CN1809379A - Disease prevention and vaccination prior to thymic reactivations - Google Patents

Abstract

The present disclosure provides methods for preventing or treating illness, improving responsiveness to immunization, and improving the efficacy of gene therapy in a patient, by disrupting sex steroid mediated signaling and reactivating the patient's thymus. In some embodiments, the patient's thymus is reactivated by interruption or ablation of sex steroid mediated signaling by the administration of LHRH agonists, LHRH antagonists, anti-LHRH receptor antibodies, anti-LHRH vaccines, anti-androgens, anti-estrogens, selective estrogen receptor modulators (SERMs), selective androgen receptor modulators (SARMs), selective progesterone response modulators (SPRMs), ERDs, aromatase inhibitors, or various combinations thereof.

Description

Disease prevention and vaccination prior to the thymus reactivate

Technical field

The present invention relates to cellular immunology, disease prevention, vaccination and field of gene.More specifically, the present invention relates to strengthen bone marrow (BM) hemopoietic and functional, strengthen BM after the hematopoietic stem cell transplantation (HSCT) and implant (engraftment) and strengthen the functional of new and existing T cell and immune other cells.

Background technology

Immune system

Immune major function is to distinguish " external " (promptly derive from health beyond any source) antigen and " self " (promptly deriving from the health) antigen, and replys in view of the above with the protection body resistance and infect.More practical saying is that immunne response also has been described to replying danger signal.These danger signal can be any variations of cell or tissue performance, and they remind this problematic cell of immune cell no longer is " normally ".Such prompting is very important on causing for example to the repulsion of foeign element as virus, antibacterial, parasite and fungal infection, and they also can be used to inducing antitumor and reply.But, such danger signal also can be the reasons that starts autoimmune disease, because self intracellular arbitrary unsuitable cell change all makes self cell become the cell (for example β islet cells of the pancreas in the diabetes) of immune system institute targeting.Perhaps, except inductive external cell or microorganism of initially replying, also can cause infringement to the unsuitable stimulation of immunocyte itself to normal self cell.

In normal immunne response, the order of incident comprises full-time antigen-presenting cell (APC) capture antigen and they is processed into little fragments of peptides that these fragments of peptides are positioned at the crack (cleft) of main histocompatibility complex (MHC) molecule on APC surface.The MHC molecule can be in I type expressed on all nucleated cell ((Tc or CTL) discerns by cytotoxic T cell) or main II type (helper T cell (Th) is discerned) expressed on immune cell.MHC II/ peptide complexes on the cell recognition APC is also replied.The factor that these cells discharged has promoted to be specific to special antigenic Tc cell or has produced one of the B cell of antibody or both activation then.In fact, HIV/AIDS has illustrated the importance of Th cell in all immunne response best, because caused serious immunodeficiency through the shortage of the caused Th cell of virus damage, finally causes opportunistic infection and death.(and on lower degree, unsuitable growth Tc) also can cause various pathological changes for example allergy, cancer and autoimmune to Th.

The inappropriate growth of these cells can be because unusual thymus, wherein the structure of thymus composition has been changed significantly, for example in many autoimmune diseasees, the required medullary epithelial cell of ripe thymic cell development by ectopic expression in the cortex that immature T cell is normally at.Therefore this may mean that developmental immature T cell accepted the ripe signal in late period and become to select signal insensitive to feminine gender prematurely, and the negative signal of selecting normally will be removed potential id reaction sexual cell.(Takeoka et al. in the NZB mice that the lupoid acne symptom takes place, (1999) Clin.Immunol.90:388) and more nearest in the NOD mice that type i diabetes takes place (Thomas-Vaslin et al., (1997) P.N.A.S.USA 94:4598; Atlan-Gepner et al., (1999) Autoimmunity 3:249-260) all found such thymus abnormalities really.How or when, the thymus abnormalities of also not knowing these forms takes place, but the aging course that their generation can be by nature or because monkey wrench for example viral infection (in AIDS patient, having described the variation of thymus), stress, chemotherapy and radiotherapy (Mackall et al., (1995) N.Eng.J.Med.332:143; Heitger et al., (1997) Blood 99:4053; Mackall andGress, (1997) Immunol.Rev.160:91).It also is possible having defective during birth.

The cell-membrane receptor of T and bone-marrow-derived lymphocyte has the antigenic ability of identification.These receptors are that the multiple rearrangement that may gene by a series of complexity generates at random, and each individual T or B cell all have unique antigen receptor like this.This huge potential multiformity means the single antigen that may run into for any body, and a plurality of lymphocytes can be with in various degree bond strength (affinity) with its identification and cause replying in various degree.Since the specificity of antigen receptor occurs at random, the problem that is occurred is why body can autoclasia not take place by the lymphocyte of anti-autoantigen so.Fortunately, there are some mechanism prevention T and B cell to carry out such autoclasia.Generally speaking, these mechanism have produced the state of a kind of immune system to self tolerance.

The effective form of self tolerance is in the position that produces potential reactive lymphocyte they physically to be removed all or kill.These positions comprise thymus that produces the T cell and the BM that produces the B cell.This is called maincenter tolerance (central tolerance).A kind of important, additional tolerance method is by regulating the Th cell, it or directly or by producing cytokine suppress the id reaction sexual cell.Suppose that all immunne response in fact all need the startup and the adjusting of t helper cell, a main target of any tolerance-induced scheme is exactly that targeting is in these t helper cells.Similarly, since the Tc cell is very important effector lymphocyte, their product is exactly a main target of for example anticancer disease and antiviral therapy strategy so.In addition, T regulate cell (Treg) for example CD4+CD25+ and NKT cell a kind of instrument is provided because they can suppress potential id reaction sexual cell.

Thymus

Thymus mainly comprises the developmental thymocyte cell (intrathymic T lymphocyte) that is dispersed in the different Interstitial cells (mainly being the epithelial cell subgroup), and these Interstitial cells constitute microenvironment and provide the optimization of T cell to grow necessary somatomedin (GF) and cell interaction.

Thymus is vitals in the immune system, because it is to produce the lymphocytic main position of T.The effect of thymus is the precursor (precursor cells) that is attracted to suitable BM source from blood as described below, and induces them to be fixed to T cell line, comprises that generation is at the necessary gene rearrangement of antigenic TXi Baoshouti (TCR).Each T cell all has single TCR type and is unique on its specificity.Generating relevant with this TCR is cell division, its increased T cell number with TCR type and so increased every kind of probability that exotic antigen all will be identified and remove.But different with the B cell, the antigenic unique characteristics of T cell recognition are that TCR only discerns the fragments of peptides that links to each other with the MHC molecular physics.Normally, this is self MHC, and selects the ability or the TCR of identification self MHC/ peptide complexes in thymus.This process is called as the forward selection and is the epithelial exclusiveness feature of a kind of cortex.If TCR can not combine with self MHC/ peptide complexes, the T cell will be died from " ignoring ", because the T cell is for its lasting survival and the ripe signal transduction through TCR that needs to a certain degree.

Since the result that tcr gene is reset is a random event, some T cells will be grown by accident for discerning the T cell of self MHC/ peptide complexes with high-affinity so.Therefore, these T cells are potential autoreactivities, and relate to autoimmune disease, for example multiple sclerosis (MS), rheumatoid arthritis (RA), diabetes, thyroiditis and systemic lupus erythematosus (sle) (SLE).Fortunately, if the affinity of TCR and self MHC/ peptide complexes is too high and the T cell has run into this specific complex in thymus, so developmental thymocyte cell is induced to the activation of suicide property and is died from apoptosis, and this process is called negative the selection.This process also is called the maincenter tolerance.The T cell death that these " have high-affinity to self " rather than reply is because they are still jejune in thymus.The strongest inducer of this negative selection is the APC that is called dendritic cell (DC) in the thymus.DC passes to the T cell with the most intensive signal, has caused intrathymic removal.But in peripheral lymphoid organs, the T cell is more sophisticated, and the DC that presents identical MHC/ peptide complexes for identical TCR will cause the activation of the T cell with TCR.

Atrophy of thymus gland and aging

Though thymus is important for the functional immunity system, discharge its every day approximately it the T cell content 1% in blood flow, but mammal and other animals in one obviously be the serious atrophy that sex steroid (sex steroid) produces this organ that is caused unusually.This atrophy progressively takes place in the year at about 5-7, about 20 years old, reach T cell output (the Douek et al. of floor level, Nature (1998) 396:690-695), this atrophy is different with the inductive reversibility atrophy of institute during to the stress response of glucocorticoid.Structurally, atrophy of thymus gland comprises the increase of the subsiding of carrying out property disappearance, cortex epithelium network, cell epimatrix material of lymphocyte content and adipose cell (lipocyte) and the lipid deposition thing infiltration (Haynes et al., (1999) J.Clin.Invest.103:453) to body of gland.This process can start from young child (for example about 5 years old age, Mackallet al., (1995) N.Eng.J.Med.332:143), but it is the most tangible when the sex steroid level reaches the adolescence of summit.

Can reflect the influence of atrophy of thymus gland in periphery, it has reduced the output of thymus to the T cell pool, and TCR storehouse (because having only the new cell of T originally just can provide) of less variation has been provided for this.Altered cytokine value (Hobbs et al., (1993) J.Immunol.150:3602 have also been observed; Kurashimaet al., (1995) variation of CD4+ and CD8+ subgroup Int.Immunol.7:97),, to the decline of the responsibility that stimulates with the bias (Mackall et al., (1995) N.Engl.J.Med.332:143) of the memory T cell that originally the T cell is opposite and to antigen or mitogenesis.

Since thymus is the generation in periphery T cell pond and the main position of keeping, it is the main cause that old people's the sickness rate based on the disease of immunity increases that this atrophy is regarded as widely.Especially; the immunodeficiency that pathological changes is for example general, to the weak response of opportunistic infection and vaccine and the increase of autoimmune disease frequency; as multiple sclerosis, rheumatoid arthritis and lupus (Doria et al., (1997) Mech.Age.Dev.95:131-142; Wey and et al., (1998) Mech.Age.Dev.102:131-147; Castle, (2000) Clin Infect Dis 31 (2): 578-585; Murasko et al., (2002) Exp.Gerontol.37:427-439) their sickness rate and seriousness have all been increased along with the age.Often the attenuating by T cell dependent immunity function (for example cytolytic T lymphocyte is active and mitogenetic replying) illustrates these immune defectives.Though homeostatic mechanism has kept the T cell number of healthy individual, when for example behind AIDS and chemotherapy or radiotherapy, a large amount of forfeiture of T cell being arranged, adult patient height susceptible is in opportunistic infection, because these all pathological changes all comprise the disappearance of T cell and/or other hemocytees (as follows).Lymphocytic recovery also is serious the hysteresis.The thymus of atrophy can not be reconstituted in the CD4+T cell (Douek et al.Nature (1998) 396:690-695) that is lacked between the HIV infection period, and with the preadolescence patient relatively, the CD4+T cell after the chemotherapy will spend 3 to 4 times of longer times just can return to normal level (Mackall et al. (1995) N.Engl.J.Med.332:143-149) after adolescence among the patient.Therefore, these patients lack to reply and infect necessary cell, and they serious immunosuppressant (Mackall et al., (1995) N.Eng.J.Med.332:143 occur; Heitger etal., (2002) Blood 99:4053).Increase (Hirokawa, (1998) " Immunity and Ageing, " in that cancer and tumor load are also arranged in later stage of life PRINCIPLES AND PRACTICE OF GERIATRIC MEDICINE, (M.Pathy, ed.) John Wiley andSons Ltd; Doria et al., (1997) Mech.Age.Dev.95:131; Castle, (2000) Clin.Infect.Dis.31:578).

But, nearest work ((1998) Nature 396:690) such as Douek has shown the generation that still has thymus output in old people's (for example even after over-65s and old HIV patient's the antiretroviral therapy), although have only very small amount of output (be about adolescence's level 5%).Have TXi Baoshouti deletion ring (T Cell Receptor Excision Circles, TREC; TREC forms as the part at the production process of antigenic TCR, and only is found on the T cell of new generation) the existence of T cell for example understand this point.In addition, Timm and Thoman ((1999) J.Immunol.162:711) are although shown the aged mouse CD4+T cell of having regenerated after bone marrow transplantation (BMT), but because old and feeble periphery microenvironment, these T cells have demonstrated to the bias of memory cell and with the generation of T cell originally of weak thymus.Also by analysis the TREC level after the hematopoietic stem cell transplantation (Douek et al., (2000) Lancet355:1875).

Thymus and neuroendocrine axis

Thymus is subjected to the influence (Kendall of the two-way exchange of itself and neuroendocrine system to a great extent, (1988) " Anatomical and physiological factors influencing the thymicmicroenvironment; " in THYMUS UPDATEI, Vol.1. (M.D.Kendall, and M.A.Ritter, eds.) Harwood Academic Publishers, p.27).Hypophysis particularly importantly, between adrenal gland and the gonad to the interaction of thymus function, comprise Nutrition (trophic effects) (thyrotropin or TSH and growth hormone or GH) and atrophy effect (atrophic effects) (lutropin or LH, follotropin or FSH, with thyroliberin or ACTH) (Kendall, (1988) " Anatomical and physiological factors influencingthe thymic microenvironment; " in THYMUS UPDATEI, Vol.1. (M.D.Kendall, and M.A.Ritter, eds.) Harwood Academic Publishers, p.27; Homo-Delarche et al., (1993) Springer Sem.Immunopathol.14:221).In fact, the physiological characteristic feature of thymus is the sexual involution that carries out of 26S Proteasome Structure and Function, the increase that circulation sex steroid when this and adolescence produces is suitable, (Hirokawa and Makinodan, (1975) J.Immunol.114:1659 that this normally progressively takes place before age 12-14 year the mankind; Tosi et al., (1982) Clin.Exp.Immunol.47:497; And Hirokawa, etal., (1994) Immunol.Lett.40:269).

Thymus mainly comprises the developmental thymocyte cell that is scattered in the different Interstitial cells (being mainly the epithelial cell subgroup), and these Interstitial cells have constituted microenvironment and provide the optimization of T cell to grow necessary somatomedin and cell interaction.Determined the mechanism that the accurate target spot of hormone and they are induced atrophy of thymus gland and are improved immunne response.Yet, the inspection of testicular feminization mutant mice shown to express on the Interstitial cell of thymus just atrophy can take place when functional sex steroid receptor is arranged.Common growth relation (Boyd et al. between thymocyte cell and their differentiation of control and the sophisticated epithelial cell subgroup, (1993) Immunol.Today 14:445) mean that sex steroid suppresses to occur in the level of any cell type, this will influence the state of other cell types then.In the gerontal patient, the number of bone marrow stem cell is reduced and is being different qualitatively.HSC can go into nest thymus again, although be with the degree lower than young patient.Therefore, the principal element that influences atrophy of thymus gland thymus intrinsic factor seemingly.In addition, the thymocyte cell in the aged animal (for example＞18 month animal) remains with to a certain degree differentiation capability (George andRitter, (1996) Immunol.Today 17:267 at least; Hirokawa et al., (1994) ImmunologyLetters 40:269; Mackallet al., (1998) Eur.J.Ilnmunol.28:1886).Yet, the nearest work of Aspinall has been presented in the old mice, the defective that has thymocyte cell to generate, and it shows as the blocking-up in the three negative precursor groups, be CD44+CD25+ (TN2) phase (Aspinall et al., (1997) J.Immunol.158:3037).

In the special circumstances of AIDS, immune major defect is the destruction of less degree of the myeloid cell of the destruction of CD4+ cell and macrophage and dendritic cell (DC).Do not have the immune system of these cells to benumb, the patient very easily feels in opportunistic infection, and death is common result.Existing treatment to AIDS is to kill or remove HIV virus according to multiple antiviral drugs.These treatments become more and more effective now, and they are reduced to the point that the patient can be considered to be in alleviation significantly with viral load.Yet the subject matter of immunodeficiency still exists, because still have only considerably less functional T cell, they are recovering but recover very slowly really.Therefore, the time period of immunodeficiency is still very long, and in some case, immune defence mechanism may never have effectively and recovered.

Hemoposieis

Bone marrow and hematopoietic stem cell

Immune main cell is B and T lymphocyte (a kind of leukocytic main type), and antigen-presenting cell (APC).All immunocytes all derive from hematopoietic stem cell and their filial generation (common lymph CFU-GM (CLP) and common medullary system CFU-GM (CMP), the both generates) basically in BM.Some precursors are migrated thymus and are converted into the T cell and thymus DC.DC acts on inducing bringing into play on the self tolerance.

Have only the HSC of ratio seldom (being nearly 0.01% in young animal) to be released from BM and find the approach that arrives thymus through the blood supply, here they divide and are ripe to form the T cell, turn back to then in the body circulation.These new thymus recently (RTE) cell of moving out has formed immune major part, and they mainly be Th with the Tc cell and in the constant supply that keeps the new T cell with different TCR storehouses are important to start on nearly all immunne response.Before leaving thymus, Th and Tc cell can be distinguished exotic antigen effectively, and reason as mentioned above.The T cell by " selections ", makes that staying those is identified as the T cell that also under normal circumstances can not replying of self resisted self cell with the intravital all cells of machine in thymus.

The B cell finally also derives from HSC, and grows in BM before entering the periphery immune system leaving BM.With T cell and immune other cell interaction after, the B cell development become to produce and discharges the plasma cell of lot of antibodies, these antibody help body to eliminate infectious organism and abnormal cell.

Other HSC that BM produced also is used to generate other all hemocytees for example cell, neutrophilic granulocyte, basophilic granulocyte and eosinophilic granulocyte, dendritic cell, mononuclear cell, macrophage, hemocyte and the erythrocyte in NK cell, regulating cell, common medullary system CFU-GM source.

Hematopoietic stem cell transplantation

Usually the hematopoietic stem cell transplantation (HSCT) that is also referred to as bone marrow transplantation (BMT) is a kind of for example treatment of the immune recovery of some serious cancerous lesion that is used for strengthening.Can exchange use " HSCT " and " BMT " and " transplanting ", they are defined as being transplanted at this divides graft in receptor, its contain or enrichment HSC, BM cell, stem cell and/or any other the cell that can generate blood, thymus, BM and/or any other immunocyte, include but not limited to the interstital stem cell of HSC, epithelial cell, common lymph CFU-GM (CLP), common marrow lymph CFU-GM (CMLP), multi-lineage progenitor cells (MLP) and/or bone marrow.In some embodiments, transplanting can be autologous peripheral blood stemcell transplant (PBSCT).Can mobilize out the HSC of BM, from blood, collect then, or HSC is contained in the BM that physically extracts from donor.HSC can be purified, concentrate, or is the part of collected BM or blood, and HSC is injected in the receptor then.Transplanting can be heterogenic, from body, isogenic or xenogeneic, and the transplanting that can comprise the cell of any number comprises " little transplanting (mini-transplants) ", it includes only fewer purpose cell.In some embodiments, before sex steroid suppresses, with it simultaneously or carry out HSCT afterwards.

HSC is nonrestrictive cell type of demonstrating, and it can be transplanted and/or by genetic modification, as used in full at the application's book.Yet, those skilled in the art are readily understood that when putting into practice in the invention that this provided, do not need undue experimentation, HSC just can be substituted by any (or multiple) cell type in many alternative cell types, these cells comprise, but be not limited to BM cell, stem cell and/or other any cells that can generate blood, thymus, BM and/or any other immunocyte, these cells include but not limited to the interstital stem cell of HSC, epithelial stem cell, CLP, CMLP, MLP and/or bone marrow.In some embodiments, HSC derives from tire liver and/or tire spleen.

Chemotherapy and radiotherapy can both be eliminated cancerous cell.But in default of specificity, healthy cell has also been killed in these treatments, in fact comprises the HSC among all leukocyte (WBC) and the BM.Caused the demand of patient because of this destruction exactly to HSCT to WBC and HSC.HSCT allows that for example healthy stem cell substitutes by for example chemotherapy or stem cell that radiotherapy damaged and their daughter cell, and healthy stem cell finally can generate the required hemocyte of patient.

HSCT is for example leukemia and lymphoma (cancer of blood and immune system cell) and for example serious combined immunodeficiency of non-pernicious immunological diseases of multiple neoplastic hematologic disorder, the Fanconi anemia, myelodysplastic syndrome, amyloidosis, aplastic anemia, Diamond Blackfan anemia, bite hemocyte lymphohistocysis disease, the Kostmann syndrome, the Wiskott-Aldrich syndrome, thrombocytopenia and hemoglobinopathy such as drepanocytosis and thalassemic base therapy.Usually exhaust treatment leukemia and lymphoma to remove the cancerous cell body with bone marrow removing or bone marrow, usually back to back is that HSCT is to recover immunologic function.Method of the present invention separately or associating (simultaneously or successively) grant HSC mobilization agent for example cytokine (as G-CSF or GM-CSF) or medicine, allow faster and better implant, also can allow to give more heavy dose of and/or more frequent chemotherapy and radiotherapy.

The transplanting (allogene HSCT) of the HSC that derives from another donor blood is often adopted in the current clinical medicine operation, the advantage that it had is the effect (graft leukemia (GVL)) of the anti-host's cancerous cell of donor T cell, but this advantage is restricted by other the anti-host's of T donor T cell effect (graft-versus-host (GVH) disease) usually, and the latter can be fatal.Since the success of HSCT and patient's in view of the above survival rate is all directly related with the time of the number of the HSC that is injected and implantation, use method of the present invention to mean that existing HSCT scheme will be more successful and will have than existing possible more patient and can accept HSCT.

Enhancing mobilizes out the mechanism of HSC for guaranteeing that it is important collecting HSC as much as possible from donor from bone marrow.GM-CSF and G-CSF have been used to this purpose at present.But other medicine for example chemotherapy and cytokine also to have demonstrated be effective.More effectively mobilize the ability of HSC to have the application that exceeds hematology's reparation.It is polyenergic and the reparation that can be used to damaged tissues that research has recently demonstrated HSC, for example cardiac muscle, skeletal muscle, liver, skeleton, connective tissue, epithelial tissue, pancreas and vascular system.

A restriction of existing HSCT strategy is that the infection that is caused with long-time immunodeficiency is relevant, particularly virus, fungus and the infection of capsular bacterium is arranged, and this is still the major reason of adult's transplanting sequela rate and mortality rate.The infection relevant with HSCT normally is very difficult to control, even with modern antimicrobial agents.The child recovers immunocompetence (Parkman et al. usually in the several months behind HSCT, (1997) Immunol.Rev.157:73), this delay with adult receptor's lymphocyte recovery has different, and this delay can last for several years and even can be the reflection of the non-constant of young Optimum (young optimum).Adult's this delay depends on multiple factor, still mainly is because T that generally acknowledges and B cell produce the degeneration (Parkman et al., (1997) Immunol.Rev.157:73) with the age to the susceptibility that infects.

In addition, the speed that graft is implanted is also being brought into play and is being acted on, and wherein the speed of graft implantation is slow more, and the probability that opportunistic infection take place is just big more.

When the cell of being transplanted " repulsion " donor's cells, second restriction of existing HSCT appearred.This is called " graft versus host disease " (GVHD) clinically.Autotransplantation can be avoided GVHD.But, comparing with allotransplantation, autoplastic total anticancer success rate is lower.In the cancer patient, the shortcoming that autotransplantation has is that they can not produce Graft Versus Tumor (GVT) effect (this is similar to the GVH effect), and has cancerous cell and may get back to danger among the patient with transplanting.Have been found that sex steroid in mice allogene HSCT model suppresses and the castrating receptor of allogene HSCT rebuilds after having improved the transplanting that medullary system and lymph two are cell.The remarkable increase that data show T shown here and B cell are rebuild, and do not have deterioration and the active disappearance of GVT (seeing for example embodiment 19) of GVHD.

Another restriction of HSCT treatment is the donor that lacks all potential candidates of treatment.Although Cord blood (UCB) has been utilized quite limited degree, each donor all has only cell seldom, so this mainly has been used to the child, total less (number of required HSC is relevant with weight in patients) of wherein required HSC.Except UCB, the supply of donor is limited and must be acceptable MHC coupling, otherwise the danger of GVH is just high.If only need less cell, because the implantation that improves or need more undemanding coupling, therefore just reduced and repelled or the danger of GVH, HSCT may can more extensively for example be used for the treatment of autoimmune disease with being used, and can utilize for example confession source (for example, 1.5 * 10 of umbilical blood ⁷Cell/kg is used to implant the receptor).

The T cell

The T cell is immune main component, and it generates in thymus.Most important T cell is the Th cell, because they in fact are the cells that starts all immunne response.The disappearance of these Th cells (for example HIV infection, chemotherapy, irradiation etc. are caused) has directly caused immunosuppressant and to infecting and the inevitable susceptibility of tumor, and death takes place apace.An important function of Th cell subgroup is to regulate immunne response.To the enhancing of T and B cell function and the balance between suppressing to vaccine for example whether effectively, whether whether cancer or tumor attacked or transplanted to be tolerated or repel all plays an important role.

Thymus is very effective at an early age, along with the age engenders the degeneration that size and function are exported.This is outstanding especially when pubarche.Because thymus output directly related (Scollay et al., (1980) Eur.J.Immunol.10:210 with the cell density (cellularity) of thymus; Berzinset al., (1998) J.Exp.Med.187:1839), atrophy of thymus gland that the age is relevant has caused move out (Steffens et al., (2000) Clin.Immunol.97:95 of gradually reducing of (RTEs) of thymus recently; Sempowski et al., (2002) Mol.Inzmunol.38:841-848; Sutherland et al., (submitted)) and originally to the attenuating (Ernst et al., (1990) J.Inarnunol.145:1295) of memory T cell ratio; Kurashima et al., (1995) Int.hrznmunol.7:97; Utsuyamaet al., (1992) Mech.Ageing Dev.63:57), this has caused TCR storehouse (Mosley et al., (1998) Cell.Immunol.189:10 of limited CD4+ and CD8+T cell; LeMaoult et al., (2000) J.Immunol.165:2367).So the T cell proliferation that responds to non-specific and receptor-mediated (CD3/TCR) stimulation is along with the age is wretched insufficiency (Hertogh-Huijbregts et al., (1990) Mech.Ageing Dev.53:141-155; Flurkeyet al., (1992) J.Gerontol.47:B115; Kirschmann et al., (1992) Cell.Immunol.139:426).

Along with the increase at age, human immunologic function is progressively degenerated; Replying of child is very good, and younger adult replys suitably, but elderly adult's this replying may non-constant.This degradation table is understood in all are replied the disappearance of one or more cell type of actual three kinds of main cell types that participated in or the existence of change: (i) antigen-presenting cell (their capture antigens are also presented it, and activate the T lymphocyte in view of the above); (ii) T lymphocyte; (iii) bone-marrow-derived lymphocyte.What the disappearance of any cell type of these three kinds of cell types or change can be interpreted as may be unsatisfied to the immunne response of antigenic stimulus.Disappearance or to change can be maybe can refer to quantity or functional at cellular level.Wherein, most probable disappearance is to be contained in the T cell mass, because the thymus function that influence caused of sex steroid is with the remarkable degeneration at age.This has caused outputing to the disappearance new or " originally " T cell in the blood flow, and this is necessary to replying of neoantigen.Reply the T cell number disappearance except potential, the existence of sex steroid may be suppressed to certain degree with having the T cell.

Treatment of cancer

As mentioned above, the chemotherapy and radiation that is used for the treatment of cancer often is deleterious for patient's non-cancerous cell, particularly to hemocyte.The major limitation that increases the frequency of these treatments and dosage is patient's ability of surviving in treatment and avoids that low immune system caused to the ability of the susceptibility of opportunistic infection.Therefore, if immune restoration can be faster or damage lighter, this will be very favourable to the patient.

Summary of the invention

The present invention relates to by strengthen the BM hemoposieis and functional, strengthen that BM behind the HSCT implants and increase the functional of existing T cell and other immunocytes through the signal pathway of barrier steroid and other hormones, be used for prevent disease or help the method for patient's rehabilitation.The bone-marrow-derived lymphocyte that is generated by cell of T originally that thymus function generated and the activated BM function that increases through new life and the level of immune other cells are also with the enhance immunity ability.

Have been found that the interruption to the signal pathway of sex steroid and/or other hormones has strengthened the functional of BM, HSC, T cell and immune other cells by direct effect or by indirect action.This finds to be developed to have formed the present invention, and enhancing BM hemoposieis and/or functional method are provided in one aspect of the invention.In some embodiments, improved existing BM hemoposieis and/or functional.In another embodiment, the patient accepts HSCT, and has improved patient's HSC hemoposieis and/or implantation.

In one aspect, the invention provides the method that strengthens the implantation behind the HSCT.In one embodiment, strengthened the implantation of BM.In another embodiment, strengthen the implantation and/or the reconstruction of thymus, finally induced the thymus recovery in view of the above.Still in another embodiment, the implantation and/or the reconstruction of spleen and/or other lymphatic organs, tissue and/or blood have been strengthened.In some embodiments, HSC is heterogenic, and in other embodiment, HSC is from body.In one embodiment, compare, increased the number of T cell precursors with the number that in the patient of the HSCT that does not carry out sex steroid signal pathway (sex steroidsignaling) interruption, has existed.In other embodiment, with the number that in the patient who does not carry out the HSCT that the sex steroid signal pathway interrupts, existed relatively, increased the DC, BM precursor, HSC, CLP, MLP, lymphocyte, myelocyte, granulocyte, neutrophilic granulocyte, macrophage, NK, NKT, platelet in total leukocyte, donor source, the number of the periphery B cell in periphery T cell, APC and/or the donor source in T cell, memory T cell, helper T cell, effector T cell, modulating T cell, RBC, B cell, donor and/or host source originally.Still in other embodiment, the patient has also accepted cytokine (for example IL-7, SCF, IL-11, G-CSF or GM-CSF) or hormone (for example arbitrary member of growth hormone or its mediators insulin-dependent somatomedin (IGF-1) or fibroblast growth family for example FGF7/ keratinocyte growth factor) treatment and has recovered with enhance immunity and/or implant after HSCT.In another embodiment, the present invention separately or unite and grant for example somatomedin (for example, G-CSF or GM-CSF) of short hemopoietic medicine, this allows faster and/or better implants and/or go into nest in target tissue and/or the recovery of enhance immunity cell.

Aspect second of the present invention, provide functional method of the immunocyte that strengthens the patient behind the HSCT.In one embodiment, immunocyte is the T cell.In another embodiment, improved the propagation responsiveness that the T cell stimulates TXi Baoshouti (TCR).In another embodiment, improved the responsiveness that the T cell stimulates antigen (for example, tetanus toxin (TT) or phytolacca american mitogen (PWM)).In one embodiment, improved the T cell to recalling (recall) antigenic responsiveness (promptly the T cell memory of Ti Gaoing is replied).Still in another embodiment, improved the propagation responsiveness that the T cell stimulates together or the secondary signal approach is relevant.In some embodiments, improved the kinetics of t cell response.In other embodiments, improved the T cell to antigenic replying that APC presented.In some embodiments of the present invention, after transplanting about 5,4,3 or 2 months in improved immune cell responses.In specific embodiment of the present invention, after transplanting, improved immune cell responses in about 1 month.In other embodiments of the present invention, after transplanting, improved immune cell responses in 2 weeks.In yet another embodiment of the present invention, after transplanting, improved immune cell responses in 1 week.Still in other embodiments of the present invention, improved immune cell responses in back 3 days in transplanting.In other embodiments, back 3 months of treatment or improved immunne response more than 3 months, comprise except exporting the T cell that new thymus source is also exported in existing T extracellular.

Aspect the 3rd, the invention provides the method that strengthens existing immune cell function, existing immunocyte includes but not limited to the immunocyte of periphery.In one embodiment, cell is the T cell.In another embodiment, cell is DC or other APC.Still in another embodiment, cell is NK cell or regulating cell, for example CD4+CD25+T cell and natural killer T (NKT) cell.In an embodiment of the invention, improved the propagation responsiveness of patient's T cell to the TCR stimulation.In another embodiment, improved the responsiveness that the T cell stimulates antigen (for example TT, PWM or KLH).Still in another embodiment, improved the T cell to being total to the propagation responsiveness of stimulation or secondary signal approach.In other embodiment, improved the responsiveness of T cell to APC institute antigen-presenting.In a special embodiment, the LHRH/GnRH analog has direct effect or indirect action to the responsiveness of existing immunocyte.In some embodiments, method usability steroid analog of the present invention (its agonist and antagonist), GnRH/LHRH analog for example is with the signal pathway of the sex steroid mediation of blocking immunity cell or bone marrow.In other embodiment, the steroid analog directly stimulates (functional activity that promptly directly increases it) thymus, BM and/or existing immune cell for example T cell, B cell and DC.

Aspect the 4th, the invention provides and strengthen the method that HSC among the patient implants and mobilizes or the HSC of blood, HSC or BM donor mobilizes.In one embodiment, the barrier steroid signal pathway immune precursor number of HSC, CD34+ cell, CLP or CMP and functional for example that increased periphery.An embodiment provides a kind of method that HSC mobilizes that is used to strengthen, comprise barrier steroid signal pathway, itself or carry out individually or unite and grant the HSC mobilization agent, for example other members or the IL-7 of cytokine, GM-CSF, G-CSF, CSF, chemotherapeutics, cyclophosphamide, flt-3 part, KGF/FGF7 or FGF family.

In one embodiment, the invention provides and allow and give high dose more and/or more frequent chemotherapy and radiotherapy and/or allow immune system faster recovery or method of being subjected to still less damaging behind chemotherapy and radiotherapy.

Aspect the 5th, the invention provides the method for prevention or treatment patient's pathological changes.A specific embodiments provides a kind of method that is used to prevent or reduce the danger of patient infection, pathological changes or disease, and this method comprises the signal pathway of the sex steroid mediation of blocking the patient.In a specific specific embodiments, blocked signal pathway to BM.In another embodiment, blocked signal pathway to thymus.Still in another embodiment, blocked signal pathway to spleen.Still in another embodiment, blocked signal pathway to the periphery immunocyte.

In some specific embodiments, method of the present invention is used to prevention or treats viral infection for example HIV, herpes, influenza and hepatitis.In other specific embodiments, method of the present invention is used to prevention or treats bacterial infection for example pneumonia and tuberculosis (TB).Still in another embodiment, method of the present invention is used to prevention or treatment fungal infection, parasitic infection, allergy and/or tumor and other cancers, no matter is pernicious or benign tumor.

In other specific embodiments, the HSC that the patient has accepted non-genetic modification transplants.In some specific embodiments, BM or HSC are transplanted among the patient so that the precursor composition to be provided, and these precursors finally can be used to the growth of newborn thymus.Among these HSC some have the ability that is converted into DC or other APC, and they may have to the T cell provides the effect of better antigen presentation and therefore have better immunne response (for example having increased Ab output and effector lymphocyte's number and/or function).In other embodiment, the sex steroid signal pathway was blocked in the process of reactivate (reactivation) when aged (after the adolescence) patient's atrophy thymus was in because of HSCT.The thymus of reactivate becomes and can absorb HSC, BM cell and other suitable precursor cells from blood, and they is converted into new T cell and DC in thymus.

Aspect the 6th, the invention provides and improve the method for patient the immunologic responsiveness of vaccine.

Aspect the 7th, flow to the patient and utilize HSC, lymph CFU-GM, medullary system CFU-GM or the epithelial stem cell of genetic modification or the gene therapy of their combination (group and each member at this refer to " GM cell "), to produce treatment or the useful specifc immunity power of prevention pathological changes.

In some embodiments of particular aspects of the present invention, pathological changes is a kind of pathological changes with clear and definite hereditary basis, for example pathological changes that genetic defect caused.Those skilled in the art know these heredopathias, and they comprise the too much of autoimmune disease, specific protein or disease, tumor and cancer that very few generation caused or the like.By in HSC, inserting normal gene and utilizing the method for invention to repair the genetic defect that causes disease, will all carry the correction of this gene from each cell that this HSC produced then.

In other embodiments, disease be a kind of be selected from by viral infection (for example HIV (human immunodeficiency virus) (HIV)), T cell function disease and directly or indirectly quantity or functional minimizing T cell or cause the T cell with to the deleterious mode effect of individuality any other disease or the T cell disease of the group formed of pathological changes.

Still in other embodiment, the invention provides by transplant by the GM cell of genetic modification with infection, the activity of opposing or prevention pathogen, duplicate or the like and their combination, to treat or to prevent for example method of the infection of HIV of pathogen, can be before the thymus reactivate or with it simultaneously to the patient infusion pathogen.In one embodiment, HSC modified with comprise that its product can disturb that HIV infects, the function of patient's T cell (with other the cell in HSC source) and/or the gene that duplicates.In an embodiment that has, with the virus resistance gene for example the RevM10 gene (see, Bonyhadi et al. for example, (1997) J.Virol.71:4707) or CXCR4 or PolyTAR gene (Strayer et al., (2002) Mol.Alter.5:33) genetic modification HSC.This has just given the resistance to virus to a certain degree, prevents or treat viral caused disease in view of the above.

In yet another aspect, the invention provides to sex steroid mediation to the blocking-up of the signal pathway of thymus and the reactivate (reactivation) of thymus subsequently.In one embodiment, the signal pathway that mediates with castrating barrier steroid.In a special embodiment, use chemical castration.In another embodiment, use operation castrating (for example by removing testis or passing through ovariectomy).In some embodiments, inhibition fully to the sex steroid signal pathway has taken place.In another embodiment, taken place the part of sex steroid signal pathway is blocked.In one embodiment, castrating reverses prepuberal state with thymic state, in view of the above with its reactivate.In another embodiment, castrating has changed the level of other molecules, and they pass through, and for example to the direct effect of existing immunocyte, has strengthened immune cell responses and/or propagation and/or the state of activation.

In specific embodiment, generate by granting gonadal hormone, effect, in conjunction with or the instrumentality of signal pathway can be directly or blocking-up is (for example indirectly, suppress, deactivation or make invalid) signal pathway of sex steroid mediation, these instrumentalities include but not limited to the medicine with gonadal hormone or its receptors bind, the agonist of gonadal hormone or antagonist include but not limited to GnRH/LHRH, estrogen antagonist agent and androgen antagonist agent, SERM, SARM, estrogen antagonist antibody, the androgen antagonist part, the estrogen antagonist part, the LHRH part, passive (antibody) or the inoculation of (antigen) anti-LHRH (or other sex steroid) initiatively, or the combination of their (" blocking-up things ").In one embodiment, use one or more blocking-up thing.In some embodiments, grant one or more blocking-up thing by the peptide extended release preparation.The example of peptide extended release preparation is provided in WO 98/08533, its full content together has been incorporated herein by reference at this.

Description of drawings

Figure 1A-B: castrate the thymocyte cell density of having regenerated apace.Figure 1A-B shows before the castrating and the diagram of the variation of thymic weight afterwards and thymocyte cell number statement.As the cell number (Figure 1B) of thymic weight (Figure 1A) or each thymus was measured, atrophy of thymus gland had caused the remarkable minimizing of thymocyte cell number with the age.For these research, the castrating of being performed the operation of old (promptly 2 years old is big) mice.After the big castrating of old (1 and 2 years old) and 2-4 week, analyze the dependency (Figure 1A) and the thymocyte cell density (Figure 1B) of thymic weight and body weight in (post-cx) male mice.Relatively see that thymic weight and cell density are with old remarkable attenuating with young adult (2 months big) mice.The attenuating that castrating has recovered this thymic weight and cell number is although still have the minimizing (Fig. 1 C) of significant cell number 1 week after castrating.In 2 whens week after castrating, find that cell number is increased to the level (Figure 1B) that can see that is similar in young adult mice.In 3 whens week after castrating, cell number begins significantly to increase and 4 weeks kept stable (Figure 1B) after castrating from young adult mice level.The result represents with meansigma methods ± 1SD of every group of 4-8 mice (Figure 1A) or every group 8-12 mice (Figure 1B). ^*=p≤0.01; ^{* *}Compare with the thymus of young adult (2 months big) and the thymus of the mice in castrating back 2-6 week=p≤0.001.

Fig. 2 A-D: castrating has recovered the CD4 of periphery: the ratio of CD8 T cell.For these research, old (2 years old big) mice operation is castrated and 2-6 week analysis periphery lymphocyte group after castrating.Fig. 2 A and 2B have shown the total lymphocyte number in the spleen.The lymphocyte number of spleen still keeps constant with the age with after castrating, total because homeostasis has been kept the cell number (Fig. 2 A and 2B) in the spleen.But, the cell number (Fig. 2 B) of the lymph node of exhausted old (18-24 month is big) mice.The minimizing that castrating has recovered this lymph-node cell density.B cell in Fig. 3 C demonstration spleen or the lymph node does not all have to change with age or castrating back to the ratio of T cell, does not see the variation after this ratio is with age or castrating yet.But all seen CD4+ in (compiling) lymph node and spleen: CD8+T cell ratio is with the remarkable reduction (p≤0.001) (Fig. 2 E) at age.4-6 is during week after castrating, and this reduction is restored to the level (Fig. 2 D) of young adult (promptly 2 months big).

The result represents with meansigma methods ± 1SD of every group of 4-8 mice (Fig. 2 A, 2C and 2E) or 8-10 mice (Fig. 2 B, 2D and 2F). ^*=p≤0.05; ^*=p≤0.01; ^{* *}Compare with young adult (2 months big) and castrating back mice=p≤0.001.

Fig. 3: though have atrophy of thymus gland or the castrating after regeneration, the thymocyte cell subgroup still is held in similar ratio.For these research, analyze the thymocyte cell subgroup with old (2 years old big) mice castrating and according to label CD4 and CD8.Shown representational fluorescence activated cell sorter (FACS) value of young adult (2 months big), old (2 years old) and CD4-CD8-DN, CD4+CD8+DP, CD4+CD8-and CD4-CD6+SP thymocyte cell group's old, castrating back animal (2 years old, castrating 4 weeks of back) CD4 (X-axis) to CD8 (Y-axis).Above each curve, all provided the percentage ratio of each quadrant.The ratio of the defined subgroup of any CD4/CD8 is not all seen any difference along with the age or after castrating.Therefore, the thymocyte cell subgroup is along with the age still is constant, and the synchronous amplification of thymocyte cell after castrating.

Fig. 4 A-B: the recovery of the thymocyte proliferation that castrating causes.Give the BrdU of a pulsed quantity of injected in mice (pulse) and analyze (BrdU+) thymocyte cell of breeding.For these research, with the mice castrating of old (2 years old big) and the level of bromodeoxyribouridine (BrdU) to determine to breed of injecting a pulsed quantity.Shown representative rectangular histogram figure (Fig. 4 A) with the intrathymic BrdU+ cell proportion after age and the castrating.Do not observe difference in intrathymic overall propagation ratio, this ratio still is constant (the left side figure of Fig. 4 A and Fig. 4 B) along with the age with after castrating.But, seen of the remarkable minimizing (Fig. 4 B, right figure) of BrdU+ cell number with the age.In 2 whens week after castrating, the BrdU+ cell number has been increased to the similar number (Fig. 4 B, right figure) that can see in young adult (promptly 2 months greatly).The result represents with meansigma methods ± 1SD of every group of 4-14 mice. ^{* *}Compare with the control mice and the mice in castrating back 2-6 week of young adult (2 months big)=p≤0.001.

Fig. 5 A-H: castrating has strengthened the propagation in all thymocyte cell subgroups.For these research, with mice castrating of old (2 years old) and the level of bromodeoxyribouridine (BrdU) to determine to breed of injecting a pulsed quantity.Express the analysis of carrying out the propagation in the different subgroups of thymocyte cell according to intrathymic CD4 with CD8.Fig. 5 A shows that the ratio of every kind of thymocyte cell subgroup in the BrdU+ cell mass does not change with age or castrating back.But, as shown in Fig. 5 B, see of the remarkable attenuating of the ratio of DN (CD4-CD8-) thymocyte proliferation with the age.Fig. 5 C shows that the population proportion of not seeing the BrdU+ cell (being proliferative cell) in the TN subgroup changes with age or castrating back.But see that propagation in TN1 (CD44+C25-CD3-CD4-CD8-) subgroup is with the remarkable increase (Fig. 5 F) with the age of the remarkable attenuating (Fig. 5 E) at age and the propagation in TN2 (CD44+CD25+C3-CD4-CD8-) subgroup.Recovered this variation (Fig. 5 D-F) after the castrating.The result represents with meansigma methods ± 1SD of every group of 4-17 mice. ^*=p≤0.05; ^*=p≤0.01 (significantly); ^{* *}=p≤0.001 (highly significant), mice relatively with young adult (2 months big).^=significantly is different from the mice (Fig. 5-H) in castrating back 2-6 week.

Fig. 6 A-C: castrating has increased the output of the T cell of old and feeble thymus.For these research, with old (2 years old big) mice castrating and inner injection of thymus FITC determining the output rating of thymus.After 24 hours, calculate the number of the FITC+ cell of periphery.As shown in Figure 6A, observe in 24 hours periods in the remarkable minimizing of (RTE) cell number of moving out of the detected thymus recently of periphery with the age.After the castrating, in 2 whens week after castrating, these numerical value significantly improve.Shown in Fig. 6 B, the rate of moving out (output/overall thymocyte cell density) still keeps constant with the age, but after castrating significant attenuating is arranged during 2 weeks.Seen CD4+ to the remarkable increase of CD8+RTE ratio with the age, 1 week the time reached normalization (Fig. 6 C) after castrating.

The result represents with meansigma methods ± 1SD of every group of 4-8 mice. ^*=p≤0.05; ^*=p≤0.01 (significantly); ^{* *}=p≤0.001 (highly significant), Fig. 6 A be with young adult (2 months big) mice comparison and Fig. 6 B and 6C for the comparison of all other groups.Relatively and with the old mice in castrating 1 week of back compare with old (1 years old and 2 years old big) non-castrating mice ^=p≤0.05.

