Hair loss abstract
The present invention pertains to a method for preventing and/or
treating epithelial tissue damage, such as is effected by inflammatory
reactions, ageing or cancer and/or to prevent and/or treat hair
loss. In particular, the present invention relates to substances
and/or compositions modifying, in particular blocking endogenous
CD.sub.1d function. According to another aspect the present invention
also provides a method for screening for compounds suitable for
use in the method and the composition of the present invention.
Hair loss claims
1. A substance capable to block or modify endogenous CD.sub.1d
function, obtainable by a process comprising the steps of: (a) exposing
epithehal cells to a substance of interest, (b) subjecting the epithelial
cells to a stress situation, (c) determining the effect of said
stress to said epithelial cells by screening for one or more of
the following assays, (i) epithelial hyperplasia (H&E), (ii)
epithelial proliferation (BrUd, PCNA), (iii) epithelial apoptosis
(TUNEL), (iv) p53 mutation accumulation, (v) quantitative and qualitative
assessment of epithelial lipids, (vi) co-clustering patterns of
apoptotic and non-apoptotic cell surface receptors, (vii) production
of pro-inflammatory cytokines, (viii) production of immuno-modulatory
cytokines, (ix) markers of inflammation, (x) anti-apoptotic transcription
factors, (xi) markers of ageing, (d) comparing the results obtained
with a control.
2. The substance according to claim 1, which is capable of preventing
and/or treating detrimental effects of stress to epithelial cells.
3. The substance according to claim 1, which is capable of preventing
or treating hair loss.
4. The substance according to any of the preceding claims, which
is a compound reducing the transcription and/or translation of the
CD.sub.1d gene.
5. The substance according to claim 4, which is a polynucleotide
antisense to a sequence comprised by the CD.sub.1d-gene and/or the
CD.sub.1d-mRNA.
6. The substance according to any of the claims 1 to 4, which is
a polynucleotide antisense to a sequence comprised by the glucosylceramide
synthase gene and/or the glucosyl-ceramide synthase mRNA.
7. The substance according to any of the claims 1 to 4, which is
a polynucleotide sense to a sequence comprised by the sphingomyelinase
or ceramide synthase gene and/or the sphingomyelinase or ceramide
synthase mRNA.
8. The substance according to any of the claims 1 to 4, which is
a polypeptide or peptide, binding to CD.sub.1d and essentially blocking
or modifying CD.sub.1d function.
9. The substance according to claim 8, wherein the polypeptide
is an antibody or the variable part of an antibody.
10. The substance according to any of the claims 1 to 4, which
is a lipid.
11. The substance according to claim 10, wherein the lipid is a
sphingolipid, glycosphingolipid, phospholipid, ganglioside, sterol,
fatty acid, glyceride or phosphatidylinositol phosphate.
12. The substance according to claim 10 and 11 which is derived
from plants, microbes or animals, or a phytochemicals, especially
a natural or synthetic polyphenols, or ingredients of green tee,
and a ginkolide, vitamin, amino acid or carotenoid.
13. The substance according to claim 8, which is a ceramide or
a ligand of a receptor belonging to the TNF-superfamily, in particular
CD95/APO-1/Fas.
14. The substance according to any of the preceding claims for
the preparation of a carrier for the prevention and/or treatment
of the detrimental effects of stress to epithelial cells and/or
hair loss.
15. A composition, containing at least a substance according to
any of the preceding claims.
16. A composition according to claim 15, which is a food composition,
a cosmetic composition or a pharmaceutical composition.
17. The composition according to claim 16, which is milk, yogurt,
curd, cheese, fermented milks, milk based fermented products, ice-creams,
milk based powders, infant formulae, cereal products, fermented
cereal based products, mineral water, chocolate or pet food, or
lotions, shampoos, creams, sun-screens, after-sun creams, anti-ageing
creams and/or ointments or tablets, liquid, dried oral supplement,
wet oral supplement, dry tube-feeding or wet tube-feeding or an
anti-cancer drug.
18. Use of a substance according to any of the claims 1 to 14 or
a composition according to any of the claims 13 to 15 for the prevention
and/or treatment of damages in epithelial tissues produced by a
stress situation and/or for the prevention and/or treatment of hair
loss.
19. The use according to claim 18, wherein the stress, situation
is a chemical stress, a biological stress or a physical stress.
20. The use according to any of the claims 19, wherein the chemical
stress is exerted by exposure to oxidants or carcinogens, or wherein
the biological stress is exerted by exposure to bacteria, viruses,
fungi, lipids derived from surrounding cells and/or microbes, or
wherein the physical stress is exerted by exposure to UV-irradiation.
21. The use according to any of the claims 18 to 20, wherein the
damage is skin burning and/or blistering, cataract formation, epidermal
hyperplasia, cancer, inflammation, immune suppression, skin ageing.
22. The use according to any of the claims 18 to 21, wherein the
epithelial cells are derived from the skin, gut, eye, lung, prostate,
liver, breast, kidney and/or the uterus.
23. The use according to claim 21, wherein the cancer is breast
cancer, colon cancer, prostate cancer, liver cancer, pancreatic
cancer, kidney cancer, non-melanoma and melanoma skin cancers.
24. A method for identifying CD.sub.1d blocking or modifying substances,
which comprises the following steps: (a) exposing epithelial cells
to a substance of interest, (b) subjecting the epithelial cells
to a stress situation, (c) determining the effect of said stress
to said epithelial cells by screening for one or more of the following
assays, (i) epithelial hyperplasia (H&E), (ii) epithelial proliferation
(BrUd, PCNA), (iii) epithelial apoptosis (TUNEL), (iv) p53 mutation
accumulation, (v) quantitative and qualitative assessment of epithelial
lipids, (vi) co-clustering patterns of apoptotic and non-apoptotic
cell surface receptors, (vii) production of pro-inflammatory cytokines,
(viii) production of immuno-modulatory cytokines, (ix) markers of
inflammation, (x) anti-apoptotic transcription factors, (xi) markers
of ageing, (d) comparing the results obtained with a control.
25. The method according to claim 24, wherein the stress situation
is a chemical stress, a biological stress or a physical stress.
26. The method according to claim 25, wherein the chemical stress
is exerted by exposure to oxidants or carcinogens, or wherein the
biological stress is exerted by exposure to bacteria, viruses, fungi,
lipids derived from surrounding cells and/or microbes, or wherein
the physical stress is exerted by exposure to UV-irradiation.
27. The method according to claim 24 to 26, wherein the pro-inflammatory
cytokines are selected from the group consisting of IL-1, TNF-.alpha.,
PGE-2, IL-6, IFN-.gamma. or IL-8.
28. The method according to any of the claims 24 to 26, wherein
the immuno-modulatory cytokines are selected from the group consisting
of PAF, IL-10, IL-4 or TGF-13.
29. The method according to any of the claims 24 to 26, wherein
the lipids are selected from the group consisting of phospholipids,
sphingolipids and glycosphingolipids.
30. The method according to any of the claims 24 to 26, wherein
the markers of inflammation include COX-2 and iNos.
31. The method according to any of the claims 24 to 26, wherein
the anti-apoptotic transcription factors include AP-1 and NFkappaB.
32. The method according to any of the claims 24 to 26, wherein
the markers of aging include elastases, collagenases, metalloproteinases,
gelatinases, stromelysins, telomerases.
33. Use of a substance according to any of the claims 1 to 14 or
a composition according to any of the claims 15 to 17 for decreasing
multi-drug resistance of cancers.
34. The use according to claim 33, wherein the cancer is skin,
gut or breast cancer.
35. Use of cells expressing and/or over-expressing CD.sub.1d in
an assay for screening for substances modifying and/or blocking
CD.sub.1d function.
36. Use of CD.sub.1d.sup.-/- animals as a test model for determining
the activity of substances influencing damages in epithelial tissues
produced by a stress situation and/or hair loss.
37. Use of a substance according to any of the claims 1 to 14 in
gene therapy.
Hair loss description
BACKGROUND OF THE INVENTION
[0001] The present invention pertains to a method for preventing
and/or treating epithelial tissue damage, such as is effected by
inflammatory reactions, ageing or cancer and/or to prevent and/or
treat hair loss. In particular, the present invention relates to
substances and/or compositions modifying, in particular blocking
endogenous CD.sub.1d function. According to another aspect the present
invention also provides a method for screening for compounds suitable
for use in the method and the composition of the present invention.
SUMMARY OF THE INVENTION
[0002] The most prominent epithelial tissue in living beings is
the skin, which represents the largest organ in the organism. The
system of skin integument, which comprises the epidermis, dermis
and the stratum cornium, correlates with those of internal organs
and concurrently interacts with the surroundings. Being the interface
between the environment and organism itself, the skin is heavily
influenced by external factors and also variable parameters of the
organism's inner system. The skin's regulative mechanisms need,
therefore, always be active to induce systemic changes necessary
to maintain normal pathological events concerning skin integument
morphology and activities. A great deal of processes assuring the
adequate consumption of increased affluence of energetic and plastic
substances according to the skin's needs become guarantors of morphological
and functional stability of skin structures. So, the state of integuments
determines the realization of metabolic processes necessary for
skin cell viability and activity leading to the presence of healthy
skin peculiarities such as barrier function, elasticity, turgor
properties, humidity, pigmentation etc.
[0003] During the lifetime of a living being different signs, characteristic
of ageing, appear on the skin, with the principal clinical signs
being the appearance of fine lines and deep wrinkles which increase
or are accentuated with age. Moreover, the skin's complexion is
generally modified and diffuse irritations and occasionally telangiectasias
may come into existence on certain areas.
[0004] These signs of ageing are even promoted by exposure of the
skin to exogenous influences, such as e.g. UV-radiation, pollutants,
free radicals or chemical substances.
[0005] Moderate UV exposure generally causes the well known effects
of reddening the skin with an accompanying inflammation reaction,
known as erythema. This phenomenon, often referred to as "sunburn",
is painful and commonly results in a subsequent peeling of the skin.
[0006] Moreover, excessive UV-exposure of the skin may also lead
to the onset of severe disorders, such as carcinogenesis, the most
common tumours being the basal cell carcinoma (BCC), followed by
squamous cell carcinoma (SCC), and more rarely malignant melanoma.
Apart from damages on the DNA-level also immuno-suppression caused
by UV exposure seems to account for both, non-melanoma and melanoma
cancer promotion. It is presently acknowledged that photo-induced
immuno-suppression permits the initiated tumour cell to evade recognition
and rejection by normal immunological mechanisms, to remain latent
for extended periods, and to eventually proliferate into a tumour.
This concept concurs with the findings that immuno-compromised patients,
whether genetically (xeroderma pigmentosum) or pharmacologically,
such as e.g. organ transplant recipients, have a higher incidence
of skin cancer as compared to people with a properly functioning
immune system.
[0007] In the art several means have been proposed to prevent destructive
effects of environmental factors on epithelial cells, in particular
skin epithelial cells.
