WO2006114805A2

WO2006114805A2 - Use of hmgb2 and hmgb3 proteins for medical applications

Info

Publication number: WO2006114805A2
Application number: PCT/IT2006/000293
Authority: WO
Inventors: Marco Bianchi; Tobias Pusterla
Original assignee: Fondazione Centro San Raffaele Del Monte Tabor
Priority date: 2005-04-28
Filing date: 2006-04-28
Publication date: 2006-11-02
Also published as: WO2006114805A3

Abstract

The invention discloses the HMGB3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity for medical use and immunostimulating activity, as well as the HMGB2 protein or functional part thereof for the preparation of an activating cell proliferation pharmaceutical composition.

Description

Use of HMGB2 and HMGB3 proteins for medical applications Prior art

High Mobility Group Box proteins (HMGBs) are a set of chromatin proteins made up of two basic DNA binding domains, HMG box A and B, and a C-terminal acidic tail. The family comprises three proteins (HMGBl, HMGB2 and HMGB3) with a highly conserved structure (80% amino acid identity) (Agresti 2003). HMGB l's expression is almost ubiquitous (Calogero, 1999), while HMGB2 is widely expressed during embryonic development (Ronfani,

2001) and, in adult mice, in lymphoid tissues, testis and monocytes. HMGB3 appears to be highly expressed during embryo development and in hemopoietic stem cells (Nemeth, 2004). The author's group already demonstrated that HMGBl is released by necrotic cells (Scaffϊdi,

2002) and can act as a cytokine via binding with RAGE (receptor for advandced glycation products) (Palumbo, 2004).

Major amino acid sequence differences (9 out of 21 aa.) are located in the RAGE binding site

(aa. 166-181). Notwithstanding such differences, the authors here report that HMGB2 is able to promote proliferation and migration of bovine aortic endothelial cells (BAEC). HMGB3 has an even more clear different behavior, acting only as chemoattractant factor at very low concentrations.

BAEC over-expressing dominant negative RAGE lost their capability to migrate in response to all HMGBs (-1, -2 and -3). Furthermore using antibodies directed against the region immediately upstream the acidic tail of HMGBl and HMGB2 the authors abolished their chemotactic activity.

These data taken together suggest that HMGBl, -2 and -3 are only partially redundant, act via

RAGE, and may have different applications.

Description of the invention It is an object of the invention the HMGB3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity for medical use. The protein or functional part thereof is able to exert a chemoattractive activity of cells able to restore a missed function (i.e. adult stem cells) and can be used for disease treatment. The chemoattracting portion is comprised in the region between Box B (aa. 80-180) and the acidic tail of HMGB2; by sequence comparison HMGB3 chemoattracting region is comprised in the same region, preferably between aa. 166- 181. The HMGB3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity is also advantageously used for the preparation of an immunostimulating composition, being able to attract immunomodulating cells. Preferably the HMGB3 protein or functional part thereof acts as adjuvant. In a preferred embodiment the immunostimulating composition is a vaccine.

It is another object of the invention a pharmaceutical composition comprising an effective amount of the HMGB3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity. It is another object of the invention an immunostimulating composition comprising an effective amount of the HMGB 3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity, preferably a vaccine composition.

The invention refers also to the use of the HMGB2 protein or functional part thereof for the preparation of an activating cell proliferation pharmaceutical composition, preferably for tissue regeneration, more preferably for myocardial tissue regeneration and/or peripheral artery diseases, alternatively for wound healing.

The present invention shall now be described in non limitating examples thereof, with particular reference to the following figures:

Figure 1: HMGB2 expression. HMGB2 is expressed and secreted in activated monocytes. A: immuno-staining for HMGB2, B: immuno-staining for HMGBl, C: merge HMGB1-HMGB2, D: DAPI, E: western blot for HMGB2 and actin on LPS treated and untreated monocytes.

Figure 2: Effect of HMGBs on bovine aortic endothelial cells proliferation. BAEC were grown in DMEM Medium containing no addition, 30 ng/ml of HMGBl, HMGB2 and HMGB3 each. Cells were counted At 24, 48 and 72h. HMGBl and HMGB2 induced cell proliferation while HMGB3 shows growth comparable to serum free cells. The experiment was repeated three times.

