U.S. patent application number 10/771004 was filed with the patent office on 2004-12-30 for pharmaceutical or food composition for treating pathologies associated with graft rejection or an allergic or autoimmune reaction.
This patent application is currently assigned to BIOTECH TOOLS S.A.. Invention is credited to Duchateau, Jean, Henot, Frederic, Legon, Thierry.
Application Number | 20040265322 10/771004 |
Document ID | / |
Family ID | 25663085 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265322 |
Kind Code |
A1 |
Henot, Frederic ; et
al. |
December 30, 2004 |
Pharmaceutical or food composition for treating pathologies
associated with graft rejection or an allergic or autoimmune
reaction
Abstract
The present invention is related to a process for obtaining a
composition comprising peptides bound to one or more heat shock
protein(s) and for possibly recovering from said composition the
bound peptides, wherein the peptides resulting from a previously
in-vitro hydrolysis of at least one immunogenic and antigenic
macromolecular structure, are mixed in-vitro with one or more heat
shock protein(s). The present invention is also related to the
compositions obtained by said process.
Inventors: |
Henot, Frederic; (Brussels,
BE) ; Legon, Thierry; (Korbeek Lo, BE) ;
Duchateau, Jean; (Soignies, BE) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
BIOTECH TOOLS S.A.
Brussels
BE
|
Family ID: |
25663085 |
Appl. No.: |
10/771004 |
Filed: |
February 3, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10771004 |
Feb 3, 2004 |
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09891148 |
Jun 25, 2001 |
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6709672 |
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09891148 |
Jun 25, 2001 |
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09380548 |
Oct 28, 1999 |
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6312711 |
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09380548 |
Oct 28, 1999 |
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PCT/BE98/00030 |
Mar 5, 1998 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
A61K 2039/54 20130101;
A61K 2039/55516 20130101; A61K 39/35 20130101; Y02A 50/474
20180101; A61K 39/0008 20130101; A61K 2039/541 20130101; A61K
39/0258 20130101; A61K 39/001 20130101; A61K 2039/555 20130101;
Y02A 50/30 20180101; A61K 39/35 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/185.1 |
International
Class: |
A61K 039/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 5, 1997 |
BE |
97/00199 |
Claims
1. A process for epitope mapping comprising the steps of
hydrolyzing in-vitro at least one immunogenic or antigenic
macromolecular structure to obtain peptides mixing said peptides
and at least one heat shock protein under conditions allowing the
formation of complexes removing unbound peptides recovering
peptides from said complexes.
2-17. cancelled
18. The method of claim 1 wherein said at least one heat shock
protein is a bacterial heat shock protein.
19. The process of claim 18 wherein said bacterial heat shock
protein is selected from the group consisting of the DnaK, DnaJ,
GroEL or GrpE heat shock proteins.
20. The process of claim 1 wherein the at least one immunogenic or
antigenic macromolecular structure is a protein.
21. The process of claim 1 wherein the at least one immunogenic or
antigenic macromolecular structure induces graft rejection.
22. The process of claim 1 wherein the at least one immunogenic or
antigenic macromolecular structure induces an allergic
reaction.
23. The process of claim 1 wherein the at least one immunogenic or
antigenic macromolecular structure induces an auto-immune disease
or autoimmune reaction.
24. The process of claim 1 wherein the at least one immunogenic or
antigenic macromolecular structure is denatured prior the in-vitro
hydrolysis, either by physical or by chemical methods.
25. The process of claim 1 wherein hydrolysis is a chemical
hydrolysis.
26. The process of claim 1 wherein hydrolysis is a enzymatic
hydrolysis.
27. A peptide of an epitope identified by the process of claim
1.
28. Pharmaceutical of food composition comprising the peptide of
claim 27.
Description
SUBJECT OF THE INVENTION
[0001] The present invention relates to a novel pharmaceutical or
food composition intended for treating pathologies associated with
graft rejection or an allergic or autoimmune reaction.
TECHNOLOGICAL BACKGROUND OF THE INVENTION
[0002] In the last twelve years, controlled studies have described
desensitization based on the oral administration of allergens (1).
This method is based on the fact that the oral administration of an
antigen facilitates the acquisition of an immunological tolerance
to it. The digestive route constitutes the mode of contact of the
body with antigens, of food or microbial origin. However, allergic
reactions are rare. Oral administration of sheep red blood cells
(SRBC) to rats prevents the rats from later producing anti-SRBC
antibodies after a subcutaneous injection, whereas, without the
prior oral intake, the allergic response would have been present.
This phenomenon constitutes what is referred to immunologically as
oral tolerance.
[0003] This oral desensitization method has been validated in
prospective and controlled studies, and makes it possible to reduce
the risks of anaphylaxis, in particular for birch pollen and acari.
It is already available on the vaccines market in a presentation in
drinkable form (sold by the company Laboratoire des
Stallergnes--Paris).
[0004] Moreover, it may be considered that the benefit, in terms of
protecting infants against allergy to milk, which has been observed
since the introduction of new, enzymatically predigested powdered
milk formulations, would result from the induction of immunological
tolerances by the presentation of antigens in the form of
peptides.
[0005] Document WO-A-95/24920 describes the use of complexes
consisting of the combination of a stress protein and a peptide as
a prophylactic or therapeutic vaccine against intracellular
pathogens.
[0006] That document does not in any way describe the possibility
of using such complexes in vivo in the treatment of autoimmune or
allergic diseases or graft rejections.
[0007] In the third paragraph of page 10, that document describes
that said immunogenic and antigenic macromolecular structures can
be associated with autoimmune or allergic diseases, and that, in
this case, said antigen is administered in combination with said
stress proteins in an amount which is sufficient to generate
tolerance or to inhibit a pre-existing immune response against said
antigen in an individual.
[0008] It is indicated that the amount of stress proteins required
to inhibit this immune response is assumed to be substantially
larger than the amounts required to obtain a stimulation.
[0009] However, it is difficult to predict or state the efficacy of
the desensitization. Clinical observation makes it possible, after
the event, to confirm or deny any improvement in the symptoms.
[0010] It is known, from international patent application WO
96/36880, to be able to detect and/or quantify ligands specific to
a pathology associated with an allergic or autoimmune response or
lung cancer, by means of a test of competition between ligands
present in a sample and other discriminable ligands. This test is
based on the fact that allergic and symptomatic individuals
recognize, by means of their antibodies, epitopes different from
those recognized by the antibodies of tolerant individuals on the
same specific immunogenic and antigenic macromolecular structure of
said pathology. That document also describes the possibility of
measuring the evolution of this specificity, in particular in the
case of children who are allergic to milk and the change toward the
in vivo acquisition of tolerance to milk.
[0011] Patent application WO94/29459, "Stress proteins and uses
thereof" relates to a stress protein and methods of modulating an
"individual" immune response. It also relates to a composition
comprising a stress protein joined to another component, such as a
fusion protein in which a stress protein is fuse to an antigen.
According to the invention, it is possible to modulate the immune
response in an individual, such as a human, other mammals or other
vertebrates by altering the individual's response to stress
protein. By altering the individual's immune response to the stress
protein, it is possible to enhance or induce an individual's
response to a pathogen or to cancer cells or enhance or induce an
up-regulation of an individual's immune status and to decrease an
individual's autoimmune response, such as it occurs in some forms
of arthritis.
[0012] U.S. Pat. No. 6,048,530 "Stress protein-peptide complexes as
prophylactic and therapeutic vaccines against intracellular
pathogens" describes a family of vaccines that contain stress
protein-peptide complexes which, when they are administered to a
mammal, are operative to initiate in the mammal a cytotoxic T cell
response against cells infected with a pre-selected intracellular
pathogen. The patent also discloses the preparation of stress
protein and immunogenic stress protein-peptide complexes from
infected cells or tissues, the isolation of potentially immunogenic
peptides from stress protein-peptide complexes from infected cells
or from MHC-peptides complexes. The synthesis of those peptides and
the reconstitution of heat shock protein-peptide complexes with
synthetic peptides or peptides isolated from heat shock
protein-peptide complexes isolated from infected cells is also
described. According to said document, the peptides-heat shock
protein complexes, when isolated from a eukaryotic cell infected
with a pre-selected intracellular pathogen, and then administered
to a mammal can stimulate a cytotoxic T cell response against cells
infected with the same pathogen.
[0013] Patent application WO97/06821 "Heat shock protein-based
vaccines and immunotherapies" describes a composition for inducing
a therapeutic immune response in a subject, comprising : a) a
target antigen; and b) a heat shock protein; wherein the target
antigen and the heat shock protein are combined in vitro under
conditions wherein binding of target antigen to the heat shock
protein occurs to form a target antigen-heat shock protein complex;
wherein the administration of the target antigen/heat shock protein
complex to the subject induces an immune response comprising a
cytotoxic cellular component.
[0014] Patent application WO98/23735 "Immune responses using
compositions containing stress proteins" describes a vaccine for
inducing a cell-mediated cytolytic immune response in a mammal
against an antigen. The vaccine is comprising the antigen and all
or a portion of stress protein or all or a portion of a protein
having an amino acid sequence sufficiently homologous to the amino
acid sequence of the stress protein to induce the immune response
against the antigen. In one embodiment, the antigen is an antigen
of the influenza virus. In another embodiment, the antigen is a
tumor-associated antigen. The patent also describes a mixture of
known allergenic antigens (allergens) and stress proteins or
compositions containing allergens chemically linked or fused to the
stress protein. Allergens used in allergen-stress protein fusion
proteins are necessarily of a peptidic nature; non peptidic
allergens can be used in conjugates containing an allergen and a
stress protein or, a mixture of allergens and stress proteins. Non
limiting examples for allergens include Fel d1 (cat), . . .