Fig. 7 A-B: castrating has strengthened the thymocyte cell regeneration after the T cell exhaustion.Handle (Fig. 7 A) with 3 months big mices or with cyclophosphamide (twice intraperitoneal injection 200mg/kg body weight cyclophosphamide in 2 days), or be exposed to (Fig. 7 B) in the sublethal exposure (625Rad).For the model that two kinds of T cells of being studied are exhausted, (ShCx) the reciprocity experimental subject of castrating with their vacation compares, and (Cx) mice of castrating is expressed the remarkable increase of thymus reactivate rate.The analysis of the total thymocyte cell number during to 1 week after the T cell exhaustion (TCD) and 2 weeks shows the thymus reactivate rate (being respectively Fig. 7 A and 7B) that has increased significantly with after cyclophosphamide or the sublethal exposure processing of castrating.Data are represented with meansigma methods ± 1SD of every group of 4-8 mice.For Fig. 7 A, ^{* *}Compare with contrast (age-matched, untreated) mice=p≤0.001; Compare with two castrating mice groups ^=p≤0.001.For Fig. 7 B, ^{* *}Compare with control mice=p≤0.001; ^=p≤0.001,1 when week and the mice of handling preceding 1 week castrating are organized with two castrating mices during 2 weeks relatively and after irradiation and compare after irradiation.

Fig. 8 A-B: spleen after cyclophosphamide is handled and the total lymphocyte number in the lymph node.For these research, utilize cyclophosphamide (twice intraperitoneal injection 200mg/kg body weight cyclophosphamide in 2 days) exhaust (3 months big) mice lymphocyte and with last cyclophosphamide injection on the same day with mice operation castrating or false castrating.Separate thymus, spleen and lymph node (compiling) and estimate general cell density.With contrast (age-matched, untreated) mice relatively, after processing, during 1 and 4 weeks, have cell number (Fig. 8 A) significantly still less in the spleen of the mice of false castrating.In 1 when week, all observed the remarkable minimizing (Fig. 8 B) of cell number in the lymph node of two processed group after processing.In 2 whens week after processing, the Cx mice has the cell number (Fig. 8 B) of significantly higher lymph node than ShCx mice.Each bar is all represented meansigma methods ± 1SD of every group of 7-17 mice. ^*=p≤0.05; ^*=p≤0.01; Compare with contrast (age-matched, untreated) mice.^=p≤0.05; Compare with the castrating mice.

Fig. 9: handle with a kind of chemotherapeutics cyclophosphamide and undergo surgery on the same day or chemical castration after the variation of cell number of thymus (empty bar), spleen (lath) and lymph node (secret note).When 1 week after processing with when comparing in 2 weeks, notice the rapid amplifying of the thymus that neuter is interior with non-castrating (single use cyclophosphamide) group.In addition, compare with the cyclophosphamide group, increased the spleen of castrating group and the cell number of lymph node significantly with single.(n=3-4 of each processed group and time point only).In the immune regeneration after cyclophosphamide is handled, chemical castration is suitable with the operation castrating.

Figure 10 A-C: the variation of the cell number of thymus (Figure 10 A), spleen (Figure 10 B) and lymph node (Figure 11 C) after the irradiation (625Rad) after 1 week of operation castrating.For these research, exhaust the T cell of young (3 months big) mice with inferior (625Rad) irradiation that causes death.In 1 week of pre-irradiation, mice is castrated or castrates by vacation.Observe of the remarkable increase (promptly faster thymus reactivate rate) (Figure 10 A) of thymus reactivate with castrating.When 1 week after processing with when comparing in 2 weeks, noticed the rapid amplifying of the thymus in the neuter with non-castrating (single with irradiation) group.(n=3-4 of each processed group and time point only).Do not see the difference of the cell number of spleen (Figure 10 B) or lymph node (Figure 10 C) with the castrating mice.Compare with control mice, be still long-term slowly low (Figure 10 C) lymph-node cell number 2 weeks after processing.The result represents with meansigma methods ± 1SD of every group of 4-8 mice. ^*=p≤0.05; ^*Compare with control mice=p≤0.01. ^{* *}Compare with contrast and castrating mice=p≤0.001.

Figure 11 A-C: shine on the same day and castrate after the variation of cell number of thymus (Figure 11 A), spleen (Figure 11 B) and lymph node (Figure 11 C).For these research, exhaust the lymphocyte of young (3 months big) mice with inferior (625Rad) irradiation that causes death.With irradiation on the same day, mice or by false castrating or castrated.Compare with the mice of vacation castrating, the castrating mice demonstrates obviously thymus reactivate rate (Figure 11 A) faster.When 2 weeks compared with non-castrating group after processing, noticed the rapid amplifying of the thymus of neuter.In the castrating mice, do not see the difference of the cell number of spleen (Figure 11 B) or lymph node (Figure 11 C).With control mice relatively, the cell number of lymph node is still during 2 weeks after processing and continues low (Figure 11 C).The result represents with meansigma methods ± 1SD of every group of 4-8 mice. ^*=p≤0.05; ^*Compare with control mice=p≤0.01. ^{* *}Compare with contrast and castrating mice=p≤0.001.

Figure 12 A-B: the total lymphocyte number in spleen after the treatment with irradiation and the lymph node.In sublethal exposure (625Rad) before, 3 months big mices or castrated or by false castrating.In 1 when week after processing, serious lymphopenia is significantly arranged all in spleen (Figure 12 A) and (compiling) lymph node (Figure 12 B).In 2 whens week after processing, the lymphocyte number of spleen has returned to control level (Figure 12 A), and the cell density of lymph node still significantly is lower than contrast (age-matched, untreated) mice (Figure 12 B).Between processed group, do not observe difference.Meansigma methods ± 1SD of every group of 6-8 mice is all represented on every hurdle. ^*=p≤0.01; ^{* *}Compare with control mice=p≤0.001.

Figure 13 A-B: castrating has recovered the responsiveness to the HSV-1 immunity inoculation.With 4 * 10 ⁵Pfu HSV is at the hind leg immunized mice.After infection the 5th day, analyze draining lymph node (lymphonodi poplitei) responsive cell.Figure 13 A has shown at the cell density with the lymph node after herpes simplex virus-1 (HSV-1) the palmula immunity inoculation.With old non-castrating group relatively, noticed the increase of the cell density in the mice after the old castrating.Figure 13 B has shown as FACS and has established total activating cell number that door measures cd8 cell in CD25 (be activating cell established door be the CD8+CD25+ cell).Castrating to old mice has recovered the immunne response of HSV-1 and the CTL cell that equates with young mice.The result represents with meansigma methods ± 1SD of 8-12 mice. ^*Compare with young (2 months big) and the mice of castrating=p≤0.01.

Figure 14 A-C: the expression (HSV is special) of the V β 10 on the CTL (cytotoxic T cell) of HSV-1 postvaccinal activation LN (lymph node).Although old (promptly 18 months big) mice all has normal V β 10 responsivenesss generally, in some mices, observed the disappearance fully that V β 10 expresses.Shown representational rectangular histogram figure.Notice that the clone of old mice replys weaken and castrate after the expection recovery of replying.

Figure 15 A-B: castrating has strengthened the metainfective activation of HSV-1.Figure 15 A has shown the representative FACS figure of activation (CD8+CD25+) cell that the LN of HSV-1 infecting mouse is interior.Do not see the activation CTL ratio with the age or the castrating after difference.As directed, the castrating of old mice has been recovered the immunne response of HSV-1 and the CTL number that equates with young mice.The result represents with meansigma methods ± 1SD of 8-12 mice. ^*Compare with young (2 months big) and the mice of castrating=p≤0.01.

Figure 16: to the specificity of the immunne response of HSV-1.From with Qu Chu lymphonodi poplitei cell the mice of HSV-1 immunity inoculation (infecting taking-up in back 5 days) at HSV-1, it was cultivated 3 days, check that then its cracking HSV peptide activates the ability of EL4 target cell.Carrying out CTL with the mice of immunity inoculation not as the contrast of cracked background level detects (as using ⁵¹Cr release is measured).At E: the T ratio is 10: 1 and 3: 1 o'clock, old mice all demonstrate remarkable attenuating (p≤0.01, ^*) the CTL activity, the attenuating of existing specific CTL percentage ratio in the lymph node is described.Castrating to old mice is replied the young adult level that returned to CTL, because the castrating mice shows and young adult (2 months big) the suitable responsiveness to HSV-1 of mice.The result is with the meansigma methods of 8 mices, and three times repeated trials ± 1SD represents. ^*Compare with young adult mice=p≤0.01; ^=and old control mice have significant difference (when E: T is 3: 1, p≤0.05; When E: T is 0.3: 1, p≤0.01).

Figure 17 A-B: to the V β TCR in the immunne response of HSV-1 being expressed and the analysis of CD4+T cell.Infect back 5 days Qu Chu lymphonodi popliteis at HSV-1, and the expression and the CD4/CD8T cell (Figure 17 B) of its CD25 of analyzed in vitro, CD8 and special TCR V β label (Figure 17 A).Percentages show that to or express activatory (CD25+) CD8+T cell of V β 10 or expression V β 8.1 in Figure 17 A is meansigma methods ± 1SD of every group of 8 mices.Along with not observing difference after age or the castrating.But, seen that CD4/CD8 ratio among the tranquillization LN group is with the attenuating (Figure 17 B) at age.Recovered this attenuating after the castrating.The result represents with meansigma methods ± 1SD of every group of 8 mices, ^{* *}Compare with young and castrating back mice=p≤0.001.

Figure 18 A-D: the BM Ly5 homology mice transplants (BMT) afterwards, and castrating has strengthened the regeneration of thymus (Figure 18 A), spleen (Figure 18 B) and BM (Figure 18 D), but does not strengthen the regeneration (Figure 18 C) of lymph node.Using C57/BL6Ly5.2+ (CD45.2+) adult BM cell (10 ⁶Individual cell) transplants preceding 1 day, with big, young adult, C57/BL6 Ly5.1+ (CD45.1+) mice irradiation (6.25Gy) in 3 months, castrating or false castrating.2 weeks and 4 weeks back execution mice are analyzed the immunologic reconstitution of thymus (Figure 18 A), spleen (Figure 18 B), lymph node (Figure 18 C) and BM (Figure 18 D).Determine donor/host source with anti-CD45.2 (Ly5.2), this antibody only with the leukocytoreaction in donor source.Compare with vacation castrating mice, in 2 week and 4 weeks behind BMT, obviously more donor's cells (Figure 18 A) is arranged in the thymus of castrating mice.Notice at all time points after the processing that when comparing the rapid amplifying of neuter thymus is all arranged with non-castrating group.With vacation castrating mice relatively, in 2 week and 4 weeks behind BMT, have obviously more cell (Figure 18 B and Figure 18 D) behind BMT, 2,4 and 6 when all, not have significant difference (Figure 18 C) between the cell density of lymph node in these spleens of castrating mice and the BM.When comparing with non-neuter, the castrating mice has homology (Ly5.2) cell that significantly increases.Data are represented with meansigma methods ± 1SD of every group of 4-5 mice. ^*=p≤0.05; ^*Compare with control mice=p≤0.01.

Figure 19 A-C: castrating has increased the BM behind the homology BMT and the cell density of thymus.Shown in Figure 19 A, 2 weeks and 4 whens week behind BMT, more cell is arranged obviously in the BM of castrating mice.During to 2 weeks, the medullary cell density of false castrating mice has reached untreated control level (1.5 * 10 ⁷± 1.5 * 10 ⁶).In 2 weeks and 4 whens week behind homology BMT,, the BM cell density of castrating mice has surpassed control level.When Figure 19 B is presented at behind the BMT 2 weeks and 4 weeks, more cell is arranged obviously in the thymus of castrating mice.In 2 weeks and 4 whens week behind homology BMT,, false pleura cell density of castrating mice is lower than untreated control level (7.6 * 10 ⁷± 5.2 * 10 ⁶).During 4 weeks, the cell density increase of thymus has surpassed control level in homology BMT and castrating back.Figure 19 C has shown behind BMT 2 weeks and 4 when all, and the cell density of spleen does not have significant difference.When 2 weeks, the splenocyte density of false castrating and castrating mice has all reached control level (8.5 * 10 ⁷± 1.1 * 10 ⁷).Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; Castrating is represented on real hurdle.

Figure 20: castrating has increased the HSC ratio behind the homology BMT.Representational FACS point diagram illustrates the expression of c-kit (y axle) to sca-1 (x axle).HSC is c-kit ^HiSca-1 ^HiIn 2 weeks and 4 whens week behind BMT,, the ratio of the HSC that donor is originated has had remarkable increase after castrating.

Figure 21 A-B: castrating has increased ratio and the number of the HSC behind the homology BMT.Shown in Figure 21 A, 2 weeks and 4 when all behind BMT, the ratio of HSC after castrating, had remarkable increase ( ^*P＜0.05).When Figure 21 B has shown behind BMT 2 weeks and 4 weeks, compare with the vacation castrating, the HSC number of castrating mice have remarkable increase ( ^*P＜0.05; ^*P＜0.01).Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; The representative castrating of real hurdle.

Figure 22 A-B: after BMT, the B cell CFU-GM and the B cell in significantly more donor source arranged in the BM of castrating mice.Shown in Figure 22 A, compare with vacation castrating, in the BM of castrating mice, have significantly more donor source CD45.1+B220+IgM-B cell CFU-GM ( ^*P＜0.05).Figure 22 B has shown with vacation castrating compares, in the BM of castrating mice, have significantly more donor source the B220+IgM+B cell ( ^*P＜0.05).Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; The representative castrating of real hurdle.

Figure 23: castrating does not influence the thymocyte cell ratio in the donor source behind the homology BMT.In vacation castrating and castrating back during 2 weeks, the increase of early stage thymocyte cell ratio of the jack to jack adapter (CD45.1+CD4-CD8-) in donor source is arranged.On this early stage time point, have only (CD45.1+) CD4 and the single positive cell of CD8 in considerably less donor source.In 4 whens week behind BMT, the thymocyte cell value in the donor source of false castrating and castrating mice is all similar to untreated contrast.

Figure 24: castrating does not increase the periphery B cell proportion behind the homology BMT.2 weeks and 4 whens week behind homology BMT, there is not difference between the spleen B220 expression of castrating and false castrating mice.

Figure 25: castrating does not increase the periphery B cell number behind the homology BMT.2 weeks and 4 do not have the significant difference of B cell number when all behind BMT.In 2 whens week behind homology BMT, the B cell number in the spleen of false castrating and castrating mice approaches untreated control level (5.0 * 10 ⁷± 4.5 * 10 ⁶).Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; The representative castrating of real hurdle.

Figure 26: increased by three feminine genders, two positive and the CD4 in the donor source in the castrating mice behind BMT and the number of the single positive thymocyte cell of CD8.When Figure 26 A is presented at behind the BMT 2 weeks and 4 weeks, compare with the vacation castrating, the castrating mice have three feminine genders (CD45.1+CD3-CD4-CD8-) thymocyte cell that significantly more donor originates ( ^*P＜0.05, ^*P＜0.01).When Figure 26 B is presented at behind the BMT 2 weeks and 4 weeks, compare with the vacation castrating, the castrating mice have the significantly more two positive (CD45.1+CD4+CD8+) thymocyte cells ( ^*P＜0.05, ^*P＜0.01).Shown in Figure 26 C, 2 weeks and 4 whens week behind BMT, compare with the vacation castrating, the castrating mice have the single positive of significantly more CD4 (CD45.1+CD3+CD4+CD8-) thymocyte cell ( ^*P＜0.05, ^*P＜0.01).When Figure 26 D is presented at behind the BMT 2 weeks and 4 weeks, compare with the vacation castrating, the castrating mice have the single positive of significantly more CD8 (CD45.1+CD3+CD4-CD8+) thymocyte cell ( ^*P＜0.05, ^*P＜0.01).Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; The representative castrating of real hurdle.

Figure 27: the periphery T cell that 2 weeks and 4 weeks the time have only considerably less donor to originate behind homology BMT.Shown in Figure 27 A, 2 weeks and 4 whens week behind homology BMT, the CD4+ that considerably less donor originates and the ratio of CD8+T cell are arranged all in the spleen of vacation castrating and castrating mice.There is not significant difference between the T cell number in the donor source when Figure 27 B is presented at 2 weeks and 4 weeks behind the BMT.In 4 whens week behind the homology BMT, are with contrast (CD4+:1.1 * 10 of untreated age-matched ⁷± 1.4 * 10 ⁶, CD8+:6.0 * 10 ⁶± 1.0 * 10 ⁵) relatively, false castrating and castrating mice all have CD4+ and CD8+ cell significantly still less.Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; The representative castrating of real hurdle.

Figure 28: the number of the DC in the donor source of the thymus when castrating has increased by 4 weeks behind the homology BMT.Shown in Figure 28 A, the DC in donor source is CD45.1+CD11c+MHCII+.When Figure 28 B is presented at 4 weeks behind the homology BMT castrating mice have significantly more donor source thymus DC ( ^*P＜0.05).In 2 whens week behind homology BMT, the dendritic cell number is the control level (1.4 * 10 when being untreated ⁵± 2.8 * 10 ⁴).In 4 whens week behind homology BMT, the dendritic cell of castrating mice has outnumbered control level.Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; The representative castrating of real hurdle.

Figure 29 A-C: castrating has strengthened the reconstruction of allogene HSCT receptor's immunocyte.CBA/J receptor's (3 months big) through lethal exposure (1300cGy) has accepted B10.BR TCD BM (5 * 10 ⁶) transplanting.In transplanting preceding 1 day, the receptor was castrated or by false castrating.Put to death animal at the 14th, 28 and 42 day in the mode of humanity, and estimated the cell density of BM (Figure 29 A), thymus (Figure 29 B) and spleen (Figure 29 C) organ. ^*(p＜0.05)。Every group contains 4 to 5 animals.

Figure 30 A-C: castrating has strengthened the HSC and the B cell in allogene HSCT receptor's donor source.Transplant the receptor of castrating and false castrating as described in Figure 29.Shown in Figure 30 A, behind HSCT, 14 days the time, in vacation castrating and castrating mice, all has only the HSC (Ly9.1-Lin-Sca+c-kit+) in donor source seldom; But during by the 28th day, the donor HSC number of castrating group has exceeded 4 times.In addition, shown in Figure 30 B-C, the B cell in significantly more donor source is arranged all in BM (Figure 30 B) that castrates mice and spleen (Figure 30 C).Utilized total BM or splenocyte the counting and the polychrome flow cytometry center and periphery B cell mass.According to the expression of CD45R, IgM and CD43, the B cell is divided into each period of development: total B cell (CD45R+), former B (pro-B) cell (CD43+CD45R+IgM-), preceding B (pre-B) cell (CD43-CD45R+IgM-), immature B cells (CD43-CD45R+IgM+).Determine donor/host source with anti-Ly9.1, this antibody only with the leukocytoreaction in host source.Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; The representative castrating of real hurdle. ^*(p＜0.05) expression is compared with false castrating, and the cell number of castrating group has remarkable increase.

Figure 31 A-E: castrating has strengthened the number of the DC in the reconstruction of allogene HSCT receptor's thymocyte cell and periphery T cell and host and donor source.Transplant castrating and false castrating mice as described in Figure 29.Put to death animal at the 14th, 28 and 42 day in the mode of humanity, and with cell counting and the polychrome flow cytometry thymocyte cell and the T cell mass of total thymus (Figure 31 A-F) and spleen (Figure 31 G).DC is defined as CD11chiIa-Khi.Figure 31 A has described the number of TN (CD3-CD4-CD8-) thymocyte cell.Figure 31 B has described the number of DP (CD4+CD8+) thymocyte cell.Figure 31 C has described the number of CD4+ (CD3+CD4+CD8-) thymocyte cell.Figure 31 D has described the number of CD8+ (CD3+CD4-CD8+) thymocyte cell.Shown in Figure 31 E, with the contrast receptor of vacation castrating relatively, behind allogene HSCT when 14 days and 28 days, the DC that the castrating receptor has significantly more host to originate.In addition, shown in Figure 31 F, with comparing of vacation castrating, 28 days the time, the castrating receptor has the DC in significantly more donor source behind allogene HSCT.Figure 31 G has described the number of the periphery T cell that identifies with anti-CD3, anti-CD4 and anti-CD8.Determine donor/host source with anti-Ly9.1, this antibody only with the leukocytoreaction in host source.The donor cd4 t cell is that Ly9.1-CD3+CD4+CD8-and donor cd8 t cell are Ly9.1-CD3+CD4-CD8+.Every group all contains 4 to 5 animals.False castrating is represented on empty hurdle; The representative castrating of real hurdle. ^*(p＜0.05) and ^*(p＜0.01) expression is compared with false castrating, and the cell number of castrating group has remarkable increase.

Figure 32 A-G: castrating does not change the function of the T cell in the donor source behind the allogene HSCT.Transplant castrating and false castrating receptor as shown in figure 29.Measured the T cell function in back 42 days in transplanting.Figure 32 A has shown the number of the T cell (CD3+CD4+ and CD3+CD8+) in the donor source after allogene HSCT6 week.Figure 32 B has shown the multiplication capacity not influence of castrating to the T cell behind the allogene HSCT.Figure 32 C has shown on the allogenic reaction T of MLR cell proliferation does not have difference.Splenic t-cell and (20Gy) BALCB/c spleen irritation cell (2 * 10 with each group (n=5) through shining ⁵Individual cells/well) in 96 orifice plates, hatched 5 days, and in last 20 hours that cultivate, add [ ³H]-the deoxidation fudr.Figure 32 D is presented on the lysis activity of T cell in donor source does not have difference.Figure 32 E has shown the expression of the interior IFN γ of cell of allogenic reaction T cell.At the 42nd day, from the receptor of aforesaid false castrating or castrating, collect spleen B6T cell, and with itself and (20Gy) (the 3rd part of BALCB/c) spleen irritation cell (2 * 10 through irradiation ⁵Individual cells/well) in 24 orifice plates, hatched 5 days.Collecting cell, and with TCD, through the irradiation (20Gy) (the biological internal reference of BALB/c or B10.BR) spleen irritation cell stimulate again.Behind 1st hour of irradiation, add Brefeldin A (10mg/mL).Measure the expression of IFN γ in the cell of CD3+CD8+ cell in donor source with flow cytometry.In Figure 32 E, shown representative curve, and diagram has shown the percentage ratio of the CD8+T cell in the donor source of expressing IFN γ in Figure 32 F.Figure 32 G shows that the functional of T cell strengthened in back 48 hours significantly in attack when mice is castrated at allogene HSCT the time.In 6 whens week behind allogene HSCT, vacation castrating and castrating mice are carried out DTH detect, and deduct left back palmula swelling by right back palmula and determine swelling.False castrating is represented on empty hurdle; The representative castrating of real hurdle.

Figure 33 A-B: castrating does not increase the weight of the GVHD among the allogene HSCT receptor or alleviates wherein GVL.For in the test described in Figure 33 A, through lethal exposure (1300cGy) (B6 * C3H) F1 receptor's (3 months big) accepts B6TCD BM cell (5 * 10 ⁶)+splenocyte (0.5 * 10 ⁶) transplanting.With Kaplan-Meier curve description survival rate.Empty circle representative only has TCD-BM to transplant the matched group (n=4) of (no T cell).Real circle is represented false castrating receptor, and short side piece representative castrating receptor.Every group all contains 8 animals.For the test described in Figure 33 B, through lethal exposure, 3 months big B6D2F1/J receptors have accepted P815 (H-2d) cell (1 * 10 ³), C57/BL6TCD BM cell (5 * 10 ⁶) and C57/BL6T cell (5 * 10 ⁵).With Kaplan-Meier curve description survival rate.Empty circle representative only has TCD-BM to transplant the matched group (n=4) of (no T cell).Real circle is represented false castrating receptor, and short side piece representative castrating receptor.Every group all contains 8-9 animal.

Figure 34 A-I: castrating and IL-7 treatment have accumulative action to the thymus behind the allogene HSCT.Transplant castrating and false castrating receptor as described in Figure 1.The 14th day sacrificed receptor (Figure 34 A) also accepted 10g/ days IL-7 or PBS (contrast) through intraperitoneal injection at the 0th day to 13 days.The 28th day sacrificed receptor (Figure 34 B) accepted 10g/ days IL-7 or PBS (contrast) at the 21st day to 28 days.From total cell counting, calculate the cell density of thymus. ^*When (p＜0.05) representative was compared with false castrating, the cell number of castrating group had significant increase.Contrast: false castrating, the receptor of injection PBS; CX: receptor castrating, injection PBS; IL-7: receptor false castrating, injection IL-7; And IL-7﹠amp; CX: receptor castrating, injection IL-7.During 2 weeks, whole BM is carried out semiquantitative RT-PCR in allogene HSCT and castrating back.Comparing afterwards from the expression (Figure 34 C) of the TGF β 1 of the HPRT balance template of castrating and false castrating mice and KGF.With KGF ^-/-And IL ^-/-Mice castrating and at the cell density of 2 all post analysis thymus, BM and spleen.Figure 34 D-F has shown from KGF ^-/-The result of the thymus of mice (Figure 34 D), BM (Figure 34 E), spleen (Figure 34 F).Figure 34 G-I has shown from IL ^-/-The result of the thymus of mice (Figure 34 G), BM (Figure 34 H) and spleen (Figure 34 I).

Figure 35: castrating has strengthened the implantation of the BM behind the HSCT, thymus and spleen.Preceding 1 day of homology HSCT mice is castrated.With 5 * 10 ⁶The Ly5.1+BM cell arrives in the C57/BL6 mice of irradiation (800Rad) through intravenous injection.Different time point after transplanting (2-6 week) flow cytometry BM, spleen and thymus.Shown in Figure 35 B,,, significantly more cell is arranged in the BM of castrating mice with comparing of vacation castrating at castrating and HSCT after 2 weeks.Similarly, shown in Figure 35 C, with comparing of vacation castrating, that castrates mice all has remarkable increase in thymocyte cell 2,4 and 6 weeks after transplanting.Shown in Figure 35 C, castrating receptor's the splenocyte number of periphery after transplanting 4 and 6 weeks all apparently higher than contrast.Ash hurdle representative castrating receptor.False castrating contrast is represented on black hurdle.

Figure 36 A-B: castrating has strengthened the implantation of HSC in BM behind the homology HSCT.Preceding 1 day of homology HSCT mice is castrated.With 5 * 10 ⁶The Ly5.1+BM cell arrives in the C57/BL6 mice of irradiation (800Rad) through intravenous injection.In 2 whens week after transplanting, are with the lin-c-kit+sca-1+HSC (Figure 36 A) of flow cytometry BM.With comparing of vacation castrating, transplant and castrate the back during 2 weeks at BMT, the significantly more HSC (Figure 36 B) in donor source is arranged in the BM of castrating mice.

Figure 37 A-B: castrating has strengthened homology HSCT (2.5 * 10 ⁶With 5 * 10 ⁶) implantation of back HSC in BM.Preceding 1 day of homology HSCT mice is castrated.With 2.5 * 10 ⁶(Figure 37 A-B) and 5 * 10 ⁶(Figure 37 C-D) Ly5.1+BM cell arrives in the C57/BL6 mice of irradiation (800Rad) through intravenous injection.In 2 whens week after transplanting, are with the lin-c-kit+sca-1+HSC of flow cytometry BM.Figure 37 A-D has described the percentage ratio of the common lymph CFU-GM in the BM.With comparing of vacation castrating, transplant and castrating back during 2 weeks at BMT, the ratio of HSC in the donor source of remarkable increase is all arranged in the BM of castrating mice.

Figure 38 A-B: castrating has strengthened homology HSCT (2.5 * 10 ⁶With 5 * 10 ⁶) DC in donor source, back is at intrathymic implantation rate.With 5 * 10 ⁶Ly5.1 ⁺The BM cell arrives in the C57/BL6 mice of irradiation (800Rad) through intravenous injection.In 2 whens week after transplanting, are with flow cytometry thymocyte cell (Figure 38 A).The DC in donor source is defined as CD45.1 ⁺CD11c ⁺MHC II ⁺CD11b ^{+ or-}In 2 whens week behind BMT,, the CD11b in the donor source of remarkable increase is arranged in the thymus of castrating mice with comparing of vacation castrating ⁺And CD11b ^-DC (Figure 38 B).

Figure 39 A-D: castrating has strengthened homology HSCT (2.5 * 10 ⁶With 5 * 10 ⁶) implantation rate of B cell in spleen in donor source, back.With 5 * 10 ⁶The Ly5.1+BM cell arrives in the C57/BL6 mice of irradiation (800Rad) through intravenous injection.In 2 whens week after transplanting, are with flow cytometry splenocyte (Figure 39 A-C).In 2 whens week behind BMT,, significantly more B220+B cell (Figure 39 D) is arranged in the spleen of castrating mice with comparing of vacation castrating.

Figure 40: the phenotype of peripheral blood lymphocyte of analyzing the human patients (all＞60 years old) of the LHRH agonist treatment carry out anti-carcinoma of prostate is formed.4 months analysis patients' specimen before treatment and behind the beginning LHRH agonist treatment.The total lymphocyte number of the every ml blood of all patients before treatment is all at the lower end of control value.After treatment, 6 examples among the 9 routine patients all demonstrate the obvious increase (having observed doubling of total cell number in some cases) of total lymphocyte counting.Relevant is the increase that 6 examples all have total T cell number among the 9 routine patients therewith.In the CD4+ subgroup, this increase or even more outstanding, 9 routine patients' 8 examples all confirm the increase of cd4 t cell level.Seen this more not outstanding trend in the CD8+ subgroup, 9 routine patients' 4 examples demonstrate the increase of level, though the degree of this increase is less than the CD4+T cell.

Figure 41: the analysis to the blood of the human patients before and after the LHRH agonist treatment confirms do not have significant change on the toatl proportion of T cell, cd4 cell or cd8 t cell, and the different variation of the ratio of CD4: CD8 after treatment.Obviously increased total T cell number although this has illustrated after treatment, treatment is kept the stable state of T cell subgroup has only slight influence.All numerical value is all suitable with control value.

Figure 42: the proportion grading of B cell in the peripheral blood of the human patients that carries out the LHRH agonist treatment and myelocyte (NK, NKT and macrophage) has been confirmed variation in various degree in the subgroup.Though NK, NKT still keep relative constant with the macrophage ratio after treatment, the minimizing of B cell proportion has appearred in 4 examples among the 9 routine patients.

Figure 43: the B in the peripheral blood of the human patients after the treatment and myelocytic total cell number goal analysis have been clearly illustrated the increase for the treatment of the level of back NK (4 examples among the 9 routine patients), NKT (4 examples among the 9 routine patients) and macrophage (3 examples among the 9 routine patients).The B cell number does not demonstrate tangible trend, and 2 examples among the 9 routine patients demonstrate the increase of level; Among the 9 routine patients 4 example shows the attenuating of 3 routine reveal competences in not variation and 9 examples.

Figure 44 A-B: human chemical castration has strengthened originally and memory T cell.Shown in Figure 44 A, after the LHRH-A treatment, observed the remarkable increase of (CD62L+CD45RA+CD45RO-) CD4+T cell originally.Shown in Figure 44 B, behind the LHRH agonist treatment, strengthened originally and remembered the number of (CD62L-CD45RA-CD45RO+) CD8+T cell.On behalf of meansigma methods ± 1SD of 16 routine patients, every hurdle all represent. ^*=p≤0.05; ^*=p≤0.01, with the treatment before numeric ratio.

Figure 45 A-B: human chemical castration has strengthened the peripheral blood lymphocyte number.Having analyzed the phenotype of the peripheral blood of the human patients that carries out as the LHRH-A chemical castration of the part of the conventional therapy of carcinoma of prostate (all＞60 years old) forms.Before treatment, analyze the patient in the time of 4 months with treatment.Shown in Figure 45 A, after the LHRH-A treatment, increased the total lymphocyte number of every μ l peripheral blood significantly.The remarkable increase of total T cell, CD4+ and CD8+T cell has reflected this point (Figure 45 B).

Serum testosterone has been exhausted in Figure 46 A-B:LHRH-A treatment effectively, and has increased thymus function and the output of T cell.In the test of Figure 46 A, treated patients with prostate cancer 4 months with LHRH-A.Treatment was all used facs analysis blood and is used the RIA serum analysis after 4 months with LHRH-A before treatment.Shown in Figure 83 A, in the time of 4 months, in patient's serum, do not detect testosterone in the LHRH-A treatment.The 13 routine patients' that the hurdle representative is analyzed meansigma methods.In the test of Figure 46 B, after the TREC level of analyzing 10 routine patients, found the positive evidence that the output of thymus function and T cell increases.In CD4+ and CD8+T cell mass, to LHRH-A treatment in the time of 4 months, 5 examples among the 10 routine patients all demonstrate the increase (surpass initial measured value＞25%) of absolute TREC level (every ml blood).This point also is reflected in the increase (per 1 * 10 of ratio ⁵Cell; Data do not show).6 examples that associated is among the 10 routine patients all demonstrate the overall increase of total TREC level.Only there is 1 routine patient to demonstrate the minimizing (about 30% minimizing) of total TREC.

Figure 47: human chemical castration has strengthened the NK number.Analyze before LHRH-A treatment and when treating 4 months.Observed the NK cell but be not the B cell with the remarkable increase of LHRH-A treatment.The result represents with meansigma methods ± 1SD of 13 routine patients. ^*=p≤0.01, with the treatment before numeric ratio.

Figure 48 A-B: human chemical castration does not increase the propagation of T cell.Figure 48 A-B has described the analysis that utilizes the on cell proliferation that the Ki-67 Detection of antigen carried out.In all patients, LHRH-A treatment all do not change all CD4+ (Figure 48 A) and CD8+T cell (Figure 48 B) originally, activation and the interior propagation level of memory cell subgroup.

Figure 49 A-C: the analysis of NKT (NK) cellular-restoring of the different time points behind HSCT to control patients and LHRH-A treatment patient.As Figure 49 A-B shown in respectively, in the allogene of contrast and autotransplantation receptor, all observed similar trend.On the contrary, the allogene patient who gives LHRH-A treatment before HSCT 3 weeks demonstrated number (Figure 49 C of significantly higher NKT cell in back 14 days to 5 months in transplanting; Data represent with meansigma methods ± 1SEM of 6-20 example patient, ^*=p≤0.05).

Figure 50: the facs analysis that the NKT cell of the different time points (14th, 21,28 and 35 day) of control patients behind HSCT is rebuild.In the allogene patient, observed early recovery, be mainly seen in and transplant the early stage CD8+ group in back, the regeneration approach beyond this prompting thymus.In transplanting back 1 month, the CD4+NKT cell also was tangible.

Figure 51 A-B: the B cell of control patients behind the HSCT of the different time points behind the HSCT (2-12 month) rebuild.Shown in Figure 51 B, compare with the allogene patient, autotransplantation patient's B cell is rebuild and is taken place sooner relatively (Figure 51 A).But after transplanting at least 6 months, it was all not obvious to return to control value (shade) in two groups.

Figure 52 A-B: the CD4+ cell of control patients behind the HSCT of the different time points behind the HSCT (2-12 month) rebuild.Though the B cell number has returned to control value (seeing Figure 48 A-B) in transplanting in the time of back 6 months, even in transplanting in the time of back 12 months, all be still serious attenuating from body (Figure 52 B) and allogene (Figure 52 A) receptor's CD4+T cell.

Figure 53 A-C: the CD8+ cell of control patients behind the HSCT of the different time points behind the HSCT (2-12 month) rebuild.Shown in Figure 52 A-B, at allogene with in the body receptor, the CD8+T cell number is all regenerated very soon after transplanting.But shown in Figure 53, as suggested in the increase of TCR γ δ+T cell, CD8 α α T cell and CD28-CD8+T cell, the CD8+T cell mainly is the thymus external source.

Figure 54 A-B: utilize the facs analysis of label Ki-67 to control patients propagation of the distinct group of the CD4+ of (Figure 54 A) and 28 days afterwards (Figure 54 B) and CD8+T cell before HSCT.Utilize label CD45RO and CD27 according to originally, the memory with activatory phenotype analytical cell.Most of propagation all occurs in CD8+T cell subgroup, and this also points out these cells is that thymus originates from outward and proliferative advantage occurs in the periphery T cell subgroup.

Figure 55 A-D: the regeneration of the cell of CD4+T originally of the different time points of patient behind HSCT of control patients and LHRH-A treatment.Figure 55 A has described the facs analysis to contrast (not LHRH-A treatment) patient's the cell of CD4+T originally (CD45RA+CD45RO-CD62L+), and shows the serious disappearance of these cells during whole research.Shown in Figure 55 B-C, autotransplantation patient behind HSCT 12 months, the cell of CD4+T originally of control patients just begins regeneration (Figure 55 C), but still is starkly lower than the control value (Figure 55 B) of allogene control patients.These presentation of results are because of patient's age, and the thymus of control patients can not recover the cell of T originally of enough numbers after transplanting.On the contrary, the patient who gives LHRH-A before allogene HSCT 3 weeks compared according to demonstrating significantly higher number (Figure 55 D) in transplanting in back 9 months and 12 months.This illustrative steroid is removed the regeneration that treatment has strengthened thymus dependent T cellular pathways.Data with 6-20 example patient ± 1SEM represents, ^*=p≤0.05.

Figure 56 A-D: the TREC level of control patients and the LHRH-A treatment patient different time points (1-12 month) behind HSCT.The analysis of TREC (be detected in thymus move out (RTE)) recently level has been emphasized allogene (Figure 52 A) and from the ability of thymus recovery level after transplanting of body (Figure 52 B) transplant patient.This all is that this M ﹠ M with these patients is all obviously relevant because of the disappearance of patient's age and the thymus function that atrophy of thymus gland caused.On the contrary, when before allotransplantation, treating with LHRH-A, the patient who carries out allogeneic peripheral blood stem cell transplantation show the remarkable increase of CD4+TREC+ cell/ml blood (transplant back 9 months with contrast (LHRH-A treatment) relatively, p≤0.01).The allogene patient who accepts LHRH-A treatment demonstrates comparison according to the remarkable CD4+TREC cell/ml blood of higher number when transplanting back 9 months (Figure 56 C).Patient from the LHRH-A of body treatment also demonstrates significantly higher level (Figure 56 D) in transplanting in the time of back 12 months.This illustrative steroid is removed the regeneration that treatment has strengthened thymus.Data with 5-18 example patient ± 1SEM represents, ^*=p≤0.01.

Figure 57 A-C:LHRH-A administration strengthened allogene (Figure 57 A-B) and behind body (Figure 57 C) HSCT to the responsiveness of the special stimulation of TCR.In 3 weeks before HSCT, give the patient LHRH-A.The patient who does not accept agonist is used as control patients.Different time points after transplanting (1-12 month) is with anti-CD3 of 5 μ g and the crosslinked analysis of carrying out the special stimulation of TCR of the anti-CD28 of 10 μ g.Shown in Figure 57 A-B, the patient of allogene LHRH-A treatment all time points except 6 months and 9 months (because patient's number of being analyzed this moment is few) all demonstrate comparison shine the stronger propagation of patient reply (as ³The H-thymidine mixes to be measured).When back 6 months of transplanting and 9 months, control patients have and treatment before the similar responsiveness of numerical value.But on all other times points, they all are significantly lower.On the contrary, relatively preceding with treatment, the patient of LHRH-A treatment has equal responsiveness on all time points except that 6 months.The patient of LHRH-A treatment one week back 1,3 and all demonstrated in 4 months comparison according to the stronger propagation of patient reply (as ³The H-thymidine mix measure).This has illustrated the contribution of direct periphery T cell effect, because up to transplanting the back 1-2 month, new CD4+T cell all is unconspicuous (Figure 57 B; Data represent with meansigma methods ± 1SEM of 5-12 example patient, ^*=p≤0.05; ^*=p≤0.01).Shown in Figure 57 C, from the patient of body LHRH-A treatment all time points except that 5 months all demonstrate the stronger propagation of comparison photograph patient reply (as ³The H-thymidine mixes to be measured).In the time of back 12 months, the patient who has observed contrast and LHRH-A treatment has returned to the value before the treatment in transplanting.

Figure 58 A-B:LHRH-A administration has strengthened behind the allogene HSCT responsiveness to PWM and the primary stimuli of TT mitosis.3 weeks were treated the patient with LHRH-A before HSCT.The patient that will not accept agonist is used as control patients.Utilize pokeweed mitogen (PWM) (PWM) or tetanus toxin (TT) different time points (1-12 month) after transplanting to carry out analysis to the responsiveness of mitosis primary stimuli.Patient with the LHRH-A treatment before HSCT demonstrates comparison according to the stronger responsiveness to PWM (Figure 58 A) and TT (Figure 58 B) of patient at all time points.

Figure 59 A-B:LHRH-A administration has strengthened behind body HSCT the responsiveness to PWM and the primary stimuli of TT mitosis.3 weeks were treated the patient with LHRH-A before HSCT.The patient that will not accept agonist is used as control patients.Utilize pokeweed mitogen (PWM) (PWM) or tetanus toxin (TT) different time points (1-12 month) after transplanting to carry out analysis to the responsiveness of mitosis primary stimuli.All demonstrating comparison according to the stronger responsiveness of patient (3 months time p≤0.001) with the patient of LHRH-A treatment at most of time points before the HSCT to PWM (Figure 59 A) and TT (Figure 59 B).Arrived when transplanting back 12 months, the patient of LHRH-A treatment has the responsiveness that has recovered.

The speed of the implantation of Figure 60 A-D:LHRH-A treatment enhancing the in body HSCT patient.In 3 weeks before HSCT, treat patient (Figure 60 A, C and D) with LHRH-A.The patient that will not accept agonist is used as control patients (Figure 60 B).After transplanting, measured total leukocyte (WBC) counting and granulocyte (G) counting of every μ l blood on the the 14th, 28 and 35 day.Shown in Figure 60 A, that accepts LHRH-A treatment demonstrated comparison on the 14th day according to (Figure 60 B) remarkable higher WBC number (p≤0.05) from the body patient after transplanting.On this time point, be compared to 45%, 87% of contrast and demonstrate granulocytic implantation (〉=500 cell/μ l blood).Accept LHRH-A treatment from the body patient after transplanting, also compared in 10-12 days according to the number that demonstrates significantly higher neutrophilic granulocyte (Figure 60 C, data represent with meansigma methods ± 1SEM of the routine patient of 8-20, ^*=p≤0.05).In addition, although be inapparent, what LHRH-A treated all has higher lymphocyte count (Figure 60 D) than matched group from the body patient on all time points.

The speed of the implantation of Figure 61 A-D:LHRH-A treatment enhancing the among the allogene HSCT patient.In 3 weeks before HSCT, treat patient (Figure 61 A, C and D) with LHRH-A.The patient that will not accept agonist is used as control patients (Figure 61 B).After transplanting, measured total leukocyte (WBC) counting and granulocyte (G) counting of every μ l blood on the the 14th, 28 and 35 day.Shown in Figure 61 A, the allogene patient who accepts LHRH-A treatment demonstrated comparison on the 14th day according to (Figure 61 B) remarkable higher WBC number (p≤0.05) after transplanting, on this time point, have 87% to demonstrate granulocytic implantation (〉=500 cell/μ l blood), be compared to 44% of contrast.The allogene patient who accepts LHRH-A treatment after transplanting the 9th, 12﹠amp; 19 days also comparison according to the number that demonstrates significantly higher neutrophilic granulocyte (Figure 61 C, data represent with meansigma methods ± 1SEM of the routine patient of 8-20, ^*=p≤0.05).In addition, the patient's that carries out autologous peripheral blood stemcell transplant analysis is confirmed the remarkable increase (after transplanting 10,12,13 and 17-21 days the time, p≤0.05) (Figure 61 D) of lymphocyte count when before allotransplantation, treating with LHRH-A.

Figure 62 A-F: in 1 week of castrating, strengthened TCR specificity periphery T cell propagation and replied.Mice castrating that 8 weeks are big and the T cell proliferation that the anti-CD28 of the anti-CD3/ of the 3rd day (Figure 61 A, C and E) and the 7th day (Figure 62 B, D and F) analysis stimulates after operation are replied.Stimulate and stimulated periphery (cervical region, oxter, the upper arm and groin) lymph node (Figure 62 A and B), mesenteric lymph node (Figure 62 C and D) and splenocyte (Figure 62 E and F) altogether 48 hours with the anti-CD3 of variable concentrations with the anti-CD28 of 10 μ g/ml of constant density.Handled cell 18 hours and basis with the tritiated thymidine of pulsed quantity then ³H-T mixes and measures propagation.Prismatic is represented neuter, and square is represented false castrating control animal, n=4, ^*P≤0.05 (non-parametric, non-matching, Mann-Whitney statistical test).

Figure 63: the LHRH-A administration has strengthened the chronic cancer victim and has treated the responsiveness of back to the TCR differential stimulus.Treat chronic malignant tumor patient with LHRH-A.Different time points after the LHRH-A administration (7-12 month) is with anti-CD3 and the crosslinked analysis of carrying out the TCR differential stimulus of anti-CD28.The patient of LHRH-A treatment demonstrates than the propagation of the periodic that level is stronger before the treatment and replys (usefulness ³The H-thymidine mix measure).This has reflected the administration of the agonist of every month long-acting injection.These presentation of results are to the direct influence of periphery T cell.But the enhanced variation of being seen in the time of back 12 months in treatment of replying the T cell that has reflected the thymus source was because just stopped the administration of all patients' agonist since 4 months.

Figure 64 shows that the mice when 60% sham-operation suffers from diabetes, and the castrating group be less than the linear graph that 20% patient suffers from diabetes.

Figure 65 is that the total thymocyte cell number that shows the NOD mice of castrating has showed increased, and total splenocyte does not have the bar diagram of difference.

Figure 66 A-C is that all thymocyte cell hypotypes that show the NOD mice of castrating all have remarkable increase (Figure 66 A).Compare with the NOD mice (Figure 66 C) of vacation castrating, total T of B cell and spleen or B cell all do not have to change (Figure 66 B).