[0008] As regards protection to sun radiation "sun blocks"
or "sunscreens" have been made available, which are applied
to the skin prior to sun exposure. Typically, sunscreen compositions
contain chemical agents, such as certain benzophenones, dibenzylmethanes
or substituted paraaminobenzoates, i.e. compounds absorbing ultraviolet
radiation, so that it cannot penetrate the skin. However, some of
the compounds used for this purpose have shown to lack sufficient
light stability and may even become toxic over long term application.
In addition, they must stay continuously on the surface of the skin
at the time of exposure to be effective. However, sunscreens are
easily rubbed off or washed off by sweating or swimming and can
also be lost by penetration into the skin.
[0009] Another means to prevent skin deterioration or ageing, respectively,
is to provide compounds scavenging free radicals. In this respect
EP 0 761 214 discloses singlet oxygen quenchers comprising aniline
derivatives and difurfuryl amine derivatives, which are reported
to reduce the oxidative stress to the skin.
[0010] Yet, all these means and methods are not sufficiently capable
to protect the skin from the growing challenge in our environment.
To this contributes an increased atmospheric pollution and also
social behaviour, according to which sun-tan is associated with
health, beauty and status. As a consequence many people expose their
skin to sun radiation to acquire a tan in spite of the negative
results accompanying such behaviour being well known. This problem
even gets more prominent with the ozone shield covering the earth
becoming thinner, resulting in a heavier exposure of living beings
to UV radiation.
[0011] Consequently there is a need in the art to provide a better
protection of the skin to environmental factors, such as stress
or sun radiation.
[0012] Accordingly, an object of the present invention is to obviate
the drawbacks of the prior art and to provide such means in order
to protect the skin from unfavourable influences encountered in
the environment, in particular from oxidative or chemical stress
or sun radiation.
[0013] This problem has been solved by providing a substance, that
is capable to essentially modify, in particular block the endogenous
CD.sub.1d function in epithelial cells.
[0014] Additional features and advantages of the present invention
are described in, and will be apparent from, the following Detailed
Description of the Invention and the figures.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1A. Wild-type mice exhibit skin damage (burning) following
exposure to a single dose (86 mJ/m.sup.2) of UVB radiation.
[0016] FIG. 1B. Wild-type mice exhibit skin damage (burning) following
exposure to a single dose (86 mJ/m.sup.2) of UVB radiation. (Close-up).
[0017] FIG. 1C. CD1d knockout mice show no obvious signs of skin
damage following exposure to a single dose (86 mJ/m.sup.2) of UVB
radiation.
[0018] FIG. 1D. CD1d knockout mice show no obvious signs of skin
damage following exposure to a single dose (86 mJ/m.sup.2) of UVB
radiation. (Close-up).
[0019] FIG. 2. Difference in degree of UVB-induced skin damage
between wild-type (Right) and CD.sub.1d knockout (Left) mice exposed
to two doses (86 mJ/m.sup.2) of UVB radiation.
[0020] FIG. 2A. Damaged (lesions) dorsal skin of wild-type mice
exposed to two doses (86 mJ/m.sup.2) of UVB radiation (Close-up).
[0021] FIG. 2B. Undamaged dorsal skin of CD.sub.1d knockout mice
exposed to two doses (86 mJ/m.sup.2) of UVB radiation.
[0022] FIG. 3. CD.sub.1d knockout mice exhibit increased epidermal
apoptosis in their dorsal epidermis compared to wild-type mice,
as measured by TUNEL. Wild-type (A) and CD1d knockout (B) mouse
skin not exposed to UV-irradiation. Wild-type (C) and CD.sub.1d
knockout (D) mouse skin 48 h after a single exposure (86 mJ/m.sup.2)
to UV-B radiation.
[0023] FIG. 4 a and b are graphs indicating the approximate amount
of CD.sub.1d in different tissues in mice and human.
[0024] FIG. 5 shows that CD.sub.1d protein is expressed in the
epidermis of mouse skin 72 h following exposure to a single dose
(430 mJ/cm.sup.2) of UVB radiation;
[0025] FIG. 6 shows that murine skin CD.sub.1d gene transcription
is regulated following UVB irradiation;
[0026] FIG. 7 shows that murine skin CD.sub.1d gene transcription
is regulated following solar simulated light irradiation;
[0027] FIG. 8 shows that CD.sub.1d gene transcription in immortalized
(DK7) human keratinocytes is regulated following solar UV irradiation;
[0028] FIG. 9. shows that COX-2 and TNF-alpha mRNA levels are down-regulated
in UVB-irradiated CD.sub.1d knockout mouse skin.
[0029] FIG. 10 a and b) show that mouse skin IL-6 and MIP1-alpha
protein levels 48 h after UVB irradiation are significantly decreased
in CD.sub.1d KO mice.
[0030] FIG. 11 shows that hydrocortisone suppresses CD.sub.1d transcription
in cells exposed to a chemical stress; and
[0031] FIG. 12 shows that CD.sub.1d is expressed in human hair
follicles.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention is essentially based on the finding
that CD.sub.1d, a transmembrane protein expressed by a number of
different cells, in particular epithelial cells, modulates a variety
of different responses of the cell to stress. As will become evident
from the following detailed description of the preferred embodiments,
essentially modifying, specifically blocking the endogenous CD.sub.1d
function in cells bearing said membrane molecule allows to prevent
the detrimental effects of stress, including ultraviolet radiation-induced
skin damage, e.g. as a result of burning, epidermal hyperplasia,
mutant p53 accumulation, inflammation, immune suppression and skin
ageing. Even more surprising is the finding that when essentially
blocking CD.sub.1d function in epithelial cells induction of cancer
in said cells, i.e. basal cell carcinoma, squamous cell carcinoma,
malignant melanoma, colon, breast, liver, prostate, kidney, pancreas
cancer etc., may be prevented. In addition, it has been surprisingly
found that modifying, in particular blocking CD.sub.1d function
influences hair growth and/or development.
[0033] CD.sub.1d as such is a type 1 transmembrane MHC class 1
like protein that non-covalently associates with .beta..sub.2-microglobulin.
The CD.sub.1d molecule is recognized by a T-cell receptor of natural
killer T-cells (NKT) which play a role in immune modulatory and
effectory reactions. It has been demonstrated that CD.sub.1d may
present lipids to NKT cells for their activation, which notion is
supported by the CD.sub.1d crystal structure having two highly hydrophobic
grooves, necessary for presenting hydrophobic molecules such as
lipids to the immune system.
[0034] In the studies leading to the present invention it has surprisingly
been noted that CD.sub.1d gene transcription in mouse skin is responsive
to external stress, such as UV radiation, which finding has been
confirmed in human keratinocytes. In addition it has been noted
that skin CD.sub.1d mediates UV-induced skin damage/inflammation
by inducing COX-2 and TNF-.alpha. gene transcription and also inhibiting
UV-induced apoptosis.
[0035] Without wishing to be bound to any theory it is currently
assumed that one of the endogenous tasks of CD.sub.1d in living
organisms is to directly control normal epithelial cell homeostasis.
Normal skin homeostasis is dependent on the critical and fine tuned
balance between epidermal differentiation, apoptosis, proliferation
and anti-apoptosis of epidermal cells. In the skin, these processes
are regulated via lipids, in particular by means of ceramides and
glucosylceramides (sphingolipids). While the nucleated cell layers
generate glucosylceramides (GlcCer), the proportions of GlcCer to
Cer decrease late in epidermal differentiation, with the Cer content
peaking in the stratum corneum acting as extracellular constituents
of the epidermal permeability barrier. In addition to their structural
properties, ceramides are associated with inhibition of cellular
proliferation, induction of cellular differentiation and programmed
cell death. In contrast, GlcCer induce cell proliferation and inhibit
programmed cell death.
[0036] Based on the findings in the present invention, CD.sub.1d
appears to be one of the receptors via which the above mentioned
lipids might fulfill their biological task. Specifically, CD.sub.1d
seems to negatively regulate apoptosis. In consequence, in cells
under a stress situation, e.g. when exposed to UV-radiation, CD.sub.1d
supports a continued existence of said stressed cells, even when
their genetic material is damaged and/or mutated, which damaged
cells will contribute to inflammation processes induced and eventually
account for the phenomenon of ageing or eventually tumour development.
[0037] In blocking and/or modifying endogenous CD.sub.1d function,
apoptosis of cells under stress may be promoted, instead of their
survival and propagation, with the effect that cells that have been
damaged to a certain extent, particularly at the DNA level, do not
have the chance to proliferate and in case disseminate in the body.
The cells once dead will then be extinguished by natural processes
in the body and be replaced by "healthy" epithelial cells.
Likewise, by means of blocking or modifying CD.sub.1d also an interaction
with NKT is substantially prevented or altered, wherein the phenomenon
of immune suppression during exposure to UV radiation will be essentially
reduced or barred at all. Also, this condition is supposed to assist
the organism's immune system to eradicate damaged cells, brought
about by exposure to UV.
[0038] The substance capable of blocking and/or modifying the CD.sub.1d
transmembrane molecule's activity may be any substance interfering
with the endogenous biological function of CD.sub.1d, and in particular
preventing or reducing association of CD.sub.1d with endogenous
or exogenous lipids. The substances are obtainable by a process
comprising the steps of (a) exposing epithelial cells to a substance
of interest, (b) subjecting the epithelial cells to a stress situation,
(c) determining the effect of said stress to said epithelial cells
by screening for one or more of the following assays: (i) epithelial
hyperplasia (H&E), (ii) epithelial proliferation (BrUd, PCNA),
(iii) epithelial apoptosis, (iv) p53 mutation accumulation, (v)
quantitative and qualitative assessment of epithelial lipids, (vi)
co-clustering patterns of apoptotic and non-apoptotic cell surface
receptors, (vii) production of pro-inflammatory cytokines, (viii)
production of immuno-modulatory cytokines, (ix) markers of inflammation,
(x) anti-apoptotic transcription factor activity (xi) markers of
ageing, and (d) comparing the results obtained with a control. Such
a control may e.g. be an assay, wherein the cells have been subjected
to the same stress situation, wherein, however, no substance to
be investigated had been added (negative control). Likewise a control
may also be, including a substance with a known positive effect
in the assay and determining the difference in effect achieved by
the substance investigated and the known substance (positive control).
[0039] A substance is considered to be active in the context of
this application, in case it prevents the negative effects of stress
as detailed according to any of the above assays.
[0040] It will be appreciated that CD.sub.1d activity may be blocked
and/or modified by substances acting on the genetic level or at
the protein level.
[0041] Substances acting on the genetic level are compounds influencing,
in particular preventing transcription or translation of the CD.sub.1d
gene, such as polynucleotides anti-sense to at least a part of the
CD.sub.1d gene or the CD.sub.1d-mRNA.
