CN101247820A - Chaperonin 10-induced immunomodulation - Google Patents

Abstract

The present invention relates to methods for modulating Toll-like receptor signalling in a subject, or in at least one cell, tissue or organ thereof, methods for treating or preventing a disease or condition in a subject, methods for modulating the production and/or secretion of one or more immunomodulators in a subject, or at least one cell, tissue or organ thereof, wherein said methods involve the administration of chaperonin 10, and wherein the chaperonin 10 associates with a Toll-like receptor in an activation cluster. Also contemplated are associated compositions and uses thereof.

Description

Chaperonin 10-induced immunomodulating

Technical field

The present invention relates generally to cpn10 and is regulating the conduction of Toll sample receptor signal, especially the application in TLR2, TLR3, TLR4, TLR7 and the conduction of TLR9 signal, and the method and composition that is used for treating by the inductive regulating action to these signal conduction of cpn10 disease, described disease includes but not limited to virus and bacterial infection, inflammatory diseases and autoimmune disease.

Background technology

Main component at the host defense system of invading bacteria, fungus, yeast and viral pathogen, participated in the successful identification of cell receptor to pathogen or its composition, this cell receptor brings out the signal transduction cascade reaction, causes the generation of the immune stimulation and the various kinds of cell factor.A receptor family that has critical role in this defence line is Toll-sample receptor (TLR) family.TLR expresses comprising on the following immunocyte: the cell of mononuclear cell, macrophage, dendritic cell, B cell and granulocyte and multiple other types comprises endotheliocyte and epithelial cell.TLR is by the molecule activation in pathogen or its source, brings out intracellular signal conduction, its mainly cause transcription factor NF-KB activation (Beg, 2002, Trends Immunol, 23509-12) and the adjusting that produces of cytokine.But also can excite a series of other paths, comprise kinase pathway (people such as Flohe, 2003, J Immunol, the 1702340-2348 of the mitogen-activated kinases of p38, the terminal kinases of c-Jun-N-and extracellular signal correction; Triantafilou and Triantafilou, 2002, Trends Immunol, 23301-304).

I type interferon mainly is IFN α and IFN β, is to the significant feature factor in the immunne response of antibacterial and viral infection.After these infected, the main source of I type interferon was a plasma cell sample dendritic cell (PDC), a kind of special subgroup of peripheral blood lymphocytes.PDC expresses Toll sample receptor TLR7 and TLR9, and the TLR7/TLR9 stimulation can activate PDC, and produces a large amount of I type interferon.

The factor in a large amount of pathogen source can optionally stimulate different TLR.Wherein most typical is lipopolysaccharide (LPS), a kind of glycolipid from the gram negative bacteria adventitia, and it is the agonist of TLR4.TLR2 can discern fat techoic acid (LTA), Peptidoglycan (petidoglycan, PGN) and lipopeptid; TLR3 can discern double-stranded RNA, generally is viral source; TLR7 can discern viral single stranded RNA, and TLR9 can discern unmethylated CpG dinucleotide, generally in antibacterial and viral DNA.TLR also can be stimulated by multiple synthetic agonist, the polyI:C (polyinosinic acid-polycytidylicacid (polysytidilic acid)) that TLR3 is stimulated for example, imidazoquinolie to the TLR7 stimulation, as resiquimod (resiquimod, R848) and imiquimod (imiquimod) and rich CpG oligonucleotide that TLR9 is stimulated.

Illustrate the regulatory mechanism that the adjusting of TLR signal conduction and TLR stimulating cytokine and chemotactic factor produce, will provide approach for the therapeutic modality of the treatment of developing multiple disease and disease (comprising virus and bacterial disease).

Mammal cpn10 (Cpn10) is also referred to as heat shock protein 10 (Hsp 10), general and chaperone 60 (Cpn60; Hsp60) typically characterized to participating in mitochondrion " molecular chaperones " albumen of protein folding together.Cpn10 is the congener of bacterioprotein GroES.GroES and Cpn10 oligomerization are 7 yuan of rings, are combined on the tubular structure as lid, and this tubular structure comprises 14 GroEL or 7 Hsp60 molecules, the albumen of degeneration can be strapped on the complex.Cpn10 also usually find on cell surface (people such as Belles, 1999, InfectImmun, 67:4191-4200) and in the extracellular fluid (people such as Shin, 2003, JBiol Chem, 278:7607-7616).

But Cpn10 has also shown and has had immunosuppressive activity (people such as Zhang, 2003, JNeurol Sci 212:37-46 in tentative autoimmune encephalomyelitis, delayed hypersensitivity and allograft rejection model; People such as Morton, 2000, Immunol Cell Biol 78:603-607).

In the past, the inventor is verified in the presence of TLR4 and TLR2 agonist (being respectively LPS and PAM3CysSK4), Cpn10 can reduce the NF-kB activation of TLR4-and TLR2-stimulation in dose-dependent mode, reduce TNF-α and RANTES secretion, increase IL-10 secretion (people such as Johnson simultaneously, 2005, J Biol Chem 280:4037-4047; International patent application PCT/AU2005/000041 number; WO2005/067959, its disclosure is incorporated herein by reference).

The inventor is surprised to find Cpn10 now also can regulate the signal conduction that is caused by TLR3, TLR7 and TLR9.This paper shows that Cpn10 can be in the presence of the TLR3-ligands specific, and dose response ground strengthens the generation of IFN α and IFN β, and reduces the NF-kB activation in the presence of TLR7-and TLR9-ligands specific.And the inventor is surprised to find Cpn10, but is not Cpn60 or GroES, combines at the cell surface place with TLR in the activation bunch, conducts thereby make a little activation bunch regulate a TLR signal.

Summary of the invention

According to a first aspect of the invention, the method of Toll sample receptor signal conduction in a kind of experimenter of adjusting or its at least a cell, tissue or the organ is provided, wherein said method comprises the cpn10 that gives effective dose, combining between the Toll-sample receptor during wherein Toll sample receptor signal conduction relates to cpn10 and activates bunch (association).

According to a second aspect of the invention, the method of Toll sample receptor signal conduction in a kind of experimenter of adjusting or its at least a cell, tissue or the organ is provided, wherein said method comprises the antagonist of at least a cpn10 that gives effective dose, combining between the Toll-sample receptor during wherein Toll sample receptor signal conduction relates to cpn10 and activates bunch, and wherein said antagonist stops the Toll sample receptor in cpn10 and the activation bunch to combine, and/or stops activation bunch to cause that signal conducts.

Activation bunch can be positioned on the cell surface.

Activation bunch can comprise cpn10, Toll sample receptor and randomly at least a other molecules.These at least a other molecules can comprise Toll-sample receptor stimulating agent.

In one embodiment, this activation bunch comprises cpn10, TLR2 and fat techoic acid (LTA).

In another embodiment, this activation bunch comprises cpn10, TLR3 and double-stranded RNA.

In another embodiment, this activation bunch comprises cpn10, TLR4 and LPS.

In further embodiment, this activation bunch comprises cpn10, TLR7 and single stranded RNA.

Also in another embodiment, this activation bunch comprises cpn10, TLR9 and comprises the DNA of CpG motif.

Cpn10 can be natural origin, that reorganization produces or the synthetic cpn10 that produces.Cpn10 can be the eukaryotic cell source.Cpn10 can be people's cpn10.

Cpn10 can comprise the peptide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQ ID NO:3.Chaperone can be acetylizad or non-acetylizad.

Cpn10 can be with the polynucleotide form administration of coding cpn10.The polynucleotide of coding cpn10 can be arranged in gene constructs, operationally are connected with promoter.Polynucleotide can comprise the sequence shown in the SEQ ID NO:4.

This method can further comprise and gives at least a other medicament.This medicament can be an immunomodulator.This immunomodulator can be an I type interferon, as IFN α or IFN β.

According to a third aspect of the present invention, the method of a kind of treatment or prevention experimenter's disease or disease is provided, wherein said method comprises the cpn10 that gives experimenter's effective dose, Toll-sample receptor in wherein said cpn10 and the activation bunch combines, and the formation of wherein activation bunch with to the startup of the immunne response of this disease or disease, strengthen and/or keep relevant.

According to a fourth aspect of the present invention, the method of a kind of treatment or prevention experimenter disease is provided, wherein said method comprises at least a cpn10 antagonist that gives experimenter's effective dose, Toll sample receptor during wherein said antagonist stops cpn10 and activates bunch combines, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and this disease or disease and/or make progress relevant.

Disease or disease can be selected from virus, fungus, yeast or bacterial infection, acute or chronic inflammatory disease, comprise septic shock, inflammatory bowel, arthritis, psoriasis, heart disease, atherosclerosis, chronic lung disease, cachexia, multiple sclerosis, GVHD and cancer.

In one embodiment, cpn10 is regulated the inductive TLR2 signal conduction of LTA-.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR2-.

In another embodiment, cpn10 is regulated double-stranded RNA-inductive TLR3 signal conduction.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR3-.

In another embodiment, cpn10 is regulated the inductive TLR4 signal conduction of LPS-.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR4-.

Also in another embodiment, cpn10 is regulated viral single stranded RNA-inductive TLR7 signal conduction.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR7-.

Still in another embodiment, cpn10 is regulated the inductive TLR9 signal conduction of CpG motif.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR9-.

Cpn10 can be natural origin, that reorganization produces or the synthetic cpn10 that produces.Cpn10 can be the eukaryotic cell source.Cpn10 can be people's cpn10.

Cpn10 can comprise the peptide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQ ID NO:3.Cpn10 can be acetylizad or non-acetylizad.

Cpn10 can be with the polynucleotide form administration of coding cpn10.The polynucleotide of coding cpn10 can be arranged in gene constructs, operationally are connected with promoter.Polynucleotide can comprise the sequence shown in the SEQ ID NO:4.

This method can further comprise and gives at least a other medicament.This medicament can be an immunomodulator.This immunomodulator can be an I type interferon, as IFN α or IFN β.

According to a fifth aspect of the present invention, providing a kind of is used for the treatment of or the compositions of prevent disease or disease, said composition comprises cpn10 and at least a pharmaceutically acceptable carrier, diluent or adjuvant, Toll sample receptor in wherein said cpn10 and the activation bunch combines, and the formation of wherein activation bunch with to the startup of the immunne response of this disease or disease, strengthen and/or keep relevant.

Said composition can further comprise at least a Toll sample receptor stimulating agent.Said composition can further comprise at least a immunomodulator, as I type interferon.

According to a sixth aspect of the invention, providing a kind of is used for the treatment of or the compositions of prevent disease or disease, said composition comprises at least a cpn10 antagonist, Toll sample receptor during wherein said antagonist stops cpn10 and activates bunch combines, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and this disease or disease and/or make progress relevant.

According to a seventh aspect of the present invention, provide cpn10 manufacturing be used for the treatment of or the medicine of prevent disease or disease in application, Toll sample receptor in wherein said cpn10 and the activation bunch combines, and the formation of wherein activation bunch with to the startup of the immunne response of this disease or disease, strengthen and/or keep relevant.

According to an eighth aspect of the present invention, provide the cpn10 antagonist manufacturing be used for the treatment of or the medicine of prevent disease or disease in application, Toll sample receptor during wherein said antagonist stops cpn10 and activates bunch combines, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and this disease or disease and/or make progress relevant.

According to a ninth aspect of the present invention, the generation and/or the excretory method of one or more immunomodulators in a kind of experimenter of adjusting or its at least a cell, tissue or the organ are provided, wherein said method comprises the cpn10 that gives effective dose, wherein said cpn10 with the activation bunch in Toll sample receptor combine, and wherein the activation bunch formation relevant with the generation and/or the excretory adjusting of one or more immunomodulators.

According to a tenth aspect of the present invention, the generation and/or the excretory method of one or more immunomodulators in a kind of experimenter of adjusting or its at least a cell, tissue or the organ are provided, wherein said method comprises that the cpn10 that gives effective dose picks up anti-agent, the wherein said anti-agent Toll-sample receptor in stoping cpn10 and activating bunch of picking up combines, and/or stop activation bunch to cause the signal conduction, and wherein the formation of activation bunch is relevant with the generation and/or the excretory adjusting of one or more immunomodulators.

Above-mentioned immunomodulator can be, for example TNF-α, IL-1 β, IL-6, IL-10, IL-12 or I type interferon.I type interferon can be IFN α or IFN β.

Above-mentioned aspect and embodiment have considered that the wild type of cpn10 and modified forms and total length cpn10 polypeptide and its have kept the application of the fragment or the derivant of immunoregulatory activity.

Definition

In the context of the present specification, the implication that term " comprises " is " mainly comprise, but not necessarily unique ".And the version (for example singulative (comprises) and plural form (comprise)) that " comprises " of term all has the implication of respective change.

As used herein term " treatment (treatment, treating) " and version thereof are meant treats morbid state or symptom no matter by any way, the formation that wards off disease, or prevent, stop, delay, alleviate or reverse any and all application of the progress of disease or other ill symptomses in addition.

The implication of term " effective dose " comprises the consumption avirulence of medicament or chemical compound but is enough to the treatment or the prophylactic effect that provide required as used herein.Required definite consumption will change according to following factors between the experimenter: for example the seriousness of the species that will treat, experimenter's age and general situation, the disease that will treat, will be by the concrete medicament of administration and administering mode or the like.Therefore, can not definite " effective dose " of regulation.Yet for any specified situation, those of ordinary skills only just can determine suitable " effective dose " by the test of routine.

Term " polypeptide " is meant the polymer of being made up of the aminoacid that links together by peptide bond.Term " polypeptide " and " albumen " are used interchangeably in this article, although for purpose of the present invention, " polypeptide " can constitute the part of full-length proteins.Polypeptide also can include but not limited to its fragment, analog, variant and derivant.These its fragments, analog, variant and derivant can all have specific structure and/or functional group's similarity with polypeptide.

Term " polynucleotide " is meant strand or double-chain polymer or its mixture of deoxyribonucleotide, ribonucleotide base or known analogs or natural nucleotide as used herein.The reverse complemental of polynucleotide and the fragment of polynucleotide, analog, variant and derivant and with any other polynucleotide that polynucleotide are hybridized under high stringency, all be included in the scope of term " polynucleotide ".

Term " is regulated (modulating, modulates) " and is changed body and is meant in the presence of particular adjustments molecule of the present invention or medicament as used herein, compare with activity, generation, secretion or its other functional levels of molecule under the situation that lacks adjusting molecule or medicament, can increase or reduce its activity, generation, secretion or functional level.The quantity that increases or reduce do not pointed out in these terms.Regulating action can be any degree that is enough to produce required result, and can be direct or indirect.

Term " immunomodulator " includes but not limited to by the excretory molecular media of one or more cell types as used herein, its startup in immunne response, activate, keep, strengthen, ripe, suppress, containment or increase in have critical role.

As used herein term " activation bunch " is meant the group with the molecule that produces the biological signals function.Usually, biological signals can comprise the adjusting of immunne response, particularly can comprise regulating the part of Toll sample receptor signal conduction as this adjustment process.Activation bunch can be for example, on the surface of cell or the interior combination of endosome (endosome), and can comprise, for example cpn10 and Toll sample receptor stimulating agent and Toll sample receptor.Other molecules also can be used as the activation bunch part combination, include but not limited to CD14.

Description of drawings

The present invention is described with reference to the accompanying drawings by non-limiting example now.

Fig. 1. after stimulating 23 hours with 100 μ g/ml polyI:C and add that 10-200 μ g/ml recombined human Cpn10 is pre-to be stimulated 2 hours, IFN β (pg/ml) secretion enters in the supernatant of RAW264.7 cell.Result displayed is from twice test.

Fig. 2. behind LPS or 100 μ g/ml polyI:C stimulation with 100ng/ml repurity, and add pre-the stimulation 2 hours of 10-100 μ g/ml recombined human Cpn10, I type interferon (IU/ml) secretion enters in the supernatant of RAW264.7 cell.

Fig. 3. there be (open squares) or do not existing under the situation of (filled squares) 50 μ g/ml recombined human Cpn10 (adding simultaneously) with agonist, after reporting sub-construction transfection with TLR7/TLR4 expression constructs and NF-κ B-luciferase, the NF-kB activation in the HEK293 cell (x-is doubly).Cell phorbol 12-myristinate 13-acetas (PMA; Protein kinase c activator), R848 (R848; The TLR7 agonist), CpG oligonucleotide (CpG; The TLR9 agonist) or the lipopolysaccharide (LPS of repurity; The TLR4 agonist) stimulates.

Fig. 4. there be (open squares) or do not existing (filled squares) 50 μ g/ml recombined human Cpn10 when (adding simultaneously) with agonist, after reporting sub-construction transfection with TLR9 expression constructs and NF-κ B-luciferase, the NF-kB activation in the HEK293 cell (x-is doubly).Cell phorbol 12-myristinate 13-acetas (PMA; Protein kinase c activator), R848 (R848; The TLR7 agonist), CpG oligonucleotide (CpG; The TLR9 agonist) or the lipopolysaccharide (LPS of repurity; The TLR4 agonist) stimulates.Two results (A and B) of clone have separately been shown.

Fig. 5. in the presence of CpG DNA (Cpn10 and agonist add simultaneously), recombined human Cpn10 concentration (12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml and 100 μ g/ml) (is shown as A to the effect of NF-kB activation in the HEK293 cell of reporting sub-construction transfection with TLR9 expression constructs and NF-κ B-luciferase, luciferase unit and B, the plain enzyme unit of relative fluorescence).

Fig. 6 .A. stimulates with the lipopolysaccharide (LPS) of repurity, and in the presence of the recombined human Cpn10 (adding simultaneously with agonist) of variable concentrations (1 μ g/ml, 10 μ g/ml or 50 μ g/ml) or when not having Cpn10, the TNF-α from human peripheral blood mononuclear cell (PBMC) discharges (ng/ml).B. as A, difference is at first PBMC to be consumed plasma cell sample dendritic cell (PDC).

Fig. 7 .A. stimulates with the lipopolysaccharide (LPS) of repurity, and in the presence of the recombined human Cpn10 of variable concentrations (1 μ g/ml, 10 μ g/ml or 50 μ g/ml) or when not having Cpn10, the IL-10 release (ng/ml) from human peripheral blood mononuclear cell (PBMC).B. as A, difference is at first PBMC to be consumed plasma cell sample dendritic cell (PDC).

Fig. 8. in RAW264-pNifty2-LUC cell line, Cpn10 does not reduce the NF-kB activation by Poly (I:C).

Fig. 9 .Cpn10 dose response ground promotes to be stimulated 24 hours RAW264.7 cell generation I type IFN by LPS (mainly being IFN-β) or Poly (I:C) (mainly being IFN-α).

Figure 10 .Cpn10 promotes the RAW264.7 cell that is stimulated by Poly (I:C) to produce IFN β with transcriptional level dose response ground.In the RAW264.7 cell that stimulates with LPS, also see the potentiation of IFN β in the presence of Cpn10.

Figure 11. the human PBMC of the different donors that stimulate from two usefulness, 50 μ g/ml Poly (I:C) produces the IFN-α of increase level in the presence of Cpn10.