Figure 67 A and 67B have shown the total thymocyte cell (Figure 67 A) of the NZB mice of castrating and the obvious increase of splenocyte (Figure 68 B).

Figure 68 shows by the mice of castrating and immunity inoculation comparison according to the figure that the tumor incidence that has lowered is arranged.

Figure 69 A-C is the mice comparison that shows castrating and immunity inoculation according to the bar diagram of the cell density that the spleen that has increased is arranged.

Figure 70 A-B be show castrate and the mice of immunity inoculation comparison according to the figure that has the γ IFN that increased to generate.

Figure 71 A-B is that the mice that shows castrating and immunity inoculation shows the figure that comparison is replied according to stronger antigenic specificity CTL.

Figure 72 A-E shows that thymusectomy does not influence sex steroid inhibition/BMT to total cell number (Figure 72 D) of the immature B cell (Figure 64 C) of the common lymph CFU-GM (Figure 72 A) of BM, total BM B cell (Figure 72 B), BM, spleen or to the effect of total B cell (Figure 72 E) of spleen.

The specific embodiment

Constituted the obtainable knowledge of those skilled in the art in this patent quoted and scientific literature.At this disclosed U.S., application, published foreign application and list of references of quoting, the full content that comprises the gene bank sequence all together is incorporated herein by reference with identical degree, is all pointed out to be incorporated herein by reference at this particularly and individually as each.

Present invention resides in do not have the thymus reactivate, under the situation prior to thymus reactivate or associating thymus reactivate, the trafficability characteristic steroid is removed and/or disruptive steroid signal pathway and increase the functional method of BM." increase BM function " and " it is functional to strengthen BM " comprises the precursor for example output of HSC (and so increased hemocyte) and/or the raising of output at this immunocyte that is defined as BM, comprises the enhancing of implanting behind the raising of hemoposieis and/or the HSCT.The raising of output can include but not limited to, the mobilization immunocyte of raising comprise that HSC arrives periphery or to target tissue particularly immunity or damaged tissues) ability.In one embodiment, improved the hemoposieis of HSC.In another embodiment, improved the output of HSC.In specific embodiment, increased the number of blood and/or immunocyte.Still in another embodiment, improved the implantation of the HSC behind the HSCT.In another embodiment, having improved HSC mobilizes periphery or goes into nest to target tissue.Still in another embodiment, improved multiplication capacity and/or be divided into hemopoietic or the ability of the filial generation of non-hemopoietic.

The present invention is also included within does not have the thymus reactivate, under the situation prior to thymus reactivate or associating thymus reactivate, the trafficability characteristic steroid is removed and/or disruptive steroid signal pathway and increase the method for the ability of T cell and other immunocytes.Term " immunocyte " and " immune cell " can be exchanged at this and be used, and be defined as HSC, T cell, B cell, DC and/or other hemocytees at this, include but not limited to HSC filial generation, CLP, MLP, lymphocyte, myelocyte, neutrophilic granulocyte, granulocyte, basophilic granulocyte, eosinophilic granulocyte, NK, NKT, platelet, erythrocyte, mononuclear cell, macrophage, originally T cell and top mentioned precursor.Cell can be or can not be periphery, can find these cells in any one or more tissue of BM, blood, spleen, lymph node, thymus, mucosa, skin or its hetero-organization.

" increase functional " of immunocyte or " enhanced functional " are meant that when the immunne response of expection is relatively the time usually with there not being sex steroid to remove institute, immunocyte can provide more required immunne response.In a kind of situation, immunocyte is the T cell.In other examples, immunocyte is B cell, DC and/or HSC." increase functional " includes but not limited to the killing and wounding target cell of improving, the lymphopoiesis that increases is replied, the signal transduction ability that improves, that improves goes into the nest ability, the APC activation that improves, the receptor that increases, the level of cell adhesion molecule or costimulatory molecules or activity, the apoptosis that lowers, the cytokine that increases, the release of interleukin and other somatomedin, the natural immunity of the antibody horizontal of the blood plasma that increases and the blood of increase and whole body (NKT (NK) cell for example, DC, neutrophilic granulocyte, macrophage etc.) level.Each can help to resist disease and infection directly or indirectly, increases responsiveness, resistance, treatment and prevention to for example infection of various exotic disease substances in view of the above, and increases the immunne response to vaccine.

The present invention also comprises pathological changes or disease, minimizing patient's pathological changes or dangerous or treatment patient's the pathological changes or the method for disease of disease that is used to prevent the patient.In a specific embodiments, disease is the T cell disease.In another embodiment, disease is a kind of autoimmune disease or allergy.In addition, this description also provides and has been used for the signal pathway that mediates by the barrier steroid and causes that the thymus reactivate improves the method for patient to the immunne response of vaccine antigen (for example a kind of vaccine antigen of the cause of disease).In two kinds of situations, can improve and can be by recovering periphery T cell is particularly realized in the periphery T cell pond on the level of T cell originally functional status quantitatively and qualitatively.Then these originally the T cell can respond to existing exotic antigen with bigger degree.

As mentioned above, the thymus of old and feeble (after the adolescence) immunocyte that causes health is to be lower than the horizontal operation of peak level (for example be found in young, preadolescence thymus)." after the adolescence " is defined as the period that thymus has reached obvious atrophy at this.The mankind, this occurs in about 20-25 year, but in a given individuality, this can take place more early or be more late.The period that " adolescence " thymus during this is defined as begins atrophy, but can be before the complete atrophy of thymus.The mankind, this starts from 10-20 year, but one in the given individuality, this can take place more early or be more late." preadolescence " is defined as period of increasing prior to the sex steroid in the individuality at this.The mankind, this occurs in about 0-10 year, but one in the given individuality, this can take place more early or be more late.

Term " inoculation ", " vaccination ", " vaccine ", " immunity ", " immunity inoculation " are defined herein as to the patient and use the preparation of a kind of initiation to antigenic immunne response.Vaccine can comprise preventative and therapeutic vaccine.Such as understood in the art, for example the infection of virus or other pathogen also is a kind of individual method of inoculating.

Term " raising ", " enhancing " or " increase " patient's " vaccine is replied " or " vaccine responsiveness " or " raising ", " enhancing " or " increase " " patient is to immunologic responsiveness of vaccine " and other similar statement can exchange use, and in this immunne response comparison that is defined as meaning and is not having to have taken place among the patient of barrier steroid signal pathway, the patient has been enhanced the immunne response of vaccine or vaccine antigen.

" pathological changes " and " disease " is used interchangeably and is defined as wherein at this that immunne response, defence or modified immunocyte will be useful any disease, infection or medical science pathological changes (Symptomatic or asymptomatic) to the patient.Pathological changes can by venereal infection because of, cancer, Drug therapy (for example chemotherapy), irradiation, chemical poisoning, genetic defect or other diseases caused.

As defined in this, " prevention " of disease or " prevention " disease are defined as fully and the protection of part at this, the alleviating of the severity of the clinical symptoms that will take place including but not limited to the patient.Having the immune individuality that has improved or modified will reduce and to die from or (for example suffer from tumor or cancer, allergy, autoimmune disease, contagious infection, virus, antibacterial, fungus or parasite) or the probability of pathological changes, and/or will show better the replying of vaccination (for example increased to vaccine or the level of antigen-specific antibodies (Ab) and the generation of effector T cell).For example reach degree that only needed to reduce or minimum therapeutic treatment with the generation that suppresses or reduce clinical symptoms and promptly can prevent pathological changes by activating or modify immune defence mechanism.Prevention infection comprises that also body defence venereal infection is because of for example virus, antibacterial, parasite, fungus etc. or defend the non-infectious cause of disease.This can have these causes of disease of prevention to enter the form that the intravital cell of body and/or immune widely cell (for example NK, DC, macrophage, neutrophilic granulocyte etc.) are removed the cause of disease effectively.In some cases, do not realize prevention fully to pathological changes, the substitute is and realized partial prophylaxis, wherein stronger, stronger or more effective immune system will help body to reduce degree, severity and persistent period or the clinical symptoms of pathological changes or the appearance of recovery time or delay clinical symptoms of pathological changes.

" treatment " of pathological changes comprises the symptom of the pathological changes that completely or partially alleviates the patient, when when not having sex steroid is removed or the patient of the signal pathway of barrier steroid mediation will be taken place those symptoms relatively.To suppress, to postpone or reduce the generation of clinical symptoms, the treatment to pathological changes can take place by the activate immunity defense mechanism.In an example, the patient has contacted the cause of disease or the high-risk of the contact cause of disease has been arranged.

The ability of replying, replying better or overcoming that has improved new (prevention) or existing (treatment) pathological changes comprises the immune system that improves body, this comprises the number of the factor that increases BM cell and/or thymus source and/or functional, and/or increases the number of immunocyte and functional.Immune activation also increased can respond to related diseases because of antigenic lymphocytic number, this caused antigen and/or exotic disease because of elimination (wholly or in part), formed the host and treated or tolerate and infect or the state of disease.Having the immune individuality that has improved or modified will reduce and to die from or (for example suffer from tumor or cancer, allergy, autoimmune disease, contagious infection, virus, antibacterial, fungus or parasite) or the probability of pathological changes, and/or will show better the replying of vaccination (for example increased to vaccine or the level of antigen-specific antibodies (Ab) and the growth of effector T cell).

The increase that significantly improves for example clear this immune defence that old mice infects the human herpes simplex vicus (see embodiment 3, and Figure 13-17).The old mice of castrating demonstrates the showed increased of lymphocyte to the infiltration of draining lymph node at first.This infiltration is the first step of immunne response, and it is necessary normally to increase the probability that the antigenic specificity lymphocyte contacts with antigen.Next step be antigen to the growth of lymphocytic activation and Ab and/or CTL and the release of lymphocytic cytokine, all these gangs are eliminated causes of disease.

" venereal infection because of ", " exotic disease because of " and " cause of disease " can be exchanged to be used, and comprises individual disease or any reason of pathological changes.The cause of disease including but not limited to virus, antibacterial, fungus, parasite, Protein virus, cancer, precancerous cell, chemistry or biology toxin, anaphylactogen, the cause of disease of bringing out asthma, the oneself protein that causes autoimmune disease and antigen or the like.

In a kind of situation, the cause of disease is a kind of virus, antibacterial, fungus or parasite, and for example from the envelope protein of human papillomavirus (HPV), it can cause cervical cancer; Or influenza peptides (for example hemagglutinin (HA), nucleoprotein (NP) or neuraminidase.

The non-limiting instance of infectious virus comprises: (for example, HIV (human immunodeficiency virus) is as HIV-1 (also being called HTLV-E, LAV or HTLV-III/LAV or HIV-III) and other separators, for example HIV-LP for retrovirus; Picornaviridae (for example poliovirus, hepatitis A virus, enterovirus, people Ke Shaqi virus, rhinovirus, echovirus); Calciviridae (for example causing the Strain of gastroenteritis); Togaviridae (for example equine encephalitis virus, rubella virus); Flaviviridae (for example, dengue virus, encephalitis, yellow fever virus); Coronaviridae (for example coronavirus, severe acute respiratory syndrome (SARS) virus); Rhabdoviridae (for example stomatitis herpesvirus, rabies virus), Filoviridae (for example Ebola virus); Paramyxoviridae (for example, parainfluenza virus, mumps virus, Measles virus, respiratory syncytial virus); Orthomyxoviridae family (for example, influenza virus); Bungaviridae (for example Hantaan virus, bunga virus, sand fly virus and Nairo virus); Arenaviridae (hemorrhagic fever virus); Reoviridae (for example, reovirus, Orbivirus and rotavirus); Birnavirus section; Hepadnaviridae (for example hepatitis virus B); Parvoviridae (piconavirus); Papovaviridae (papillomavirus, polyoma virus); Adenoviridae (most adenovirus); Herpetoviridae (for example herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (for example, alastrim virus, vaccinia virus, poxvirus); And Iridoviridae (for example African swine fever virus); Not the virus of typing (for example the cause of disease of the cause of disease of the pathogenic cause of disease of spongiform encephalopathy, hepatitis D (thinking the defective satellite of hepatitis B virus), viral hepatitis non-A non-B (1 type=interior propagation; (being hepatitis C), Norwalk and the correlated virus that 2 types=non-enteral is propagated, and Astrovirus).

The limiting examples of infectious bacteria comprises: helicobacter pylori, the Bai Shi spirillum, the pneumonia legionella, mycobacteria zygoblast (sporozoites) (for example, mycobacterium tuberculosis, mycobacterium avium, Mycobacterium intracellulare, mycobacterium kansasii, the Ge Dengshi mycobacteria), staphylococcus aureus, Diplococcus gonorrhoeae, Neisseria meningitidis, listerisa monocytogenes in mjme, micrococcus scarlatinae (A group B streptococcus), streptococcus agalactiae (B group B streptococcus), streptococcus (viridans group), streptococcus faecalis, bargen's streptococcus, streptococcus (anaerobism Pseudomonas), streptococcus pneumoniae, pathogenic Campylobacter, enterococcus, hemophilus influenza, Bacillus anthracis, corynebacterium diphtheriae, rod bacillus zygoblast, erysipelothrix rhusiopathiae, bacillus perfringens, clostridium tetanus, clostridium perfringen, klebsiella pneumoniae, multocida, Bacteroides, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponoma palladium, superfine treponema, leptospira, and actinomyces israelii (Actinomyces israelli).

The limiting examples of infectious fungus comprises: Cryptococcus histolyticus, Histoplasma capsulatum, Blastomyces coccidioides, Blastomyces dermatitidis, chlamydia trachomatis, Candida albicans.

Other infectious organisms (being protista) are including but not limited to Plasmodium falciparum and toxoplasma gondii.

In other specific embodiments, the cause of disease is a kind of anaphylactogen.The anaphylaxis pathological changes is including but not limited to eczema, allergic rhinitis or rhinitis, Hay Fever, rubella (urticaria) and food anaphylaxis and other atopy pathological changes.

Still in another embodiment, the cause of disease is a kind of cancer or tumor.Cancer or tumor can be virulent or nonmalignant.For example comprise in this used tumor or cancer, brain, pulmonary's (for example minicell with non-small cell) and pleura, gynecological, urogenital tract and endocrine cell (cervix uteri for example, the uterus, inner membrance, bladder, kidney, ovary, mammary gland and/or prostate), gastrointestinal tract (for example, anus, biliary tract, carcinoid, gallbladder, stomach or stomach, liver, esophagus, pancreas, rectum, small intestinal and/or colon), and other cancer, and bone, skin and connective tissue are (for example, melanoma and/or sarcoma), and/or blood system (for example, blood, myelodysplastic syndrome, myeloproliferative disease, plasma cell tumor, lymphoma and/or leukemia) tumor.

The present invention can be used to any maturation and immune animal species (comprising the people), for example mammal and marsupial that sex steroid drives that have.In some instances, invention is used to for example people of large mammal.

Term thymus " regeneration ", " reactivate " and " reconstruction " and their derivant can be exchanged use at this, and are defined as atrophy or impaired (for example by chemicals, irradiation, graft versus host disease, infection, genetic predisposition) thymus at this and return to its state of activation." state of activation " this be defined as looking like be T cell output that the patient's that has been blocked of the signal pathway of wherein sex steroid hormone mediation thymus is finished be preadolescence thymus (not arriving hebetic patient's thymus) output at least 10% or at least 20% or at least 40% or at least 60% or at least 80% or at least 90%.

" receptor ", " patient " and " host " can be exchanged at this and be used, and be defined as the object of treatment that the acceptance steroid is removed the signal pathway of treatment and/or the mediation of barrier steroid at this, in due course, for having accepted the object that HSC transplants." donor " in this source that is defined as transplanting, it can be isogenic, heterogenic or xenogeneic.In some cases, the patient can be provided for being transplanted on a little time point in evening for example his or his autogenous cell in the patient.Can use allogene SHC graft, and these allografts are the grafts between the member of not matching that occur in same species, and in xenogenesis HSC graft, donor and receptor are different plant species.Also can use the homogenic HSC graft between the coupling animal.Being defined as donor and receptor's MHC and/or less important histocompatibility label with reference to the term " coupling " of HSC graft, " unmatched ", " mispairing " and " not quite identical " at this is identical (coupling) or be not that (unmatched, mispairing with not quite identical) is identical.

In the full text of this description, term " comprises " and " comprising " will be understood to mean that the group that comprises described element, composition or step or element, composition or step, but does not get rid of the group of other any elements, composition or step or element, composition or step.

The blocking-up of the signal pathway of sex steroid mediation

The present invention also provides the method for signal pathway of blocking-up patient's sex steroid mediation, wherein patient's thymus subsequently can by or be not re-activated.In addition, the invention provides the method for the functional status of the immunocyte (for example T cell) that improves the patient.With regard to the T cell, thymus begins to increase the growth rate of early stage precursor (CD3-CD4-CD8-cell) and they is converted into CD4+CD8+ and new mature C D3 subsequently ^HiCD4+CD8-(T assists (Th) lymphocyte) or CD3 ^HiCD4-CD8+ (cytotoxic T cell (CTL)).The thymus that brings back to life has also increased it maybe can form the picked-up of the precursor of T cell to the HSC in the blood flow or other stem cell, and they are converted into DC in new T cell and the thymus.The activity of the thymus that is increased all is similar to the activity of being found in normal younger thymus (for example preadolescence) aspect a lot.The result of the thymus output of this renewal has increased the level of the cell of T originally in the blood (those did not also run into antigenic T cell).This has also increased periphery T cell to for example stimulating by with the crosslinked of anti-CD28Ab or by with the TCR stimulation of for example anti-cd 3 antibodies or the mitogen ability of replying of the stimulation of pokeweed mitogen (PWM) (PWM) for example, and the t cell response of this increase can occur in before the thymus reactivate, for example in 2,3,4,5,6,7,14 or 21 days.The compositions of this incident causes body to become to defend better the immune attack of infecting with other (for example cancer), or recovers (for example, become and can recover better) from immune system attack from chemotherapy and radiation.Therefore, method of the present invention can be used to prevention or treatment pathological changes or disease, increases the patient to the immunologic responsiveness of vaccine and optionally be used for gene therapy.

As used in this, the interruption of " sex steroid removing ", " inhibition of the signal pathway of sex steroid mediation ", " sex steroid blocking-up ", sex steroid signal pathway " term similar with other is defined as to the generation of small part barrier steroid (and/or other hormones) and/or sex steroid (and/or other hormones) signal pathway at this, no matter is direct or indirect effect.In one embodiment, interrupted arriving the sex steroid signal pathway of thymus.Be understood that easily, with the whole bag of tricks well known in the art can the barrier steroid signal pathway of mediation, at this certain methods has been described.For example, the inhibition that gonadal hormone is produced or block one or more sex hormone receptor and will reach required blocking-up, as grant sex steroid agonist and/or agonist, or (antigen) initiatively or passive (antibody) resistance steroid inoculation.

A kind of method of blocking-up of the nonrestrictive signal pathway that is used to form sex steroid mediation is by castrating.The method that is used to castrate includes but not limited to chemical castration and operation castrating.

" castrating " is defined as reducing or eliminating of sex steroid generation, activity and/or intravital distribution at this.This finally returns to the patient preadolescence state effectively, and this moment, thymus was that function is more arranged before castrating at once.Patient's gonad has been removed in the operation castrating.The method of castrating that is used to perform the operation is all known for the veterinary and the doctor that are subjected to conventional training.A kind of nonrestrictive method that is used to castrate buck has been described in the following embodiments.Other nonrestrictive methods that are used to castrate human patients comprise that uterectomy or ovariectomy operation (castrating women) and operation castrating remove testis (castrating the male).In some clinical cases, it may be suitable for good and all removing gonad through the physics castrating.

Chemical castration is a kind of more not permanent castrating form.As defined in this, " chemical castration " is to grant a kind of chemicals a period of time, caused sex steroid generation, effect and/reducing or eliminating of distributing in the body.The number of chemical thing can play a role by this way.The limiting examples of these chemicals is sex steroid mortifier and/or following described analog.During giving chemicals and after giving a period of time, patient's hormone produces and can be stopped or reduce.Stop giving or can reversing castrating of chemicals by the gonadal hormone of being correlated with.

Term " sex steroid analog ", " sex steroid scavenger ", " sex steroid inhibitor ", " inhibitor of sex steroid signal pathway ", " dressing agent of sex steroid signal pathway " term similar with other are defined as any one or more at this and can reduce, block, prevent or stop the medicinal reagent of the signal pathway of sex steroid (and/or other hormones) mediation.GnRH (also being called LHRH or GnRH/LHRH at this) and their analog are the non-limiting instance of the inhibitor of sex steroid signal pathway used in the full text of the application's book.Yet, those skilled in the art are understood that easily, when putting into practice in the invention that this provided, can substitute GnRH/LKRH described herein or their analog with any (or multiple) of (or other blockeres or physics castrating) in multiple alternate sex steroid inhibitor or the analog, and not need undue experimentation.

Any can the barrier steroid or the medicine of the signal pathway of barrier steroid mediation or other castrating method can be used to method of the present invention.For example, functional non-limiting method of inhibition steroid signal pathway, reactivate thymus and/or enhancing BM and immunocyte is to reduce the gonad generation or discharge normal sex steroid the normal effect (promptly discharging promoting sexual gland hormone, FSH and LH) of hypophysis is also final by modifying GnRH.Therefore, in a kind of situation, sex steroid is removed by granting for example GnRH analog realization of one or more gonadal hormone analog.GnRH is the short property sex gland hormones of a kind of stimulation hypophysis secretion, the hypothalamic decapeptide of metakentrin (LH) and follicle stimulating hormone (FSH).Therefore, GnRH agonist (for example form of Synarel_ or Lupron_) causes the overstimulation of receptor at first and generates FSH and LH through feedback mechanism by the quick hypophysis that will suppress of the mistake of LHRH receptor.These promoting sexual gland hormone normally act on gonad with the estrogen of release property steroid, particularly women and male's testosterone, and the disappearance of FSH and LH has reduced the release of these gonadal hormone significantly.Its direct result be sex steroid blood plasma level decline and therefore to the progressively release of the inhibition signal of thymus.For example by using the GnRH antagonist can realize the decline faster of circulation sex steroid level.

In some embodiments, by grant the sex steroid analog for example the analog of luteinising hormone-releasing hormo (LHRH) blocked the signal pathway of sex steroid mediation.Sex steroid analog and their application in treatment and chemical castration are known.The sex steroid analog be commercialization and their the treatment and chemical castration on application know.These analog include but not limited to, below the agonist of LHRH receptor (LHRH-R): buserelin (buserelin acetate for example, trade name Suprefact_ (for example 0.5-02mg/ days, subcutaneous usefulness), SuprefactDepot_, with Suprefact_ nasal spray (2 μ g/ nostrils for example, per 8 hours), Hoechst, also be described in U.S. No.4,003,884,4,118,483 and 4,275,001), Cystorelin_ (diacetic acid promoting sexual gland hormone four hydrates for example, Hoechst), deslorelin (for example, the acetic acid deslorelin, Deslorell_, Balance Pharmaceuticals), gonadorelin (for example, Factrel, trade name Factrel_ (100 μ g veins or subcutaneous usefulness), Ayerst Laboratories), goserelin (goserelin acetate, trade name Zoladex_, AstraZeneca, Aukland, NZ, also be described in and be described in U.S. No.4,100,274 and 4,128,638, GB 9112859 and GB9112825), histrelin (for example, Supprelin (Roberts)., Supprelin_ (subcutaneous 10 μ g/kg/d), Ortho, also be described in EP 217659), leuprorelin (leuprolide) (the bright third sharp moral, trade name Lupron_, or Lupron Depot_, Abbott/TAP, Lake Forest, IL, also be described in U.S. No.4,490,291,3,972,859,4,008,209,4,992,421, with 4,005,063, DE 2509783), leuprorelin (leuprorelin) (for example, the bright third sharp moral, trade name ProstapSR_ (for example, the subcutaneous or intramuscular injection of single dose 3.75mg in every month), Prostap3_ (single dose 11.25mg was subcutaneous in for example per 3 months), Wyeth, USA, also be described in Plosker et al., (1994) Drugs 48:930), lutrelin (Wyeth, USA, also be described in U.S. No.4,089,946), meterelin _ (for example, Avorelina (10-15mg slow release formulation), also be described in EP 23904 and WO 91/18016), Na Faruilin (for example, trade name Synarel_ (i.n.200-1800 μ g/d), Syntex also is described in U.S. No.4,234,571, WO 93/15722, with EP 52510), and triptorelin (for example, is pounced on love song Pu Ruilin, trade name Trelstar LA_ (11.25mg in 3 months), Trelstar LA Debioclip_ (is pre-charged with, single dose gives), LA TrelstarDepot_ (3.75mg in 1 month), and Decapeptyl_, Debiopharm S.A., Switzerland also is described in U.S. No.4,010,125,4,018,726,4,024,121, with 5,258,492, EP364819).The LHRH analog also includes but not limited to the antagonist of following LHRH-R: 1: PN: WO02056903 PAGE: 25 claimed protein (trade name Plenaxis ^TM(for example, at the 1st, 15 and 29 day intramuscular injection 100mg, per afterwards 4 all intramuscular injection 100mg), Praecis Pharmaceuticals, Inc., Cambridge, MA) and cetrorelix (for example, cetrorelix acetate, trade name Cetrotide ^TM(for example, subcutaneous 0.25 or 3mg), Zentaris, Frankfurt, Germany).Other sex steroid analog comprises that Eulexin_ (for example, flutamide (for example, 2 capsules, every day 2 times, total amount 750mg/d), Schering-PloughCorp., also be described in FR 7923545, WO 86/01105 and PT 100899) and dioxane derivatives (for example, described in EP413209) and for example at EP 181236, U.S. No.4,608,251,4,656,247,4,642,332,4,010,149,3,992,365 and 4,010, other LHRH analog described in 149.Also comprise the combination of agonist, the combination of antagonist and the combination of agonist and antagonist.A kind of nonrestrictive analog of the present invention is deslorelin (U.S. No.4,218,439 is described).For the tabulation widely of analog, see Vickeryet al. (1984) LHRH AND ITS ANALOGS:CONTRACEPTIVE﹠amp; THERAPEUTIC APPLICATIONS (Vickery etal., eds.) MTP Press Ltd., Lancaster, PA.Also can use the modified forms of every kind of analog, for example its acetic acid, citric acid and other salt, these are known for those skilled in the art.

The non-limiting instance that sex steroid is removed the administration of medicine is that subcutaneous/intradermal injection GnRH agonist " slow release " (for example stores agent (depot), 1,3 or 4 months Lupron_ injection) or subcutaneous/intradermal injection " slow release " contain GnRH implant (for example, 1 or 3 months Zoladex_, for example 3.6mg or 10.8mg implant).According to suitable dosage form, also can intramuscular (i.m.), intravenous (i.v.) or these medicines of orally give.Another example is to contain by subcutaneous injection (for example to have an appointment 30mg Lupron_, Lupron Depot_ (the storage suspension of the bright third sharp moral), TAP Pharmaceuticals Products, Inc., Lake Forest, " storage agent " IL) or " impregnability implant ".30mg Lupron_ is enough to remove sex steroid 4 months to allow that thymus recovers and export the new cell of T originally in blood flow.

The mechanism of many transplantability steroid signal pathways described herein is known, and some medicines wherein particularly the GnRH agonist be used to treat genital organ disease for example some hormone-sensitive cancers comprised breast carcinoma and carcinoma of prostate, endometriosis, reproductive disease, hirsutism, precocity, property variation and control fertility a lot of years.

In some instances, sex steroid is removed and/or the interruption of the signal pathway of sex steroid mediation or patient's the thymus of having blocked final reactivate.In some cases, blocking-up has reversed patient's hormone state.The method according to this invention, receptor's hormone state has been reversed, and makes receptor's hormone state near the preadolescence level.The level of the sex steroid hormone by reducing the receptor has reduced the signal pathway of these hormones to thymus, allows reactivate thymus in view of the above.The patient can be hebetic or postpubertal, or the patient had (or once having) disease to small part atrophy thymus.Same, the patient had (or once having) treatment of diseases, wherein treatment of diseases to small part was caused patient's atrophy of thymus gland.These treatments can be antiviral, immunosuppressant, chemotherapy and/or radiotherapy.In other embodiment, the patient be menopause or taked other modes for example institute such as wound, medicine reduced sex steroid (or other hormonal readinesses).

Sex steroid is removed or the blocking-up of the signal pathway of sex steroid mediation is had one or more direct effect to BM and/or immune cell, has wherein improved functional.These effects can take place before the thymus reactivate or with it simultaneously.

In some embodiments, by individually or unite a kind of LHRH analog or any other castrating method and grant a kind of androgen antagonist for example the androgen blocker is (for example, bicalutamide, trade name Cosudex_ or Casodex_, 5-500mg for example, the oral QID of 50mg for example, AstraZeneca, Aukland NZ) realizes that sex steroid is removed or to the inhibition of sex steroid signal pathway.Also can be by individually or unite a kind of LHRH analog or any other castrating method is granted cyproterone acetate (trade name, Androcor_, Shering AG, Germany, 10-1000mg for example, 100mg bd or tds, or IM 300mg weekly), the 17 that act as progesterone realizes that sex steroid is removed or to the blocking-up of sex steroid signal pathway.The androgen antagonist that can use other (for example, the antifungal drug of imidazoles, liarozole (Liazol_ for example, for example, 150mg/ days, a kind of aromatase inhibitor) and ketoconazole, flutamide (trade name Euflex_ and Eulexin_, Shering Plough Corp, N.J, for example 250 or the oral QID of 750mg), megestrol acetate (Megace_, for example 480-840mg/ days, or nilutamide (trade name Anandron_ and Nilandron_, Roussel, France, for example 150-300mg/ days).Androgen antagonist often is important in treatment, because they are normally used for alleviating the short-term potentiation that the GnRH analog is caused.Some androgen antagonist play a role by the displacement that suppresses androgen receptor, and this has disturbed negative feedback, have caused the increase of testosterone levels and the minimum disappearance of libido/ability.The androgen antagonist that is used for another kind of type of the present invention be selective androgen receptor instrumentality (SARMs) (for example, quinoline, bicalutamide (trade name Cosudex_ or Casodex_, the same) and flutamide (trade name Eulexin_, for example oral 250mg/ days)).Other androgen antagonist of knowing comprise 5 alpha reductase inhibitors (for example, dutasteride's (for example, oral 0.5mg/ days), it suppressed two kind of 5 alpha-reductase isozyme and cause more and faster DHT suppress; Finasteride (trade name Proscar_, 0.5-500mg, for example every day oral 5mg), it has suppressed the generations of 5 alpha-reductases 2 and the final DHT of inhibition, but has only very little or not effect to testosterone or LH level).

In other embodiment, by individually or unite that a kind of LHRH analog or any other castrating method grant that estrogen antagonist realizes that steroid is exhausted or to the inhibition of the signal pathway of sex steroid mediation.Some estrogen antagonists (for example, Anastrozole (trade name Arimidex_ and fulvestrant (trade name Faslodex_, 10-1000mg, every month 250mg IM for example) plays a role by combining with estrogen receptor (ER), and finally suppressed combining of estrogen and receptor with the high-affinity that is similar to estradiol.Also targeting is in the conformation change of receptor and downward modulation estrogen receptor in the combination of Faslodex_, and FSH or LH level do not have significant change.Antiestrogenic other non-limiting instance are tamoxifen (trade name Nolvadex_); Clomifene (trade name Clomid_), for example 50-250mg/ days, a kind of nonsteroidal ER part with mixed agonist/antagonist performance, it has stimulated the release of promoting sexual gland hormone; Diethylstilbestrol (trade name Stilphostrol_), for example 1-3mg/ days, it demonstrates similar to estrone but stronger estrogen activity, therefore it is considered to a kind of estrogen agonist, but it can combine with androgen and estrogen receptor to induce the FSH of hypophysis and the feedback suppression of LH generation, diethylstilbestrol diphosphate for example 50 to 200mg/ days; And danazol, droloxifene and Iodoxyfene, each all act as antagonist.Can be separately or unite another kind of estrogen antagonist that other castrating method uses be selective estrogen receptor instrumentality (SERMs) (for example, toremifene (trade name Fareston_, 5-1000mg, the oral QID of 60mg for example), Raloxofene (trade name Evista_) and tamoxifen (trade name Nolvadex_, 1-1000mg, the oral bd of 20mg for example), it act as the antagonist of the estrogen receptor of the agonist of estrogen receptor of bone and cardiovascular system and mammary gland).Estrogen receptor downward modulation thing (ERD) (for example tamoxifen (trade name Nolvadex_)) also can be used to the present invention.

Other non-limiting instance of can be individually or uniting the method for the inhibition steroid signal pathway that other castrating method uses comprise aromatase inhibitor and other gonad blocker (for example, aminoglutethimide, formestane, vorazole, exemestane, Anastrozole ((trade name Arimidex_; 0.1-100mg, the oral QID of 1mg for example), it has reduced estradiol and has increased LH and testosterone), letrozole (trade name Femarat_, for example oral QID of 2.5mg) and exemestane (trade name Aromasin_, 1-2000mg, for example 25mg/ days); Aldosterone antagonists (for example, spironolactone (trade name Aldactone_), for example 100 to 400mg/ days), it act as blocking-up androgen cytochrome P-450 receptor); And eplerenone, a kind of selectivity aldosterone receptor antagonist); Gestation (medroxyprogesterone acetate for example, for example 5mg/ days, it is synthetic and LH is synthetic that it has suppressed testosterone); And Progesterone and anti-Progesterone for example selective progesterone reply instrumentality (SPRM) (for example, megestrol acetate, for example 160mg/ days, mifepristone (RU486, Mifeprex_, for example 200mg/ days); And other has the active chemical compound of estrogen/estrogen antagonist (for example, phytoestrogen, flavone, isoflavone and coumestan derivant, a lignanoid and have the industrial chemical (for example DDT) of phenol ring.Also can use anti-GnRH vaccine (to see Hsu et al. for example, (2000) Cancer Res.60:3701; Talwar, (1999) Immunol.Rev.171:173-92) or other any medicines that can simulate the effect that said medicine produces.In addition, also can develop and use the instrumentality based on steroid receptor, it can be special in thymus and/or BM by targeting.Number of mechanisms in the mechanism of these inhibition steroid signal pathways is all known.Also can use the modified forms of every kind of medicine, for example their acetic acid, citric acid and other salt, these are known for those skilled in the art.

Because the feedback mechanism of the braiding complexity of hormone system and mutual, sex steroid grant the inhibition that may cause the sex steroid signal pathway.For example, estradiol has reduced the generation of promoting sexual gland hormone and to the sensitivity of GnRH effect.But higher levels of estradiol causes promoting sexual gland hormone to discharge (surge) in a large number.Same, progesterone influences frequency and the number that LH discharges.The male, testosterone has suppressed the generation of promoting sexual gland hormone.The estrogen of granting among the male has reduced LH and testosterone, and estrogen antagonist has increased LH.

In other embodiments, the lactotropin that has suppressed the patient.Another method of the signal pathway of inhibition steroid mediation can be the method by direct or indirect adjusting lactotropin level.Lactotropin is a kind of synthetic as incretogenous single chain protein hormone.The range of normal value of male and non-pregnant female lactotropin is normally from about 0 to 20ng/ml, but in pregnancy is female, scope normally about 10 to 300ng/ml.Reported total hundreds of different effect of lactotropin.Lactotropin stimulates female development of breast and milk to produce.The known exception lactotropin is involved in pituitary tumor, menoxenia, sterile, sexual impotence and lactogenic (milk generation).The effect of lactotropin in the immunne response of normal and pathology inquired in a considerable amount of researchs step by step.Find that lactotropin all has regulating action in the many aspects of immunologic function, illustrate on evidence that also hyperprolactinemia is immunosuppressant (Matera L, Neuroimmunomodulation.1997Jul-Aug; 4 (4): 171-80).Grant the lactotropin and the decline of the survival rate of infecting mouse and inhibition relevant (Oberbeck R, the J Surg Res.2003 Aug of cellular immune function of pharmaceutical dosage; 113 (2): 248-56).Also there is multiple medicine can damage and causes hyperprolactinemia the dopaminergic inhibition of lactotropin.The dopamine antagonist medicine (for example comprises haloperidol, fluphenazine, dogmatil, metoclopramide and short digestive tract power medicine, bromopride, clebopride, domperidone and levosulpiride), they are used for the treatment of upper gastrointestinal motion pathological changes by clinical.

Responding to inhibin A and the B peptide that promoting sexual gland hormone generated in gonad has reduced hypophysis and has suppressed FSH.Activin (Activin) raises the GnRH receptor usually and stimulates FHS synthetic, but excessive generation can stop the generation of sex steroid.Therefore these hormones also can be the target spots to the inhibition of the signal pathway of sex steroid mediation.

In some embodiments, a kind of LHRH-R antagonist being administered to the patient, is a kind of LHRH-R agonist subsequently.For example, can grant antagonist to cause the castrating (this is normal for for example Abarelix) in 5-8 days with the single injection of sufficient dosage.When sex steroid has reached this castrating level, give agonist.This scheme is eliminated or has been limited at sex steroid and produces any spike that the sex steroid before reducing produces, and grants agonist and may produce such spike.In another alternate embodiments, used to produce LHRH-R agonist very little or that do not produce sex steroid generation spike, this can use or not use the LHRH-R antagonist in advance.

Inhibition to the sex steroid signal pathway

Gonadal hormone comprises the hormone molecule of a large amount of androgens, estrogen and Progesterone family.The nonrestrictive member of the Progesterone family of C21 steroid comprises progesterone, 17 α-hydroxyprogesterone, 20 α-hydroxyprogesterone, pregnanedione, pregnanediol and pregnenolone.The nonrestrictive member of the androgen family of C19 steroid comprises testosterone, androstenedione, dihydrotestosterone (DHT), androstanedione, Androstandiol, dehydroepiandrosterone and 17 α-hydroxyandrostenedione.The nonrestrictive member of the estrogen family of C17 steroid comprises estrone, estradiol-17 α and oestradiol-17.

The signal pathway of sex steroid is the net result of complicated consequence that comprises the approach ingredient of biosynthesis, secretion, metabolism, compartmentation (compartmentalization) and effect.The part of this approach is not also understood fully, yet, still have multiple existing and potential mechanism to be used to realize inhibition to the sex steroid signal pathway.In one aspect of the invention, by the obtainable sex steroid level of the horizontal modified biological of cellular level promptly so-called " dissociating ", by change biosynthesis or metabolism, with target cell on or interior sex steroid receptor combine and/or the cell of sex steroid in signal pathway can realize inhibition to the sex steroid signal pathway.

It is possible influencing signal pathway directly or indirectly.Direct method comprise the biosynthesis that influences sex steroid and metabolism, with corresponding receptor combine and the cell of signal in the method for modifying.The method that indirect method comprises the generation of those known effect sex steroid hormones and effect is existing peptide hormone and somatomedin in hypophysis body of gland and the gonad for example.The latter includes but not limited to the inhibin that FSH, LH and activin and gonad generated, activin and the insulin-like growth factor-i (IGF-1) that the hypophysis body of gland is generated.

Those skilled in the art will recognize by in the level of relevant hormone, enzyme, receptor, binding molecule and/or part or by comprising coding to the direct effect of molecule or by the precursor to molecule or regulating its nucleic acid, maybe can modify and carry out above-mentioned modification in the effect of molecule of sex steroid effect inhibition to the sex steroid signal pathway can take place.

The direct method that suppresses signal pathway

Biosynthesis

Biosynthetic speed is that steroid hormone produces and " dissociate " the main rate-limiting step of bioavailability of hormone of serum.To key enzyme early stage in the approach for example the inhibition of P450 cholesterol side chain cleavage (P450scc) will reduce the generation of all main sex steroids.On the other hand, the enzyme of back in the approach for example is converted into androgen estrogenic P450 aromatase (P450arom) or testosterone is converted into the inhibition of 5 alpha-reductases of DHT, will only influence the generation of estrogen or DHT respectively.Biosynthetic another the important aspect of sex steroid hormone is the oxidoreduction enzyme family of changing mutually between the inactive and bioactive steroid of catalysis, for example mutual conversion of 17OHS dehydrogenase (17-HSD) catalysis androstenedione and testosterone or estrone and estradiol-17.These enzymes are catalytic reduction tissue and cell-specific and common or oxidation reaction, for example only found 3 types, 17 β HSD in the Leydig of testis cell, and found 1 type, 17 β HSD in ovary.Therefore they provide the probability that reduces the generation of androgen or estrogenic activity form specifically.

There is the inhibitor of the enzyme in the known steroid biosynthesis pathway of many kinds to be used to clinical practice or in development.Some examples in these inhibitor and their treatment pattern have been enumerated below.Importantly exceedingly not influence other steroid be the important adrenal glucocorticoid and the generation of mineralocorticoid to metabolic stability for example in the effect of these enzyme inhibitors.When using these inhibitor, it may be essential alternate glucocorticoid being provided and mineralocorticoid is provided sometimes to the patient.

The biosynthesis of sex steroid occurs in a plurality of positions and utilizes number of ways, mainly is to produce in ovary and testis, but some are arranged is to produce in the adrenal gland, and at other the synthesis of derivatives in the fat of for example organizing.Therefore, may need a plurality of mechanism of inhibition steroid signal pathway to guarantee to suppress fully to realize the present invention.

Metabolism and compartmentation (compartmentalization)

The half-life of sex steroid in blood is short, only is several minutes usually, and this is because of tachymetabolism, particularly liver, and the removing of kidney and fat.Metabolism comprises glycosylation and Sulfated association reaction, and reduction reaction.In these metabolite some remain with biologic activity, this biologic activity or incretogenous for example estrogen sulfate, or for example go back proandrogens by intrinsic biologic activity.Can both influence " dissociating " level of sex steroid hormone to any interference of accretion rate.But, also do not obtain the method for the realization this point the same with influencing biosynthetic method.

Another method that reduces " free " sex steroid hormone level is the albumen bonded compartmentation of sex hormone binding globulin, hydrocortisone hormonebinding globulin, albumin and testosterone-estradiol binding globulin for example in trafficability characteristic steroid hormone and the serum.With the sex steroid part for example combining of carrier molecule make and can not obtain sex steroid with receptors bind.The carrier level for example SHBG increase or introduce other with the bonded part of sex steroid for example soluble recepter can cause bonded increase.Perhaps, the carrier molecule of reduction level can be so that sex steroid be more responsive to degraded.

Resisting the active or the passive immunity inoculation of special sex steroid hormone is a kind of compartmentation of form.In this method has successfully increased the document of ovulation rate of animal after estrogen antagonist or androgenic immunity inoculation, example is arranged.From the secretion vesicle, secrete sex steroid.Inhibition or modification to mechanism of secretion are the another kind of methods of inhibition steroid signal pathway.

Signal pathway in receptor and the cell

Sex steroid through in the cell or as the special receptor acting on target cell membrane shown in recently in cell.

Intracellular receptor is the member of nuclear receptor superfamily.They be positioned at cell cytoplasm and be transported to nuclear after sex steroid hormone combines, here they have changed transcribing of specific gene.The receptor of sex steroid hormone exists in a variety of forms.What know in the literature is the progesterone receptor of two kinds of forms, PRA and PRB, and the estrogen receptor of three kinds of forms, ER α, ER β 1 and ER β 2.Can modify the bonded gene transcription of steroid response element that responds in sex steroid hormone receptor and the gene promoter region with several different methods.Be present in coactivator in the target cell nuclear and corepressor and can both modify combining and influence in view of the above and transcribing of sex steroid-receptor complex and DNA.To the multiple evaluations in these auxilliary exciting things and the corepressor is known, and to modify them be the focus of research at present to the method for the effect of sex steroid receptor.The example of the transcription factor that sex steroid hormone is comprised on is NF-1, SP1, Oct-1 and TFIID.The effect fully of steroid needs these auxiliary adjustment things.The methods of effect of modifying these nuclear instrumentalities may relate to the balance between exciting thing and the mortifier, by using antagonist or through the expression of gene of control coding and regulating thing.In addition, c-AMP.

Recently, identified the special receptor of estrogen and progesterone on cell membrane, their structure is different from PR in the cell.Do not resemble the classical genomic steroid receptor that acts on, these receptors by approach transmission in the cell of also not being familiar with fully fast, the effect of non-genomic group.A report shows, has activated the sphingol approach relevant with cell proliferation with the interactional estrogen of membrane receptor.

The method that the effect of obtainable or developing cytoplasm receptor change sex steroid through sex steroid has been arranged.In this situation, with the interactional androgen antagonist of special steroid receptor, estrogen antagonist and gestation be in the literature know and be used in the clinical practice, as described above.Their effect may be competition or level, sensitivity, conformation, associating or the signal pathway of blocking receptor, modified receptor.These medicines can be various forms of, steroid with nonsteroidal, emulative and noncompetitive.Relevant especially is the selective receptor instrumentality, SARM, SERM and SPRM, they are targeting in particular tissues and as above illustrated.

Have two kinds of methods can realize downward modulation to receptor: the first, by excessive agonist (steroid part), and the second, the transcribing of the corresponding gene by suppressing the coding receptor.By use selective agonist for example tamoxifen can realize first method.

The indirect method that suppresses signal pathway

Biosynthesis

Wherein a kind of indirect method of inhibition steroid signal pathway comprises by the biosynthesis to the modification downward modulation related steroid of the availability of the biosynthetic pituitary gonadotropic hormone FSH that be responsible for to drive the sex steroid hormone in the gonad and LH or effect.The excretory inhibitor of a kind of FSH that has set up is an inhibin, and a kind of gonad responds to the hormone that FSH produces.Be shown to animal and granted the FSH level that inhibin has reduced serum, because the minimizing of hypophysis secretion FSH.The best known method of realizing the minimizing of two kinds of promoting sexual gland hormone is through hypothalamic hormone GnRH/LHRH, and it drives synthetic justacrine FSH of hypophysis and LH.Therefore the agonist and the antagonist that have reduced the secretion of FSH and LH and reduced the GnRH that the steroid of gonad produces have been used to clinical practice now, and be as the described herein.

