2.1 U78733 /FEATURE
= mRNA #1 /DEFINITION = HSSMAD2S8 Homo sapiens mad protein homolog
Smad2 gene, exon 11 2.2 Cluster Incl. L07540:Human replication
factor C, 36-kDa subunit mRNA, complete cds /cds = (9,1031) /gb =
L07540 2.2 Cluster Incl. AF040958:Homo sapiens lysosomal
neuraminidase precursor, mRNA, complete cds /cds = (129,1376) 2.2
D00596 /FEATURE = cds /DEFINITION = HUMTS1 Homo sapiens gene for
thymidylate synthase, exons 1, 2, 3, 4, 5, 6, 7, 2.2 Cluster Incl.
A1659108:tu08c09.x1 Homo sapiens cDNA, 3 end /clone = IMAGE-
2250448 /clone_end = 3 /gb = A1659108 2.2 Cluster Incl.
AF042083:Homo sapiens BH3 interacting domain death agonist (BID)
mRNA, complete cds /cds = (140,727) 2.2 Cluster Incl. W28907:53e12
Homo sapiens cDNA /gb = W28907 /gi = 1308855 / ug = Hs.111429 /len
= 989 2.3 Cluster Incl. AF073362:Homo sapiens endo/exonuclease
Mre11 (MRE11A) mRNA, complete cds /cds = (0,2126) 2.3 Escherichia
coli /REF = J04423 /DEF = E coli bioD gene dethiobiotin synthetase
/ LEN = 676 (-5 and -3 represent transcript 2.3 Cluster Incl.
D59253:Human mRNA for NCBP interacting protein 1, complete cds /
cds = (36,506) /gb = D59253 2.3 M21154 /FEATURE = mRNA/DEFINITION =
HUMAMDHuman5- adenosylmethionine decarboxylase mRNA, complete cds
2.3 Proto-Oncogene C-Myc, Alt. Splice 3, Orf 114 2.3 Cluster Incl.
W26787:15d8 Homo sapiens cDNA /gb = W26787 /gi = 1306078 / ug =
Hs.195188 /len = 768 2.4 L12002 /FEATURE = /DEFINITION = HUMITGA4A
Human integrin alpha 4 subunit mRNA, complete cds 2.4 Cluster Incl.
M55536:Human glucose transporter pseudogene /cds = UNKNOWN / gb =
M55536 /gi = 183299 /ug = Hs.121583 2.4X98743/FEATURE = cds /
DEFINITION = HSRNAHELC H.sapiens mRNA for RNA helicase
(Myc-regulated dead box protein) 2.4 S75881 /FEATURE = /DEFINITION
= S75881 A-myb = DNA-binding transactivator {3 region}[human,
CCRF-CEM T leukemia line, mRNA Partial, 831 nt] 2.4 Cluster Incl.
AF050110:Homo sapiens TGFb inducible early protein and early growth
response protein alpha genes, complete cds /cds = (123,1565) /gb =
AF050110 /gi = 3523144 /ug = Hs.82173 / len = 2899 2.5 Cluster
Incl. M86667:H.sapiens NAP (nucleosome assembly protein) mRNA,
complete cds /cds = (75,1250) /gb = M86667 / gi = 189066 /ug =
Hs.179662 /len = 1560 2.5 U17743 /FEATURE = /DEFINITION = HSU17743
Human JNK activating kinase (JNKK1) mRNA, complete cds 2.5 Cluster
Incl. U90549:Human non-histone chromosomal protein (NHC) mRNA,
complete cds /cds = (691,963) /gb = U90549 / gi = 2062699 /ug =
Hs.63272 /len = 1981 2.5 Cluster Incl. U31382:Human G protein
gamma-4 subunit mRNA, complete cds / cds = (98,325) /gb = U31382
/gi = 995916 / ug = Hs.32976 /len = 670 2.5 Cluster Incl.