Figure 12 .Cpn10 but be not GroES, the reactive increase of inductive dose in Poly (I:C)-inductive IFN-β.

Figure 13 .Cpn10 preincubate washs then, reduces the increase at the aborning Cpn10 dose response of Poly (I:C)-inductive IFN-β.

Figure 14 .Cpn10 do not strengthen from IFNAR1-/-IFN beta response among the BMM of mice.

Figure 15. macrophage needs the adjusting (n=2) of Cpn10 to the TLR4 reaction by the startup of I type interferon path.

Figure 16. induce serum TNF α and IFN β level in the mice of systemic inflammatorome with LPS.

Figure 17. infect with SFV 6-9 days ages newborn C57BL/6 mice survival rate.6-9 days ages, the use of newborn C57BL/6 mice was as follows: A group: SFV (n=13); B group: SFV+Cpn1020 μ g (n=13); C group: SFV+Cpn1050 μ g (n=13).B group and C group lumbar injection 20 and 50 μ g Cpn10, (after 3 hours) inject 50 μ l SFV (30x TCID50) to all three groups then.According to shown in legend survival rate is mapped.

Figure 18. induce the Cpn10 promoter by heat shock (HS) stimulation and TLR stimulation.Representative certificate of analysis heat shock is restored and can be induced the Cpn10 promoter activity significantly in 6 hours, returns to baseline values (n=2 time repeat to chemically examine) then behind the HS in 18 hours (A).On the contrary, stimulate, produce 2.7-promoter doubly by 24 hours stimulations and induce, and in the time of 30 hours, keep constant (A) with LTA (TLR2 agonist).With TLR2 (B, C), TLR7 (D, E) or TLR9 (F, G) part titration irritation cell, induce Cpn10 promoter (n=2-6 repetition) in the dose dependent mode.Identical data among C, E and the data reflection shown in the data shown in the G and B, D and the F, but the multiple that shows as on the baseline changes.Error bar is expressed as a SD of mean.The top level of TLR agonist and cytokine induction Cpn10 promoter different (H).For every kind of agonist, the Cpn10 promoter activity is expressed as with the standardized luciferase cps of baseline values.Best agonist concentration and chemical examination number of repetition: TLR2: bacillus subtilis LTA 100 μ g/ml, n=12; TLR3:100 μ g/ml poly (I:C), n=6; TLR4: ultrapure Escherichia coli LPS 10ng/ml, n=12; TLR7: imiquimod R83710 μ g/ml, n=2; TLR9, CpG ODN 1mM, n=2; IFN γ 1ng/ml, n=3, IL-1 β (1ng/ml), n=2, TNF-α (100ng/ml), n=2 (H).Estimate the significance (p＜0.02) and the significance (F) (p＜0.05) of (A) with ANOVA, and be expressed as * .cps=per second counting.In the untransfected RAW264.7 cell that stimulates with LTA, measure Cpn10 by PCR in real time (I)

The increase of the concentration dependent that the mRNA level is temporary transient.With contrast house-keeping gene (18S and Pbgd) standardization is carried out in gene expression.

Figure 19 .TLR agonist and heat shock induction Cpn10 produce, but only use the TLR agonist to cause that the extracellular of Cpn10 discharges.By ELISA the full cellular lysate of RAW264.7 that LTA-stimulates is carried out quantitative analysis, the Cpn10 level changes very little (A) between display process cell and the control cells, but the analytical proof of culture supernatant extracellular Cpn10 after 100 μ g/ml LTA stimulate 30 hours increases 3-doubly (B).Supernatant C pn10 carries out standardization with the cell number in every hole.The Cpn10 promoter activation that time-history analysis proof continues surpasses 30 hours, as to the bonded reaction of TLR2.Protein concentration carries out standardization (C) to the result per sample.Slight, the moderate or the severe heat shock of RAW264.7 cell can not induce the extracellular of Cpn10 to discharge (D).Cpn10 level in RA patient's blood circulation is significantly higher than MS or psoriatic or normal healthy controls experimenter.Adopt Tukey check analysis significance afterwards by one-sided ANOVA, and be expressed as ^*(p＜0.01) or ^*(P＜0.001) (E).

Figure 20 .Cpn10 restriction agonist is through the inductive signal conduction of a plurality of TLR.With the RAW264.7 cell of HIV-LTR-LUC promoter construction stable transfection, before with multiple TLR ligand stimulation with the Cpn10 preincubate, caused suppressing in these cells luciferase activity (index of NF-kB activation) (A).Agonist concentration and incubation time are: CpG ODN16.25 μ g/ml, 4h; Imiquimod 10 μ g/ml, 2h; Poly (I:C) 100 μ g/ml, 4h; Zymosan 10 μ g/ml, 2h; PGN 10 μ g/ml, 2h; PGN 10 μ g/ml, 2h; Ultrapure LPS10ng/ml, 2h; LPS 5ng/ml, 2h.RAW264.7 cell (B, C) or human PBMC (D) and Cpn10 preincubate, carrying out LPS then stimulates (be 10ng/ml in the RAW264.7 cell, be 100ng/ml among the PBMC) to cause the dose response reduction of phosphorylation MAPK signal level.The human PBMC stimulates in the presence of Cpn10 with the part of TLRs 2,2/6,4,7,9, causes the reduction of TNF-α, IL-1 β, IL-6 and IL-10 level, and the increase imiquimod-and the inductive IFN α generation of CpG ODN-(E).The ligand concentration that uses is: 10 μ g/mlPGN, 10 μ g/ml LTA, 10 μ g/ml zymosans, 0.12ng/ml LPS, 6.5 μ g/ml CpGODN, 10 μ g/ml imiquimods.

Figure 21. the RAW264 cell of growing in suspension was hatched 4 hours in the presence of 100 μ g/ml Cpn10 or isopyknic dilution buffer liquid, dyeed then existing with proof TLR4.

Figure 22. the RAW264 cell of in suspension, growing in the presence of 100 μ g/ml Cpn10 or isopyknic dilution buffer liquid, with or do not hatch 4 hours with 2ng/ml LPS (adding in back 2 hours that handle at Cpn10), dye then existing with proof TLR4.

Figure 23. the RAW264 cell of in suspension, growing in the presence of 100 μ g/ml Cpn10 or isopyknic dilution buffer liquid, with or do not hatch 4 hours with 20ng/ml LPS (adding in back 2 hours that handle at Cpn10), carry out TLR4 dyeing afterwards.(average fluorescent strength: orange: 4.39, black: 6.14, blueness: 25)

Figure 24. the RAW264 cell of in suspension, growing in the presence of 100 μ g/ml Cpn10 or isopyknic dilution buffer liquid, with or not with 2ng/ml LPS (adding in back 2 hours that handle at Cpn10) overnight incubation, carry out TLR4 dyeing then.Average fluorescent strength: blue-25, red-33, orange-11.7, black-20.

Figure 25. the RAW264 cell of in suspension, growing in the presence of 100 μ g/ml Cpn10 or isopyknic dilution buffer liquid, with or not with 20ng/ml LPS (adding in back 2 hours that handle at Cpn10) overnight incubation, carry out TLR4 dyeing then.(average fluorescent strength: orange: 3.9, black: 7.9)

Figure 26. representational TLR part and Cpn10 are to the inhibition of luciferase activity.Data among A, C, E, G, the I have been described the luciferase unit among the CPS, and B, D, F, H, J to be the inhibition percentage ratio of luciferase activity in the RAW264-HIV-LTR-LUC cell that stimulates with LPS represent these data.

Figure 27. by the inductive inhibition of the activatory maximum Cpn10 of the HIV-LTR of a large amount of TLR ligand stimulations, as the indirect index of NF-kB activation.

Figure 28. in 30 minutes cell of LPS stimulation, Cpn10 dose response ground reduces p38 (A), the ERK1/2 of phosphorylation and the level of JNK1/2 (B).As being shown in the RAW264.7 to mouse macrophage, in the human PBMC of fresh separated, Cpn10 dose response ground reduces the level (C) of the inductive p38 of LPS-, ERK1/2 and JNK1/2 phosphorylation.Because the inflammatory cascade reaction that the activation of map kinase path and the inducing cell factor produce is closely related, so the variation that the inductive cytokine of part of these data show Cpn10-mediation produces is from the variation in the reaction of TLR signal cascade.

Figure 29. (PGN, LTA), TLR2, the part of 6 (zymosans), TLR4 (LPS), TLR7 (imiquimod) or TLR9 (CpG ODN2216) stimulates the human PBMC in the presence of Cpn10, causes production of cytokines to reduce with TLR2.

Figure 30 .Cpn10 preincubate washs then, adds LPS still limits NF-κ B in the dose response mode activation then.

Figure 31 .Cpn10 preincubate stimulated 6 hours with IFN-γ then, and the TNF-α that causes the RAW264.7 cell to produce increases.

The SDS-PAGE of Figure 32 .Cpn10 binding molecule analyzes.Combine also the therefrom proteic SDS-PAGE gel of cellular lysate of eluting with the Cpn10 affinity column.

The 2D gel electrophoresis of Figure 33 .Cpn10 binding molecule.Combine also the therefrom proteic 2D gel of cellular lysate of eluting with the Cpn10 affinity column.

The toxicity curve of PolyI:C in three days in Figure 34 .RAW264 (A) and the HeLa cell (B).

Figure 35. in RAW264 (A) and HeLa cell (B) in three days Cpn10 to the influence of PolyI:C.

Figure 36 .Cpn10 interacts at cell surface and human antigen's presenting cell, and endocytosis is to the endolysosome cell.The crosslinked Cpn10 of the PBMC of purification and fluorophor was hatched under 4 ℃ 10 minutes, measured surface fluorescence (A) by flow cytometer.The associating of Cpn10 and mDC and pDC is the closest, and mononuclear cell is presented at the combination increase (B) that LTA stimulates fluorescence Cpn10 after 4 hours.The people pDC of RAW264.7 cell and purification and fluorescence Cpn10 were hatched under 37 ℃ 30 minutes, carried out co-focusing imaging then.Hatch altogether with the fluorescent marker of mitochondrion and acid cell and to show in the Cpn10 and swallow (C) in the endolysosome cell.

The amino acid sequence of wild type people Cpn10 (GenBank registration number X75821) is provided among the SEQ ID NO:1. The amino acid sequence of two kinds of modified forms of Cpn10, it has extra amino acid residue in the N-end, is provided in SEQ ID NO:2 and 3. Its coding nucleotide sequence is provided among the SEQ ID NO:4. The Oligonucleolide primers that uses among the disclosed embodiment in this article is provided among the SEQ ID NO:5-10.

The specific embodiment

Shown in the past Cpn10 can reduce the generation of proinflammatory cytokine in cell culture and the body such as TNF-α and RANTES (referring to, people such as Johnson for example, 2005, J Biol Chem280:4037-4047 and International Patent Application PCT/AU2005/000041, WO2005/067959, these disclosures are incorporated herein by reference).These find to support use Cpn10 as healing potion to treat various inflammatory diseasess, comprise, for example, multiple sclerosis, rheumatoid arthritis and graft versus host disease.

Regulate the method that conduction of Toll sample receptor signal and immunomodulator produce

As disclosed herein, reference Cpn10 is when with TLR3 agonist polyI:C administration now for the inventor, and concertedness ground increases the generation of IFN β and IFN α significantly.In addition, by measuring the minimizing of NF-kB activation, prove that also Cpn10 can be through TLR7 and the conduction of TLR9 conditioning signal.

And the inventor it has surprisingly been found that after stimulating with LPS, and Cpn10, but be not Cpn60 or GroES unites with TLR4 form with " activation bunch " on cell surface.The inventor further is presented at fat techoic acid (LTA) stimulates back Cpn10 and TLR2 to unite, and is stimulating back and TLR7 to unite with viral single stranded RNA and/or imiquimod, and is stimulating back and TLR9 to unite with CpG DNA.In each case, the form of this associating all is Cpn10 and " activation bunch " interacts, and should " activate bunch " comprise specific TLR and agonist thereof.

Therefore, the invention provides the method for regulating Toll sample receptor signal conduction in experimenter or its at least a cell, tissue or the organ, wherein said method comprises the cpn10 that gives effective dose, the associating between the Toll-sample receptor during wherein Toll sample receptor signal conduction relates to cpn10 and activates bunch.

The conduction of Toll sample receptor signal also can be regulated by following mode: the antagonist that gives at least a cpn10 of effective dose, associating between the Toll-sample receptor during wherein Toll sample receptor signal conduction relates to cpn10 and activates bunch, and wherein saidly pick up anti-agent and stop the Toll sample receptor in cpn10 and the activation bunch to be united, and/or stop activation bunch to cause that signal conducts.

Activation bunch can be positioned on the cell surface or for example on the surface of the cell vesicle of Inclusion.Activation bunch can comprise cpn10, Toll sample receptor and optional at least a other molecule.These at least a other molecules can comprise Toll sample receptor stimulating agent.

In one embodiment, this activation bunch comprises cpn10, TLR2 and fat techoic acid (LTA).In another embodiment, this activation bunch comprises cpn10, TLR3 and double-stranded RNA.In another embodiment, this activation bunch comprises cpn10, TLR4 and LPS.In further embodiment, this activation bunch comprises cpn10, TLR7 and single stranded RNA.Also in another embodiment, this activation bunch comprises cpn10, TLR9 and comprises the DNA of CpG motif.

Cpn10 can be natural origin, that reorganization produces or the synthetic cpn10 that produces.Cpn10 can be the eukaryotic cell source.Cpn10 can be people's cpn10.Cpn10 can comprise the peptide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQ IDNO:3.Cpn10 can be acetylizad or non-acetylizad, and can comprise or not comprise the terminal extension of single or multiple N-.Cpn10 can be with the polynucleotide form administration of coding cpn10.The polynucleotide of coding cpn10 can be arranged in gene constructs, operationally are connected with promoter.Polynucleotide can comprise the sequence shown in the SEQ IDNO:4.

This method further comprises and gives at least a other medicament.This medicament can be an immunomodulator.This immunomodulator can be, for example, and TNF-α, IL-1 β, IL-6, IL-10, IL-12 or I type interferon.I type interferon can be IFN α or IFN β.

Usually, the adjusting activity of Cpn10 is brought into play by following manner: at first be that TLR contacts with Cpn10, add one or more TLR agonist then, as the inventor and this paper disclosure (related content that for example, relates to the immunomodulating test of RAW264.7 cell) proved like that.Agonist can be antibacterial, fungus, yeast or viral pathogen, from its deutero-or consequent molecule or composition or composition or synthetic chemical compound.This TLR agonist also can be the product from the human damaging cells relevant with autoimmune disease, and as for TLR9, the example is that people DNA is as agonist.The limiting examples of TLR3 agonist comprises double-stranded RNA and polyI:C.The limiting examples of TLR7 agonist comprises the imidazoquinolie of single stranded RNA, for example resiquimod and imiquimod and the micromolecule agonist of isatoribine (isatoribine) for example.The limiting examples of TLR9 agonist comprises the DNA that contains unmethylated rich CpG motif and the oligonucleotide of the synthetic CpG of comprising.But those skilled in the art understand at an easy rate and the invention is not restricted to these identical TLR excitements.

The present invention also provides the generation and/or the excretory method of regulating one or more immunomodulators in experimenter or its at least a cell, tissue or the organ, wherein said method comprises the cpn10 that gives effective dose, wherein said cpn10 with the activation bunch in Toll sample receptor unite, and wherein the activation bunch formation with the adjusting one or more immunomodulators generation and/or secrete relevant.

The generation of one or more immunomodulators and/or excretory adjusting also can reach by following method: the cpn10 antagonist that gives effective dose, wherein said antagonist stops the Toll-sample receptor in cpn10 and the activation bunch to be united, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and one or more immunomodulators of adjusting and/or secrete relevant.

Immunomodulator can be, for example, and TNF-α, IL-1 β, IL-6, IL-10, IL-12 or I type interferon.I type interferon can be IFN α or IFN β.

Treatment or prophylactic method

The application of Cpn10 in Clinical Processing various diseases and disease supported in the inventor's who is provided discovery in this article, and these diseases and disease comprise acute and slow virus and bacterial disease.Therefore, the invention provides treatment or prevention virus that caused by virus or bacterial infection or relevant with virus or bacterial infection and bacterial disease and handicapped method, this method comprises and gives Cpn10.

In addition, the stimulation of TLR3, TLR7 and/or TLR9 is relevant with natural reaction and/or therapeutic treatment to multiple other diseases, and these diseases comprise asthma, anaphylaxis, inflammatory bowel and systemic inflammatory.Therefore the present invention also relates to treatment or prevent these diseases and handicapped method, this method to comprise give Cpn10.

Therefore, the invention provides treatment or prevention experimenter's the disease or the method for disease, wherein said method comprises the cpn10 that gives experimenter's effective dose, wherein the Toll sample receptor in this cpn10 and the activation bunch is united, and the formation that wherein activates bunch with to the startup of the immunne response of this disease or disease, strengthen and/or keep relevant.

Described method also can comprise at least a cpn10 antagonist that gives experimenter's effective dose, wherein said antagonist stops the Toll sample receptor in cpn10 and the activation bunch to be united, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and this disease or disease and/or make progress relevant.

Above-mentioned disease or disease can include but not limited to, virus, fungus, yeast or bacterial infection, acute or chronic inflammatory disease comprise septic shock, inflammatory bowel, arthritis, psoriasis, heart disease, atherosclerosis, chronic lung disease, cachexia, multiple sclerosis, GVHD and cancer.Any disease or disease, its generation and/or progress or at its immunne response startup, strengthen and/or keep and relate to the associating of Toll sample receptor in cpn10 and the activation bunch, all be considered within the scope of the invention.

In one embodiment, cpn10 is regulated the inductive TLR2 signal conduction of LTA-.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR2-.In the another one embodiment, cpn10 is regulated double-stranded RNA-inductive TLR3 signal conduction.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR3-.In another embodiment, cpn10 is regulated the inductive TLR4 signal conduction of LPS-.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR4-.In further embodiment, cpn10 is regulated the inductive TLR7 signal conduction of viral single stranded RNA.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR7-.Still in further embodiment, cpn10 is regulated the inductive TLR9 signal conduction of CpG motif.The generation and/or the secretion of the inductive immunomodulator of cpn10 scalable TLR9-.

Compositions and application thereof

The invention provides and be used for the treatment of or the compositions of prevent disease or disease, said composition comprises cpn10 and at least a pharmaceutically acceptable carrier, diluent or adjuvant, Toll sample receptor in wherein said cpn10 and the activation bunch is united, and the formation that wherein activates bunch with to the startup of the immunne response of this disease or disease, strengthen and/or keep relevant.

Said composition can further comprise at least a Toll sample receptor stimulating agent.Said composition can further comprise at least a immunomodulator, as I type interferon.

Be used for the treatment of or other compositionss of prevent disease or disease, said composition comprises at least a cpn10 antagonist, wherein said antagonist stops the Toll sample receptor in cpn10 and the activation bunch to be united, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and this disease or disease and/or make progress relevant.