The another kind of biosynthetic indirect method that reduces sex steroid hormone is the effect of modifying FSH and LH in the gonad level.Antibody by using facedown FSH and LH or be designed for their molecule of associated receptor that produces on the gonadal cell of sex steroid hormone with FSH and LH competition and can realize this point.Another kind of FSH and the LH of modifying is auxiliary adjustment thing by a kind of promoting sexual gland hormone effect to the method for the effect of gonadal cell.For example, activin can weaken the theca cell of ovary and the Leydig cell response LH of testis produces androgenic ability.

Modification can occur in the level of hormone precursor for example to for example cracked inhibition of the signal peptide of GnRH of signal peptide.

Signal pathway in receptor and the cell

The indirect method that changes the signal pathway effect of sex steroid hormone comprises that downward modulation causes the genome of steroid and the receptor pathway of non-genomic group effect.Its example is the ability of the ER level of progesterone downward modulation target tissue.Following method comprises the processing of utilization to the molecule of the auxiliary adjustment thing of the endonuclear receptor of known effect, causes the reduction of cell to the ability of replying of steroid.

Other factors

Though be used for, BM lymphocyte production functional and immune cell function to BM directly and the stimulation of indirect action mainly be to be dependent on to the inhibition of the effect of sex steroid and/or the direct effect of LHRH analog, can act as and strengthen together or increase (add up, collaborative or replenish) thymus, BM and/or immunocyte effect and functional additament also may be useful.Can use or not use additament.These chemical compounds comprise, but be not limited to cytokine and somatomedin, for example interleukin-2 (IL-2,100,000 to 1,000,000IU, for example 600,000IU/kg, per 8 hours repeated doses), interleukin-7 (IL-7,10ng/kg/ days to 100ng/kg/ days, according to the treatment needs), interleukin-15 (IL-15; 0.1-20m μ g/kg/ days), interleukin-11 (IL-11; 1-1000 μ g/kg); Epithelium and fibroblast growth family member, stem cell factor (SCF; Also be known as steel factor (steel factor) or c-kit part; 0.25-12.5mg/ml), granulocyte colony-stimulating factor (G-CSF; 1 and 15 μ g/kg/ days, IV or SC), granulocyte macronucleus colony stimulating factor (GM-CSF; G/ square metre/day of 50-1000 μ, SC or IV), insulin-dependent somatomedin (IGF-1) and keratinocyte growth factor (KGF; 1 μ g/kg was by 100mg/kg/ days) (see for example Sempowski et al., (2000) J.Immunol.164:2180; Andrew and Aspinall, (2001) J.Immunol.166:1524-1530; Rossi et al., (2002) Blood 100:682); Erythropoietin (EPO; 10-500 unit/kg, IV or SC).Be used for the present invention simultaneously or other suitable Hemopoietic factors of granting altogether continuously, CSF, cytokine, lymphokine, the example of the non-exclusionism of hemopoietic growth factor and interleukin comprises Meg-CSF (megakaryocyte colony stimulating factor is called as the c-mpl part recently), MIF (macrophage inhibition factor), LIF (leukaemia inhibitory factor), TNF (tumor necrosis factor), IGF, platelet derived growth factor (PDGF), M-CSF, IL-1, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-12, IL-13, LIF, flt3/flk2, the human growth hormone, Bcell growth factor, B cell differential factor and eosinophilic granulocyte's differentiation factor or their combination.

When initial LHRH analog (or other castrating method) is used, can give once one or more of these additional compounds.Other any forms that can unite agonist, antagonist or sex steroid blocking-up give every kind of treatment.Because somatomedin has the fast relatively half-life (for example a few hours), they may need administration every day (for example administration every day totally 7 days or the longer time).As manufacturer's defined, the somatomedin/cytokine that can give optimised form is to preserve their biological activity, as the form of purifying protein.But the additional dose that can give any of these materials or combination at any time is functional with further stimulation BM and other immunocytes.In some cases, carry out simultaneously with the administration of additional cytokine, somatomedin or their combination that sex steroid is removed or to the blocking-up of sex steroid signal pathway.In other cases, carry out successively with the administration of additional cytokine, somatomedin or their combination that sex steroid is removed or to the blocking-up of sex steroid signal pathway.

Term " mobilization agent " is defined as being meant for example SDF (for example AMD3100), growth hormone, GM-CSF, G-CSF and chemotherapy medicaments such as (for example cyclophosphamide) at this, and they have strengthened the mobilization of BM stem cell.

Known G-CSF and GM-CSF can mobilize the generation of granulocyte (mainly being neutrophilic granulocyte) and macrophage respectively, and the generation that also causes the DC that has increased BM, this helps to provide nonspecific immune response (Janeway et al. to the patient who is in the antigen attack, (2001) Immunobiology 5th Ed., p.325).For example, use G-CSF and GM-CSF to reduce to accept the incidence rate of the infection (showing as the heat generation agranulocytosis) of the non-medullary system malignant tumor patient of bone marrow depression antitumor drug clinically, this is relevant with the significant sickness rate of serious agranulocytosis and heating usually.In addition, confirm that clinically these two kinds of medicines can both prevent to accept HSCT patient's infection.G-CSF and GM-CSF are used to carry out the patient that the peripheral blood precursor is collected or treated at present.Stimulate the differentiation of BM stem cell and/or the colony stimulating factor (CSF) of propagation to produce a lot of interest, because they are to the treatment ability that is subjected to the inhibition level of the cell in recovery hematopoietic stem cell source.According to their activity, identified and distinguished out the CSF of people and mice system.For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the external formation of neutrophilic granulocyte and macrophage colony respectively, and GM-CSF and interleukin-3 (IL-3) have activity widely, and their stimulate the formation of macrophage, neutrophilic granulocyte and eosinophilic granulocyte's colony.IL-3 also stimulates mastocyte, megalokaryocyte and formation pure and that mixed red assembly falls (when adding erythropoietin).GM-CSF has quickened the recovery of neutrophilic granulocyte and has kept that it is functional, but platelet recovery is had only very little verifiable effect.On the contrary, IL-3 has promoted the recovery of neutrophilic granulocyte and monocytic more slow increase, but has quickened hematoblastic recovery.

Therefore, in some embodiments of the inventive method, G-CSF and/or GM-CSF have been used.Sex steroid with G-CSF and/or GM-CSF treatment is removed (successively or simultaneously) and has been caused the lymph of BM and the increase of myelocyte output, and this has improved short-term and the long-term results of suffering from the patient that maybe may suffer from infection conversely significantly.In another method, behind chemotherapy or radiotherapy, granted CSF in 3-4 days.By the interruption to the sex steroid signal pathway also greatly strengthened with use CSF relevant clinical effectiveness.Especially, use method of the present invention to allow to control better infection with CSF to the patient who accepts tumor radiotherapy for example or chemotherapy.In addition, if can be effectively and promptly " restart " immune system, can use the chemotherapeutics or the radiotherapy that have increased dosage and frequency.This can betide and be with or without allogene or from the introducing of body HSC, the introducing of HSC is with the timely recovery of enhance immunity system functionality further.Castrating also will cause the more essential transplanted HSC of low number, when obtaining the HSC of limited quantity or when navel blood stem cell is used to transplant, this will be useful from donor.

For example, use two kinds of different types of drugs (for example a kind of GnRH analog, for example Lupron_ simultaneously; With a kind of androgen blocker, for example Cosudex_) can allow identical immune regeneration, but may need to have reduced the G-CSF or the GM-CSF of dosage.Similarly, when G-CSF that utilizes same dose or GM-CSF, use these two kinds of different types of drugs can allow the rejuvenation (rejuvenation) bigger or the longer time of immune system cell simultaneously.In addition, use two kinds of different types of drugs can allow the immune system cell rejuvenation equally simultaneously, reduced exhausting or the medicine of disruptive steroid signal pathway or the combination of medicine of dosage when dosage comparison (promptly use with " usually " that be used for the treatment of carcinoma of prostate, endometriosis or breast carcinoma reduce to some extent) but utilized.In addition, when utilized reduce dosage be used to exhaust or during the combination of the medicine of disruptive steroid signal pathway or medicine, use simultaneously two kinds of different types of drugs allow immune system cell more or the rejuvenation of longer time.

Indication

The known medicine that causes sex steroid removal or barrier steroid signal pathway, can be separately or with or do not unite use with top mentioned somatomedin and cytokine, be used for: the minimizing of the infection relevant with multiple therapeutic scheme, exhaust that the rejuvenation of BM (is seen after treating, for example embodiment 19), (see as the auxiliary treatment that a kind of HSC of promotion implants, for example embodiment 22), (for example see as a kind of allogene or from the auxiliary treatment of effective processing of body organ or cell transplantation, embodiment 21 and 22, and it is unsettled jointly, the U.S. No.10/419 that has altogether, 039 and 10/749,119), the vaccination scheme (is seen, embodiment 10-13 for example, 29 and have altogether, common unsettled U.S. No.10/418,747 and 10/748,450), the processing of various autoimmune diseasees (is seen, embodiment 32-35 and having altogether for example, common unsettled U.S. No.10/419,066 and 0/749,118), the treatment or the processing of the consequence of various infectious disease (are seen, embodiment 3 and 14 for example), the prevention of various cancers and the raising of treatment (are seen, embodiment 13 and having altogether for example, common unsettled U.S. No.10/418,727 and 10/749,122), (see embodiment 14 and have altogether with the range gene therapeutic scheme, common unsettled U.S. No.10/419,068 and 10/748,851).

The application of these medicines in these diseases will cause more effective therapeutic outcome maybe will cause more effective overall therapeutic scheme.In addition, described as embodiment 25 and 26, dosage or the administration (or radiocurable dosage) that can change various chemotherapeutics make their produce now side effect still less and/or cause the result of patient's better quality of life.In addition, using altogether of various cytokines and somatomedin can be allowed the necessary HSC number of minimizing transplanting.For example, utilize method of the present invention, the adult HSCT of end user's umbilical blood may be possible now, obtains to implant required cell number because reduced.

Pharmaceutical composition

Can or can carrier-free use the used chemical compound of the present invention with any pharmaceutically useful vector administration.The dosage form that can prepare Pharmaceutical composition according to the method for standard (is seen Remington for example, The Science and Practice of Pharmacy.Gennaro A.R., ed., 20th edition, Williams﹠amp; Wilkins PA, USA (2000)).The non-limiting instance of pharmaceutically suitable carrier comprises peplos, solvent and diluent compatible mutually on the physiology., intravenous outer, subcutaneous for intestinal and intramuscular administration, compositions can be subjected to the protection that for example encapsulates.Perhaps, can allow that the carrier that these compositions slowly discharge provides compositions (plural number) time with the protection active component.Known in the art have a variety of polymer and copolymer to be used to prepare the dosage form that regularly discharges, for example various version of lactic acid/ethanol copolymer.See, U.S. No.5 for example, 410,016, it is used as biodegradable coating with modified polyethylene glycol polymer.

The dosage form that plan is used for oral administration can be prepared as liquid, capsule, tablet or the like.These compositionss for example can comprise, the coating that excipient, diluent and/or protection active component are not degraded.Such dosage form be know (see Remington for example, The Science and Practiceof Pharmacy, Gennaro A.R., ed., 20th edition, Williams﹠amp; Wilkins PA, USA (2000)).

In any dosage form of the present invention, can comprise not active other chemical compounds (promptly can not block the chemical compound of the ability of LHRH analog barrier steroid hormone signal pathway) that can negativity influence the LHRH analog.Example is various somatomedin described herein and other cytokines.

Dosage

The dosage that can easily determine used sex steroid analog of the present invention or inhibitor barrier steroid hormone signal pathway through the doctor or the veterinary of routine training, also can it (for example be determined by the inquiry medical literature, THE PHYSICIAN ' S DESK REFERENCE, 52NDEDITION, Medical Economics Company, 1998).

The attending doctor in conjunction with various factors with the modified medicaments effect for example severity, administration time and other clinical factors of patient's pathological changes, body weight, sex and diet, any pathological changes will determine the method that is used for the treatment of above-mentioned pathological changes relevant dosage.By the results of regular determination physiochemical indice for example differential blood count or the like can monitor the patient's that treats progress.

The dosage of being narrated above adjusting is with other compositions in the compensation therapeutic combination.These compositions comprise with the co-administered of other CSF, cytokine, lymphokine, interleukin, hemopoietic growth factor, with the co-administered of chemotherapeutics and/or radiation and various patients that the attending doctor recognized for example factor of modified medicaments effect, for example severity of patient's pathological changes, body weight, sex and diet, any pathological changes, administration time and other clinical factors of relevant content.

Except above-mentioned dosage, for example, can grant LHRH analog and other sex steroid analog with the single dose that will continue for some time (for example 3 to 6 months).In some cases, dosage form was with effective 1 to 2 months.Standard dose is different with the type of used analog, but this is decisions easily to those skilled in the art, does not need undue experimentation.Generally speaking, dosage is between about 0.01mg/kg and about 10mg/kg, or between about 0.01mg/kg and the about 5mg/kg.

The length of the treatment time of sex steroid inhibition or LHRH/GnRH analog is different along with the degree of atrophy of thymus gland and damage, and this is decisions easily to those skilled in the art, does not need undue experimentation.As if the patient is old more, or they have been exposed to the T cell and exhaust thing for example chemotherapy or radiotherapy are many more, and they need the treatment of GnRH for example just long more.Usually be considered to be enough to detect the new T cell in the blood in four months.The method that detects the new T cell in the blood is known in this area.For example, be a kind of method of T cell detection by the existence of determining TXi Baoshouti deletion ring (TREC), when having formed TREC when TCR forms, and it lacks in cell when cell division.Therefore, TREC only sees in new (originally) T cell.The TREC level is an indication of human thymus function.In WO/00 230,256, described these and other method in detail.

Dosage is because of used sex steroid inhibitor or for example resistance steroid vaccine or other blockeres are different.In some cases, a dosage can be prepared as and continue the same long time with the infectious persistent period of periodicity.For example, " influenza season " usually occur in the month in winter.A kind of dosage form that can just begin preparation as described herein when influenza begins season and give the LHRH analog to be protecting one period more than 2 months, whenever gives additional dose more than 2 months, lowers or disappears up to the danger of infecting.

Can prepare the dosage form of enhance immunity system.Perhaps, can prepare the infection dosage form of enhance immunity system simultaneously that stops influenza virus for example specifically.The latter's dosage form can comprise by genetic engineering processing produced toleration (as follows) to influenza virus through (GM) of genetic modification cell.Can be with sex steroid analog or LHRH analog or spatially and/or all grant the GM cell on the time dividually.With the same, can use multidose to produce protection and prevention infection between the influenza seasonal period to the patient along with the time to influenza virus with non-GM cell.

Those skilled in the art can understand that the effective time of the method for at least some barrier steroid signal pathways is only the same with the time of granting the suitable combination thing long.Therefore, in case the advantage of some embodiment of the present invention is to have realized immunological effect required for the present invention, just can stopped treatment (2-3 month), and the reproductive system of these objects will return to normally.

Using of chemical castration medicine

Can medicine is administered to intravital method grant sex steroid and remove to remove medicine by any.Therefore, according to invention, can include but not limited to that intravenous, Intradermal, subcutaneous, intramuscular, part and oral route of administration grant sex steroid and remove medicine with any approach.

Except above-mentioned method, can finish giving of the chemical compound that is used for method of the present invention through the known several different methods of those skilled in the art.One is used to grant chemical inhibitor has utilized effective 3 months single dose with the standard method of the signal pathway of inhibition steroid mediation LHRH agonist.To this, the simple intravenous of single time or intramuscular injection all are not enough, and therefore agonist was just removed away in patient's body before past three months.The substitute is, can use remover liquid injection or implant or any other the instrument of allowing slow release inhibitor of storing.Similarly, can use to be used to increase the method that the inhibitor half-life in vivo is retained in this required function simultaneously, for example by modifying chemicals.

The useful mechanism of using includes, but not limited to the laser irradiation to skin.At unsettled U.S. No.10/418 that have altogether, common, this embodiment has been described in more detail in 727 and also at U.S. No.4,775,361,5,643,252,5,839,446,6,056,738,6,315,772 and 6,251,099.The another kind of useful mechanism of using is included in and produces high-voltage pulse transient state (also being called stress wave or pulse transient state) on the skin.At unsettled U.S. No.10/418 that have altogether, common, this embodiment has been described in more detail in 727 and also at U.S. No.5,614,502 and 5,658,822.By with chemical compound and carrier or do not have carrier to be placed on identical position can to finish or carry out every kind of method.A method of this placement is to be placed on and to be held on skin in the pad of persistent period of one section treatment.

Opportunity

In a situation, exhaust that the administration of the medicine (or other castrating method) of sex steroid or barrier steroid signal pathway occurs in before the chemotherapy or illumination scheme that for example may cause some BM medullary cells exhaustion and/or damage circulation immunocyte.

Cell

Or under the situation that does not have the thymus reactivate or prior to the thymus reactivate or in the thymus reactivate, the hematopoietic cell (it is desirable to from body) that the injection hemopoietic forebody cell for example clearly is defined as CD34+ can strengthen the degree and the kinetics of thymus reactivate and/or increase immunocyte and BM is functional and implant.HSC also can also be defined as low Thy-1 and CD38-, CD34+CD38-; The cell of low Thy-1 that lacks the label of other cell lines (lin feminine gender) is more primary HSC, and it can continue or have more secular ability of going into nest again for more time.

Can replenish the method for various inventions described herein by adding CD34+HSC for example and/or epithelial stem cell.In a kind of situation, these cells are from body or isogenic and obtained from patient or twin children before the thymus reactivate.Can obtain HSC by sorting patient's blood and/or CD34+ or the CD34lo cell of BM.Can there be several different methods to be used to strengthen the number of HSC, include, but is not limited to by before collecting cell to the patient use G-CSF (Neupogen, Amgen), collected cell culture is used G-CSF to the patient in SCSF and/or after additional CD34+ cell.Perhaps, if strengthened the CD34+ cell mass, so just needn't from blood or BM, sub-elect CD34+ by using G-CSF to the patient before.

HSC can be used to genetic modification.These HSC can derive from BM, peripheral blood or umbilical cord or any other HSC source and can be from body or non-from body.Also usefully lymph or medullary system CFU-GM, the interstital stem cell of also finding in bone marrow and epithelial stem cell also can be from body or non-from body.Stem cell also can comprise Cord blood.They also can comprise the stem cell with the potential that is divided into multiple different cell type, for example embryonic stem cell and now at many adult stems of being found in for example BM, pancreas, brain and the olfactory system of organizing.

In the situation of using non-(donor) cell from body, during the thymus reactivate or produced toleration afterwards to these cells.During the signal pathway of beginning barrier steroid mediation or afterwards, relevant (through (GM) of genetic modification or without genetic modification) donor's cells is transplanted in the receptor.These cells, it is desirable to stem cell or CFU-GM by thymus combination and accept, wherein they have generated toleration to donor by eliminating any T cell that active antagonism donor's cells's new generation can be arranged once in a while.They " belong to the receptor " and can be called the new T cell of thymus and the part of the generation of DC then.Formed T cell all is identified as self to receptor and donor, generated in view of the above to from the toleration of the graft of donor (see, unsettled U.S. No.10/419 that have altogether, common, 039 and PCT/IBO1/02740).

In another embodiment, receptor stem cell of being granted or CFU-GM (through genetic modification or without genetic modification) comprise cell from a plurality of individualities, make the receptor develop toleration to one group of MHC type, make the receptor be considered to suitable candidate easier or cell, tissue or organ transplantation fast, because they have the MHC that mates with wider donor.

The present invention also provides and has been used for external DC is incorporated into the intrathymic method of patient.Can realize this point by grant the donor's cells to the receptor with the toleration that forms in the receptor.The donor's cells can be DC, epithelial stem cell, adult or embryonic stem cell or hemopoietic forebody cell.The donor's cells can be CD34+HSC, lymph CFU-GM or medullary system CFU-GM.In some cases, the donor's cells is CD34+ or CD34lo HSC.Donor HSC can grow in the receptor and be DC.The donor's cells can be granted the receptor and by the peripheral blood system or directly or in BM is moved to the thymus of reactivate.Integrate for the thymus that strengthens inducing tolerance, also can unite by usability steroid inhibitor for example the LHRH/GnRH analog to the activation of thymus reactivate, toward the inner injection of thymus stem cell.As if microenvironment at thymus reaches in the content of wherein suitable cell growth factor, even non-HSC also can be induced formation DC.

Inhibition or disappearance to sex steroid have increased the picked-up of thymus to hemopoietic forebody cell significantly.These cells are integrated in the thymus and by the mode identical with donee's cells and produce DC, NK, NKT and T cell.The result is the chimera of T cell, DC and other cells.The donor's cells means that in the intrathymic integration of receptor T cell that this thymus produces with selected, makes them can tolerate the donor's cells.Such toleration is allowed cell, tissue and the organ of further transplanting it from the receptor, has reduced the needs to immunosuppressive drug, because institute's materials implanted will be the material of self by receptor's immune system recognition.

Randomly to the genetic modification of stem cell or CFU-GM

This description also comprises immune method and the gene therapy methods that is used for randomly changing individuality.By the GM cell being administered to receptor and can realizing this point by the signal pathway of barrier steroid mediation.Invention also comprises gene therapy methods, strengthens the functional of BM and/or immunocyte by combining and regenerating thymus, or with the thymus reactivate simultaneously or before this or do not have to carry out under the situation of thymus reactivate.

The cell of genetic modification can be HSC, epithelial stem cell, embryo or adult stem cell or medullary system or lymph CFU-GM.In a specific embodiments, the cell of genetic modification is CD34+ or CD34lo HSC, lymph CFU-GM or medullary system CFU-GM.In another embodiment, the cell of genetic modification is CD34+ or CD34lo HSC.The cell of genetic modification is applied to the patient and through PBF and moves to thymus.When lacking sex steroid, increased the picked-up of thymus significantly to these hemopoietic forebody cells.These cells are integrated in the thymus and produce dendritic cell and the T cell that carries from the genetic modification of reformed cell.The result is that the T cell mass that has required hereditary change circulates in the peripheral blood of receptor, and the increase that is accompanied by patient's the caused cell of thymus reactivate, tissue and organ group.

3-4 at the signal pathway that begins the mediation of barrier steroid (begins the near 2-3 week after LHRH treats) in week, has occurred initial new T cell in the blood flow.But the growth fully of T cell mass is 3-4 (or more a plurality of) moon possibly.

This description also comprises the gene therapy methods of hematopoietic stem cell, lymph CFU-GM, medullary system CFU-GM, epithelial stem cell or their combination (GM cell) that utilize genetic modification.These cells of previous conveying that other people carried out have been unsuccessful as the trial of gene therapy, cause the level of the not count enable of modifying cell.This description provides a kind of new method that is used to carry these cells, and it has promoted the picked-up of cell and has been divided into required T cell.With the injection cell modified in the patient.Stem cell and the CFU-GM that the thymus picked-up is modified also is converted into the cell that produces in T cell, dendritic cell and other thymus.Each cell of these new cells all contains the genetic modification of parental generation stem/progenitor cells.

In one embodiment, the method for invention uses HSC, lymph CFU-GM, medullary system CFU-GM, epithelial stem cell or their combination (jointly being called as the GM cell) of genetic modification to produce the resistance that immune system is attacked special antigen.

A kind of suitable gene or polynucleotide (that is the nucleotide sequence of definition specific protein) that generate or induce the resistance of or other causes of disease infectious to one or more are worked in stem cell and/or the CFU-GM genetic engineering.By specific gene being incorporated in the HSC cell differentiation precedent such as APC, the albumen of its expression conduct expressed peptide in MHC I type or II type background.This expression will greatly increase the number of " presenting " required antigenic APC than the number of normal generation, increase suitable T cell recognition specific antigen and the chance of replying in view of the above.

Used multiple antitumor or cytotoxic cancer therapy drug all caused the bone marrow toxicity (for example, vinblastine, cisplatin, methotrexate, alkylating agent, antifolic thing, vinca alkaloids and anthracycline antibiotics) of moderate to severe in clinical now.In another embodiment of the invention, drug resistant gene can be incorporated in the HSC to give its drug resistance to cancer therapy drug.These genes comprise for example dihydrofolate reductase.The cytotoxicity that the invention provides these chemotherapeutics has the usage of chemical sproof HSC can pass through to reduce myelosuppressive incidence rate and severity, allow more use and/or side effect still less (Podda et al., (1992) Proc.AM.Acad.Sci.USA 89:9676 of these medicines; Banerjee et al., (1994) Stem Cells 12:378).

Thymus can absorb the stem cell and the CFU-GM of modification and be translated into the cell that T cell, DC and other thymus are produced.Each cell of these new cells all contains the genetic modification of the stem/progenitor cells of parental generation, and the infection or the damage that can tolerate the cause of disease wholly or in part in view of the above and caused.In 2 weeks of for example castrating, also increased for example number of the B cell in spleen and the lymph node of bone marrow, blood and peripheral lymphoid organs.In a specific embodiments, the patient contacted with the cause of disease or be in highly dangerous that the cause of disease contacts in.

The patient can be given the sex steroid analog activating their thymus, and/or improves their marrow function, and this comprises the ability that has increased picked-up and produced HSC.The patient can be by themselves HSC of injection, or can be by the HSC of injection from appropriate donors, and they for example by 3 days (twice subcutaneous injection every day) of G-CSF treatment, collected HSC subsequently from blood at the 4th and 5 day.HSC can or transduce to produce required albumen or antigen by gene (for example encoding from albumen, peptide or the antigen of the cause of disease) transfection.After in being expelled to patient's body, HSC enters bone marrow and some HSC develop into whole body antigen-presenting cell (APC) everywhere at last.Antigen is expressed in the background of the I type MHC on surface of these APC and/or II type MHC molecule.By expressing required antigen, APC has improved the activation to T and bone-marrow-derived lymphocyte.Transplanted HSC also can enter thymus, develops into DC and related antigen is to pass developmental T lymphocyte.If have only the DC (for example＜0.1% thymocyte cell) of peanut, DC can to the selection bias of new T cell to antigen reactive those T cells.If there is a large amount of special DC, can with the deletion of identical principle may with antigen reactive new T cell, this can be used to the prevention or the treatment of autoimmune disease.

In one embodiment, the patient infection HIV.In a special specific embodiments, the step that provides more in detail below the method that is used for the treatment of this patient comprises: (1) to reduce virus titer, continues this treatment to prevent or to reduce the infection of new T cell with highly active antiretroviral therapy (HAART) treatment in whole process; (2) remove T cell (immunosuppressant); (3) by granting for example signal pathway of LHRH analog barrier steroid mediation of sex steroid analog; (4) when thymus begins reactivate, grant by modification contain that expression will prevent that HIV infects, prevention HIV duplicates, deactivation HIV virus or other can stop HIV to infect the GM cell of proteic gene of the effect of T cell; (5) if the GM cell not from body, before the thymus reactivate or grant the donor's cells with it simultaneously and make immune system that the donor's cells is identified as self cell; (6) be established and new mature T cells group when having begun to break away from thymus when the thymus chimera, reduce and finally eliminated immunosuppressant.

During the castrating step or afterwards, can will be transplanted in the receptor patient from the hematopoietic stem cell of donor or CFU-GM or epithelial stem cell.It is to belong to the cell of receptor and begin to become the new T cell of thymus and the part of DC product that thymus is accepted these cells.Formed T cell mass all is identified as self with donor and receptor (for non-autoplastic situation).In receptor, also can generate toleration from the graft of donor.Graft can be cell, tissue or the organ of donor or their combination.

In one embodiment, the method for the present invention graft that can use thymus is with the implantation that improves the donor's cells or to the toleration of the graft of donor.In some specific embodiments, thymic graft is when when the patient is athymism, when patient's thymus has resistance to regeneration or to fast rapid regeneration resistance being arranged.In some specific embodiments, used the xenograft (seeing U.S. No.5 for example, 658,564) of the thymus of inducing tolerance.In other specific embodiments, can use heterogenic thymic graft.

As defined in this, term in the patient " generation toleration " or " inducing tolerance " and other similar term all refers to completely and the part induction of tolerance (for example, compare with the patient who is not treated by method of the present invention, the patient more tolerates or tolerates fully graft).Utilize methods known in the art for example can test induction of tolerance by the MLR reaction.

Therefore, in one approach, the patient is during castrating or accept HSCT afterwards.In a kind of situation, the patient has been injected they self HSC.In another kind of situation, the patient has been injected the HSC from appropriate donors.Can with or without G-CSF pretreatment patient or donor (for example, every day, twice subcutaneous injection was totally three days, collected HSC subsequently from blood at the 4th and 5 day).In a kind of situation, before the thymus reactivate or with it simultaneously, supply with hematopoietic cell to the patient, this has increased the immunocompetence of patient's body.The cell of being transplanted can by or not by genetic modification.The cell of being transplanted can be HSC, epithelial stem cell or hemopoietic progenitor cell.The cell of being transplanted can be CD34+HSC, lymph CFU-GM or medullary system CFU-GM.In some cases, the cell of being transplanted is CD34+ or CD34lo HSC.HSC can by or not by genetic modification.

In some method, use gene (for example, coding is from albumen, peptide or antigenic gene or other relevant genes of the cause of disease) transfection or transduction HSC to produce relevant albumen or antigen.In an example, method of the present invention has used HSC, lymph CFU-GM, medullary system CFU-GM, epithelial stem cell or its combination (being called " GM cell " jointly) of genetic modification to produce the immune system (seeing that for example embodiment 14) that the special antigen of tolerance is attacked.At the U.S. No.09/758 that has altogether, this method has been described in more detail in 910,10/419,068 and 10/399,213.

When lacking sex steroid, increased the picked-up of thymus significantly to HSC.These cellular integration are in the thymus and produced DC and the T cell that carries from the genetic modification of change cell.The result is that the T cell mass with required hereditary change circulates in the peripheral blood of receptor, and the increase that is attended by the caused cell of reactivate thymus, tissue and organ group, and they can respond to antigen fast, specifically.Initial new T cell can appear in (in nearly 2 to 3 weeks after the beginning LHRH treatment) in blood flow in 3 to 4 weeks of the signal pathway that beginning barrier steroid mediates.The growth fully of T cell pool 3 to 4 months possibly.

After in being injected into the patient, HSC enters bone and the bone marrow from blood, and some disengagings are got back in the blood then, and finally is converted into whole body T cell, DC and APC everywhere.Antigen is expressed in the background of the I type MHC on these APC surfaces and/or II type MHC molecule.For the GM cell, by expressing required antigen, APC has improved the activation of T and bone-marrow-derived lymphocyte.The HSC that is transplanted also can enter thymus, grows to be DC, and related antigen is passs developmental T lymphocyte.

If have only the DC (for example＜0.1% thymocyte cell) of peanut, DC can will be partial to and antigen reactive those T cells the selection of new T cell.If there is a large amount of special DC, can with the deletion of identical principle may with antigen reactive new T cell, this can be used to the prevention or the treatment of autoimmune disease.In 2 weeks of for example castrating, also increased for example number of the B cell in spleen and the lymph node of bone marrow, blood and peripheral lymphoid organs.

For HSC is that each new cell all contains the genetic modification of parental generation stem/progenitor cells when having the GM of specific performance, and tolerates infection or infringement in the cause of disease in view of the above wholly or in part.

Those skilled in the art know and are used to separate and the method for transduce stem cell and CFU-GM.For example at PCT document WO 95/08105, WO 96/33281, WO96/33282, U.S. No.5, the example of these type methods has been described in 681,559,5,199,942,5,559,703,5,399,493,5,061,620.

Antisense polynucleotides

Term " antisense " is defined as being complementary to the polynucleotide sequence of polynucleotide of the present invention at this.Polynucleotide can be DNA or RNA.Can generate antisense molecule with any method, method comprises being connected to by the related gene with inverted orientation and allows synthetic on the synthetic viral promotors of complementary strand.In case be introduced in the cell, this is transcribed chain and combines the formation duplex with the native sequences that cell is generated.These duplexs have been blocked and have further been transcribed or translate then.In this way, can generate the phenotype of sudden change.

Catalytic nucleic acid

Term " catalytic nucleic acid " is defined as discerning specifically the dna molecular of chemical modification of a kind of special substrate and catalytic substrate or the molecule (also being called " ribozyme ") that contains molecule (also being called " DNAzyme " or " DNA enzyme " in this area) or the RNA molecule of DNA or contain RNA at this.Nucleic acid base in the catalytic nucleic acid can be base A, C, G, T and U, and their derivant.The derivant of these bases is known in the field.

Catalytic nucleic acid contains the antisense sequences that is useful on the specific recognition target nucleic acid and the active nucleic acid of lyases usually.Specific site in the catalysis chain cracking target nucleic acid.Useful especially in the present invention ribozyme type is hammerhead ribozyme (Haseloff and Gerlach (1988) Nature 334:585), Perrimanet al., Gene 113:157) and hairpin ribozyme (Shippy et al. (1992), (1999) Mol.Biotechnol, 12:117).

dsRNA

Double-stranded RNA (dsRNA) is useful especially for suppressing special proteic generation specifically.Though be reluctant to be limited by theory, a group provides a kind of model (Dougherty and Parks, (1995), Curr.Opina.Cell Biol.7:399) that is used for can reducing with dsRNA by it mechanism of albumen generation.Recently this model is modified and is expanded (Waterhouse etal., (1998) Proc.Natl.Acad.Sci.USA 95:13959).This technology depends on and has the dsRNA contain with the sequence of the mRNA basically identical of related gene, the wherein polypeptide of mRNA coding first aspect of the present invention.Expediently, can read in the frame in the single exploitation of recombinant vector or host cell and generate dsRNA, wherein be connected with justice and antisense sequences with irrelevant sequence side, this makes has justice and antisense sequences hybridization to form the dsRNA molecule, has the ring structure that the nothing to do with sequence forms.To the design of suitable dsRNA molecule of the present invention and to produce all be in those skilled in the art's limit of power, particularly be incorporated into Dougherty and Parks, (1995), Curr.Opin.Cell Biol7:399; Waterhouse et al., (1998) Proc.Natl.Acad.Sci.USA 95:13959; With PCT publication No.WO 99/32619, WO99/53050, WO99/49029 and WO01/34815.

Anti-HIV construct

Those skilled in the art can be developed and to be used for suitable anti-HIV construct of the present invention.In fact, developed and multiple anti-HIV antisense constructs and ribozyme, and be described in U.S. No.5,811,275,5,741,706 and 5,144,019 and PCT publication No.WO 94/26877 and Australian patent application No.56394/94 in.

Gene

Comprise that in method of the present invention useful gene used among the GM HSCT and genetic fragment (polynucleotide) comprise that those encode T cells are to the gene of special venereal infection because of the resistance of infection.These venereal infections are because of comprising, but are not limited to HIV, T chronic myeloid leukemia virus and other cause the virus of lymphocytic hyperplasia disease.

For HIV/AIDS, can use several genes and/or genetic fragment, including but not limited to: the nef transcription factor; A kind of coding cuts the gene for example tat and the rev (Bauer et al., (1997) Blood 89:2259) of the ribozyme of HIV gene specifically; The trans-dominant mutant form of HIV rev gene, RevM10, it has demonstrated inhibition HIV and has duplicated (Bonyhadi et al., (1997) J.Virol.71:4707); The overexpression construct of HIV-1rev response element (RRE) (Kohn et al., (1999) Blood 94:368); Encode its expression can suppress HIV infection cell or the RNA that duplicates or proteic any gene; With their fragment and compositions.

But can use the form of the stably express of these genes or genetic fragment.Term " but stably express " is defined in after gene or genetic fragment be transferred to cell at this, can be at least at the product (RNA and/or albumen) of expressing gene on the semipermanent basis of host cell and in the cell filial generation after division and/or differentiation or genetic fragment (" function fragment ").This gene or genetic fragment that requires whether to be included in the carrier all will have the suitable signal sequence that is used for DNA is transcribed into RNA.In addition, when gene or the coded albumen of genetic fragment be that DNA also encodes and translates signal when influencing patient's the bioactive molecule of state.

In most situations, gene or genetic fragment are contained in the carrier.The general personnel in this area know can be used to express required RNA or proteic expression vector.

Expression vector is to instruct transcribing and the carrier of translating of formed RNA in this contained DNA sequence.Expression vector can be in time multiplexed cell system, can be by genetic modification, and it comprises plasmid, phage, virus and mini-chromosome.Similarly, gene or genetic fragment can become the integrated part of the chromosomal DNA of cell.Recombinant vector and methodology are normally known.

Be used to express proteic expression vector of the present invention and can comprise origin of replication.Suitably the expression vector that makes up comprises the origin of replication that is used for intracellular self-replicating, maybe can be incorporated in the chromosome of host cell.These carriers also can comprise the selected marker thing, a limited number of useful Restriction Enzyme site, high copy number order and strong promoter.Promoter is to instruct RNA polymerase to combine with DNA and start the synthetic DNA sequence of RNA; Strong promoter has caused high-frequency startup.

In one embodiment, the dna vector construct comprises promoter, enhancer and a polyadenylation signal.Promoter can be selected from by HIV, nonrestrictive group of being constituted of for example long terminal repetition (LTR), simian virus 40 (SV40), Epstein Bar virus (EBV), cytomegalovirus (CMV), Rous sarcoma virus (RSV), moloney virus, MMT virus (MMTV), human actin, human myoglobulin, human hemoglobin, people's muscle creatinine, human metal thioalbumen (metalothionein).In another example, used inducible promoter, the number and the opportunity that make the expression to control the gene that inserted or polynucleotide.

Enhancer can be selected from include but not limited to human actin, human myoglobulin, human hemoglobin, people's muscle creatinine and virus enhancer for example those from the group of the enhancer of CMV, RSV and EBV.Promoter can be from identical or different genes with enhancer.

For example, can from the group of being formed by LTR polyadenylation signal and SV40 polyadenylation signal, particularly wherein SV40 minimal adenoviral thuja acid signal, select polyadenylation signal.

Expression vector of the present invention can operably be connected on code book the invention used RNA or proteic DNA, and promptly carrier can instruct duplicating of the dna molecular that connected and RNA or proteic expression that dna molecular is coded.Therefore, for albumen, expression vector must have the transcription initiation signal in the upstream of the dna molecular that is connected, and keeps correct reading frame to allow the expression of dna molecular under the control of control sequence and the generation of the coded desirable proteins of dna molecular.Expression vector can include but not limited to, cloning vehicle, modified cloning vehicle and specially designed plasmid or virus.Can use inducible promoter, the number and the opportunity that make the expression to control the gene that inserted or polynucleotide.

Those skilled in the art can be created on the DNA construct that function is arranged in the cell.In order to test expression, utilize to can be by the tissue culture of the cell of those cell same types of genetic modification at the expression of external test genetic constructs.

The method of genetic modification

Can genetic modification be incorporated in the cell that just is used to gene therapy with the recombination method of standard.For example, to the transduction of the retroviral vector of the HSC that cultivates be a kind of successful methods known in the art (Belmont and Jurecic (1997) " Methods for EfficientRetrovirus-Mediated Gene Transfer to Mouse Hematopoietic Stem Cells; " inGene Therapy Protocols (P.D.Robbins, ed.) Humana Press, pp.223-240; Bahnson et al., (1997) " Method for Retrovirus-Mediated Gene TransfertoCD34+-Enriched Cells, " and in Gene Therapy Protocols (P.D.Robbins, ed.), Humana Press, pp.249-263).Other carrier includes but not limited to the carrier in those adenovirus sources or the slow virus source and moloney murine leukemia virus source.

Following method also is useful to the genetic modification of HSC: particle gun (Yang is for example used in the gene transfer of granule mediation, N.-S.and P.Ziegelhoffer, (1994) " The ParticleBombardment System for Mammalian Gene Transfer; " In PARTICLEBOMBARDMENT TECHNOLOGY FOR GENE TRANSFER (Yang, N.-S.and Christou, P., eds.), Oxford University Press, New York, pp.117-141), liposome-mediated gene transfer (Nabel et al., (1992) Hum.Gene Ther.:3:649), coprecipitation of genetically modified vectors with calcium phosphate (Graham and Van Der Eb, (1973) Virol.52:456), electroporation (otter et al., (1984) Proc.Natl.Acad.Sci.USA 81:7161), and microinjection (Capecchi, cell22:479), and other can be stably transfer to HSC and other and make gene be expressed part-time at least method in by the cell of genetic modification carrying intravital gene or oligonucleotide (1980) with being.

Developmental TCR storehouse deflection or depart from specific antigen: allergy and autoimmune disease

Enhancing thymus means performance and the type that can handle dendritic cell to the ability of the picked-up of hematopoietic stem cell.For example, can be with the specific gene transfection stem cell in the dendritic cell that finally is expressed in thymus (and intravital other positions).These genes can comprise the gene of those coding specific antigens, may be deleterious at the immunne response of these specific antigens, for example in autoimmune disease and allergy.

This part of the present invention is to be based on a discovery, be exactly that sex steroid suppresses that functional and immune cell function has direct effect to BM, and finally will help to overcome the autoimmune disease that the patient suffers from the reactivate of autoimmunity patient's thymus.This identical principle also is applied to suffering from patient hypersensitive.When thymus is re-activated, formed immune system new or that modify, they no longer discern and/or respond to autoantigen.

According to the present invention, can use following scheme.Patient's immunosuppressant that at first will be diagnosed as autoimmune disease (for example type i diabetes) is to stop progression of disease.Can be by granting immunosuppressant (for example cyclosporin or rapamycin) realization this point individually or with anti-T cell and anti-B cell antibody (for example removing the anti-CD3 of T cell or anti-CD19, CD20 or the CD21 of anti-T cell gamma globulin and removal B cell).The patient simultaneously, can grant sex steroid analog (or other castrating method) by immunosuppressant.Can mobilize the T cell of himself then with G-CSF.If his autoimmunity is caused by the cross reaction of its T cell and the cause of disease that he was run into before, the removing of T cell will be removed autoreactive T cell so, and the cell (for example his β islet cells) that new T cell of growing will can not continue to discern him is external cell.In this way, alleviated himself immune disease.In addition,, can stop sex steroid and remove treatment, recover patient's fertility thus in case himself immune disease is alleviated.

In another non-limiting instance of the present invention, rebuild autoimmune disease with the allogene stem cell.In some specific embodiments, these allogene stem cell are cord blood cells, and it does not comprise mature T cells.

In some embodiments, therefore the HSC that is transplanted can cause HSC transplanting completely (for example, to transplant every kg body weight 5 * 10 after bone marrow removing completely or bone marrow exhaustion at every turn ⁶Cell).In some specific embodiments, only need to realize less bone marrow removing, for example after 2-3Gy irradiation (or 300rad), grant about 3-4 * 10 ⁵Individual cell/kg body weight.In some specific embodiments, used the T cell to remove the method (seeing that for example embodiment 2) of (TCD) and/or the removing of another kind of immunocyte.Minimum just may be enough to the allergy of reduction of patient or the symptom of autoimmune disease to 10% chimera.In some specific embodiments, donor HSC is from Cord blood (for example, 1.5 * 10 ⁷Cell/kg is used for the implantation of receptor).

In other embodiments, removed chemotherapy preceding 45 days up to bone marrow, the patient begins to accept Lupron and accepts Lupron simultaneously continuously with BMT, the feasible length overall (equal 3 injections, the per injection administration can be kept 3 months) about 9 months that is exposed to medicine.In the different interval during studying, can collect the number (particularly thymus is moved out recently) and the function (concrete is external replying the T cytositimulation) of blood sample analysis T cell.This embodiment also is applicable to the HSCT (for example HSCT after cancer radiation and the chemotherapy) of other purposes described herein usually.

In other embodiments, can after removing, lymph serum transplant HSC.In some embodiments, can selective clearing T cell and B cell, remove cell (for example those relate to autoimmunity and cell hypersensitive) as required.Selection can comprise removes activating cell or relates to autoimmune or the cell type of anaphylactic response.Can be according to cell surface marker thing for example CD4, CD8, B220, thy1, TCR, CD3, CD5, CD7, CD25, CD26, CD23, CD30, CD38, CD49b, CD69, CD70, CD71, CD95, CD96, antibody specificity or Ig chain or go up key cell factor receptor person for example IL-2R B chain and TGF β select cell.A kind of method of knowing that is used to exhaust is to use antilymphocyte globulin.Other are selected and the method for sorting cells is known, and comprise that magnetic is separated with fluorecyte, centrifugal and more particularly plasmapheresis, leukocyte exclusion and lymphocyte exclusion.

In some embodiments, bone marrow is removed, bone marrow is exhausted, lymph is exhausted, the T cell removes and/and other carry out HSCT when optionally immunocyte is removed not having.

In other embodiments, method of the present invention also comprises by for example bestowing immunosuppressant (for example, cyclosporin, prednisone, Ozothioprine, FK506, according to Drymotaenium miyoshianum (Mak.) Mak. and methotrexate) with patient's immunosuppressant (seeing U.S. No.5,876,708).In one embodiment, when no HSCT, carry out immunosuppressant.In one embodiment, carry out immunosuppressant in conjunction with HSCT (for example before it, with it simultaneously and afterwards).In another embodiment, do not have that bone marrow is removed, lymph serum removes, the T cell remove and/and other optionally immunocyte remove, remove and carry out immunosuppressant when exhausting.Still in another embodiment, in conjunction with bone marrow remove, lymph serum removes, the T cell removes and/and other optionally immunocyte removings, removal and exhaustion carry out immunosuppressant (for example before it, with it simultaneously and carry out afterwards).