[0042] The terms oligonucleotide and polynucleotide, which are
interchangeably used herein, include linear oligomers/polymers of
natural or modified monomers or linkages, including desoxyribonucleosides,
ribonucleosides, .alpha.-anomeric forms thereof, polyamide nucleic
acids, and the like, capable of specifically binding to the target
nucleic acid by way of a regular pattern of monomer-to-monomer interactions,
such as Watson-Crick type of base pairing, Hoogsteen or reverse
Hoogsteen types of base pairing, or the like. Usually the monomers
are linked by phosphodiester bonds or analogs thereof to form oligonucleotides
ranging in size from a few monomeric units, e.g. 3-5, to several
100 or even thousands of monomeric units.
[0043] The (anti-)sense oligo-/polynucleotides may also contain
pendent groups or moieties, to enhance specificity, nuclease resistance,
delivery, or other property related to efficacy, such as e.g. cholesterol
moieties, duplex intercalators such as acridine, poly-L-lysine,
"end capping" with one or more nuclease-resistant linkage
groups such as phosphorothioate, and the like. The corresponding
oligonucleotide may be used for blocking transcription, RNA processing
and/or translation of the mRNA, Consequently, the oligonucleotide
may comprise exon, but also intron sequences of the CD.sub.1d-target
gene, as desired.
[0044] The nucleotide sequence of the human CD.sub.1d gene or mRNA
is obtainable from NCBI (Accession numbers: AP002532 and NM.sub.--001766,
respectively). Based on his general knowledge and skill, the skilled
person may select at least a portion of the coding region of the
CD.sub.1d gene and design an appropriate anti-sense polynucleotide,
that prevents transcription and/or translation of the CD.sub.1d
gene. Likewise, also a part of the non-coding region of the CD.sub.1d
gene may serve as an agent for preventing transcription or reducing
the number of transcripts, respectively, of the CD.sub.1d gene.
Here, in particular parts of the promotor region may serve as a
template for preparing an antisense polynucleotide, but likewise
transitions regions from introns and exons and vice versa. According
to a preferred embodiment such a substance may be an DNA or a cRNA
(RNA-interference).
[0045] Yet, apart from the CD.sub.1d gene being the target, also
the activity of a number of regulatory molecules which control epithelial
homeostasis such as ceramides and/or glucosylceramides, may be modified
such, that they exert the desired effect on the CD.sub.1d molecule.
To this end, the number of the glucosylceramide synthase transcripts
may be reduced by designing an polynucleotide antisense to at least
a part of the glucosylceramide synthase gene or glucosylceramide
synthase mRNA, so that eventually the signal to epithelial cells
to proliferate is turned down. The nucleotide sequence of the glucosylceramide
synthase gene is disclosed in Ichikawa et al., PNAS 93 (1996), 4638-4643,
which document is incorporated herein by way of reference. Likewise,
non coding regions may serve as a template for the antisense polynucleotide,
such as the promotor region and/or transitions from introns to exons
and vice versa. According to a preferred embodiment such a substance
may be a DNA or a cRNA (RNA-interference).
[0046] Apart from reducing the proliferation signal also the signal
driving epithelial cells to apoptosis via the CD.sub.1d molecule
may be enhanced. In this respect the number of corresponding transcripts
may be increased, which may be effected by providing a higher number
of polynucleotides encoding a sequence comprised by the sphingomyelinase
or ceramide synthase gene and/or the sphingomyelinase or ceramide
synthase mRNA.
[0047] Apart from the genetic level, the biological activity of
the CD.sub.1d molecule may also be modified, in particular blocked
at the protein level, in particular by any substance binding to
the CD.sub.1d receptor on or in epithelial cells and blocking the
endogenous biological functionality thereof.
[0048] According to a preferred embodiment the substance capable
of modifying, in particular blocking biological CD.sub.1d function
is a polypeptide or a peptide, in particular hydrophobic peptides,
more preferably an antibody, or a part thereof, that binds to the
CD.sub.1d receptor and blocks its biological function, such as the
interaction with NKT. As parts thereof, in particular mini-antibodies
are envisaged lacking the F.sub.c-part. According to an alternative
embodiment the substance capable of blocking the biological CD.sub.1d
function may also be a soluble CD.sub.1d receptor, that is, that
part of the polypeptide lacking the region, anchoring the polypeptide
in the membrane. The soluble CD.sub.1d receptor will scavenge the
in vivo ligands that promote survival of the stressed cells, thus
promoting apoptosis. In addition, binding of the natural killer
cells to CD.sub.1d in vivo will be reduced, thus preventing activation
of the T-cells and consequently inflammatory and/or immunosuppressive
reactions.
[0049] According to a preferred embodiment the substance capable
of blocking and/or modifying biological CD.sub.1d function is a
lipid derived from a plant, microbe or animal, including a phospholipid,
ganglioside, sphingolipid, glycosphingolipid, phosphatidylinositol
phosphate, sterol, polyphenol, glyceride or fatty acid. These lipids
may influence CD.sub.1d function by directly binding the CD.sub.1d
molecule or indirectly by influencing CD.sub.1d gene expression.
[0050] According to an alternative embodiment the substance capable
of blocking and/or modifying biological CD.sub.1d function is a
ceramide, such as ceramide 8 or sphingosine phosphocholine or a
ligand of a receptor belonging to the TNF-superfamily, in particular
CD95/APO-1/Fas, which induces apoptosis thus interfering with the
anti-apoptotic function of CD.sub.1d. In another embodiment the
objective substance is an organic compound obtained by chemical
synthesis.
[0051] It is well established that ceramide glycosylation, via
glucosylceramide synthase, and the subsequent build up of glucosylceramides
allows cellular escape from stress-induced programmed cell death,
conferring cancer cell resistance of a variety of cancers including
breast, skin, colon and epitheliod carcinomas, to cytotoxic anti-cancer
agents. As CD.sub.1d can bind glucosylceramide and is over-expressed
by the same multi-drug-resistant cancer cells (e.g squamous cell
carcinoma), it is envisioned that the anti-apoptotic activity of
CD.sub.1d regulates cancer cell resistance to cytotoxic drugs, possibly
at the level of protein-glucosylceramide binding. Thus, in principle
the substances of the present invention that block and/or modify
endogenous CD.sub.1d function strongly decrease multi-drug resistance
of a variety of cancers including skin, gut and breast cancers.
[0052] In principle, the substances of the present invention may
also influence the bi-directional trafficking of CD.sub.1d to and
from the membrane.
[0053] The substances may be included in any composition suitable
for administering the substance to an individual, in particular
a food composition, a cosmetic composition or a pharmaceutical composition.
[0054] The pharmaceutical compositions containing at least one
of the substances capable of blocking or modifying the CD.sub.1d
surface molecule according to the invention can be administered
for prophylactic and/or therapeutic treatments. In therapeutic applications,
compositions are administered to a patient already suffering from
a disease, as described herein under, in an amount sufficient to
cure or at least partially arrest the symptoms of the disease and
its complications. An amount adequate to accomplish this is defined
as "a therapeutically effective dose". Amounts effective
for this will depend on the severity of the disease and the weight
and general state of the patient.
[0055] In prophylactic applications, compositions containing at
least one of the substances capable of blocking or modifying the
CD.sub.1d surface molecule according to the invention are administered
to a patient susceptible to or otherwise at risk of a particular
disease. Such an amount is defined to be "a prophylactic effective
dose". In this use, the precise amounts again depend on the
patient's state of health and weight.
[0056] The compounds of the invention are preferably administered
with a pharmaceutical acceptable carrier, the nature of the carrier
differing with the mode of administration, for example parenteral,
intravenous, oral and topical (including ophthalmic) routes.
[0057] The desired formulation can be made using a variety of excipients
including, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin cellulose, magnesium
carbonate. This composition may be a tablet, a capsule, a pill,
a solution, a suspension, a syrup, a dried oral supplement, a wet
oral supplement, dry tube-feeding, wet tube-feeding etc. In order
to control the drug release, sustained-release formulations can
also be used.
[0058] The kind of the carrier/excipient and the amount thereof
will depend on the nature of the substance and the mode of drug
delivery and/or administration contemplated. E.g., for formulations
containing weakly soluble antisense oligonucleotides, micro-emulsions
may be employed, for example by using a non-ionic surfactant such
as Tween 80 in an amount of about 0.04-0.05% (w/v), to increase
solubility. Other components may include antioxidants, such as ascorbic
acid, hydrophilic polymers, such as, monosaccharides, disaccharides,
and other carbohydrates including cellulose or its derivatives,
dextrins, chelating agents, such as EDTA, and like components well
known to those in the pharmaceutical sciences. These various components
utilized provide a variety of functions, including regulation of
drug concentration, regulation of solubility, chemical stabilization,
regulation of viscosity, absorption enhancement, regulation of pH,
and the like. For example, in water soluble formulations the pharmaceutical
composition preferably includes a buffer such as a phosphate buffer,
or other organic acid salts, preferably at a pH of between about
7 and 8.
[0059] It will be appreciated that the skilled person will, based
on his own knowledge select the appropriate components and galenic
form to target the active compound to the tissue of interest, e.g.
the colon, stomach, skin, kidney or liver, taking into account the
route of administration which may be by way of injection, topical
application, intranasal administration, administration by implanted
or transdermal sustained release systems, and the like.
[0060] The objective substance may also be formulated in a cosmetic
product, such as lotions, shampoos, creams, sun-screens, after-sun
creams, sun-blocker, anti-ageing creams, ointments and/or anti-hair
loss liquids. This proves in particular advantageous for essentially
blocking CD.sub.1d function in the skin and to prevent the adverse
effect of sun radiation, photo-ageing and exposure of the skin to
free radicals. Thus, e.g. by providing a sun-screen containing in
addition to a common agent, absorbing UV-light a substance as defined
herein, a protection to the sun may be provided, which by far exceeds
anything known so far. This feature is based in particular on the
fact that the objective substance will penetrate the skin and exert
its effect after having reached the target molecules. Since this
effect will stay for a while, protection to the sun will even be
present in case the sun-screen has been rubbed off or has been washed
off, as e.g. during sport etc. Yet, apart from sun-screens the objective
substances may be included in common day-creams, lotions etc. to
prevent negative effects of the daily environment, including pollution,
oxidative stress etc. It will be appreciated that the present cosmetic
products will contain a mixture of different ingredients known to
the skilled person, ensuring a fast penetration of the objective
substance into the skin and preventing degradation thereof during
storage.
[0061] Another high important composition according to the present
invention is food material. In our present society a great deal
of food is ingested, such as sausages, salted or grilled meat etc.,
that contains preservatives, ingredients or substances, that are
injurious to the gut. E.g. grilled meat contains aliphatic and aromatic
compounds known to be cancerogenic. Also preservatives, that kill
micro-organisms contained in food material (e.g. sausages) by manipulating
their DNA, will exert a similar effect to cells of the gut. In fact,
the number of intestinal cancer is steadily increasing in our society,
which may be attributed at least in part to the type of food taken
by humans.