The present invention also considered cpn10 manufacturing be used for the treatment of or the medicine of prevent disease or disease in application, Toll sample receptor in wherein said cpn10 and the activation bunch is united, and the formation that wherein activates bunch with to the startup of the immunne response of this disease or disease, strengthen and/or keep relevant.

The present invention also considered the cpn10 antagonist manufacturing be used for the treatment of or the medicine of prevent disease or disease in application, wherein saidly pick up anti-agent and stop the Toll sample receptor in cpn10 and the activation bunch to be united, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and this disease or disease and/or make progress relevant.

Usually, the suitable groups compound of Shi Yonging can be prepared according to method and the step that those of ordinary skills knew in the method according to the invention, therefore can comprise pharmaceutically acceptable carrier, diluent and/or adjuvant.

Therapeutic alliance

Those of skill in the art will appreciate that, the method according to this invention, and Cpn10 can be separately or is combined administration with one or more other medicament.For example, Cpn10 can stimulate one or more TLR agonist administration among TLR2, TLR3, TLR4, TLR7 and the TLR9 with one or more.In addition, the present invention has considered to use Cpn10 to combine with other Therapeutic Method with treatment disease and handicapped therapeutic alliance.For example, Cpn 10 can be used for treating viral disease, and this disease responds to the treatment of I type interferon (as IFN β or IFN α).In addition, owing to there was the activation energy of report TLR7 of agonist induction and TLR9 to strengthen tumor to radiocurable reaction in the past, so Cpn10 can combine with radiotherapy and is used for the treatment of cancer.

For this therapeutic alliance, every kind of component administration at one time of therapeutic alliance, or with any order successive administration, or in the different time administration, so that obtain required effect.Perhaps, these components can be formulated together as combination product in single dose unit.When individually dosed, these components can not done although do not need so preferably by identical route of administration administration.

Cpn10

According to various aspects of the present invention and embodiment, the experimenter of needs treatment is given the Cpn10 of effective dose.In special embodiment, the experimenter that be treated is the people, and therefore, the Cpn10 polypeptide is a people Cpn10 polypeptide.Those skilled in the art will be appreciated that, can change according to many factors according to the accurate homology of Cpn10 used in the present invention, and the species that will be treated for example, the Cpn10 of Xuan Zeing can derive from the species that will be treated like this.

Cpn 10 generally is recombinant C pn 10.People such as Morton, 2000 (Immunol Cell Biol78:603-607), people such as Ryan, people such as 1995 (J Biol Chem 270:22037-22043) and Johnson, the method of describing among 2005 (the J Biol Chem 280:4037-4047) is the example of the proteic suitable production method of recombinant C pn10, be not limited to employed purification or production method although the technical staff will understand the present invention, and any other method can be used to produce the Cpn10 that is used for the method according to this invention and compositions of the present invention.

Cpn10 polypeptide used according to the invention and fragments of peptides can use the recombinant nucleic acid technology of standard to obtain, or for example use traditional liquid phase or solid phase synthesis technique to synthesize.Available one or more protease, as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease, the digestion polypeptide produces the Cpn10 peptide.The fragments of peptides that has digested can come purification by for example high performance liquid chromatography (HPLC) technology.

Embodiments of the present invention have also been considered the administration of the polynucleotide of coding Cpn10.In this case, polynucleotide generally operationally are connected with promoter, produce suitable peptide sequence like this after giving the experimenter with polynucleotide.Polynucleotide can give the experimenter in carrier.Carrier can be plasmid vector, viral vector or any other suitable carriers that is suitable for inserting exogenous array, and these carriers are directed in the eukaryotic cell and express the sequence that is imported into.General carrier is a carrier for expression of eukaryon, and can comprise expression control and job sequence, as promoter, enhancer, ribosome binding site, polyadenylation signal and transcription terminator.Nucleic acid construct thing to be administered can comprise that exposed DNA maybe can be the form of compositions, in conjunction with one or more pharmaceutically acceptable carriers.

The Cpn10 polypeptide can have, but is not limited to, the aminoacid sequence shown in SEQ ID NO:1.The nucleotide sequence of polynucleotide of coding Cpn10 can be shown in SEQ ID NO:4 or show the sequence homology enough with it and with the sequence hybridization of SEQ ID NO:4.In in addition optional embodiment, the nucleotide sequence of polynucleotide can have at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 96%, 97%, 98% or 99% homology with the sequence shown in the SEQ ID NO:4.

Be included in interior its fragment and the variant in addition of scope of term as used herein " polypeptide " and " polynucleotide ".Only as an example, the fragments of peptides of the Cpn10 described in WO 95/15338 can be used for according in various aspects of the present invention and the embodiment.

Term " fragment " is meant the coding proteic ingredient of total length Cpn10 or as the nucleic acid or the peptide sequence of the proteic ingredient of total length Cpn10.With regard to polypeptide, fragment has the biologic activity identical with full-length proteins character.The biological active fragment of Cpn10 used according to the invention can typically have the immunoregulatory activity of about at least 50% corresponding full-length proteins, more typically has about at least 60% this activity, about at least 70% this activity more typically, about at least 80% this activity more typically, more typically about at least 90% this activity and more typically about at least 95% this activity.

Term as used herein " variant " is meant similar basically molecule.Usually, variable nucleic acid sequence allosome encoded polypeptides has identical in nature biologic activity.Usually, the peptide sequence variant also has identical in nature biologic activity.In addition, these peptide sequence variants also can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology.

And, polypeptide variants can comprise analog, wherein the polypeptide of term " analog " indication is the derivant of Cpn10, and derivant comprises interpolation, removes, replaces one or more aminoacid, and this polypeptide has kept the function substantially the same with natural Cpn10 like this.Well known in the art is that some aminoacid in the polypeptide can be changed and do not change the activity (conservative substitution) of polypeptide.Term " conservative amino acid replacement " is meant in polypeptide chain (proteic basic sequence), an amino acid replacement or replace with another aminoacid with similar characteristic.For example, to be replaced into the aminoacid aspartic acid (Asp) with similar electric charge will be conservative amino acid replacement to charged aminoacid glutamic acid (Glu).Aminoacid addition can be from the fusion of Cpn10 polypeptide or its fragment and second peptide species or peptide, as polyhistidine labelling, maltose-binding protein merge, glutathione s-transferase merges, green fluorescent protein merges or add FLAG for example or the epi-position labelling of c-myc.For example, wild type people Cpn10 polypeptide can comprise additional GSM three peptide moieties (the SEQ ID NO:2 of N-end; Referring to, for example WO 95/15338, its disclosure is incorporated herein by reference) or additional alanine (A) residue (the SEQ ID NO:3 of N-end; WO2004/041300, its disclosure is incorporated herein by reference).Other mutant forms of Cpn10 are included in those disclosed in No. the 2005904765th, the Australian temporary patent application, and the disclosure of this application is incorporated herein by reference.The present invention has also considered the application of polynucleotide of Cpn10 of these modified forms of coding Cpn10.

The Cpn10 variant can produce by the mutation of proteic mutation of Cpn10 or code nucleic acid, for example uses the method that well known to a person skilled in the art to pass through random mutagenesis or direct mutagenesis.These methods are found in, for example " molecular biological universal method (Current Protocols InMolecular Biology) " (the 9th chapter), people such as Ausubel, 1994, John Wiley﹠amp; Sons, Inc., New York, its disclosure is incorporated herein by reference.Variant and analog also comprise with polypeptide, fusion rotein or the post translational modification of other chemical part complexations.The example of suitable modification is at common unsettled International Patent Application PCT/AU2005/000041; Describe among the WO2005/067959, its disclosure is incorporated herein by reference.

In addition, Cpn10 polypeptide or its fragment can have other post translational modification effect, comprise the side chain modification, for example acetylation, amidatioon (amidination), carbamylation, reduction alkanisation and other modifications known in those skilled in the art.

The Cpn10 polypeptide can further include, but not limited to any polynucleotide of hybridizing with Cpn10 polynucleotide as herein defined under high stringency.As used herein term " high stringency " is meant two interfertile conditions of polynucleotide, and can comprise, for example, the concentration of salt and/or detergent in the solution, in two multi-nucleotide hybrid processes the temperature of employed solution and the persistent period of hybridization.Therefore, as used herein term " high stringency " is meant the solution condition that only helps two multi-nucleotide hybrids when two polynucleotide have high homology.The homology degree can include but not limited to, about scope of 50% to 99%.Therefore, " high stringency " condition can relate to but be not limited to, in about 60 ℃-70 ℃ temperature range, use lavation buffer solution, this lavation buffer solution comprises sodium lauryl sulphate and/or the 0-1x sodium chloride-sodium citrate of 0-10%, or any other combination of the time bar of buffer, temperature or generation " high stringency " solution of being used to hybridize.

Route of administration

Compositions can be carried out administration by standard way.Usually, compositions can be passed through parenteral (for example, intravenous, spinal column is interior, subcutaneous or intramuscular), per os or topical routes.Administration can be general, zonal or partial.The concrete route of administration that will use under any specified situation will depend on many factors, the required dosage of the particular compound that comprise the seriousness of character, disease of the disease that will treat and degree, will be sent and the potential side effect of this chemical compound.

The form that compositions of the present invention can be form, the dosage form (for example capsule, tablet, Caplet (caplet), elixir) that is fit to oral absorption, the ointment, emulsifiable paste or the lotion form that are fit to topical that are fit to drug administration by injection, be fit to send with eye drop, be fit to the inhalation aerosol form of (as sucking by intranasal or per os sucks), be fit to parenteral the form of (promptly subcutaneous, intramuscular or intravenous injection).

For the administration as Injectable solution or suspension, avirulent parenteral acceptable diluent or carrier can comprise Ringer's mixture, isotonic saline solution, phosphate buffered saline (PBS), ethanol and 1,2 propylene glycol.

Diluent, carrier, adjuvant and excipient

Usually, suitable compositions can prepare according to the known method of those of ordinary skills, and can comprise pharmaceutically acceptable diluent, adjuvant and/or excipient.With regard to compatible with other compositions of said composition, diluent, adjuvant and excipient must be " acceptable ", and harmless to its receiver.

The example of pharmaceutically acceptable carrier or diluent includes but not limited to, demineralized water or distilled water, saline solution, plant based oil such as Oleum Arachidis hypogaeae semen, safflower oil, olive oil, Oleum Gossypii semen, Semen Maydis oil, Oleum sesami, Oleum Arachidis hypogaeae semen or Oleum Cocois; Silicone oil comprises polysiloxanes, as methyl polysiloxane, phenyl polysiloxane and methyl phenyl silicone; Volatile silicone; Mineral oil is as liquid paraffin, soft paraffin or squalane; Cellulose derivative is as methylcellulose, ethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose or hydroxypropyl emthylcellulose; Low-level chain triacontanol, for example ethanol or isopropyl alcohol; Rudimentary aralkyl alcohol (aralkanol); Rudimentary ployalkylene glycol or rudimentary alkylene glycol, for example Polyethylene Glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3 butylene glycol or glycerol; Fatty acid ester is as isopropyl palmitate, isopropyl myristate or ethyl oleate; Polyvinylpyrrolidone; Agar; Carrageenin; Tragacanth or arabic gum and vaseline oil.Generally, carrier or variety carrier will account for the 10wt%-99.9wt% of compositions.

Some examples that are used for oral suitable carriers, diluent, excipient and adjuvant include, but are not limited to Oleum Arachidis hypogaeae semen, liquid paraffin, sodium carboxymethyl cellulose, methylcellulose, sodium alginate, arabic gum, Tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatin and lecithin.In addition, these oral formulations can contain suitable flavoring agent and coloring agent.When being used for capsule form, the chemical compound of capsule available energy delay disintegration wraps quilt, as glyceryl monostearate or distearin.

Adjuvant generally comprises but is not limited to, lubricant, emulsifying agent, thickening agent, antiseptic, antibacterial and buffer agent.

The solid form that is used for oral administration can contain, but is not limited to, acceptable binding agent, sweetener, disintegrating agent, diluent, flavoring agent, coating dress material, antiseptic, lubricant and/or delay agent in people and the pharmacopedics for animals practice.Suitable bonding includes but not limited to, arabic gum, gelatin, corn starch, Tragacanth, sodium alginate, carboxymethyl cellulose or Polyethylene Glycol.Suitable sweetener comprises sucrose, lactose, glucose, aspartame (aspartame) or glucide.Suitable disintegrants includes but not limited to, corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar.Suitable diluent includes but not limited to, lactose, sorbitol, mannitol, dextrose, Kaolin, cellulose, calcium carbonate, calcium silicates or dicalcium phosphate.Suitable flavoring agent includes but not limited to, Oleum menthae, wintergreen oil, Fructus Pruni pseudocerasi, Fructus Citri junoris or Fructus Rubi flavoring agent.Suitable coating dress material includes but not limited to, the polymer of acrylic acid and/or methacrylic acid and/or its ester or copolymer, wax, aliphatic alcohol, zein, Lac or glutelin.Suitable antiseptic includes but not limited to, sodium benzoate, vitamin E, alpha tocopherol, ascorbic acid, methyl parahydroxybenzoate, propyl p-hydroxybenzoate or sodium sulfite.Examples of suitable lubricants includes but not limited to, magnesium stearate, stearic acid, enuatrol, sodium chloride or Talcum.Suitable delay agent comprises glyceryl monostearate or distearin.

The liquid form that is used for oral administration can contain liquid-carrier except above-mentioned medicament.Suitable liquid-carrier includes but not limited to, water, oils are as olive oil, Oleum Arachidis hypogaeae semen, Oleum sesami, Oleum helianthi, safflower oil, Oleum Arachidis hypogaeae semen, Oleum Cocois, liquid paraffin, ethylene glycol, propylene glycol, Polyethylene Glycol, ethanol, propanol, isopropyl alcohol, glycerol, aliphatic alcohol, triglyceride or its mixture.

The suspension that is used for oral administration can further comprise dispersant and/or suspending agent.Suitable suspending agent includes but not limited to, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl emthylcellulose, polyvinylpyrrolidone, sodium alginate or acetyl alcohol.Suitable dispersant includes but not limited to, the polyoxyethylene ester of lecithin, fatty acid (as stearic acid), polyoxyethylene sorbitol be single-or two-oleate ,-stearate or-laurate, polyoxyethylene Sorbitan list-or two-oleate ,-stearate or-laurate and analog.

The Emulsion that is used for oral administration can further comprise one or more emulsifying agents.Suitable emulsifying agent comprises but is not limited to, and as above dispersant of being given an example or natural gum are as guar gum, arabic gum or Tragacanth.

But the method for compositions that is used to prepare parenteral is conspicuous for those skilled in the art, its more details is described in, for example, " Lei Mingdun pharmaceutical science (Remington ' sPharmaceutical Science) ", the 15th edition, Mack Publishing Company, Easton, Pa. in, this book is incorporated herein by reference.

Topical formulations of the present invention comprises active component and one or more acceptable carriers and any other therapeutic component randomly.The preparation that is fit to topical includes but not limited to, is fit to liquid or the semi-liquid preparations of skin permeation to the position of needs treatment, as the drop of liniment, lotion, emulsifiable paste, ointment or paste and suitable eye, ear or nasal administration.

Can comprise sterile aqueous or oily solution or suspension according to drop of the present invention.These preparations can by with solubilization of active ingredient at antibacterial and/or antifungal and/or any other suitable antiseptic and randomly comprise in the aqueous solution of surfactant and preparing.The solution that generates can be transferred in the suitable containers and sterilization by filtering clarification subsequently.Sterilization can be finished by following method: at 90 ℃ of-100 ℃ of following autoclavings or keep half an hour, or by filtering, adopt the sterile working to be transferred in the container then.Being fit to be included in the antibacterial in the drop and the example of antifungal is phenylmercuric nitrate or phenylmercuric acetate (0.002%), benzalkonium chloride (0.01%) and acetic acid hibitane (0.01%).The suitable solvent that is used to prepare oily solution includes but not limited to glycerol, rare pure and mild propylene glycol.

Lotion according to the present invention comprises the lotion that is fit to spread on skin or eye.Eye wass can comprise the aseptic aqueous solution that randomly contains antibacterial, and can be by preparing with above-mentioned similar approach about the preparation drop.The lotion or the liniment that are used to spread on skin also can include but not limited to, medicament (for example alcohol or acetone) and/or the wetting agent (as glycerol) or the oil (as Oleum Ricini or Oleum Arachidis hypogaeae semen) of rapid draing and cooling skin.

According to emulsifiable paste of the present invention, ointment or paste is the semi-solid preparation of the active component of external.They can be by will be separately or active component in solution in aqueous or non-aqueous liquid or the suspension, microgranule or powder type, mixes with oils and fats or non-oils and fats substrate and make.This substrate can comprise hydrocarbon, as hard, soft or liquid paraffin, glycerol, Cera Flava, metallic soap; Mucus; The oil of natural origin is as Semen Armeniacae Amarum, corn, Semen arachidis hypogaeae, Semen Ricini or olive oil; Lanoline or derivatives thereof or fatty acid (as stearic acid or oleic acid) and alcohol (as propylene glycol and Polyethylene Glycol).

Compositions can add any suitable surfactant, as anion, cation or nonionic surfactant, as sorbitan ester or its polyoxyethylene deriv.Also can comprise suspending agent, as natural gum, cellulose derivative or inorganic substances, as siliceous silicate (silicaceous silicas) and other components, as lanoline.

Compositions also can be with the form administration of liposome.Liposome generally derives from phospholipid or other lipid materials, and list in the aqueous medium-or the multilamellar aqua liquid is brilliant forms by being dispersed in.Can use can form liposome any avirulent, the physiology is acceptable and metabolizable lipid.The compositions of liposome form can contain stabilizing agent, antiseptic, excipient and analog.Preferred lipid is phospholipid and phosphatidylcholine (lecithin), all is natural in synthetic.The method that forms liposome is well known in the art, concrete document about this is seen: Prescott, Ed., " cell biology method (Methods in Cell Biology) ", XW volume, AcademicPress, New York, N.Y. p.33 (1976) reach followingly or the like, and its content is incorporated herein by reference.

Compositions can with a series of Polyethylene Glycol (PEG) derivant conjugation.It is a kind of being used to of determining very much to reduce the protein plasma clearance that PEG is added to (PEGization) in the albumen, thereby increases the method (people such as Nucci, 1991, Adv.Drug Del.Rev.6:133) of its effect.Other benefits of PEGization can comprise, protein advantages of higher stability, immunogenicity reduce, dissolubility increases and proteoclastic susceptibility descends (Sheffield W.2001, Curr DrugTargets Cardiovasc Haematol Disord.1:1-22).The PEG molecule contains-(OCH ₃CH ₂) _nThe basic repetitive structure of-OH, and be divided into several groups according to its molecular weight.PEG derivant and protein conjugation are to increase its hydrodynamic radius, and usually, the increase of its half-life directly relevant (Sheffield W.2001, Curr DrugTargets Cardiovasc Haematol Disord.1:1-22) with the size of the PEG chain that adheres to.