[0015] Epitope mapping technology is the identification and
localization of the specific regions of macromolecules that are
recognized by the immune system. An epitope is a small region of a
macro molecule that the immune system recognizes and responds to. T
cell epitopes are linear fragments of the original protein molecule
whereas B cell epitopes can be either linear fragments or folded
three-dimensional regions of the intact macro molecule.
[0016] Several methods for epitopes mapping have been previously
described:
[0017] Classical Epitope Mapping
[0018] Defined fragments of the cDNA for the antigens of interest
are expressed as recombinant (fusion)proteins and probed with
autoantisera in various assays such as Western blot or ELISA.
[0019] Phage Display Technology
[0020] Small random fragments of the cDNA for the antigen of
interest are cloned into the phage protein pIII or pVIII of the
filamentous phage and are displayed on the surface of the phage.
Epitope-displaying phages can be captured with antibodies in a
procedure called "bio-panning". Sequencing of the inserts of the
corresponding phages gives some information on the epitopes. This
procedure is in principle capable to identify conformational
epitopes.
[0021] Peptide Scan Technology
[0022] Small overlapping oligopeptides that ideally cover the
complete amino acid sequence of the antigen of interest are
synthesized on a solid support and probed with antisera. This
method allows the identification of linear epitopes on the amino
acid level. It also allows rapid mutational studies.
[0023] Prior art provides neither a method to prepare in-vitro a
mixture between at least one heat shock protein and peptides
resulting from the in-vitro hydrolysis of at least one immunogenic
protein, nor a method to identify from such a mixture the peptides
that can bind to the stress proteins and/or the peptides that do
not bind to the stress protein.
[0024] Prior art describes neither compositions according to the
invention comprising a mixture of at least one heat shock proteins
and peptides resulting from the in-vitro hydrolysis of at least one
immunogenic protein and antigenic macromolecular structure, nor a
method to prepare such a composition.
[0025] Prior art describes neither compositions according to the
invention comprising a mixture of heat shock proteins, complexes
between the heat shock proteins and the peptides resulting from
said in-vitro hydrolysis, and free peptides resulting from the
in-vitro hydrolysis nor a method to prepare such a composition.
[0026] Prior art describes neither a composition according to the
invention comprising purified complexes between heat shock proteins
and some of the peptides resulting from said in-vitro hydrolysis
nor a method to prepare such a composition.
[0027] Prior art describes neither compositions according to the
invention comprising peptides that do not bind to heat shock
proteins, these peptides resulting from the said in-vitro
hydrolysis nor a method to prepare such a composition.
[0028] Prior art describes neither compositions comprising the
peptides isolated from the complexes made between Heat Stock
Proteins (HSP) and peptides resulting from said in-vitro hydrolysis
nor a method to prepare such a composition.
[0029] Aims of the Invention
[0030] The aim of the present invention is to provide a novel
composition, which may be of pharmaceutical or food type, designed
to modify the immune response of patients toward a pathology
associated with an allergic or autoimmune reaction or toward graft
rejection phenomena, such that the immune response of said patients
comes close to the natural tolerance manifested by normal
individuals (who remain free of symptoms although they are also
liable to be exposed to this pathology).
[0031] The present invention is also directed toward providing an
inexpensive pharmaceutical or food composition which is easy to
administer and which can be used in a prophylactic and/or
therapeutic manner.
[0032] A further aim of the invention is to provide with an easy,
reliable and fast method to prepare in vitro, characterize and
possibly recover immunogenic peptides. The invention is based on
mixing in vitro at least one heat shock protein with peptides
resulting from a in-vitro hydrolysis of at least one immunogenic
and antigenic macromolecular structure.
[0033] Another aim of the invention is to provide with an easy,
reliable and easily up-scalable method to prepare in-vitro and to
characterize and possibly to recover a mixture of at least one heat
shock protein with peptides resulting from the in-vitro hydrolysis
of the said immunogenic and antigenic macromolecular structure.
[0034] Another aim of the invention is to provide with a method to
separate and recover the peptides that do not bind to heat shock
protein and the peptide-heat shock protein complexes, from a
mixture according to the invention. Another aim of the invention is
to characterize and possibly recover such unbound peptides.
[0035] Another aim of the invention is to provide with a method to
isolate from the peptides-heat shock protein complexes according to
the invention, the peptides that bind to the heat shock protein, to
characterize and possibly to recover such peptides.
[0036] Characteristic Elements of the Invention
[0037] The present invention relates to a pharmaceutical or food
composition comprising an adequate pharmaceutical or food vehicle,
a stress protein (also known as "heat shock protein" or HSP) and at
least one of the epitopes (conformational or sequential epitope) of
an immunogenic and antigenic macromolecular structure, said
structure inducing graft rejection, an allergic reaction or an
autoimmune reaction. Preferably, the pharmaceutical or food vehicle
of the composition is adequate for mucosal (in particular oral) or
cutaneous administration.
[0038] Advantageously, the stress protein and the epitope naturally
form in vitro a complex naturally (i.e. without formation of a
covalent bond), as described by Roamn et al., Febs (1994), Fouri et
al., The Journal of Biological Chemistry, Volume 269 No. 48, pp.
30470-30478 (1994), Palleros et al., The Journal of Biological
Chemistry, Volume 269 No. 48, pp. 13107-13114 (1994), Grageroov and
Gottesman, Journal of Molecular Biology, No. 241, pp. 133-135
(1994), and Schmid et al., Science, Volume 260, p. 1991 (1994)
incorporated below by way of reference.
[0039] Advantageously, the stress protein is a bacterial stress
protein present, for example, in saprophytic bacteria, such as E.
coli.
[0040] Among the stress proteins of the present invention, mention
may be made of the stress protein GroEL, the stress proteins GrpE,
DnaK or DnaJ as described in particular by Hendrick and Hartl
(Annual Review of Biochemistry, No. 62, p. 349 (1993)) or the heat
shock proteins HSP 60, 70, etc.
[0041] The expression "phenomenon of graft rejection or allergic or
autoimmune reaction" means hypersensitivity reactions of immediate
or delayed type brought about by contact in particular with an
allergen (this reaction can be immediate and specific (anaphylaxis,
urticaria, etc.) or delayed over time) or autoimmune diseases and
disorders of the immune system of immediate or delayed type
associated with graft rejections of host against graft type and
graft against host type.
[0042] Autoimmunity is a state of immunization of an individual
against his or her own constituents, and the phenomenon of graft
rejection is a state of immunization of an individual against
foreign constituents (bodily fluids such as blood, cerebrospinal
fluid, etc., cells, tissues, organs, antibodies, etc.) deliberately
implanted into the patient. These phenomena are observed in
particular in pathologies selected from the group consisting of
infections associated with SLE (Systemic Lupus Erythematosus
disease), Gougerot-Sjogren syndrome (or Sjogren's disease) and
rheumatoid polyarthritis, as well as pathologies such as
sarcoidosis and osteopenia, spondylarthritis, scleroderma, multiple
sclerosis, amyotrophic lateral sclerosis, hyperthyroidism,
Addison's disease, autoimmune hemolytic anemia, Crohn's disease,
Goddpasture's syndrome, Graves' disease, Hashimoto's thyroiditis,
idiopathic purpural hemorrhage, insulin-dependent diabetes,
myasthenia, pemphigus vulgaris, pernicious anemia,
poststreptococcal glomerulonephritis, psoriasis and spontaneous
sterility, as well as immediate or delayed phenomena observed
during graft rejections.
[0043] The expression "immunogenic and antigenic macromolecular
structure which induces graft rejection or an allergic or
autoimmune reaction" means macromolecules such as allergens made of
peptides, lipides, polysaccharides and/or nucleic acids, preferably
selected from the group consisting of the major allergic antigens
present in chemicals (latex), in foods such as eggs, soya and milk,
in particular bovine beta-lactoglobulin (BLG) from cow's milk, the
major allergic antigens present in plants, molds, medicaments (in
particular antibiotics or vaccines) and pollens, the major allergic
antigens present in animals, in particular in hairs, and venom, in
particular wasp and other insects venom, the major antigens of the
allergic reaction to acari, to the mite present in house dust
(antigen P1 Dermatophagoides pteronyssinus), the major antigen of
Aspergillus fumagatus, and staphylococcal enterotoxin B (SEB).
[0044] Other non-limiting examples of allergens or mixtures of
allergens have also been described in the publication
ISBN-91-970475-5-4 by Pharmacia AB, which is incorporated herein by
way of reference.
[0045] The "macromolecular structure" can also be an antigenic
complex made of peptides, liquids, saccharides and/or nucleic acids
which induces an autoimmune disease. Preferably, this immunogenic
and antigenic macromolecular structure is specific to lupus (SLE)
or Sjogren's disease, in particular the plasma membrane or a
portion of this membrane containing membrane DNA with a weight of
greater than 100 KD, in particular as described in patent
application WO 96/13723, the publication number of which is
incorporated by way of reference.