[0062] Consequently, the present invention provides a food composition
that prevents the onset and/or development of such gut disorders,
such as a composition selected from the group consisting of milk,
or fermented milk products, such as e.g. yogurt, curd, cheese, milk
based fermented products, ice-creams, milk based powders, infant
formulae, cereal products and fermented cereal based products, mineral
water, chocolate or pet food containing at least a substance capable
of essentially blocking and/or modifying CD.sub.1d function. Since
the objective compound will be contained in a food material in amounts,
that do not affect the original taste thereof, the consumer will
not notice any change in the product, but will experience the beneficial
effects thereof, namely a protective or even curing effect. Once
the food material has been ingested the objective substances will
arrive at the target cells, which may be epithelial cells of the
gut, i.e. of the stomach or the intestine, and will bind to the
CD.sub.1d receptor and exert its activity. As a consequence, cells,
that are already damaged will preferably go to apoptosis instead
of being maintained in said damaged form.
[0063] Since epithelial cells bearing CD.sub.1d have been found
in a number of organs, such as the liver, the small intestine, the
colon, the kidney, the prostate, the uterus, the pancreas, breast,
skin and conjunctiva, the choice of the composition as detailed
above will, by and large depend on the target tissue. As will be
understood, for skin a cosmetic product might be the composition
of choice, while in case of delivering the objective substance directly
to the gut or the colon, a food product may be first choice. However,
a food product may also be suitable for delivering the objective
substance or substances to other organs, such as the kidney or the
liver, which will depend on the stability of the substance in the
body and its capacity of being absorbed by the body in the gut.
Since food is a daily ingested material such a product offers a
great variety of different possibilities. Yet, in case the objective
substance is prone to degradation in the gut a pharmaceutical composition
may be selected, providing e.g. encapsulation or other galenic forms
to deliver the objective substance to the target tissue/to target
cells.
[0064] It will be understood that the concept of the present invention
may likewise be applied as an adjuvant therapy assisting in presently
used medications. In this respect the pharmaceutical composition
of the present invention may be administered together with e.g.
cytostatika so as to prevent escape of the tumor treated from the
treatment, which sometimes occurs in long term treatments of certain
tumors or to assist in killing residual cancer cells not captured
with the pharmaceutical regimen. Since the substance(s) of the present
invention may easily be administered together with food material
special clinical food may be applied containing a high amount of
the objective substances. Also melanoma may be directly treated
with an antibody medication against melanoma together with a pharmaceutical
composition or a cosmetic product as described herein. It will be
clear that on reading the present specification together with the
appending claims the skilled person will envisage a variety of different
alternatives to the specific embodiments mentioned herein.
[0065] In principle, the substances according to the present invention
may be used for the treatment and/or prevention of damages in epithelial
tissues, such as e.g. in the skin, gut, eye, lung, liver, prostate,
breast, kidney and/or in the uterus, which are produced by a stress
situation, e.g. by means of a chemical, biological or a physical
stress, e.g. by exposure to oxidants or carcinogens, exposure to
bacteria, viruses, fungi, lipids derived from surrounding cells
and/or microbes, or exposure to UV-irradiation. Likewise, the substances
may be utilized for preventing and/or treating hair loss.
[0066] Consequently, the substances and/or compositions according
to the present invention may be utilized for treating and or preventing
damages of the skin, in particular actinic and ageing damages of
the skin such as dryness, actinic keratoses, irregular pigmentation
(notably comprising freckling, lentigines, guttate hypomelanosis
and persistent hyperpigmentation), wrinckling (notably comprising
fine surface lines and deep furrows), stellate pseudoscars, elastosis,
inelasticity, telangiectasia, venous lakes, purpura, comedones,
sebaceous hyperplasia, acrochordon, cherry angiogema, seborrhea
keratosis, lentigo, basal cell carcinoma and squamous cell carcinoma,
skin burning and/or blistering, cataract formation, epidermal hyperplasia,
inflammation, immune suppression, and cancer, e.g. non-melanoma
and melanoma skin cancers.
[0067] In order to arrive at additional substances having the above
characteristics the present invention also provides a method for
screening for such substances. In this method epithelial cells are
utilized that may be in the form of a primary culture, i.e. directly
derived from an individual or in the form of a cell line. For carrying
out the method a cell culture is particularly preferred, since it
allows for the continuous supply of epithelial cells during the
experiments. Care must be taken that the cell culture of epithelial
cells used exhibit the same phenotypic traits as do cells of a primary
culture or epithelial cells directly obtained from a tissue sample.
It will be understood that the person skilled in the art will select
the starting material depending on the assay. Hence, if a first
round assay is to be carried out a cell culture design seems to
be most appropriate, while in case for further rounds, i.e. assessing
the activity of potential candidates, the tissue or even the animal
model seems to be more appropriate.
[0068] The epithelial cells are exposed to a substance of interest
for a time period sufficient to ensure a contact of the substance
with the cells. In a next step the epithelial cells are exposed
to a stress situation, which may be effected e.g. by irradiating
the cells with different dosages of UV light, or adding hydrogen
peroxide or toxic chemicals to the cell culture. However, the type
of stress is not critical as long as the cells are challenged to
initiate processes, normally started under stress situations, such
as e.g. the production of pro-inflammatory cytokines e.g. IFN-g,
TNF-a, IL-1, IL-6, IL-8, apoptosis, altered lipid metabolism, increased
production of p53, altered cell signaling as a result of altered
patterns of cell surface receptor co-clustering, NF-.kappa.B activation,
AP1 activation, showing hyperproliferation (anti-apoptosis), altered
barrier function etc. It will be understood that also more than
one substance may be tested at the same time, that is a cocktail
of one or more substances, which might prove beneficial for the
second or further round of assaying.
[0069] In a next step the effect of said stress on the epithelial
cells is determined by assessing one or more of the following features,
for example: epithelial proliferation (PCNA: Ouhtit et al., American
Journal of Pathology [2000], 156: 201-207; BrUd: Lu Y-P et al.,
Cancer Research [1999], 59: 4591-4602); epithelial apoptosis (Tunel
Assay; modification of protocol outlined by Ouhtit et al., American
Journal of Pathology [2000], 156: 201-207); p53 mutation accumulation
(Allele-specific polymerase chain reaction [AS-PCR] and single-strand
conformation polymorphism [SSCP], Ananthaswamy et al., Nature Medicine
[1997], 3: 510-514); production of pro-inflammatory and immuno-modulatory
cytokines (e.g. TNF-.alpha., PGE-2, IL-1, IL-6, IL-8, IL-4, IL-10,
Platelet Activating Factor, TGF.beta.); markers of inflammation
(e.g. COX-2, iNos); and anti-apoptotic transcription factor (including
AP-1, NFkappaB) activity by TaqMan Real-time RT-PCR, ELISA, and
Immunohistochemistry; qualitative and quantitative assessment of
phospholipids, glycosphingolipid and sphingolipid content (Electron-Spray
Tandem Mass Spectrometry); analysis of co-clustering patterns of
epithelial cell surface receptor molecules including cytokine receptors
(e.g. IL-6), molecules of the TNF-superfamily of receptors (e.g.
CD95/APO-1/Fas) and growth regulating receptors (e.g. EGF, Insulin)
by fluorescence resonance electron transfer analysis (FRET); markers
of ageing, e.g. elastases, collagenases, metalloproteinases, gelatinase,
stromelysins, telomerase.
[0070] The results obtained are then compared with a control, which
may simply be an assay, wherein the same type of cells are exposed
to the same stress conditions with the proviso, that no compound
to be assessed for its CD.sub.1d blocking capacity is provided.
As for the animal model a positive control is represented by a CD.sub.1d.sup.-/-
animal, wherein CD.sub.1d activity is lacking at all.
[0071] The following examples illustrate the invention in more
detail without restricting the same thereto.
EXAMPLE 1
[0072] Generation of CD.sub.1d Mutant Mice
[0073] Mouse CD.sub.1d is encoded by two genes, CD.sub.1d1 and
CD.sub.1d2, that share a high degree of nucleotide sequence identity
(Bradbury et al., EMBO J., 7 (1988), 3081-3086). The product of
the CD.sub.1d1 gene is recognized by all anti-CD.sub.1 antibodies
that have been described, whereas surface expression of the CD.sub.1d2
product has not yet been demonstrated. In addition, the predicted
.alpha.2 domain of the CD.sub.1d2 gene product lacks an intra-domain
disulfide bond that is found in the .alpha.2 domain of all published
classic and non-classic MHC class I molecules (Bradbury, supra).
This disulphide bond is thought to be critical for the folding of
the antigen-binding groove. Thus, the CD.sub.1d2 gene may not encode
a functional antigen-presenting molecule, and all functions previously
attributed to mouse CD.sub.1 may be effected by the product of the
CD.sub.1d1 gene. For this reason, it was decided to introduce a
targeted mutation into the CD.sub.1d1 gene, while leaving CD.sub.1d2
intact.
[0074] The CD.sub.1d gene was isolated from a strain 129/Sv phage
library with a probe generated by polymerase chain reaction. The
targeting construct was prepared using a 2.8 kb Apa1 fragment containing
the 5' region of the CD.sub.1d gene, a 3.2 kb BamHI-NotI fragment
containing the 3' region of the CD.sub.1d gene (the NotI site in
this fragment comes from the pBluescript vector into which phage
DNA was initially subcloned), a neomycin resistance gene (neo),
and the pBluescript plasmid (Stratagene). This construct was designed
to delete a fragment of about 200 bp from the exon encoding the
.alpha.2 domain of CD.sub.1d1. The strain 129/Sv-derived embryonic
stem (ES) cell line TL1 was transfected with the NotI-linearized
targeting vector. G418-resistant colonies were selected and isolated
as described in Van Kaer et al., Cell 71 (1992), 1205-1214. Genomic
DNA from individual clones was digested with EcoRI and hybridized
with a 2.3 kb CIaI-EcoRI probe from the 5' end of the CD.sub.1d1
gene. Recombination was confirmed by digestion with KpnI and hybridization
with a 700 bp BamHI-EcoRI probe from the 3' end of the CD.sub.1d1
gene. Chimeric mice were mated with C57BL/6 mice to score for germline
transmission, and heterozygous mutant mice were intercrossed to
obtain (C57BL/6.times.129/Sv) F2 homozygous mutants. Mice were typed
for their CD.sub.1d1 status by genomic southern blotting with the
5' probe. Mutant mice were healthy and bred normally.
[0075] Because the ES cells and mouse strain used to generate mutant
animals differ in their TL status (129/Sv is a TL+ stain and C57BL/6
is a TL-strain) all mice used in this study were genotyped for TL.
To type mice for their TL status, tail DNA was digested with BglII
and hybridized with a TL-specific probe that detects a polymorphism
between strains 129/Sv (TL+) and C57BL/6 (TL-) (Pontarotti et al.,
Proc. Natl. Acad. Sci. USA 83 (1986), 1782-1786). This probe was
generated by polymerase chain reaction using a set of primers designed
on the basis of published sequences (Pontarotti, supra): TABLE-US-00001
5'-TATACAGAGCTCCGTAGGAC-3'; and 5'-AGTTGTCTGCAGCCACGAAC-3'.