Compositions also can be with the form administration of microgranule.By polyactide (PLA), polylactide-co-glycolide copolymer (polylactide-co-glycolide, PLGA) and 6-caprolactone (

-caprolactone) Biodegradable microparticle of Xing Chenging has been widely used as pharmaceutical carrier with the increase plasma half-life, and therefore prolongs effect (R.Kumar, M., 2000, JPharm Pharmaceut Sci.3 (2) 234-258).Microgranule is used for sending multiple drug candidates by preparation, and these material standed fors comprise vaccine, antibiotic and DNA.And these preparations have been developed and have been used for multiple route of delivery, comprise parenteral subcutaneous injection, intravenous injection and suction.

Compositions can add controlled release matrix, and this substrate is made up of Sucrose acetoisobutyrate (SAIB) and organic solvent or ORGANIC SOLVENT MIXTURES.Polymeric additive can be added in the media as discharging dressing agent with the further increase viscosity and the rate of release that slows down.SAIB is known food additive.It is the sucrose derivative of very hydrophobic, complete esterification, and the specified ratio of its isobutyrate and acetate group is 6: 2.As mixed ester, SAIB can crystallization but is existed with limpid thick liquid.SAIB and pharmaceutically acceptable organic solvent (as ethanol or benzyl alcohol) mixing energy are fully reduced the viscosity of mixture to allow injection.The active drug component can be added in the SAIB delivery vehicle to form SAIB solution or suspension preparation.When the preparation percutaneous was injected down, solvent diffused out from substrate, makes SAIB-medicine or SAIB-drug-polymer mixture set up formed in situ bank (forming depot).

For the purposes of the present invention, molecule and medicament can be used as therapeutic or prophylactic compositions gives the experimenter.In therapeutic was used, compositions gave to suffer from the patient of disease, and its consumption is enough to cure or suppress at least in part disease and complication thereof.Said composition should provide the molecule of capacity or medicament to treat the patient effectively.

Dosage

To depend on multiple factor for effective dosage level on any specific patient treatment, comprise: the dysfunction of being treated and this handicapped seriousness; The molecule that uses or the activity of medicament; The compositions of using; Patient's age, body weight, general health situation, sex and diet; Administration time; Route of administration; The chelating of this molecule or medicament (sequestration) rate; The persistent period of treatment; With this medicine for the treatment of associating or using simultaneously, and known other correlative factors in the medical science.

Those skilled in the art can need determine the medicament of the suitable disease of treatment or effective, the avirulent consumption of chemical compound by the test of routine.

Usually, effective dose is desirably in about 0.0001mg to the per 24 hours scope of about every kg body weight of 1000mg; Typically be per 24 hours of the extremely about every kg body weight of 750mg of about 0.001mg; Approximately the extremely about every kg body weight of 500mg of 0.01mg is per 24 hours; Approximately the extremely about every kg body weight of 500mg of 0.1mg is per 24 hours; Approximately the extremely about every kg body weight of 250mg of 0.1mg is per 24 hours; Approximately the extremely about every kg body weight of 250mg of 1.0mg is per 24 hours.More typically, effective dosage ranges is desirably in about 1.0mg to the per 24 hours scope of about every kg body weight of 200mg; Approximately the extremely about every kg body weight of 100mg of 1.0mg is per 24 hours; Approximately the extremely about every kg body weight of 50mg of 1.0mg is per 24 hours; Approximately the extremely about every kg body weight of 25mg of 1.0mg is per 24 hours; Approximately the extremely about every kg body weight of 50mg of 5.0mg is per 24 hours; Approximately the extremely about every kg body weight of 20mg of 5.0mg is per 24 hours; Approximately the extremely about every kg body weight of 15mg of 5.0mg is per 24 hours.

Perhaps, effective dose can reach about 500mg/m ²Usually, effective dose is desirably in about 25 to about 500mg/m ²Scope in, preferably approximately 25 to about 350mg/m ², more preferably about 25 to about 300mg/m ², also more preferably about 25 to about 250mg/m ², even more preferably about 50 to about 250mg/m ², even also more preferably about 75 to about 150mg/m ²

Typically, in therapeutic was used, treatment will be at the persistent period of morbid state.

In addition, it should be apparent that for those of ordinary skills, will determine the optimal number of individual dosage and blanking time by the nature and extent of the morbid state of being treated, form, approach and the position of administration and the character of the particular individual that quilt is treated.This optimum condition also can be determined by routine techniques.

Also it should be apparent that for those of ordinary skills, the best time-histories of treatment, the medication number of times of for example specified compositions every day continues the natural law of regulation, can use the conventional process of treatment confirmed test to determine by those skilled in the art.

Cpn10 agonist and antagonist

The present invention has also considered the application and the screening of Cpn10 agonist and antagonist and has produced these agonist and pick up the method for anti-agent.

Cpn10 agonist and antagonist can design specifically or screen conduction of Toll sample receptor signal and the excretory effect of immunomodulator according to it.

Antibody can be used as the agonist or the antagonist of Cpn10 or its fragment or analog.Preferably give proteinase activity and/or chaperone or substrate zone or fragment, prepare suitable antibody in conjunction with character from discontinuity zone or fragment, especially those participations of Cpn10 polypeptide.Antigenicity Cpn10 polypeptide contains about at least 5, and preferably about at least 10 aminoacid.

The method that produces suitable antibodies is readily appreciated that for those skilled in the art.For example, anti--the Cpn10 monoclonal antibody, typically contain the Fab part, can use " antibody-laboratory manual (Antibodies-A Laboratory Manual) ", Harlow and Lane edit, ColdSpring Harbor Laboratory, the hybridoma technology described in the N.Y. (1988) prepares.

In fact, in the MONOCLONAL ANTIBODIES SPECIFIC FOR at Cpn10 or its fragment or analog, can use to provide any technology of being produced antibody molecule by the continuous cell line of cultivating.These technology comprise by people such as Kohler, 1975, Nature, the hybridoma technology of the initial exploitation of 256:495-497 and trioma (trioma) technology, people B-quadroma technology (people such as Kozbor, 1983, Immunology Today, 4:72) and EBV-hybridoma technology (people such as Cole, " monoclonal antibody and treatment of cancer (Monoclonal Antibodies and CancerTherapy) ", pp.77-96, Alan R.Liss, Inc., (1985)) the generation human monoclonal antibodies.The immortal cell line that produces antibody can be set up by the other technologies outside merging, and for example transforms bone-marrow-derived lymphocyte with carcinogenic dna direct, or uses the Epstein-Barr virus transfection.Referring to, for example, people such as M.Schreier, " hybridoma technology (Hybridoma Techniques) " (1980); People such as Hammerling, " monoclonal antibody and T-quadroma (Monoclonal Antibodiesand T-cell Hybridomas) " (1981); People such as Kennett, " monoclonal antibody (MonoclonalAntibodies) " (1980).

In a word, by producing the means of hybridoma (it produces monoclonal antibody), myeloma or other self immortalized cell line and lymphocyte merge, and this lymphocyte is from its recognition factor-bound fraction or recognition factor or the hyperimmune mammiferous spleen acquisition of its specific DNA bound fraction of originating.Be used to implement the hybridoma of generation monoclonal antibody of the present invention, the ability that special transcriptional activity in immunoreactive ability and its inhibition target cell takes place by itself and this recognition factor is identified.

Be used to implement monoclonal antibody of the present invention, can comprise that the monoclonal hybridoma culture of the Nutrient medium that contains hybridoma produces by startup, this hybridoma secretion has the specific antibody molecule of suitable antigen.Be enough to make hybridoma secretory antibody molecule to the condition of culture medium with keep culture under the persistent period.Collect the culture medium that contains antibody then.Antibody molecule can further separate by technique known then.

Equally, known in this area have plurality of step to can be used for producing polyclonal antibody.For the generation of anti-Cpn10 polyclonal antibody, multiple host animal can come immunity with Cpn10 or its fragment or analog injection, and these animals include but not limited to horse, milch cow, rabbit, chicken, mice, rat, sheep, goat etc.In addition, Cpn10 polypeptide or its fragment or analog can with immunogenic carrier, for example bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) conjugation.Various adjuvants also can be used to increase immunological response, include but not limited to Freund adjuvant (fully with incomplete), the mineral coagulant of for example aluminium hydroxide, surfactant, as LYSOLECITHIN SUNLECITHIN A, polyether polyol (pluronic polyol), polyanion, peptide, oil emulsion, keyhole limpet hemocyanin, dinitrophenol and people's adjuvant of coming in handy, as BCG (bacillus calmette-guerin vaccine) and Corynebacterium.

Screening required antibody also can finish by multiple technologies as known in the art.The specific immunity of antibody can include but not limited in conjunction with test, radioimmunoassay, ELISA (elisa), sandwich immunoassay (sandwich immunoassay), immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion is measured, the original position immunoassay, the Western trace, precipitation, CA, complement is in conjunction with mensuration, immunofluorescence assay, protein A is measured, measure with immunoelectrophoresis, with similar test (referring to, for example, people such as Ausubel, the chief editor, 1994, " molecular biological universal method (Current Protocols in MolecularBiology) ", Vol.1, John Wiley﹠amp; Sons, Inc., New York).The combination of antibody can detect by the detectable label on the one-level antibody.Perhaps, antibody can suitably the secondary antibody or the combining of reagent of labelling be detected with quilt by it.Be known in the art many methods and in immunoassay, be used to detect combination, and these methods within the scope of the invention.

The antibody of cultivating at Cpn10 or its fragment or analog (or its fragment) has the binding affinity with Cpn10.Preferably, the binding affinity of antibody (or its fragment) or affinity are greater than about 10 ⁵M ^-1, more preferably greater than about 10 ⁶M ^-1, more preferably also greater than about 10 ⁷M ^-1, and most preferably greater than about 10 ⁸M ^-1

With regard to obtain appropriate amount according to regard to the antibody of the present invention, can use with the serum-free medium batch fermentation and make antibody.After fermentation, antibody can be through combining the multistep step purification of chromatography and inactivation of virus/removing step.For example, antibody can at first separate by the protein A affinity chromatography, handles with any lipid envelope virus of deactivation with solvent/detergent then.Be further purified (typically by anion and cation-exchange chromatography) and can be used to remove remaining protein, solvent/detergent and nucleic acid.Antibody purified can further be used the gel filtration column purification and be formulated in 0.9% saline.Then can be with batch formulation sterilization and the filtration virus and the distribution of making.

The present invention also comprises agonist and the antagonist outside the antibody.Can be by identifying candidate's agonist or pick up anti-agent with Toll sample receptor and the ability that the Toll sample receptor stimulating agent of choosing wantonly forms molecular complex.In addition, can identify candidate's antagonist by the ability that stops or destroy the molecular complex that comprises Cpn10 and Toll sample receptor or Toll sample receptor stimulating agent to form.

The technology and the step that are used to identify and produce agonist and antagonist are known in those skilled in the art, comprise that for example screening molecular library, computer-aided screening structural database, Computer Aided Modeling and/or the design of synthetic compound library (for example combinatorial library) or more traditional biophysics of detection molecules binding interactions learn a skill.

Come further to describe in more detail the present invention referring now to following specific embodiment, these specific embodiments should not be construed as by any way and limit the scope of the invention.

Embodiment

Recombined human Cpn10

For the test described in the following embodiment 1 to 4, as people such as Johnson, described in 2005 (the J BiolChem 280:4037-4047), recombined human Cpn10 (GenBank registration number X75821) produces in escherichia coli, and its N-end is an alanine residue.Measure purity＞97% by SDS-PAGE.Only before using, the aliquot sample of Cpn10 is thawed.In the rhodanese refolding chemical examination of GroEL mediation, the Cpn10 of all batches has shown the mole active (data not shown) identical with escherichia coli GroES.

Embodiment 1-produces antiviral cell factor IFN β in the presence of PolyI:C

Test the selective agonist of working as with TLR3 to measure, during PolyI:C (polyinosinic acid-polycytidylicacid) administering drug combinations, the effect of the generation of Cpn10 enantiopathy poison cell factor interferon-beta (IFN β).

As people such as Johnson, described in 2005 (the J BiolChem 280:4037-4047), be to test among the RAW264.7 (ATCC registration number TIB71) at mouse macrophage.In brief, the RAW264.7 cell is with 2 * 10 ⁵Cells/well is seeded in 24 orifice plates, and overnight incubation (37 ℃, 5%CO ₂).Recombined human Cpn10 (with the concentration between 10 μ g/ml to the 200 μ g/ml) or buffer are added in the cell 2 hours with double, and (San Diego is CA) to the final concentration of 100 μ g/ml for tlrl-pic, InvivoGen to add polyI:C then.After 24 hours, collect supernatant and use description (Cat No.42400-1, the R﹠amp of specific ELISA according to production firm; DSystems Inc, Minneapolis MN) analyzes IFN β level.

As shown in fig. 1, the RAW264.7 cell that does not stimulate does not have and can discharge in the culture supernatant by detected IFN β.Do not having under the situation of Cpn10, stimulating with the polyI:C of 100 μ g/ml the IFN β of 292pg/ml is discharged in the RAW264.7 culture supernatant.Adding causes the IFN β level in the dose dependent ground increase culture supernatant from the ever-increasing Cpn10 of dosage of 10 μ g/ml to 200 μ g/ml, reaches the top level of 900pg/ml when 150 μ g/ml Cpn10.

The antiviral activity of embodiment 2-RAW264.7 cell conditioned medium liquid

The cell culture supernatant that will obtain from the test described in the top embodiment 1 is freezing and be kept at-20 ℃, is used for reducing (CPER) in L cell pathological changes effect afterwards and measures analyzing total antiviral activity (total burst size of I type interferon).

In brief, 3 * 10 ⁴The L cell inoculation is in the 96-orifice plate, and it is adherent to carry out to leave standstill 4h at 37 ℃.Double IFN standard substance and detection supernatant are added in the culture plate with half-log10 serial dilutions.The IFN field of activity of standard substance diluent is from 1-10 ⁴IU/ml.Culture plate overnight incubation 15h removes culture medium, and with Semliki Forest virus (Semliki Forestvirus) with tissue culture ID ₅₀100 times of titres be added in each hole.Culture plate was hatched under 37 ℃ 3 days, and the pair cell vigor is marked then.Every culture plate contains not to be had IFN or detects the contrast of two row's cells of supernatant as maximum cell death.Measure the IFN titre with respect to NIH's standard (National Institutes of Health standard) Ga02901511, as the IFN concentration that 50% cell is provided protection.As shown in Figure 2, add Cpn10 and cause in the presence of polyI:C, dose response increase significantly (being issued to maximum at 100 μ g/ml Cpn10) takes place in the release of I type interferon.

Shown data show that (wherein viral nucleic acid can be released) gives Cpn10 under the situation of viral infection among the embodiment 1 and 2, or when with TLR3 agonist co-administered, cause stimulating the antiviral immunity of higher level.

NF-kB activation in the HEK293 cell of embodiment 3-expression TLR7

Research people Cpn10 regulates the ability via the signal conduction of Toll sample receptor TLR7 in human embryonic kidney cell line HEK293.People such as the stable cell lines of the HEK293 of expression TLR7 and TLR4 such as Latz set up described in 2002 (the J Biol Chem 277:47834-47843), and with every hole 2 * 10 ⁴The density of cell is seeded in the 96-well culture plate.After the grow overnight, in order to measure the NF-kB activation, cell reports that with NF-κ B-luciferase construction is (according to people such as Latz, 2002, JBiol Chem 277:47834-47843) transient transfection, this construction comprise by drive 5NF-kB site that the Lampyridea luciferase genes expresses and structural active Renilla luciferase report subbase because of artificial report formed of polymer.

After cell culture spends the night, give cell simultaneously with recombined human Cpn10 separately or with one of following reagent with the final concentration of 50 μ g/ml: TLR7 selective agonist resiquimod R848, thiophosphate CpG oligonucleotide (MWG Biotech) (TLR9 selective agonist), TLR4 selective agonist lipopolysaccharide (LPS) or protein kinase C activation thing phorbol 12-myristinate 13-acetas (PMA), in contrast.

Dual by using then-luciferase assay report subsystem (Dual-LuciferaseAssay Reporter System), description (Promega according to production firm, MadisonWI) and assay plate reading photometer (Perkin Elmer) measure uciferase activity, measured NF-kB activation and quantitatively in back 5 hours in stimulation.

The result is presented among Fig. 3, is expressed as NF-kB activation multiple.When not stimulating, (use culture medium separately), do not induce the NF-kB activation.On the contrary, in the concentration range of 0.32-40 μ M, TLR7 selective agonist R848 induces the NF-kB activation doubly above 5-through TLR7.But in the presence of 50 μ g/ml Cpn10, this activation has reduced about 2-doubly.

NF-kB activation in the HEK293 cell of embodiment 4-expression TLR9

Also in the HEK293 cell, study the ability of people Cpn10 adjusting via the signal conduction of Toll sample receptor TLR9.Measure as described in example 3 above.

Measure among the clone separately for two at the HEK293 cell of the expression TLR9 of stable transfection.The result is presented among Fig. 4.Do not having not induce the NF-kB activation under the situation about stimulating (using culture medium separately).But with 0.08-10 μ M concentration range activation HEK-TLR9, induce NF-kB activation doubly up to about 33-with CpG DNA.In the presence of 50 μ g/ml Cpn10, this activation has reduced 5-to 10-doubly.

For further research Cpn10 to the HEK293 cell of expressing TLR9 reactive effect to CpG DNA, use Cpn10-12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml and the 100 μ g/ml of variable concentrations to be similar to above-mentioned test (see figure 5).Observed Cpn10-mediation effect (Fig. 4) above these results have confirmed, and illustrated the dose response of Cpn10-mediation effect to the conduction of TLR9 signal.

Embodiment 5-human peripheral blood mononuclear cell's release of cytokines

In order to determine that Cpn10 regulates the ability of release of cytokines in the former generation people cell, uses isolating human peripheral blood mononuclear cell (PBMC) from healthy volunteer's donor.As people such as Johnson, 2005 (J Biol Chem 280:4037-4047) are described, separate PBMC by the buoyant density gradient centrifugation from the blood of heparinization.

As people such as Latz, described in 2004 (the Nature Immunol 5:190-198), use separation PBMC that anti--BDCS-2-APC post (Miltenyi) handles half to remove plasma cell sample dendritic cell (PDC).PDC is the main source of I type interferon IFN α in the PBMC colony.Known PDC expresses TLR7 and TLR9, and is the key cells type that the mankind are used to discern single-chain nucleic acid.

For the PBMC of PBMC that contains PDC and removal PDC, two kinds of cells are all with 10 ⁶The density of living cells/ml is seeded in the 96-orifice plate with 200 μ l, and as people such as Johnson, cultivates described in 2005 (the J Biol Chem 280:4037-4047).In brief, add the final concentration of Cpn10 to 1 μ g/ml, 10 μ g/ml or 50 μ g/ml, culture plate is hatched 1 hour before adding LPS (non-repurity or repurity).Cell is at 37 ℃ and 5%CO then ₂Further hatched 20 hours down.Collect supernatant and use the description (R﹠amp of specific ELISA according to production firm; D Systems, Inc., Minneapolis, MN) generation of analysis proinflammatory cytokine TNF-α (Fig. 6) and anti-inflammatory cytokines IL-10 (Fig. 7).