[0046] Other non-limiting examples of antigenic complexes which
induce autoimmune diseases have also been described by Roitt I. M.
(Essential Immunology, Blackwell Scientific Publication (ch. 14)
ISBN 0-632-01994-8) and by Humbel R. L. (Auto-anticorps et maladies
auto-immunes [Autoantibodies and autoimmune diseases], Ed.
Scientifiques Elsevier (1994) ISBN 2-906077-58-5).
[0047] This immunogenic and antigenic macromolecular structure can
also be major histocompatibility loci (MHC I and/or MHC II) or
minor histocomptability immunogenic and antigenic macromolecular
structures present at the surface of blood cells and involved in
the inducement of the immune system (or portions thereof), which
are specific to an individual and are involved in graft rejection
phenomena (including bodly fluid transfusions).
[0048] The appropriate pharmaceutical or food vehicle according to
the present invention may be any suitable additive or support, such
as a nontoxic compatible substance for administration of the
composition according to the invention to a patient. The type of
appropriate pharmaceutical or food vehicle used will depend on the
mode of administration selected. In particular, for oral
administration, these vehicles can consist of aqueous solutions,
syrups, lozenges, capsules, etc. Other pharmaceutical vehicles such
as creams or ointments may be selected depending on the type of
administration, in particular for cutaneous administrations.
[0049] A person skilled in the art can also adapt the
pharmaceutical vehicle as a function of a subcutaneous,
intradermal, intravenous, intramuscular or parenteral
administration, via nasal or oral inhalation, etc.
[0050] The peptide and epitope according to the invention are
advantageously (possibly) added to a specific emulsion composition,
more preferably an oil in water emulsion which is suitable for
specific mucosal administrations (especially an oral
administration).
[0051] The percentage of active compound present in the composition
according to the invention will depend on the type of patient, the
pathology treated and the route of administration. The doses will
be limited only by the patient's tolerance to the product, as well
as by the administration rates.
[0052] The administration concentrations will be chosen in
particular such that the abovementioned pathological signs and
symptoms are reduced, preferably eliminated, by the administration
doses envisaged by the posology. The preferred concentrations for a
human are: mg(active compounds)/Kg(patient).
[0053] The inventors have discovered, unexpectedly, that the use of
the pharmaceutical and/or food composition according to the
invention makes it possible to modify the immune response of a
patient induced with said immunogenic and antigenic macromolecular
structure. The modification of a patient's immune response can be
detected and quantified in particular according to the process and
technique described in patent application WO 96/36880 or by any
method of clinical analysis of the treated patient (including
prophylactic methods) which is well known to those skilled in the
art.
[0054] Another aspect of the present invention relates to the use
of the composition according to the invention for the preparation
of a medicament designed to modify a patient's immune response
toward an immunogenic and antigenic macromolecular structure which
induces graft rejection or an allergic or autoimmune reaction. In
particular, the present invention relates to the use of the
pharmaceutical and/or food composition according to the invention
for the preparation of a medicament intended to desensitize atopic
or non-atopic allergies.
[0055] Another aspect of the present invention relates to the use
of the pharmaceutical and/or food composition according to the
invention for the preparation of a medicament intended for the
prevention or treatment of the abovementioned allergic reactions
and autoimmune diseases, for the treatment or prevention of graft
rejections, optionally in combination with a specific product for
reducing or neutralizing allergic reactions, autoimmune reactions
and graft rejection phenomena (in particular the administration of
immunosuppressants such as azathioprine, steroids, antilymphocyte
globulins, cyclosporin A, rapamycin, KF-506 (tacrolimus) or
lymphokines (in particular IL-10), and the analogs and agonists
thereof which are well known to those skilled in the art.
[0056] The terms "analogs" and "agonists" of these molecules means
other molecules, or derivatives of these molecules, which act on
the same receptor or via the same mechanism of action as the
abovementioned specific products.
[0057] The present invention also relates to a process for the
therapeutic or prophylactic treatment of a patient, comprising the
step of administration of the composition according to the
invention to said patient so as to modify the patient's immune
response toward an immunogenic and antigenic macromolecular
structure which induces graft rejection or an allergic or
autoimmune reaction.
[0058] The invention is related to any product comprising a mixture
of at least one carrier molecule solected from the group consisting
of heat shock proteins, antibodies, major locus histocompatibility
complex (MHC) or similar molecules and peptides resulting from the
in-vitro hydrolysis of at least one immunogenic protein.
[0059] The invention is related to any product comprising a mixture
according to the invention of:
[0060] peptides resulting from said in-vitro hydrolysis of at least
one immunogenic protein,
[0061] complexes between said carrier molecule and the peptides
resulting from said in-vitro hydrolysis uncomplexed carrier
molecules.
[0062] The invention is also related to any product comprising
purified complexes between at least of said carrier molecule and
peptides resulting from said in-vitro hydrolysis.
[0063] The invention is related to any product comprising the
peptides isolated from the complexes between a said carrier
molecule and peptides resulting from the said in-vitro
hydrolysis.
[0064] The invention is also related to any pharmaceutical or food
compositions comprising any product according to the invention. In
a preferred embodiment, the products according to the invention are
associated with immuno-modulators like but not limited to
cytokines, anti-cytokines, corticoids, vaccine adjuvants,
antibodies, thymic peptides, nanoparticles, emulsions and
liposomes. In another embodiment, the composition comprise at least
two carrier molecules complexes according to the invention.
[0065] The invention is also related to any pharmaceutical or food
compositions according to the invention able to enhance an immune
response against the entire protein through, but not limited to,
T-cells cytotoxic immune response or to down-regulating immune
response against the entire protein or any autoimmune diseases
associated protein through, but not limited to, the mechanisms
described by H. L. Wiener in Encyclopedia of Immunology, 2.sup.nd
edition Academic Press (Ed P. J. DELVE & I. M. ROITT) pages
1893-1899 or by W. O Weigle in Encyclopedia of immunolgy 2.sup.nd
edition Academic Press (Ed P. J. DELVE & I. M. ROITT) pages
2359-2361 or by D. W Scott in Encyclopedia of Immunology 2.sup.nd
edition Academic Press (Ed P. J. DELVE & I. M. ROITT) pages
2362-2367. In a preferred embodiment the tolerance mechanism is a
low dose mechanism. In another preferred embodiment the composition
according to the invention induces TGF-.beta. producing T-cells
named Th3 T-cells.
[0066] The invention is also related to any diagnosis kit including
any product according to the invention. In a preferred embodiment,
the product according to the invention included in the diagnosis
kit comprises at least two peptides-carrier molecule complexes
according to the invention.
[0067] In a preferred embodiment of the invention, the proteins
are:
[0068] any protein or other macromolecular structure or recombinant
protein or mutated protein or combination thereof related to an
autoimmune disease among other insulin, thyroglobulin, type II
collagen, gliadin, GAD65, proteolipid protein, S-antigen,
acetylcholin receptor, haptenized colonic proteins,
interphotoreceptor retinoid binding protein, myelin, peripheral
nerve P2, LDL, HDL
[0069] any protein or other macromolecular structure or recombinant
protein or mutated protein or combination thereof related to
allergy like the allergens according to Diagnostic Testing of
Allergic disease Clinical Allergy and Immunology S. F. Kemp &
R. F Lockey ISBN 0-8247-0303-0 pages 13-44.
[0070] any protein or other macromolecular structure or recombinant
protein or mutated protein or combination thereof related to
allograft and/or xenograft rejection like xenoantigen, alloantigen,
MHC-peptides and blood group antigens.
[0071] any protein or other macromolecular recombinant protein or
mutated protein or combination thereof from pathogens responsible
of any infectious or parasitic diseases like virus, bacteria,
fungi, protozoa and helminth.
[0072] any carrier molecule (heat shock protein related to
autoimmune diseases or any recombinant protein or mutated protein
or combination thereof or blood proteins).
[0073] In another preferred embodiment, the macromolecular
structure, especially the protein, the recombinant protein, the
mutated protein or the combinations thereof are extensively washed
in an adequate solution through, but not limited to,
ultra-filtration or dialysis to remove any low molecular weight
material loosely associated with it.
[0074] Hydrolysis can be performed by enzymatic digestion with at
least one protease or other suitable enzyme of any living organism.
The proteases could be selected among the list according to the
Nomenclature Committee of the International Union of Biochemistry
and Molecular Biology at
http://www.chem.qmw.ac.uk/iumbmb/enzyme/EC34, and the list of the
MEROPS database http://www.merops.co.uk and of the Rawlings ND and
Barret AJ MEROPS: the peptidase database; Nucl. Acids Res. 28
323-325 (1998), and of Barret AJ, Rawlings ND Woessner JF (eds)
1998 Handbook of Proteolytic Enzymes, Academic Press London.
[0075] In a preferred embodiment, macromolecular structures,
especially proteins, known to be resistant to in-vitro or in-vivo
hydrolysis are denatured either by physical (e.g. heating, high
mechanical pressure) or by chemical methods (e. g. reductive
reagents, urea, guanidinium chloride).
[0076] In another preferred embodiment, the structures resistant to
hydrolysis are allergens (e.g. OVA).
[0077] In another preferred embodiment, the macromolecular
structures with a proteolytic activity are denatured or
specifically inhibited before enzymatic digestion (e. g; Der
p1).