[0076] The CD.sub.1d1 mutant and wild-type mice were housed in
a specific-pathogen-free barrier animal facility, accredited by
the American Association for Accreditation of Laboratory Animal
Care (AAALAC). Animals were used between 12-16 weeks of age at the
start of the experiments. They were housed in filter-protected cages
with a 12 h light-dark controlled cycle, and provided with autoclaved
NIH open formula mouse chow and water ad libidum. The institutional
Animal Care and Use Committee approved all procedures. Within each
experiment all mice were aged- and sex-matched.
[0077] It is underlined that other genetic backgrounds can be used
for creating a CD.sub.1d mutant mouse, such as Balb/C genetic background.
EXAMPLE 2
[0078] UV Irradiation of Mice
[0079] A bank of five Philips TL-40W/12 sunlamps (Philips, The
Netherlands) was used to irradiate the mice. These lamps emit a
spectrum from 270 to 400 nm; 54% of the irradiation was within the
UVB range (280-315 nm) of the solar spectrum, with 45% being in
the UVA (315-400 nm) region and less than 1% in the UV-C (240-280
nm) range. The irradiance of the five bulbs averaged 10 W/m.sup.2,
as measured by a UVB PMA research radiometer.
[0080] The dorsal hair of the mice was removed with electric clippers
and the mice were placed into a plexiglass box separated into individual
compartments by Plexiglas dividers and covered with a wire top which
decreased the incident dose by 14%. For each UV-irradiation, the
box was placed each time in the same position under the lamps to
compensate for the uneven distribution of energy along the length
of the bulbs. The mice were exposed once or twice to an incident
dose of 86 mJ/cm.sup.2 UVB from five Philips TL-40 W/12 sunlamps.
Mice were exposed to a second dose of UVB radiation 96 h after the
first exposure. All mice were analyzed for signs of skin damage
24, 48, 72 and 96 h after their last UVB exposure.
[0081] Visually, a clear difference in the degree of skin damage
was observed between wild-type and CD.sub.1d knockout mice following
UVB-irradiation of their shaved dorsums. Whilst clear and significant
skin damage (burning, skin lesions) was exhibited by UV-irradiated
wild-type mice, no obvious signs of skin damage were detected in
UV-irradiated CD1d knockout mice.
EXAMPLE 3
[0082] Measurement of Apoptosis in Epidermis
[0083] Apoptotic cell death was detected using the DeadEnd.TM.
Fluorometric TUNEL System (Promega) which measures the fragmented
DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP
at 3'-OH DNA ends using the enzyme Terminal Deoxynucleotidyl Transferase
(TdT). TdT forms a polymeric tail using the principle of the TUNEL
assay. Briefly, formaline fixed paraffin embedded tissue sections
on slides were deparaffinized twice in fresh xylene for 5 min at
room temperature. They were washed in 100% ethanol for 5 min and
then rehydrated sequentially by immersing the sections through graded
(100%, 95%, 85%, 70%, 50%) ethanol washes for 3 min. Afterwards,
the sections were immersed in 0.85% NaCl for 5 min, washed in PBS
and then fixed in 4% paraformaldehyde for 15 min followed by two
washes in PBS. After removing residual fluid from the sections by
tapping, each tissue section was covered with 20 .mu.g/ml proteinase
K (Sigma) for 8-10 min at room temperature. After proteinase K treatment,
tissue sections were rinsed in PBS and then fixed by immersing in
4% paraformaldehyde for 5 min. This was followed by a wash in PBS,
removal of residual fluid by tapping and incubation of the sections
in equilibrium buffer (Promega) for 5-10 min. After equilibration,
the sections were incubated in a humidified chamber with TdT enzyme
for 1 h at 37.degree. C. Sections were soaked in stop buffer (SSC;
Promega) for 15 min to terminate the reactions and then rinsed in
three changes of PBS. After rinsing, sections were stained with
propidium iodide solution freshly diluted to 1 .mu.g/ml in PBS for
15 min in the dark. They were then washed three times in deionized
water for 5 min, and afterwards, excess fluid wiped off the area
surrounding the cells. The sections were then immediately examined
under a fluorescence microscope.
[0084] At 2, 6, 24, 48, 72 and 96 h after UV exposure (acute/chronic)
a TUNEL Assay (modification of protocol outlined by Ouhtit et al.,
American Journal of Pathology [2000], 156: 201-207), of the skin
taken from CD.sub.1d.sup.-/- and wild-type mice was carried out.
The results revealed that epidermal cells within the skin of CD.sub.1d.sup.-/-
mice were undergoing a high degree of apoptosis compared to wild-type
mice. In contrast, in wild-type skin the epidermal cells were undergoing
significantly less apoptosis.
EXAMPLE 4
[0085] Measurement of Epidermal Hyperplasia
[0086] Dorsal skin biopsies were fixed overnight in 4% paraformaldehyde
and paraffin embedded. Sections were stained with hematoxylin and
eosin (H&E) and viewed by light microscopy.
[0087] Following UVB exposure, CD.sub.1d.sup.-/- mice exhibited
significantly reduced epidermal hyperplasia 48 h after the last
UVB treatment compared to UV-irradiated wild-type mice.
EXAMPLE 5
[0088] Gene Profiling
[0089] In order to elucidate CD.sub.1d function a gene profiling
assay comparing wild-type and CD.sub.1d knockout mouse gene expression
had been performed.
[0090] Skin tissue was extracted from 5 individual wild-type and
CD.sub.1d knockout mice and extracted separately using Trizol kit
(Invitrogen AG, Basel, Switzerland) and then Qiagen RNeasy mini-kits
(Basel Switzerland) according to manufacturer instructions with
DNase I treatment to remove any genomic DNA contamination. RNA samples
were quantified by OD then analyzed via dynamic gel electrophoresis
with the Agilent Bioanalyser for intact 28S and 18S rRNA (All 28/18
ratio's were between 1.6 and 2.0). Study samples were judged to
contain sufficient amounts of high-quality RNA for hybridization
to GeneChips. As another quality control measure, prior to hybridization
with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, Calif.),
we confirmed that all samples gave strong signals for pre-selected
genes, using Affymetrix test chips (Test chip 5'/3' ratios were
less than 3.0).
[0091] For skin, 10 .mu.g total RNA was the starting material for
all individual mouse samples. In general, total RNA was converted
to biotinylated cRNA, hybridized in the Affymetrix probe array cartridge,
stained, and then quantified. First and second strand cDNA synthesis
was performed using the SuperScript Choice System (Invitrogen AG,
Basel, Switzerland), according to manufacturer instructions, but
using an oligo-dT primer containing a T7 RNA polymerase binding
site. Labeled cRNA was prepared with the RNA Transcript Labeling
kit (Enzo Biochem Inc., NY). Biotinylated CTP and UTP were used
together with unlabeled NTPs in the reaction, and unincorporated
nucleotides were removed with Nucleospin columns (Macherey-Nagel,
Duren, Germany).
[0092] cRNA (20 .mu.g) was fragmented at 94.degree. C. for 35 min
in buffer containing 200 mM Tris-acetate pH 8.1, 500 mM KOAc, 150
mM MgOAc. Prior to hybridization, fragmented cRNA in hybridization
mix (Buffer containing 100 mM MES, 1M NaCl, 20 mM EDTA, 0.01% Tween
20, 0.5 ng/.mu.l BSA, 0.1 ng/.mu.l herring sperm and Affymetrix
controls), was heated to 95.degree. C. for 5 min, cooled to 45.degree.
C. and loaded onto an Affymetrix probe array cartridge. The probe
array was incubated for 16 h at 45.degree. C. at constant rotation
(60 rpm), then exposed to Affymetrix washing and staining protocol.
[0093] This protocol included: [0094] one wash with non-stringent
buffer (6.times. SSPE, 0.01% Tween 20, 0.005% antifoam) [0095] one
wash with stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% tween
20) [0096] First stain with 0.01 mg/ml streptavidin-phycoerythrin
conjugate (Molecular Probes) in buffer containing 100 mM MES, 1M
NaCl, 0.05% Tween 20, 4 mg/ml of BSA. [0097] one wash with non-stringent
buffer (6.times. SSPE, 0.01% Tween 20, 0.005% antifoam) [0098] Second
stain with 3 .mu.g/ml of biotinylated anti-streptavidin +0.2 mg/ml
of IgG in buffer containing 100 mM MES, 1M NaCl, 0.05% Tween 20,
4 mg/ml of BSA. [0099] Third stain with 0.01 mg/ml streptavidin-phycoerythrin
conjugate (Molecular Probes) in buffer containing 100 mM MES, 1M
NaCl, 0.05% Tween 20, 4 mg/ml of BSA. [0100] one wash with non-stringent
buffer (6.times. SSPE, 0.01% Tween 20, 0.005% antifoam)
[0101] A mathematical method was developed and applied to the raw
GeneChip data for the selection of differentially regulated genes.
This method moves beyond setting a single fold change cut-off by
considering the standard deviation (SD) in the context of absolute
expression, or absolute difference intensity (ADI).
[0102] The method include the following steps: (A) data processing
by the commercially available "MAS5" Affymetrix program
(Santa Clara, Calif., USA) and rescaling, (B) logarithmic transformation
to distribution normality of the rescaled data, (C) multiple hypotheses
(one per gene) analysis of variance (ANOVA) testing, (D) the determination
of the robust mean within condition SD (equation 1), within bins
of 200 genes ordered by mean ADI levels, to determine a significance
limit SD between condition, named the REGExpress function (equation
2 from Genome Biology 2001 2(12): preprint0009.1-0009.31); and (E)
subsequent ranking of genes by the p value of the REGExpress and
ANOVA, to help focus at effect importance. The selection is made
with the p value resulting from multiple hypotheses (one per gene)
ANOVA testing and/or with the p value resulting from REGExpress.
[0103] Probe arrays were scanned at 488 nm using an Argon-ion Laser
(made for Affymetrix by Agilent). Readings from the quantitative
scanning were analyzed with Affymetrix Gene Expression Analysis
Software.
[0104] The findings are summarized in the tables I to III below.