As shown in Fig. 6 and 7, when lacking LPS, use culture medium or Cpn10 not to induce the generation of TNF-α or IL-10 separately.

The LPS (0.01-10ng/ml) that adds repurity in untreated PBMC culture causes the generation of TNF-α, and in the presence of up to 10 μ g/ml Cpn10, this generation dose response ground reduces (maximum 32%) (Fig. 6 A).But in the PBMC that removes PDC, Cpn10 has lost the influence of the generation of TNF-α (Fig. 6 B).Therefore, Cpn10 needs the existence of PDC to the effect of the generation of the inductive TNF-α of LPS-, and this effect disappears when lacking PDC.This discovery is somewhat wondrous, because PDC does not express TLR4, therefore the stimulation of LPS is not reacted, thereby shows that Cpn10 may be indirect to the regulating action of the signal conduction of TLR4 guiding.

Under the situation of IL-10 (Fig. 7), supernatant from PBMC proves when Cpn10 exists, the generation of IL-10 increases, although prove from the supernatant of the PBMC that removes PDC, the dose response of the generation of IL-10 (until 10 μ g/ml Cpn10) increase (Fig. 7 B) than the PBMC that is untreated with PDC in viewed increase (Fig. 7 A) degree high.

Embodiment 6-Cpn10 is to the activatory adjusting of TLR3

This research is to be used for determining to compare with other TLR, and whether Cpn10 differentially regulates the bonded reaction to TLR3, but the interactional target of its indication molecule.

Specific agonist can excite the activation of two kinds of downstream signal pathways to the stimulation of TLR, MyD88-dependency or dependent/non-dependent.MyD88 is a kind of for the very general adapter molecule of all TLR except that TLR3.The function of this molecule is that IL-1R-associated kinase (IRAK) and TNFR-correlation factor 6 (TRAF6) are raised together with activation I κ B (IKK) α β γ kinases complex, and this complex phosphorylation I κ B α causes that nuclear translocation and DNA combine with NF-κ B then.The MyD88-dependent/non-dependent activation of TLR relates to the activation of the adaptive son (TRIF) that contains the Toll/IL-IR domain of inducing IFN-β.This has caused the activation of NF-κ B and IFN regulatory factor 3 (IRF3) to postpone, thereby causes generation and the IFN-inductivity expression of gene of IFN-β.The combination of TLR3 mainly activates the TRIF path, and TLR4 can activate MyD88-and two kinds of paths of TRIF-dependency.One of purpose of this research is to determine to compare with other TLR, and whether Cpn10 can regulate because the kinetics of the reaction that the TLR3 activation causes, thereby and provides some information for target and the model of action of Cpn10.

6.1 general material

Cpn10 lot number CH003 and GroES (Invivogen) use or use not together with LPS (Sigma) and Poly (I:C).The cell line of using comprises RAW264-pNifty2-LUC and RAW264.7.

6.2 result

6.2.1Cpn10 do not suppress the NF-kB activation by TLR3

The mouse macrophage like cell is the RAW264.7 plasmid stable transfection that driven by ELAM1 promoter (RAW264-pNifiy2-LUC).The ELAM1 promoter is near 5 NF-kB sites and respond the activation of nuclear translocation and NF-κ B transcription factor in the cell and drive luciferase and report sub-expression of gene.As shown in Figure 8, the existence of very wide concentration range Cpn10 can not change the kinetics of the inductive NF-kB activation of TLR3-in the RAW264-pNifty2-LUC cell that stimulates with Poly (I:C).Poly (I:C) is the synthetic analogues of double-stranded RNA, and its molecular pattern is relevant with viral infection, activates NF-κ B by the interaction with TLR3.Dose response ground increases the generation of I type IFN 6.2.2Cpn10 respond the combination of TLR3 and TLR4

RAW264.7 cell and Cpn10 preincubate 2 hours stimulated 24 hours with LPS or Poly (I:C) then.Point is collected cell conditioned medium liquid at this moment, and (people such as Hamilton, (1996) J Immunol 156 (7): 2553) use cytopathy to reduce bioassay (cytopathic effect reduction bioassay) and analyze IFN α/β antiviral activity as mentioned previously.As shown in following Fig. 9, in the presence of Cpn10, by TLR4 (LPS) or TLR3[Poly (I:C)] stimulate and can raise I type IFN in 24 hours.

The RAW264.7 cell is with 2.5 * 10 then ⁵/ ml is seeded in aseptic 24 orifice plates, and adherent 16-20 hour.Pair cell preincubate in the presence of titrating Cpn10 uses LPS (TLR2/4) or Poly (I:C) to stimulate then 4 or 6 hours.The collecting cell lysate, and down freezing or handle immediately and be used to use Dynabead directly to carry out mRNA to separate at-70 ℃.MRNA carries out RT-PCR with reverse the transcribing of oligomerization (dT) primer with Superscript III First-Strand SynthesisSystem.The cDNA that obtains uses mice IFN β special primer to increase in PCR.In order to confirm that the equivalent template is added in each reaction and confirms to have carried out consistent amplification, each sample is transcribed GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and increased.On agarose gel, separate the PCR product, and after bromination second pyridine dyeing, develop as shown in Figure 10.

Except data from mouse macrophage cell line RAW264.7, as shown in Figure 11, proved that also the human PBMC who stimulates with Poly (I:C) shows, in the presence of Cpn10, the generation of I type IFN (IFN-α) increases.

6.2.3Cpn10 escherichia coli homologue, GroES can not increase Poly (I:C)-inductive IFN-β and produce

The specificity that increase Poly (I:C)-inductive IFN-β produces in order to determine the Cpn10-mediation detects escherichia coli Cpn10 homologue, GroES.As shown in Figure 12, GroES can not regulate Poly (I:C) by the inductive IFN-β generation of TLR3.

6.2.4 wash then with the Cpn10 preincubate, eliminated the dose response increase of the generation of Poly (I:C)-inductive IFN-β

Stimulate in the mensuration at LPS, before adding LPS, wash by PBS then, do not influence the ability that Cpn10 regulates the NF-kB activation with the Cpn10 preincubate.But as shown in Figure 13, in Poly (I:C) measured, RAW264 cell and Cpn10 preincubate had greatly reduced the generation of the IFN-β of Cpn10 dose response increase Poly (I:C) stimulation then by the PBS washing.

6.2.5Cpn10 to Poly (I:C)-or the adjusting of LPS-induced reaction need the positive feedback of I type interferon path

The feature that has the mice of null mutation in the IFNAR1 of I type IFN receptor (interferon associated receptor 1) composition is, increase corresponding with susceptibility to viral infection, lack antiviral response fully, and lost the reaction of the antiproliferative of IFN α/β people such as (, (1995) Proc.Natl.Acad.Sci.USA 92:11284) Hwang.IFNAR1 deficient mice (IFNAR1/-) lacks constitutive character IFN activity, and have aspect the ratio of the hematopoietic cell that comprises macrophage and the reaction unusual.

Use from wild type (wt) or IFNAR1-/-mice the macrophage (BMM) of isolating derived from bone marrow, come the effect of earlier external back interior evaluating Cpn10 in IFN " pre-swash (priming) " reaction.Separate BMM and in the presence of the Cpn10 of dose titration, activate with one group of TLR agonist (Poly (I:C)-TLR3 or LPS-TLR4).Collect supernatant after 24 hours, and use BD Mus inflammation CBA and Mus IFN β ELISA test kit analysis of cells factor level.Result from these mensuration shows that Cpn10 produces the regulating action of (Figure 14) and the inductive TNF-α generation of LPS-(Figure 15) to Poly (I:C)-inductive IFN β, and it is essential reacting through the positive feedback of IFNAR1 receptor.

These data link together IFN feedback network and Cpn10-mediation activity, have further confirmed from result in the body of Mus endotoxemia research.In this pyemic mouse model, mice with 100 μ g Cpn10 through intravenous administration pretreatment 30 minutes, intravenous injection 10 μ g LPS then.1.5 collect serum after hour, and use the generation of BD Mus inflammation CBA and IFN β elisa assay proinflammatory cytokine.As shown in Figure 16, with the remarkable reduction (p＜0.05) of the common mice serum TNF alpha levels of handling of Cpn10, relevant with the remarkable reduction (p＜0.01) of serum I FN β level.

6.3 discuss and conclusion

At least a exception that the NF-κ B of Cpn10-mediation regulates pattern relates to the activating cell by TLR3 part Poly (I:C).In this TLR3 system, find Poly (I:C) stimulation of Cpn10 response cell and the NF-kB activation is not had effect.

In addition, find that Cpn10 can respond Poly (I:C) and induce concertedness ground to increase the generation of I type interferon.Be that Cpn10 has participated in the adjusting of sharp in advance or feedback circuit reaction to this obviously unusual a kind of explanation.For example, I type interferon has been presented in the LPS-regulation and control reaction or directly or (for example, pass through SOCS1) indirectly and play a significant role.This is the major progress of understanding in the mechanism of action of Cpn10, because it shows that Cpn10 swashs in advance and reduces proinflammatory cytokine by alleviating the early stage cell that is caused by I type IFN, as TNF α.

The adjusting that embodiment 7-Cpn10 infects mice Semliki Forest virus (SFV)

This series of studies is used for determining whether the administration of Cpn10 influences the fatality rate of Semliki Forest virus in the mouse model (SFV), and whether Cpn10 responds the TLR activation and the generation of adjusting I type IFN.

This body inner model relates to the systemic infection of Semliki Forest virus (SFV) with the preliminary adjusting of determining I type IFN antiviral activity.Can in newborn mice, carry out this model, all infected comprising brain at all interior organs, in 4-6 days, cause death according to dosage, so advantage is sensitivity and the violent degree that infects.Perhaps, this model can detect in adult rats, because the generation of I type IFN is arranged, adult rats is less to the susceptibility of virus.It being understood that behind viral infection in first 24-48 hour that the generation of IFN causes that virus titer reduces.This model can carry out " chronic " response analysis, comprises serum analysis and immunne response.

This in vitro study comprises the influence that a series of mensuration regulating action that the pair cell factor produces to detect Cpn10 response TLR activation is swashed by macrophage in advance, comprises relatively the bone marrow macrophage from WT and IFNAR deficient mice fresh separated.

7.1 material and method

(in 50mM Tris, 150mM NaCl, endotoxin＜0.01EU/ml) uses Cpn10 lot number CH003 with 5.0mg/ml.Dilution preparation buffer comprises 50mM Tris and 150mM NaCl.Pam3cys and polyI:C are from Invivogen.Mice is C57BL/6.

For studying in the body, the perspective study that relates to titration of virus in mice is to determine suitable viral lot number and dosage.For whole research, every group is used 10-13 Mus, and each viral dosage uses 3 groups of mices.Each organizes following the processing: virus (A) is only arranged; (B) Cpn10 of virus+concentration A; (C) Cpn10 of virus+concentration B.Approximately the newborn mice intraperitoneal injection Cpn 10 (or preparation buffer) and the SFV in 6-9 days ages, and foster by its mother.In addition, adult rats (wt type and IFNAR deficiency) the intraperitoneal injection SFV in age in 6-8 week, per 12 hours intraperitoneal injection Cpn10 in first week of virus injection, its dosage uses fixed dosage.

For in vitro study, adopt Cpn10 response TLR activation and influence and comparison WT and IFNAR defective macrophage that regulating action that the pair cell factor produces is swashed by macrophage in advance.Macrophage (BMM) from the derived from bone marrow of WT and IFNAR deficient mice is handled under the Cpn10 of range of doses with TLR agonist pam3cys (TLR2), polyI:C (TLR3) and LPS (TLR4).

7.2 result

The data of ex vivo newborn rat viral infection research show, the survival rate of the SFV infecting mouse of handling with Cpn10 significantly increases.This research comprises the following newborn C57BL/6 mice in 6-9 days ages of respectively organizing: A group: SFV (n=13); B group: SFV+Cpn10 20 μ g (n=13); C group: SFV+Cpn10 50 μ g (n=13).

B group and C group lumbar injection 20 and 50 μ gCpn10, (after 3 hours) inject 50 μ l SFV (30x TCID50) to all three groups then.Mice is housed in its mother, and carefully nursing in entire test.The standard indication of monitoring these newborn mice health status blanking time (12/24 hour) carefully with rule comprises activity situation, health, behavior etc.

In the time of 72 hours, 54% and 69% 20 and 50 μ g Cpn10 handle mice and still survive, and survival rate is 15% (Figure 17) in untreated mice and compare.Notice that also the mice in the maximum dose level group gets around, other two groups then very unhealthy, lies on one side.In the time of 84 hours, all mices of only handling with SFV all die from the infection of virus, and 31% B group and C group mice still survive.

7.3 discuss and conclusion

As 72 hour datas as seen, the administration of Cpn10 has prolonged the survival rate behind the viral infection in dosage dependence mode.In infection back 96 hours, with the mice of Cpn10 injection 18% survival is arranged, and all untreated mice are all dead in the time of back 88 hours in infection.In two Cpn10 processed group, be 50% and 70% at the mice survival rate of infection in the time of back 72 hours, compare that survival rate is 18% in the untreated fish group, this species diversity is very significant under the situation that gives heavy dose of virus.It should be noted that also this result obtains behind the Cpn10 single-dose.These data show that Cpn10 has induced the antiviral protective effect.

Embodiment 8-Cpn10 and bunch associating of CD14-dependent/non-dependent LPS receptor

The plasma membrane of cell is made up of the heterogeneous thing in a plurality of sides (lateral heterogeneity), speckle (patch) and micro structure territory.These film micro structure territories or lipid raft are rich in glycosyl sphingolipid and cholesterol, and relevant with the cell processes of film sorting and signal transduction and so on.The inventor thinks that in the process that the LPS signal clusters Cpn10 can concentrate on the lipid raft to destroy the signal conduction by directly combining with TLR4 or with one of other members that cluster.

8.1 material and method

The cell marking that is used for FRET.MonoMac 6 cells (human monocyte cell line) 100 μ l mixture labellings of donor cross-linking antibody (Cy3) and receptor cross-linking antibody (Cy5).For the labelling of Cpn10, use anti--Cpn10 rabbit polyclonal antibody.Cell is used 4% formaldehyde fixed 15min then with PBS/0.02%BSA rinsing twice.Fixed cell is in order to prevent the contingent reorganization again of protein in the test process.

Co-focusing imaging.Cell is at Carl Zeiss, and Inc.LSM510 Laser Scanning Confocal Microscope (Axiovert 200 fluorescence microscopies are housed) is gone up and used the imaging of 1.4NA 63x Zeiss object lens.Use LSM2.5 image analysis software (Carl Zeiss, Inc.) analysis image.Use suitable filter set to detect Cy3 and Cy5.Use typical exposure frequency (less than 5s) for image acquisition, use the Cy5 optical filter not observe fluorescence, use the Cy3 filter set also not detect Cy5 fluorescence from the sample of Cy3-labelling.

FRET measures.FRET is the Noninvasive imaging technique, is used for determining the molecule nearness.FRET can take place on the distance of 1-10nm, and can effectively increase the resolution of optical microscope to molecular level.It relate to energy from the excited state non-radiation type of donor molecule be transferred to (Wu and Brand (1994) Anal.Biochem.218:1-13) on the suitable receptor.6 powers of distance (Kenworthy and Edidin (1998) Journal of Cell Biology 142:69-84 that is inversely proportional between the speed that energy shifts and donor and the receptor; Kenworthy and Edidin (1998) Gelb, M.H. (Ed.) " molecular biological method (Methods in molecular Biology) " .HumanaPress Inc, Totowa, NJ pp 37-49).Energy shifts the efficient of (E) according to r and R ₀(distinctive Distance) define:

E＝1/[1+(r/R ₀) ⁶][1]

In this research, use (Kenworthy and Edidin (1998) Journalof Cell Biology 142:69-84 as mentioned previously; Kenworthy and Edidin (1998) Gelb, M.H. (Ed.) " molecular biological method (Methods in molecular Biology) " .Humana PressInc, Totowa, NJ pp 37-49) method measure FRET.In brief, sample shifts the increase (taking off cancellation) that detects to donor fluorescence behind the complete photobleaching of acceptor molecule with donor and receptor cross-linking antibody labelling, energy.Use is only faded the required minimum time of (bleach) Cy3 so that measure with the cell of 26ic-Cy3 probe mark.Adopt arc light to use the Cy3 optical filter Cy3 to be faded by the continuous agitation that is set at 5 minutes.Under these conditions, Cy5 is not faded.The FRET image is calculated in increase by following formula donor fluorescence behind the receptor photobleaching:

E (%) * 100=10,000 * [(Cy3 fade back-Cy3 fade before)/Cy3 fade back] [2]

The conversion coefficient of use 10,000 extends to E the scale of 12-bit image.

8.2 result and discussion

In first group of data shown in the table 1, FRET is used for determining to participate in the acceptor molecule concentration of the inductive cell activation of LPS (MonoMac6 cell) in the lipid raft.By carrying out FRET experiment, might study Cpn10 (lot number P003-04 or CH003), Hsp60, escherichia coli GroES and other molecules whether with lipid raft co (co-localising).FRET measures is to the cancellation of taking off of donor fluorescence behind the complete photobleaching of acceptor fluorescence group.The increase of donor fluorescence shows because energy shifts after destroying receptor, donor fluorescence in the presence of receptor by cancellation.The use positive control comes the energy transfer efficiency in the detection system, i.e. transfer between the different epi-positions on mAbs to GM1 (ganglioside, the related molecule of the lipid raft) molecule shows that maximum energy transfer efficiency (E%) is 37 ± 1.0.Can use the negative control between FITC-GM1 and the rhodamine-MHC, it shows does not have tangible energy to shift (3 ± 0.4) yet.This background FRET value is considered to because two kinds of species all exist with high concentration by FRET was produced at random.

Table 1. D-A between the energy transfer efficiency value

Behind the receptor photobleaching, the energy that detects between the different pairings from the increase of donor fluorescence shifts.Data are expressed as the mean ± standard deviation of a plurality of independent trialss.

Data presented proves in the table 1, and CD14 is arranged in the lipid raft, but does not find that before LPS stimulates TLR4 and lipid raft unite, and is to stimulate at LPS then to be raised to this place obviously.

Next group data description reuse the nearness that Cpn10 that FRET obtains and TLR4 cluster.In table 2, positive control is TLR4 itself, obtains 36 ± 2.0 maximum energy transfer efficient (E%).

Table 2. D-A between the energy transfer efficiency value

Behind the receptor photobleaching, shift from the energy between the different pairings of increase detection of donor fluorescence.Data are expressed as the mean ± standard deviation of a plurality of independent trialss.

As shown in table 2, when lacking LPS, Cpn10 and Hsp70, Hsp90 or CXCR4 are irrelevant, but proof stimulates the member of back and these LPS activation bunch to get in touch at LPS.Though proof before LPS stimulates Cpn10 whether associated data are in this not demonstration with TLR4, in the LPS stimulating course, Cpn10 and TLR4 have very strong getting in touch.

