Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/515—Angiogenesic factors; Angiogenin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
  • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• PEPTIDES HAVING AN ACTIVITY OF STIMULATION OF THE IMMUNE RESPONSE AND OF TISSUE REGENERATION.
• the present invention relates to a new family of peptide molecules having the capacity in particular to stimulate the expression of cytokines of inflammation and to promote tissue regeneration.
• the invention therefore also relates to pharmaceutical compositions containing at least one of the peptides.
• angiogenic growth factors are known in the prior art such as the factors HARP, MK, FGF-1, FGF-2, VEGF, HIVl-tat, HIV2-tat, HGF, HB-EGF or even angiogenin.
• HARP Heparin Affin Regulatory Peptide
• PTN Pleiotrophin
• HB-GAM heparin binding-growth associated molecule
• the growth factor HARP is a polypeptide of 168 amino acids containing a hydrophobic N-terminal motif of 32 amino acids corresponding to a signal peptide.
• HARP is a secreted protein of 136 amino acids, in its short form, or 139 amino acids, in its long form, whose apparent molecular weight, determined in SDS-PAGE under reducing conditions, is 18 kDa.
• HARP was originally isolated from the brains of newborn rats as a molecule inducing neuritic growth in vitro (3) suggesting that this polypeptide is involved in the maturation of neuronal cells (4). Later, studies have shown that this polypeptide is also present in non-neuronal tissues, including the heart (5), the uterus (6), the cartilages (7), and the bone extracts (8), demonstrating that the function of HARP is not limited to an activity promoting neuritic growth as previously reported (3).
• HARP is able to stimulate the growth of fibroblast, epithelial and endothelial cells in vitro (6, 9). This mitogenic activity has since been confirmed by the use of recombinant proteins produced from eukaryotic expression systems (9, 12). HARP also induces the formation of pseudo-capillaries in vitro (12). In vivo, in different tissue models, the localization of HARP is notably associated with the endothelial cells of the blood capillaries (16). At present, data relating to HARP suggests that this polypeptide plays a role in the complex mechanisms involved in angiogenesis and neoangiogenesis in tumors. A great deal of research has been done in this direction to determine the involvement of HARP in tumor progression, in particular in hormone-dependent tumors such as the breast or the prostate.
• HARP like MK
• human purified recombinant proteins from HARP have been shown to be mitogenic to endothelial cells (9,12), and exert in angiogenic activity (12).
• Numerous studies have shown the involvement of HARP and MK in development methods (10, 13, 14).
• HARP protein mRNA distribution studies during embryonic and postnatal development suggest important functions in cell growth and differentiation (15). However, little is known about the in vivo physiological functions of these molecules.
• the presence of HARP transcripts in adult tissues including the meninges, iris, testes and uterus also indicate a physiological role during adulthood.
• the research carried out in the context of the present invention has focused on numerous angiogenic growth factors, such as FGF-1, FGF-2, VEGF, HIVl-tat, HIV2-tat, HB-EGF, Angiogenin, HARP and MK , and enabled the inventors to identify peptide sequences which are found in several of these factors. From these, the inventors have constructed peptide molecules rich in basic amino acids lysine (K) and arginine (R).
• K basic amino acids
• R arginine
• Table I below reports portions of sequences rich in basic amino acids of several growth factors where the positions of basic amino acids are substantially aligned.
• n and m are each an integer from 0 to 20 the sum of which n + m is between 0 and 20, preferably between 0 and 15 and most preferably between 0 and 10,
• Al is a basic amino acid and more particularly lysine (Lys) or arginine (Arg),
• A2 is an amino acid chosen from: basic amino acids, glutamic acid (Glu), glycine (Gly), aspartic acid (Asp),
• A3 is an amino acid chosen from: basic amino acids, proline (Pro), glutamic acid (Glu), glutamine (Gin),
• A4 is an amino acid chosen from: basic amino acids, glutamic acid (Glu), glycine (Gly), serine (Ser), valine (Val).
• peptides of formula (I) will also be designated “pAHA” for "angiogenic HARP peptide”.