S81916:phosphoglycerate kinase {alternatively spliced} [human,
phosphoglycerate kinase deficient patient with episodes of musci,
mRNA Partial Mutant, 307 nt] /cds = (0,143) /gb = S81916/ gi =
1470308 /ug = Hs.169313 /len = 307 2.5 Cluster Incl. M64595:Human
small G protein (Gx) mRNA, 3 end /cds = (0,542) / gb = M64595 /gi =
183708 /ug = Hs.173466 / len = 757 2.5 Serine
Hydroxymethyltransferase, Cytosolic, Alt. Splice 3 2.5 U88629
/FEATURE = cds /DEFINITION = HSU88629 Human RNA polymerase II
elongation factor ELL2, complete cds 2.5 Cluster Incl. U72518:Human
destrin-2 pseudogene mRNA, complete cds / cds = (268,798) /gb =
U72518 /gi = 1673523 / ug = Hs.199299 /len = 1057 2.5 Cluster Incl.
L14595:Human alanine/serine/cysteine/threonine transporter (ASCT 1)
mRNA, complete cds 2.5 Cluster Incl. AB014584:Homo sapiens mRNA for
KIAA0684 protein, partial cds / cds = (0,2711) /gb = AB014584 / gi
= 3327181 /ug = Hs.24594 /len = 4124 2.5 Cluster Incl.
AI924594:wn57a11.x1 Homo sapiens cDNA, 3 end /clone = IMAGE-
2449532 /clone_end = 3 /gb = A1924594 / gi = 5660558 /ug =
Hs.122540 /len = 685 2.5 U68111 /FEATURE = mRNA /DEFINITION =
HSPPP1R2E6 Human protein phosphatase inhibitor 2 (PPP1R2) gene,
exon 6 2.5 Cluster Incl. AL009179:dJ97D16.4 (Histone H2B) /cds =
(25,405) /gb = AL009179 / gi = 3217024 /ug = Hs.137594 /len = 488
2.6 Cluster Incl. AF091077:Homo sapiens clone 558 unknown mRNA,
complete sequence /cds = (1,300) /gb = AF091077 / gi = 3859991 /ug
= Hs.40368 /len = 947 2.7 Cluster Incl. M28211:Homo sapiens
GTP-binding protein (RAB4) mRINA, complete cds /cds = (70,711) /gb
= M28211 / gi = 550067 /ug = Hs.234038 /len = 735 2.6 X69549
/FEATURE = cds /DEFINITION = HSRHO2 H.sapiens mRNA for rho
GDP-dissociation Inhibitor 2 2.6 Cluster Incl. Z85986:Human DNA
sequence from clone 108K11 on chromosome 6p21 Contains SRP20 (SR
protein family member), Ndr protein kinase gene similar to yeast
suppressor protein SRP4O, EST and GSS /cds = (0,932) /gb = Z85986
gi = 4034056 /ug = Hs.152400 /len = 933 2.6 Zinc Finger Protein,
Kruppel-Like 2.7 D10656 /FEATURE = /DEFINITION = HUMCRK Human mRNA
for CRK-II, complete cds 2.7 M28211 /FEATURE = /DEFINITION =
HUMRAB4A Homo sapiens GTP-binding protein (RAB4) mRNA, complete cds
2.7 Cluster Incl. AB019435 :Homo sapiens mRNA for putative
phospholipase, complete cds /cds = (72,3074) /gb = AB019435 / gi =
4760646 /ug = Hs.125670 /len = 3088 2.8 U39318 /FEATUIRE =
/DEFINITION = HSU39318 Human E2 ubiquitin conjugating enzyme UbcH5C
(UBCH5C) mRNA, complete cds 2.9 Cluster Incl. X78711:H.sapiens mRNA
for glycerol kinase testis specific 1 / cds = (26,1687) /gb =
X78711 /gi = 515028 / ug = Hs.1466 /len = 1838 2.8 Cluster Incl.