These data prove that also before or after the LPS irritation cell, Hsp60 and TLR4, CD14 or lipid raft are irrelevant.Importantly, the escherichia coli homologue GroES of Cpn10, it is conventionally used as negative control in the active detection test of Cpn10 as immune response modifier, and it is also irrelevant with other members of lipid raft or CD14-dependent/non-dependent LPS activation bunch.

Embodiment 9-Cpn10 and the TLR interaction that clusters, And be positioned at the lipid raft on PBMC surface

For determine Cpn10 that endogenous produces whether on the surface of PBMC with the component interaction in TLR, TLR-correlation molecule and lipid micro structure territory, the reaction that the adherent mononuclear cell of people that the inventor uses anti--Cpn10 antibody of fluorophor labelling to adopt FRET to analyze fresh separated stimulates LPS.Owing to there was not recombinant C pn10 to be added in the cell culture before imaging, therefore the Cpn10 that discerns on cell surface will export the bank in cell.

9.1 material and method

The cell marking that is used for FRET.From healthy donors blood, use density gradient centrifugation to separate PBMC through Ficoll-Hypaque, and the preparation attached cell.The flushing cell is so that remove non-adherent cell.Remaining attached cell monolayer (1-2 * 10 ⁵Mononuclear cell/hole) in 24 orifice plates, added in the serum-free medium (Gibco) of 0.01%L-glutamine and 40 μ g/ml gentamycins and cultivated.Portion is rich in monocytic preparation estimates purity (＞80% purity) routinely by flow cytometer.Perhaps, according to the scheme of having delivered (Bender, people such as A, 1996.JImmunol Methods 196:121-135) before FRET measures, from the adherent peripheral blood lymphocytes of richness with DC and the GM-CSF and the IL-4 (R﹠amp of cells of monocytic origin; D Systems) cultivated 5 days.

Basically (Triantafilou, K 2001.Nat Immunol2:338-345) is used for the cell marking that FRET analyzes as delivering in the past.The person monocytic cell of fresh separated or the DC of cells of monocytic origin use 100ng/ml LPS (from S.minnesota, List Labs, the Re-LPS of CA), 10ng/ml staphylococcus aureus LTA is (by Thomas Hartung, Konstanz, the Germany present), 0.1 μ g/ml poly (I:C), the 20ng/ml imiquimod, 1 μ MCpG ODN (Invivogen), 25 μ g/ml are from the ssRNA of Coxsackie B virus 3 purification, or buffer control stimulation 10min, wash, use 1: 1 mixture labelling of donor cross-linking antibody (Cy3) and receptor cross-linking antibody (Cy5) then.The again reorganization of fixed cell to prevent that protein is possible then.

Antibody.The antibody that uses in FRET analyzes comprises: Cpnl0 (Johnson, B.2005.J.BiolChem 280:4037-4047), Hsp70, CXCR4, TLR3, TLR7, TLR9 (SantaCruz), TLR2 (Genentech), Hsp90 (Bioquote), CDl4 (26ic, ATCC, from the HB246 hybridoma), GMl (GMl-1, Calbiochem, GMl-2b, North StarBioproducts, Liverpool, UK), (MCAl 15 for I class MHC, Serotec), (HTAl 25, HyCult) for TLR4.Antibody uses FluoroLink labelling kit (AmershamPharmacia) to carry out labelling with Cy3 or Cy5.

FRET measures.Emission excites Cy3 at the 543nm place with helium/Ne laser, and emission excites Cy5 at the 633nm place with helium/Ne laser.Cell is at Carl Zeiss, and Inc.LSM510META Laser Scanning Confocal Microscope (Axiovert 200 fluorescence microscopies are housed) is gone up and used the imaging of 1.4NA 63x Zeiss object lens.Use LSM 2.5 image analysis software (Carl Zeiss, Inc.) analysis image.Use suitable filter set to detect Cy3 and Cy5.Image acquisition is used typical exposure frequency (less than 5s), do not observe fluorescence from the sample of Cy3-labelling.Only continuously track is scanned with a laser, each detector channel all is in open mode when each scanning.FRET measures is to the cancellation of taking off of donor fluorescence behind the complete photobleaching of acceptor fluorescence group.Energy shifts (E) efficient according to r and R ₀(distinctive Forster distance) defines: E=1/[1+ (r/R ₀) ⁶] (29).This FRET measuring method once had description (30) in the past.

9.2 result

As shown in table 3, find that after LPS stimulated, the Cpn10 that endogenous produces and other members of TLR4 and TLR4 signal complex got in touch.The interaction of Cpn10 that LPS identifies before stimulating and lipid raft part shows that it discharges from dead cell, or because the caused rise of physical operations of pair cell in the purge process.The proof of endogenous Cpn10 in lipid raft domain shown that it is delivered on the cell surface and the mode of cell exocrine.

Table 3. is measured as FRET, on endogenous Cpn10 and the PBMC

The lipid raft and the film of TLR4 signal conduction complex are united

Data are expressed as the mean ± standard deviation of several independent trialss.

The inventor also attempts to use FRET to analyze and the cell of fresh separated determines that whether Cpn10 directly gets in touch with other TLR.FRET measures is to the cancellation of taking off of donor fluorescence behind the complete photobleaching of acceptor fluorescence group.As shown in table 4, have part but be not to lack under the situation of part, Cpn10 and be arranged on the cell surface and most of TLR of the endosome of the DC (MoDC) of cells of monocytic origin between (except TLR3) observe the very big cancellation of taking off.(note going up the positive FRET signal most probable explanation of TLR7 and 9, in the MoDC of cell colony, have pDC in a small amount with MoDC.)

Table 4. is measured as FRET, exists at part but is not to lack under the situation of part, and the TLR in Cpn10 and cell surface and the people MoDC endosome gets in touch.

Behind the receptor photobleaching, shift (E ± Δ E (%)) from the energy between the different pairings of increase detection of donor fluorescence.For each pairing, donor (Cy3) is Cpn10, and exception is that donor in the controlled trial (Cy3) is TLR4; Receptor (Cy5) is TLR.FRET:E% * 100=10 is calculated in increase by following formula donor fluorescence behind the receptor photobleaching, and the 000[donor fades before back-donor fades)/donor fades afterwards].Data are expressed as the mean ± standard deviation of several independent trialss.

Embodiment 10-TLR is in conjunction with the effect to Cpn10

Whether in order to determine the relation between TLR combination and the Cpn10, especially detecting Cpn10 is immune endogenous regulator, and the inventor has checked whether endogenous Cpn10 is inductive by cellular stress with TLR combination or the activatory form of cytokine.The inventor has also checked when having stood suitable stimulation, and whether Cpn10 can discharge from cell, and whether Cpn10 can limit the inductive signal of part of other TLR.

10.1 material and method

Reagent.Basically as previously mentioned (Johnson, B.2005.J.BiolChem 280:4037-4047) is by BresaGen Limited (Adelaide, Aus) the recombined human Cpn10 of production GMP specification.Measure the purity of Cpn10 by SDS-PAGE and want＞99%, and (people such as Brinker, 2001.Cell 107:223-233) shows the mole activity (data not shown) identical with GroES in the rhodanese refolding chemical examination of GroEL-mediation.For facs analysis, cysteine residues is machined on the C-end of Cpn10, and according to the description labelling of production firm with AlexFluor647 (Invitrogen).The LPS that measures Cpn10 by Endosafe chemical examination (Charles RiverLaboratories) pollutes common wanting＜0.2EU/mg albumen.TLR part and cytokine comprise: Escherichia coli LPS (bacterial strain 055:B5, Sigma), the ultrapure LPS of escherichia coli (0111:B4Sigma), staphylococcus aureus LTA, bacillus subtilis LTA, staphylococcus aureus PGN, bacillus subtilis PGN, zymosan cell wall from saccharomyces cerevisiae, poly (I:C), imiquimod R837, CpG ODN 1826 and ODN2216, (all are all from Invivogen in GpC ODN1826 contrast, San Diego, CA), reorganization Mus IFN γ (Chemicon), IL-1 β (PeproTech) and reorganization humanTNF-(Invivogen).Antibody.Cultivation is at the rabbit polyclonal antibody of Cpn10, and by the CBio affinity purification.Fluidic cell bead microarray art (the CytometricBead Array of identification phosphorylation signal protein (people and Mus all have), CBA) Flex Sets is from BD, and above-mentioned signal protein comprises ERK1/2 (T202/Y204), JNK1/2 (T183/Y185), p38/MAP kinases (T180/Y182), CD14 and FcBlock (CD16/CD32).

Transfection, cytositimulation and promoter Analysis.(La Trobe University Melbourne) provides pCAT/LUC-CPN promoter construction by M.Ryan and N.Hoogenraad.Use GeneJuice (Novagen) transient transfection RAW264.7 cell.Harvesting behind the 24h is estimated cell viability by trypan blue dye exclusion test.Cell inoculation is in 24 orifice plates, and heat shock (43 ℃ of 15min (slightly), 30min (moderate) or 60min (severe)) then 37 ℃ of recoveries, or stimulates behind the 18h.Cell is at cell culture lytic reagent (Cell Culture LysisReagent, CCLR, Promega) washing and results in use luciferase assay system (Promega) and Wallac Victor 2 multiple labeling calculating instruments (Perkin-Elmer) to measure Lampyridea luciferase signal.

PCR in real time.The RAW264.7 cell is with 2.5 * 10 ⁵/ ml is seeded in aseptic 24 orifice plates, and before stimulating adherent 16-20 hour.Every kind of condition is measured repeat samples.Use Dynabeads (Dynal) separating mRNA, carry out RT-PCR (Invitrogen) with oligomerization (dT) primer and Superscript III First-StrandSynthesis System reverse transcription.The cDNA that generates increases in 10 μ l qPCR reaction with double, every part of justice and antisense primer that contains each 10 μ M of (20%) cDNA template, 6.25 μ l Platinum SYBR Green qPCR Supermix-UDG (Invitrogen) and 0.25 μ l of 2.5 μ l dilution.Reaction condition is as follows: 95 ℃ 2 minutes, carried out 40 circulations in 20 seconds with 95 ℃ 10 seconds, 60 ℃ 15 seconds and 72 ℃, last fusing step be 72-95 ℃ 50 seconds.On Rotor-Gene 3000 instruments (CorbettResearch), carry out the qPCR reaction.Expression of target gene contrasts according to house-keeping gene and carries out standardization, and analyze as previously mentioned (Livak, KJ., and T.D.Schmittgen.2001, Methods25:402-408).Primer: 18S justice CGG CTA CCA CAT CCAAGG AA (SEQID NO:5), 18S antisense GCT GGA ATT ACC GCG GCT (SEQ ID NO:6); Cpn10 justice GCC GAAACT GTAACC AAA GG (SEQ ID NO:7), Cpn10 antisense CAG GCT CAA TCT CTC CAC TC (SEQ ID NO:8); Pbgd justice CCT GGT TGT TCA CTC CCT GA (SEQ ID NO:9), Pbgd antisense CAACAG CAT CAC AAG GGT TTT (SEQ ID NO:10).

Endogenous Cpn10's is quantitative among cell culture supernatant, cellular lysate and the patients serum.The RAW264.7 cell of untransfected is handled as mentioned above, concentrates culture supernatant.Estimate the vigor of cell, the cell under every kind of condition is concentrated in together (n=4), be centrifuged into lamellar, and be suspended among the CCLR again.By improvement Bradford algoscopy (BioRad) total cell protein content is carried out quantitatively.By the Cpn10 level in ELISA (detection limit 0.195ng/ml) analysis of cells supernatant and the extract (each sample 25 μ g).Detect circulation Cpn10 level in the clinical trial experimenter serum by ELISA entering when research.According to the GCP guideline carry out these the test, and before sample detection every Informed Consent Form that subjects signed is written.Human Ethics Committee valid experimentation scheme by local center.

RAW264.7-HIV-LTR-LUC measures.Carry out RAW264.7-HIV-LTR-LUC as previously mentioned and measure (Johnson, B.2005.J.Biol Chem 280:4037-4047).Cell and Cpn10 or contrast buffer preincubate 2 hours add the TLR part of range of doses then.In each determination test, the detectable concentration scope of Cpn10 is 25-100 μ g/ml, the ligand concentration that uses will form sharp-pointed dose-effect curve, be that Escherichia coli LPS is 0.2-5ng/ml, ultrapure Escherichia coli LPS is 5-10ng/ml, and LTA and PGN are 10-100 μ g/ml, zymosan is 10-100 μ g/ml, poly (I:C) is 10-100 μ g/ml, and imiquimod is 1-10 μ g/ml, and CpG ODN is 3.25-16.25 μ g/ml.Optimization is enough on background to guarantee signal with the incubation time of every kind of agonist.As the negative control of people Cpn10, the conventional escherichia coli homologue (carrying out purification (Brinker, 2001.Cell 107:223-233) basically as mentioned previously) of using the Cpn10 that is called GroES.In measuring, these are used to induce the shown level (Johnson, B.2005.J.Biol Chem 280:4037-4047) of this cell line of using under the activatory concentration of HIV-LTR before known being less than of endotoxin contaminants level of attention in Cpn10 and GroES preparation and TLR ligand reagent.

Human PBMC's cytokine assay.From healthy volunteer's blood, separate PBMC by carrying out the buoyant density gradient centrifugation at Ficoll-PaquePlus (Amersham Biosciences).PBMC uses agonist at 37 ℃ and 5%CO with Cpn10 (or buffer contrast) pretreatment 1h then ₂Under stimulate 20h, collect culture supernatant then.Use CBA (BD) or ELISA (Bender MedSystems) analysis of cells factor level.

The detection of protein phosphorylation.RAW264.7 or PBMC are hatched 2h with Cpn10, stimulate 30min with LPS then, and be centrifugal, dissolves tablet with 0.1%SDS/PBS.Lysate boils 5min, and cooling by centrifugal that cell debris is agglomerating, is as above measured the protein concentration of supernatant.The BD CBA Flex Sets that use is used for p38, ERK1/2 and JNK1/2 detects the signal protein of phosphorylation.Perhaps, RAW264.7 handles with Cytofix/Cytoperm (BD), and interior p38 of double staining cell and cell surface CD14.Use BD FACSArray Bioanalyzer with FCAP array software analysis sample.

Statistical analysis.Use one-sided ANOVA and Tukey multiple comparisons check (Tukey ' sMultiple Comparison Test) under the best agonist concentration more not incentive condition and incentive condition with the evaluation cytositimulation after the variation of promoter activity.Use one-sided ANOVA and Tukey multiple comparisons to check (Tukey ' s Multiple Comparison Post Test) to come significance between the group of the Cpn10 in experimenter's blood serum sample is tested afterwards.

10.2 result

10.2.1.TLR zygotic induction Cpn10 promoter

The RAW264.7 cell carries out transfection with the sub-construction of report, and the generation of luciferase is under the control of Cpn10 promoter in this construction.Reorganization IFN γ, TNF-α and the IL-1 β of purification detect as non-TLR agonist.(43 ℃, 30min) cell of handling proves that the Cpn10 promoter has significantly induces, and turns back to baseline values (Figure 18 A) during 18h when putting the recovery time of 6h with the moderate heat shock.Induce the Cpn10 promoter with determining TLR agonist dose dependent subsequently, observe maximum activation (Figure 18 A and Figure 19 C) after 20 hours.Optimize agonist concentration then to induce maximum promoter reaction (Figure 18 B-G).

Relatively the reaction to TLR and non-TLR agonist shows that the Cpn promoter activity has unique characteristic curve (Figure 18 H).Observe as the reaction to TLR2,7 and 9 part, Cpn10 has tangible rise, shows relevant with MyD88-dependent T LR signal transduction pathway.Use can adopt the TLR part (TLR3 and 4) of TRIF-dependent signals pathway then not observe the Cpn10 promoter significant rise.And non-TLR agonist IFN γ, IL-1 β and TNF-α can not induce the substance of Cpn10 promoter to raise, and show the specificity that has aspect the control to a certain degree of transcribing at Cpn10.

Another evidence of transcribing as the inductive Cpn10 of TLR-, use the PCR in real time analysis to show that LTA has induced Cpn10mRNA to the stimulation of RAW264.7 cell with having caused concentration dependent, when 18h, reach maximum and induce, stimulate back 24h to be back near baseline values (Figure 18 I).

10.2.2. discharge from cell in conjunction with back Cpn10 at TLR

In order to check when standing suitable stimulation, whether Cpn10 discharges from cell, and the inventor stimulates the RAW274.7 cell with LTA, the existence by ELISA monitoring endogenous Cpn10 and be released into Cpn10 in the culture medium by cell.The analysis of cellular lysate shows that the cell of LTA processing and the interior Cpn10 level of cell of control cells change very little (Figure 19 A).But the analysis of culture supernatant shows that then the more unprovoked control cells of solubility Cpn10 has increased by 3 times (Figure 19 B) behind 30h.In addition, the extracellular Cpn10 level that produces of irritation cell changes in time with the Cpn10 promoter activity and the increase seen is relevant (Figure 19 C).These data show that the new synthetic Cpn10 of a part that produces is discharged by cell under the condition of immune stimulating.Accept the culture supernatant analytical proof of the cell (the RAW264.7 cell of RAW264.7 or pCAT/LUC-CPN-transfection) of slight, moderate or severe heat shock (HS), with compare the gathering seldom of extracellular Cpn10 (Figure 19 D) with LTA stimulated cells supernatant.These data show that Cpn10 is induced with assistance mitochondrial protein misfolding by HS, and specific immunity excites the extracellular accumulation that causes Cpn10.

In order to obtain the evidence that Cpn10 can be conditioned in disease, the inventor has detected the patient's who suffers from chronic inflammatory disease Cpn10 baseline serum level, and itself and healthy volunteer's cohort are compared.As shown in Figure 19 E, the Cpn10 cyclical level of suffering among the patient of rheumatoid arthritis (is respectively 1.32ng/ml ± 1.41, n=23 apparently higher than multiple sclerosis patients, psoriatic or health volunteer; 0.44ng/ml ± 0.46, n=46; 0.55ng/ml ± 0.52, n=25; 0.45ng/ml ± 0.32, n=24).Therefore each Cpn10 serum levels and individual occurent inflammation and/or disease levels are relevant.No matter whether be to cause, be clear that very Cpn10 accumulates in the extracellular environment, and some cytositimulation can strengthen its discharge owing to the post-stimulatory zest of immunocyte in passive release after the lysis or the inflammatory excitation process discharges.

10.2.3.Cpn10 the kinetics of restricted T LR signal conduction

The inventor has checked recombinant C pn10 whether can limit the inductive signal of part of other Toll sample receptors.The Cpn10 preincubate of this report daughter cell system and optium concentration 2 hours, use various TLR parts (its scope is from gram-positive bacterium Peptidoglycan (PGN) and fat techoic acid (LTA) to synthetic RNA and DNA motif) to stimulate then, stimulated cells is compared when lacking exogenous Cpn10, causes the inductive luciferase activity of NF-κ B-obviously to reduce.Except synthetic TLR3 part polyinosinic acid-polycytidylicacid (poly (I:C)), in the cell culture with every other tested TLR ligand stimulation, the existence of Cpn10 causes the inhibition (Figure 20 A) of luciferase activity 20-80%.These results show that Cpn10 can regulate the intensity by the immunne response signal that part produced of most of TLR.