• the invention more particularly contemplates the peptides of the following formulas:
• the peptide of formula (II) has been defined more particularly from the sequence of HARP (1-14).
• the peptide of formula (III) has been defined more particularly from the sequence of HIV tat (70-92).
• the peptide of formula (IV) has been defined more particularly from the sequence of HB-EGF (85-114).
• the peptide of formula (V) has been defined more particularly from the sequence of HARP (108-132).
• the peptide of formula (VI) has been defined more particularly from the sequence of VEGF (145-170).
• the peptide of formula (VII) has been defined more particularly from the sequence of HIV tat (41-65).
• the peptide of formula (VIII) has been defined more particularly from the sequence of MK (1-11).
• the peptides of the invention can be prepared by chemical synthesis or by gene expression techniques from the corresponding polynucleotide sequence by techniques known to those skilled in the art.
• the inventors have demonstrated that the pAHA peptides have angiogenic and healing properties like HARP and at comparable doses (ED50 # 5-50 ng / ml). They observed the remarkable activity of these peptides on vascular ischemia (angiogenesis) and muscle regeneration and scarring. They have also shown that the peptides of the invention are capable of stimulating the expression of the cytokines of inflammation and are therefore useful for preventing or treating diseases linked to immunosuppression, and in particular AIDS.
• the invention therefore also relates to a pharmaceutical composition containing one or more of the preceding peptides, associated in said composition with one or more pharmaceutically acceptable vehicles.
• the invention relates more particularly to a composition comprising one or more pAHA peptides, and optionally another compound, useful for promoting cell regeneration and growth, such as muscle growth , and therefore in wound healing.
• the invention relates very particularly to a composition comprising one or more pAHA peptides, and optionally another compound, useful for preventing or treating vascular ischemia.
• a composition comprising one or more pAHA peptides, and optionally another compound, useful for preventing or treating vascular ischemia.
• the research carried out in the context of the invention has made it possible to demonstrate unexpected properties of pAHAs on the proliferation of circulating blood cells, and more particularly of peripheral blood mononuclear cells.
• This property has made it possible to show the stimulating properties of pAHA on certain cytokines, in particular the cytokines of inflammation.
• HARP protein mRNA has in fact been observed in cells of blood vessels, both endothelial cells and smooth muscle cells, as well as in the human mammary glands (16). In addition, it has been reported that HARP is an angiogenic growth factor (12) and that it is synthesized and localized in vascular endothelial cells (16).
• the inventors therefore evaluated the in vi tro activity of this growth factor on PBMCs (human mononuclear cells from peripheral blood) freshly isolated by incubating PBMCs with the HARP factor or with a pAHA peptide.
• PBMCs human mononuclear cells from peripheral blood
• the results obtained show that HARP and pAHA are capable of stimulating the incorporation of tritiated thymidine into the nucleus of PBMCs.
• These results therefore demonstrate that the HARP molecule as well as the pAHA peptide strongly stimulate the proliferation of human mononuclear cells of the peripheral blood, and more particularly after one week an increase in the population of T lymphocytes is observed.
• HARP is active on the proliferation of lymphocytes at very low concentrations, of the order of 10 ⁇ M. This surprising result has led the inventors to consider that the HARP factor must bind to its receptor on PBMCs with a high affinity.
• HARP does not act on the production of IL2 interleukins, but that HARP, and pAHA, bind with a strong affinity to a specific receptor present on the lymphocytes, and induce the activation of the interleukin sites, in particular of the IL2 sites.
• the HARP receptors are very little known.
• Kd 600 ⁇ M
• HARP binding sites have also been found in several cell types, including rat kidney cells, human mammary adenocarcinoma cells, human epidermal carcinoma cells, human hepatocarcinoma cells, neuroblasts mouse, and pheochromocytoma cells. It is commonly accepted that no biological response transmitted by HARP has been observed on this type of cells, and therefore that these binding sites cannot be considered as functional receptors.