W27594:34h4 Homo sapiens cDNA /gb = W27594 /gi = 1307542 / ug =
Hs.8258 /len = 702 2.8 X05360 /FEATUiRE = cds /DEFINITION = HSCDC2
Human CDC2 gene involved in cell cycle control 2.8 V00568 /FEATURE
= cds /DEFINITION = HSMYC1 Human mRNA encoding the c-myc oncogene
2.8 Cluster Incl. L24804:Human (p23) mRNA, complete cds /cds =
(232,714) / gb = L24804 /gi = 438651 /ug = Hs.75839 /len = 782 2.1
0Cluster Incl. Y09443:H.sapiens mRNA for
alkyl-dihydroxyacetonephosphate synthase precursor /cds = (15,1991)
/ gb = Y09443 /gi = 1922284 /ug = Hs.22580 /len = 2074 2.8 Cluster
Incl. Z82200:Human DNA sequence from clone 333E23 on chromosome
Xq21.1 Contains putative purinergic receptor P2Y10 /cds = (0,1019)
/gb = Z82200 /gi = 2370075 /ug = Hs.166137 / len = 1020 2.9 L05624
/FEATURE = /DEFINITION = HUMMKK Homo sapiens MAP kinase kinase
mRNA, complete cds 2.9 Cluster Incl. D88357:Homo sapiens mRNA for
CDC2 delta T, complete cds / cds = (27,749) /gb = D88357 /gi =
3126638 / ug = Hs.184572 /len = 780 Up regulated genes due to LQGV
treatment 4.9 Cluster Incl. AF043324:Homo sapiens
N-myristoyltransferase 1 mRNA, complete cds(cds = (10,1500)/gb =
AF043324 /gi = 3005062 /ug = Hs.111039 /len = 4378) 3.3 Cluster
Incl. L08096:Human CD27 ligand mRNA, complete cds /cds = (150,731)
(gb = L08096 /gi = 307127 /ug = Hs.99899 /len = 926) Cluster Incl.
AF043325 :Homo sapiens N-myristoyltransferase 2 MRNA, complete cds
/cds = (46,1542) /gb = AF043325 / gi = 3005064 /ug = Hs.122647 /len
= 2838 2.1 Cluster Incl. AL031681:dJ862K6.2.2 (splicing factor,
arginine/serine-rich 6 (SRP55-2)(isoform 2)) /cds = (106,513) 2.1
Cluster Incl. X87838:H.sapiens mRNA for beta-catenin /cds =
(214,2559) / gb = X87838 /gi = 1154853 /ug = Hs.171271 2.2 Cluster
Incl. AW024285:wt69d06.x1 Homo sapiens cDNA, 3 end /clone = IMAGE-
2512715 /clone_end = 3 /gb = AW024285 2.2 Cluster Incl.
D38524:Human mRNA for 5-nucleotidase /cds = (83,1768) / gb = D38524
/gi = 633070 /ug = Hs.138593 2.2 Cluster Incl. L38935:Homo sapiens
GT212 mRNA /cds = UNKNOWN /gb = L38935 / gi = 1008845 /ug =
Hs.83086 /len = 1165 2.5 Cluster Incl. L12711:Homo sapiens
transketolase (tk) mRNA, complete cds / cds = (98,1969) /gb =
L12711 /gi = 388890 2.6 Cluster Incl. AF026029:Homo sapiens poly(A)
binding protein II (PABP2) gene, complete cds /cds = (1282,2202)
2.8 Cluster Incl. X70683:H.sapiens mRNA for SOX-4 protein /cds =
(350,1774) / gb = X70683 /gi = 36552 /ug = Hs.83484
[0403] Further Examples of Use
[0404] Examples of different receptor-intracellular signalling
pathways involved in different disease pathogenesis where
signalling molecules according to the invention find their use
are:
[0405] LPS stimulation of antigen presenting cells (like DC,
macrophages, monocytes) through different Toll-like receptors
activates different signalling pathways including, MAPK pathways,
ERK, JNK and p38 pathways. These pathways directly or indirectly
phosphorylate and activate various transcription factors, including
Elk-1, c-Jun, c-Fos, ATF-1, ATF-2, SRF, and CREB. In addition, LPS
activates the IKK pathway of MyD88, IRAK, and TRAF6. TAK1-TAB2 and
MEKK1-ECSIT complexes phosphorylate IKKb, which in turn
phosphorylates IkBs. Subsequent degradation of IkBs permits nuclear
translocation of NFkB/Rel complexes, such as p50/p65. Moreover, the
P13K-Akt pathway phosphorylates and activates p65 via an unknown
kinase. Some of these pathways could also be regulated by other
receptor signalling molecules such as hormones/growth factor
receptor tyrosine kinases (PKC/Ras/IRS pathway) and cytokine
receptors (JAK/STAT pathway). In the genomic experiment with the
T-cell line several of these genes appeared to be downregulated or
upregulated by the peptide used (LQGV). It is now clear that other
peptides in T cells and the same and other peptides in other cell
types similarly down-regulate or up-regulate several of these
transcription factors and signalling molecules. In DC and
fertilized eggs experiments NMPF had the ability to modulate growth