Figure 3: HMGB2 chemotactic activity on BAEC. BAEC were subjected to chemotaxis assays with 1, 3,10, 30, 100 ng/ml HMGB2. HMGB2 shows concentration-dependent chemoattractant effect on endothelial cells. Figure 4: Chemotactic effect of HMGB2 fragments on bovine aortic endothelial cells. A BAEC were subjected to chemotaxis assays with HMGB2 Box A (1-80), Box B (80-180), HMGB2A+B (1-180) and the wild type protein at 0.1, 1, 10, 100 ng/ml of concentration each. HMGB2A+B and Box B have a chemotactic effect comparable to wt HMGB2. In contrast Box A has no significant chemotactic activity. B Schematic representation of wt full length, tailless (HMGB2A+B), Box A and Box B HMGB2 fragments tested for migration assay. Figure 5: Chemotactic effect of HMGb3 on bovine aortic endothelial cells. BAEC were subjected to chemotaxis assays with 1, 3, 10, 30, 100 ng/ml of HMGB3. HMGB3 has a chemotactic effect on cells although it seems not comparable to HMGBl and HMGB2. HMGB3 shows migration-inducing ability at a ten fold lower concentration compared to other HMGBs although the amount of cells migrating per field is lower compared to other HMGBs. Figure 6: Chemotactic effect of HMGBs on bovine aortic endothelial cells. Migration assay was performed with the most migration-promoting concentration of each protein: HMGBl (30ng/ml),HMGB2 (3ng/ml) and HMGB3 (lng/ml).

Figure 7: Antibody against HMGB2 blocks BAEC migration. A Antibody directed against residues 150-183 at 1, 10, 100 ng, 1 and 10 ug put in the lower compartment of Boyden chambers with 30 ng/ml of HMGB2 shows a decrease in cell migration. Treatment with lOOng of HMGB2 antibody shows basal levels of endothelial cells migration. B shows migration assay with increasing concentrations of HMGB2 (1, 3, 10, 30, 100 ng/ml) with or without lOOng of blocking anti-HMGB2 anibody. Figure 8: BAEC over-expressing dominant negative RAGE loose their migrating ability. A BAEC cotransfected with dn-RAGE expressing plasmid or control pCDNA3 and YFP expressing plasmid in a 1:3 ratio were assayed for chemotaxis in response to 20% serum, 30ng/ml of HMGB2 and lng/ml of HMGB3. Cells transfected with dn-RAGE exhibited a significant decrease in migration in comparison to cells transfected with control plasmid pCDNA3. B Control migration performed with non-transfected cells synchronized with transfected cells under stimulus of 20% serum, 30ng/ml HMGB2 and lng/ml HMGB3.

Figure 9: Residues 1-300 of RAGE present on BAEC cell membrane was blocked. Chemotaxis was performed with HMGBl (30ng/ml), HMGB2 (3ng/ml) and HMGB3 (lng/ml).BAEC in the upper compartment were added of lμg of anti-RAGE H300 antibody. Cells whose receptor is blocked by H300 antibody show basal levels of migration compared to untreated cells. Figure 10: Human and murine HMGBs alignment.

Materials and methods

Intra-peritoneal washes C57bl6 mice were injected intraperitoneally with ImI of 3% thioglycollate broth (Fluka).

After 72 hours, intraperitoneal activated monocytes were collected with a syringe with 5ml of

PBS. Cells were washed twice with serum free RPMI medium and cultured in RPMI with 10%

FCS.

Cells/cell stimulation Bovine Aorta Endothelial Cells (BAEC) were isolated from a section of the thoracic aorta of a freshly slaughtered calf as described (Palumbo et al., 2002). Mouse monocytes were obtained by intraperitoneal washes and cultured in RPMI medium additioned with 10% FCS. Cells were stimulated with lOμg/ml of LPS for 16 hours.

Cell supenatant The conditioned medium of LPS-treated monocytes was collected, precipitated for 16h in acetone 80% at -20°C, centrifuged (13,000 rpm for 30 min), air dried and resuspended in

RIPA buffer. Samples were run on SDS-page gel and blotted on Immobilon transfer membrane (Millipore). Western blot was performed as described (Degryse et al., 2001), with anti-HMGB2 (1:3000) and anti-β-actin (1:1000) (Sigma). Films were scanned on an Agfa StudioStar scanner.