• Parallel studies describe interactions between HARP and heparan proteoglycan sulfate, such as syndecan-1 and syndecan-3. Syndecan-3 has been shown to interact with HARP with an apparent Kd of 800pM. This heparan proteoglycan sulfate is involved in the neuritic growth activity of HARP since anti-syndecan-3 antibodies can block this activity (21).
• MK Midkine
• HARP binds to different surface receptors, which has also recently been 'demonstrated (24, 25).
• the two molecules are expressed and isolated using analogous experimental methods, including the same recombinant expression system and the same purification techniques. The fact that the incorporation of tritiated thymidine is observed only with HARP, and not with MK, excludes the presence of a bacterial contaminant exhibiting mitogenic activity with respect to PBMCs.
• the invention relates more particularly to a pharmaceutical composition comprising one or more pAHA peptides, and optionally another compound, useful for stimulating the proliferation of mononuclear cells of the blood, and in particular T lymphocytes. Stimulation of the proliferation of T lymphocyte cells is very particularly useful in the treatment of immunosuppressed patients.
• Example 4 Another observation was made on cells from the blood of AIDS patients.
• the inventors show how the stimulation of the patient's blood cells by the peptides of the invention makes it possible to amplify the replication of the HIV virus in vitro and thus to promote its detection and therefore its typing.
• Compositions comprising peptides of the invention are therefore also useful for the diagnosis of infection by HIV viruses.
• the effectiveness of the anti-viral agents will be enhanced by administering them before or simultaneously with one or more peptides of the invention.
• the peptides of the invention will promote the replication and release of HIV viruses in vivo, in particular residual HIV viruses, which remain present in the body after antiviral treatment.
• the administration of the peptides according to the invention, by activating these viruses, would thus make them more accessible to antiviral agents, and more easily destructible.
• cytokines in general play a role in cell proliferation, more particularly in blood cells. The inventors therefore sought to demonstrate the role of HARP and pAHA on the expression of cytokines. Inducing properties of the expression of inflammation cytokines have thus been demonstrated.
• inflammation cytokines is meant TNFalpha, IL1, IL6, and INFgamma.
• the inventors therefore tested the induction of the expression of three cytokines of inflammation
• TNFalpha, IL1 and IL6 by PBMCs cells treated with HARP, and the induction of expression of IL6 by two pAHA peptides in accordance with the present invention, the sequences of which are reported in Example 5 below.
• the results obtained with the HARP molecule indicate that HARP is capable of inducing in a dose dependent manner the expression of the cytokines TNFalpha, IL1 and IL6.
• results obtained with the two pAHA peptides show that they are capable of stimulating the expression of IL ⁇ .
• An increase in the expression of these cytokines is also detected after addition to the cells of other peptides in accordance with the present invention, derived from angiogenin and the tat protein (see Table I). No expression of these inflammatory cytokines is detected when the HARP molecule is denatured.
• the invention relates very particularly to a pharmaceutical composition
• a pharmaceutical composition comprising one or more pAHA peptides, and optionally another compound, useful for stimulating the production of cytokines of inflammation.
• Such a composition according to the invention is therefore particularly indicated in the prevention or treatment of diseases linked to immunosuppression.
• peptides of the invention or a composition containing them to promote growth and differentiation of cells in culture, in particular of lymphoid cells, such as endothelial cells and T lymphocytes. Indeed, the culture of these cells is often practiced in the context of diagnostic tests.
• the peptides of the invention can be produced by genetic expression of a polynucleotide sequence encoding said peptides.
• the invention therefore also relates to a nucleic acid molecule constituted by or comprising at least one polynucleotide sequence coding for a peptide defined above.
• nucleic acid molecules are more particularly vectors, such as plasmids which can be used to transform host cells in vitro or in vivo.
• host cells is meant for example bacteria allowing the production of the peptides of the invention.
• the invention therefore also relates to compositions comprising as active principle at least one nucleic acid molecule or cells defined above.
• FIG. 1 shows the stimulation of the incorporation of tritiated thymidine in PBMCs stimulated or not by HARP.
• White bar cells no stimulated
• hatched bar cells stimulated with 100 ng / ml of HARP.