The fold increase (+) or decrease (-) is the statistically significant
relative fold increase or decrease of a gene expressed in CD1d knockout
mice compared to the same gene expressed in wild-type mice. It becomes
clearly evident that blocking CD1d upregulates genes controlling
hair follicle development, and down-regulates genes involved in
inflammation and cancer development. TABLE-US-00002 TABLE I Genes
which regulate hair follicle development Fold increase/ Mean Mean
Biological Gene Name decrease Wt CD1d-/- Function mmu-crystalline
+27.0 0.922 4.251 thyroid binding protein regulating hair follicle
development PPatched homolog 2 +2.763 3.810 4.826 hair follicle
development
[0105] TABLE-US-00003 TABLE II Genes which regulate inflammation
Fold increase/ Mean Mean Gene Name decrease Wt CD1d-/- Biological
Function Disease Association TGF beta +1.743 3.630 4.186 Signalling
molecule of activated Kinease the p38-MAPKinase and the Stress activated
protein Kinase (SAPK) pathways. Rel-A(NfKappaB) -0.735 7.350 7.043
anti-apoptotic, induction inflammatory disorders of inflammatory
cytokines. Cytochrome beta -0.738 6.829 6.525 superoxide generation
Plasminogen activator -0.357 3.906 2.875 Serine protease inhibitor.
inflammatory disorders inhibitor (PAI-1) Regulates fibrolysis. MRP14
-0.202 3.943 2.344 Ca.sup.++ dependent regulatory acute and chronic
protein in inflammatory inflammatory responses responses. e.g. Psoriasis
Mast cell protease -0.661 6.427 6.014 proteolysis and peptidolysis.
inflammation P-Selectin -0.685 6.439 6.060 Cell adhesion inflammation
TFII-1 +1.373 5.707 6.024 Transcription factor which regulates c-Fos
activity Interleukin-6 -0.268 1.882 0.565 cytokine: multi-functional
inflammation
[0106] TABLE-US-00004 TABLE III Genes which regulate cancer growth/development:
Fold increase/ Mean Mean Gene Name decrease Wt CD1d-/- Biological
Function Disease Association v-Rel(NfKappaB) -0.735 7.350 7.043
oncogenic- cancer transforms cells Plasminogen activator -0.357
3.906 2.875 serine protease metastatic tumors inhibitor (PAI-1)
inhibitor P-Selectin -0.685 6.439 6.060 adherence facilitates tumor
metastasis. Cathepsin S -0.697 7.545 7.184 Cysteine protease malignancy
Proliferin -0.234 1.870 0.419 regulates mouse fibrosarcomas angiogenesis
Interleukin-6 -0.268 1.882 0.565 secreted by basal cell carcinomas
and malignant melanomas. CSF-1 receptor -0.750 7.196 6.909 Growth
factor Cancer regulating cell proliferation.
EXAMPLE 6
Evaluation of the Inflammatory Response Induced by a Single Topical
Administration of TPA
[0107] Phorbol-12-myristate-13-acetate (TPA) provided by Sigma
Aldrich (L'Isle d'Abeau Chesnes BP701, 38297 Saint Quentin Fallavier,
France) is dissolved in acetone at the dose of 0.01% (W/V) and 20
.mu.l of the solution is applied topically onto the internal face
of the right ear of CD.sub.1d.sup.-/- mice or wild-type mice in
order to induce an acute inflammatory response.
[0108] The animals are maintained in individual cages with a standard
pellet diet in an animal room with a 12-hour light-dark cycle. The
facilities provide a filtered air with a temperature of 22+/-2.degree.
C. and a relative humidity of 55+/-10%.
[0109] The inflammatory response is quantified 6 hours, 24 hours
and 48 hours after application by measuring the ear oedema using
a micrometer (<(<<oditest >> provided by Kroeplin
Gmbh, Postfach 1255 D36372 Schluchlern, Germany).
[0110] The oedema is calculated as follows:
[0111] (oedema=ear thickness of the treated group-ear thickness
of the acetone group).
[0112] The mean value of CD.sub.1d.sup.-/- group is compared to
the mean value of the wild-type group using the Student's t-test.
EXAMPLE 7
Evaluation of the Inflammatory Response Induced by a Single Topical
Administration of Arachidonic Acid
[0113] Arachidonic acid (5-8-11-eicosatetraenoic acid) provided
by Sigma Aldrich (L'Isle d'Abeau Chesnes BP701, 38297 Saint Quentin
Fallavier, France) is dissolved in acetone at the concentration
of 140 nM and 25 .mu.l of the solution is applied topically onto
the internal face of the right ear of CD.sub.1d.sup.-/- mice or
wild-type mice in order to induce an acute inflammatory response.
[0114] The animals are maintained in individual cages with a standard
pellet diet in an animal room with a 12-hour light-dark cycle. The
facilities provide a filtered air with a temperature of 22+/-2.degree.
C. and a relative humidity of 55+/-10%.
[0115] The inflammatory response is quantified 1 hour, 2 hours,
and 4 hours after application by measuring the ear oedema using
a micrometer (<<oditest >> provided by Kroeplin Gmbh,
Postfach 1255 D36372 Schluchlern, Germany).
[0116] The Oedema is Calculated as Follows:
[0117] (oedema=ear thickness of the treated group-ear thickness
of the acetone group).
[0118] The mean value of CD.sub.1d.sup.-/- group is compared to
the mean value of the wild-type group using the Student's t-test.
EXAMPLE 8
[0119] Evaluation of the DTH (Delayed-Type Hypersensivity) Reaction
Induced by Oxazolone
[0120] Oxazolone (4-ethyoxymethylene-2-phenyl-oxazol-5-one) provided
by Sigma Aldrich (L'Isle d'Abeau Chesnes BP701, 38297 Saint Quentin
Fallavier, France) is dissolved in acetone at the concentration
of 1% (WN) and 50 .mu.l of the solution is applied once daily for
4 days on the abdominal skin of shaved CD.sub.1d.sup.-/- mice or
shaved wild-type mice.
[0121] 4 days later the animals are challenged by a single administration
(20 .mu.l) onto the internal face of the right ear of oxazolone
dissolved in acetone at the dose of 0.3%. The post-challenge response
is quantified 24 hours and 48 hours after application by measuring
the ear oedema using a micrometer (<<oditest>> provided
by Kroeplin Gmbh, Postfach 1255 D36372 Schluchlern, Germany).
[0122] The Oedema is Calculated as Follow:
[0123] (oedema=ear thickness of the treated group-ear thickness
of the acetone group).
[0124] The mean value of CD.sub.1d.sup.-/- group is compared to
the mean value of the wild-type group using the Student's t-test.
EXAMPLE 9
[0125] Evaluation of Skin Damages Induced by UV Irradiation Using
a Solar Simulator
[0126] A solar simulator (Oriel 81050) equipped with an UVC filter
is used to irradiate CD.sub.1d.sup.-/- mice or wild-type mice.
[0127] Irradiation: UVB+UVA doses and to be precised
[0128] Effect on epidermis: SBC counts, epidermal hyperplasia measurement
[0129] Effect on the dermis: MMP1 and MMP3 expression with immuno-histochemical
methods
EXAMPLE 10
Regulation of CD.sub.1d Gene Transcription by UV Radiation and
Role of CD.sub.1d in Regulating UV-Induced COX-2 and TNF-.alpha.
Gene Transcription
Mice
[0130] Specific pathogen-free male outbred 129/C57BL/6 wild-type
and 129/C57BL/6 CD.sub.1d knockout mice were obtained from L. Van
Kaer, Vanderbilt University Medical Center (Nashville, TN, USA).
The animals were maintained in facilities in accordance with current
Swiss regulations and standards. They were housed in filter-protected
cages, and ambient lighting was controlled to provide 12 h light/12
h dark cycles. Autoclaved open-formula mouse chow and water were
provided ad libidum. All animal procedures were reviewed and approved
by the Institutional Animal care and Use Committee. Within each
experiment all the mice were matched for age and sex. The mice were
16 weeks at the start of each experiment.
UV Light Source
[0131] The UVB source was a bank of five Philips TL-40 W/12 sunlamps
(Philips, The Netherlands). These lamps emit a spectrum from 270
to 400 nm; 54% of the irradiation was within the UVB range (280-315
nm) of the solar spectrum, 45% in the UVA (315-400 nm) region and
less than 1% in the UV-C (240-280 .mu.m) range. The irradiance of
the five bulbs averaged 10 W/m.sup.2, as measured by a UVB PMA research
radiometer. Solar simulated light (UVA+UVB) was produced by a 1000
W exon UV solar simulator (Solar Light Company, PA, USA) equipped
with a WG-320 atmospheric attenuation filter (1 mm thick), a visible/infrared
band pass blocking filter (UG-5; 5 mm thick), and a dichroic mirror
to further reduce visible and infrared energy.
UV Irradiation of Mice
[0132] The dorsal hair of the mice was removed with electric clippers.
For mice being exposed to UVB radiation they were placed into a
Plexiglass box separated into individual compartments by Plexiglass
dividers and covered with a wire top which decreased the incident
dose by 14%. For each UVB-irradiation, the box was placed each time
in the same position under the lamps to compensate for the uneven
distribution of energy along the length of the bulbs. For mice being
exposed to solar UV radiation (UVA+UVB) the mice were anaesthetized
to immobilize them prior to being exposed to the beam of the solar
simulator. The mice were exposed once to an incident dose of 86,
215 or 430 mJ/cm.sup.2 UVB from five Philips TL-40 W/12 sunlamps.
Mice exposed to solar light (UVA+UVB) were exposed once to an incident
dose of 1680 (1 min), 16,800 (10 min) or 33,600 mJ/cm.sup.2 (20
min) solar radiation.
UV Irradiation of Keratinocyte Cell Cultures
[0133] Confluent cultures of keratinocytes grown in sterile 6-well
plates (Corning, Netherlands) were submitted to a single dose (570
mJ/cm.sup.2) of solar UV irradiation. Treatment was performed without
plastic lids after having removed medium and replaced it by sterile
HBSS. Control cultures were not irradiated. After UV exposure, HBSS
was removed and medium put back on cultures. The cells were incubated
at 37.degree. C. with 5% CO.sub.2 and at various time points thereafter
harvested for RNA.
Immunostaining of Mouse Tissue
[0134] Biopsies of wild-type mouse skin were fixed in formaline
before being embedded in paraffin. Cross sections (5 .mu.m thick)
of paraffin embedded tissues were made, deparaffinized by gentle
heating, de-hydrated and rehydrated using the following procedure:
2 times for 3 min in Xylol, 3 min in Ethanol 100%, 3 min in Ethanol
95%, 3 min in Ethanol 80% and 3 min in PBS IX. Fresh skin was also
embedded in Tissue-Tek (4583, Sakura Finetek, Torrance, USA) and
frozen in liquid nitrogen. The sections were then rehydrated in
PBS 1.times. for few minutes. Sections were stained using the anti-mouse
CD1d 1H1 primary mAb and developed using the mouse Histostain-plus
kit (ZYMED Laboratories Inc., San Francisco, USA).
Extraction of Total RNA from Skin or Cell Lysates
[0135] Treated and control cultures of human keratinocytes grown
in 6-well plates were placed on ice and washed twice using PBS 1.times.
at 4.degree. C. Cell lysate was obtained by scraping the cells in
350 .mu.l of lysis buffer RLT (74104, Qiagen AG, Basel, Switzerland)
supplemented with 1% of .beta.-mercaptoethanol and by briefly vortexing
them. QIAshredder columns (79656, Qiagen AG, Basel, Switzerland)
were used to homogenize cell extracts by centrifugation at 13,000.times.g
for 2 min. Total RNA was then prepared using RNAeasy kits (74104,
Qiagen AG, Basel, Switzerland) according to the manufacturer's protocols.