As mentioned above, bone marrow is removed, bone marrow is exhausted, lymph serum removes, immunosuppressant, T cell remove and/and other optionally immunocyte to remove be the non-limiting instance type that the immunocyte that all is used in the full text of the application's book is removed.Generic term " immunocyte exhaustion " is defined as comprising each method in these methods at this, and promptly bone marrow is removed, bone marrow is exhausted, lymph serum removes, the T cell is removed and/or other optionally immunocyte is removed (for example B cell and NK cell are exhausted).Those skilled in the art are understood that easily, in practice during in the invention that this provided, can replace any in these " exhaustion " methods with any (or multiple) other " exhaustion " method.

In one embodiment, exhausted the NK cell.NK antibody is used to exhaust that the NK cell mass is known in this area.For example, anti-NK antibody anti-blood plasma of polyclone that the source is anti-human thymocyte.U.S. No.6,296,846 have described the method for NK and T cell exhaustion and the formation that non-bone marrow is removed treatment and chimeric lymph hematopoietic cell group, and all these all can be used to method of the present invention.

In some embodiments, method of the present invention for example also comprised before HSCT, the natural antibody in the organ (for example liver or kidney) that is obtained from donor by blood perfusion in the absorption receptor blood.

In another embodiment, the present invention also comprises the treatment (T cellhelp-reducing treatment) that reduces t helper cell, for example increase direct and indirect (for example by stimulation or inhibition to the second cytokine activity level) promotion is to the activity level of the cytokine (for example IL-10, IL-4 and TGF β) of the toleration of graft, with (for example lower to promote the activity of the cytokine of the repulsion of graft, the cytokine (for example, IFN β, IL-1, IL-2 and IL-12) of a kind of opposing and inhibition toleration.In some embodiments, bestow cytokine to promote toleration.Cytokine can derive from the donor species and derive from receptor's species (seeing that for example, U.S. No.5,624,823, it has described the DNA of the coding porcine interleukin-10 that is used for this purposes).The mature T cells that the persistent period of helper-inducer treatment could be approximately equal to and be less than receptor's species starts the time required to antigenic repulsion (at human these normally 8-12 days) after first by antigenic stimulus.In other embodiments, the persistent period is to be approximately equal to or the mature T cells that is less than the receptor starts to the required time of antigenic repulsion 2,3,4,5 and 10 times after first by antigenic stimulus.When existing and lacking the mature T cells that can stimulate the receptor and discharge the treatment of cytokine, for example lack prednisone (17,21-dihydroxypregna-1,4-diene-3,11, can bestow the auxiliary minimizing treatment of short-term in the time of 20-trione).Can be before introducing graft or during approximate time, begin the auxiliary treatment that reduces.The auxiliary minimizing treatment of short-term can be before the art and postoperative.In some embodiments, donor and receptor are I type couplings.

Still in further specific embodiments, wherein antigen is not a kind of autoantigen but a kind of exotic antigen (for example, pollen and marine product) can adopt similar strategy.If allergy is that some opportunistics activation of abnormal T and B cell clone is caused, the immunosuppressant of removing T cell and B cell so, and (with it simultaneously) thymus reactivate subsequently causes removal in the cell of anaphylactic response.Because allergy is that the opportunistic activation of abnormal T and B cell clone is caused, it just occurs once more unlike meeting so, and new regenerated thymus also can generate adjusting T cell.Though may still have autoreactivity IgE to circulate in patient's body, they finally can disappear, because removed the cell of secreting them effectively.In case immune system is rebuilt, can stops sex steroid and remove treatment, and recover patient's fertility.

The invention provides the method that is used for the treatment of autoimmune disease, this method or do not carry out BMT as described herein or carry out BMT or use the GM cell.Method of the present invention can also comprise that organ and cell transplantation are to repair and replacement damaged cells, tissue and organ.For example, in IDDM, the patient may need islet cell transplantation to replace impaired islet cells.Utilize method of the present invention can realize prevention to the clinical symptoms of autoimmune disease, wherein the patient have clinical before symptom and family's susceptibility.

In further specific embodiments of the present invention, if known autoimmune disease and the antigen hypersensitive of relating to can adopt the genetic modification to HSC.For example, in multiple sclerosis, antigen can be myelin glycoprotein (MOG), myelin oligodendroglia albumen, myelin basic protein and proteolipid protein(PLP).In pernicious anemia, antigen can be the proton pump of stomach.In type i diabetes, antigen can be proinsulin (J Clin Invest. (2003) 111:1365., GAD or anislet cell antigen.T cell epitopes of type II collagen have been described withrheumatoid arthritis in (Ohnishi etal. (2003) Int.J.Mol.Med.1:331).For struma lymphomatosa, antigen is thyroid peroxidase, and for the Graves disease, antigen is thyrotropin receptor (Dawe etal., (1993) Springer Semin.Immunopathol.14:285).Systemic lupus erythematosus (sle) antigen comprises for example H1 histone of DNA, histone, ribosome, snRNP, scRNP.Especially, Ro (SS-A) is relevant with systemic lupus erythematosus (sle) (SLE) and rheumatoid arthritis with La (SS-B) ribonucleoprotein antigen (for example Ro60 and Ro52).Myasthenia gravis antigen comprises acetylcholine receptor alpha chain and some at Atassi eta l., the T cellular antigens determinant described in (2001) Crit.Rev.Immunol.21:1.Same, some anaphylaxis is reply (for example in the cat allergy, to cat saliva antigen allergy) to known antigens.In these situations, by before being bestowed to receptor, can be at first with donor HSC genetic modification with antigen expressed.The expression of CD34 according to them can be isolated HSC.Bestow these cells together can for the patient and strengthen the inhibitor GnRH analog for example of the signal pathway of the functional sex steroid mediation of BM.Therefore, the HSC that heritability is modified not only develops into DC and the therefore new T cell that forms of tolerance, and they also enter BM as DC and delete new, id reaction and B cell hypersensitive.Therefore, in thymus and bone marrow, all realized maincenter tolerance, alleviated patient's autoimmune disease or symptom hypersensitive in view of the above autoantigen or anaphylactogen.In some specific embodiments, also use immunocyte to exhaust and inhibition.

Be used for exhausting the example of high quick T cell at another, when target antigen is known antigens, can need be to the epithelial stem cell (for example from the body epithelial stem cell) of the gene transfection thymus of the specific antigen of its toleration with coding.By it being separated (seeing Gill etal., (2002) Nat.Immunol.3:635) from thymus (particularly embryo's thymus) to the labelling of thymus epithelial precursor with Ab MTS 24 and its human homologue.

Therefore, the ultimate principle of invention is that autoimmune disease and the prevention high predicted case (for example familial distribution) that the immune system with the toleration that T cell and/or B cell (if suitable) are exhausted, subsequently reconstruction is new stops just suffering from develops into autoimmune disease.Diagnosis of autoimmune disease at first, and determine whether to exist family's (heredity) susceptibility.Next step determines whether there has been recent long-time infection in the patient by the antigen mimicking and the autoimmune disease that clonal activation caused of chance.In fact the cause of disease of determining disease may be impossible.Next step carries out the T cell and exhausts, and if suitable, carry out the B cell and exhaust (exhausting), the antibody (anti-IgE) of combined chemotherapy, radiotherapy and/or anti-B cell reagent (for example, CD19, CD20 and CD21) or special Ig hypotype with other immunocytes.By bestowing GnRH, improved the functional of bone marrow and immunocyte to the patient.Inject HSC with it simultaneously, in the gene institute transfection of the external autoantigen that is encoded, HSC enters the thymus of rejuvenation and is converted into DC HSC, to give developmental T presented by cells autoantigen, inducing tolerance in view of the above.The HSC of institute's transfection also will produce antigen and give developmental B cell with antigen presentation in bone marrow, cause their deletion in view of the above, this to betide thymus T cell on similar.The usage of immunosuppressant scheme (anti-T, B treatment) will overcome the activation that any discomfort of the potential autoreactivity T that is pre-existing in and B cell is worked as.In addition, in the situation of no obvious genetic susceptibility, GnRH can unite G-CSF injection with increase in the blood from the level of body HSC to strengthen the thymus reactivate.

In some embodiments, will be transplanted in the receptor body to increase the final reproduction speed of thymus from the hemopoietic of donor (donor that matches as MHC) and lymphoid stem cell and/or CFU-GM.In another embodiment, these cells of transplanting are from no autoimmune disease or healthy donor hypersensitive, and they have replaced patient's unusual stem cell and/or CFU-GM.

In one embodiment, eliminated patient's autoimmune disease by T cell mass to the small part of removing the patient.The signal pathway of sex steroid mediation is blocked., still can not induce the abnormal T cell of toleration of self is removed from the T cell mass after the hiving off again of peripheral blood at new T cell.

In another embodiment, exhaust treatment by identical lymphocyte, and by being blocked, the signal pathway of sex steroid mediation strengthens thymus T cell development simultaneously, to allow that " clean " T cell mass moves in again to PBF, eliminated causing of patient immune system cell hypersensitive.In other cases, after barrier steroid signal pathway, because allergy and autoimmune disease have been alleviated in functional increase of BM and other immunocytes.

Prevention

The present invention also provides by associating thymus reactivate, perhaps prior to the thymus reactivate or under the situation of the reactivate that does not have thymus, strengthen the functional of BM and/or other immunocytes, infect the method for resistance and treatment patient's infection with prevention infection, increase.In this stage, patient's immune system is enhanced, rejuvenation and reactivate, has increased it in view of the above to exotic antigen replying of virus and antibacterial for example.For example, as confirming, in Figure 13-17, shown of the immune effect of thymus reactivate to mice with virus (HSV) attack.The mice that had before had the thymus reactivate shows the resistance that HSV is infected, and does not have those mices of thymus reactivate (old and feeble thymus) to have higher levels of HSV to infect.The immune system of known mice is the model that is similar to very much people's immune system and is used as human diseases.Therefore, the result of mice can be used to show human replying.Shown that the thymus reactivate verified this point once more to the data of people's effect.In embodiment 23 illustrated increase functional ability of immunocyte, wherein 3-7 days and before the thymus reactivate, increased the responsiveness and the propagation of castrating the TCR of mice the earliest to the castrating back.

The raising that vaccine is replied

This description relates to the field of " initiatively vaccination ", wherein antigen is bestowed to the patient, and its immune system responds to antigen by forming anti-antigenic immunne response then.Vaccination can comprise preventative and therapeutic vaccine.As known in the art, use method of the present invention, in fact can suppress, do not need undue experimentation with the method associativity steroid of any vaccination.In some embodiments, can be prior to the thymus reactivate or give vaccination with it simultaneously.Also can use a plurality of dosage (for example reinforced immunological inoculation) as required.

In one embodiment, vaccine is dead and or the vaccine (for example through showing tremendous enthusiasm other chemicals) of deactivation.In another embodiment, vaccine is attenuated vaccine (for example poliomyelitis and an antismallpox vaccine).In one embodiment, vaccine is subunit vaccine (for example Hepatitis B virus vaccine, wherein hepatitis B virus surface antigen (HBsAg) is an antigenic specificity albumen).

In another embodiment, vaccine is a recombiant vaccine.One type recombiant vaccine be that wherein the cause of disease (for example virus) is deleted the special viral toxic gene that causes make virus be nontoxic attenuated vaccine.The recombiant vaccine of another kind of type has adopted usage infective but virus-free toxic carrier, and carrier is the gene that has inserted the coding target antigen by genetic modification.The example of recombiant vaccine is the vaccinia virus vaccine.

Still in another embodiment, vaccine is a dna vaccination.Vaccine based on DNA uses bacterial plasmid being expressed protein immunogen by among the host who inoculates usually.Be cloned in the carrier for expression of eukaryon with the recombinant DNA technology former cDNA of related immune that will encode.Amplification vaccine plasmid in antibacterial then, and be purified, be integrated directly in the host of being inoculated.Can inoculate DNA by the DNA in the needle injection saline or by the particle gun equipment that the gold bead with the DNA coating is transported in the skin.Those skilled in the art know and understand easily preparation and use the method for these vaccines.

There are some can influence the performance of immunne response and the parameter of degree: the position of antigenic level and type, vaccination, the availability of suitable APC, the general health situation of individuality and the state of T and B cell pool.Wherein, the T cell is a most fragile because the significant sex steroid that after pubarche, becomes the minimizing of inductive tangible thymus output and sex steroid to the generally inhibition of t cell response.Only presenting the existence of the cell of T originally of specificity component widely and having normal Th1 when all being optimized on the ratio of Tc cell, could implement any vaccination project in logic Th2 cell and Th when the potential T of replying cell.The antibody whether type of T cell helps to support the immunne response decision to be generated is that the defence of C ' dependency antibody and macrophage dependent/non-dependent will be mobilized (1 type is replied), or deny the antibody that generated be that C ' dependent/non-dependent antibody and the dependent defence of macrophage will be mobilized (2 types are replied) (summary is seen, Fearon andLocksley (1996) Science 272:50; Seder and Paul (1994) Annu.Rev.Immunol.12:635).Traditionally, 1 type is replied relevant with the generation of cytotoxic T cell and 2 types are replied with production of antibodies relevant.Therefore, the level of the cytokine that is generated and type also can be amplified to is enough to deal with required replying (for example, some diseases requires Th1 to reply, and ask for something Th2 replys, for example protective immunity).This comprises and gives Th1 and Th2 cytokines (for example, giving the DNA of the recombinant cytokine and the Codocyte factor) immune response pattern with the conversion patient auxiliaryly.Immunostimulating CpG oligonucleotide has been used to that also the immunne response to various vaccine dosage forms is transformed to more Th1 type and has replied.

By method described herein, sex steroid suppresses to have caused the increase of BM and immune cell function, do not have the thymus reactivate or prior to the situation of thymus reactivate under or with the thymus reactivate simultaneously, it allows the immunologic responsiveness of generation to vaccine.

The immune system that this method can be united any other form stimulates, and comprises adjuvant, additional molecule and cytokine therapy.For example, useful cytokine comprises, but is not limited to interleukin-22 (IL-2) and IL-15 as common immune somatomedin, will replys the IL-4 that is partial to Th2 (humoral immunization) and will reply IFN γ, the IL-12 that promotes Th1 that is partial to Th1 (cell-mediated inflammatory response) and the IL-10 that promotes the Th2 cell.Additional molecule is including but not limited to the inhibitor of CTLA4, and it is by promoting that B7.2 stimulation approach has strengthened immunne response normally by the CD28/B7.1 that CTLA4 suppressed.

The method of vaccination that recombinant gene expression vector can be used to invent.Recombinant vector can be plasmid or cosmid, and it comprises the polynucleotide of the invention of coding for antigens, but also can be virus and retrovirus.Used carrier can be " naked " (promptly for example liposome, colloidal particle etc. are not relevant with transport vehicle) in polynucleotide vaccine.For convenience's sake, used in this manual term " plasmid " will refer to plasmid or cosmid, and which is to be suitable for expressing related peptides (size of the gene of the required peptide of wherein encoding has determined selection between the two) according to.Plasmid commonly used can be pBR322.

Utilizable various viral vector comprise for example retrovirus of adenovirus, herpesvirus, vaccinia virus and RNA viruses in method of vaccination of the present invention.Retroviral vector can use mice and the retroviral derivant of bird.Retroviral example comprises, but is not limited to moloney murine leukemia virus (MoMuLV), Harvey mouse sarcoma virus (HaMuS-V), MMT virus (MuMTV) and Rous sarcoma virus (RSV).Plasmid and viral vector in other method of vaccination that can be used for inventing are known in the vaccine field.

Effect to BM and HSC

The function that the invention provides the BM that is used to increase the patient comprises generation that increases HSC and the method that strengthens hemoposieis.These methods all are useful in a plurality of application.For example, no matter be to be used for cancer or other purpose, the side effect of wherein a kind of difficulty of chemotherapy and radiation is exactly their negativity influences to patient B M.According to the dosage of chemotherapy, BM can be damaged or the generation of removing and hemocyte can be hindered.According to the present invention, the sex steroid analog (for example LHRH analog) of bestowing a dosage behind chemotherapeutic treatment helps the recovery of chemotherapy to the damage of BM and hemocyte.Same, bestow the LHRH analog several weeks before giving chemotherapy has increased HSC and other hemocyte group, feasible some illeffectss that reduced chemotherapy.

For example, the raising of BM function can be applied to suffer from the patient of hematologic disease.Term " hematologic disease " includes but not limited to for example leukemia of the disease relevant with hemopoietic in this any disease and disease that is defined as involving directly or indirectly the cell of blood system.Therefore, for example, method of the present invention is used in the new cell used among the allogene HSCT from the receptor (coupling with unmatched), or behind body HSCT with the cancerous cell of patient's oneself cell replacement blood system.

The HSC that BM increased produces and has caused erythrocytic increase, and it is useful to handling the RBC generation conversely.Can easily determine this point by seeking the hematocrit value that for example increases.

In some instances, the HSC that is increased is CD34+ or CD34lo HSC.The HSC that is mobilized (for example using G-CSF) can help " reparation " and rejuvenation to organize for example heart tissue and lung tissue.HSC has the ability that generates non-hemopoietic tissue.Though in the external work of having carried out a lot of this respects, a small amount of myocardial cell after the research at Mayo Clinic Rochester has been presented at BMT is the donor source.Similar, impaired cardiac muscle is repaired with HSC by the Beuamont hospital of Michigan, although HSC whether becomes the myocyte and blood vessel is not clear.Mouse test has also demonstrated the ability that HSC becomes the β cell of insulin-producing.Other work have demonstrated HSC can become skeletal muscle (myocyte), liver (hepatocyte), skeleton, connective tissue, epithelial tissue (for example epithelial tissue of lung, gastrointestinal tract and skin), blood vessel, neuron and beta Cell of islet.

Method described herein can be used for repairing to the infringement of BM and/or helps to replace to be damaged or destructive hemocyte by various treatments (for example cancer chemotherapeutic drug, radiotherapy) and disease (for example HIV, chronic renal failure).

In some chemotherapy regimens, for example treat the high-dose chemotherapy of any neoplastic hematologic disorder, be essential effect to the removing of BM.The method that can at once use invention after removing generation is to stimulate BM and to increase HSC and the generation of their filial generation hemocyte, to shorten patient's recovery time.After bestowing chemotherapy, in patient's body, removed the common needs of chemotherapeutics one day or more days, bestow the LHRH analog of a dosage of method described herein to the patient.This can unite and bestows from body or allogene BM or hematopoietic stem cell or CFU-GM and other the factor for example colony stimulating factor (CSF) and stem cell factor (SCF).

Perhaps, the patient can have bad (or " fatigue ") BM function and may not produce HSC abundant or normal number and other hemocyte.Multiple pathological changes can cause this point, comprise normal aging, infect for a long time, after the chemotherapy, after the radiotherapy, the chronic disease state comprises the inductive immunosuppressant of cancer, genetic abnormality and transplanting.In addition, shine for example total irradiation and can significant impact be arranged the production capacity of BM.These pathological changes also can or be treated in advance minimizing negative effect (for example chemotherapy and/or radiocurable negative effect), or after generation by treatment with reverse effect.

Effect to the T cell

The blocker (for example GnRH agonist) of the signal pathway by picked-up sex steroid mediation can temporarily suppress male and female sex steroid.The disappearance of known class sterin has caused the reactivate of thymus function and has strengthened the generation of T cell originally.In addition, even before those new T cells had an opportunity to leave thymus, original T cell was more responsive to stimulation, has caused more efficiently immunne response.The responsiveness that is increased is tangible (seeing that for example embodiment 23) in a couple of days of disappearance sex steroid.This may be because there has not been the inhibitory action of sex steroid.This may be that they can the associating external irritant stimulate the T cell altogether because of reactivate and " assisting " that thymus produced and " assisting a ruler in governing a country " that be re-activated factor.Also because immune many cells all have the surface receptor of GnRH, GnRH itself provides other stimulation can for the T cell.Because the disappearance of sex steroid is so fast to the effect of periphery T cell, can give GnRH as single therapy when for example giving vaccine.One month dosage form is useful, and it has the good effect that immune stimulatory replys but does not have the side effect of the longer time disappearance of sex steroid.Also can bestow antigenic " reinforcement " injection subsequently.

Sex steroid inhibitor (for example GnRH analog) all is useful for the immunization therapy of the form of ownership of strengthening the cancer patient, to the removing of the cancerous cell of fleeing from chemotherapy and operation, also is useful to prevention opposing opportunistic infection particularly.Can these analog of preventative use be designed for that prevention is for example infected or the immunne response of the vaccination plan of cancer with raising.

Method of the present invention has been utilized the inhibition to the sex steroid signal pathway.Sex steroid has suppressed the T of thymus, BM and whole body and the function of bone-marrow-derived lymphocyte, and these lymphocytes accumulate in the main lymph zone of health, includes but not limited to blood, lymph node, mucosal tissue (for example respiratory tract, gastrointestinal and reproductive tract).Be surprised to find to the removing of sex steroid and/or to the blocking-up of the signal pathway of the sex steroid mediation thymus (and number of T cell therefore and " quality ") that not only can be used to regenerate, can also be used to or under the situation that does not have the thymus reactivate or, improve T cell (with immune other cells) functional of original and new generation prior to the thymus reactivate or in the thymus reactivate.

The immunne response of difference can have fast and clinically important result.This can mean increases, the susceptibility of cancer and tumor is increased and/or poor to the responsiveness of vaccination the susceptibility of common infection (for example influenza).

The number of the cell of T originally in total T cell pool and/or the increase of ratio all have positive therapeutical effect fast to various clinical (subclinical) pathological changes and disease, these pathological changes and disease include but not limited to cancer, immunodeficiency (particularly viral infection, for example acquired immune deficiency syndrome (AIDS) (AIDS) and SARS (Severe Acute Respiratory Syndrome) (SARS) and influenza), autoimmunity, transplanting, allergy and the general efficient that improves the vaccination plan.At that have altogether and common unsettled U. S. application No.10/418, all described each application in these applications in 747,10/419,039,10/418,953,10/418,727,10/419,066 and 10/419,068 in detail.

About the application of method of the present invention on prophylaxis of viral infections, observed the example recently of the immune effect of function difference in SARS virus temporarily.Although this virus is relevant with cold virus, but very different, make sophisticated immune system can not discern it.The T cell infection SARS for example that can do with previous the unknown originally only.But,, generally be grown up, and as if the child can do with this disease all to this disease height susceptible because have only a spot of cell of T originally and can not produce rational additives amount.Can reflect this demography on mortalogram, the mean age at death is nearly 50 years old, does not record prepuberal death.

In an example, the inhibition (for example using the LHRH/GnRH analog) of sex steroid signal pathway is used to strengthen T and the bone-marrow-derived lymphocyte responsiveness to antigenic stimulus.This stimulation can be to enter the intravital microorganism of people (for example antibacterial, virus, fungus, parasite etc.).

In another example, the inhibition (for example using the LHRH/GnRH analog) of sex steroid signal pathway is used to strengthen immunne response to vaccine antigen.

In a non-restrictive example, the inhibition (for example using the LHRH/GnRH analog) of sex steroid signal pathway is occurred in before the immune system attack.This allows the time that the sex steroid disappearance occurs.Also can be used for direct enhance immunity and reply with acting as (through the mediation of the signal pathway of cell surface, increased cytokine, reduced mortifier etc.) stimulus object administration simultaneously or finish inhibition (for example using the LHRH/GnRH analog) successively to the sex steroid signal pathway.The snap action that also can gradation gives the inhibition (for example using the LHRH/GnRH analog) to the sex steroid signal pathway is because strengthened the functional of original lymphocyte (and non-lymphocyte).Along with the minimizing of the sex steroid of time has increased the generation of T cell, B cell and APC and functional, they add up ground, continue to strengthen and reply synergistically and complementally.Then can for example the increase of APC and original T cell, B cell and APC produce more efficiently immunne response to primary infection, secondary infection, vaccination etc. to the increase of the sensitivity that stimulates with new T cell, B cell and other immunocytes.

Therefore, the method according to this invention, with the blocker of sex steroid signal pathway by starting these activity of immune cells or functional increase has caused clinical positive acting, even before these medicines can cause significant thymus reactivate.

According to the present invention, these medicines also can be used to help to replace and may be damaged and destructive hemocyte including but not limited to cancer chemotherapeutic drug and/or radiotherapy and for example chronic renal failure of disease by various other treatment and diseases.As this other local institutes in greater detail, the usage that sex steroid suppresses medication combined G-CSF, GM-CSF, erythropoietin (EPO), SCF or other hormones and cytokine also can be used to further improve these chemical compounds are strengthened the effect that hemocytees produce.

Term " modify T cell mass form " is defined as changing character and/or ratio by functional defined and the defined T cell of expression subgroup by the characteristic molecule at this.The example of these characteristic molecules is including but not limited to TXi Baoshouti, CD4, CD8, CD3, CD25, CD28, CD44, CD45, CD62L and CD69.

" increase the number of cell ", for example increase the number of T cell, be defined as a thymus and/or a for example absolute increase of the number of the T cell in lymph node, gastrointestinal tract, urogenital tract and the respiratory tract of circulation and/or spleen and/or BM and/or peripheral tissues in the object at this.The relative increase of T cell also represented in this speech, for example when comparing with the B cell.

" have be subjected to T cell mass that press down or unusual or the object of function " comprises the individuality that HIV (human immunodeficiency virus) infects, particularly suffer from the virus of AIDS or any other attack T cell or the individuality that infects, or suffer from the individuality of any T cell disease that identifies dcc gene.In addition, this speech comprises after any adolescence individual, particularly has the old people of the increase of the attenuating of the immunologic responsiveness that atrophy of thymus gland caused after the adolescence and disease incidence.

Infecting at object has in the situation of HIV, with the HSC heritability modify make they and they filial generation particularly T cell, macrophage and the DC infection and/or the destruction that can tolerate HIV virus be useful.Genetic modification can comprise that the nucleic acid molecules with one or more prevention virus replications, assembling and/or infection is incorporated in the HSC.These nucleic acid molecules can be gene, antisense constructs, ribozyme, dsRNA and the catalytic nucleic acid molecules of coding antiviral protein.

Have in the situation of defective T cell at object, HSC can be modified by heritability and makes defective normalization.For disease T chronic myeloid leukemia for example, modification can comprise that introducing nucleic acid construct or gene makes HSC normalization and suppress or reduce the probability that they become cancerous cell.

Those skilled in the art understand that it all may be useful that the present invention has in any T cell disease of clear and definite hereditary basis in treatment.

Method of the present invention also is useful for treatment AIDS, wherein treatment comprise reduce viral load, increase the T cell number and functional, by inhibition reactivate thymus function to the sex steroid signal pathway.The patient can accept the HSC that modified by heritability, makes all filial generations (for example T cell and DC) can tolerate the further infection of HIV.This means that not only the patient has been eliminated HIV virus, and the patient no longer susceptible is in common infection because the T cell has returned to normal level, and the new T cell that can tolerate HIV can be removed the cell that any residual virus infected.In theory, similar strategy can be applied to any T cell defect and any targeting in the gene therapy of the HSC of the viral infection of T cell.

Effect to the B cell

The same with other immune cell, the function of B cell has also been lowered along with old, and this part is because minimizing that the T cell produces and the auxiliary shortage of T cell that is caused.But, also have relevant B cell function of significant age variation (Hu et al., (1993) Int, Immunol.5:1035-1039).Although because firm homeostasis mechanism of regulating, the B cell number is still constant relatively in whole life, but reduced the output of BM, the clonal expansion of periphery B cell, and the weakening of immunne response (LeMaoult et al., (1999) J.Immunol.162:6384-6391).It mainly is to ascribe the auxiliary minimizing of T cell (Hu et al. to that the attenuating of in old-age group external antigenic antigen being replied is considered to, (1993) Int.Immunol.5:1035-1039, LeMaoult et al., (I999) J.Immunol.162:6384-6391).But, the selectivity disappearance of classification conversion defective (Weksler et al., (2000) Vaccine 18:1624-1628) and high-affinity antibody may have effect (Nicoletti et al., (1993) J.Immunol.150:543-549).

Castrating to old mice has caused the B cell CFU-GM of IL-7 sensitivity to comprise the increase (Ellis et al., (2001) Int.Immunol.13:553-558) of pro B lymphocyte in late period, pre B lymphocyte and immature B cells.The increase major part of this circulation B cell is because the BM (CD45R that moves out recently ^LoCD24 ^Hi) increase of number, and these cells are up to the back level (Ellis et al., (2001) Int Immunol.13:553-558) that still was in rising in 54 days of castrating.

Therefore, can not have the thymus reactivate or prior to the situation of thymus reactivate under or with the thymus reactivate simultaneously, with the number of method increase B cell of the present invention and functional.This for increase to normal patient and defective immune patient for example those controls (by prevention or treatment) of accepting the bacterial infection among the patient of disease treatment scheme may be useful.B cell number that is increased and after surgery functional and/or burn survivor and wherein patient's immune system be may also be useful in impaired other situations.

Effect to DC

The present invention also provides the method that increases the functional and/or DC number of DC.After sex steroid is removed (after for example giving the LHRH analog), increased the DC of thymus and periphery, periphery DC may also help the T cytositimulation.DC is important antigen-presenting cell, and number that is increased and/or function may be useful on the responsiveness of cancer for example improving the cause of disease.The DC that has strengthened is functional to realize that to the cause of disease may also be useful on the toleration of anaphylactogen or autoantigen (for autoimmune disease) for example.

To hematoblastic effect

The present invention also provides platelet increasing number and/or functional method.Thrombocytopenia is common and Different types of etiopathogenises is arranged, including but not limited to barren BM and splenomegaly.Pathological changes causes the hemorrhage that is very difficult to treat usually.The modal disease relevant with thrombocytopenia comprises leukemia, aplastic anemia, liver cirrhosis and Gaucher disease.Usually need a large amount of blood replacings, because the hematoblastic half-life of the storage blood that is used for transfusing blood is very short.

Effect to NK, NKT and Treg cell

The present invention also provides number and/or the functional method (seeing for example embodiment 17 and Figure 43 and 49) that increases NK and NKT cell.This may be useful in crohn, the syndromic treatment of Chediak-Highashi for example to the disease that shows the NK defective.In comprising the patient of lupus and rheumatoid arthritis, connective tissue disease reported impaired NK cell function.The NK cell defence cancer and venereal infection because of on be important.The nonrestrictive disease relevant with NK cell defect (number and/or function) of other that may be benefited from the method for invention comprises herpesvirus infection (varicella zoster virus (VZV for example, chickenpox, herpes zoster), HSV, CMV and EBV), other viral infection (for example, measles, mumps virus, influenza virus and HIV, think that now these all are to be controlled by the NK cell to small part), mycobacterial infections (mycobacterium tuberculosis for example, mycobacterium avium, these obviously are subjected to the control of NK and macrophage), (for example, solid tumor comprises melanoma to other malignant tumor, breast carcinoma and rectal cancer), with the BMT of the identical or unmatched receptor of monoploid to acute myeloid leukemia.The NKT cell is the important instrumentality of immunne response, because they are Producers of the very high yield of cytokine, and does not need activation in advance.They not only have effect on the prevention autoimmune disease, also promoted antitumaous effect.Treg (often being defined as CD4+CD25+) also is the main instrumentality of immunne response, mainly is the cytokine product by them.

Macrophage

The present invention also provides the method that is used to increase macrophage number and function (seeing, for example embodiment 17 and Figure 43), and it is helping to remove venereal infection because of particularly bringing into play main effect on the antibacterial.

Embodiment

The following examples partly provide the specific embodiment of the method for invention, but it can not be interpreted as invention is restricted to the content of embodiment.

Embodiment 1: reverse the old and feeble atrophy of thymus gland that causes

Material and method

Animal: obtain CBA/CAH and C57B16/J male mice from the center animal service center of Monash university, and with its stable breeding in normal condition.Center animal service center, Walterand Eliza Hall medical research institute (Parkville from Monash university, Vicotoria) and A.R.C. (Perth, Western Australia) obtain C57B16/J Ly5.1, and with its stable breeding in normal condition.Month very much not wait in age from 4-6 week to 26, can indicate the age in relevant place.

Operation castrating: through lumbar injection 0.3ml 0.3mg xylazine (Rompun_; BayerAustralia Ltd., Botany NS W is Australia) with 1.5mg ketalar (Ketalar_; Parke-Davis, Caringbah, NSW, saline solution Australia) is with mouse anesthesia.By the scrotal incision castrating that undergos surgery: appear testis, with suture with its ligation, then with the periphery fatty tissue with its taking-up.Order with operation and to close wound.False castrating is not taken out testis exactly after above-mentioned steps, and is used as the contrast of all researchs.

Bromodeoxyuridine (BrdU) mixes: mice accepted twice BrdU (Sigma ChemicalCo., St.Louis, intraperitoneal injection MO), each dosage is the BrdU of the 100mg/kg body weight in the 100 μ l PBS, separates 4 hours at (i.e. 4 hours interval).Control mice is only accepted vector injection.After injecting 1 hour for the second time, cut thymus, or make cell suspending liquid and be used for facs analysis, or at once it is embedded in (O.C.T.compound among the Tissue Tek, Miles Inc., Indiana), flash freezing and storing-70 ℃ in liquid nitrogen up to use.

Flow cytometry: use CO ₂Suffocate and put to death mice, and take out thymus, spleen and mesenteric lymph node.Wash organ through one 200 μ m sieve lightly at cold PBS/1%FCS/0.02%Azide, centrifugal (650g, 5 minutes, 4 ℃), and be resuspended among arbitrary PBS/FCS/Az.With splenocyte in the erythrocyte splitting buffer 4 ℃ hatched 10 minutes, wash and be resuspended among the PBS/FCS/Az.Utilize hematimeter and bromination second pyridine/acridine orange and at fluorescence microscope (Axioskop; CarlZeiss, Oberkochen, Germany) under observation replication cell concentration and vigor.

For trichroism immunofluorescence, with anti-α β TCR-FITC, anti-CD4-PE and anti-CD8-APC (all available from Pharmingen, San Diego, CA) labeled cell is followed by flow cytometry.Or with α β TCR-FITC/CD4-PE/CD8-APC or with B220-B (Sigma) and CD4-PE and CD8-APC labelling spleen and lymph node suspension.Use Laboratories available from Caltag, Inc., Burlingame, the streptavidin of CA-trichroism coalition (conjugate) demonstrates B220-B.

BrdU for cell detects, and with CD4-PE and CD8-APC surface markers cell, is foregoing fixing and permeabilization (Carayon and Bord, (1989) J.Imm.Meth.147:225) subsequently.Simply, staining cell at 4 ℃, is fixedly spent the night among 1% paraformaldehyde (PFA)/0.01%Tween-20.(100Kunitz units, Roche USA), are hatched 30 minutes with denatured DNA under 37 ℃ at 500 μ l DNA enzymes with the cell after the washing.At last, cell and anti-BrdU-FITC (Becton-Dickinson) were at room temperature hatched 30 minutes, washing and re-suspended cell are used for facs analysis.

BrdU for the TN subgroup analyzes, and cell is all established door outside the Lin of APC cell, detects CD44-biotin and CD25-PE subsequently before BrdU detects.All antibody all from Pharmingen (San Diego, CA).

For four color immunofluorescences, with thymocyte cell labelling CD3, CD4, CD8, B220 and Mac-1, (Amersham U.K.) detects jointly, and establishes an analysis negative cells (TN) with anti-Mus Ig-Cy5.They also are colored CD25-PE (Pharmingen, San Diego, CA) and CD44-B (Pharmingen, San Diego, CA), be foregoing streptavidin-trichroism (Caltag, CA) (Godfrey and Zlotnik, (1993) Immunol.Today 14:547) subsequently.The aforesaid then detection of carrying out BrdU.

At FACSCalibur ^TM(Becton-Dickinson) go up analytical specimen.With 90 degree scattered light numerical value different lymphocytes is established door according to 0 degree, and use CellQuest ^TMSoftware (Becton-Dickinson) analytical data.

Immunohistology: (Leica) cuts out refrigerated thymus section with cryostat, and it is fixed in 100% acetone at once.

For the dichromatism immunofluorescence, dye section with one group of monoclonal antibody is two: at MTS6,10,12,15,16,20,24,32,33,35 and 44 (Godfrey et al., (1990) Immunol.70:66 that this laboratory produced; Table 1), and with polyvalent rabbit anti-cell keratin antibody (Dako, Carpinteria CA) measure the coexpression that epithelial cell determines thing.With bonded sheep anti mouse Ig (the Silenus Laboratories of FITC, Victoria Australia) demonstrates bonded mAb, and with bonded goat antirabbit Ig (the Silenus Laboratories of TRITC, Victoria Australia) demonstrates antikeratin antibody.

BrdU for section detects, or with antikeratin antibody and back to back anti-rabbit TRITC or with can be with anti-Mus Ig-Cy3 (Amersham, Uppsala, Sweden) the shown special mAb stained that goes out.Carry out BrdU then as previously mentioned and detect (Penit et al., (1996) Proc.Natl.Acad.Sci, USA 86:5547).Simply, will cut into slices and in 70% ethanol, fix 30 minutes.Half-dried section is hatched in 4M HCl, by washing neutralization in borate buffer (Sigma), is washed twice in PBS subsequently.Detect BrdU with anti-BrdU-FITC (Becton-Dickinson).

For trichroism immunofluorescence, with special MTS mAb and antikeratin antibody labelling section together.As above carrying out BrdU then detects.

With Leica fluorescence and Nikon Laser Scanning Confocal Microscope analysis section.

Migration research (promptly to thymus recently move out the analysis of (RTE)): through lumbar injection 0.3ml0.3mg xylazine (Rompun_; Bayer Australia Ltd., Botany NSW is Australia) with 1.5mg ketalar (Ketalar_; Parke-Davis, Caringbah, NSW, saline solution Australia) is with mouse anesthesia.

Ins and outs and other local described those countings similar (Scollay et al., (1980) Proc.Natl.Acad.Sci, USA 86:5547 to FITC labelling thymocyte cell; Berzins et al., (1998) J.Exp.Med.187:1839).Simply, expose lobes of thymus, every leaf is injected nearly 10 μ l350 μ g/ml FITC (among the PBS).Order with operation and to close wound.Heat mice up to recovery fully from anesthesia.After injection nearly 24 hours, use CO ₂Suffocate and put to death mice, and take out lymphatic organ and analyze.

After cell counting,, use flow cytometry then with anti-CD4-PE and anti-CD8-APC stained preparation.Migrating cell is identified as the FITC+ cell (with cell and the doublet that omits autofluorescence) of establishing door of the work (live) of expressing CD4 or CD8.The percentage ratio that adds FITC+CD4 and cd8 cell provides total migration percentage ratio to give lymph node and spleen respectively.As Berzinset al., (the described calculating of carrying out output rating every day of (1998) J.Exp.Med.187:1839.

The data of using non-paired student t check or non-parametric Mann-Whitney U check to be analyzed are used to determine contrast and carried out significance,statistical between three times the test result of test at least.Represent that with following test bit and contrast know significant difference is arranged: ^*P≤0.05; ^*P≤0.01 He ^{* *}P≤0.001.

The result

I. the age is to thymocyte cell group's effect

(i) thymic weight and thymocyte cell number

Along with the increase at age, the thymic weight of mice (Figure 1A) and total thymocyte cell number (Figure 1B) all have the decline (p≤0.001) of highly significant.The meansigma methods of the relative thymic weight (mg thymus/g health) of young adult is 3.34, and 18-24 month mice just drops to 0.66 (deposition of fat has limited accurate calculating).The thymus that the decline of thymic weight can ascribe the minimizing of total thymus number: 1-2 month big (being young adult) to contains has an appointment 6.7 * 10 ⁷Thymocyte cell is to having reduced to about 4.5 * 10 24 months the time ⁶Removed the effect of sex steroid to thymus by castrating, recovered the number of thymocyte cell, in 4 whens week after castrating, thymus all equals young adult on weight (Figure 1A) and cell density (Figure 1B).What is interesting is that 2 whens week increased thymocyte cell number (1.2 * 10 significantly after castrating ⁸) (p≤0.001), having arrived the castrating back during 4 weeks, the thymus number has returned to the level of normal young mice.

The minimizing of the T cell number that does not reflect thymus and produced in periphery, the splenocyte number is along with the age is still constant (Fig. 2 A and 2B).The homeostasis mechanism of periphery is conspicuous, because the B cell of spleen and lymph node is not subjected to the influence at age to the ratio of T cell, and the minimizing thereupon (Fig. 2 C) that arrives the T cell number of periphery.But, CD4+ to the ratio of CD8+T cell along with the age significantly descends, 1: 1 (Fig. 2 D) when from 2 months when big 2: 1 to 2 years old are big.After the T cell number of castrating and arrival periphery increases thereupon, do not observe the variation of periphery T cell number: the B of splenic t-cell number and spleen and lymph node: the ratio of T cell is along with castrating does not all have to change (Fig. 2 A-C).The CD4 of the periphery that reduces along with the age: be still obvious attenuating CD8 ratio 2 weeks after castrating, but to 4 weeks of castrating back just having recovered (Fig. 2 D) fully.

(ii) along with the thymocyte cell subgroup after age and the castrating

For the thymocyte cell number determining to be seen along with whether the minimizing at age is the result of the exhaustion of special cell mass, with specific markers substance markers thymocyte cell to analyze different subgroups.In addition, this allows the analysis of dynamics to the thymus complex group after the castrating.The cell subsets ratio of the thymus of the ratio of main thymocyte cell subgroup and those young adults (2-4 month) is compared (Fig. 3), find that this ratio is along with the age still is consistent.In addition, the further subgroup typing of thymocyte cell is found by the expression of α β TCR the ratio of these groups is along with the age does not change.In 2 weeks and 4 whens week after castrating,, the thymocyte cell subgroup still is identical ratio, though the thymocyte cell number after castrating has increased by 100 times, this explanation is the synchronous amplification of all thymocyte cell subgroups, rather than the amplification of different progresses.

Therefore, the minimizing of the cell number seen in the thymus of old (2 years old) animal shows it is the balance minimizing of all cells hypotype, the not significant change of the T cell mass that detects to some extent.The thymus reactivate occurs in a synchronous manner, simultaneously rather than successively all T cell subsets of restock.

II. the propagation of thymocyte cell

Shown in Fig. 4 A-4B, in the time of 2-4 month, there is the thymocyte cell of 15-20% to breed.Major part in these cells (about 80%) is two male (DP) (being CD4+CD8+), and three feminine genders (TN) (being CD3-CD4-CD8-) subgroup has constituted and accounts for about 6% second jumpbogroup (Fig. 5 A).These TN cells are intrathymic least sophisticated cells and comprise precursor in the thymus.Therefore, the visible most division of immunohistology is under peplos and in the cortex.Some divisions are found in facs analysis institute localized medullary substance zone, and facs analysis has shown the ratio (9%CD4+T cell and 25%CD8+T cell) (Fig. 5 B) of single positive (being CD4+CD8-or CD4-CD8+) cell young (2 months), splitted thymus.

Although the cell number in the old mouse thymus (2 years old big) has reduced with being limited, the toatl proportion of the thymocyte cell of propagation is still constant (Fig. 4 B and 5C), but the ratio of the somatoblast among the CD4-CD8-has decline, and the propagation (data do not show) that has also reduced the CD4-CD8+T cell significantly.Immunohistology shows that the distribution of somatoblast in the time of 1 years old reflected the finding in young adult (2-4 month), and still, to 2 years old the time, it is outer and in the blood vessel periphery that propagation is mainly seen in cortex, and the division in medullary substance seldom.

The earliest to castrating back during 1 week, the remarkable increase (data do not show) of the ratio of the CD4-CD8-cell of propagation and CD4-CD8+ cell is arranged.Castrating has obviously overcome the blocking-up of age to the propagation of these cells.There is the corresponding ratio of propagation CD4+CD8-cell to descend (data do not show) after the castrating.In 2 whens week after castrating, although increased the thymocyte cell number significantly, the toatl proportion of the thymocyte cell of breeding is variation not, at this synchronous amplification (Fig. 4 A, 4B and 5C) of cell has been described.The position of the thymocyte cell amplification when immunohistology has shown 2 weeks after the castrating and the degree of somatoblast all approach the state of 2 months big thymus.

Except postthymic precursor cell, the DN subgroup also comprises α β TCR+CD4-CD8-thymocyte cell, think them in the process that is converted into the SP cell, all reduced two kinds of co-receptors (Godfreyand Zlotnik, (1993) Immunol.Today 14:547).By the door of establishing to these mature cells, it is possible analyzing real TN part (CD3-CD4-CD8-) and the subgroup of their expression CD44 and CD25.Fig. 5 E-H has illustrated the interior propagation degree of each TN subgroup in young, old and the castrating mice.This has shown the remarkable attenuating (p＜0.001) of the propagation of TN1 subgroup (CD44+CD25-CD3-CD4-CD8-), 10% to 18 months big about 2% (Fig. 5) from normal young mice, and 1 week just recovered after castrating.

III. thymocyte cell is moved out

In young mice, nearly 1% T cell move out in the thymus (Scollay et al., (1980) Proc.Natl.Acad.Sci, USA 86:5547) all there is every day.Find rate that the castrating mice is moved out equal 14 months big normal young mice and even 2 years old big mice, although reduced (p≤0.0001) (Fig. 6 A and 6B) on the number significantly.Recently the CD4 that moves out of thymus: the CD8 ratio increases to some extent, from 2 months big 3: 1 to 26 months big 7: 1 (Fig. 6 C).In 1 when week after castrating, this ratio is normalization (Fig. 6 C).During 2 weeks, the cell number of the periphery of moving out significantly increases to the castrating back, and total rate of moving out has reduced to 0.4%, and this has reflected the amplification (Fig. 6 B) of thymus.