• FIG. 2 represents the dose response effect of HARP tested on PBMCs.
• A) The cells are cultured in the absence or in the presence of different concentrations ranging from 0.1 to 100 ng / ml of HARP (• - •) or of Midkine (MK) (O-O). Each of the values represents the average of the cpm obtained ⁇ the standard deviation.
• B) The cells are incubated with tetanus toxin (TT) at 1800 UT / ml, phytohemagglutinin (PHA) at 2.5 ⁇ g / ml, interleukin-2 (IL2) at 50 IU / ml, or untreated ( NT) as internal stimulation controls.
• TT tetanus toxin
• PHA phytohemagglutinin
• IL2 interleukin-2
• NT untreated
• FIG. 3 represents the dose response effect of HARP on PBMCs treated with an anti CD3.
• the cells are cultured as previously described. It should be noted that if the cells are treated with 100 ng / ml of HARP in the presence of CD3, high cell mortality is then observed. Black bar; HARP alone, white bar; HARP + anti CD3.
• FIG. 4 represents the dose response effect of HARP on PBMCs treated with tetanus toxin.
• the cells are cultured as previously described. It should be noted that if the cells are treated with 100 ng / ml of HARP in the presence of tetanus toxin (800 IU / ml) a high cell mortality is then observed. Hatched bar: HARP only; white bar: HARP + tetanus toxin.
• FIG. 5 represents the effect of the HARP protein on the replication of the HIV virus by measuring the immunoreactivity p24.
• PBMCs from a patient infected with HIV are incubated according to the protocol described in ⁇ 1 for 3 days with variable concentrations of HARP (0.1-100 ng / ml).
• the viral replication is estimated by measuring the immunoreactivity associated with the p24 protein present in the culture medium. (*) low cell mortality; (**) high cell mortality.
• the production of the virus is evaluated by the "Abott HIV Ag monoclonal" test which is an immunoenzymatic assay on a solid phase of the sandwich type.
• the viruses present in the test sample are lysed with Triton X100 and then the lysate is incubated with polystyrene beads coated with anti-p24 monoclonal antibody. After incubation, the beads are washed, and the presence of specific immunoglobulins is revealed by incubation with a second anti-mouse Ig antibody coupled to peroxidase. The revelation is made by adding a substrate of the peroxidase; 1 orthophenylenediamine
• Figure 6 shows the activation of peripheral blood mononucleotide cells by the HARP peptides.
• the PBMCs are cultured for 7 days in RPMI culture medium containing 10% fetal calf serum in the absence or in the presence of 1 ng / ml of the HARP protein or of 1 ⁇ g / ml of the peptides 1 or 2.
• L incorporation of tritiated thymidine is determined as described above.
• Figure 7 shows the study of the effect of
• FIG. 8 represents the effect of HARP on the expression of TNF ⁇ after 3 days (white bars) or
• FIG. 9 represents the effect of HARP on the expression of IL6 after 3 days (white bars) or 7 days of culture (black bars).
• the assay of these cytokines is carried out using an ELISA test marketed by R&D.
• FIG. 10 represents the expression of IL6 by peptides 1 and 2 corresponding to the NH 2 parts
• COOH of the HARP polypeptide COOH of the HARP polypeptide.
• the assay of these cytokines is carried out using an ELISA test marketed by R&D after 7 days of incubation.
• Figure 11 shows the effect of HARP peptides on muscle regeneration.
• the adult rat soleus muscle is crushed, treated or not with the peptides and then removed after 4 days of regeneration: 1: treated with PBS (50 ⁇ l)
• Figure 12 illustrates the angiogenic effect of peptide 1 tested in the CAM.
• Figure 13 is a schematic representation of the plasmid used to produce the peptide corresponding to the N-terminal part (residues 1 to 14) of HARP.
• Human mononuclei cells from different healthy donors are isolated from peripheral blood after centrifugation on a cushion.
• Ficoll-Hypaque (Pharmacia Biotech) as described by the manufacturer.
• the cells are washed, then cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (56 ° C, 30 min), 100 units / ml of penicillin and 100 ⁇ g / ml of streptomycin.