factor (GM-CSF, VEGF) and LPS signalling. Some diseases associated
with dysregulation of NF-kB and related transcription factors are:
Atherosclerosis, asthma, arthritis, anthrax, cachexia, cancer,
diabetes, euthyroid sick syndrome, AIDS, inflammatory bowel
disease, stroke, (sepsis) septic shock, inflammation,
neuropathological diseases, autoimmunity, thrombosis,
cardiovascular disease, psychological disease, post surgical
depression, wound healing, burn-wounds healing and
neurodegenerative disorders.
[0406] PKC plays an essential role in T cell activation via
stimulation of for example AP-1 and NF-kB that selectively
translocate to the T cell synapse via Vav/Rac pathway. PKC is
involved in a variety of immunological and non-immunological
diseases as is clear from standard text books of internal medicine
(examples are metabolic diseases, cancer, angiogenesis, immune
mediated disorders, diabetes etc.)
[0407] LPS and ceramide induce differential multimeric receptor
complexes, including CD14, CD11b, Fc-gRIII, CD36, TAPA, DAF and
TLR4. This signal transduction pathway explains the altered
function of monocytes in hypercholesterolemia and lipid
disorders.
[0408] Oxidized low-density lipoproteins contribute to stages of
the atherogenic process and certain concentrations of oxidized
low-density lipoproteins induce apoptosis in macrophages through
signal transduction pathways. These pathways are involved in
various vascular diseases such as atherosclerosis, thrombosis
etc.
[0409] Bacterial DNA is recognized by cells of the innate immune
system. This recognition requires endosomal maturation and leads to
activation of NF-kB and the MAPK pathway. Recently it has been
shown that signaling requires the Toll like receptor 9 (TLR9) and
the signalling adaptor protein MyD88. Recognition of dsRNA during
viral infection seems to be dependent on intracellular recognition
by the dsRNA dependent protein kinase PKR. TLRs play an essential
role in the immune system and they are important in bridging and
balancing innate immunity and adaptive immunity. Modulation of
these receptors or their down-stream signalling pathways are
important for the treatment of various immunological conditions
such as infections, cancer, immune-mediated diseases, autoimmunity,
certain metabolic diseases with immunological component, vascular
diseases, inflammatory diseases etc.
[0410] Effect of growth factor PDGF-AA on NF-kB and proinflammatory
cytokine expression in rheumatoid synoviocytes; PDGF-AA augmented
NF-kB activity and mRNA expression of IL-1b, IL-8 and MIP-1a.
Therefore, PDGF-AA may play an important role in progression of
inflammation as well as proliferation of synoviocytes in RA.
[0411] Dendritic cell (DC) activation is a critical event for the
induction of immune responses. DC activation induced by LPS can be
separated into two distinct processes: first, maturation, leading
to upregulation of MHC and costimulatory molecules, and second,
rescue from immediate apoptosis after withdrawal of growth factors
(survival). LPS induces NF-kB transcription factor. Inhibition of
NF-kB activation blocked maturation of DCs in terms of upregulation
of MHC and costimulatory molecules. In addition, LPS activates the
extracellular signal-regulated kinases (ERK), and specific
inhibition of MEK1, the kinase which activates ERK, abrogate the
ability of LPS to prevent apoptosis but do not inhibit DC
maturation or NF-kB nuclear translocation. This shows that ERK and
NF-kB regulate different aspects of LPS induced DC activation. Our
DC data and NF-kB data also show the various effects of NMPF
peptide on DC maturation and proliferation in the presence or
absence of LPS. NMPF peptides modulate these pathways and are novel
tools for the regulation of DC function and immunoregulation. This
opens new ways for the treatment of immune diseases, particularly
those in which the immune system is in disbalance (DC1-DC2,
Th1-Th2, regulatory cell etc.)