Discuss

Although having shown old and feeble thymus is serious atrophy, along with the age is still keeping its Functional Capability, the level that the T cell proliferation takes place, break up and move out equals young adult mice.Although thymus function is subjected to the adjusting of nerve-endocrine-more intermediary complex interactions of immunity axle, the degree of the thymus reactivate after the castrating has illustrated that sex steroid produces inductive atrophy the most significant and long effect is arranged.

As previously shown, thymic weight is along with the age has been reduced (Hirokawa andMakinodan, (1975) J.Immunol.114:1659, Aspinall, (1997) J.Immunol.158:3037) significantly, and this remarkable minimizing with the thymocyte cell number is relevant.Castrating operation the inductive further atrophy that stress cause thymus because of the effect of glucocorticoid, this point can be ignored by the influence that 2 weeks after castrating remove sex steroid, and it makes the cell density of thymus be increased to the 20-30 before the castrating doubly.Arrived 3 weeks of castrating back, old and feeble thymus demonstrates the remarkable increase on thymus size and the cell number, is better than the thymus of young adult, and prediction is the effect that 2 months big mices itself has been applied because of sex steroid.

Data validation previous discovery, pay attention to the ability (Aspinall, (1997) J.Immunol.158:3037) that thymus continues differentiation along with the age and keeps constant subgroup ratio.In addition, thymocyte cell differentiation shown with castrating after take place simultaneously, the synchronous amplification of thymocyte cell subgroup has been described.Because the thymocyte cell number, has been analyzed the propagation of thymocyte cell along with the remarkable minimizing at age to determine whether it is the priming factors of atrophy of thymus gland.

The influence that remove sex steroid inductive atrophy of thymus gland of age or castrating back does not all have influence on the propagation of thymocyte cell, and 14% of all thymocyte cells is breeding approximately.But this splitted position is all differences along with the age: 2 months big mouse thymus demonstrate under the whole coating and the cortex zone in the division of enriching (TN and DPT cell), and also have some divisions to occur in medullary substance.Because the thymus epithelial structure is along with the age disintegrates, value-added position is nondescript, but shows as the pattern still less more unified than the mice of youth, and the cortex skin of dividing a word with a hyphen at the end of a line.In 2 whens week after castrating, somatoblast can both detect in whole cortex, and is significantly in medullary substance, has and the similar distribution of big thymus in 2 months.

Do not change as the phenotype of analyzing determined propagation group with CD4 and CD8 with age and castrating back.But, the remarkable attenuating of the propagation of TN and CD8+ cell with the age found in the analysis of the propagation in the thymus subgroup.According to label CD44 and CD25 the remarkable attenuating that TN1 (CD44+CD25-) group breeds has been found in the further analysis in the T cell subgroup, remedied this point by TN2 (CD44-CD25+) group increase.In the TN group these have reflected the discovery of Aspinall ((1997) J.Immunol.158:3037) unusually.Surprised is, 2 when all to the castrating back, and the TN subgroup is bred with normal level, and this has illustrated the fast reaction of this group to the inhibition of sex steroid effect.In addition, in 2 weeks and 4 whens week after castrating,, the ratio of the CD8+T cell of propagation will may illustrate their effects in the foundation of peripheral t cell pool apparently higher than contrasting thymus.

Demonstration is along with the age, the thymus migration has taken place in the thymocyte cell of constant ratio, this point and Scollay et al., ((1980) Proc.Natl.Acad.Sci, USA 86:5547) previous data contradicts, they show that thymocyte cell moves to 10 times minimizing of the mobility of periphery.These results' difference may be the effect of FITC being absorbed because of difficulty or lipidosis to FITC labelling in the thymus of 2 years old big thymus.But as Scollay found, the absolute number of the T cell of migration had been reduced significantly, had caused the remarkable attenuating of RTEs to the ratio in periphery T cell pond.This will cause periphery mainly to influence the change (Mackall et al., (1995) N.Eng.J.Med.332:143) of T cell bank.Previous article (for example, Mackall et al., (1995) N.Eng.J.Med.332:143) shown the T cell bank along with the age bias to memory rather than T cell phenotype originally.If but individuality has run into new antigen, reduced T cell bank may not be dealt with, and this may cause the immunodeficiency in the senior people.It is evident that the needs of rebuilding the T cell pool in the immunoincompetent individuality are arranged.Castrating allows that thymus passes through to increase significantly originally the output of T cell and realizes periphery regeneration.

B as spleen and lymph node: as shown in the T ratio, the T cell number of periphery still is a constant level, may be because the homeostasis of periphery.(Mackall?et?al.，(1995)N.Eng.J.Med.332：143；Berzins?et?al.，(1998)J.Exp.Med.187：1839)。But, the interference that peripheral cells is formed is along with the thymus of aging is tangible, show as CD4: the remarkable decline of CD8 ratio, from 1: 1 of 2: 1 to 2 years old mices of young adult, the increase that the CD4+T cell produces the CD8+T cell of the more responsive performance at age or thymus external source may be described.Arrived the castrating back during 2 weeks, this ratio has reflected the rapid answer of immune system to the operation castrating by normalization at this.

Above-mentioned discovery has shown old and feeble thymus energy functionating first, and its character is equal to preadolescence thymus.Aspect this, though the T cell number has been reduced significantly, the ability of thymocyte cell differentiation is not blocked.Their total propagation and the ability of finally moving to periphery are not subjected to the influence of relevant atrophy of thymus gland of age yet.But, noticed two important discoveries.The first, demonstrate illeffects on to the T ability of cell proliferation to the T cell, this discovery with Aspinall ((1997) J.Immunol.158:3037) is relevant.This defective can ascribe the latent defect of thymocyte cell itself to.Although still shown that in this data presented and previous work Differentiation-of Thymocytes reduces to some extent but still has generation, and from the influence (Hirokawa that moves into and be not subjected to the age of the stem cell of BM, (1998), " Immunity and Ageing " in PRINCIPLESAND PRACTICE OF GERIATRIC MEDICINE, (M.Pathy, ed.) John Wileyand Sons Ltd; Mackall and Gress, (1997) ImmunoL Rev.160:91).The second, along with the age has reduced the multiplication capacity of CD8+T cell significantly, and after castrating, with 2 months mice relatively, increased the ratio of propagation CD8+T cell significantly.Final step (Suda and Zlotnik, (1991) J.Immunol.146:3068) before the propagation of mature T cells is considered to move, so the remarkable minimizing of CD8+ propagation may illustrate the attenuating of their transfer ability.CD4 among the RTEs: the cd8 t cell ratio is along with this hypothesis has been supported in the discovery of the increase at age, and this has pointed out the minimizing of the cd8 t cell of migration.Same, if keeping to cd8 t cell, thymus epithelial provides key factor, no matter be the influence of matter molecule or cytokine between lymph, produce by the sex steroid that increases and may block this factor.By removing the influence of sex steroid, can be this optimization ground propagation cd8 t cell group.

The disappearance of the propagation defective prompting cortex epithelium of viewed TN1 subgroup influences the growth of thymocyte cell on the critical stage that tcr gene is reset, wherein the cortex epithelium provides thymocyte cell to generate the necessary factor for example IL-7 and SCF (Godfrey and Zlotnik, (1993) Immunol.Today 14:547; Aspinall, (1997) J.Immunol.158:3037).In fact, IL-7-/-with IL-7-/+mice demonstrates and similar thymus form (Wiles et al., (1992) Eur.J.Immunol.22:1037 seen in old mice; Zlotnik and Moore, (1995) Curr.Opin.Immunol.7:206); Von Freeden-Jeffry, (1995) J.Exp.Med.181:1519).

Generally speaking, old and feeble thymus still keeps its Functional Capability, but the growth of the thymocyte cell in the old mice is not under the strictness control of the thymic epithelial cell seen in the normal young mice, and this is because the disappearance of the structural intergrity of thymus microenvironment.Therefore, the propagation of these cells, differentiation and migration will be under optimized adjustings, and may cause the release of the autoreactivity/immunoincompetent T cell that has increased periphery.Defective in TN and particularly the CD8+ group all may cause the variation with the age in the peripheral t cell pool.Castrating will provide a kind of periphery T cell pond that is used to regenerate also therefore to be used to rebuild important tool immunosuppressant, immunodeficiency or the individuality that the immunity mistake is compensatory to the reconstruction of thymus function.

Embodiment 2: the atrophy of thymus gland that reverses chemotherapy or radiotherapy-induced

Material and method

Material and method are as described in Example 1.In addition, used following method.

BM rebuilds: recipient mice (3-4 month big C57BL6/J) has been accepted 5.5Gy irradiation twice, 3 hours at interval.After second time exposure dose 1 hour, give injection 5 * 10 in the mouse vein ⁶Donor BM cell.By with tibia and the femur of RPMI-1640 medium flow, gather in the crops cell collected in culture medium then and obtain the BM cell through donor (2 months big homologous C57BL6/J Ly5.1+) mice.

Irradiation: a 3-4 month big mice is accepted the total irradiation of 625Rad.

Use the T cell of cyclophosphamide to exhaust: to give old mice (for example 2 years old big) injection cyclophosphamide (the 200mg/kg body weight is in 2 days) and with its castrating.

The result

Castrating has strengthened the regeneration after the serious T cell exhaustion (TCD).Model (using the chemotherapy of cyclophosphamide or the sublethal exposure of use 625Rad) for two kinds of T cells exhaustion of being studied, castrate comparing of (SHCx) with their vacation, the castrating mice all demonstrates the remarkable increase (Fig. 7 A and 7B) of thymus reactivate rate.Arrived 1 week of treatment back, castrating mice even just demonstrate the regeneration (Fig. 7 and 9-11) of thymus in early days.In comparison, neuter does not demonstrate DN and the serious disappearance of DP thymocyte cell (somatoblast fast) and the increase of CD4 subsequently and cd8 cell (anti-radiation) ratio.With in addition after 1 week after the treatment just demonstrating the thymus size and increase at least 4 times castrating the thymocyte cell number of animal difference best illustration this point.When 2 weeks, neuter does not demonstrate the normalization of relative thymocyte cell, with the regeneration of DN and DP thymocyte cell.But the ratio of thymocyte cell still is not equal to the thymus of young adult contrast.In fact, in 2 whens week, castrating and not castrate greatest differences between the regulation rate between the mice be maximum (to 4 weeks time, the number of the thymocyte cell between the treatment group is identical).

After cyclophosphamide treated for 1 week, it is (untreated, age-matched that the thymocyte cell density of SHCx mice will be less than contrast significantly; P≤0.001) and Cx mice (p≤0.05) (Fig. 7 A).On this time point, 1 the week before the castrating mice and and the treatment on the same day the castrating mice between do not observe difference on the thymus reactivate rate, two groups of cell numbers that all demonstrate them are the twice (Fig. 7 A and 7B) of SHCx mice at least.Similar, during 2 weeks, two groups of Cx mices have the number (p≤0.001) (Fig. 7 A) of (5-6 doubly) thymocyte cell significantly higher than SHCx mice in cyclophosphamide treatment back.In control mice, in 4 time-of-weeks, recovered the thymocyte cell number gradually, but castrating has obviously strengthened this point, even in 1 week (data do not show).In 1 week after castrating, similarly increased the cell number (data do not show) of castrating interior spleen of mice and lymph node.

By 1 week of pre-irradiation (Figure 10) or with irradiation on the same day the castrating of (Figure 11) detected the effect that castrating recovers thymus opportunity.When castrating before 1 week, castrating recovers to have effect (comparison of Figure 10 A and Figure 11) faster to thymus.When 2 weeks, the thymus reactivate in the mice that 1 week castrated before treatment " has been caught up with " in castrating on the same day.In two kinds of situations, separately spleen or lymph node all there is not big effect (Figure 10 B and 10C, and Figure 11 B and 11C).

After irradiation, all thymocyte cell density will be less than control mice (p≤0.001) (Fig. 7 B and 11A) significantly and the mice (p≤0.01) (Fig. 7 B) of 1 periderm castrating before treatment demonstrating with the treatment mice being castrated and castrate by vacation on the same day.In 2 whens week after treatment, the castrating scheme is as broad as long to the recovery of thymocyte cell number, and two groups of castrating mices all show postradiation thymocyte cell than the remarkable enhancing in the SHCx mice (p≤0.001) (Fig. 7 B, 10A and 11A).Therefore, castrating has strengthened the thymus reactivate after serious T cell is exhausted significantly, can castrate at least 1 week before prior to compromised immune.

What is interesting is that the thymus size shows the baseline of " having surpassed " contrast thymus.This has pointed out intrathymic rapid amplifying, also not migration of the thymocyte cell of these new formation (thymocyte cell need about 3-4 move out and enter periphery).Therefore, although the ratio in each subgroup is identical before being released to periphery, set up the number of thymocyte cell.

After young mice (about 2-3 month big) cyclophosphamide is handled, although reduced total lymphocyte number in the spleen of Cx mice, whole analysis the time all do not have significant difference (data do not have demonstration) with control mice in mutually.But, ShCx mice 1 week and all demonstrate total spleens cell number purpose 4 weeks and significantly reduce (p≤0.05) (Fig. 8 A) after treatment.In lymph node, 1 week after treatment, all observed the remarkable minimizing (p≤0.01) (Fig. 8 B) of cell density in false castrating and the castrating mice, may reflect stress steroid influence.To treating the back during 2 weeks, lymph-node cell density and the control mice of castrating mice are suitable, but false castrating mice did not recover their lymph-node cell number up to the treatment back in 4 weeks yet, and its cell density will be less than the Cx mice (Fig. 8 B) in (p≤0.05) contrast and treatment 2 weeks of back significantly.These results show that castrating can strengthen the speed of the recovery of total lymphocyte number after the cyclophosphamide treatment.

Serious lymphopenia has been induced in sublethal exposure (625Rad), and like this 1 when week after treatment, the cell density that treatment group (Cx and SHCx) portion demonstrates spleen and lymph node will be less than (p≤0.001) control mice (Figure 12 A and 12B) significantly.To shining the back during 2 weeks, castrate and false splenocyte number similar to control value (Figure 12 A) of castrating mice, and the lymph-node cell number still is markedly inferior to control value (false castrating mice p≤0.001; Castrating mice p≤0.01) (Figure 12 B).Between Cx and ShCx mice, do not observe significant difference.

Figure 19 for example understands the usage and the comparison of operation castrating in enhancing T cell regeneration of chemical castration.Inject used in this embodiment 4 weeks of chemicals deslorelin (LHRH-A), and demonstrate the suitable of the reproduction speed after the cyclophosphamide treatment and the castrating of performing the operation.Potentiation equates in the thymus amplification, also equates in the recovery of spleen and lymph node.The kinetics of chemical castration is slower than operation, that is to say, mice need spend about above time in 3 weeks to reduce their sex steroid level.But chemical castration is still the same effective with the operation castrating, can think to have identical effect.

Discuss

In the animal model of immunodepletion, inquired into castrating to the structure of thymus and the influence of T cell generation.Concrete, embodiment 2 has checked the effect of castrating the immune recovery after sublethal exposure and the cyclophosphamide treatment.The immunodepletion of these forms act on suppress DNA synthetic and therefore targeting in quick splitted cell.In thymus, these cells are very jejune cortical thymocytes, but all subgroups all have been subjected to influence (Fredrickson and Basch, (1994) Dev.Comp.Immunol 18:251).In the older animals of normal health, the difference quantitatively and qualitatively of periphery T cell seldom causes pathological state.But, after the serious exhaustion of T cell serious problem has appearred, because reduced the ability of the T cell regeneration of thymus.These results occur among the HIV/AIDS, particularly after the chemotherapy and radiotherapy of treatment of cancer (Mackall et al., (1995) N.Eng.J.Med.332:143).

In the mice of sublethal exposure and cyclophosphamide treatment, castrating has obviously strengthened the thymus reactivate.In order to estimate mainly is the effect, operation castrating of the glucocorticoid inducible stress response to the thymus reactivate, castrating on the same day and before 7 days of immunodepletion.Although the increase of just seeing thymocyte cell density and organizational structure 1 week the earliest after the immunodepletion, 2 weeks were observed bigger difference after castrating.This whether immunodepletion on the same day or the situation of castrating before 1 week.

Immunohistology confirms that in all situations, it is normal that the thymic tissue structure in castrating 2 weeks of back shows as phenotype, and the organizational structure of not castrating mice has been disintegrated.Full epithelium label shows that immunodepletion has caused subsiding of cortex epithelium and to the generally blocking-up of the thymic tissue structure in the thymus of not castrating mice.The medullary substance label is supported this discovery.What is interesting is, castrate inductive regenerated wherein a kind of first feature and be the obvious rise of the extracellular matrix that MTS 16 identified.

The flow cytometry tables of data is understood the remarkable increase of the cell number of all the thymocyte cell subgroups in the castrating mice.On each time point, have in immunodepletion and all CD4, CD8 after castrating and the synchronous increase of the defined subgroup of α β TCR.This is rare but consistent result, because the T cell development is a progressive process, expectation will have the initial increase of precursor (be included in CD4-CD8-door in) and it may occur in before first time point.And, because precursor is being represented very total thymocyte cell of small scale, may not detect the conversion on their number.Also analyzed castrating other cells have been comprised macrophage and granulocytic effect.Usually, intrathymic macrophage and granulocyte number have only very little variation.

In the model of the irradiation of immunodepletion and cyclophosphamide, the thymocyte cell number all after treatment, peaked in 2 weeks and 4 week the back reduce.Almost or chemotherapy after at once, thymic weight and cell density lower and began the first phase of thymus reactivate theatrically after nearly 5 days.The regeneration of the thymocyte cell of anti-irradiation has caused first ripple of rebuilding (5-14 days), and it can generate all thymus subgroups (Penit and Ezine, (1989) Proc.Natl.Acad.Sci, USA 86:5547).Viewed second attenuating is because the limited multiplication capacity of the cell of anti-radiation has reduced the generation (also irradiated influence) of postthymic precursor cell with BM between the 16th and 22 day.Second regeneration period be because the precursor of BM origin to the restock (Huiskamp et al., (1983) Radiat.Res.95:370) of thymus.

In the adult mice, the growth from HSC to the mature T cells will be spent nearly 28 days (Shortmanet.al., (1990) Sem.immunol.2:3).Therefore, until 4 weeks of treatment back just see the very little variation of periphery T cell.Periphery is subjected to the support of some thymus output, but estimates to be cloned in the amplification that does not exist when being exhausted cell until the major part of the T cell of finding in periphery treatment 4 weeks of back is the anti-cyclophosphamide or the irradiation of propagation.Some secular change of periphery after estimating to castrate comprise that most important thymus output increases the variation in caused TCR storehouse.

Embodiment 3: the thymus reactivate behind the inhibition steroid makes that defective periphery T cell function is extensive Multiple

Material and method

Material and method are as described in embodiment 1 and 2.In addition, used following method.

HSV-1 immunity inoculation: operation castrating old (〉=18 months) mice.At 6 weeks after the castrating (behind the thymus reactivate).After the anesthesia, on mouse hind leg, inject 4 * 10 among the aseptic PBS with No. 20 syringe needles ⁵The HSV-1 of plaque forming unit (pfu) (KOS strain).Metainfective mice is housed in independently in the cage, and put to death mice in the mode of humanity on the 5th day after immunity inoculation, Qu Chu popliteal (drain) lymph node is used for analyzing simultaneously.

Virus is from Assoc.Prof.Frank Carbone (Melbourne University).Virus is stored strain growth and titration, and (Gibco-BRL is on the VERO cell monolayer among MEM Australia) in being added with 5%FCS.

After infection, carried out popliteal on the 5th day to drain () analysis of lymph node.For HSV-1 research, with anti-CD25-PE, anti-CD8-APC and the biological uniformly dyeing color of anti-V β 10-lymphonodi poplitei cell.For the detection of DC, using CD45.1-FITC, I-A ^bUse the FcR blocking-up before-pE and the dyeing of CD11c-biotin.Detect all biotinylated antibody with streptavidin-PerCP.For the detection of HSC, by the BM cell being established door in the Lin-cell with the common dyeing of anti-CD3, CD4, CD8, Gr-1, B220 and Mac-1 (all combines with FITC).Detect HSC by dyeing with CD11c-APC and Sca-1-PE.Analyze for the TN thymocyte cell, all cells is established door detect in Lin-group and by dyeing with CD44-biotin, CD25-PE and c-kit-APC.

The cytotoxicity of lymph-node cell is detected: with lymph-node cell at 37 ℃, 6.5%CO ₂In hatched 3 days.Determine specificity with the non-transfected cell strain (EL4) that is added with gB498-505 peptide (gBp), the EL4 cell in contrast separately.Use 30: 1 initial effector: the target ratio.With plate at 37 ℃, 6.5%CO ₂In hatched 4 hours, then at 650g _MaxCentrifugal 5 minutes down.From each hole, collect supernatant (100 μ l) and transfer in the glass fermentation tube, be used for the mensuration of Packard Cobra automatic gamma counter.

The result

For the functional consequence of determining the thymus reactivate (for example whether castrating can enhance immunity replys), detected herpes simplex virus (HSV) immunity inoculation, it allows the research to progression of disease and CTL effect.Find that the castrating mice has the responsiveness to virus of quantitative and qualitative raising.

Analyzed popliteal (drain) lymph node on the 5th day at the palmula immunized mice and after immunity inoculation.In addition, get palmula and homogenization to determine the virus titer on the special time point during the whole test.Having detected regional (popliteal) lymph node replys (Figure 13-17) to what HSV-1 infected.

Observed of the remarkable minimizing (Figure 13 A, 13B and 14) of lymph-node cell density with the age.After immunity inoculation the 5th day (promptly 5 days), the castrating mice has the lymph-node cell density significantly higher than old mice (data do not show).Although the difference of the ratio of not seeing activation (CD8+CD25+) cell after with age or castrating, when with old comparing, the activating cell number in the lymph node is along with castrating has increased (data do not have demonstration) significantly.In addition, quite (data do not show) found in activating cell number and the young adult, the CTL in the prompting castrating mice just is being activated bigger degree, but because the B cell activation, and young adult may have a lymph node that has increased.Confirmed this point (Figure 16) with detection with the CTL detection of the special cracked ratio of age and the appearance of castrating back.With young adult (2 months big) mice relatively, old mice demonstrates at effector: target is the cracked remarkable minimizing of targeted cells (Figure 16) of 10: 1 and 3: 1 o'clock.Castrating has recovered mice and has generated the ability (Figure 16) that the metainfective specific CTL of HSV is replied.

In addition, though total expression of the V β of activating cell along with the age still keeps constant (data not show), the subgroup of old (18 months) mice demonstrates the minimizing (Figure 14 A-C) that this clone replys.During 6 weeks, the number of the total number of the lymph-node cell of infiltration and activation CD25+CD8+ cell has been increased to young adult level (data do not show) to the castrating back.But, the more important thing is that castrating has strengthened the CTL responsiveness (Figure 16) of the target cell that HSV is infected of greatly having been lowered significantly and recovered the CD4 in the lymph node in old mice: CD8 ratio (Figure 17 B).In fact, compare with young adult and castrating mice,, so illustrated in whole life along with the age has all been seen the minimizing (Figure 17 B) of the CD4+T cell in the draining lymph node, to the very important needs of the generation of the T cell that increases thymus, in order to reach maximum immunologic responsiveness.

Embodiment 4: the inhibition to sex steroid has strengthened the picked-up of thymus to new hemopoietic forebody cell, makes Become the chimeric mixture of host and donor lymphocyte (T, B and DC)

Material and method are as described in the embodiment 1-3.In addition, used following method.

Previous test has shown that microchimera is formed on organ transplantation and accepts to have played important function.DC also has been presented on the antigenic toleration of graft integration being arranged.Therefore, studied the influence of castrating to formation of thymus chimera and dendritic cell number.

In order to measure stem cell picked-up effect, carried out the BM reconstruction as embodiment 2 is described to the thymus reactivate.

For the test of homogenic animal, each treatment group is all used 3 months big mices (n=4).All contrasts all be age-matched with untreated.

Compare total thymocyte cell number of the contrast of castrating and false castrating reconstruction mice and untreated age-matched, in Figure 18 A, summarized this point.In the mice of preceding 1 day of reconstruction castrating, with vacation castrating control mice relatively, significantly the increasing of thymus reactivate rate (p≤0.01) arranged.The thymocyte cell density of false castrating mice will be lower than the untreated control level (7.6 * 10 in 2 weeks and 4 weeks behind the homology BMT ⁷± 5.2 * 10 ⁶), surpassed control level (Figure 18 A) and the time be increased in the thymus density of castrating mice 4 weeks behind BMT.When 6 weeks, cell number still is lower than control level.But the cell number of castrating mice is than false castrating mice high 3 times (p≤0.05) (Figure 18 A).

After 4 weeks, significantly more cell (p≤0.05) (Figure 18 D) is arranged also in the BM of castrating mice at BMT.To 2 whens week, the BM cell density of false castrating has reached untreated control level (1.5 * 10 ⁷± 1.5 * 10 ⁶), and the BM cell density of castrating mice 2 weeks and all surpass control level (Figure 18 D) 4 weeks behind homology BMT.In irradiation and reconstruction 2 weeks of back, the mesenteric lymph node cell number in castrating and the uncastrated mice has all reduced; But to during the time point in 4 weeks, cell number has reached control level.There is not statistically-significant difference (Figure 18 C) at the lymph-node cell number of castrating and do not castrate between the treatment group.The splenocyte density of false castrating and castrating has arrived untreated control level (1.5 * 10 when 2 weeks ⁷± 1.5 * 10 ⁶), but false castrating group occurs after week reducing at 4-6, and the castrating mice still has high-caliber splenocyte (Figure 18 B).With uncastrated mice relatively, the castrating mice all demonstrates more lymphocyte (p≤0.05) at these time points (promptly rebuilding 4 week and 6 weeks of back), although total do not see the difference (Figure 18 B) of castrating and not castrating the splenocyte between the treatment group when 2 weeks.

Therefore, in the mice of reconstruction castrating in preceding 1 day, compare, the remarkable increase (p≤0.01) (Figure 18 A) of thymus reactivate rate is arranged with vacation castrating (ShCx) control mice.In 2 weeks and 4 whens week behind homology BMT,, false thymocyte cell density of castrating mice will be lower than untreated control level (7.6 * 10 ⁷± 5.2 * 10 ⁶), and behind BMT 4 whens week, the thymocyte cell density of castrating mice has increased and has surpassed control level (Figure 18 A).Castrating mice ratio not neuter has significantly more homology cell (Ly5.2).

In not castrating mice, the remarkable minimizing of thymocyte cell number was arranged in the time in 4 weeks, there is seldom or do not have regenerated evidence (data do not show).But in the castrating group, had thymocyte cell generation widely during to 2 weeks, returned to control level during to 4 weeks, this is higher more than 10 times than not castrating mice.When being confirmed for 4 weeks, the flow cytometry of the CD45.2 of thymus (antigen in donor source) in not castrating group, do not detect the cell in donor source, but be that all thymocyte cells of the castrating mice on this time point all are (data do not show) in donor source in fact significantly.Suppose the hemopoietic forebody cell of the extensive enhancing of this thymocyte cell generation, determine whether the T cell differentiation normally has been important so from the donor source.With flow cytometry CD4, CD8 and the defined subgroup of TCR.The difference that on the thymocyte cell subgroup ratio in 2 weeks after the reconstruction, does not match (data do not show).This observation is impossible when 4 weeks, because do not castrate the reconstruction that mice does not still have the cell in donor source.But on this time point, the thymocyte cell ratio of castrating mice shows as normally.

In one group of parallel test, BMT castrating in preceding 1 day or false castrating 3 months big, young adult C57/BL6 mices.For homology BMT, mice has been accepted 800RAD TBI and IV injection 5 * 10 ⁶The Ly5.1+BM cell.2 weeks and 4 weeks back execution mice, and the immunologic reconstitution of analysis BM, thymus and spleen.Determine donor/host source with anti-CD45.1 antibody, this antibody only with the leukocytoreaction in donor source.

In Figure 19-28, shown result from this group parallel test.

Figure 20 and 21 has shown the number of HSC in the donor source among the BM of neuter and the increase of ratio.This has illustrated implantation that improves and the recovery faster of pointing out BMT.

Figure 22 has shown the B cell CFU-GM in the donor source among the BM that castrates mice and the increase of B cell.But Figure 24 and 25 has shown the number or the ratio of the periphery B cell when castrating does not change 2 weeks of castrating back and 4 weeks.

Figure 26 shows the number of TN, DP, CD4 and the cd8 cell of castrating the donor source that has increased thymus.But Figure 23 shows that castrating does not change the ratio of the CD4 and the cd8 cell of donor thymocyte cell.In periphery, considerably less CD4 or cd8 cell are arranged, and on the time point of being considered, these cells do not increase with castrating.

Importantly, Figure 28 has shown to the increase of castrating back number of the donor DC of thymus during 4 weeks.

Discuss

Embodiment 4 has shown that castrating is to influence homogenic and that homology BM transplants.Starzl et al., (1992) Lancet 339:1579 have reported the good prognostic indicator that lymph and non-adenoid tangible microchimera are allotransplantations.Being known as them is necessary (Starzl et al., (1992) Lancet 339:1579) for inducing the tolerance to graft.The DC in donor source is present in these chimeras and is considered to be in the effect (Thomson and Lu, (1999) Immunol.Today 20:20) of avoiding bringing into play on the transplant rejection integration.Known DC is that the feminine gender of thymus is selected the key factor in the step, if in the DC existence in donor source and the receptor's thymus, just can remove graft reaction T cell.

In order to determine whether castrating can increase chimera and form, and has carried out a research with isogenic fetal liver transplantation.The result has shown that the regeneration of the thymus of castrating mice has been enhanced.When with homologous (Ly5) mice repeated trials, these trend have been seen once more.Because the existence of homology label, the chimera state of measuring mice is possible.Early rebuild the back during 2 weeks to the tire liver, just can detect the dendritic cell in donor source in thymus, the number of castrating mice is not castrate 4 times of mice.4 whens week after reconstruction, do not castrate the reconstruction that mice does not show the cell in donor source, illustrate that castrating in fact can increase the probability of chimera formation.Suppose the thymus reactivate after castrating can not only increase lethal exposure and the reconstruction of tire liver, it has also increased the number of the dendritic cell in donor source in the thymus, and for stem cell transplantation, this method has increased the probability that graft is accepted.

Embodiment 5: immunocyte is exhausted

In order to prevent existing T cell among the potential graft receptor patient to the interference of graft, the patient has carried out the T cell and has exhausted (removing).A standard method of this step is as follows.The patient accepts to inject every day 15mg/kg Atgam (xenogeneic anti-T cytoglobin, Pharmacia Upjohn) anti-T-cell antibody of form is 10 days, use in conjunction T cell activation inhibitor cyclosporin A, 3mg/kg continuous infusion 3-4 week then is a tablet of taking 9mg/kg every day as required.This treatment does not influence the intrathymic earlier T cell development of patient, can not be transported in the thymus because of the size of human thymus and configuration because have the antibody of the necessary amount of such effect.Thymus after nearly 4-6 week of continued treatment lacks with the admissibility steroid is rebuild.

Inhibitor that also can use in conjunction secondary signal level carries out the prevention to t cell responses, these inhibitor for example interleukin, additional molecule (for example blocking for example antibody of CD28), strengthen the T cell is removed or other immunocytes are removed signal pathway molecule or cell adhesion molecule.Thymus rebuild the phase will with the injection of donor HSC in conjunction with (from blood, obtaining when obtaining relevant organ or tissue, with G-CSF (every day, subcutaneous injection was 2 times, totally 3 days) mobilization in advance from blood; Or directly from donor BM, gathered).The level of the circulation HSC that has strengthened will promote the picked-up (the GnRH level institute of the disappearance of sex steroid and/or raising is activated) of thymus.These donors HSC will develop into DC in the thymus and what cause any new formation will be the removal of " donor is reactive " T cell once in a while.This will set up any repulsion that the maincenter tolerance of donor's cells and tissue is also prevented or farthest minimizes the host in view of the above.The growth of new T cell bank also will overcome the T cell and exhaust the caused immunodeficiency of scheme.

The exhaustion of periphery T cell makes the danger of transplant rejection minimize because it exhausted non-specificly all T cells comprise those potentially with the T cell of external donor reaction.But because of the disappearance of T cell, this method has been induced general immune deficiency state, this means that viral infection is hypersusceptible to the patient to infecting particularly simultaneously.

Embodiment 6: sex steroid is exhausted treatment

The patient has accepted to give the sex steroid exhaustion treatment of LHRH agonist form.Give Leucrin_ and (stored remover liquid injection; 22.5mg) or Zoladex_ (implant; 10.8mg) the LHRH agonist of form, each all is effective 3 months of single dose.This can reduce to the level that is enough to reactivate thymus with the sex steroid level effectively.In some cases, the inhibitor of granting adrenal gland's product of sex steroid also is necessary.Also can in exhausting the persistent period for the treatment of, sex steroid give a slice Cosudex_ (5mg/ days or 50mg/ days) every day.Perhaps, give for example hypodermic cetrorelix of patient GnRH antagonist or 1: PN: WO02056903 PAGE: 25 claimed protein.

After the operation castrating, the sex steroid in the blood reduces to minimum needs 1-3 week, and will spend 3-4 week behind the chemical castration.In some cases, treatment need be extended to second injection/implantation of 3 months.Strengthen (by injecting G-CSF to the host) as allogene donor (for the graft of external organization) or from the blood HSC of the HSC of body the time and can increase the thymus amplification so that these HSC are mobilized thymus from BM.

Embodiment 7: other medication

The medication that stored agent or implant in 3 months that can replace the LHRH agonist with other method.In an example, can be with for example laser irradiation patient's of Er:YAG laser skin, to melt or to change skin, to reduce cuticular hindrance function.

At U.S. No.6, laser ablation or change have been described in 251,100,6,419,642 and 4,775,361.

In another example, administration is the mode by the pressure wave of laser generation.With suitable vessel for example the LHRH agonist of a dosage in the soft packing ring of plastics (about 1/16 inch of diameter about 1 inch and thickness) be placed on the site of generation pressure wave of skin.The target material of the use-case black polystyrene sheet that 1mm is thick according to appointment covers this site then.Solid-state ruby laser (in 20ns pulse duration, each pulse can produce and be up to 2 joules energy) with the Q conversion generates individual pulse transient state, this transient state impact target material.Black polystyrene target material absorbs laser irradiation fully, makes skin only be exposed to pulse transient state, and is not exposed to laser irradiation.Can repetitive operation every day, or by required frequent repetition, to keep the circulation blood levels of agonist.

Embodiment 8: the donor's cells uses

Under feasible situation, by before cell collection, (for example injecting 10 μ g/kg granulocyte colony-stimulating factors (G-CSF) 2-5 days to donor, inject 10 μ g/kg every day once or twice, 2-5 days altogether) strengthen the level of the hematopoietic stem cell (HSC) in the donor blood.Also can before gathering, (before 7-14 days) give donor LH injection RH agonist and/or cytokine for example G-CSF or GM-CSF, to strengthen level or the quality of the stem cell in the blood.For example from donor blood or BM, be purified into the CD34+ donor's cells by flow cytometry or immunomagnetic beads.With the bonded antibody of people CD34 specificity be commercial (from for example Research Diagnostics Inc., Flanders, NJ; Miltenyi-Biotec, Germany).It is the CD34+ cell that the HSC that flow cytometry is originated donor is defined as.Also can by use feeder cell (for example fibroblast), somatomedin for example stem cell factor (SCF) and preventing be divided into these CD34+HSC of cultured and amplified in vitro of the LIF of specific cell type.Nearly 3-4 week after giving the LHRH agonist (be thymus just begun regenerated before or simultaneously) time, give patient infusion donor HSC, optimum dose is about 2-4 * 10 ⁶Cell/kg.Also can inject G-CSF to help the amplification of donor HSC to the receptor.If because the order of severity of clinical pathological changes make this time scheme can not the time, can grant HSC and GnRH simultaneously.Nearly 2-3 gives second dosage after week HSC may be necessary, and this helps the growth (particularly in thymus) of thymus reactivate and donor DC.In case HSC is implanted to (promptly being attached to) and/or moves to BM and thymus, effect will be significantly, because HSC is a self renewal.

Positive reactivate or reactivate thymus picked-up donor HSC and they are converted into the T cell and the DC of donor type, and receptor's HSC is converted into receptor's type T cell and DC.Induce removal by cell death, or by through the immunity regulatory cell inducing tolerance, donor and host DC can tolerate any new T or the NK cell that can may react with donor or donee's cells.

Embodiment 9: the transplanting of graft HSC

In an embodiment of the invention, the T cell that still continues as the receptor is exhausted and/or other immunocyte is exhausted and/or during immunosuppressant therapy, an organ or one group of cell of partly having been exhausted the donor T cell are transplanted in the receptor patient from donor.Receptor's thymus is activated by the GnRH treatment and is soaked into by external HSC.

In week, first batch of new T cell has appeared in receptor's the blood flow at the about 3-4 of LHRH treatment.But,, can continue the about 3-4 of immunosuppressant data month in order to allow the stable chimera that produces host and donor hematopoietic cell.Because in reactivate thymus, exist donor and host's DC, the intracellular possible donor reactivity of new T and the cell of host response have been removed.Select through the positive of host's thymus epithelial, the T cell has kept the ability of replying to normal infection by the peptide that the host APC in identification receptor's the peripheral blood is presented.Donor DC has set up in fact consistent with independent host's immune system immune system state to the combination of receptor's lymphatic organ, except the toleration to donor's cells, tissue and organ.Therefore, exist normal immune regulation mechanism.These also can comprise regulates the T cells whose development, and it utilizes cytokine, and for example IL-4,5,10, TGF-β, TNF α open or close immunne response.

Embodiment 10: the immunity inoculation and the prevention of viral infection (influenza)

Influenza virus is segmental RNA viruses, and it has caused the acute respiratory infection of hyperinfection.The subject matter relevant with the vaccine development of influenza is, these viruses are escaped immunological surveillance and are retained in ability among the host group by changing hemagglutinin (HA) and neuraminidase (N) through so-called antigenic drift and antigenic shift phenomenon by the antigen site on the membrane glycoprotein, having had.Main correlative to resisiting influenza virus is the neutralizing antibody that resists the HA of the strong selection of having experienced antigenic drift and transformation.But, more conservative antigenic cross-reaction (Shu et al., (1993) J.Virol.67:2723) to different strains of influenza viruses has for example taken place between the nucleoprotein (NP) at integrated protein.Previous CTL and the protective effect (Ulmer et al. (1993) Science 259:1745) that has shown after the immunity inoculation of the polynucleotide of using coding NP to the influenza attack.

Material and method

Operation castrating: be dissolved in brinish 5ml 100mg/ml ketalar (Ketalar_ by intraperitoneal injection 30-40 μ l; Parke-Davis, Caringbah, NSW Australia) adds 1ml20mg/ml xylazine (Rompun_; Bayer Australia Ltd., Botany NSW, mixture anesthesia BALB/c mouse Australia).As in other local described castratings that undergos surgery, by scrotal incision, appear testis, it is used the suture ligation, take out testis with the periphery fatty tissue then.And perform the operation to order and close wound.Will be by above-mentioned but do not take out the prepared vacation castrating mice of the method for testis in contrast.

Chemical castration: to the Lupron_ (a kind of GnRH agonist) of mouse muscle injection 10mg/kg as 1 month slow release formulation.Otherwise just give injected in mice GnRH antagonist (for example cetrorelix or 1: PN: WO02056903 PAGE: 25 claimed protein).According to product description, carry out affirmation to the sex steroid disappearance with the standard radioimmunoassay of plasma specimen.To castrating back normally should reach castrating level (＜0.5ng testosterone or estrogen/ml) 3-4 week.

The preparation of influenza A/PR/8/34 subunit vaccine: influenza A/PR/8/34 (H1N1) the subunit vaccine preparation for preparing purification according to methods known in the art.Simply, from influenza A/PR/8/34 granule, extract hemagglutinin (HA) and neuraminic acid glycosidase (NA) antigen with non-ionic detergent (7.5%N-octyl group-β-o-sulfo-pyranglucoside).After centrifugal, the supernatant (55%HA) that is rich in HA/NA is used as subunit vaccine.

Influenza A/PR/8/34 subunit immunity inoculation: about 8 weeks behind operation castrating nearly 6 weeks of back or chemical castration, with influenza reassortant virus (about 7000HAU) immunized mice of hypodermic 100 μ l formalin deactivations.

Behind initial immunization, during about 4 weeks (or more late), can randomly carry out the reinforced immunological inoculation.Freund's complete adjuvant (CFA) is used to the reinforced immunological inoculation that initial immunization and incomplete Freund are used to choose wantonly.

Perhaps, can directly intramuscular injection be in musculus quadriceps for example with influenza A/PR/8/34 subunit vaccine preparation (on seeing), what dosage was about 1 μ g to about 10 μ g is diluted in the interior bacterin preparation of 40 μ l, 0.9% saline.

Plasmid DNA: to the plasmid DNA preparation of expression vectors is (seeing of knowing in this area, Current Protocols In Immunology for example, Unit 2.14, John E.Coligan et al., (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002).Simply, will be completely influenza A/PR/8/34 nucleoprotein (NP) gene or hemagglutinin (HA) coded sequence be cloned into expression vector for example among the pCMV, it is under the transcriptional control of cytomegalovirus (CMV) immediate early promoter.

Empty plasmid (pCMV that does not for example have insert) is used as negative control.With the standard counting plasmid is cultivated in bacillus coli DH 5 alpha or HB101 cell, and (Chatsworth CA) is purified with the Qiagen_Ultra-Pure_-100 post according to product man description.Verify all plasmids with suitable restricted enzyme and agarose gel electrophoresis.Be used in optical density that 260nm and 280nm read and determine the purity of DNA product.All plasmids are resuspended in the TE buffer and at-20 ℃ to descend to store up to use.

The dna immunization inoculation: the method for dna immunization inoculation is known in this area.For example, at Current Protocols In Immunology, Unit 2.14, John E.Coligan et al., (eds), Wiley and Sons, New York, NY (1994), and yearly updates including 2002) describes the method for (particle gun) dna immunization inoculation of Intradermal, intramuscular and granule mediation in detail.

Randomly use the DNA of the Codocyte factor, immunne response is converted to required Th1 or Th2 type immunne response.Induce hereditism's adjuvant of Th1 to comprise for example IFN γ and IL-12.Induce hereditism's adjuvant of Th2 to comprise for example IL-4, IL-5 and IL-10.The preparation of hereditism's adjuvant of inducing Th1 and Th2 and the summary of the usage in induce immune response thereof are seen for example Robinson, et al., (2000) Adv.Virus Res.55:1).

Whether the influenza reassortant virus is attacked: can be than their vacation castrating contrast better to the attack of resisiting influenza virus (have and do not have immunity inoculation), with 10 of 50 μ l in order to determine the castrating mice ^-4Doubly be diluted in the allantoic fluid (50-100LD50 that contains influenza A/PR/8/34 (H1N1) influenza virus of PBS/2%BSA; 0.25HAU) intranasal vaccination attacks the mice of methoxyflurane anesthesia.Weighing every day mice, and before body weight disappearance＞20% is attacked after the body weight with its execution.In the virus attack of this dosage, 100% originally mice will yield to influenza infection at 4-6 days.

Randomly infect with the activation memory T cell, but be to use 10 with inferior causing death ^-7Viral dilution liquid.Also can be randomly with inferior cause death to infect determine non-immune, castrating mice and whether have better immunne response than vacation castrating contrast, as following listed ELISA, cytokine assay (Th), CTL measure or the like determined.Before being used for these tests, can the optimization lethal and the virus titer of inferior lethal infection.

Enzyme-linked immunosorbent assay: the different time sections of (or before infecting and afterwards) before immunity inoculation and afterwards, with every group mice blood-letting, and the serum of measuring individual mice with the quantitative enzyme-linked immunosorbent assay (ELISA) of standard is to detect anti-HA or the NP specific IgG level in the serum.Can randomly measure IgG1 and IgG2a level, known they are relevant with Th1 type antibody response with Th2 respectively.

Be used for the preparation and the stimulation of the splenocyte that cytokine produces: the aseptic collection spleen of mice on the same group and it is collected in the p60 culture dish that contains the 4ml RPMI-10 culture medium of having an appointment (RPMI-1640,10% hyclone, 50 μ g/ml gentamycins) never.Method with standard prepares spleen and cracking RBC.Counting cells and it is resuspended in contains 80U/ml Mus IL-2 (Sigma, St.Louis among RPMI-10 MO), make that final cell concentration is 2 * 10 then ⁷Cell/ml.100 microlitre cells are distributed in each hole of 96 hole tissue culturing plates, make that final concentration is 2 * 10 ⁶Cells/well.100 μ l are dissolved in suitable peptide among the RPMI-10 or inactivating influenza virus is implemented to stimulate by adding.Or use K ^d-restricted HA _533-541Peptide (IYSTVASSL; SEQ ID NO:1)) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or use K ^d-restricted NP _147-155Peptide (TYQRTRALV; SEQ ID NO:2) (Rotzschke et al., (1990) Nature 348:252) stimulates the CD8+T cell.Influenza virus (every hole 13 with deactivation, the influenza virus that 000HAU boils adds every hole 13, the influenza virus of 000HAU formalin deactivation) adds that anti-CD28 (1 μ g/ml) and anti-CD49d (1 μ g/ml) stimulate CD4+T cell (Waldrop et al., (1998) J.Immunol.161:5284).List carries out negative control with culture medium to stimulate.Incubated cell then, as described below detects the extracellular cytokine or detects the cell within a cell factor with FACS with ELISA.