• the cells seeded at 10 6 cells per ml in a round bottom 96-well culture dish (Costar), are cultured for 7 days in the presence or not of the recombinant human HARP protein produced in Coli, at a concentration of 100 ng / ml.
• the HARP polypeptide is capable of stimulating the incorporation of tritiated thymidine in the nucleus of PBMCs.
• the stimulation index defined as the ratio of the radioactivity incorporated in the cells treated with HARP to that of the control cells, not treated with HARP, varies from 2.3 to 51.7 times (see results of experiment N ° 4 and N ° 7).
• FIG. 1 The histogram presented as an insert in FIG. 1 shows that treatment with 100 ng / ml of HARP induces a 2.9-fold increase in the number of cells compared to the untreated control, demonstrating that the incorporation of thymidine observed is indeed proportional to the number of cells.
• This cell count was carried out with the cells used for the incorporation of tritiated thymidine from experiment No. 7.
• the dose response curve of the HARP protein (0.1 to 500 ng / ml) tested on the PBMCs is presented in FIG. 2A.
• the positive stimulation controls were performed using phytohemagglutinin (PHA) 2.5 ⁇ g / ml and tetanus toxin (TT) 1800 IU / ml (fig. 2B). No stimulation is observed after addition of IL2 showing • an absence of activation of the cells used for these tests.
• PHA phytohemagglutinin
• TT tetanus toxin
• Example 2 Role of the HARP protein as a co-stimulator of the specific immune response.
• T lymphocytes can be obtained via the activator of the antigen receptor (TCR) associated with the major histocompatibility system (MHC).
• TCR antigen receptor
• MHC major histocompatibility system
• This activation requires, in addition to the specific recognition TCR-MHC / antigen, the action of adhesion molecules playing a role of coactivation and amplification of the response.
• HARP could amplify cell proliferation either by stimulation of the receptor to T lymphocytes using an anti CD3 or a memory antigen, tetanus toxin.
• Activation of the T lymphocyte receptor is obtained by treating lymphocytes with a monoclonal anti CD3 antibody (1/100, I munotech).
• the effect of HARP on the cellular proliferation of PBMCs is tested by adding an optimal concentration of HARP (1 ng / ml, cf. example 1) in the presence or not of anti CD3.
• the cultures as well as the quantification of the incorporated tritiated thymidine are carried out as described in Example 1. The results obtained are presented in FIG. 3.
• the anti CD3 antibody (1/100) stimulates, after 7 days of incubation with the cells, the incorporation of tritiated thymidine by 25 times (control; 600 ⁇ 60 cpm, anti CD3; 15000 ⁇ 200 cpm).
• control; 600 ⁇ 60 cpm, anti CD3; 15000 ⁇ 200 cpm At a dose of 1 ng / ml of HARP and in the absence of anti CD3, an amplification of 5.8 times is observed for this donor compared to the control (control; 600 ⁇ 60 cpm, HARP; 3500 ⁇ 200 cpm).
• PBMCs cells cultivated under the conditions described in ⁇ 1 are stimulated by tetanus toxin (1800 IU / ml, Mérieux) alone or in combination with the HARP protein used at a concentration ranging from 0.1 to 100 ng / ml.
• the tetanus toxin will specifically amplify a subpopulation of memory T lymphocytes.
• Example 3 Determination of the amplified cell population after treatment with HARP in a culture of PBMCs.
• the cells were isolated from the peripheral blood of normal subjects, blood donors, taken from Vacutainer tube containing EDTA.
• the mononuclear cells were separated by ficoll gradient, counted and adjusted to 10 6 cells per ml.
• the cells are incubated for 5 days at 37 ° C. in a humid atmosphere with 5% CO 2 in the presence of HARP or other peptides at concentrations which are mentioned in each of the examples described.
• Table II presents the effects of the HARP molecule tested at a concentration of 1 ⁇ g / ml on the proliferation of lymphoid cells.
• Example 4 Action of the HARP molecule on mononuclear cells from peripheral blood from individuals infected with HIV.