HMGB2. HMGB3 and antibodies

Expression and purification of the full-length HMGB2 and HMGB3 protein and fragments thereof was performed as described previously for HMGBl (Muller et al., 2001). Endotoxins were removed by passage through Detoxy-Gel columns (Pierce). Rabbit polyclonal anti-HMGBl and HMGB2 antibodies were from Pharmingen BD, polyclonal antibodies against HMGBl were from MBL, anti-RAGE H300 rabbit antibody was from Santa Cruz.

Immunofluorescence

LPS-stimulated monocytes were fixed in 4% paraformaldehyde, permeabilized, saturated and processed for immunofluorescence with mouse monoclonal anti-HMGBl (dilution 1:500) and rabbit anti-HMGB2 antibody (dilution 1:1000) followed by Alexa Fluor 488/546-conjugated anti-rabbit/anti-mouse Ig (Molecular Probes). Images were taken with a Zeiss SlOO TV microscope equipped with a Zeiss 32x/ 0.4 Ph2 LD Achroplane objective. Proliferation assay

Cells were seeded in 6- well plates (1x10⁵ cells/well) and grown in DMEM supplemented with 10% FCS. After 24 hours the medium was replaced with serum-free DMEM for 16 hours. Subsequently the cells were grown with medium alone, or medium with the addition of 10% FCS or HMGBl, HMGB2 or HMGB3 at the concentration of 30 ng/ml. Cells were counted after 1, 2 and 3 days, and Trypan blue dye exclusion was used as indicator of cell viability. All experiments were performed three times in duplicate. Chemotaxis assay

Cell migration was assayed using Boyden chambers (Degryse et al., 2001). Briefly, PVP-free polycarbonate filters with 12 μm pores (Costar) were coated with 5 μg/ml porcine skin gelatin (Sigma). Serum-free DMEM (negative control), DMEM containing 1, 3, 10, 30 or 100 ng/ml of HMGBl, HMGB2, HMGB3 or 0.1, 1, 10, lOOng/ml of HMGB2 mutants and DMEM with 10% serum (positive control) were placed in the lower chambers. Chemotaxis blocking- experiments were performed by adding in the lower chamber 30ng/ml of HMGB2 and 0.001, 0.01, 0.1, 1, lOμg of anti-HMGB2 antibody or in the upper chamber lμg of anti-RAGE antibody. BAEC cells were grown in DMEM plus 10% FCS, starved overnight, washed twice with PBS to eliminate any floating cells, and harvested with trypsin. Fifty thousand cells resuspended in 200 μl DMEM were placed in the upper chambers and incubated at 37°C in 5% CO₂ for 5 h. Cells remaining on the upper surface of the filters were mechanically removed, those which had migrated to the lower surface were fixed with ethanol, stained with Giemsa Stain Modified (Sigma) and counted at 40Ox magnification in ten random fields per filter. Assays were performed in triplicate and repeated three times in independent experiments.

Cell transfection

Dominant negative RAGE is a RAGE truncation lacking the entire intracytoplasmic domain (Hofmann et al., 1999). One million BAEC cells were co-transfected with 1 mg pNLSl-YFP (pNLSl-YFP was constructed like pNLSl-GFP (Bonaldi, 2003)) and 3 mg of plasmid encoding dominant negative RAGE (dnRAGE) or pCDNA3 empty vector, using FuGene reagent (Roche). After 24 hours, the medium was replaced with DMEM plus 10% FCS; after additional 24 hours the chemotaxis assay was carried out as described above. Only YFP- positive cells were considered. Protein alignment

Protein sequences of both human and murine HMGBl, HMGB2 and HMGB3 were aligned with ClustalW.