The chromium release assay of CTL: utilize method well known in the art to measure CTL replying of influenza HA and NP (seen, Current Protocols In Immunology for example, John E.Coligan etal., (eds), Unit 3, Wiley and Sons, New York, NY (1994), and annual renewal version comprises 2002).Synthetic peptide HA _533-541IYSTVASSL (SEQ ID NO:1) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or NP147-155TYQRTRALV (SEQ ID NO:2) (Rotzschke et al., (1990) Nature 348:252) be used as peptide in the system target step.With the type of the replying splenocyte of every animal of RPMI-10 washing and with it by 6.3 * 10 ⁶The final concentration of cell/ml is resuspended in and contains 10U/ml Mus IL-2 (Sigma, St.Louis is among RPMI-10 MO).From originally, make the stimulating type splenocyte the isogenic mice, and with it by 1 * 10 ⁷The concentration of cell/ml is suspended among the RPMI-10.Adding final concentration is the ametycin of 25 μ g/ml.With cell at 37 ℃/5%CO ₂In hatched 30 minutes, then with RPMI-10 washing 3 times.Then the stimulating type cell is resuspended among RPMI-10 culture medium and the 10U/ml IL-2, making its concentration is 2.4 * 10 ⁶Cell/ml, and the adding final concentration is 9 * 10 ^-6The HA peptide of M or final concentration are 2 * 10 ^-6The NP peptide of M is at 37 ℃/5%CO ₂In hatched 2 hours.With peptide activated (pulsed) stimulating type cell (2.4 * 10 ⁶) and reply type cell (6.3 * 10 ⁶) 37 ℃/5%CO in the SM culture medium (RPMI-10,1mM non essential amino acid, 1mM Sodium Pyruvate) of the 2ml of 24 orifice plates volume ₂Hatched altogether 5 days.Measure the activated mouse hypertrophy cell tumor of responsive cell (being called the effector lymphocyte now) cleavage of peptide P815 cell (the MHC coupling, ability H-2d) of stimulated in vitro with the chromium release assay method.The P815 that is dissolved in RPMI-10 by taking out 0.1ml also adds 25 μ l FBS and 0.1mCi is dissolved in radiolabeled sodium chromate in the 0.2ml normal saline (MEN, Boston MA), use ⁵¹Cr labelling P815 cell.With target cell at 37 ℃/5%CO ₂In hatched 2 hours, with RPMI-10 washing 3 times and be resuspended in and contain RPMI-10 and add HA (9 * 10 ^-6M) or NP (1 * 10 ^-6M) in the 15ml polypropylene test tube of peptide.With target cell at 37 ℃/5%CO ₂Hatched 2 hours.To be dissolved in radiolabeled, the activated target cell of peptide among the RPMI by every hole 5 * 10 ⁴Cell joins in each hole of 96 orifice plates.Collect the responsive cell (being called the effector lymphocyte now) of the irriate in each immunity inoculation group,, and it is joined in each hole of 96 orifice plates that the final volume that contains target cell is the 0.2ml/ hole with RPMI-10 washing 3 times.Effector: the target cell ratio is 50: 1,25: 1,12.5: 1 and 6.25: 1.With cell at 37 ℃/5%CO ₂Hatched 5 hours, and measured the lysis of 25 μ g supernatant with liquid scintillation counting technique.Be lower than given effector lymphocyte's specimen, the cracked percentage ratio of the specificity of labels targets cell is [100x (spontaneity of the Cr release-specimen in the specimen discharges)/(spontaneity of maximum Cr release-specimen discharges)].It is from radioactive amount that target cell discharged when not adding the effector lymphocyte that spontaneous chromium discharges.It is to be radioactive amount that the target cell cracking is discharged after 1% adding Triton-100 to final concentration that maximum chromium discharges.Spontaneous release should surpass 15%.

ELISA is to the IFN γ of a large amount of culture supernatant or the detection of IL-5: can measure the IFN γ and the IL-5 cytokine levels of a large amount of culture supernatant, known they reply relevant with Th1 and Th2 type respectively.The splenocyte that will compile is at 37 ℃/5%CO ₂Hatched 2 days, and collected then and compile supernatant.The all ELISA antibody and the cytokine of purification all available from Pharmingen (San Diego, CA).50 microlitres are cushioned liquid (0.1M NaHCO3 at bag, pH 8.2) in be diluted to the purification of 5 μ g/ml (rat anti-mouse IFN γ) or 3 μ g/ml (rat anti-mouse IL-5) the antibacterial agent monoclonal antibody be distributed to 96 orifice plate (Corning, Corning is in each hole NY) and 4 ℃ of overnight incubation.With PBS-T washing, sealing and wash plate again.Reference material (the mouse cell factor of reorganization) is joined in the hole by the different diluted concentration in RPMI-10 with specimen, and 4 ℃ of overnight incubation to reach maximum sensitivity.With PBS-T wash plate 6 times.Biotinylated rat anti-mouse cytokines measurement antibody is diluted to final concentration in PBS-T be 2 μ g/ml, adds 100 μ l toward every hole.Plate was hatched 1 hour at 37 ℃, then with PBS-T washing 6 times.(Gibco BRL, Grand Island add 100 μ l NY) by dilution in 1: 2000, and toward every hole with streptavidin-AP according to product description.Plate was hatched 30 minutes, wash again 6 times with PBS-T.(BioRad, Hercules CA) were also hatched 50 minutes under the room temperature again, and plate is developed by adding every hole 100 μ l AP developing solutions.By adding 100 μ l/ hole NaOH cessation reactions and at OD ₄₀₅The place reads data.With 2.21 editions computer softwares of Softmax Pro (Molecular Devices, Sunnyvale, CA) analytical data.

Cell within a cell factor dyeing and facs analysis: can measure IFN γ and IL-5 cytokine levels in the cell of splenocyte, known they reply relevant with Th1 and Th2 type respectively.The splenocyte that will compile contains 5%CO at 37 ℃ ₂Humid air in hatched 5-6 hour.According to product description, (Pharmingen, SanDiego CA), and are hatched 5-6 hour (Waldrop et al., (1998) J.Immunol.161:5284) again with cell to add Golgi transport inhibitors Monensin by 0.14 μ l/ hole.Cell is thoroughly resuspended and transfer in the U type base plate in 96 holes.Unless other explanations, all (San Diego CA), carries out all FACS staining procedures on ice with ice-cold reagent to all reagent (GolgiStop test kit and antibody) available from Pharmingen.With FACS buffer (1 * PBS, 2%BSA, 0.1%w/v Hydrazoic acid,sodium salt) wash plate 2 times.With rat anti-mouse CD8 β-APC of 50 μ l ,-D69-PE and-CD16/CD32 (Fc γ III/RII; " Fc blocking-up ") the dilute solution padding cell of 1: 100 FACS buffer.For tetramer dyeing (as follows), the FACS buffer of usefulness CD8 β-TriColor, CD69-PE, CD16/CD32 and HA-or the NP-tetramer-APC is staining cell similarly.Cell was in the dark hatched 30 minutes, with FACS buffer washing 3 times.By cell being resuspended in up hill and dale in the 100 μ l Cytofix/Cytoperm solution of every hole and in the dark hatching 20 minutes, cell permeabilization is handled.With Permwash solution washing cell 3 times.Finished cell inner dyeing in 30 minutes by in the dark hatching with the diluent of 1: 100 the Permash solution of rat anti-mouse IFN γ-FITC of every hole 50 μ l.Wash 1 time with Permwash solution washing cell 2 times and with the FACS buffer.Cell fixation in 1% paraformaldehyde solution of 200 μ l, and is transferred in the micro tube by 96 hole format permutation.Be wrapped in pipe in the paper tinsel and be stored in 4 ℃ up to analyzing (being less than 2 days).At FACScan flow cytometer (BectonDickenson, San Jose, CA) middle analytical specimen.Utilization with the CD8-FITC of rat anti-mouse ,-PE ,-TriColor or-the painted control cells of singly dying of APC compares.With 2.7 editions softwares of FlowJo (Tree Star, San Carlos, CA) analysis result.

The tetramer: can be with HA and the specific CD8+T cell response of NP after HA and quantitative HA of the NP tetramer or the NP immunity inoculation.Basically according to the previous described preparation tetramer (Flynn et al., (1998) Immunity 8:683).Present embodiment has utilized and has been compounded with synthetic influenza reassortant virus peptide HA _533-541(IYSTVASSL; SEQ ID NO:1) (Winter, Fields, and Brownlee, (1981) Nature 292:72) or NP _147-155(TYQRTRALV; SEQ IDNO:2) H-2K of (Rotzschke et al., (1990) Nature 348:252) ^dI type MHC molecule.

As long as be noted that and do less variation, just applicable to detectable widely, this point is easy to determine for those skilled in the art described in the present embodiment method.

Embodiment 11: the immunity inoculation and the prevention of parasitic infection (malaria)

Circumsporozoite protein (CSP) is the target spot (Hoffman et al., (1991) Science 252:520) of the immunity of this proerythrocyte ring.In the rodent model of Plasmodium yoelii (Plasmodium yoelli); passive transfer P.yoelii CSP monoclonal antibody specific (Charoenvit et al.; (1991) J.Immunol.146:1020); and adopting property transfer P.yoelii CSP specific C D8+T cell (Rodrigues et al.; (1991) int.Immunol 3:579; Weiss et al.; (1992) J.Immunol.149:2103) and CD4+T cell (Renia et al., (1993) J.Immunol.150:1471) protective effect is all arranged.Estimated multiple vaccine by the designed protection mice antagonism zygoblast of the immunne response of inducing anti-P.yoelii CSP.

Can use and can be used for the known in the art any plasmodial sporozoite protein that to induce plasmodial protective effect of the present invention, for example plasmodium falciparum, Plasmodium vivax, malariae and Plasmodium ovale CSP; SSP2 (TRAP); Pfs16 (Sheba); LSA-1; LSA-2; LSA-3; MSA-1 (PMMSA, PSA, p185, p190); MSA-2 (Gymmnsa, gp56,38-45kDa antigen); RESA (Pf155); EBA-175; AMA-1 (Pf83); SERA (p113, p126, SERP, Pf140); RAP-1; RAP-2; RhopH3; PfHRP-II; Pf55; Pf35; GBP (96-R); ABRA (p101); Exp-1 (CRA, Ag5.1); Aldolase; The Duffy of Plasmodium vivax is conjugated protein; Reticulocyte is conjugated protein; HSP70-1 (p75); Pfg25; Pfg28; Pfg48/45; And Pfg230.

Material and method

Castrating: as above undergo surgery and/or chemical castration.

Parasite: 17XNL (nonlethal) strain (U.S. No.5,814,617) of use P.yoelii as discussed previously.

The preparation of P.yoelii zygoblast through irradiation: before described the preparation that is used for immunity inoculation (seeing Franke et al. for example, (2000) Infect.Immun.68:3403) through the P.yoelii zygoblast of irradiation.Simply, with the discontinuous gradient technology from being subjected to 10,000rads ( ¹³⁷Ce) isolate zygoblast (Pacheco et al., (1979) J.Parisitol.65:414) in Zhao She the infected Anopheles stephen mosquito.

With the immunity inoculation of P.yoelii zygoblast through irradiation: in operation castrating back nearly 6 all or behind chemical castration about 8 weeks, with 50,000 zygoblasts through tail vein intravenous immunized mice.In 4 week and 6 weeks behind primary vaccination, the reinforced immunological that randomly gives 20,000 to 30,000 zygoblasts is inoculated (seeing Franke et al. for example, (2000) Infect Immun.68:3403).

Plasmid DNA and dna immunization inoculation: the plasmid DNA of coding total length P.yoelii CSP is known in this area.For example, can use al., pyCSP carrier described in detail in ((1998) Proc.Natl.Acad.Sci.USA 95:7648) at Sedegah et.

The method of dna immunization inoculation is also known in this area.For example, at CurrentProtocols In Immunology, Unit 2.14 (John E.Coligan et al., (eds), Wiley andSons, New York, NY (1994), and annual renewal version comprises 2002) described the method for dna immunization inoculation of (particle gun) of Intradermal, intramuscular and granule mediation in detail.

The peptide immunity inoculation: the method for P.yoelii CSP peptide preparation is known (seeing Franke et al. for example, (2000) Infect Immun.68:3403) in this area.

The chromium release assay method of CTL: because the CD8+CTL of anti-P.yoelii has been shown as is adopting property transfer protection (Weiss et al.; (1992) J.Immunol.149:2103); and the CD8+T cell be through the immunity inoculation of the zygoblast of irradiation the needed cell of protective effect (the Weiss et al. of inductive anti-P.yoelii; (1988) Proc.Natl.Acad.Sci USA 85:573); therefore must determine P.yoelii CSP vaccination (for example, zygoblast through shining; CSP peptide or CSPDNA immunity inoculation) whether caused the CSP specific CTL.

With method well known in the art measured CTL reply (see, CurrentProtocols In Immunology for example, Unit 2.14, John E.Coligan et al., (eds), Wiley andSons, New York, NY (1994), and annual renewal version comprises 2002).Other local described common methods that are used for influenza HA and NP have been used, except with synthetic P.yoelli CSP peptide (281-296 in this description; SYVPSAEQILEFVKQI; SEQ ID NO:3) active cell.

Mensuration to the inhibition of liver stage of development: before described liver stage of development mensuration and passed through original position collagenase perfusion collection (Franke et al., (1999) Vaccine 17:1201 to the mouse liver cell of mouse liver; Franke et al., (2000) Infect.Immun.68:3403).Hepatocyte is pressed every chamber 1 * 10 ⁵Individual cell is seeded on the Lab-Tek plastic slide of Room 8, and with 7.5 * 10 ⁴Individual P.yoelli zygoblast was hatched 3 hours.Wash culture then and at 37 ℃/5%CO ₂Cultivated again 24 hours.As above obtain and add the effector lymphocyte of the chromium release assay that is used for CTL, with itself and the about 24-48 of infected liver cell culture hour.Wash culture then, and in ice-cold absolute methanol fixing locellus slide glass 10 minutes.Before hatching, the parasitic monoclonal antibody of liver stage (NYLS1 or NYLS3, both have been described in U.S. No.5,814,617) of locellus slide glass and direct anti-P.yoelii is hatched then with the anti-Mus Ig of FITC labelled goat.Count the number of the schizont in three liver stages in the repeated trials then with epifluorescence microscope.Calculate inhibition percentage ratio with formula [((contrast-test)/contrast) * 100].

Infect and attack:, must before tentative attack, determine the ID of P.yoelli zygoblast for lethal hit dosage ₅₀But it also is possible that menophania tail vein is given the dosage (nonlethal, 17XNL strain) of about 50 to 100 the P.yoelli zygoblasts of injection in the mouse vein.Intravenous inoculation was put to death mice and is taken out liver after 42 hours again.Prepare hepatocellular single celled culture medium suspension, and in each hole in 10 holes of multicell slide glass, all put into 2 * 10 ⁵Individual hepatocyte.Can and be chilled in-70 ℃ with the slide glass drying up to analysis.In order to count the number of zygoblast, slide glass is dry and hatched with NYLS1 earlier before hatching with the anti-Mus Ig of FITC labelled goat, with the number of the every indoor liver stage zygoblast of fluorescence microscope counting.

Reduced infected hepatocellular number in case confirm castrating and/or immunity inoculation, obtained blood smear to determine whether immunity inoculation protects the infection that has resisted the blood stage.If in the 5-14 after attack days, in blood smear, do not find parasite just to think that mice is shielded.

In order to check the protection curative effect of independent castrating (no immunity inoculation) to the primary infection of P.yoelli zygoblast, aforesaid infection has also been analyzed the castrating mice.False castrating mice is taken as contrast.

The human research: after having set up the effectiveness in the mice, in double blinding placebo field trial, immunity inoculation a lot of people.

Embodiment 12: to immunity inoculation and the prevention of bacterial infection (TB Ag85)

Tuberculosis (TB) is the chronic infectious disease of the caused pulmonary of a kind of mycobacterium tuberculosis cause of disease, is that one of worldwide clinical most important infection (is seen U.S. No.5 for example, 736,524; Summary is seen Bloom and Murray, (1993), and Science 257,1055).

Mycobacterium tuberculosis is a kind of intracellular pathogen that infects macrophage.The effector lymphocyte who the immunity of TB is comprised some types.Cytokine for example IFN γ is a kind of effective ways that minimize mycobacterium intracellulare propagation to the activation of macrophage.

The acquisition of the protective effect of anti-TB needs CD8+ and CD4+T cell (seeing Orme etal. for example, (1993) J.Infect.Dis.167:1481).Known these cell responses are in infecting secretion Th1 cytokines, for example IFN γ, and the cytotoxic activity with antigenic specificity.In fact, to reply for against mycobacterium tuberculosis protective effect be useful (seeing Flynn et al. for example, (1992) Proc.Natl.Acad.Sci.USA 89:12013) to CTL known in the art.

The main T cellular antigens of TB are those mycobacterias secreted albumen during they stay in macrophage.These T cellular antigens comprise, but be not limited to antigen 85 albumen compositions (85A, 85B, 85C) (Wiker and Harboe, (1992) Microbiol.Rev.56:648) and ESAT-6 (Andersen, (1994) Infect.Immunity, 62:2536).Also describe other T cellular antigens in this area and (seen Young and Garbe for example, (1991) Res.Microbiol.142:55; Andersen, (1992) J.Infect.Dis.166:874; Siva and Lowrie, (1994) Immunol.82:244; Romain et al (1993) Proc.Natl.Acad.Sci.USA 90:5322; And Faith et al., (1991) Immunol.74:1).

Cloned and every kind of proteic gene of three kinds of antigen 85 albumen (A, B and C) that checked order (seeing Borremans et al. for example, (1989) Infect.Immunity 57:3123); DeWit et al., (1994) DNA Seq.4:267), and shown they infect and vaccination after can cause intensive t cell response.

Material and method

The castrating of mice: as above carry out operation and/or chemical castration to BALB/c or C57BL/6 mice.

Protein immunization inoculation: the common method that is used for mycobacterium tuberculosis (TB) purification and immunity inoculation is knownly (to see in this area, Current Protocols In Immunology for example, Unit 2.14, John E.Coligan et al., (eds), Wiley and Sons, New York, NY (1994), and annual renewal version comprises 2002).The TB that can prepare purification with preliminary SDS-PAGE.Nearly 2mg TB albumen application of sample is in the hole with the formed standard 1.5mm slab gel of big stripping fork.The edge that can take out gel behind electrophoresis also dyes to identify the TB protein band in the gel.Downcut from gel then and contain the gel area of TB protein band, and place it among the PBS, final concentration is the TB albumen of every ml 0.5mg purification, and is stored in 4 ℃ up to use.Can be used for immunity inoculation with the purified TB albumen of isopyknic Freund's complete adjuvant (CFA) emulsifying then.

Nearly 6 weeks after operation castrating or about 8 weeks behind the chemical castration are with the TB (0.5ml/ml PBS) of 2ml CFA emulsifying 2ml purification and be stored in 4 ℃.Inhale lentamente through the 3ml glass syringe that is connected with No. 19 syringe needles and to push away the TB/CFA mixture, must avoid over-drastic bubble.In case when emulsion is homogenizing concentration, substitute No. 19 syringe needles, discharge all bubbles with No. 22 syringe needles.Give the TB/CFA emulsion (also can carry out immunity inoculation) of injection 50 μ l volumes in castrating and the false castrating mouse muscle through Intradermal or subcutaneous route.Bacterin preparation also can use mycobacterium bovis BCG BCG.

Can behind initial immunization, randomly implement the reinforced immunological inoculation by 4-8 (or more late).Prepare TB adjuvant emulsion by above-mentioned identical mode, be used for all reinforced immunological inoculations except replace CFA with incomplete Freund (IFA).Can also carry out the reinforced immunological inoculation after this 2-4 week (or more late interval).

Plasmid DNA: before described the DNA sequence of the coding Ag85 that is suitable for and carrier (seeing U.S. No.5 for example, 736,524).Those skilled in the art can easily determine other expression vectors that is suitable for.

Antigen 85DNA immunity inoculation: the method for dna immunization inoculation is also known in this area.For example, at for example Current Protocols In Immunology, Unit 2.14 (John E.Coliganet al., (eds), Wiley and Sons, New York, NY (1994), and annual renewal version comprises 2002) described the method for dna immunization inoculation of (particle gun) of Intradermal, intramuscular and granule mediation in detail.

Randomly use the DNA of the Codocyte factor, immunne response is converted to required Th1 or Th2 type immunne response.The adjuvant of inducing Th1 to take place comprises for example IFN γ and IL-12.The adjuvant of inducing Th2 to take place comprises for example IL-4, IL-5 and IL-10.The preparation of inducing the adjuvant that Th1 and Th2 take place and the summary of the usage in induce immune response thereof are seen for example Robinson, et al., (2000) Adv.Virus Res.55:1).

In operation castrating back nearly 6 weeks or behind chemical castration about 8 weeks, be diluted in 200 μ g DNA in the 100 μ l saline for injection in the mouse muscle.

The inoculation of DNA reinforced immunological is randomly used during 2 weeks in 4 weeks and reinforcement back behind primary vaccination.

Enzyme-linked immunosorbent assay: the different time sections of (or before infecting and afterwards) before immunity inoculation and afterwards, with every group mice blood-letting, and measure the specific IgG level of the serum of individual mice with the quantitative enzyme-linked immunosorbent assay (ELISA) of standard with the anti-Ag85 in the detection serum.Can randomly measure IgG1 and IgG2 level, known they are relevant with Th1 type antibody response with Th2 respectively.

Before the primary vaccination and afterwards and the different time points after strengthening collect serum, and the existence of the anti-Ag85 specific antibody in the serum analysis.In other places of this description basic ELISA method has been described, except using the Ag85 albumen of purification.

Cytokine assay: analyzed from being responded to the Ag85 secretion of the stimulated cells factor again by the splenocyte of inoculation mice, at Huygen et al., (1992) Effect.Immunity 60:2880 and U.S. No.5 are described in 736,524 as for example.Simply, splenocyte is hatched with culturing filtrate albumen or the C57BL/6T cellular antigens determinant peptide (aminoacid 241-260) of the Ag85A of mycobacterium bovis BCG BCG purification.

4 weeks and strengthening back 2 weeks (or more late) behind primary vaccination, measure cytokine, measure IL-2, IFN γ and IL-6 with the standard bioassay method, utilize method well known in the art to measure IL-4 and IL-10 with ELISA.See, Current Protocols In Immunology for example, Unit 6 (John E.Coligan et al., (eds), Wiley and Sons, New York, NY (1994) and the annual version that upgrades comprise 2002).

Mycobacterial infections and attack: in order to check the curative effect of vaccination, the mycobacterium bovis BCG BCG (0.5mg) that lives with intravenous injection attacks mice.Different time point after attack is analyzed the propagation of BCG in mice spleen and lung.Positive control is a mice originally (by being required to be castrating and/or false castrating mice) of having accepted challenge dose.

For the inspectability steroid is removed preventing the curative effect of primary infection, the mycobacterium bovis BCG BCG that described similarly injection is lived as above-mentioned challenge trial.False castrating mice is taken as contrast.

Estimate that the spleen of mice under fire, vaccination and the number of the colony forming unit (CPU) in the lung lung interior and castrating, the primary infection mice all will be markedly inferior to the negative control animal, this has illustrated the protective effect in the mycobacterium bovis BCG attack model of living.

Embodiment 13: the immunity inoculation of cancer and prevention

For the deterministic class sterin remove prophylaxis of cancer and/whether be effectively on the postvaccinal protective immune response of cause cancer antigen vaccine, carried out following research.

Material and method

Castrating: as above carry out operation and/or chemical castration to the C57BL/6 mice.

CEA immunity inoculation: about 8 weeks behind operation castrating nearly 6 weeks of back or chemical castration, with coding hCEA (CEA) gene (MC38-CEA-2) (Conry et al., (1995) adenovirus vector Cancer GeneTher.2:33) for example is at U.S. No.6, AdCMV-hcea inoculation mice described in 348,450.Perhaps utilize in will the encode plasmid DNA of people CEA gene of the method for other local described various dna vaccinations inoculations of this description and be expelled to (for example, intramuscular injection is interior to musculus quadriceps) in the mice.

Tumor challenge: in order to measure the curative effect of sex steroid removing to the anti-tumor activity of CEA mice immunized, mice has been accepted tumor challenge.On the different time points after the immunity inoculation, the homogenic tumor cell (Conry et al., (1995) CancerGene Ther.2:33) of expressing human CEA gene (MC38-CEA-2) is inoculated in the mice.Every other day observe the growth of the palp tumor nodule of mice.When the tumor nodule diameter surpasses 1cm, put to death mice.Time between inoculation and the execution is the time for survival.

For the curative effect of inspectability steroid removing prophylaxis of tumours, the tumor cell inoculation of using expressing human CEA gene is in mice castrating, not vaccination, and is as listed above.False castrating mice is when comparing.

Embodiment 14: the transplanting of genetic modification HSC (gene therapy)

The I.SCID-hu mouse model

Material and method

Mice: as discussed previously basically passing through (sees people's tire liver and thymus fragment operation transplantation for example in the preparation SCID-hu mice in the CB-17scid/scid mice, Namikawa et al., (1990) J.Exp.Med.172:1055 and Bonyhadi et al., (1997) J.Virol.71:4707).The method that makes up SCID-hu Thy/Liv mice also is found in for example Current Protocols InImmunology, and Unit 6, John E.Coligan et al., (eds), Wiley and Sons, New York, NY (1994) and the annual version that upgrades comprise 2002.

The operation castrating of mice: be dissolved in brinish 5ml 100mg/ml ketalar (Ketalar_ by intraperitoneal injection 30-40 μ l; Parke-Davis, Caringbah, NSW Australia) adds 1ml20mg/ml xylazine (Rompun_; Bayer Australia Ltd., Botany NSW, mixture anesthesia SCID-hu mice Australia).As in other local described castratings that undergos surgery, by scrotal incision, appear testis, it is used the suture ligation, take out testis with the periphery fatty tissue then.And perform the operation to order and close wound.Will be by above-mentioned but do not take out the prepared vacation castrating mice of the method for testis in contrast.

Chemical castration: as above carry out chemical castration.

The separation of people CD34+HSC: collecting also with the known technology of those skilled in the art, handler's umbilical blood (CB) HSC (sees, DiGusto et al. for example, (1997) Blood, 87:1261 (1997), Bonyhadi et al., (1997) J.Virol.71:4707).The part of each CB specimen has all been carried out the HLA phenotype typing of MA2.1 surface molecular.Utilize the described method of Bonyhadi et al. ((1997) J.Virol.71:4707) immunomagnetic beads enrichment CD34+ cell.Simply, (QBEND-10 Immunotech) is hatched, then washing and with 2 * 10 with CB cell and anti-CD34 antibody ⁷The final concentration of/ml is resuspended.Then according to product description goat anti-mouse IgG 1 magnetic bead (Dynal) enrichment CD34+ cell.Then CD34+ cell and 50 μ l glucoproteinases (O-sialoglycoprotein endopeptidase) are hatched, this enzyme causes the release of CD34+ cell from the immunomagnetic beads.Remove magnetic bead with Magnet, then cell be used to utilize the flow cytometry of bonded anti-CD34-PE antibody, to determine the aggregate level of existing CD34+ cell in the cell mass.Perhaps, cell is by with anti-CD34 antibody magnetic mark and be stored in autoMACS ^TMIn.AutoMACS ^TMCan be used to before further flow cytometry sorting, carry out the pre-sorting of magnetic of pair cell.For example, the MACS_MicroBeads that in the past adds anti-FITC or anti-PE in the painted cell of FITC or PE.AutoMACS then ^TMMagnetic mark sorting cells according to cell.Can select positive or negative partly to be used for the sorting of flow cytometry.

Randomly, can use IL-3, IL-6 and or with SCF or LIF (every kind of 10ng/ml) amplification in vitro HSC.

The preparation of (GM) HSC of RevM10 carrier and genetic modification: RevM10 is known in this area, and in that having been described this carrier widely, the research of the GM HSC of the T cell survival that is used for the HIV infected patient (sees, for example, Woffendin et al., (1996) Proc.Natl.Acad.Sci.USA, 93:2889; For review, see Amado et al., (1999) Front.Biosci.4:d468).Known HIV Rev albumen can influence the virus lays dormant phase in the HIV infection cell, and HIV is duplicated is vital.RevM10 is the derivant of Rev because with the sudden change in the leucine zone of being rich in of the interactional Rev of cytokine.RevM10 has Aspartic Acid on 78 sites to the replacement of the leucine on leucic replacement and 79 sites to glutamic acid.These results of mutation are that RevM10 can compete and the combining of Rev response element (RRE) effectively with wild type HIV Rev.

Can use any RevM10 gene transfer vector known in the art and that be described.For example, retroviral RevM10 carrier, pLJ-RevM10 is used to transform HSC.The pLJ-RevM10 carrier has demonstrated behind the individuality that is transported to the HIV infection can strengthen T cell implantation (Ranga et al., (1998) Proc.Natl.Acad.Sci.USA 95:1201).Additive method that makes up and the retroviral vector that is applicable to preparation GM HSC are (see, for example, Bonyhadi et al., (1997) J.Virol.71:4707) known in this area.

In another example, pRSV/TAR RevM10 plasmid is used to utilize the gene transfer of granule mediation that non-virus carrier is transported in the isolating targeting HSC of institute, basically with Woffendin etal., (1994) Proc.Natl.Acad.Sci.USA, 91:11581 is described.Also can use pRSV/TAR RevM10 plasmid (the Woffendin et al. that contains Rous sarcoma virus (RSV) promoter and be used to express the open reading frame of RevM10 from-18 to+72 the tat activating reaction element (TAR) of HIV, (1994) Proc.Natl.Acad.Sci.USA, 91:11581; Liu et al., (1997) Gene Ther.1:32).Previous shown this plasmid to the in-vitro transfection of people PBL provide resistance that HIV is infected (Woffendin et al., (1994) Proc.Natl.Acad.Sci.USA, 91:11581).

A kind of marker gene for example Lyt-2 α (mice CD8 α) gene also can be incorporated in the RevM10 carrier, with the convenient purification of GM HSC and the facs analysis of analysis (are seen in later step, for example, Bonyhadi et al., (1997) J.Virol.71:4707).

The Δ Rev10 that has made up the disappearance that contains methionine (Met) start codon (ATG) and comprised the junctional complex of the termination codon in a series of BgIII sites that in framework, are inserted into the RevM10 gene, and be used as negative control and (see, Bonyhadi et al. for example, (1997) J.Virol.71:4707).

GM HSC is injected in the mice: implant the back at the thymus regulating liver-QI and analyzed the SCID-hu mice in about 4 months.For the scavenger cell group, be confirmed as having accepted the total irradiation (TBI) of nearly 400rads with the mice of people's donor HSC HLA unmatched (MA2.1).After the TBI, as discussed previously rebuilds mice (DiGusto et al, (1997) Blood, 87:1261, Bonyhadi et al., (1997) J.Virol.71:4707) with RevM10GM HSC (on seeing).To the HSC of control mice injection unmodified or used the HSC of Δ RevM10 gene or irrelevant genetic modification.

Flow cytometry is to the analysis of GM HSC: rebuild nearly 8 to 12 weeks of back at GM HSC, take out the Thy/Liv graft, its HLA phenotype (MA2.1) and CD4+, CD8+ and Lyt2 (" label " of the flow cytometry that obtains thymocyte cell and know easily with those skilled in the art and the methods analyst of facs analysis, the mice homologue of CD8 α) distribution of surface expression (is seen, Bonyhadi et al. for example, (1997) J.Virol.71:4707; Also see Current Protocols InImmunology, Unit 6, John E.Coligan et al., (eds), and Wiley and Sons, New York, NY (1994) and the annual version that upgrades comprise 2002).Utilize standard pcr to measure the transgenic DNA of thymocyte cell with the primer that is specific to the RevM10 gene.

GM HSC is infected the analysis of resistance to HIV: rebuild nearly 8 to 12 weeks of back at GM HSC, take out the Thy/Liv graft and from the SCID-hu mice that GM HSC rebuilds, obtain thymocyte cell.(Bonyhadi et al., (1997) J.Virol.71:4707) as discussed previously is with the JR-CSF molecular separation object external stimulus of HIV-1 and infect thymocyte cell.Simply, contain in sodium selenite medium supplement (Sigma), 40U people rIL-2/ml and 2 μ g/ml lectins (PHA) the RPMI culture medium (Sigma) of 10%FCS, 50 μ g/ml streptomycins, 50U/G benzylpenicillin, 1 * MEM vitamin solution, 1 * transfer insulin, at the allogene feeder cell (10 that has through irradiation ⁶PERIPHERAL BLOOD MONONUCLEAR CELL/ml and 10 ⁵During JY cell/ml), the stimulated in vitro thymocyte cell.Per approximately 10 days as before at Vandekerckhove et al., described in (1992) J.Exp.Med.1:1033 with feeder cell and PHA irritation cell again.After stimulation nearly 5 days the time, according to HLA phenotype (MA2.1) and Lyt2 (" label ", the mice homologue of CD8 α) sorting cells.The cell of institute's sorting is stimulated and can be amplified the increase cell to form above about 90% purity again.Sub-elect the CD4+/Lyt2+ cell then, with a part nearly 5 * 10 ⁴The cell of individual institute sorting is put in a plurality of holes of tissue culturing plate at the bottom of the 96 hole U.In each hole, add about 200TCD ₅₀EW, HIV-1 primary separation thing, or 1000TCD ₅₀JR-CSF, HIV-1 molecular separation thing.The previous method of having described viral storage liquid preparation (Bonyhadi et al., (1993) Nature, 363:728).Changed culture medium from the 3rd day to 12 day every day.Collect the supernatant part every other day and be stored in-80 ℃ up to use.Analyze with p24ELISA according to product description (Coulter) then and organize culture supernatant.

The treatment of II.HIV infected individuals

Material and method

The separation of people CD34+HSC: because most HIV infected patients have the HSC of very low titre, it is possible therefore supplying with cell with donor.In practice, by before collecting cell, injecting 10 μ g/ml granulocyte colony-stimulating factors (G-CSF) 2-5 days, strengthened the HSC level in the donor blood to donor.

In this embodiment, collect with technology well known in the art and handler's umbilical blood (CB) HSC (sees DiGusto et al. for example, (1997) Blood, 87:1261; Bonyhadi et al., (1997) J.Virol.71:4707).The part of each CB specimen is used to carry out HLA phenotype typing, for example is purified into the CD34+ donor's cells with flow cytometry or immunomagnetic beads from donor blood (or BM), basically as above-mentioned.The HSC in the donor source of identifying with flow cytometry is the CD34+ cell.

Randomly, can use IL-3, IL-6 and or with SCF or LIF (every kind of 10ng/ml) the amplification HSC that exsomatizes.

The preparation of (GM) HSC of RevM10 carrier and genetic modification: can use at arbitrary RevM10 gene transfer vector known in the art and that be described, comprise the carrier described in those superincumbent mice study.Utilize the gene transfer of GM reverse transcription carrier or utilize the method for gene transfection of the conveying of granule mediation also to know, and describe to some extent in other places of this description in this area.

As mentioned above, can make up the trans-dominant mutant form Rem10 that retroviral vector contains the HIV-1rev gene, this mutant form has shown that can suppress HIV duplicates (Bonyhadi et al., (1997) J.Virol.71:4707).Generated the supernatant that contains two-way carrier by the infection that is used for producing the filtering supernatant of cell from the close preferendum (ecotropic) of carrier transfection.

In the LCTM culture medium that is added with IL-3, IL-6 and SCF or LIF (every kind of 10ng/ml), the collected CD34+ cell of randomly pre-stimulation enters cell cycle with inducing cell.

In this embodiment, utilize (" particle gun " shifts) of granule mediation the HSC that is rich in CD34+ to be carried out transfection with linear RevM10 plasmid, basically as at Woffendin et al., (1996) Proc.Natl.Acad.Sci.USA, described in the 93:2889.But, if carry out retrovirus transduction, just in cell, added carrier-containing supernatant 2-3 days repeatedly, transduce in the cell with allowable carrier.

HAART treatment to the HIV infected patient: beginning HAART treatment before T cell exhaustion and sex steroid removing all keeps treatment with the minimizing virus titer in whole process.

The T cell is exhausted: as giving in embodiment 5, carry out the T cell and exhaust to remove many HIV infection cells as much as possible.

Sex steroid is removed treatment: as described in the embodiment 6, give HIV infected patient sex steroid and remove treatment.

GM HSC is expelled in patient's body: before injection, GM HSC is being comprised that (Chiron cultivates nearly 10 days of amplification to IL-2 in X-Vivo15 culture medium 300IU/ml).Nearly 1-3 week after giving the LHRH agonist, before thymus just begins reactivate or simultaneously, give the HSC of patient infusion genetic modification, optional dosage is about 2-4 * 10 ⁶Individual cell/kg.Also can randomly inject G-CSF, help the amplification of GM HSC to the receptor.

Before giving patient's infusion, at once, wash GM HSC 4 times with Dulbecco PBS.Cell is resuspended in the saline that 100ml contains 1.25% human albumin and 4500U/ml IL-2, and in 30 minutes, is infused in patient's body.

Give HIV infected patient sex steroid remove and injection GM HSC after, all new T cells (and DC, macrophage etc.) all tolerate in the infection subsequently of this virus.Mean that HSC will enter thymus for the patient infusion allogene HSC that carries out the thymus reactivate.Positive reactivate or the thymus picked-up genetic modification of reactivate SHC and be translated into donor type T cell and DC, and receptor's HSC is converted into receptor's type T cell and DC.Induce deletion by cell death, or by the immunity regulatory cell inducing tolerance, donor DC will tolerate any T cell that may react with the receptor.

When having set up the thymus chimera, new mature T cells group has begun to leave thymus, has occurred that immunosuppressant alleviates and final disappearance.

Treat behind the infusion: after infusion, utilization is to restricted dilution PCR results of regular determination retention time and the half-life of GM HSC in the HIV infected patient of the PBL specimen that obtains from the patient, basically as at Woffendin et al., (1996) Proc.Natl.Acad.Sci.USA describes among the 93:2889.Compared the relative level of GM HSC among infected patient and the negative control patient who accepts the ARevM10 carrier.

Also utilize method well known in the art to carry out hematological (for example CD34+T cell counting) of various standards, immunologic (for example NAT) and virological (for example virus titer) research.

Immunosuppressant stops: as termination immunosuppressant given in embodiment 16.

Embodiment 15: alternative plan

Aspect required time of the transplanting of shortening donor's cells, tissue or organ, revised used timeline among the embodiment 1-14.Beginning the removing of T cell or other immunocytes exhaustion and sex steroid simultaneously removes.The T cell is removed or other immunocytes are exhausted lasting about 10 days, and sex steroid was removed lasting about 3 months.In one embodiment, after the beginning therapeutic alliance about 10-12 days, when thymus begins reactivate, carry out HSC and transplant.

In one even shorter timetable, the exhaustion and the HSC that begin two types simultaneously transplant.In this case, the T cell is removed or other immunocytes exhaustion have continued 3-12 month, for example 3-4 month.

Embodiment 16: immunosuppressant stops

When having set up thymus chimera and new mature T cells group when beginning to leave thymus, from the patient, get blood, and the disappearance to donor's cells's responsiveness of vitro examination T cell (is seen in the mixed lymphocyte reaction of standard, for example, Current Protocols InImmunology.John E.Coligan et al., (eds), Wiley and Sons, New York, NY (1994) and the annual version that upgrades comprise 2002).If do not reply, gradually reduce immunosuppressant therapy to allow defense reaction to infecting.If there is not the sign of repelling,, finally stop immunosuppressant therapy fully as existing the part prompting of existing activating T cell in the blood.Because HSC has the ability of very strong self renewal, formed hemopoietic chimera will all be stable (people tolerance and that do not implant normally just) at the lifelong of patient in theory.

Embodiment 17: use LHRH agonist reactivate people thymus

Material and method

In order to show that people's thymus can be by method of the present invention institute reactivate, these methods are used to use the patient of chemotherapeutic treatment carcinoma of prostate.

Patient: selected 16 routine I-III phase patients with prostate cancer (prostate specific antigen (PSA) integration with them is estimated).All objects all be the age 60 and 77 years old between the male, these patients are before the local radiotherapy of accepting carcinoma of prostate as required, all stood the androgen blocking treatment of the associating of standard, treatment is according to every month injection GnRH agonist, every month 3.6mg goserelin acetate (Zoladex_) or 7.5mg leuprorelin (Lupron_) treatment 4-6 month altogether.

Facs analysis: (20 μ l) joins in the 200 μ l whole bloods with suitable antibody cocktail, and at room temperature hatched 30 minutes the dark place.Cracking RBC, the washing remaining cell also is resuspended in it and is used for facs analysis among 1%PFA.With CD19-FITC, CD4-FITC, CD8-FITC, CD27-FITC, CD45RA-PE, CD45RO-CyChrome, CD62L-FITC and CD56-PE (all from Pharmingen, San Diego, antibody staining specimen CA).

Statistical analysis: by before the treatment relatively and the result of treatment back, each patient is as internal reference, and checks or the rank test analysis result of Wilcoxon labelling with pairing student t.

The result

Before sex steroid is removed treatment and 4 months post-evaluation patients with prostate cancer.In Figure 19-23, summarized the result.Data have jointly confirmed the quantitative and raising qualitatively of the T cell state in many patients.

The I.LHRH treatment is to lymphocyte and the wherein effect of the sum of T cell subgroup

Analyzed the patient's (all＞60 years old) of the LHRH agonist treatment that carries out carcinoma of prostate the phenotype of peripheral blood lymphocyte and formed (Figure 40).4 months analysis patients' specimen before treatment and behind beginning LHRH agonist treatment.The total lymphocyte number of the every ml blood of all patients before treatment is all in the lower bound of control value.

After treatment, 6 examples among the 9 routine patients demonstrate the remarkable increase (having observed the double of total cell number in some cases) of total lymphocyte counting.6 examples that relevant therewith is among the 9 routine patients have the increase of total T cell number.In the CD4+ subgroup, in this 8 examples that are increased in 9 routine patients or even more tangible, confirmed the increase of CD4+T cellular level.In 9 routine patients' 4 routine patients, seen the more not outstanding trend in the CD8+ subgroup, shown as the increase of level, though the degree that increases is less than the CD4+T cell usually.

II.LHRH treats the effect to the ratio of T cell subgroup:

To confirming do not have significant change and CD4+ on the population proportion of T cell, CD4+ or CD8+T cell with the analysis of afterwards blood samples of patients before the LHRH agonist treatment: the CD8+ ratio has different variation (Figure 41) after treatment.In this explanation treatment maintenance very little effect is arranged, although total increased the T cell number significantly after the treatment to the homeostasis of T cell subgroup.All numerical value are all suitable with control value.

The III.LHRH treatment is to the effect of B cell and myelocytic ratio

The analysis of the ratio of B cell in the peripheral blood of patients of carrying out the LHRH agonist treatment and myelocyte (NK, NKT and macrophage) is confirmed to have in various degree variation (Figure 42) in subgroup.NK, NKT still keep relative constant with the macrophage ratio after treatment, and the decline of B cell proportion has appearred in 4 examples among the 9 routine patients.

The IV.LHRH agonist treatment is to the effect of B cell and myelocyte sum

B in the peripheral blood after the treatment and myelocytic total cell number goal analysis are clearly illustrated the level (Figure 43) that has increased the cell number of NK (5 examples among the 9 routine patients), NKT (4 examples among the 9 routine patients) and macrophage (3 examples among the 9 routine patients) after the treatment.The B cell number does not demonstrate outstanding trend, and wherein 2 examples among the 9 routine patients demonstrate the increase of level, and 4 examples among the 9 routine patients do not demonstrate and change, and 3 examples among the 9 routine patients demonstrate the minimizing of level.

The V.LHRH treatment is to the effect of naive cell with respect to the level of memory cell

The main variation of being seen behind the LHRH agonist treatment is in the T of peripheral blood cell mass.Particularly, the increase of the ratio of (CD45RA+) CD4+ cell is arranged originally, and in CD4+T cell subgroup, 6 examples among the 9 routine patients (CD45RA+) occurred originally to the increase (data do not show) of the ratio of memory (CD45RO+).

VI. conclusion

Therefore can inference: the LHRH agonist treatment that animal is for example had the people of atrophy thymus can be induced the regeneration of thymus.Shown the generally raising of the blood T lymphocyte state of these patients with prostate cancer that acceptance steroid removing is treated.As if these cells are to derive from thymus, because described other sources that do not have (the TCR α β+CD8 α β chain) of main flow T cell.Gastrointestinal tract T cell mainly is TCR γ δ or CD8 α α chain.

Embodiment 18: people's the regeneration of periphery immunocyte pond after hematopoietic stem cell transplantation

I. Allogene and from body HSCT

This embodiment relates to the clinical trial of carrying out with HSCT patient.In order to estimate the thymus that recovers the people and the clinical probability of marrow function, by analysis the routine patients with prostate cancer (＞60 years old) that carries out removing treatment based on the sex steroid of LHRH agonist (chemical castration).When beginning treatment and at this moment all patients' serum testosterone concentration all be in back 4 months of the treatment of castrating level, inspection patient.

Material and method:

The patient: 82 routine patients are because malignant disease or marrow failure have carried out high-dose chemotherapy (HDT) and PBSCT (allogene control patients 22 examples, allogene LHRH-A treatment patient 20 examples; Contrast 20 examples from body; From body LHRH-A treatment patient 20 examples).In 3 weeks before body or allogeneic stem cell transplantation, give test patient 3.6mg (effective 4 weeks) Zoladex (LHRH-A), injection in every month is 4 totally months then.Before treatment, transplant the back in 5 weeks weekly and analyzed all patients every month after this up to 12 months.Obtain ethics permission (test number 01/006) from Alferd human research Ethics Committee.

Facs analysis to full periphery blood: (20 μ l) joins in the 200 μ l whole bloods with suitable antibody cocktail, and at room temperature hatched 30 minutes the dark place.Cracking RBC, the washing remaining cell also is resuspended in it and is used for facs analysis among 1%PFA.With CD19-FITC, CD4-FITC, CD8-FITC, CD27-FITC, CD45RA-PE, CD45RO-CyChrome, CD62L-FITC and CD56-PE (all from Pharmingen, San Diego, antibody staining specimen CA).