• T lymphocytes and monocytes Activation of T lymphocytes and monocytes by cytokines induces production and / or activation of nuclear factors of the host cell capable of reactivating viral transcription.
• This viral reactivation induced by IL1 and TNF ⁇ partly depends on the activation of factor NF- ⁇ b.
• the secretion by circulating monocytes of cytokines IL1, IL6 and in particular TNF ⁇ which are capable of inducing or increasing the replication of HIV in T lymphocytes / monocytes suggests that these cytokines may increase the progression of the disease.
• CD4 ⁇ 200 / mm 3 activated only by the HARP protein, are capable of producing the HIV virus measured by the production of the viral antigen p24. This production is maximum for a HARP concentration of 1 ng / ml. At a concentration of 100 ng / ml, significant cell death is observed.
• HARP can make it possible to amplify in expression the expression of P24 and be used for typing of the HIV viral strains, and on the other hand in vivo as 1) inducer of the replication of the virus, thus facilitating the action of antiviral agents on quiescent infected lymphocytes or 2) at large doses (corresponding to the effects observed in in vitro at 100 ng / ml, induce the death of chronically activated T cells (see the previous examples illustrated in FIGS. 3 and 4)
• Example 5 Activation of lymphocytes by HARP peptides.
• Peptide 1 NH 2 -AEAGKKEKPEKKVKKSDCGEW-COOH; 21 amino acids.
• Peptide 2 NH 2 -AESKKKKKEGKKQEKMLD-COOH; 18 amino acids.
• Example 6 Induction of the expression of TNF ⁇ , IL6 and IL1 by PBMCs cells treated with HARP.
• the cells were isolated from the peripheral blood of normal subjects, blood donors, taken from a Vacutainer tube containing EDTA. The mononuclear cells were separated by ficoll gradient, counted and adjusted to 10 6 cells per ml. The cells are incubated for 3 or 7 days at 37 ° C. in a humid atmosphere with 5% of CO 2 in the presence of a variable HARP concentration ranging from 1 to 1000 ng / ml or of various peptides which are mentioned in the legends of the figures.
• FIGS. 7, 8 and 9 The results are represented in FIGS. 7, 8 and 9. The analysis of these results indicates to us that the HARP molecule is capable of inducing in a dose-dependent manner the expression of the cytokines ILl ⁇ (FIG. 7), TNF ⁇ ( fig. 8) and IL6 (fig. 9).
• peptides 1 and 2 are capable of stimulating the expression of IL6.
• An increase in the expression of these cytokines is also detected after adding tat peptides or other molecules (angiogenin, tat protein; result not shown) having a homologous protein domain to the cells. No expression of these inflammatory cytokines is detected in this system when the HARP molecule is denatured or when the cells are treated with LPS.
• Example 7 Effect of HARP peptides in muscle regeneration.
• FIG. 11 The results are presented in FIG. 11. The analysis of these sections indicates that the muscle treated with 1 ⁇ g of peptide 1 has a much greater number of mononuclear cells (FIG. 11.2) than the muscles treated with peptide 2 (FIG. 11.3) or injected only with 50 ⁇ l of PBS (fig. 11.1). This observation indicates that the injection of peptide 1 into a crushed muscle, induces after 4 days of treatment an increase in the number of mononucleated cells present in the endomysial tubes promoting tissue regeneration.
• Example 8 Effect of HARP peptides on angiogenesis.
• the "Chicken allantoic membrane test” (CAM test) was used in this study to evaluate in vivo the effect of the peptides HARP 1 and 2 on the induction of angiogenesis.
• the structure of these peptides is presented in Example 5. The experimental procedure is as follows:
• FIG. 12 The effect of peptide 1 on the induction of angiogenesis in CAM is illustrated in the following example (FIG. 12).
• Example 9 Expression and assay of the mitogenic activity of the peptide corresponding to the N-terminal part of the HARP molecule (residue 1 to 14).
• N terminal peptide (amino acids 1-14) of human HARP is obtained by recombination in a eukaryotic expression system.