[0078] Hydrolysis can also be performed with at least one chemical
agent like, but not limited to, mild acid (70% formic acid at
40.degree.), hydroxylamine, cyanogen bromide, iodosobenzoic acid or
2-nitro-5-thiocyanobenzoate followed by alkali.
[0079] In a preferred embodiment, the peptides or glycopeptides
resulting from in-vitro hydrolysis of at least one macromolecular
structure can be separated on the basis of their molecular weight
e.g. by ultra-filtration or gel filtration or dialysis prior to the
mixing with the heat shock proteins. In a preferred embodiment, the
cut-off of the filter or of the membrane is about 10 kDa and at
most 10 kDa (other suitable filters to be used have cut-offs of
about 5 or 50 kDa).
[0080] In a preferred embodiment, the peptides or glycopeptides
resulting from the in-vitro hydrolysis can be separated on the
basis of their hydroplylic/hydrophobic properties e.g. by phase
separation or solvent extraction before the mixing with the carrier
molecule. Prior to the mixing with the carrier molecule, the
organic phase is removed e.g. by solvent stripping or solvent
evaporation or lyophilisation and hydrophobic peptides are
dissolved in an adequate water solution e.g. a suitable buffer to
make the complexes with the HSP.
[0081] In a preferred embodiment, the peptides or glycopeptides
resulting from the in-vitro hydrolysis are bound to a solid support
e.g. by covalent link, hydrophobic interactions, interactions
between biotin or biotin derivatives and an avidin selected among
the avidin protein families.
[0082] Characterization of the peptides or glycopeptides resulting
from said hydrolysis can be performed either by ion exchange
chromatography, or by gel electrophoresis, gel filtration or
electro-elution, or by reverse phase high pressure liquid
chromatography (HPLC).
[0083] Peptides that were bound to the HSP can be individually
collected after HPLC or ion exchange chromatography elution, or by
gel electrophoresis, gel filtration or electro-elution.
[0084] N-terminal peptides sequence analysis can be performed by
procedures based upon the Edman degradation reaction. C-terminal
peptides sequence analysis can be performed by using non specific
carboxypeptidases (A, B, P, Y) . In another embodiment said peptide
sequence can be performed by mass spectrometry analysis (e. g.
MALDI or MALDI-TOF or ESI or LC-MS or MS/MS or FAB).
[0085] Peptides-carrier molecule complexes according to the
invention can be characterized directly from the crude mixture by
mass spectrometry e.g. MALDI or MALDI-TOF.
[0086] Carrier molecules, especially the heat shock proteins
according to the invention are molecular chaperones according to
Mary-Jane Gething "Guidebook to molecular chaperones and protein
folding catalysts" A Sambrook & Tooze publication at Oxford
University Press and "Molecular Chaperones and Folding
Catalysts--Regulation, Cellular Function and Mechanisms" B.
Bukau--Harwood Academic publishers. In a preferred embodiment of
the invention, the heat shock proteins are purified and depleted of
any bound-peptides according to the method known by the person
skilled in the art, e.g. incubation in a low-pH buffer or in an
ATP-containing buffer.
[0087] In another preferred embodiment, the heat shock proteins are
partial or total recombinant heat shock proteins or any homologous
sequences of said HSP described previously. In another preferred
embodiment the heat shock protein are microbial heat shock protein
(bacteria, fungi and yeast).
[0088] In a preferred embodiment, the carrier molecules are bound
to a solid support, e.g. by covalent links, hydrophobic reactions,
interactions between biotin and biotin derivatives and an avidin
chosen among the avidin protein families, gelatin, ATP--or ADP
affinity column.
[0089] The mixture is obtained by mixing in vitro at least one
solution of peptides resulting from an in vitro hydrolysis of at
least one protein with at least one solution containing at least
one carrier molecule. In this case it is bound to a solid support,
at least one solution of peptides resulting from the hydrolysis of
at least one carrier molecule is put into contact with the solid
support. In the case of peptides resulting from the in-vitro
hydrolysis of at least one protein are bound to a solid support, at
least one solution of at least one carrier molecule is put into
contact with the solid support. In a preferred embodiment, the
mixing is performed under conditions allowing the formation of
peptide-carrier molecule complexes. Such conditions are described
in the prior art.
[0090] The compositions according to the invention are prepared as
a galenic form to modulate the immune response.
EXAMPLES
Example 1
Epitopes Mapping
[0091] Materials
[0092] Laboratory bench microcentrifuge
[0093] Water bath 37.degree. C.
[0094] Water bath 25.degree. C.
[0095] Water bath 20.degree. C.
[0096] Centricon YM-10 (Millipore)
[0097] Tris.HCl 20 mM pH 8.0
[0098] Tris.HCl 1.0 M pH 6.8
[0099] Distillated water pH 2.0 (pH adjusted with HCl 0.1 N)
[0100] Centricon YM-10 (filtre cut-off 10 kDa)
[0101] HEPES 25 mM ; KCl 10 mM ; MgCl.sub.23 mM ;
.beta.-mercaptoethanol 5 mM ; pH 7.5 : buffer 1
[0102] ATP (Sigma, A-2383)
[0103] ADP (Sigma, A-6646)
[0104] Pepsin (Sigma, P-6887)
[0105] Trypsin, TPCK-treated (Sigma, T-1426)
[0106] Proteins of Interest
[0107] Bovine beta-lactoglobulin (Sigma, L-0130) or BLG
[0108] Bee venom phospholipase A2 (Sigma, P-9279) or PLA2
[0109] Ovalbumin (Sigma, A-5503) or OVA
[0110] Myelin basic protein (Sigma, M-1891) or MBP rec Alt a 1
(Altermono Altanotx allergen) (Biomay Produktions und
HandelsgesmbH, Dr Bohr-Gasse 7b, A-1030 Wien)
[0111] rec Bet v 1a Biotech allergen (Biomay Produktions)
[0112] HSP Preparation
[0113] The DnaK protein was purified from E coli BR1640 transformed
with the plasmid BR1639/pBM19 carrying the coding sequence of DnaK.
DnaK was purified from a crude extract of the bacteria grown in LB
medium containing tetracyclin. Protein expression was induced with
1 mM IPTG during four hours. The protein was purified according to
the literature in three steps (ion exchange chromatography,
hydroxyapatite column and gel filtration). The purity of the
protein was checked by a SDS page gel electrophoresis. Overloading
of the gel with the protein shows a purity of at least 98%.
[0114] Protein Preparation
[0115] The protein of interest is extensively washed with
distillated water pH 2.0 in case of pepsin digestion or with
trypsin digestion buffer in case of trypsin digestion by
centrifugation through a centricon YM-10 assembly to remove any low
molecular weight material loosely associated with it.
[0116] Pepsin Digestion
[0117] One to five milligrams of the protein of interest is
dissolved in 1 to 5 mL of distillated water pH 2.0 (final
concentration of 1 mg/mL) . The solution is incubated for 10
minutes at 100.degree. C. The solution is then rapidly cooled off
at 4.degree. C., and 20 to 100 .mu.L of pepsin solution (10 mg/mL
of distillated water pH 2.0, final ration protein/protease of 0.2)
is added to the protein solution. The resulting solution is
incubated at 37.degree. C. for six hours. A 200 .mu.L aliquote is
removed at the end of the incubation and placed into 200 .mu.L of
20 mM Tris.HCl pH 8.0. The sample is then centrifuged through a
centricon YM-10 assembly to remove the remaining protein and
pepsin.
[0118] Trypsin Digestion
[0119] One to five milligrams of the protein of interest is
dissolved in 1 to 5 mL of Tris.HCl 40 mM pH 8.0 (final
concentration of 1 mg/mL). The solution is incubated for 10 minutes
at 100.degree. C. The solution is then rapidly cooled off at
4.degree. C., and 18 to 90 .mu.L of .beta.-mercaptoethanol is added
(1.8% v/v). The resulting solution is incubated at 37.degree. C.
for 10 minutes and 20 to 100 .mu.L of trypsin solution (10 mg/mL of
Tris.HCl 40 mM pH 8.0, final ration protein/protease of 0.2) is
added to the protein solution. The resulting solution is incubated
at 37.degree. C. for six hours. The solution is then centrifuged
through a centricon YM-10 assembly to remove the remaining protein
and trypsin.
[0120] DnaK.ATP Complex Preparation
[0121] 25 .mu.L of ATP solution (4.5 mg/mL of buffer 1) is added to
400 .mu.L of DnaK (2mg/mL of buffer 1). The solution is incubated
at 20.degree. C. for one hour, and then is centrifuged through a
centricon YM-10 assembly to remove any low molecular weight
material loosely associated with Dna K. The large molecular weight
fraction is removed, and washed extensively with buffer 1 by
ultrafiltration using a centricon YM-10.
[0122] In Vitro Production of Stress Protein-Peptide Complexes.
Isolation of the Bound Peptides
[0123] The ultrafiltrated and neutralized pepsin or trypsin
digestion is mixed with the ATP-pretreated DnaK to give at least a
1:1 (w:w) DnaK:peptides ratio. Then, ADP is added (1 mM final) and
the mixture is incubated for one hour at 25.degree. C. in the
suitable buffer 1. The preparations are centrifuged through a
centricon YM-10 assembly to remove the remaining unbound peptides.
The low and the large molecular weight fractions are recovered. The
large molecular weight fraction containing DnaK-peptide complexes
is washed extensively with buffer 1 containing 1 mM ADP by
ultrafiltration using a centricon YM-10. The bound peptides are
eluted by incubating the HSP-peptide complexes in a low pH buffer.