Can trigger MyD88-dependency and the reaction of dependent/non-dependent signal cascade with the LPS irritation cell.MyD88-dependency cascade reaction is that all TLR are shared except TLR3, and relates to NF-κ B and mitogen-activated protein(MAP) (MAP) kinases (MAPK), comprises the terminal kinases (JNK) of p38, extracellular signal-regulated kinase (ERK) and c-Jun N-.The terminal point of two paths is startup and dynamic (dynamical) adjustings of immunne response.Cpn10 regulates the NF-kB activation and cytokine produces the incident that is experienced in order to illustrate behind the irritation cell, and the inventor has analyzed the phosphorylation level that participates in the key protein of map kinase signal path.As shown in Figure 20 B-D, in the human PBMC of mouse macrophage cell line (RAW264.7) and fresh separated, Cpn10 dose response ground reduces the phosphorylation level of the inductive p38 of LPS, ERK1/2 and JNK1/2.Because inducing of the inflammatory cascade reaction that the activation of MAPK path and cytokine produce is closely related, these digital proofs in the inductive cytokine of part produces, the variation during the variation of Cpn10-mediation is reacted from the TLR signal cascade.

In order to confirm the activity of Cpn10 generation by the caused cytokine of people's cell of the fresh separated of multiple TLR ligand stimulation, the inventor is with Cpn10 or without the PBMC 1h of Cpn10 pretreatment from healthy donors, and the TLR ligand stimulation of the selected scope of usefulness is 20 hours then.Use the cytokine levels in ELISA or fluidic cell bead microarray art (CBA) the analysis of cells culture supernatant then.As shown in Figure 20 E, in the presence of Cpn10, be suppressed by the inductive cytokine generation of agonist (comprising TNF-α, IL-1 β, IL-6 and the IL-10) institute of TLR2, TLR2/6 or TLR4 (PGN, LTA, zymosan, LPS).Although it should be noted that Cpn10 mediation to slightly the inducing of the generation of the inductive IL-10 of LPS-, it responds digital proof other TLR part and reduces the generation of IL-10.Equally, compare, in the presence of Cpn10,, cause that the generation of TNF-α reduces, and increase IFN α with agonist and the imiquimod or the CpG ODN stimulation PBMC of TLR7 or 9 with the control cultures that does not have Cpn10.

10.3 discuss

The Cpn10 that this research has not been recognized before having disclosed is as the effect of immune endogenous regulator.Through the TLR of receptor system rather than by TNF-α, IFN γ or IL-1 beta receptor irritation cell, induced Cpn10 promoter and can the proteic generation of detected Cpn10 in the culture medium of extracellular.The human PBMC of cell activation (estimating by the NF-kB activation), MAPK phosphorylation and mouse cell line and fresh separated that the addition agent quantitative response ground restricted T LR-of recombinant C pn10 drives is in external generation cytokine.

The result of Cpn10 is induced in these proof immunologic injuries (insult), and the data that show the restriction inflammatory reaction size of Cpn10-mediation, provides strong evidence to prove, Cpn10 is the important member to the very important negative feedback path of immunne response network.

Embodiment 11-stimulates the inductive change that TLR4 is expressed of back Cpn10-at LPS

The inventor has managed to determine the influence that Cpn10 expresses TLR4 in the RAW264 cell, so that check stimulates back Cpn10 whether can influence the surface expression of TLR4 at LPS.

11.1 material and method

The white escherichia coli in LPS source (Sigma), anti--Mus TLR4/MD-2-biotin and streptavidin-phycoerythrin are from eBioscience, and anti--Mus IFN-γ is from BD.

Exist or do not have in the suspension of 100 μ g/ml Cpn10 the specified time of cell culture.Cell transfer is to the 10ml test tube then, and counting gathers group and also is suspended in the fresh culture again.100 μ l cell suspension are transferred to Eppendorf pipe (10 ⁶Cell/pipe) in.Carry out 4 hours Cpn10 at the Eppendorf pipe that places incubator and handle and the LPS stimulation, mixed lightly in per 15 minutes.Cell washs 3 times with PBS/0.2%FBS.Anti--Mus TLR4-biotin (1 μ l is in 50 μ l PBS/0.2%FBS) or control antibodies (anti--Mus IFN-γ-biotin) were hatched on ice 1 hour with cell.Cell washs 3 times in 1ml PBS/0.2%FBS, seals 45min with 50 μ l PBS/10%FBS down at 4 ℃ then.Add streptavidin-PE with 1/100 diluent then, and continued to hatch 1 hour on ice.After 3 washings, cell fixation is in 1% paraformaldehyde/PBS, and use Cell Quest software is analyzed on FACS Caliber.

11.2 result

Cpn10 and RAW264 cell are hatched and were not changed the TLR4 expression in 4 hours.Use anti--Mus TLR4/MD-2-biotin to use streptavidin-PE to be easy on the surface of RAW264 cell, detect TLR4 (Figure 21) then.Control antibodies can not show tangible dyeing (data not shown).Cpn10 handled 4 hours or the processing of contrast buffer all can not change the TLR4 expression.

The influence of LPS downward modulation TLR4 and Cpn10.Add 2ng/ml LPS and make the TLR4 of adding LPS after 2 hours express slight minimizing (Figure 22), 20ng/ml LPS causes that TLR4 expresses almost completely disappear (Figure 23).Cpn10 is hatched the behavior that change to add TLR4 behind the LPS in 4 hours indistinctively.Extend to when spending the night when Cpn10 handles, to compare with the cell (Figure 24 and 25) that buffer is handled, TLR4 expresses and reduces (Figure 24 and 25) slightly.When giving cell 2ng/ml LPS, the minimizing more remarkable a little (Figure 24) that the TLR4 of LPS-mediation expresses in the cell that Cpn10 handles.When cell was handled with 20ng/ml LPS, TLR4 expressed disappear basically (Figure 25) in all groups.

11.3 discuss and conclusion

By active minimizing of LUC that Cpn10 processing in 4 hours is mediated, cause that the inductive NF-kB activation of LPS-reduces 30-50%.This effect can not be because the TLR4 down-regulated expression of Cpn10-mediation is caused, because this processing is very little to the expression change of TLR4.May be remarkable a little after 4 hours Cpn10 handle by the downward modulation that 2ng/ml LPS is mediated, more remarkable certainly after the Cpn10 processing is spent the night.These results show that Cpn10 can change and add the kinetics that TLR4 expresses behind the LPS.

Embodiment 12-Cpn10 restriction is to the cell effect of broad-spectrum TLR agonist

The inventor to prove Cpn10-mediation to many TLR parts the inhibiting popularity of inductive NF-kB activation.

12.1 material and method

Escherichia coli LPS 055:B5 is from Sigma, and Escherichia coli LPS 0111:B4 ultrapure (1 * 10 ⁶EU/mg), staphylococcus aureus fat techoic acid (LTA) (12.5EU/mg), aureus peptide polysaccharide (PGN) (＜0.125EU/mg), bacillus subtilis fat techoic acid (LTA) (12.5EU/mg), bacillus subtilis Peptidoglycan (PGN) (0.25EU/mg), the saccharomyces cerevisiae zymosan (＜0.125EU/ml), imiquimod R837, CpGODN1826 (Mus), CpG ODN2216 (people) and GpC ODN1826 (non-irritating contrast) are from Invivogen.Reorganization Mus IL-1 β is from R﹠amp; D Systems, reorganization Mus IFN γ and reorganization Mus TNF α are from Chemicon.Fluidic cell bead microarray is from BD.

The RAW264-HIV-LTR-LUC bioassary method.Basically as Johnson, people such as B. carry out bioassay described in the 2005.JBiol Chem 280:4037-4047.In each is measured, Cpn10 detects with the concentration range of 25-100 μ g/ml, the stimulus object concentration of using will form sharp-pointed dose-effect curve, be that Escherichia coli LPS is 0.2-5ng/ml, ultrapure Escherichia coli LPS is 5-10ng/ml, staphylococcus aureus and bacillus subtilis LTA and PGN are 10-100 μ g/ml, and the saccharomyces cerevisiae zymosan is 10-100 μ g/ml.Be noted that, contaminated with endotoxins level in Cpn10 preparation and TLR part, in measuring, these are used to induce the shown level (Johnson, people 2005.J.Biol Chem 280:4037-4047 such as B.) of this cell line of using under the activatory concentration of HIV-LTR before known being less than.Below in the data presented, be noted that the reading of stimulation of describing this cell line with the NF-kB activation, it is based on following understanding: HIV LTR is known to have the reactivity of height to the NF-kB activation, is the activatory indirect measurement of this transcription factor therefore.

Human peripheral blood mononuclear cell (PBMC) cytokine assay.From healthy volunteer's heparinized blood, separate PBMC through Ficoll-Paque Plus (AmershamBiosciences) by the buoyant density gradient centrifugation.PBMC perhaps is kept at mother solution in the cryovial standby in liquid nitrogen as the cell of fresh separated.PBMC is with 8 * 10 ⁵The density of living cells/ml with 200 μ l/ holes be dispensed to 96 hole tissue culturing plates (Greiner Bio-One, Kremsmuenster, Austria) in.Add Cpn10 and kept 1 hour, use agonist then at 37 ℃ and 5%CO ₂Under stimulated 20 hours, collect culture supernatant then.Use commercially available ELISA test kit (R﹠amp; DSciences) and fluidic cell bead microarray (BD) analysis of cells factor level.

The phosphorylation level of detection signal molecule.RAW264.7 or PBMC and Cpn10 are hatched 2h, and LPS stimulates 30min then, and centrifugal, tablet dissolves with 0.1%SDS/PBS.Lysate boils 5min, and cooling is agglomerating by the centrifugal cell debris that makes, and as above detects the protein concentration in the supernatant.The BD CBA Flex Sets that use is used for p38, ERK1/2 and JNK1/2 detects the phosphorylation signal protein.Perhaps, handle RAW264.7 with Cytofix/Cytoperm (BD), and interior p38 of double staining cell and cell surface CD14.Adopt BD FACSArrayBioanalyzer to use FCAP array software analysis sample.

12.2 result

The activation of the HIV-LTR of TLR agonist induction.Digital proof among Figure 26 the Cpn10 ability of in mouse macrophage RAW264-HIV-LTR-LUC cell, regulating the NF-kB activation, this cell is with the TLR ligand stimulation of finite concentration scope.We have detected the part of a large amount of TLR, comprise the LPS (being described as stimulating TLR2 and TLR4 simultaneously) of ultrapure LPS (only TLR4) and non-repurity, from the LTA (TLR2) of two kinds of gram-positive bacteriums (bacillus subtilis and staphylococcus aureus) with from the PGN (TLR2) of above-mentioned two kinds of gram-positive bacteriums.In addition, cell is used from the isolating preparation of the cell wall of yeast saccharomyces cerevisiae, zymosan (TLR2/TLR6) and synthetic double-stranded RNA, and poly (I:C) stimulates, and above-mentioned poly (I:C) can be in conjunction with TLR3 and by its irritation cell.Cell also detects after stimulating with CpG DNA (TLR9) or imiquimod (R837), and imiquimod (R837) is a kind of low-molecular-weight synthetic molecules, its in the presence of Cpn10 through TLR7MyD88-dependency path activating cell.Data show is after detected four kinds of TLR (TLR2,4,7,9) combination, and Cpn10 can induce the activation of regulating NF-κ B.

Figure 27 has described the largest percentage by the inductive luciferase activity inhibition of Cpn10 in the RAW264-HIV-LTR-LUC cell of various TLR ligand stimulations.Although the reaction pattern of tested most of parts is all closely similar, promptly on average suppress 50% HIV-LTR activation, as if Cpn10 do not respond poly (I:C) and change the inductive NF-kB activation of TLR3-.And Cpn10 is limited to the NF-κ B inhibitory action of the activatory cell performance of yeast cell wall preparation zymosan, is approximately 20%.

The influence of the phosphorylation of signal protein in the Cpn10 pair cell.LPS has stimulated RAW264 cell activation signal transduction pathway in several cells comprise IkappaB kinases (IKK)-NF-κ B path and three kinds of mitogen-activated protein kinases (MAPK) path: extracellular signal-regulated kinase (ERK) 1 and 2, the terminal kinases (JNK) of c-Jun N-and p38.In farther downstream, these signal transduction pathway activate many transcription factor, comprise NF-κ B and AP-1, and they induce the gene of coding inflammatory mediator in phase then.In order to estimate the influence of Cpn10 to these signaling systeies, the inventor has measured and has existed or do not exist under the situation of Cpn10, with p38, the JNK1/2 of phosphorylation and the level of ERK1/2 in 30 minutes the cell of LPS stimulation.The inventor has shown each these member's of Cpn10 dose response ground restricting signal pathway the inductive phosphorylation of LPS-(Figure 28).

In order to confirm Cpn10 to produce the activity of cytokine with fresh separated people's cell of multiple TLR ligand stimulation, the inventor uses the PBMC 1h of Cpn10 pretreatment from healthy donors, uses the TLR ligand stimulation 20h of selected scope then.Use ELISA or fluidic cell bead microarray to measure cytokine levels in the cell culture supernatant then.As shown in Figure 29 A-D, in the presence of Cpn10,, comprise that TNF-α, the inductive cytokine of IL-1 β, IL-6 and IL-10 institute produce and reduced by the agonist of TLR2 or TLR4 (PGN, LTA, zymosan, LPS).Equally, the data indication among Figure 29 E is compared with the control cultures of not using Cpn10, with agonist and the imiquimod or CpG (ODN2216) the stimulation PBMC of TLR7 or 9, causes that production of cytokines reduces in the presence of Cpn10.

The Cpn10 preincubate washs the ability that does not influence the inductive NF-kB activation of Cpn10 restriction LPS-then.The inventor was verified in the past, used Cpn10 and RAW264-HIV-LTR-LUC cell preincubate 2 hours before stimulating with LPS, made the dose response reduction of luciferase activity reach optimization.As shown in figure 30, proved also now before LPS stimulates and Cpn10 preincubate 2 hours that carry out the PBS washing then, the luciferase activity (as measuring of NF-kB activation) that does not change the Cpn10-mediation reduces.

Non-TLR swashs the moving inductive NF-kB activation of agent of fclfcl.In order further to characterize the activity of Cpn10, the inventor has detected the regulating action of Cpn10 to many non-TLR agonist.As the part of this non-TLR testing program, in the presence of Cpn10, the RAW264.7 cell stimulates 6 hours (Figure 31) with low endotoxin (2.24EU/mg) the reorganization Mus IFN-γ of 1-10ng/ml.The collecting cell supernatant also uses elisa assay TNF-α.Find that Cpn10 can promote the inductive TNF-α of IFN-γ to produce.When cell and Cpn10 preincubate before IFN-γ stimulates in the time of 2 hours, this remarkable synergistic function of the generation of TNF-α is enhanced.Exist at Cpn10 under the situation of 2 hours or 4 hours, in lysate, also be measured to the higher inductive luciferase level of NF-κ B-with the RAW264-HIV-LTR-LUC cell of IFN-γ stimulation.

12.3 discuss and conclusion

The dynamic (dynamical) ability of Cpn10 adjusting macrophage that the inventor is verified to the reaction of multiple TLR (comprising TLR2,4,7 and 9)-ligands specific, therefore and proved the influence of phosphorylation of the signal transduction molecule of the upstream that Cpn10 produces NF-kB activation and cytokine, above-mentioned signal transduction molecule is the MAPK family member of signal conductive protein, comprises ERK, JNK and p38.The inventor also has been presented at by in the activatory cell of LPS, and Cpn10 dose response ground reduces the level of endocellular phosphorus acidify MAPK.

Digital proof in the present embodiment is adjusted in the kinetics that the innate immunity in the macrophage of the many TLR ligand stimulations of external use is replied the Cpn10 dose response.By the inductive maximum horizontal that suppresses the NF-kB activation of the Cpn10-of these molecules triggerings be: after with TLR7 agonist (R837) stimulation RAW264-HIV-LTR-LUC cell line, be about 80%.Under another kind of numerical range, adding Cpn10 with the concentration range of 10-100 μ g/ml can not influence the bonded kinetics of yeast cell wall preparation zymosan and TLR2/6 above 20%.

The pattern that reduces at least a exception of NF-kB activation for Cpn10 comprises the caused activation of polyinosinic acid-polycytidylicacid (poly (I:C)).Poly (I:C) is the synthetic analogues of double-stranded RNA, and its molecular pattern is relevant with the viral infection that activates NF-κ B through the interaction with TLR3.Cpn10 responds this agonist and the generation of dose response ground increase I type interferon, and does not limit the activation signals of NF-κ B.

Embodiment 13-cell surface receptor and the crosslinked bonded evaluation of Cpn10 in solid matrix

13.1 material and method

Separate on the host cell receptor in conjunction with Cpn10.Use Cpn10 (lot number CH003 is provided by CBio Ltd) preparation affinity column (the activatory Hi-Trap of NHS-) (AmershamPharmacia).Cpn10 dialyses in the bicarbonate buffer of pH 8.3, and crosslinked on post according to the description of production firm.

Use NP-40 dissolving buffer (0.05%w/v) dissolving person monocytic cell.All cellular lysate is washed unconjugated molecule by the Cpn10 affinity column off with PBS.Use b-octyl group-glucoside (b-OG) elution buffer (15mM triethanolamine, pH 11.5,140mM NaCl and 30mM b-OG) eluting Cpn10-bind receptor molecule, use 1M 2-(N-morpholino) the ethyl sulfonic acid neutralization of pH 5, and dialyse through PBS.In contrast, cellular lysate is also by the pillar of crosslinking protein not on it.

Use Centriprep YM-10 (Millipore) concentrate eluant, be concentrated into 1ml and also analyze by SDS-PAGE (Figure 32) or 2D gel (Figure 33) electrophoresis.For the 2D gel electrophoresis, and this spissated elute soln use rehydration buffer before analyzing (8M carbamide, 2%CHAPS, 10mM DTT, 0.2%Bio-Lyte) (Biorad) carries out buffer-exchanged.Downcut purpose band or speckle, trypsinization is also sent to and is carried out the MALDI-TOF analysis.