Results

HMGB2 is expressed in monocytes

Mouse monocytes were obtained with intra-peritoneal washes of wild type c57bl/6 mice. Cells seeded were cultured with RPMI medium and activated with LPS. hnmuno-fluorescence assay with anti-HMGB2 antibody was performed on activated monocytes. Fig. 1 shows how HMGB2 is expressed in mouse monocytes at nuclear level. Furthermore HMGB2 is also present in localized spots in the cytosol, suggesting a relocalization from the nucleus into secretory vesicles in the activated cells. ESTs analysis and RT-PCR confirmed HMGB2 expression in myeloid cells.

HMGB2 stimulates endothelial cells proliferation (^"not HMGB3)

Endothelial cells were taken from aorta of adult bovines. Primary colture cells were seeded in

DMEM medium with 10% FCS. Cells were starved for 16h in the absence of fetal serum to syncronize the cell population. Endothelial cells seeded in different plates were stimulated with 30 ng/ml of HMGBl, HMGB2 and HMGB3. As Fig. 2 shows already 24h after the treatment an increase in the number of endothelial cells stimulated with both HMGBl and HMGB2 was noticed. BAEC still responded to HMGB land HMGB2 48h after the stimulus and actually reach the proliferating peak at 72h. Surprisingly these data contrast with what seen by Palumbo, 2004 on mesangioblasts. HMGBl was able to promote mesangioblast proliferation 24h after the stimulus, whereas only slight proliferation was seen at 48 and 72h. These cells exposed every 24h to HMGBl continued to proliferate, suggesting a depletion of the protein present in the medium. Our data suggest that BAEC do not deplete the stimulus from the medium or have a prolonged response to it. Although Bl and B2 curves were comparable (Fig.2), BAEC had a higher response to HMGBl suggesting its higher activity. Fig.2 shows clearly that HMGB3 has no proliferating effect: stimulated cells show levels comparable to serum free treated cells over the 72h assay.

These data indicate that HMGBl and HMGB2 act as growth factors for endothelial cells and suggest that different cell types may have different proliferating responses to these proteins. Interestingly HMGB3 showed no proliferating effect although HMGBs share 80% homology of sequence suggesting that these proteins are only partially redundant.

HMGB2 induces BAEC migration

Previously it was shown that HMGBl acts not only as proliferating stimulus but as a chenioattractant for mouse mesangioblasts too (Palumbo, 2004), hence we investigated whether HMGB2 and HMGB3 could share the same capability.

Fig 3 shows how in a chemotaxis assay using modified Boyden chambers, HMGB2 stimulates migration of bovine endothelial cells. BAEC responded to protein concentrations raging from 1 ng/nil to 100 ng/ml in a concentration-dependent way. The migration peak was seen at 3-10 ng/ml of HMGB2.

In order to identify the chemoattractant portion, individual HMGB2 domains were also tested for their ability to promote cell migration (fig.4A). HMGB2A+B, the protein lacking the acidic tail (residues 1-180), showed a slightly higher capability of promoting cell migration compared to the wild type protein. HMGB2 Box B (residues 80-180) showed a behavior comparable to HMGB2A+B. HMGB2 Box A alone (1-80) induced basal levels of migration. The active portion of the protein should be thus limited in the region between Box B and the acidic-tail-upstream linker region.

Compared together HMGBl and HMGB2's data show a similar action of the two proteins although, utilizing same concentrations, cells seem to be more responsive towards HMGB2's stimulus.

These data, taken together with experiments performed with HMGBl by Huttunen, 2002 and Palumbo, 2004, tell that the protein portion inducing chemotaxis should be identified in the region comprised between Box B and the acidic tail, though the region is the most divergent, having 9 out of 21 different amino acids. The same chemotaxis experiment was used to see whether HMGB3 too could induce endothelial cells migration. The protein did promote chemotaxis but, as showed in Fig. 5, seems to act in a slightly different way. In particular HMGB3 seems to be active at lower concentrations, in fact the chemotaxis peak was identified at a concentration ten fold lower compared to both HMGBl and -2: 1 ng/ml. Increasing amounts of protein show a decrease in cell migration. Furthermore at 10ng/ml, the chemotactic peak for HMGBl and-2, cells treated with HMGB3 show basal levels. Higher concentrations (100ng/ml) showed levels lower than controls suggesting a toxicity of the protein.