A last ultrafiltration using a centricon YM-10 is removing the
large molecular weight fraction. The incubation of the chaperone in
ATP solution is not required to form complexes between chaperones
and peptides.
[0124] HPLC Analysis
[0125] The resulting low molecular weight fractions are
fractionated by reverse phase high pressure liquid chromatography
(HPLC) using a Vydac C18 reverse phase column (HP32, 201TP52 C18,
250/2.1 mm,5 .mu.m) equilibrated with buffer A (99.0% H.sub.2O; TFA
0.5/1000 v/v). The bound material is eluted at a flow rate of 0.2
mL/min by developing the column with a gradient of 0 to 100% of
buffer B (1.0% CH.sub.3CN; TFA 0.45/1000 v/v) . The elution of the
peptides can be monitored at both OD 214 nm and OD 280 nm.
[0126] Characterization
[0127] Peptides that were bound to DnaK can be individually
collected after HPLC elution. Both their mass and aminoacid
sequences can be determined.
[0128] The FIGS. 1 to 6 present the results of reverse-phase HPLC
separation of peptides. Chromatograms generated by monitoring
absorption at 214 nm. OVA: ovalbumin; BLG: beta-lactoglobulin ,
PLA2: phospholipase A2 from bee venom; MBP: myelin basic protein;
Alta1=major allergen of Alternaria Alternata; Betula=major allergen
of Birch.
[0129] FIG. 1.1: peptides (MW<or=10 kDa) generated by
pepsin-cleavage of OVA
[0130] FIG. 1.2: unbound peptides (MW<or=10 kDa) to DnaK, from
the pepsin-cleavage of OVA.
[0131] FIG. 1.3: pepsin generated-OVA peptides (MW<or=10 kDa)
that were bound to DnaK.
[0132] FIG. 2.1: peptides (MW<or=10 kDa) generated by
pepsin-cleavage of PLA2.
[0133] FIG. 2.2: unbound peptides (MW<or=10 kDa) to DnaK, from
the pepsin-cleavage of PLA2
[0134] FIG. 2.3: pepsin generated-PLA2 peptides (MW<or=10 kDa)
that were bound to DnaK.
[0135] FIG. 3.1: peptides (MW<or=10 kDa) generated by
pepsin-cleavage of MBP.
[0136] FIG. 3.2: unbound peptides (MW<or=10 kDa) to DnaK, from
the pepsin-cleavage of MBP
[0137] FIG. 3.3: pepsin generated-MBP peptides (MW<or=10 kDa)
that were bound to DnaK.
[0138] FIG. 4.1: peptides (MW<or=10 kDa) generated by
trypsin-cleavage of BLG.
[0139] FIG. 4.2: trypsin generated-BLG peptides (MW<or=10 kDa)
that were bound to DnaK.
[0140] FIG. 5.1: peptides (MW<or=10 kDa) generated by pepsin
digestion of Alt a 1.
[0141] FIG. 5.2: unbound peptides (MW<=10 kDa) to Dna-K, from
the pepsin cleavage of Alt a 1.
[0142] FIG. 5.3: pepsin generated-Alt a 1 peptides (MW<=10 kDa),
that were bound to DnaK.
[0143] FIG. 6.1: peptides (MW<or=10 kDa) generated by pepsin
digestion of Bet v 1a.
[0144] FIG. 6.2: unbound peptides (MW<=10 kDa) to the HSP, from
the pepsin cleavage of Bet v 1a
[0145] FIG. 6.3: pepsin generated-Bet v 1a peptides (MW<=10
kDa), that were bound to the HSP.
[0146] FIG. 7: chromatogram of any potential material, including
ADP and/or ATP, bound to ATP-treated DnaK.
Example 2
Diagnosis Test Including a Mixture of Peptides Resulting from the
Pepsin Digestion of Beta-Lactoglobulin and E. coli HSP
[0147] Pools of purified IgG from milk allergic children
(Allergics) and from healthy controls (HC) were labeled with biotin
and allowed to react with solid phase bound peptides. These
peptides were obtained after pepsin digestion of beta lactoglobulin
(BLG) and ultrafiltration (<10 kD). Antibody binding was
assessed with streptavidin-peroxidase conjugate, and appropriate
substrate coloration.
[0148] Interference on the antibody binding was examined by mixing
peptides (i.e. pepsin digested BLG=pB), HSP or HSP-pB mixture (w/w)
together with the antibody solution.
[0149] The presence of peptides alone had no effect on the actual
binding efficiency of Allergics or HC IgG. Neither GroEL alone nor
GroEl-peptides complexes were inhibitors.
[0150] The presence of DnaK-pB complexes but not DnaK alone was
inhibiting the binding of allergic IgG but not those from HC,
indicating that conformational modifications of allergenic peptides
through DnaK complex formation was competing with solid phase bound
allergenic peptides for allergic IgG. Other results with DnaJ and
GrpE were unconclusive here as both HSP alone or in peptide
complexed form were inhibitors for both allergics and HC.
[0151] The DnaK example is demonstrating the potential use of
HSP-peptide complexes in allergic diagnostic tests as discriminant
from normal non allergic subjects.
[0152] The enclosed FIG. 8 represents the relative % of binding
efficiency of pools of purified IgG from milk allergic children
(Allergics) and from healthy controls (HC), expressed as the ratio
of the measured optical density in the presence of potential
interfering substances and the control binding in the presence of
the dilution buffer alone (PBS) (pB:peptides resulting from
beta-lactoglobulin pepsin digestion (<10 kD) . HSP were : GroEl,
DnaK, DnaJ, GrpE as recombinant proteins from E. Coli. (+pB+HSP)
are mixtures of peptides resulting from beta-lactoglobulin pepsin
digestion (<10 kD) and the above mentioned HSP (w/w)).
Example 3
Antigenic Nature of HSP-Peptides Complexes
[0153] 1. Betalactoglobulin
[0154] Peptides obtained from pepsin digestion of betalactoglobulin
(BLG) and ultrafiltrated (<10 kD) were shown to remain antigenic
in vitro as attested by their capacity to stimulate the lymphocyte
proliferation of any sensitized human subject, in a similar way to
that induced by the intact whole BLG molecule. Detectable levels of
lymphocyte stimulation were obtained after 5 days cultures and
assessed by H3 thymidine incorporation. The lowest stimulant
concentration of BLG or its pepsin digested peptides falled to 10
ng/ml. Using a non stimulant concentration of DnaK (100 ng/ml) in
culture, the admission to the mixture of pepsin digested peptides
lowered the stimulant antigenic treshold by a factor of 10 (i.e. 1
ng/ml) . This is indicating the adjuvant effect of DnaK when joined
to sub treshold levels of antigen.
[0155] 2. Allogenic Peptides
[0156] Membranes of lymphomononuclear cells were isolated from C3H
mice spleens, homogenized, submitted to pepsin digestion and
ultrafiltrated to keep peptides <10 kDa. When used as antigen to
stimulate lymphocytes from the allogenic strain of Balc/c mice,
their was no response at any tested concentration. Mixtures of
these peptides with DnaK allowed a significative stimulation
manifested by increased H3 thymidine incorporation, as could be
induced by intact C3H cells. The response was not visible if the
responding cells were of the same strain origin as the peptide
donor. The specificity of these responses is entirely superposable
to those of mixed lymphocyte cultures and excludes a role for any
unspecific stimulation that could have occurred, as from the DnaK
for example. Indeed DnaK alone was not stimulant. This is
documenting that DnaK complexes are able to present allogenic
peptides in an available form to responding lymphocytes, and that
it renders possible to stimulate cells with a soluble form of
antigens that are usually presented in a cellular form.
Example 4
Pharmaceutical Compositions
[0157] A. Basis of the Model Considered
[0158] a) Using the Oral Route
[0159] Oral administration allows an induction of immunological
tolerances, and is increasingly widely applied in the field of
antiallergic desensitization. However, it requires the use of
larger amounts of antigens than via the parenteral route, and has
to extend over periods of at least several years (2, 3).
Optimization of the dose administration regime and of their
periodicity can be adapted by a person skilled in the art so as to
avoid syndromic reactions (replication of the allergic
symptomatology in the case of an overdose), which are frequent but
not dangerous on account of the slow progression in the increase of
the doses administered (2).
[0160] b) Using Peptide-Stress Protein Complexes
[0161] Stress proteins (heat shock proteins (HSP)) constitute a
series of protein families, which have been highly conserved during
evolution from bacteria through to man, and which have the capacity
to bind to peptides or to proteins whose conformational structure
is altered or on course to its final conformation (4).
[0162] They have several roles, including participation in
intracellular transport leading to polypeptide assembly for the
synthesis of certain proteins or their elimination. Some are
expressed at the surface of different cells and can contribute to
the antigenic presentation, in particular to T lymphocytes to the
receptors for antigen of gamma-delta type, which colonize the
mucous membranes and lymphoid organs associated with the digestive
mucosa.
[0163] The antigenic presentation via HSPs of the family HSP70, to
gamma-delta (.gamma., .delta.) T lymphocytes makes it possible to
dispense with the presentation dependent on the type II major
histocompatibility complex.