[0412] DC mediate NK cell activation which can result in tumour
growth inhibition. DC cells and other antigen presenting cells
(like macrophages, B-cells) play an essential role in the immune
system and they are also important in bridging and balancing innate
immunity and adaptive immunity. Modulation of these cells or their
down-stream signalling pathways are important for the treatment of
various immunological conditions such as infections, cancer,
immune-mediated diseases, autoimmunity, certain metabolic diseases
with immunological component, vascular diseases, inflammatory
diseases etc. There is also evidence in the literature that mast
cells play important roles in exerting the innate immunity by
releasing inflammatory cytokines and recruitment of neutrophils
after recognition of infectious agents through TLRs on mast
cells.
[0413] In murine macrophages infected with Mycobacterium
tuberculosis through JAK pathway activate STAT1 and activation of
STAT1 may be the main transcription factor involved in
IFN-g-induced MHC class II inhibition.
[0414] Recognition of mannose-binding lectin (MBL) through TLRs
influences multiple immune mechanisms in response to infection and
involved in innate immunity. Balance between innate and adoptive
immunity is crucial for balanced immune system and dysregulation in
immune system lead to different spectrum of diseases such as,
inflammatory diseases, autoimmunity, infectious diseases, pregnancy
associated diseases (like miscarriage and pre-eclampsia), diabetes,
atherosclerosis and other metabolic diseases.
[0415] Nuclear factor-kappaB (NFkappaB) is critical for the
transcription of multiple genes involved in myocardial
ischemia-reperfusion injury. Clinical and experimental studies have
shown that myocardial ischemia-reperfusion injury results in
activation of the TLRs and the complement system through both the
classical and the alternative pathway in myocardial infarction,
atherosclerosis, intestinal ischaemia, hemorrhagic shock pulmonary
injury, and cerebral infarction etc.
[0416] Peroxisome proliferator-activated receptors (PPARs) are
ligand-activated transcription factors which function as regulators
of lipid and lipoprotein metabolism, glucose homeostasis, influence
cellular proliferation, differentiation and apoptosis and
modulation of inflammatory responses. PPAR alpha is highly
expressed in liver, muscle, kidney and heart, where it stimulates
the beta-oxidative degradation of fatty acids. PPAR gamma is
predominantly expressed in intestine and adipose tissue, where it
triggers adipocyte differentiation and promotes lipid storage.
Recently, the expression of PPAR alpha and PPAR gamma was also
reported in cells of the vascular wall, such as
monocyte/macrophages, endothelial and smooth muscle cells. The
hypolipidemic fibrates and the antidiabetic glitazones are
synthetic ligands for PPAR alpha and PPAR gamma, respectively.
Furthermore, fatty acid-derivatives and eicosanoids are natural
PPAR ligands: PPAR alpha is activated by leukotriene B4, whereas
prostaglandin J2 is a PPAR gamma ligand, as well as of some
components of oxidized LDL, such as 9- and 13-HODE. These
observations suggested a potential role for PPARs not only in
metabolic but also in inflammation control and, by consequence, in
related diseases such as atherosclerosis. More recently, PPAR
activators were shown to inhibit the activation of inflammatory
response genes (such as IL-2, IL-6, IL-8, TNF alpha and
metalloproteases) by negatively interfering with the NF-kappa B,
STAT and AP-1 signalling pathways in cells of the vascular wall.
Furthermore, PPARs may also control lipid metabolism in the cells
of the atherosclerotic plaque. PPARs are also involved in a variety
of immunological and non-immunological diseases as is clear from
standard text books of internal medicine (examples are metabolic
diseases, cancer, angiogenesis, immune mediated disorders, diabetes
etc.)