By 1D gel electrophoresis protein isolate.The solution of eluting is handled with 2x SDS-PAGE reduction buffer (4ml 10%SDS, 2.5ml 0.5M Tris-HCl PH:6.8,5g glycerol, 1ml 14.3M mercaptoethanol and 5mg bromophenol blue), and places boiling water 5 minutes.In order to move sample, use Criterion Tris-HCl Precast 4-20% gradient gel (Biorad).In gamut RAINBOW molecular weight marker (2 μ l) first hole of packing into, the remaining hole 40 μ l samples of packing into.Gel places electrophoresis tank then, and adds running buffer.Running buffer is made up of 0.025M Tris, 0.2M glycine, 0.1%SDS, and is adjusted to pH 8.3.Carried out electrophoresis 50 minutes with the 200V constant voltage.Behind the electrophoresis, take out gel, use fixedly buffer (40% ethanol, 10% acetic acid, 50%H ₂O) fixedly spend the night, with Coomassie blue dyestuff (50% Coomassie blue (0.3w/v), 25% methanol, 5% acetic acid, 20%H ₂O) dyeing is 1.5 hours.This gel is used destaining solution (10% ethanol, 10% acetic acid, 80%H at last ₂O) washing is several times with decolouring.

By 2D gel electrophoresis protein isolate.Use non-linear curing pH gradient (3-10ReadyStripIPG adhesive tape 11cm) as first to.The 200 μ l samples of drawing five equilibrium are to the focusing dish, and are then that the IPG adhesive tape is placed on it, under the glue side direction, immerse and contain in the solution of sample and buffer.Mineral oil places on the IPG adhesive tape to avoid the carbamide precipitation.Adhesive tape under passive condition 20 ℃ of rehydration 12 hours.Adhesive tape is with constant every adhesive tape 50 μ A then, and the 6000-7000 volt focuses on, and is about 52,000v-hr.For prepare be used for second to ReadyStrip IPG adhesive tape, it is taken out from the focusing dish, and contains 50mM Tris HCl, balance in the buffer of pH 8.8,6M carbamide, 2%SDS, 30% glycerol, 1%DTT at 5ml.In the same buffer of 5ml volume, carry out the balance second time, substitute DTT with 1.5% iodoacetamide in this buffer.After the balance, ReadyStrip IPG adhesive tape is placed on the Criterion gel (4-20% gradient), and cover to have 0.5% agarose of micro-mercaptoethanol.This gel moves 60min with 200V in Criterion electrophoresis tank (cell).Take out gel behind the electrophoresis, use fixedly buffer (40% ethanol, 10% acetic acid, 50%H ₂O) fixedly spend the night, with Coomassie blue dyestuff (50% Coomassie blue (0.3w/v), 25% methanol, 5% acetic acid, 20%H ₂O) dyeing is 1.5 hours.This gel is used destaining solution (10% ethanol, 10% acetic acid, 80%H at last ₂O) washing is decoloured several times.

13.2 result

Conjugated protein by affinity chromatography and SDSPAGE electrophoretic separation Cpn10-.The inventor uses method as described below to carry out preliminary research, and uses mass spectral analysis to identify that several Cpn10-are conjugated protein, and is as shown in table 5.

Table 5. uses mass spectrum to identify the Cpn10-binding molecule

The protein title	Registration number
		78kDa glucose regulated protein precursor (GRP 78)	GRP78_MESAU
The serum albumin precursor	ALBU_BOVIN
		EF-T u-B	EFTU2_PSEPK
Annexin A2	ANXA2_HUMAN
		Phosphotriose isomerase	TPIS_PANTR
Profilin-1	PROF1_HUMAN
		10kDa heat shock protein mitochondrion	CH10_HUMAN
Ig γ-1-chain C district	IGHG1_HUMAN
		Ig α
2 chain C districts	IGHA2_HUMAN
		α-1-antitrypsin precursor	A1AT_HUMAN
Peptidyl-propyl cis-trans isomerase A (Ciclosporin A-conjugated protein)	PPIA MACMU

In order to verify these results and determine not certified proteic homology that repeatedly separation of C pn10-is conjugated protein by affinity chromatography for the inventor, and analyzes the molecule of eluting to gel electrophoresis (Figure 33) by SDS-PAGE (Figure 32) and 2-.

Controlled trial proof does not have protein band (data not shown) on SDSPAGE, in this controlled trial, cellular lysate is passed through on the affinity column of crosslinking protein not having.

13.3 discuss and conclusion

This mass spectrometric data can be identified several albumen, these albumen for Cpn10 and host cell combine and the cell of Cpn10 in transportation have potential importance.Especially profilin (profilin), a kind of very little actin binding protein, the kinetics of polymerization that it can modulate actin, its seemingly a kind of Cpn10-is conjugated protein.Another very important molecule is GRP78 in regulating the Cpn10 reaction.GRP78 is the chaperone that participates in the Cpn10 transportation.

Embodiment 14-Cpn10 is to the cell with the polyI:C stimulated in vitro The influence of anti-ross river virus reaction

Whether the inventor wants to use the extremely sensitive virus of known antiviral effect to IFN α/β to detect Cpn10 can influence the inductive antiviral toleration of polyI:C-.

14.1 material and method

Cpn10。Cpn10 is dissolved in the Tris-brine buffer solution of pH7.6, and it is freezing in dry ice to be divided into a plurality of aliquots, is stored under-20 ℃.Before use aliquot is thawed.PolyI：C。For data presented among Figure 34, PolyI:C (being provided by Invivogen) is dissolved in the Sterile Saline with 5mg/ml, and stored frozen is under-20 ℃.Bottle is thawed, and be diluted in the culture medium.For buffer contrast, the Sterile Saline of equal volume is added in the cell culture hole.For data presented among Figure 35, PolyI:C is dissolved in the Sterile Saline with 5mg/ml, carries out five equilibrium with 25 μ l/500 μ l then, adds 62.5 μ l, 100% ethanol (2.5 times of volumes) with precipitation dsRNA.These test tubes are kept under-70 ℃.Measuring the same day, these five equilibrium reagent with maximum speed (13,000rpm) 40 ℃ centrifugal 30 minutes down.With the agglomerate washing once, agglomerating in the distilled water that 70% ethanol DEPC-handles, remove supernatant, in cover,, and be suspended in again among the 100 μ l RPMI 1640 agglomerate drying (about 15 minutes).Use the RNA quantitative characteristic of BioPhotometer (Eppendorf) to measure, the dsRNA response rate of mensuration is about 50%.Specified in the drawings all dsRNA concentration all are assumed to be 50% response rate.

Antivirus test.The RAW264.7 cell is cultivated in bottle becomes the adhere-wall culture thing, it is scraped (that is, non-trypsinized), is seeded to then in the 96-orifice plate.The HeLa cell is carried out trypsinized in the mode of standard to be inoculated then.Cell is seeded in the 96-orifice plate (for RAW264.7 cell 5 * 10 with double ³Cell is 10 for the HeLa cell ⁴, every hole 50 μ l culture medium).After 4 hours, add Cpn10 (being diluted in the culture medium), add polyI:C (being diluted in the culture medium) after other 30 minutes with 50 μ l with 50 μ l.Cell overnight incubation in 96 orifice plates adds RRV (in 50 μ l, for RAW264.7 cell MOI=1, for HeLa cell MOI=10) then then.After 3 days, fixed cell is used violet staining, washing, drying, with 100% methanol extraction dyestuff, and as described in the document at A595nm place reading (people such as Antalis, J Exp Med 1998,187 (11): 1799).

14.2 result

PolyI:C is to the effect of RAW264.7 cell.Originally, polyI:C with the scope titration of 0-100 μ g/ml to the RAW264.7 cell.PolyI:C concentration＞5-10 μ g/ml makes cell show tangible toxicity sign, and cell rounding is also grown very poor in three days minute.Then do not observe this toxicity (Figure 34) for the HeLa cell.

Cpn10 and polyI:C.Carry out titration to determine for RAW264.7 cell and the required dosage of HeLa cell induction antiviral toleration.Then, use the polyI:C of suitable concn scope, the hypothesis that whether can increase the antiviral activity of dsRNA to Cpn10 is tested.As shown in figure 35, detected polyI:C and Cpn10 are under various concentration in the tests in these three days of measuring cell viability, exist or do not exist under the situation of Cpn10, RAW264.7 cell or HeLa cell are not all showing any difference aspect its antiviral toleration.

14.3 discuss and conclusion

In this research, it is inductive at α virus-the induce antiviral toleration of CPE that Cpn10 does not influence synthetic dsRNA-.Viewpoint below this data presented can not be supported: Cpn10 can influence the signal conduction by dsRNA.The signal conduction can take place through TLR3, PKR, RIG-1/mda-5 and/or ATF-2/c-Jun in dsRNA, triggers other signal transduction pathway by Cytoplasm dsRNA, is the main sensor of extracellular dsRNA although it is believed that TLR3.

Embodiment 15-recombinant C pn10 interacts with APC on cell surface

The inventor has considered that extracellular Cpn10 regulates the mode that immunne response may be taked.In order to discern leukocyte subgroup in conjunction with the cell of Cpn10, the inventor has used the Cpn10 of fluorophor-labelling on C-terminal cysteine residue, and prove its kept with wild type Cpn10 at external identical chaperone and immunoregulatory activity (data not shown).With the Cpn10 of labelling and PBMC under 4 ℃, and one group be used to identify the antibody incubation of independent leukocyte subgroup after, prove that the interaction of Cpn10 and APC is the strongest, especially medullary system DC (CD3 ^-CD4 ^LowCD14 ^-CD11c ⁺), plasma cell sample DC (CD3 ^-CD4 ^LowCD14 ^-CD11c ^Low) and mononuclear cell (CD4 ^LowCD14 ⁺), with the affinity lower (Figure 36 A) of T cell.When PBMC stimulates with LTA, with unprovoked CD14 ⁺Cell is compared, fluorescence Cpn10 and CD14 during 4h ⁺Cell surface in conjunction with level about 1.6-increase (Figure 36 B) is doubly arranged.The outer Cpn10 of these tables of data clear-cellss is to the pivotal regulatory cell of innate immune system, and APC has brought into play its biologic activity.

The inventor has also studied fluorescence Cpn10 by confocal microscopy and has entered picked-up path in the immunocyte.As people pDC or mouse macrophage and fluorescence Cpn10, and when being used for that the reagent of cell is hatched together in the recognizing cells, prove that Cpn10 promptly is ingested, and be transported in the acidifying cell intracellular vesicle and do not enter kytoplasm or mitochondrion interior (Figure 36 C).These data show Cpn10 albumen and immune competent cell are united, and concentrate on the zone that signal shows the existence of TLR activation motif.

The compositions that embodiment 16-is used for the treatment of

According to the enforcement that this paper provided best mode of the present invention, summarized concrete preferred composition below.Below example only as the illustrative embodiment of compositions, and the scope that does not limit the present invention in any way.

Embodiment 16 (a)-the be used for compositions of parenteral

The compositions that is used for intramuscular injection can be formed into and contain aseptic buffered water of 1ml and 1mg suitable combination thing.

Equally, the compositions that is used for intravenous infusion can comprise aseptic Ringer's mixture of 250ml and 5mg suitable combination thing.

Embodiment 16 (b)-injectable parenteral compositions

Be suitable for being prepared in propylene glycol that the compositions of drug administration by injection can be by being blended in 1wt% suitable combination thing 10vol% and the water.This solution passes through filtration sterilization.

Embodiment 16 (c)-capsule composition

The compositions of the suitable combination thing of capsule form can be prepared by inserting to the two-piece type hard gel capsule of standard with the 50mg medicament of powder type or chemical compound, 100mg lactose, 35mg Talcum and 10mg magnesium stearate.

Embodiment 16 (d)-eye drop composition

The exemplary composition of sending as eye drop is summarized as follows:

Suitable compound 0.3g

Methyl hydroxybenzoate 0.005g

Nipasol 0.06g

Pure water is to the 100.00ml.

Methyl hydroxybenzoate and nipasol are dissolved under 75 ℃ in the 70ml pure water, the solution that cooling generates.Add suitable compound then, by membrane filter (0.22 μ m aperture) after filtration to this solution sterilization, and in the sterile chamber of packing under the aseptic condition.

Embodiment 16 (e)-the be used for compositions of inhalation

For capacity is the aerosol container of 20-30ml: the mixture of 10mg suitable combination thing and 0.5-0.8wt% lubricant (as polysorbate85 or oleic acid) is dispersed in the propellant (as freon), and packs into and be used for the suitable aerosol container of intranasal or per os inhalation.

Embodiment 16 (f)-ointment compositions

The exemplary composition of sending as ointment comprises 1.0g suitable combination thing, and paraffinum molle alba disperses to produce slick homogenizing product to 100.0g.

Sequence table

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＜120〉Chaperonin 10-induced immunomodulating

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Claims (38)

1. regulate the method that Toll sample receptor signal conducts in experimenter or its at least a cell, tissue or the organ for one kind, wherein said method comprises the cpn10 that gives effective dose, combining between the Toll-sample receptor during wherein Toll sample receptor signal conduction relates to described cpn10 and activates bunch.

2. regulate the method that Toll sample receptor signal conducts in experimenter or its at least a cell, tissue or the organ for one kind, wherein said method comprises the antagonist of at least a cpn10 that gives effective dose, combining between the Toll-sample receptor during wherein Toll sample receptor signal conduction relates to described cpn10 and activates bunch, and wherein said antagonist stops the Toll sample receptor in cpn10 and the activation bunch to combine, and/or stops activation bunch to cause that signal conducts.

3. according to claim 1 or the described method of claim 2, wherein said activation bunch comprises cpn10, Toll sample receptor and randomly at least a other molecules.

4. method according to claim 3, wherein said at least a other molecules comprise Toll-sample receptor stimulating agent.

5. according to any described method in the claim 1 to 4, wherein said activation bunch comprises cpn10, TLR2 and fat techoic acid (LTA).

6. according to any described method in the claim 1 to 4, wherein said activation bunch comprises cpn10, TLR3 and double-stranded RNA.

7. according to any described method in the claim 1 to 4, wherein said activation bunch comprises cpn10, TLR4 and LPS.

8. according to any described method in the claim 1 to 4, wherein said activation bunch comprises cpn10, TLR7 and single stranded RNA.

9. according to any described method in the claim 1 to 4, wherein said activation bunch comprises cpn10, TLR9 and comprises the DNA of CpG motif.

10. according to any described method in the claim 1 to 9, wherein said cpn10 comprises the peptide sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQ ID NO:3.

11. according to any described method in claim 1 or 3 to 9, wherein said cpn10 is with the form administration of the polynucleotide of coding cpn10.

12. method according to claim 11, wherein said polynucleotide comprise the sequence shown in the SEQID NO:4.

13. according to any described method in the claim 1 to 12, it further comprises and gives at least a other medicament.

14. method according to claim 13, wherein said at least a other medicament is the immunomodulator that is selected from IFN α or IFN β.

15. method for the treatment of or preventing experimenter's disease or disease, wherein said method comprises the cpn10 that gives experimenter's effective dose, Toll-sample receptor in wherein said cpn10 and the activation bunch combines, and the formation of wherein activation bunch with to the startup of the immunne response of described disease or disease, strengthen and/or keep relevant.

16. method for the treatment of or preventing experimenter's disease or disease, wherein said method comprises the antagonist of at least a cpn10 that gives experimenter's effective dose, Toll sample receptor during wherein said antagonist stops cpn10 and activates bunch combines, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and described disease or disease and/or make progress relevant.

17. according to claim 15 or the described method of claim 16, wherein said disease or disease are selected from virus or bacterial infection, acute or chronic inflammatory disease, comprise septic shock, inflammatory bowel, arthritis, psoriasis, heart disease, atherosclerosis, chronic lung disease, cachexia, multiple sclerosis, GVHD and cancer.

18. according to any described method in the claim 15 to 17, wherein said cpn10 is regulated the inductive TLR2 signal conduction of LTA-.

19. method according to claim 18, wherein said cpn10 is regulated the generation and/or the secretion of the inductive immunomodulator of TLR2-.

20. according to any described method in the claim 15 to 17, wherein said cpn10 is regulated double-stranded RNA-inductive TLR3 signal conduction.

21. method according to claim 20, wherein said cpn10 is regulated the generation and/or the secretion of the inductive immunomodulator of TLR3-.

22. according to any described method in the claim 15 to 17, wherein said cpn10 is regulated the inductive TLR4 signal conduction of LPS-.

23. method according to claim 22, wherein said cpn10 is regulated the generation and/or the secretion of the inductive immunomodulator of TLR4-.

24. according to any described method in the claim 15 to 17, wherein said cpn10 is regulated viral single stranded RNA-inductive TLR7 signal conduction.

25. method according to claim 24, wherein said cpn10 is regulated the generation and/or the secretion of the inductive immunomodulator of TLR7-.

26. according to any described method in the claim 15 to 17, wherein said cpn10 is regulated the inductive TLR9 signal conduction of CpG motif.

27. method according to claim 26, wherein said cpn10 is regulated the generation and/or the secretion of the inductive immunomodulator of TLR9-.

28. according to any described method in the claim 15 to 27, it further comprises and gives at least a other medicament.

29. method according to claim 28, wherein said at least a other medicament is the immunomodulator that is selected from IFN α or IFN β.

30. one kind is used for the treatment of or the compositions of prevent disease or disease, described compositions comprises cpn10 and at least a pharmaceutically acceptable carrier, diluent or adjuvant, Toll sample receptor in wherein said cpn10 and the activation bunch combines, and the formation of wherein activation bunch with to the startup of the immunne response of described disease or disease, strengthen and/or keep relevant.

31. compositions according to claim 30, it further comprises the agonist and/or at least a immunomodulator of at least a Toll sample receptor.

32. one kind is used for the treatment of or the compositions of prevent disease or disease, described compositions comprises the antagonist of at least a cpn10, Toll sample receptor during wherein said antagonist stops cpn10 and activates bunch combines, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and described disease or disease and/or make progress relevant.

33. cpn10 manufacturing be used for the treatment of or the medicine of prevent disease or disease in application, Toll sample receptor in wherein said cpn10 and the activation bunch combines, and the formation of wherein activation bunch with to the startup of the immunne response of described disease or disease, strengthen and/or keep relevant.

34. the antagonist of cpn10 manufacturing be used for the treatment of or the medicine of prevent disease or disease in application, Toll sample receptor during wherein said antagonist stops cpn10 and activates bunch combines, and/or stop activation bunch to cause the signal conduction, and the generation of the formation of wherein activation bunch and described disease or disease and/or make progress relevant.

A 35. generation and/or excretory method of regulating one or more immunomodulators in experimenter or its at least a cell, tissue or the organ, wherein said method comprises the cpn10 that gives effective dose, wherein said cpn10 with the activation bunch in Toll sample receptor combine, and wherein the activation bunch formation relevant with generation and/or excretory adjusting to one or more immunomodulators.

A 36. generation and/or excretory method of regulating one or more immunomodulators in experimenter or its at least a cell, tissue or the organ, wherein said method comprises the antagonist of the cpn10 that gives effective dose, Toll-sample receptor during wherein said antagonist stops cpn10 and activates bunch combines, and/or stop activation bunch to cause the signal conduction, and wherein the formation of activation bunch is relevant with generation and/or excretory adjusting to one or more immunomodulators.

37. according to claim 35 or the described method of claim 36, wherein said immunomodulator is selected from TNF-α, IL-1 β, IL-6, IL-10, IL-12 or I type interferon.

38. according to the described method of claim 37, wherein said I type interferon is IFN α or IFN β.

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