[0164] Parenteral injection of HSP-peptide complexes into
experimental animals makes it possible to obtain a noteworthy
adjuvant effect (5, 6) which determines or amplifies the antigenic
power of these peptides.
[0165] Certain bacterial HSPs of the families HSP60 and HSP70 are
the target of immune responses which have a protective role with
regard to infection with these microorganisms.
[0166] It has recently been proposed to perform desensitization
orally by giving peptide extracts of E. coli containing HSP60 to
patients suffering from rheumatoid arthritis, with a certain
beneficial effect (5, 6). Considering the negligible side effects,
the authors propose to attempt the test on other inflammatory
complaints in order to manipulate a response directed against one
of these microbial HSPs itself, which is considered as an
auto-antigen substitute.
[0167] The inventors have discovered, unexpectedly, that stress
proteins constitute a noteworthy vector for presenting peptides to
lymphoid systems of the digestive tract and inducing a tolerance.
The stress proteins of saprophytic bacteria appear to be the ones
which are the most abundant in nature in the digestive lumen. It is
also probable that the peptides derived from the digestion of food
constitute the most abundant mass of antigenic fragments available
for the formation of HSP-peptide complexes. However, the abundance
of the peptides generated, and the presumed limited amount of
bacterial HSPs makes the formation of an immunologically efficient
amount of these HSP-antigenic peptide complexes uncertain, and all
the more so since the absorbed amount of antigen with desensitizing
intent is very low (a few tens of .mu.g) with regard to the food
protein load.
[0168] The inventors have proposed to promote the formation of
these complexes before arrival in the digestive tract, i.e. in
vitro, by using purified E. coli stress proteins and peptides
derived from the digestion of BLG with pepsin.
[0169] c) Using Tests of Competition Between Serum Antibodies and
Monoclonal Antibodies for BLG
[0170] The two monoclonal antibodies referred to below as M6 and M7
each recognize a different conformational epitope on the BLG
molecule. Their different qualitative properties are used as
recognition markers for single epitopes in a competition with all
of an individual's serum antibodies. It emerges from clinical
studies that symptomatic individuals and asymptomatic individuals
recognize on this molecule epitopes which, for at least a part, are
different (7), which are referred to below as epitopic
profiles.
[0171] The epitope recognized by M6 is recognized particularly well
by allergic and symptomatic individuals. Binding of the M6 antibody
to intact BLG is actually better inhibited by the sera of children
who are allergic to milk than by the sera of non-allergic
individuals, whether these are children or adults in good health
(blood donors).
[0172] The epitope recognized by M7 is better recognized by
asymptomatic individuals than by allergic individuals. Binding of
the M7 antibody to BLG is better inhibited by asymptomatic
individuals than by allergic individuals.
[0173] Antigenic binding competitively against M6 is used as a
specificity index representing the epitopic profile recognized by
the allergic individuals, and in a complementary manner, the
competition against M7 as a specificity index representing the
epitopic profile recognized by the asymptomatic individuals.
[0174] Validation of this interpretation was confirmed
longitudinally by clinical studies. The acquisition of a state of
tolerance to milk is accompanied by a conversion of the fine
specificity of the serum antibodies, toward the standard profile of
asymptomatic individuals.
[0175] It is this epitopic discrimination expressed at the level of
the circulating antibodies which serves here as an analytical tool
for influencing oral antigenic modulation.
[0176] B. Experimental Model
[0177] Syngenic mice received, in their drinking water, very small
amounts of peptides derived from the peptic digestion of
beta-lactoglobulin (BLG), which were precoupled or otherwise with
purified stress proteins and whose functional capacity was intact
(capacity to bind to impaired peptides or proteins).
[0178] a) Animal Origin and Rearing Conditions
[0179] 40 individuals 8 to 16 weeks old were taken from a rearing
stock of Balbc mice fed for several generations on a diet poor in
cow's milk: 13 .mu.g of beta-lactoglobulin/gram of nutrient
granules.
[0180] b) Preparation of the Antigenic Complexes
[0181] BLG was digested on contact with pepsin coupled to agarose
(Sigma) under incomplete digestion conditions, and then filtered on
a 10,000 dalton filter. The concentration of the digestion product
(pB) was measured by spectrophotometry (yield of 30 to 50% of
intact protein).
[0182] 1 .mu.g/ml phosphate buffer solutions (PBS) were incubated
with 1 .mu.g/ml solutions of the following E. coli stress proteins:
DnaK, DnaJ, GroEL, GrpE (Stressgen) for at least one hour at
ambient temperature. 1 ml aliquots of each combination were
frozen.
[0183] c) Treatment Groups and Oral Posology of the Complexes
[0184] A solution of complexes (1 ml) was added, after thawing, to
the 100 ml water bottle given daily to each cage of four mice. Each
type of complex is administered to 8 mice. A control group receives
the non-complexed pB antigen.
[0185] The solution was added 3 times a week for two weeks (i.e.
six times), from time zero.
[0186] d) Antibody Response
[0187] An individual sample of blood was taken from the
retro-orbital plexus at time zero and after 4 weeks. The animals
are anaesthetized with ether and then exsanguinated, after 8
weeks.
[0188] The specificity of the serum antibodies was examined by an
ELISA type competitive test.
[0189] e) Test of Antibody Specificity by Competition
[0190] Polystyrene multiwell plates are passively covered, by
absorption at ambient temperature, with a small amount of BLG (0.3
.mu.g/ml in bicarbonate buffer) and then saturated with gelatin
(1%, :weight/vol--Haemacel (R)).
[0191] The mouse serum is diluted 100-fold with dilution buffer
(PBSdil) consisting of: PBS-EDTA (10 mM)--Tween 20 (0.05%)--gelatin
(Haemacel--1%).
[0192] Two murine monoclonal antibodies produced were selected for
their specificity with regard to conformational epitopes of BLG.
They were biotinylated and are used at their limit dilution for
antigenic binding, defined in the following way: the dilution which
allows a maximum signal but which is sensitive to any reduction in
the antigen load, at its specific dilution, and which can be
competitively inhibited with a pool of sera from untreated mice.
The reason for this is that the latter produce natural antibodies
against BLG, in relation to exposure to the food antigen, even
minimal exposure.
[0193] 100 .mu.l of diluted serum and of biotinylated antibody are
mixed together in a well, in duplicate.
[0194] After incubation overnight at ambient temperature, the
binding of the monoclonal antibody is measured by the proportional
retention of biotin revealed by uptake of streptavidin coupled to
horseradish peroxidase. This peroxidase colors an
ortho-phenylenediamine substrate. The optical density (O.D.) is
measured by spectrophotometry. The background noise (b.n.) is
measured in antigen-free wells. The maximum binding is defined
either in the absence of competition (monoclonal antibody alone) or
in the presence of relatively non-inhibiting serum.
[0195] The results are expressed as a percentage of inhibition of
binding of the monoclonal antibody by:
% inhibition=100.times. (test O.D.-b.n. O.D.)/(maximum O.D.-b.n.
O.D.)
[0196] The correspondence between the profile of epitopes
recognized on the antigen and the individual's clinical state
(tolerance or otherwise) is confirmed by other examples:
[0197] model of allergy to acari: the evolution of the fine
specificity of anti-acari antibodies in allergic children shows the
existence of an epitopic profile under the effect of the
desensitization induced both parenterally and orally,
[0198] the evolution of the antibodies
[0199] g) Results
[0200] FIG. 9 summarizes the experimental data:
[0201] Inhibition of the M6 Antibody
[0202] The left-hand side shows the changes in the averages of
inhibition (+ standard deviation) of the binding of the M6
monoclonal antibody by the individual sera, for the various
treatment groups.
[0203] The numerical data are compiled in Table 1.
[0204] The control group receiving the peptides digested with
pepsin (pB) shows an increase in its average inhibition capacity
from 45 to 60% and 59% after 4 and 8 weeks. This variation is
significant (p<0.05-paired T test) relative to the start, but
stable after 4 weeks.
[0205] For the group receiving the DnaK-pB complexes, this capacity
rises from 48 to 56% and then 77% over the same period, the latter
being greater than in the control group (p<0.01-non-paired T
test) and very significant relative to the time zero
(p<0.001-paired T).
[0206] Similarly, the groups receiving the complexes DnaJ-pB,
GroEL-pB and GrpE-pB show a very significant increase after 4 weeks
and which rises further at the eighth week (this value is
considerably higher than the value of the control group for the
complexes GroEL-pB and GrpE-pB at the corresponding moment).
[0207] Inhibition of the M7 Antibody
[0208] The right-hand side of FIG. 9 shows the changes in the
averages of inhibition (+ standard deviation) of the binding of the
M7 monoclonal antibody, by the individual sera, for the various
groups treated.
[0209] The numerical data are compiled in Table 2.
[0210] The control group receiving the BLG peptides digested with
pepsin (pB) have an average inhibition capacity which falls from 70
to 52% and 57% in 4 and 8 weeks. This variation is significant
(p<0.01-paired T test), although stable after 4 weeks.
[0211] For the group receiving the DnaK-pB complexes, this capacity
already reduces significantly at the fourth week, falling from 68
to 51%, as in the control group.
[0212] However, the result collapses at 17% at the eighth week
(p<0.001-paired T), which is markedly lower than that of the
control group for the corresponding sample (p<0.01-non-paired T
test).
[0213] The change is parallel to that for the group treated with
the DnaJ-pB complexes.