In order to compare the chemotactic activity of the three proteins a migration assay was set up utilizing each protein at its migration-promoting peak concentration: 30 ng/ml HMGBl, 3 ng/ml HMGB2 and 1 ng/ml HMGB3. Fig. 6 shows the migratory effect of HMGBs: at their peak HMGBl and HMGB2 seem to have the same activity whereas HMGB3 seems a little less active working at a lower concentration.

Anti-HMGB2 blocks cell migration

Residues 150-183, the linker region between box B and the acidic tail, were identified as the HMGBl segment promoting migration through its interaction with RAGE (Huttunen, 2002).

Because of its high homology with HMGBl, we analyzed whether HMGB2 linker region too could be implied in chemotactic response.

HMGB2 polyclonal antibody specifically recognizes as epitope the amino acid stretch comprised between HMGB2 box B and the acidic tail (residues 166 and 181) which is the likely region implied in chemotaxis. In order to examine if HMGB2's activity is comparable to HMGBl, we performed a chemotactic assay using HMGB2 blocked by an anti-HMGB2 antibody.

Fig. 7 shows blocking assays on cell migration. Two kinds of experiments were performed.

Increasing amounts of HMGB2 antibody were tested on HMGB2 concentration 30ng/ml to find the antibody blocking concentration (Fig. 7A). lOOng of anti-HMGB2 antibody was seen to be the optimal amount inhibiting cell migration. A second migration assay was performed with increasing concentrations of HMGB2 and lOOng of blocking anti-HMGB2 antibody. Fig.

7B explains how chemotaxis assay performed with anti-HMGB2 shows a decreased amount of migrating cells compared to untreated cells. These data identify HMGB2 chemoattractant region between residues 166 and 181. DN-RAGE transfected BAEC show basal levels of proliferation

HMGBl and HMGB2's proven redundancy suggest that HMGB2 chemoattractant function should be mediated by the binding with RAGE such as with HMGBl(Huttunen, 2002). Western blot and RNA profiling confirmed RAGE expression in Bovine Aortic Endothelial Cells. We created a plasmid vector expressing the receptor for advanced-end glycosylation end products (RAGE) without its cytoplasmic region; this receptor works as a dominant- negative RAGE.

We cotrasfected BAEC with plasmid vector expressing nuclear YFP together with dominant negative RAGE (dn-RAGE) or with the empty control vector (pCDNA3). The entire cell population was subjected to chemotaxis assay both with HMGB2 and -B3 stimulus but only yellow fluorescent cells were counted. Endothelial cells over-expressing dominant negative RAGE were severely impaired in their capability to respond to extracellular HMGB2 and HMGB3 compared to cells trasfected with control pCDNA3 (Fig. 8). These data, taken together with experiments performed with blocking antibodies, confirm that all HMGBs are able to bind and signal through RAGE.

Anti RAGE H300 antibody blocks BAEC migration

All these data taken together suggest that RAGE is able to promote endothelial cell migration via its binding with all three members of the HMGB family. RAGE extracellular portion is composed of 400 residues, blockage of this portion should inactivate the receptor because of the occlusion of the binding domain and thus not induce the activation of the migratory pathway.

In order to confirm RAGE-HMGBs interaction a migration assay was performed. HMGBs, each used at its highest migration-promoting concentration (30 ng/ml HMGBl, 3 ng/ml HMGB2 and 1 ng/ml HMGB3), were used as chemotactants. Bovine aortic endothelial cells were treated with 1 μg of H300, a polyclonal antibody that recognizes and binds the first 300 residues of the extracellular portion of RAGE.

The migration assay shows that H300-treated BAEC lose their ability to respond to the

HMGBs chemotactic stimulus: cells with blocked-RAGE in the presence of HMGBl, HMGB2 or HMGB3 show basal levels of migration, whereas untreated cells do migrate (Fig

9).

This result confirms that all three members of the HMGB family bind to RAGE and that this binding stimulates cell migration.

HMGB-RAGE binding motif

The redundancy among the three proteins was analyzed more in detail. Utilizing bio- informatic tools we aligned both human and murine HMGBs (Fig. 10). Comparing our experiments with data from Palumbo, 2004 and Huttunen, 2002, we restricted the HMGBs- RAGE interacting region to residues 160-181. This linker region seems to be divided into two parts: a similar N-terminus and a more diverse c-terminus region.