Genomic DNA contamination was removed with on-column DNase digestion
using a RNase-free DNase Set (79254, Qiagen AG, Basel, Switzerland).
Skin samples of 1 cm.times.1 cm were cut into small pieces and homogenized
in 1 ml of TRIZOL Reagent (15596-026, Invitrogen AG, Basel, Switzerland)
using a rotor-stator homogenizer (Polytron, Kinematica, Luzern,
Switzerland). The supernatant obtained after centrifugation at 12,000.times.g
was recovered in a fresh tube and incubated for 5 min at room temperature.
0.2 ml of chloroform was added to the tube, which was vigorously
shaken for 15 sec and incubated at room temperature for 2-3 min.
Samples were centrifuged at 12,000.times.g for 15 min at 4.degree.
C. The upper aqueous phase was transferred to a fresh tube and total
RNA precipitated using 0.5 ml of isopropyl alcohol for 10 min at
room temperature. The RNA pellet obtained by centrifugation at 12,000.times.g
for 10 min at 4.degree. C. was washed with 1 ml of 75% EtOH followed
by centrifugation at 7,500.times.g for 5 min at 4.degree. C. The
RNA pellet was finally dried at room temperature and dissolved in
40 .mu.l of RNase free water by incubating the samples 10 min at
55-60.degree. C. Possible DNA contamination was removed with on-column
DNase digestion using a RNase-free DNase Set.
Semi-Quantitative RT-PCR
[0136] A. Reverse Transcription-Polymerase Chain Reaction and PCR
Reaction
[0137] 5 .mu.g of total RNA were reverse transcribed by oligo-dT
priming to first strand cDNA using the Superscript First-Strand
Synthesis System for RT-PCR (11904-018, Invitrogen AG, Basel, Switzerland)
according to the manufacturer's instructions. PCR of cDNA was performed
to either detect specific expression of a single gene (single PCR)
or multiple genes (Multiple gene PCR). For single PCR, 48111 of
PCR master mix containing 5 .mu.l PCR Buffer, 31 .mu.l 25 mM MgCl.sub.2,
1 .mu.g 10 mMdNTPs, 0.5 .mu.l 50 .mu.M of both sense and anti-sense
oligonucleotides, 3 .mu.l DMSO, 34.51 .mu.l of water and 0.5 .mu.l
of 5 U/.mu.l Taq DNA Polymerase was added to 2 .mu.l of cDNA. All
the reagents were purchased from Invitrogen (15558-026 and 18427-013,
Basel, Switzerland). The number of cycles and the annealing temperature
applied to amplify cDNA samples were specific to each gene tested,
one cycle consisting of 30 s at 94.degree. C., 30 s at x.degree.
C. and 30 s at 72.degree. C., each amplification being preceded
by 2 min at 94.degree. C. and finished by 3 min at 72.degree. C.
Kit MP-70211 (Maxim Biotechnologies, San Francisco, USA) for multiple
gene PCR of genes implicated in apoptosis and inflammation were
used as instructed. Prior to using each kit, the condition for running
multiple reverse and forward primers at the same time to detect
multiple genes was determined. The conditions for using kit MP-70211
which contained primers for detecting TNF-.alpha. and COX-2 genes,
were as follows: 96.degree. C. for 1 min and 60.degree. C. for 4
min (cycles 2.times.); 94.degree. C. for 1 min and 60.degree. C.
for 2 min (cycles 29.times.); 70.degree. C. for 10 min (cycle 1.times.)
and 25.degree. C. soak. The DNA sequence of the reverse and forward
primers used the MPCR kit for detecting multiple genes under one
set of conditions were proprietary and thus are not described in
this report.
[0138] Primer Sequences and Number of Cycles of Amplification TABLE-US-00005
Annealing No. of Product Gene Primer Sequence (5'-3') temper. cycles
Size GAPDH sense AAT CCC ATC ACC ATC TTC CA 52 16 558 antisense
GTC ATC ATA TTT GGC AGG TT CD1d sense GCT CAA CCA GGA CAA GTG GAC
GAG 66 27 452 antisense AGG AAC AGC AAG CAC GCC AGG ACT
[0139] Mouse: TABLE-US-00006 Annealing No. of Product Gene Primer
Sequence (5'-3') temper. cycles Size GAPDH sense TTC ACC ACC ATG
GAG AAG GC 60 22 236 antisense GGC ATG GAC TGT GGT CAT GA CD1d.1
sense ACG TCC TGG CAG ACA GTC CCA GG 60 24 706 antisense TTA ATG
TTG AAA AGA GCG TAC TGG C
B. Relative Quantification of mRNA Levels
[0140] Amplification of the genes was analyzed by loading 10 .mu.l
of the PCR products on a 3% agarose gel which was run in a 1.times.TAE
buffer containing 2% Ethidium Bromide at 150V for 30 min. The PCR
products were visualized as fluorescent bands under UV light. Gels
were scanned using a Kodac DC 120 Camera and fluorescence intensity
of the bands was quantified using the Software Scion Image .beta.
4.02 Win (Scion Corporation, Maryland, USA).
[0141] While studies of CD.sub.1d proteins in the murine system
suggest a widespread and constitutive expression on many hematopoietic
cell types as well as intestinal epithelial cells, and hepatocytes,
it was not known whether this molecule is expressed by normal and/or
UV exposed mouse skin cells, especially keratinocytes. To address
this question, mouse skin from unirradiated and UVB-irradiated wild-type
mice was fixed in formaline, sectioned and stained using an anti-mouse
CD1d mAb (1H1) to detect CD.sub.1d protein. Detection of CD.sub.1d
protein in normal unirradiated skin was negative (data not shown).
However, CD1d protein was detected (brown color) in the epidermis
and dermis of UVB-irradiated mouse skin (FIG. 5). Staining was largely
confined to the more differentiated layers of the skin (stratum
granulosum and stratum corneum) and at the cellular level was localized
to the cytoplasm and nuclear membrane.
[0142] Thus, UVB-induced mouse skin damage/burning may be directly
regulated at the level of the mouse keratinocyte rather than by
antigen-presenting cells (locally or systemically).
CD1d Gene Transcription is Regulated by UV Radiation
[0143] Having demonstrated that UVB-induced skin damage is regulated
by CD.sub.1d and that CD.sub.1d protein is expressed by mouse epidermal
cells (keratinocytes), it was next important to establish whether
skin CD.sub.1d expression is regulated by UV radiation. Any indication
that skin CD.sub.1d expression is regulated by UV radiation would
suggest that modulation of CD.sub.1d levels in skin is a critical
factor responsible for regulating UV-induced skin damage. To address
this question, the shaved dorsum of wild-type mice was exposed to
a single dose (86 mJ/cm.sup.2) of UVB radiation and at various times
post irradiation (6, 24, 48, 72 and 96 h) the irradiated skin was
excised, RNA extracted and purified, and CD.sub.1d mRNA levels determined
by semi-quantitative RT-PCR. As a control, normal non-irradiated
mouse skin was excised, RNA extracted and purified and CD1d mRNA
levels determined by semi-quantitative RT-PCR.
[0144] As shown in FIG. 6, the level of CD.sub.1d mRNA in whole
mouse skin which decreased as early as 6 h after UVB exposure was
significantly reduced 24 h post irradiation compared to levels detected
in normal non-irradiated skin. In contrast, 48, 72 and 96 hours
following UVB exposure CD.sub.1d mRNA levels were raised above the
levels detected in normal unirradiated control skin. To further
validate our studies on the effect of UVB radiation on skin CD.sub.1d
gene transcription, we exposed the shaved dorsum of wild-type mice
to varying doses of solar UV irradiation (UVB+UVA)-1680 mJ/cm.sup.2
(1 min), 16,800 mJ/cm.sup.2 (10 min) or 33,600 mJ/cm.sup.2 (20 min)
of solar UV. At 6 and 72 h post irradiation the irradiated skin
was excised, RNA extracted and purified, and CD.sub.1d mRNA levels
determined by semi-quantitative RT-PCR (FIG. 7). As with UVB exposure,
we observed a similar decrease and increase in CD.sub.1d mRNA levels
6 and 72 h following solar UV irradiation, respectively, regardless
of the UV dose, suggesting that the response of skin CD.sub.1d gene
transcription to UV radiation is an important event in the skin's
response to the damaging effects of UV exposure.
Human Keratinocyte CD1d Gene Transcription is Regulated by Solar
UV Radiation
[0145] In an attempt to address whether human CD.sub.1d gene transcription
is regulated by UV radiation we investigated whether cultured human
keratinocytes exhibit a similar gene transcription kinetic profile
in response to UV irradiation. At different time points following
exposure of triplicate DK7 cell keratinocyte cultures to a single
dose of 5700 mJ/cm.sup.2 solar UV radiation, the cells were harvested
for RNA and semi-quantitative RT-PCR performed to determine the
relative level of CD.sub.1d mRNA (FIG. 8). As observed with UV irradiated
(UVB or solar) mouse skin, CD.sub.1d mRNA levels decreased 6 h post-irradiation
compared to normal non-irradiated controls. Analysis of CD.sub.1d
mRNA levels 10 h post-irradiation revealed that these levels had
further decreased compared to the levels detected in normal non-irradiated
cell cultures. Between 16 and 48 h hours after UV exposure, the
level of CD.sub.1d mRNA increased proportionally; a pattern also
observed in the skin of UV-irradiated whole wild-type skin suggesting
that UV-induced CD.sub.1d gene transcription in mouse skin was likely
being regulated at the level of the keratinocyte.
[0146] Human keratinocyte CD.sub.1d gene transcription is responsive
to UV radiation and appears to be regulated in a similar manner
to mouse skin CD.sub.1d implying that a) skin CD.sub.1d plays a
critical role in regulating the response of skin to UV irradiation
and b) modulation of CD.sub.1d levels in skin is a critical factor
responsible for regulating UV-induced skin inflammation/damage.
Gene Transcription of Key Genes which Regulate Skin Inflammation
is Diminished in UVB irradiated CD1d Knockout Mouse Skin
[0147] To further validate CD.sub.1d as a critical molecule responsible
for regulating UVB-induced skin inflammation/damage we next investigated
whether COX-2 and TNF-.alpha. gene transcription (key genes responsible
for regulating UVB-induced skin inflammation/damage) is deregulated
in UV-irradiated CD.sub.1d knockout mouse skin. It was found that
COX-2 and TNF-.alpha. mRNA levels in CD.sub.1d knockout mouse skin
were inhibited 48 and 72 h after UV irradiation (FIG. 9). Since
UV-induced skin damage/inflammation in wild-type mice is observed
at 48 and 72 h after UV exposure these data demonstrate that skin
CD.sub.1d mediates UV-induced skin inflammation/damage by inducing
COX-2 and TNF-.alpha. gene transcription.
EXAMPLE 11
[0148] Inflammatory cytokines synthesis in UV-irradiated skin of
CD.sub.1d Knockout mice is decreased compared to wild-type control
mice.