[0214] For the group receiving GroEL-pB complexes, the reduction in
the inhibitory power is immediately maximal, reaching 28% from the
fourth week, and remains at the same level, 30%, at the eighth
week.
[0215] In the group receiving GrpE-pB complexes the reduction in
the inhibitory power is also immediately maximal, falling from 72
to 22% from the fourth week, but appears subsequently to diminish,
returning to an average level of 41%.
[0216] h) Conclusion
[0217] The administration of peptides derived from the enzymatic
digestion of a major milk antigen, in this instance
beta-lactoglobulin, in the form of complexes associated with stress
proteins according to the invention, and via the oral route,
results in a radical and very rapid modification in the profile of
the epitopes recognized by the circulating antibodies. These
antibodies are naturally present in all the individuals exposed to
the antigen via their food. In a model of mice, chronically exposed
to a small amount of the antigen via this route, the dose of
antigen administered over a brief period of time is very low, far
below the amount ingested naturally (estimated at 0.25 .mu.g per
individual and per day of treatment in the form of complexes and
150 .mu.g per individual and per day in the common diet).
[0218] The speed of the change is all the more noteworthy since the
half-life of the serum antibodies, mainly IgGs, is 3 weeks, which
means that at the eighth week, there should still be a quarter of
the antibodies present at the end of the treatment of only 2 weeks.
All the stress proteins used were efficient. In a second experiment
with DnaK-pB complexes, an attempt to determine a lower limit dose
was unsuccessful, despite the use of doses as much as 10 times
lower (0.1 .mu.g/100 ml bottle/3 days per week).
[0219] C. Orally Induced Tolerance with Regard to Major
Histocompatibility Antigens
[0220] 1. Experimental Model
[0221] Syngenic animals (Balbc mice) receive a protein preparation
dissolved in their drinking water. It contains fragments of
histocompatibility antigens from syngenic mice of another strain,
any graft from which they would reject (C3H mice).
[0222] The tolerance-generating effect is expected to be enhanced
when these fragments are combined with a bacterial stress protein
(in this case Dnak from E. coli).
[0223] As a control, a group of mice receive, in the same manner, a
complex of Dnak with peptide fragments similarly obtained from
beta-lactoglobulin (major antigen of milk).
[0224] It should be expected that the oral sensitization would
specifically attenuate the lymphocyte reactivity with regard to a
strain of foreign lymphocytes of the same type as those used for
the oral preparation and not with respect to an unrelated third
strain.
[0225] 2. Materials and Method
[0226] a) Animals:
[0227] 3 groups of 12 mice are taken from a rearing stock of
syngenic mice of Balbc strain are reared in cages of 6 animals.
Each group receives, for 2 weeks, one of the following preparations
in the bottle of drinking water at a rate of 3 distributions per
week (every other day and not at the weekend) and a dose of 1 .mu.g
of complex per 100 ml of water:
[0228] a complex of Dnak-beta-lactoglobulin peptides (control
preparation)
[0229] a solution of pepsin-digested spleen lymphocyte membrane
peptides (containing fragments of histocompatibility antigens) from
CH3 mice
[0230] a complex of these peptides associated with purified Dnak
from E. coli (Stressgen).
[0231] b) Test of Acquired Tolerance (in vitro)
[0232] This test is based on a monodirectional mixed lymphocyte
culture.
[0233] The responding cells are isolated from the spleen of the
test animals. The lympho-monocyte cells are obtained after
centrifugation on a density gradient with a ficoll-isopaque mixture
(Pharmacia). They are resuspended using 4 million cells/ml in RPMI
1640 culture medium buffered with Hepes and with bicarbonate,
supplemented with 2-mercaptoethanol, glutamine, geomycin and 10%
calf serum.
[0234] The stimulating cells are obtained in the same way from mice
of different strains, from their MHC: the C3H strain is a domestic
(DOM) strain.
[0235] They are incubated for one hour in the presence of mitomycin
in order to block their ability to multiply. They are then
resuspended under the same conditions as the responding cells.
[0236] Lymphocyte Culture:
[0237] An equal volume of suspension (0.1 ml) of responding cells
and of stimulating cells are mixed, in triplicate, in
round-bottomed wells of polystyrene multiwell culture plates in
order to be subsequently incubated in an air/CO.sub.2 (95/5%;
vol/vol), humidified incubator at 37.5.degree. C. for 5 days.
[0238] Each microculture well receives 2 .mu.c of 2 C/mM tritiated
thymidine (Amersham) 16 hours before stopping the culturing, which
is carried out using a MASH II machine which filters each
microculture on a glass-fiber membrane which retains the cell
nuclei.
[0239] The nuclear radioactivity of each pellet, which reflects the
de novo incorporation of thymidine into DNA, is measured by liquid
scintillation counting (Packard Tricarb).
[0240] The results are expressed in counts per minute and represent
the average of 3 samples of the same culture at the individual
scale.
[0241] c) Experimental Procedure
[0242] The samples are taken at 2 different times:
[0243] during the 3rd week following the start of the oral
administration of one of the preparations,
[0244] during the 7th week.
[0245] The animals are sacrificed 3 times per period.
[0246] Each culture experiment comprises 2 animals per group
treated.
[0247] The mixed lymphocyte culture is prepared in parallel:
[0248] a) with respect to mitomycin-treated cells of C3H origin
[0249] b) with respect to mitomycin-treated cells of DOM origin
[0250] d) Preparation of the Peptides
[0251] Lymphocytes (20 million) of a suitable mouse strain are
isolated from the spleen. This is a mixture of T and B lymphocyte
in approximately equal amounts and thus bearing antigens of type I
and II. They are treated with ultrasound (3.times.10 sec) and then
centrifuged at 1000.times. g for 10 minutes. The supernatant is
collected and recentrifuged in the same way. Next, the supernatant
is centrifuged twice at 8000.times. g. The final supernatant is
enriched in cell membranes and freed of cell nuclear debris and
Golgi apparatus. It is then subjected to digestion with pepsin
coupled to agarose beads, at pH 2 in glycine buffer, for 1 h 30 min
at 37.degree. C. After gentle centrifugation to separate the
agarose beads, and neutralization at pH 7 with TRIS buffer, the
mixture is filtered through a filter (Millipore, limit 10 kD). The
yield is about 500 .mu.g of peptide (determination by
spectrophotometry) referred to as LMp.
[0252] A solution of 50 .mu.g of peptide is mixed with a solution
of 50 .mu.g of Dnak (Stressgen) to form a Dnak-LMp complex.
[0253] The Dnak complex with beta-lactoglobulin peptide (Bp) is
made in the same way using peptides derived from the peptic
digestion of purified beta-lactoglobulin (cf. above).
[0254] 3. Results
[0255] After Treatment for 3 Weeks (FIG. 10)
[0256] The group of mice which received the Dnak-LMp complex
responds the worst to the stimulation of mitomycin-treated C3H
cells (C3Hm).
[0257] This is different (p<0.02; T-test) from the group which
received the peptide LMp alone, and equal to that which received
the Dnak-Bp control complex (p<0.01; T-test).
[0258] It should be noted, however, that administration of the
peptide alone (without Dnak) also has an effect, since this group
responds significantly less well than the control group (p<0.01;
T-test).
[0259] However, the specificity of the response inhibition is
guaranteed by the fact that the lymphocyte reactivity of the three
groups is equivalent with regard to mitomycin-treated cells of a
third, unrelated strain (DOMm).
[0260] After 7 Weeks (FIG. 11), or 4 Weeks After Stopping the Oral
Administration
[0261] The differences between the 3 groups remain quite
pronounced. The group treated with Dnak-LMp is the most inhibited
with regard both to the group which received the membrane peptide
alone (p<0.02; T-test) and to the control group (p<0.01;
T-test).
[0262] Administration of the peptide alone again allows an
attenuation of response with respect to the control group
(p<0.01; T-test).
[0263] The specificity of the response is once again verified by
the parallel test with respect to mitomycin-treated cells of an
unrelated strain (DOMm) and in which the 3 differently treated
groups react in the same way.
[0264] The administration of peptides obtained by peptic digestion
of spleen lymphocytes from mouse strains characterized by an
incompatibility in the H-2 system both at the K and D levels and
the A-E levels in extremely low amounts and for two weeks, has the
effect of strongly attenuating the unconditional response of
immunocompetent lymphocytes, in vitro, which usually indicates this
incompatibility.
[0265] This attenuation is enhanced by the presentation of this
type of peptide in the form of peptide-Dnak complexes.
[0266] This attenuation is specific and in no way reaches the
capacity of response with regard to a different variety, which
bears no relation to the strain used for the tolerization.
[0267] D. Tolerance of Syngenic Mice with Regard to a Graft of
Allogenic Cells
[0268] 1. Model and Experimental Scheme
[0269] Mouse strains:
[0270] Balbc for the animals made tolerant
[0271] C3H for the animals donating cells to be grafted (allogenic)
and stimulating cells in mixed lymphocyte culture (MLC).
[0272] They are reared in cages of 6 animals. Each group consists
of 12 animals per treatment.
[0273] The oral treatment is carried out according to the previous
procedure.