References

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Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B, Stern DM, Nawroth PP. Understanding RAGE, the receptor for advanced glycation end products.J MoI Med. 2005 Nov;83(ll):876-86. Epub 2005 Aug 24. Review.

Bonaldi T, Talamo F, Scaffidi P, Ferrera D, Porto A, Bachi A, Rubartelli A, Agresti A, Bianchi ME. Monocytic cells hyperacetylate chromatin protein HMGBl to redirect it towards secretion. EMBO J. 2003 Oct 15;22(20):5551-60. Calogero S, Grassi F, Aguzzi A, Voigtlander T, Ferrier P, Ferrari S, Bianchi ME. The lack of chromosomal protein Hmgl does not disrupt cell growth but causes lethal hypoglycaemia in newborn mice. Nat Genet. 1999 Jul;22(3):276-80.

Deane R, Du Yan S, Submamaryan RK, LaRue B, Jovanovic S, Hogg E, Welch D, Manness L, Lin C, Yu J, Zhu H, Ghiso J, Frangione B, Stern A, Schmidt AM, Armstrong DL, Arnold B, Liliensiek B, Nawroth P, Hofman F, Kindy M, Stern D, Zlokovic B. RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat Med. 2003 Jul;9(7):907-13. Degryse B, Bonaldi T, Scaffidi P, Muller S, Resnati M, Sanvito F, Arrigoni G, Bianchi ME. The high mobility group (HMG) boxes of the nuclear protein HMGl induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells. J Cell Biol. 2001 Mar 19; 152(6): 1197- 206. Gardella S, Andrei C, Ferrera D, Lotti LV, Torrisi MR, Bianchi ME, Rubartelli A. The nuclear protein HMGBl is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway. EMBO Rep. 2002 Oct;3(10):995-1001. Epub 2002 Sep 13. Hori O, Brett J, Slattery T, Cao R, Zhang J, Chen JX, Nagashima M, Lundh ER, Vijay S, Nitecki D, et al. The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system. J Biol Chem. 1995 Oct 27;270(43):25752-61. Huttunen HJ, Fages C, Kuja-Panula J, Ridley AJ, Rauvala H. Receptor for advanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis. Cancer Res. 2002 Aug 15;62(16):4805-l l. Nemeth MJ, Cline AP, Anderson SM, Garrett-Beal LJ, Bodine DM. Hmgb3 deficiency deregulates proliferation and differentiation of common lymphoid and myeloid progenitors. Blood. 2005 Jan 15;105(2):627-34. Epub 2004 Sep 9.

Palumbo R, Gaetano C, Antonini A, Pompilio G, Bracco E, Ronnstrand L, Heldin CH, Capogrossi MC. Different effects of high and low shear stress on platelet-derived growth factor isoform release by endothelial cells: consequences for smooth muscle cell migration. Arterioscler Thromb Vase Biol. 2002 Mar l;22(3):405-ll.

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Claims

I . The HMGB3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity for medical use.

2. Use of the HMGB3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity for the preparation of an immunostimulating composition.

3. Use of the HMGB3 protein or functional part thereof according to claim 2 as adjuvant.

4. Use according to claim 3 wherein the immunostimulating composition is a vaccine.

5. A pharmaceutical composition comprising an effective amount of the HMGB3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity.

6. An immunostimulating composition comprising an effective amount of the HMGB3 protein or functional part thereof exerting a chemoattracting and not a proliferating activity.

7. The immunostimulating composition according to claim 6 being a vaccine composition.

8. Use of the HMGB2 protein or functional part thereof for the preparation of an activating cell proliferation pharmaceutical composition.

9. Use of the HMGB2 protein or functional part thereof according to claim 8 for the preparation of an activating cell proliferation pharmaceutical composition for tissue regeneration.

10. Use of the HMGB2 protein or functional part thereof according to claim 9 for the preparation of an activating cell proliferation pharmaceutical composition for myocardial tissue regeneration and/or peripheral artery diseases.

I 1. Use of the HMGB2 protein or functional part thereof according to claim 9 for the preparation of an activating cell proliferation pharmaceutical composition for wound healing.