• This peptide is produced from the cDNA of human HARP under the EcoRI clone in the eukaryotic expression vector PcDNA-3 (InVitroGen) by creation of a stop codon at amino acid number 15 (site-specific mutagenesis kit, QuickChange, Stratagene US).
• eukaryotic cells are transfected with this construct (FInstitute, Roche, NJ USA). The expression is followed by Western blotting from the culture media conditioned by the cells transfected using the anti N terminal antibody to HARP (residues 1-15, marketed by Santa Cruz, Ca, USA). The cells are cultured for 72 h in the presence of butyrate then the conditioned medium is recovered. After purification of the peptide involving cation chromatography and a reverse phase (Waters, Symmetry®, C18, 5 ⁇ m, 4.6 x 250 mm). Elution from the column is carried out by a linear gradient of acetonitrile. The presence of peptide in the eluted fractions is monitored by measuring the optical density at 220 nm. The assay of the mitogenic activity induced by the peptide thus purified is carried out according to the following protocol:
• the cells used are HUVEC cells (Clonetics) used between passages 1 to 5.
• Each of the wells of a 48-well culture dish (Costar) are incubated overnight at 4 ° C. with a solution of HARP (100 ng / ml), purified HARP peptide (100 ng / ml) or only buffer in negative control.
• HARP 100 ng / ml
• purified HARP peptide 100 ng / ml
• the cells are seeded at the rate of 2 ⁇ 10 4 cells per cm 2 in DMEM culture medium containing 2% of fetal vow serum.
• Each assay is carried out in triplicate.
• the induction of cell proliferation is estimated by counting the cells after 72 hours of culture.
• HBNF and MK members of a novel gene fa ily of heparin-binding proteins with potential roles in embryogenesis and brain function. Prog. Growth Factor Res. 3: 143.
• Pleiotrophin is an abundant protein in dissociative extracts of bovine fetal epiphyseal cartilage and nasal cartilage from newborns. J. Orthop. Res. 11: 479.
• HB-GAM heparin-binding growth-associated molecule
• Peng HB Ali AA, Dai Z, Daggett DF, Raulo E and Rauvala H. 1995.
• Mitsiadis TA Salmivirta M, Muramatsu T, Muramatsu H, Rauvala H, Lehtonen E, Jalkanen M and Thesleff I. 1995.
• heparin-binding cytokines midkine (MK) and HB-GAM (pleiotrophin) is associated with epitheliai-mesenchymal interactions during fetal development and organogenesis. Development 121: 37.
• Midkine induces tumor cell proliferation and binds to a hight affinity signaling receptor associated with JAK tyrosine kinases. J. Biol. Chem. 273: 3654

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Public Health (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Molecular Biology (AREA)
• Engineering & Computer Science (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Zoology (AREA)
• Toxicology (AREA)
• Virology (AREA)
• Immunology (AREA)
• Vascular Medicine (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Dermatology (AREA)
• Heart & Thoracic Surgery (AREA)
• Cardiology (AREA)
• Urology & Nephrology (AREA)
• AIDS & HIV (AREA)
• Tropical Medicine & Parasitology (AREA)
• Neurology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Physical Education & Sports Medicine (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=9550838

Family Applications (1)

Country Status (7)

Families Citing this family (14)

Citations (1)

Family Cites Families (6)

• 1999
  • 1999-10-12 FR FR9912714A patent/FR2799465B1/fr not_active Expired - Fee Related
• 2000
  • 2000-10-06 JP JP2001530354A patent/JP2004502636A/ja active Pending
  • 2000-10-06 AU AU77955/00A patent/AU7795500A/en not_active Abandoned
  • 2000-10-06 CA CA002387604A patent/CA2387604A1/fr not_active Abandoned
  • 2000-10-06 EP EP00967971A patent/EP1222201A2/de not_active Ceased
  • 2000-10-06 WO PCT/FR2000/002786 patent/WO2001027136A2/fr not_active Ceased
• 2002
  • 2002-04-04 US US10/116,391 patent/US6932973B2/en not_active Expired - Fee Related

Patent Citations (1)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events