[0274] Experimental Scheme:
[0275] Complexes administered in the drinking water:
[0276] Days 0, 2, 4, 7, 9
[0277] Allogenic graft: 20.times.10.sup.6 intraperitoneal C3H
spleen cells:
[0278] Day 16
[0279] Sacrifice, collection of spleens and culturing of the spleen
cells:
[0280] 15 weeks after the graft
[0281] Detection and Counting of the Allogenic Cells
[0282] a) By the Presence of Cells Bearing MHC Type II
[0283] Functional test in bidirectional syngenic mixed culture.
[0284] The spleen cells of treated and grafted mice are cultured
with cells from untreated (naive) syngenic mice (Balbc).
[0285] Normally, there is no proliferative response to be expected
if the content of the spleen cells of treated and grafted animals
is composed solely of syngenic cells.
[0286] On the other hand, the presence of allogenic cells,
signaling the in vivo taking of the graft, should result in a
proliferation of MLC type by the so-called naive, intolerant cells,
which is proportionately greater the larger the number of foreign
cells.
[0287] In order to evaluate the order of magnitude of the taking of
the graft, an attempt at relative quantification of the response is
carried out with reference to a dose/response curve obtained by
adding known and increasing amounts of C3H allogenic cells to an
identical amount of naive and responding cells (200.times.10.sup.3
cells/well).
[0288] b) By the Presence of Cells Bearing MHC Type I
[0289] Direct counting by flow cytofluorometry immunofluorescence
using a mouse monoclonal antibody specific for MHC type I of the
C3H mouse : H-2 kk (Serotec), coupled to fluorescein or to
phycoerythrin on a suspension of spleen cells.
[0290] 2. Results
[0291] As seen in FIG. 12, 15 weeks after the peritoneal graft, the
spleen cells of the group treated with DnaK-C3H mouse
lymphomonocyte membrane peptide complexes are incapable of
responding to stimulation with mitomycin-treated C3H cells. This is
evidence of the induction of an allogenic tolerance.
[0292] The group treated with the peptide alone is also tolerated
to a lesser extent.
[0293] The group treated with DnaK-beta-lactoglobulin peptide is
not tolerant at all.
[0294] Moreover, the mixed cultures stimulated with another
allogenic population, originating from a histoincompatible strain
other than C3H, are all similar. This is evidence that no treatment
has impaired or altered the MLC response capacity, and that the
effect of the treatment is quite specific to the mouse strain from
which the membrane peptides originate.
[0295] As represented in FIG. 13, the MLC response of cells from
animals which are neither treated nor grafted is used to reveal the
existence of foreign cells in a mixture of spleen cells from
grafted animals, which would thus be of C3H origin in this
case.
[0296] It appears that the spleens of mice treated orally before
grafting with LMp and DnaK-LMp contain allogenic components since
they give rise to a very significantly proliferative response which
is different from the response of the control group treated with a
DnaK-irrelevant peptide (beta-lactoglobulin) complex. The latter
has a response which does not differ from the background noise.
[0297] For comparative purposes, a series of mixed cultures were
carried out in parallel with known and increasing amounts of C3H
cells (FIG. 14). They give rise to a proliferative response which
is proportional to the amount of foreign cells.
[0298] The average response level recorded with spleen cells from
grafted animals and tolerized with DnaK-LMp complexes is thought to
correspond to a content of about 30% of C3H cells.
[0299] The presence of cells of grafted type in the spleen is
measured by immunofluorescence using an antibody which is specific
for the MHC I of C3H (H-2kk) (Table 5). The group treated with
DnaK-LMp contains 14.7% of this antibody on average, this value
being significantly higher than that of the other two groups.
[0300] This presence of allo-antigens can only be observed
providing that the grafted animal spleen cells are left to stand at
37.degree. C. in the absence of serum, which suggests that it is
essential to be able to re-express these antigens whose presence
would thus be modified in vivo, quite probably by anti-H-2kk
antibodies. This adds another mechanism of tolerance of the graft
to the tolerance purely attributed to the responding T cells.
1TABLE 1 Inhibitions of the M6 monoclonal antibody which binds nBLG
by means of individual mouse sera: change as a function of time as
a function of the type of complex administered orally. % of
inhibition of the binding of M6 Standard Number Average deviation
of cases Group 1: Control (dBLG only) SAMPLE 1 45.0100 8.4146 8
SAMPLE 2 60.6750 3.9304 8 SAMPLE 3 59.6338 17.4714 8 Group 2:
dBLG-DnaK complexes SAMPLE 1 48.4350 7.0540 8 SAMPLE 2 56.3675
5.6146 8 SAMPLE 3 77.0975 3.8966 8 Group 3: dBLG- SAMPLE 1 23.7350
15.3990 8 SAMPLE 2 65.7013 6.2958 8 SAMPLE 3 65.3863 4.7270 8 Group
4: dBLG- SAMPLE 1 24.9538 4.7972 8 SAMPLE 2 56.0100 4.3929 8 SAMPLE
3 80.0287 1.9401 8 Group 5: dBLG- SAMPLE 1 37.3313 6.4248 8 SAMPLE
2 56.6962 5.5641 8 SAMPLE 3 87.1525 7.8731 8
[0301]
2TABLE 2 Inhibitions of the M7 monoclonal antibody which binds nBLG
by means of individual mouse sera: change as a function of time as
a function of the type of complex administered orally. % of
inhibition of the binding of M7 Standard Number Average deviation
of cases Group 1: Control (dBLG only) SAMPLE 1 70.0658 3.5541 8
SAMPLE 2 52.8224 2.3458 8 SAMPLE 3 57.0592 7.8996 8 Group 2:
dBLG-DnaK complexes SAMPLE 1 68.9145 2.6698 8 SAMPLE 2 51.6908
3.0857 8 SAMPLE 3 17.2697 8.0473 8 Group 3: dBLG- SAMPLE 1 78.4276
3.4832 8 SAMPLE 2 50.8553 3.9778 8 SAMPLE 3 26.9148 3.2069 8 Group
4: dBLG- SAMPLE 1 73.9671 3.1679 8 SAMPLE 2 28.5132 8.6072 8 SAMPLE
3 30.2829 14.2174 8 Group 5: dBLG- SAMPLE 1 72.8355 4.7722 8 SAMPLE
2 22.2961 9.5040 8 SAMPLE 3 41.3684 6.4331 8
[0302]
3TABLE 3 Anti(native) nBLG antibody titers after logarithmic
transformations: change as a function of time as a function of the
type of complex administered orally. Ln of titers (A.U.) Standard
Number Average deviation of cases Group 1: Control (dBLG only)
SAMPLE 1 3.8526 .4547 8 SAMPLE 2 4.2162 .3395 8 SAMPLE 3 4.2059
.2946 8 Group 2: dBLG-DnaK complexes SAMPLE 1 3.9738 .7957 8 SAMPLE
2 4.3749 .6353 8 SAMPLE 3 3.2562 .5057 8 Group 3: dBLG- SAMPLE 1
3.7073 .4435 7 SAMPLE 2 4.1348 .5475 8 SAMPLE 3 4.3917 .5047 8
Group 4: dBLG- SAMPLE 1 4.3714 .4215 8 SAMPLE 2 3.6419 .4704 8
SAMPLE 3 3.9964 .2724 8 Group 5: dBLG- SAMPLE 1 4.1526 .6401 8
SAMPLE 2 4.1739 .4464 8 SAMPLE 3 3.6126 .4873 8
[0303]
4TABLE 4 Differences in inhibition between the binding of the M6
and M7 antibodies to nBLG: % of inhibition of the binding of M6 - %
of inhibition of the binding of M7 Standard Number Average
deviation of cases Group 1: Control (dBLG only) SAMPLE 1 -25.0558
9.0035 8 SAMPLE 2 7.8526 5.6470 8 SAMPLE 3 2.5745 19.8676 8 Group
2: dBLG-DnaK complexes SAMPLE 1 -20.4795 8.5253 8 SAMPLE 2 4.6767
6.1131 8 SAMPLE 3 59.8278 9.2686 8 Group 3: dBLG- SAMPLE 1 -54.6926
13.8705 8 SAMPLE 2 14.8460 6.4665 8 SAMPLE 3 38.4718 5.6903 8 Group
4: dBLG- SAMPLE 1 -49.0134 3.9824 8 SAMPLE 2 27.4968 10.6337 8
SAMPLE 3 49.7459 14.6732 8 Group 5: dBLG- SAMPLE 1 -35.5043 4.8959
8 SAMPLE 2 34.4002 13.4093 8 SAMPLE 3 45.7841 8.7931 8
[0304]
5TABLE 5 Persistant allogenic cells (H-2kk+) in the spleens of
Balbc mice grafted with C3H cells: Oral treatment Lymphocyte
membrane DnaK-Bp peptide (LMp) alone DnaK-LMp 1.0% 0.9% 14.2% 1.5
2.8 25.0 0.5 3.6 9.2 0.5 3.7 10.4 Average ET Average ET Average ET
0.9% 0.5 2.7% 1.3 14.7% 7.2
[0305] Coupled T-tests:
[0306] DnaK-LMp/DnaK-Bp:p=0.026
[0307] DnaK-LMp/LMp:p=0.052
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239-264 (1994)
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[0310] 3. Staines, U. et al., J. Rheumatol., Vol. 54 (3), pp.
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[0311] 4. Polla, B. S. et al., Clin. Exp. Allergy, Vol. 23 (7), pp.
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[0312] 5. Healy, A. M. et al., Ann. N. Y. Acad. Sci., Vol. 663, pp.
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* * * * *
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