UV Irradiation of Mice
[0149] The mice were exposed once to an incident dose of 200 mJ/cm.sup.2
UVB radiation. Three month old female inbred 129/C57BL/6 WT and
129/C57BL/6 CD.sub.1d KO mice were involved in this study (n=4).
Cytokine Quantification
[0150] 8 mm punch biopsies of skin were harvested at 0 h, 24 h,
48 h, 72 h, 96 h and 168 h post-irradiation and immediately frozen
in liquid nitrogen. IL-6 and MIP1-alpha were quantified in skin
homogenates using classical ELISA methods.
[0151] In WT mice UV-B irradiation induces a high up-regulation
of inflammatory cytokines synthesis, 48 hours post-irradiation,
whereas in CD.sub.1d KO mice synthesis of IL-6 and MIP1-alpha protein
is significantly reduced. This demonstrates a major role for CD.sub.1d
in UVB-induced cutaneous inflammation.
EXAMPLE 12
[0152] Hydrocortisone down-regulates chemical stress-induced CD.sub.1d
gene transcription.
[0153] Primary human keratinocytes were grown in complete KGM before
being exposed to 300 .mu.M H.sub.2O.sub.2 as a stress factor. 48
hours after seeding, KGM medium was replaced by KGM without hydrocortisone
and cultures were treated with 300 .mu.M of H.sub.2O.sub.2. Total
RNA was extracted at different time after treatment and CD.sub.1d
mRNA quantified by Real Time PCR.
[0154] For Taq Man Assays, Applied Biosystems recommends to use
10 to 100 ng of initial RNA quantity per well. Consequently, first
strand cDNA synthesis was performed in a 20 .mu.l volume using 1
.mu.g of total RNA and 150 .mu.g of random hexamers following the
manufacturer's recommendations (Superscript.TM. First-Strand Synthesis
System for RT-PCR, 11904-018, Invitrogen). 1 .mu.l of the resulting
cDNA samples was used for amplification by Real Time PCR.
[0155] The sets of primers and probes used for detection of CD1d
cDNA were provided by Applied Biosystems as Assays on Demand (respectively
Hs00174321_ml and Hs00166289_ml). The primers and probes for the
housekeeping gene GAPDH were provided as PDARS (4310884E, Applied
Biosystems).
[0156] All the cDNA samples were tested in triplicate. PCR reaction
mixtures were prepared on ice in micro centrifuge tubes. For one
replicate, a pre-mix of 24 .mu.l was made using 1.25 .mu.l of 20.times.
Target or Control mix, 10.25 .mu.l of water and 12.5 .mu.l of 2.times.
TaqMan Universal Master Mix, and added to 1 .mu.l of cDNA. The PCR
reaction mixtures were gently and quickly centrifuged before being
aliquoted at the rate of 25 .mu.l per well of a 96-wells plate.
The plate was sealed, centrifuged at 2000 rpm for 30 seconds and
placed in a 5700 Sequence Detection System for thermal cycling and
fluorescence analysis using the following PCR program:
[0157] 2 min at 50.degree. C.
[0158] 10 min at 95.degree. C.
[0159] 40 cycles of 15 sec at 95.degree. C. and 1 min at 60.degree.
C.
[0160] Results were analyzed using the GeneAmp.RTM. 5700 SDS software.
Amplification plots showing amplification of cDNA of interest in
function of the number of cycles were obtained.
[0161] The gene expression fold changes obtained in the test condition
compared to the control condition were determined by the comparative
Ct method using the following formula: Fold .times. .times. changes
= 2 - .DELTA..DELTA. .times. .times. Ct = 2 - ( .DELTA. .times.
.times. Ct .times. .times. test - .DELTA. .times. .times. Ct .times.
.times. control ) where .DELTA. .times. .times. Ct = Ct target .times.
.times. gene - Ct housekeeping .times. .times. gene
[0162] As shown in FIG. 11, modulation of CD.sub.1d gene expression
is observed over time when cells are exposed to H.sub.2O.sub.2.
In presence of Hydrocortisone, the pattern of CD.sub.1d gene expression
obtained following H.sub.2O.sub.2 challenge differed in that hydrocortisone
suppressed CD.sub.1d transcription.
[0163] Thus, Hydrocortisone is able to down-regulate CD.sub.1d
expression in cells subjected to a stress.
EXAMPLE 13
[0164] Phospholipid levels are disregulated in the skin and the
intestine of CD.sub.1d knockout mice compared to wild-type skin.
[0165] Mice: Female inbred C57BL/6 CD1d -/- and wild-type C57BL/6
mice aged 5 months were sacrificed and the respective tissue excised.
The pieces of skin/intestinal tissue from each mouse were snap frozen
using liquid nitrogen. They were then analysed for lipid content.
[0166] The main family of lipids regulated by CD.sub.1d in skin
were phospholipids. TABLE-US-00007 nMolar per gram of skin tissue
CD1d-/-* Wild-type Spingomyelin 33 +/- 30.4 176 +/- 116 Lysophosphatidylcholine
3.8 +/- 3.7 10.0 +/- 4.1 Phoshatidylcholine 35.1 +/- 21.1 8.5 +/-
12.9 Phosphatidylserine 67 +/- 30.4 115 +/- 33 *The values are statistically
significantly different from wild-type control (p < 0.05)
Sphingomyelin
[0167] It is a ubiquitous component of animal cell membranes, where
it is by far the most abundant sphingolipid. Indeed, it can comprise
as much as 50% of the lipids in certain tissues, though it is usually
less abundant than phosphatidylcholine. For example, it makes up
about 10% of the lipids of brain. It is the single most abundant
lipid in erythrocytes of most ruminant animals, where it replaces
phosphatidylcholine entirely. In this instance, there is known to
be a highly active phospholipase A that breaks down the glycerophospholipids,
but not sphingomyelin. Like phosphatidylcholine, sphingomyelin tends
to be most abundant in the plasma membrane, and especially in the
outer leaflet, of cells.
[0168] Now, it is known that sphingomyelin (and other sphingolipids)
and cholesterol may be located together in specific sub-domains
(`rafts` or related structures termed `caveolae`) of membranes.
As sphingolipids containing long, largely saturated acyl chains,
they pack more tightly together, thus giving sphingolipids much
higher melting temperatures than membrane glycerophospholipids.
This tight acyl chain packing is essential for raft lipid organization,
since the differential packing facility of sphingolipids and phospholipids
is believed to lead to phase separation in the membrane, giving
rise to sphingolipid-rich rafts (`liquid-ordered` phase) surrounded
by glycerophospholipid-rich domains (`liquid-disordered` phase).
Interactions between specific cellular proteins and lipids in these
rafts are believed to be important in signalling mechanisms implicating
an important role for sphingomyelin in regulating cell signaling.
Sphingomyelin is a Key Lipid in Signal Transduction Processes Involved
in Apoptosis.
[0169] Also, sphingomyelin serves as a precursor for ceramides,
long-chain bases and sphingosine-1-phosphate, as part of the `sphingomyelin
cycle`, and many other important sphingolipids. (see figure below).
Some of these have functions as intracellular messengers, and others
are essential membrane constituents Lysophosphatidylcholine [0170]
a phospholipid that is pro-inflammatory [0171] elevated in lesional
psoriasis [0172] intracutaneous injection induces skin inflammation
[0173] formed by the action of phospholipaseA2 which is the rate
limiting step in the production of arachadonic acid. (link to regulation
of COX-2 by CD.sub.1d).
[0174] For the large intestine the following results were obtained:
TABLE-US-00008 Large Intestine nMoles per gram of Tissue Lipid Class
Saturation Fatty Acid Family CD1d-/-* Wild-type Cardiolipin Saturated
Stearic Acid 405 .+-. 89 950 .+-. 442 Total 405 950 Unsaturated
Vaccenic Acid 74 .+-. 21 167 .+-. 45 Oleic Acid 216 .+-. 42 377
.+-. 86 .alpha. Linolenic Acid 23 .+-. 9 43 .+-. 9 DHA 164 .+-.
92 596 .+-. 278 Linoleic Acid 781 .+-. 210 2066 .+-. 727 DGLA 36
.+-. 12 60 .+-. 16 Total 1294 3309 Total Cardiolipin 1699 4259 Lysophosphatidylcholine
Saturated Myristic Acid 78 .+-. 41 27 .+-. 5 Arachidic Acid 7 .+-.
3 3 .+-. 0.5 Total 85 30 Unsaturated Eicosenoic Acid 9 .+-. 5 3
.+-. 2 Erucic Acid 234 .+-. 143 17 .+-. 36 Eicosadienoic Acid 153
.+-. 98 30 .+-. 61 DGLA 11 .+-. 5 4 .+-. 1 Docosadienoic Acid 12
.+-. 8 2 .+-. 3 Total 419 56 Total Lysophosphatidylcholine 504 86
Free Fatty Acids Saturated Pentadecanoic acid 31 .+-. 10 17 .+-.
5 Total 31 17 Unsaturated Nervonic acid 9 .+-. 5 2 .+-. 4 Total
9 2 Total Free Fatty Acids 40 19 Cholesterol Ester Unsaturated Eicosapentaenoic
acid 5 .+-. 3 1 .+-. 3 Palmitelaidic acid 49 .+-. 27 13 .+-. 8 Total
54 14 Diglyceride Unsaturated Eicosenoic acid 11 .+-. 8 33 .+-.
16 Total 11 33 Phosphatidylcholine Unsaturated Linoleic acid 2551
.+-. 2321 6398 .+-. 2862 Total 2551 6398 Phosphatidylserine Unsaturated
Linoleic acid 501 .+-. 105 1102 .+-. 338 Total 501 1102 *All values
were statistically significantly different from wild-type groups
(p < 0.05)
[0175] For the small intestine the following results were obtained:
TABLE-US-00009 Small Intestine Fatty Acid nMoles per gram of Tissue
Lipid Class Saturation Family CD1d-/-* Wild-type Cardiolipin Unsaturated
Eicosenoic 12 .+-. 7 21 .+-. 3 acid DHA 178 .+-. 124 480 .+-. 121
Total 190 501 Phosphati- Saturated Behenic acid 13 .+-. 10 27 .+-.
10 dylcholine Total 13 27 Unsaturated Mead acid 7 .+-. 4 15 .+-.
4 Eicosate- 2 .+-. 3 7 .+-. 3 traenoic acid DGLA 154 .+-. 96 310
.+-. 115 Docosadie- 5 .+-. 4 10 .+-. 3 noic acid Total 168 342 Total
Phosphatidylcholine 181 369 *All values were statistically significantly
different from wild-type groups (p < 0.05)
[0176] This above data support a role for CD.sub.1d in the regulation
of phospholipid metabolism which controls inflammatory processes.
[0177] It should be understood that various changes and modifications
to the presently preferred embodiments described herein will be
apparent to those skilled in the art. Such changes and modifications
can be made without departing from the spirit and scope of the present
invention and without diminishing its intended advantages. It is
therefore intended that such changes and modifications be covered
by the appended claims.
|