[0417] As mentioned above the nuclear receptor PPARg is important
in adipogenesis, lipid storage and involved in atherosclerosis.
While expressed in adipose tissue this receptor is also expressed
in macrophages and in the colon. In addition, PPARg is implicated
in a number of processes such as cancer and inflammation. Moreover,
microbes, via its cognate receptors, typified by the TLRs, possess
the capacity to regulate PPARg dependent metabolic functions and as
such illustrates the intricate interplay between the microbial
flora and metabolic control in the alimentary tract.
[0418] Cyclo-oxygenase 2 (COX2), an inducible isoform of
prostaglandin H synthase, which mediates prostaglandin synthesis
during inflammation, and which is selectively overexpressed in
colon tumours, is thought to play an important role in colon
carcinogenesis. Induction of COX2 by inflammatory cytokines or
hypoxia-induced oxidative stress can be mediated by nuclear factor
kappa B (NF-kappaB). So, inhibition of NF-kB modulate COX pathway
and this inhibition of NF-kB can be therapeutically useful in
diseases in which COXs are involved, such as inflammation, pain,
cancer (especially colorectal cancer), inflammatory bowel disease
and others.
[0419] Neuronal subsets in normal brains constitutively express
functionally competent C5a receptors. The functional role of C5a
receptors revealed that C5a triggered rapid activation of protein
kinase C and activation and nuclear translocation of the NF-kappa B
transcription factor. In addition, C5a was found to be mitogenic
for undifferentiated human neuroblastoma cells, a novel action for
the C5aR. In contrast, C5a protects terminally differentiated human
neuroblastoma cells from toxicity mediated by the amyloid A beta
peptide. This shows that normal hippocampal neurons as well as
undifferentiated and differentiated human neuroblastoma cells
express functional C5a receptors. These results show the role of
neuronal C5aR receptors in normal neuronal development, neuronal
homeostasis, and neuroinflammatory conditions such as Alzheimer's
disease.
[0420] Activation of the complement system plays also an important
role in the pathogenesis of atherosclerosis. The proinflammatory
cytokine interleukin (IL)-6 is potentially involved in the
progression of the disease. Here the complement system induces IL-6
release from human vascular smooth-muscle cells (VSMC) by a
Gi-dependent pathway involving the generation of oxidative stress
and the activation of the redox sensitive transcription factors
NF-kB and AP-1. Modulation of complement system is important for
broad ranges of disorders such as blood disorders, infections, some
metabolic diseases (diabetes), vascular diseases, transplant
rejection and related disorders, autoimmune diseases, and other
immunological diseases.
[0421] Different transcription factors like NF-kB and intracellular
signaling molecules such as different kinases are also involved in
multiple drug resistance. So, it is reasonable to believe that NMPF
peptides will be effective against multiple drug resistance.
Moreover, our genomic data shows that a number of genes and
signalling molecules involved in tumorogenesis and metastasis are
modulated. In addition since oligopeptides have also effect on
angiogenesis, thus these peptides will also be used for the
treatment of cancer and related diseases whereby angiogenesis
requires modulation.
[0422] Proliferative diabetic retinopathy (PDR) is one of the major
causes of acquired blindness. The hallmark of PDR is
neovascularisation (NV), abnormal angiogenesis that may ultimately
cause severe vitreous cavity bleeding and/or retinal detachment.
Since NMPF peptides have angiogenesis stimulatory as well as
inhibitory effects and have the ability to modulate intracellular
signaling involved in growth factors (like insulin), pharmacologic
therapy with certain NMPF peptides can improve metabolic control
(like glucose) or blunt the biochemical consequences of
hyperglycaemia (through mechanisms such as in which aldose
reductase, protein kinase C (PKC), PPARs are involved). For this
metabolic control or diabetes (type 2) NMPF (LQGV, VLPALP, VLPALPQ,
GVLPALPQ, AQG, LAG, LQA, AQGV, VAPALP, VAPALPQ, VLPALPA, LPGC, MTR,
MTRV, LQG, CRGVNPVVS are recommended. The angiogenesis in PDR could
be also treated with above mentioned oligopeptides.
* * * * *