Ki67 analyzes: in order to detect proliferative cell, with CD27-FITC, CD45RO-CyChrome, and CD4-or CD8-APC (Pharmingen, San Diego, CA) padding specimen.Behind erythrocyte splitting, specimen is changed solution (Becton-Dickinson, USA thoroughly at 500 μ l 1X FACS; From the 10X solution of RO water, make 1X solution) middle dark place, hatched under the RT 20 minutes, with washed specimen (2ml FACS buffer, 5 minutes, 600g _Max, RT) at room temperature hatched 30 minutes the dark place with anti-Ki-67-PE or anti-Ki67-FITC (or the contrast of suitable homotype).Wash specimen then and it is resuspended in and be used among the 1%PFA analyzing.

The preparation of PBMC: be used for detection of T cytositimulation and TREC analysis by Ficoll-Hypaque separation and the centrifugal lymphocyte of preparing purification subsequently, take out plasma layer, before analytical steroid levels, it is stored in-20 ℃.The cell that is not used to the detection of T LS is resuspended in the freezing culture medium, and before TREC analyzes it is stored in the liquid nitrogen.

The T LS detects: for the mitogenesis primary stimuli, by 1 * 10 in every hole 100 μ lRPMI-FCS ⁵The concentration of individual cell is tiled in the lymphocyte of purification in the 96 hole circle base plates.With cell and dosage from the PHA of 1-10 μ g/ml at 37 ℃, 5%CO ₂Hatch together down.For the TCR differential stimulus, cell was hatched 48 hours in the plate of using anti-CD3 of purification (1-10 μ g/ml) and anti-CD28 (10 μ g/ml) coating in advance.Form back (48-72 hour) in plaque, in every hole, add 1 μ Ci ³The H-thymidine, and plate hatched 16-24 hour again.Collecting board in bag filter (mats), and at the β enumerator (Packard-Coulter USA) goes up with liquid scintigraphy mensuration ³The situation of mixing of H-thymidine.

TREC analyzes:

Cell sorting: in ice, dyeed 30 minutes with the quick thawing of refrigerated specimen and with anti-CD4-FITC and anti-CD8-APC, fix (stirring down) with its washing (2ml FACS buffer) and with 3% formalin PBS solution.Hatched specimen again 30 minutes, washing also is resuspended in the FACS buffer that 500 μ l are used for sorting with it.In MoFlo_ cell sorter (Cytomation Inc.), sub-elect CD4+ and CD8+ cell mass.

DNA separates: sorting cells also is resuspended in E.C. 3.4.21.64 (PK) the digestion buffer (2 * 10 with it ⁵Individual cell/20 μ l 0.8mg/mL solution).Specimen was hatched 1 hour under 56 ℃, under 95 ℃, hatch 10 minutes subsequently with inactivated proteases.

Utilize the PCR in real time of molecular beacon: (Zhang et al., (1999) J.Exp.Med.190:725) as discussed previously is used to analyze the PCR in real time of selected intracellular TREC content.Primer is: adopted primer is arranged, 5 '-GGATGGAAAACACAGTGTGACATGG-3 ' (SEQ IDNO:4), antisense primer, 5 '-CTGTCAACAAAGGTGATGCCACATCC-3 ' (SEQ IDNO:5).Carrying out 1 circulation degeneration (95 ℃ 10 minutes), is 45 circulation amplifications (94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ 30 seconds) subsequently.Cell equivalent among the standardization input DNA detects quantitative CCR5 coded sequence with PCR in real time separately, and it does not contain pseudogene.

Statistical analysis: carry out statistical analysis with Instat II software.For carcinoma of prostate HSCT research, carry out Mann-Whitney U check.For the human research, by before the treatment relatively and the result after the treatment, each patient is as internal reference, and checks or the wilcoxonsigned order is closed the check analysis result with paired student t.

The result:

Figure 49 has described different time points (2-8 week) behind HSCT to the analysis of NKT (NK) cellular-restoring of control patients.As Figure 49 A-B shown in respectively, in the allogene of two contrasts and autotransplantation receptor, all observed similar trend.On the contrary, the allogene patient who before HSCT, accepts LHRH-A treatment 3 weeks from transplant back the 14th day all demonstrated by 5 months significantly higher NKT cell (V α 24+V β 11+) number (Figure 49 C, data are expressed as meansigma methods ± 1SEM of 6-20 example patient, ^*P≤0.05).According to their V α 24+V β 11+ phenotype analytical NKT cell.

Figure 50 has described the facs analysis that the different time points (the 14th, 21,28 and 35 day) behind HSCT is rebuild the NKT cell of control patients.Observed early recovery in the allogene patient, this is mainly seen in transplants in the early stage CD8+ group in back, and this has illustrated the regeneration of the outer approach of thymus.From transplanting back 1 month, the CD4+NKT cell also is tangible.

Figure 51 has described the B cell of the control patients of the different time points (2-12 month) behind HSCT and has rebuild.Shown in Figure 51 B, B cell among the autotransplantation patient is rebuild than taking place sooner relatively (Figure 51 A) among the allogene patient.But after transplanting at least 6 months, the recoveries to control value (dash area) in two groups were still not obvious.

The CD4+ that Figure 52 has described the control patients of the different time points (2-12 month) behind HSCT rebuilds.When the B cell number when transplanting returned to control value in back 6 months (seeing Figure 48 A-B), be still serious attenuating from (Figure 52 B) of body and the CD4+T cell number among heterogenic (Figure 52 A) receptor, even in transplanting in the time of back 12 months.

Figure 53 has described the CD8+ cell regeneration of the control patients of the different time points (2-12 month) behind HSCT.Shown in Figure 53 A-B, correspondingly at allogene with in the body receptor, the CD8+T cell number is all regenerated very soon after transplanting.But shown in Figure 53 C, the CD8+T cell mainly is the thymus external source, as suggested in the increase of TCR γ δ+T CD8+T cell, CD8 α α T cell and CD28-CD8+T cell.

Figure 54 described utilize label Ki-67 to (Figure 54 A) before the HSCT with 28 days after the facs analysis of propagation of the different CD4+ of control patients of (Figure 54 B) and CD8+T cell mass.Utilize label CD45RO and CD27, according to originally, memory and activation phenotype analytical cell.Most propagation occurs in CD8+T cell subgroup, and this illustrates that also these cells are that the propagation thymus external source and main all occurs in the periphery T cell subgroup.

Figure 55 has described the control patients of the different time points (2-12 month) behind HSCT and LHRH-A treatment patient's the cell regeneration of CD4+T originally.Figure 55 has described the facs analysis of CD4+T cell (CD45RA+CD45RO-CD62L+) originally, and shows the serious disappearance of these cells in whole research process.Shown in Figure 55 B-C, the cell of CD4+T originally among the autotransplantation patient begins regeneration (Figure 55 C) 12 months the time behind HSCT, but still is markedly inferior to control value (Figure 55 B) among the allogene patient.These presentation of results thymus can not recover the number of the abundant cell of T originally after transplanting because of patient's age.On the contrary, before allogene, accepted in 3 weeks among the patient of LHRH-A, to transplanting back 9﹠amp; All demonstrate the cell of comparing according to remarkable higher number of CD4+T originally in the time of 12 months and (compare 9﹠amp after transplanting with contrast (LHRH-A treats); 12 months p≤0.05) (Figure 55 D).The regeneration of this explanation thymus dependent T cellular pathways is removed treatment along with sex steroid and is strengthened.

Figure 56 has described the TREC level of the control patients of different time points behind HSCT (1-12 month).To being detected in that thymus recently moves out that the analysis of TREC level of cell (RTE) focuses on allogene (Figure 52 A) and from the incapability of thymus recovery level after transplanting of body (Figure 52 B) patient.This is ascribed to patient's age once more, and because the disappearance of the thymus function that atrophy of thymus gland caused has thought that this M ﹠ M to these patients all has suitable influence.On the contrary, when before allotransplantation, accepting the LHRH-A treatment, the patient who carries out allogeneic peripheral blood stem cell transplantation shows the remarkable increase of CD4+TREC+ cell/ml blood (relatively, transplanting back 9 months p≤0.01 with contrast (LHRH-A does not treat)).The allogene patient who accepts the LHRH-A treatment demonstrates the CD4+TREC+ cell/ml blood of comparison according to remarkable higher number in transplanting in the time of back 9 months.Patient from the LHRH-A of body treatment also demonstrated significantly higher level (Figure 56 D) in back 12 months in transplanting.This explanation thymus reactivate is removed the enhancing of treatment with sex steroid.Data are expressed as meansigma methods ± 1SEM of 5-18 example patient, ^*P≤0.01.

The LHRH-A administration has increased the NK of peripheral blood significantly but has not been the number of B cell.In general, do not observe the significant change (Figure 47) of B cell number with the LHRH-A treatment.But, observed the NK cell number (Figure 47) with the remarkable increase (p≤0.01) for the treatment of.Therefore, the removal of sex steroid has caused the remarkable increase of T cell and NK cell number.

With previous research consistent (Garzetti et al., (1996) Obstet.GynecoL 88:234-40 to the patient that treated with the LHRH-agonist; Oliver et al., (1995) Urol.Int, 54:226-229; Umesaki et al., (1999) Gynecol.Obstet.Invest.48:66-8), observed the remarkable increase (Figure 45 and 47) of total lymphocyte, T cell (mainly being CD4+) and NK cell.To the more detail analysis of T cell mass found originally the CD4+T cell and originally with the remarkable increase (Figure 44) of memory CD8+T cell number after the LHRH-A treatment.

In order to determine originally whether the increase of T cell is (to see example (the Soares et al. that for example uses the IL-7 administration by the periphery amplification, J.Immunol. (1998) 161:5909-5917)) or as the direct result of thymus reactivate, carried out the analysis (Hazenberg et al., (2001) J.Mol.Med.79:631-40) of on cell proliferation (Ki-67 antigen+) and TREC level.CD4+ (Figure 48 A) and CD8+T cell (Figure 48 B) originally, among activation or the memory cell group, do not see that all the propagation level is with any variation of agonist treatment (still for 2-4% low-level).This explanation treatment does not have the propagation of direct inducing T cell, and the hyper-proliferative of periphery can not influence the level of TREC.This is not except that the probability that increases in the periphery of time point more early.But, estimate the increase that this will only cause activation/memory cell level.After analysis, found the positive evidence (Figure 46 B) of the increase of thymus function and T cell number to 10 routine patients' TREC level.In CD4+ and CD8+T cell mass, 5 among 10 routine patients example to the LHRH-A treatment demonstrate in the time of 4 months absolute TREC level (every ml blood) increase (above initial show value＞25%).This also is reflected in the increase (per 1 * 10 of ratio ⁵Individual cell).6 examples among the Xiang Guan 10 routine patients all demonstrate the overall increase of total TREC level therewith.Only 1 routine patient demonstrates the minimizing of total TREC (reducing about 10%).Because can in thymus, take place as mitosis (the Hazenberg et al. after the normal cytocerastic part of T or the output, (2001) J.Mol.Med.79:631-40) diluted TREC (Zhang et al., (1999) J.Exp.Med.190:725-732), absolute TREC level will be represented the low valuation very to the output of T cell.Therefore, the regeneration of the remarkable increase of total TREC+ cell of the periphery behind the agonist treatment and thymus dependent T cellular pathways (Douek et al., (1998) Nature 396:690-695 (1998); Douek et al., (2001) J.Immunol.167:6663-8; Hochberg et al., (2001) Blood 98:1116-21) in full accord.Total, these data acknowledgements sex steroid suppressed to have improved the adult thymus output ability and the basis of the cell number of T originally after the serious T cell that recovers in the various clinical pathological changes is exhausted is provided.

Embodiment 19: sex steroid is removed the immunologic reconstitution after the hematopoietic stem cell transplantation that has strengthened mice

Carry out this test and be suppressing to improve the hypothesis of immunologic reconstitution after their transplanting for the sex steroid of checking allogene HSCT receptor.Therefore, these tests are intended to set up the hemopoietic recovery after whether the sex steroid removing influences allogene HSCT.14 days the time, the castrating mice has increased the thymocyte cell number significantly than false castrating contrast behind HSCT.During by 28 days, these cells are still increasing, and the splenocyte density that castrate mice this moment also has increase.In thymus, T cell precursors and DC have remarkable increase after HSCT and castrating.After HSCT and castrating, BM precursor and developmental B cell have also been increased significantly.The peripheral t in the donor source behind these centers increases changing into allogene HSCT and the remarkable increase of B cell.Each immunostimulant strategy has all brought the danger that increases the weight of to take place graft versus host disease (GVHD).In the allogene group, castrating mice when inducing GVHD.When relatively mice was castrated in castrating and vacation, GVHD incidence rate or severity did not all have significant difference.In addition, when lacking sex steroid, GVT is active not to disappear.The previous lymphocyte that has strengthened allogene HSCT receptor after IL-7 treats that has been presented at recovers.The association list of IL-7 treatment and castrating reveals the thymus behind the HSCT is had accumulative action.The hemopoietic that the removing of castrating of these presentation of results and the sex steroid that caused has strengthened behind the allogene HSCT recovers, and does not increase the weight of GVHD and kept GVT.

Material and method

Reagent: the antigenic antibody of anti-mice of anti-mice CD16/CD32FcR sealer (2.4G2) and the fluorogen labelling below all from Pharmingen (San Diego, CA): Ly-9.1 (30C7), CD3 (145-2C11), CD4 (RM4-5), CD8.2 (53-5.8), TXi Baoshouti β (TCR-β; H57-597), CD45R/B220 (RA3-6B2), CD43 (S7), IgM-FITC (R6-60.2), CD11b (M1/70), Ly-6G (Gr-1) (RB6-8C5), c-kit (2B8), Sca-1 (D7), CD11c (HL3) I-A ^k(11-5.2), homotype tester: Mus IgG2a-k (R35-95), Mus IgG2a-1 (B39-4), Mus IgG2b-(A95-1), Mus IgGl-k (R3-34), logical sequence Mus IgG-group l-k (A19-3), logical sequence Mus IgG-group 2-1 (Ha4/8) and 2.4G2 and Fcr (FcR sealer).Avidin streptococcus-FITC, PercP-phycoerythrin (PE) also from Pharmingen (SanDiego, CA).(Cytheris, Vanves France) provide recombined human IL-7 by Michel doctor Morre.

In order to confirm that the people IL-7 that recombinates can stimulate mouse cell, carried out thymidine with IL-7 dependency mice pre B lymphocyte strain 2E8 and mixed propagation and detect, people IL-7 used in the concurrent present research has the proliferation function that equates with mice IL-7 to mouse cell.Tissue culture medium (TCM) is made of the RPMI 1640 that is added with 10% heat-inactivated fetal bovine serum, 100U/ml penicillin, 100mg/mL streptomycin and 2mM L-glutaminate (and the 50mM 2 mercapto ethanol that is used to cultivate 32Dp210 cell and propagation detection).

Mice and HSCT: male C57BL/6J (B6, H-2b), C3FeB6Fl/J ([B63C3H] F1; H-2b/k), B10.BR (H-2k), B6D2F1/J (H-2b/d), CBA/J (H-2k), Balb/c (H2-d), IL7-/-and KGF-/-mice is all from Jackson laboratory (Bar Harbor, ME), and when they be 8 to 12 weeks big between the time use it in the test.Use 4 months to 7 months KGF-broad in the middle/-and IL7-/-mice.The HSCT scheme has obtained the animal treatment of Memorial Sloan-Kettering Cancer center institute and has used the permission of committee.Aseptic taking-up BM cell from femur and tibia.By hatching 30 minutes at 4 ℃ with anti-Thy-1.2 antibody, and then (ON Canada) was hatched 1 hour at 37 ℃, had taken out the T cell of donor BM for Cedarlane Laboratories, Hornby with low TOX-M rabbit complement.By the purification on nylon fine hair post and subsequently with ammonia chloride erythrocyte splitting buffer removal erythrocyte, obtained splenic t-cell (being used for GVHD analyzes).Cell (is with or without splenic t-cell and leukaemia's 5 * 10 ⁶Individual BM cell) be resuspended in Dulbecco improvement minimal medium (Life Technologies, Grand Island, NY) in, and it is transplanted among the receptor of lethal exposure by tail vein infusion (0.25mL cumulative volume) at the 0th day.Before transplanting the 0th day, the receptor accepted the 1300cGy total irradiation ( ¹³⁷The Cs source), gradation gives, between each dosage 3 hours at interval (to reduce gastrointestinal toxicity).Mice is housed in aseptic little isolation cage, and accepts normal food and autoclaved hypochlorination's drinking water (pH 3.0).

Operation castrating: with mouse anesthesia and cut out little scrotal incision, take out with the testis ligation and with the fatty tissue of periphery to appear testis.Order with operation and to close wound.False castrating requires identical operation technique except taking out testis.Transplant at BM and to castrate in preceding 1 day, be used for immunologic reconstitution and GVHD research.

The administration of IL-7: or the from the 0th to 13 day or the from the 21st to 27 day intraperitoneal give 10 μ g/ days IL-7, is used for immunologic reconstitution research.Inject PBS at identical time point to control mice.

Flow cytometry: in FACS buffer (phosphate-buffered saline (PBS)/2% bovin serum albumin (BSA)/0.1% triazo-compound), wash BM cell, splenocyte or thymocyte cell, and 1-3 * 106 cell was hatched under 4 ℃ 30 minutes with the CD16/CD32FcR sealer.Then cell and first antibody were hatched under 4 ℃ 30 minutes, and with FACS buffer washed twice.As required, cell and bonded streptavidin were hatched under 4 ℃ 30 minutes again.The painted cell of institute is resuspended in the FACS buffer and at FACSCalibur ^TM(Becton Dickinson, San Jose use CellQuest on CA) to flow cytometer ^TMSoftware is analyzed.

Propagation detects: with splenocyte (4 * 10 ⁵Individual cells/well) in 96 holes with as stimulus object through the irradiation (2000cGy) BALB/C splenocyte (2 * 10 ⁵Individual cells/well) hatches 5 days, and stimulate splenocyte (4 * 10 with α CD3 (145-2c11) and α CD28 (37.51) (final concentration is 2.5mg/mL) ⁵Individual cells/well) 4 day.During in the end 18 hours, usefulness 1mCi/ hole [ ³H] thymidine excitement (pulse) culture, and collect DNA with Harvester 96 (Packard).Stimulation index (SI) is calculated as irritation cell (cpm) to the ratio of irritation cell (cpm) not.

⁵¹Cr discharges algoscopy: at 37 ℃ and 5%CO ₂Use 100mCi down, ⁵¹Cr labelling 2 * 10 ⁶The target cell of individual cell/mL 2 hours.After washing three times, the target cell of institute's labelling is pressed 2.5 * 10 ³Individual cells/well be tiled in the U base plate (Costar, Cambridge, MA) in.With final volume is 200mL's and cultivate 2 days splenocyte in different effectors through the BALB/C splenocyte (1: 2 ratio) of irradiation: the target cell ratio joins in 4 to 6 holes, and at 37 ℃ and 5%CO ₂Under hatched 4 to 6 hours.At last, from each hole, take out the 35mL supernatant, and counting discharges to determine test on the gamma calculating instrument.From the hole of only accepting target cell and culture medium, obtain spontaneous release, from the hole of accepting 5%Triton X-100, always discharged.Calculate cytotoxicity percentage ratio with following formula: toxicity percentage ratio=100 * [(test release-spontaneous release)/(total release-spontaneous release)].

Dye to the detection of allogenic reaction T cell clone with IFN-γ in the cell: simply, the luxuriant and rich with fragrance moral bacterium A of cell and mine-laying (10mg/mL) is hatched 12 to 15 hours (for [TCD] that exhaust with the T cell, stimulate through the allogene of the stimulating type cell of irradiation), collect and washed cell, use the bonded antibody staining of first (surface) fluorogen (FITC, PerCP and APC) then, fix, also change cell thoroughly with Cytofix/Cytoperm test kit (Pharmingen), and at last with α IFN γ-PE dyeing.Carry out facs analysis by establishing door for the group who sets.Use as following mentioned flow cytometer and software.

Irritated the sending out of delayed should be detected: 42 days the time, inject 200 μ l, 0.01% sheep red blood cell (Colorado Serum, Denver, false castrating of PBS solution sensitization CO) and castrating mice through tail vein behind allogene BMT.In the time of the 46th day, attack the right back palmula of sensitized animal with 50 μ l, 20% sheep RBC suspension, simultaneously left back palmula accepted equal volume 50 μ l PBS solution in contrast.Quantifier (Mitutoyo, Kanagawa, Japan) the palmula swelling of mensuration after 24 hours and 48 hours with sign thickness.The measured value that palmula measured value by the test right side deducts the left side palmula of PBS injection determines the intensity of reaction.

Judgement to GVHD: with the severity of at first judging GVHD by the described clinical GVHD integrating system of Cooke et al. ((1996) Blood 88:3230-9).Simply, give 5 clinical parameters of animal individualization ground score (lose weight, attitude, vigor, fur and skin) of the ear's labelling in the coding cage weekly, score value from 0 to 2 minute.Integration (0-10) by whole 5 conditions that add up draws clinical GVHD index.Monitor survival rate every day.Integration is considered to dying more than or equal to 5 minutes animal, and is put to death by the mode with humanity.

The evaluation of the death that GVHD is caused to the evaluation of GVT-P815 (H-2d) mastocytoma inducing action and to mastocytoma death: B6D2F1/J receptor is at allogene HSCT (5 * 10 ⁶(TCD) BM cell and 5 * 10 that individual T cell is exhausted ⁵The T cell in individual C57/BL6 source) the 0th day intravenous accepted 1 * 10 ³Individual P815 (H-2d) cell.Detect survival rate every day, usefulness as discussed previously becomes celestial and determines the cause of death behind the HSCT.Simply, dying from leukemic feature is that hepatosplenomegaly and microscopy find to have the mastocyte oncocyte in liver and spleen, be defined as lacking leukaemia in hepatosplenomegaly and liver and the spleen and die from GVHD, and the clinical symptoms that when death, has the GVHD that clinical GVHD integrating system estimated.

Sxemiquantitative RT-PCR: with Superscript II reverse transcriptase (Life Technologies, Rockville, USA) total cell RNA of the full BM of reverse transcription.With PCR Master Mix (Promega, Madison, USA) 35 circulations of pcr amplification cDNA (94 ℃ 30 seconds; 56 ℃ 30 seconds; 72 ℃ 60 seconds).HPRT:5 ' CACAggACTAgAACACCTgC3 ' and gCTggTgAAAAggACCTCT3 '; TGF β 1:5 ' CTACTgCTTCAgCTCCACAg3 ' and 5 ' TgCACTTgCAggAgCgCAC3 '; KGF:5 ' gCCTTgTCACgACCTgTTTC3 ' and 5 ' AgTTCACACTCgTAgCCgTTTg 3 '.

To IL7-/-enzymic digestion of thymus: IL7-/-mice includes the CD45-thymus Interstitial cell of significant proportion, each thymus is all at 0.125% (w/v) collagenase/Bacillus polymyxa Neutral proteinase (Roche AppliedSciences, Indianapolis, USA) and in 0.1% (w/v) DNA enzyme accept enzymic digestion, from thymus, discharge most between matter and hematopoietic cell, to allow the cell density that accurately calculates thymus.Differentiate the CD45-Interstitial cell with anti-CD45 antibody.

Statistics: all data all are represented as meansigma methods ± SEM.Close check analysis existence data with Mantel-Cox logarithm order, with the every other statistical analysis of Mann-Whitney U check carrying out non-parametric, non-matching.It is significant that P value less than 0.05 is considered to statistics.

The result

I. castrating has increased the cell density of BM, thymus and spleen behind the allogene HSCT.At allogene HSCT castrating in preceding 1 day thymus CBA mouse.Mice accepted the 1300cGy total irradiation and subsequently 5 * 10 ⁶Individual B10.BR TCD BM cell.Early behind HSCT 14 days the time (Figure 29 A-B), with the vacation castrating compare that (proportion by subtraction is 9.5 * 10 ⁶± 3.0 * 10 ⁵With 25 * 10 ⁶± 2.6 * 10 ⁶), the BM (16 * 10 of castrating mice ⁶± 1.4 * 10 ⁶) and thymus interior (55.4 * 10 ⁶± 1.8 * 10 ⁶) significantly more cell arranged.28 days the time, these numbers still have significant raising (BM:22 * 10 in the castrating mice behind HSCT ⁶± 4.0 * 10 ⁶To 14 * 10 ⁶± 2.2 * 10 ⁶Thymus: 72 * 10 ⁶± 5.9 * 10 ⁶To 45 * 10 ⁶± 2.9 * 10 ⁶).In the time of the 28th day, the splenocyte density in the castrating mice also increases the splenocyte number (253 * 10 that has surpassed false castrating significantly ⁶± 28.4 * 10 ⁶To 126 * 10 ⁶± 13.9 * 10 ⁶) (Figure 29 C).During by 28 days, the castrating mice begins near the cell density before transplanting.When having arrived behind the HSCT 42 days, thymus and splenocyte density between castrating mice and the false castrating mice no longer include significant difference.Because vacation castrating receptor is a young mice, lymphocyte generated after they had active transplanting, but compared with the castrating receptor, prolonged the required time of normal cell density that generates elementary and secondary lymphoid tissue that reaches significantly.

II., the HSC that significantly more donor source is arranged among the BM of 28 days castrating mice behind HSCT.Some researchs have shown that sex steroid has suppressed propagation and/or differentiation (Thurmond et al., (2000) Endocrmol.141:2309-2318 of early stage hemopoietic forebody cell; Medina et al., (2001) Nat.Immunol.2:718; Kouro et al., (2001) Blood 97:2708).Therefore, inquired into the influence of castrating to the HSC number in the allotransplantation group.The HSC number in donor source all is low-downly (to be respectively 2.98 * 10 in 14 days vacation castrating and the castrating mice behind allogene HSCT ²± 1.25 * 10 ²With 2.66 * 10 ²± 8.8 * 10 ¹) (Figure 30 A).But, during by the 28th day, compare (1.1 * 10 with the vacation castrating ³± 4.1 * 10 ²), the castrating mice has the HSC (4.8 * 10 in significantly more Ly9.1+Lin-Sca-1+c-kit+ donor source ³± 1.1 * 10 ³) (Figure 30 A).

III. strengthened the recovery of the B cell in donor source prior to the castrating of allogene HSCT.In analysis, distinguish the cytocerastic three phases of B: pro B lymphocyte (CD45R+CD43+IgM-), pre B lymphocyte (CD45R+CD43-IgM-) and immature B cells (CD45R+CD43-IgM+) to the B cellular-restoring.Behind allogene HSCT 14 days the time, with vacation castrating contrast (2.08 * 10 ⁶± 5.0 * 10 ⁴) relatively, significantly more pre B lymphocyte (5.5 * 10 is arranged in the BM of castrating mice ⁶± 1.7 * 10 ⁶) (Figure 30 B).In the time of 28 days, significantly more pre B lymphocyte (sham-cx:3.1 * 10 are arranged also in the BM of castrating mice ⁶± 3.7 * 10 ⁵C.f.cx:6.6 * 10 ⁶± 6.6 * 10 ⁵) and immature B cells (sham-cx:1.3 * 10 ⁶± 2.6 * 10 ⁵C.f.cx:3.0 * 10 ⁶± 3.4 * 10 ⁵) (Figure 30 B).Behind HSCT 28 days the time, the increase of BM B cell and their CFU-GM is converted into remarkable increase (sham-cx:64.9 * 10 of immature B cells number of the spleen of castrating mice ⁶± 6.4 * 10 ⁵C.f.cx:112.0 * 10 ⁶± 10.0 * 10 ⁵) (Figure 30 C).These results and previous result coincide, and illustrates that the B cell that castrating has strengthened BM produces and exports.

IV. the T cell that strengthened behind the allogene HSCT of castrating is rebuild.According to the expression of CD3, CD4 and CD8, thymocyte cell and peripheral cells are divided into the stage of development: three feminine genders (TN) (CD3-CD4-CD8-), two positive (DP) (CD4+CD8+), single positive CD4 (SP CD4) CD3+CD4+CD8-and single positive CD8 (SP CD8) CD3+CD4-CD8+ (Figure 31 A-D).Early 14 days the time, compare behind the allogene HSCT, significantly more TN, DP, SP CD4 and SP CD8 thymocyte cell are arranged in the castrating mice with the vacation castrating.28 days the time, DP in the castrating group and CD4SP cell number still have significantly and improve behind HSCT.During by the 42nd day, false castrating is all identical with all thymocyte cell subgroups in the castrating mice.The DC in host and donor source is considered to avoiding bringing into play on the transplant rejection effect (Morelli et al., (2001) Semin.Immunol.13:323-335) completely.Behind allogene HSCT, 14 days the time, the CD11 in significantly more host source is arranged in the thymus of castrating mice ^ChIDC.28 days the time, the DC in intrathymic host of castrating mice and donor source has increases (Figure 31 E-F) significantly behind allogene HSCT.In the time of the 28th day, to compare with the vacation castrating, the increase of the thymocyte cell number in the castrating mice transforms for the ripe CD4+ in the source of the donor in the spleen of castrating mice and the remarkable increase (Figure 31 G) of CD8+T cell number.

V. with regard to each cell, there is not significant difference between the T cell of false castrating and castrating mice.Function potential for the periphery T cell of determining the castrating mice behind the allogene HSCT has carried out a series of vitro detection.Figure 32 A has showed the number of T cell in the donor source of the castrating mice in 6 weeks behind the allogene HSCT and false castrating contrast.Measured the multiplication capacity of splenic t-cell in two ways: α CD3/ α CD8 crosslinked (Figure 32 B) and 3 ^RdPart MLR (use through the irradiation the BALB/C splenocyte as stimulus object) (Figure 32 C).When relatively more false castrating and castrating mice in these two groups, on the multiplication capacity of periphery T cell, all there is not significant difference.6 weeks behind the allogene HSCT are with splenocyte and the BALB/C splenocyte (3 through shining ^RdPart) cultivated 5 days.After allogene stimulated 5 days, the most cells in the cultivation all were the CD8+T cells.The half of these cells be used to CTL ( ⁵¹Cr) detect to determine the false cytotoxicity of castrating and castrating the splenocyte of mice.Tested at different effectors: the splenocyte of target cell ratio kills and wounds and has ⁵¹The ability (Figure 32 D) of the A20 of Cr (BALB/C B cell lymphoma tumor cell line) cell.Aspect cytotoxicity, there is not significant difference between false castrating and the castrating mice.Again stimulate other half the cell of having cultivated 5 days to determine the output of IFN γ with 3rd part (BALB/C) or isogenic (B10.BR) through the splenocyte of irradiation and brefeldin A.Figure 32 E has shown the output of the IFN γ of CD8+T cell behind BALB/C stimulation originally and BALB/C or B 10.BR secondary stimulus (contrast) in donor source.Figure 32 F has diagrammatically showed this point.When relatively more false castrating and castrating mice, the ratio of the CD8+ cell in the donor source of product IFN γ does not have significant difference.

For the interior evaluating immunologic function, used the DTH detection, wherein behind castrating and allogene BMT, 42 days the time, use the sRBC sensitized mice.At the 46th day, attack mice and after 24 and 48 hours, measure palmula swelling.When simultaneously emasculated mice is compared with false castrating with allogene HSCT, strengthened significantly and attacked back 48 hours DTH and reply (Figure 32 G).

These functional detections confirm that with regard to each cell, castrating receptor's T cell is suitable with false castrating receptor's T cell, and can respond to new antigen, has complete propagation, cytotoxicity and production of cytokines.But, even after transplanting 6 weeks, the significantly faster T cell of castrating among the receptor rebuild and changed the enhancing of replying for DTH.

VI. the castrating prior to allogene HSCT does not increase the weight of GVHD and has kept the GVT activity.GVHD and GVT are that the T cell of mainly being originated by the allogenic reaction donor that allograft changed over to is mediated.Any treatment that is used for the enhance immunity reconstruction all might increase the weight of GVHD or weaken the GVT activity on the contrary.In order to set up castrating the allogenic reaction T cell in donor source there is not stimulation, by adding allogene donor T cell induction GVHD toward allograft in.When relatively mice is castrated in castrating and vacation, because sickness rate that GVHD caused or mortality rate all do not have significant difference (Figure 33 A).In order to estimate castrating, when transplanting, mastocyte tumor cell strain P815 (H-2d) is expelled among the B6D2F1/J receptor the active effect of GVT.Become celestial the animal of duration of test death, and determine dead reason (tumor is to GVHD).The mortality rate that mastocytoma caused changes (9 mice in 6) yet after castrating, when comparing with the vacation castrating (8 mice in 5).The GVT that this explanation castrating is not eliminated behind the HSCT replys (Figure 33 B).

VII. IL-7 and the castrating behind the allogene HSCT has accumulative action.Before shown that the IL-7 treatment can increase the T of the animal that does not have other treatment and the number of B cell, and recovery (Alpdogan et al., (2001) the Blood 98:2256 that also can strengthen the lymphatic system after cyclophosphamide, irradiation, the homogenic and allogene HSCT treatment; Bolotin et al., (1996) Blood 88:1887; Faltynek etal., (1992) J.Immunol.149:1276; Morrissey et al., (1991) J.Immunol.146:1547).Known IL-7 has increased the T cell number by increasing thymus activity and periphery.

Therefore, determine in the receptor of allogene HSCT, to unite castrating and grant IL-7.When treating back 14 days, in castrating mice and those are accepted the thymus of mice of therapeutic alliance (castrating and IL-7 administration), significantly more cell is arranged.On this early stage time point, in the castrating mice castrating contrast PBS treatment, false and IL-7 treatment, false, all do not see difference.In the castrating group with accept also not see significant difference between the mice of therapeutic alliance, illustrate in allogene HSCT, IL-7 treatment and when castrating back 14 days, this only is the effect (Figure 34 A) that castrating is caused.On this later time point of 28 days behind the allogene HSCT, the cell density of castrating group and single thymus with the IL-7 group all is higher than matched group significantly separately.When analysis in 28 days behind allogene HSCT, the therapeutic alliance of IL-7 treatment and castrating has accumulative action (Figure 34 B) to thymocyte cell density.

VIII. the sxemiquantitative RT-PCR of IL-7, TGF β 1 and KGF is found the increase of the KGF after allogene HSCT and the castrating and the minimizing of TGF β 1.The RT-PCR of full medullary cell found not have during 6 weeks evening the level of can detected IL-7 transcribing in vacation castrating and castrating mice behind allogene HSCT.When the template used from mice contrast, that do not transplant, detected IL-7 (data do not show).Known TGF β 1 and KGF are the crucial mediators of hemopoietic.2 whens week behind castrating and allogene BMT, the minimizing of use HPRT equibrated template for displaying TGF β 1 and the increase (Figure 34 C) of KGF.

IX. KGF-/-mice rather than IL-7-/-seen the variation that the castrating back is taken place in the mice.For research further be hidden in immunologic reconstitution back that castrating back institute strengthened may be machine-processed, castrated KGF-/-and IL-7-/-mice, 2 all post analysis thymus, spleen and BM (TGF β 1-/-mice is in preadolescence death, Shull et aL (1992) Nature 359:693).When the KGF-of relatively more false castrating and castrating/-during mice, increased the cell density of thymus significantly.Although the difference on total cell density of not seeing BM and spleen on this early stage time point seeing in the wild-type mice, has been seen variation (Ellis et al. (2001) Int.Immunol.13:553 as previous in the B of BM groups of cells; Data do not show).Because IL-/-major part of the intrathymic cell of mice all is the fact of CD45-Interstitial cell, when using these mices, has obtained cell suspending liquid with enzymic digestion.By carrying out enzymic digestion, more cell is released in the suspension, and this has caused the higher a little thymocyte cell density (vonFreeden-Jeffry et al. (1995) J.Exp.Med.181:1519) in the previous document of being seen in this test of ratio.When relatively castrating mice and false castrating contrast, IL-/-all do not see difference among thymus (Figure 34 G), spleen (Figure 34 H) or the BM (Figure 34 I) of mice.These find enhancing that explanation IL-7 is seen behind castrating and allogene HSCT immunologic reconstitution in important function.

Discuss

The receptor of allogene HSCT has experienced the immunodeficiency phase of segment length's time, and this is relevant with fatefulue infection.Along with the increase at receptor's age, the danger of this infection increases because completely immunology to rebuild the required time longer.The immunodeficiency phase behind the HSCT can surpass 1 year, and long term studies confirms that older HSCT patient has reduced TREC+CD4+T cell (Storek et al., (2001) Blood 98:3505 than their donor recently; Lewin etal., (2002) Blood 100:2235).The damage of this explanation thymus and the decline of T cell yield subsequently may be longer than what imagined.The infection behind the HSCT of the overwhelming majority and the shortage relevant (Storek etal., (1997) Am.J.Hematol.54:131) of periphery CD4+T cell.Therefore, the increase of the periphery T cell number that the castrating back is taken place may reduce these incidence of infection, causes the increase of total survival rate of transplant patient.

A plurality of groups all concentrate on the emphasis of their research on the hematopoietic reconstitution behind the HSCT, and most results likely are from the application of hemopoietic growth factor and cytokine.For example, G-CSF is used to mobilize donor stem cell (Dreger et al., (1993) Blood 81:1404).Demonstrations such as Noach have caused the enhancing ((2002) Blood 100:312) of donor's cells's implantation with the pretreatment of SCF and IL-1II or SCF and Flt-3 part.KGF shows implantation and the reconstruction that has strengthened homogenic and allogene group and has improved GVHD (Panoskaltsis-Mortari et al., (2000) Blood 96:4350; Kriianovski, et al., (1999) Blood 94:825).IL-7 has strengthened immunologic reconstitution (the Bolotin et al. behind the homogenic HSCT, (1996) Blood 88:1887), and the immunologic reconstitution that can strengthen behind the allogene HSCT does not increase the weight of GVHD (Alpdoganet al., (2001) Blood 98:2256) with keeping the GVT activity.

Some researchs have shown number (Ellise et al., (2001) Int.Immunol.13:553 of sex steroid removing the having increased BM and the spleen B cell of operation or the male mice that chemical castration caused; Erben et al., (2001) Hor7n.Metab.Res. (2001) 33:491; Wilson et al., (1995) Blood 85:1535; Masuzawa et al., (1994) J.Clin.Invest.94:1090).The increase of periphery B cell number mainly is because B220 ^LoCD24 ^HiRecently the BM increase (Ellis et al., (2001) Int.Immunol.13:553) of moving out.Olsen etc. have confirmed that androgen has strengthened the output of the TGF β of Interstitial cell in the BM, and this has suppressed B cell development (J.Clin.Invest. (2001) 108:697) conversely.In addition, the external neutralization to TGF β has reversed dihydrotestosterone to the B cell inhibiting.The IL-7 of matter produced and has suppressed subsequently the propagation (Tang et al., (1997) J.Immunol.159:117) of B cell precursor between TGF β had also demonstrated and reduced.Therefore, in this situation, a possible explanation of the effect that castrating/androgen is removed is it has suppressed TGF β behind allogene HSCT generation, has strengthened the B cell development conversely, has explained that the castrating mice has increased the B cell number of BM and spleen than false castrating contrast.

TGF β has also regulated the propagation of hematopoietic stem cell.Batard etc. have confirmed that the TGF β of physiological concentration has suppressed in-vitro multiplication and the differentiation ((2000) J.Cell.Sci.113:383-90) of HSC.In addition, the survival and the propagation (Fan et al., (2002) J.Immunol.168:755-62) of these cells the blocking-up of the TGF signal beta approach of HSC (through the temporary transient expression of the II receptor of sudden change) have been strengthened.Therefore, the increase of allogene HSCT and the back HSC number of being seen in 28 days of castrating may be because the minimizing of the TGF β output of BM Interstitial cell is possible.

Estrogen and androgen can both influence differentiation and propagation (Thurmond et al., Endocrinol., (2000) 141:2309-18 of HSC; Medina et al., (2001) Nat.Immunol.2:718-24; Kouro et al., (2001) Blood 97:2708-15).Estrogen has directly suppressed propagation and differentiation (Medina et al., (2001) Nat.Immunol.2:718 of HSC and some lymph CFU-GM subgroups; Kouro et al. (2001) Blood 97:2708).Estrogen receptor of HSC expressive function (ER) and estrogen administration have reduced number (Thurmond et al., (2000) Endocrinol.141:2309 of Lin-c-kit+Sca-1+HSC; Kouro et al., Blood (2001) 97:2708).The research explanation that Thurmond etc. carried out has been blocked the conversion between c-kit+Sca-1+ precursor and the more sophisticated subgroup (c-kit+Sca-1-and c-kit-Sca-1-) when having ERoc in the hematopoietic cell of BM (Endocrinol. (2000) 141:2309).ER also exist with the BM Interstitial cell in (Girasole et al., (1992) J.Clin.Invest.89:883; Smithson et al., (1995) J.Immunol.155:3409), illustrating that estrogen also may produce somatomedin to Interstitial cell effect is arranged, this has influenced HSC propagation and/or differentiation conversely.Although the indirect action of most evidence explanation estrogen confrontation HSC through between BM has functional estrogen receptor and does not get rid of directly effect in the lymphatic system of BM.

Olsen etc. display functionality androgen receptor are present on the thymus epithelial rather than on the thymocyte cell, this receptor all is crucial (Olsen et al., (2001) Endocrinol.142:1278) for relevant involution of thymus of age and the regeneration of removing through sex steroid subsequently.

Although involution of thymus and/or regenerated molecular mechanism are still unclear, some possible candidate mechanism are arranged here.Thymus IL-7 level lowers (Aspinall, et al., (2000) Vaccine 18:1629 along with the age; Andrew et al., (2002) Exp.Gerontol.37:455; Ortman et al., (2002) Int.Immunol.14:813).It is still unclear that whether this is to lower because the minimizing of the cell number of product IL-7 or existing cell produce the ability of cytokine.But, can reverse the increase of relevant thymus apoptosis of age and strengthen hemoposieis (Andrew et al., (2001) J.Immunol.166:1524) the IL-7 treatment of Aged Mice.Stem cell factor of mouse thymus (SCF) and M-CSFmRNA express and also reduce (Andrew et al., (2002) Exp.Gerontol.37:455) along with the age.In intracellular signal pathway level, reduced transcription factor E2A to DN thymus development key, also reduced and be present on the thymic epithelial cell, and related to the propagation of this cell and the transcription factor Foxnl of growth (whn) (Ortman et al., (2002) Unit.Immunol.14:813).Sempowski etc. have monitored old people's mRNA steady-state level, and demonstrate the remarkable increase (J.Immunol. (2000) 164:2180) of leukaemia inhibitory factor (LIF), oncostatin M, IL-6 and SCF mRNA.

Above-mentioned research has illustrated that to replying of castrating be multifactorial.Castrating IL-/-increase that the test of mice explanation IL-7 produces is the important component part of castrating effect.But, when with heavy dose of IL-7 and castrating treatment mice, observed accumulative action to thymocyte cell density, this explanation castrating provides than increasing the effect that the more thymus of IL-7 level generates separately.

DC is intrathymic negative crucial vehicle (Jenkinson et al., (1985) Transplantat.39:331 that selects; Matzinger et al., (1989) Nature 338:74-60), and in temporary transient the setting, be involved in allogene antigen and removal donor specific induced t cell graft acceptance by presenting thymus after transplanting.Tomita has confirmed the receptor's of MHC I type mispairing the cell-mediated removal (Tomita et al., (1994) J.Immunol.153:1087-1098) of the reactive cell of donor in donor source of thymus.They show that also the DC in the thymus source of intravenous injection has been transported in host's thymus.In addition, shown that the inner injection of thymus host cell adds that heterogenic antigen, donor's cells or donor soluble peptide have all increased graft acceptance (GalTovillo et al., (1999) Transplantation68:1827; Ali et al., (2000) Transplantation 69:221; Garrovillo et al., (2001) Am.J.Transplant.1:129; Oluwole et al., (1993) Transplantation 56:1523-1527).These results show that castrating has increased the number of the DC in the host of the thymus behind the allogene HSCT and donor source significantly.That is to say that sex steroid is removed differentiation and/or the propagation that has strengthened thymus DC.Therefore, the castrating with hematopoietic stem cell and solid organ transplantation use in conjunction has increased the graft acceptance.

Conclusion

Current research have been found that the barrier steroid is removed bone marrow and HSCT after immunologic reconstitution have significant positive acting.HSC and B and T CFU-GM are strengthened significantly.Strategy after this provides the important platform of an efficient that is used to increase implantation and has depended on the transplanting of complete hemopoietic system, for example the vaccination of antitumor or microbial antigen or targeting are in the gene therapy of donor HSCT.The increase of DC is important to inducing and keeping the antigenic toleration of allogene in the thymus.In addition, in the castrating receptor, do not increase the weight of GVHD and elimination GVT activity.These results confirm that the prophylactic treatment that temporary transient sex steroid removing (for example using the LHRH analog) is rebuild as enhance immunity is useful.

Embodiment 20: sex steroid is removed and has been strengthened the TCR differential stimulus after the hematopoietic stem cell transplantation

In order to estimate the functional characteristic of regenerated T cell, analyzed the responsiveness of patient to the TCR differential stimulus.

Material and method

The patient: give test patient Zoladex (LHRH-A) before stem cell transplantation 3 weeks, injection in every month is 1 time 4 totally months then.Before treatment, transplant the back in 5 weeks weekly with analyzed all patients every month up to 12 months.Obtain ethics permission (test number 01/006) from Alfred human research Ethics Committee.

The preparation of PBMC: the lymphocyte of purification is used for detection of T cytositimulation and TREC analysis, and as above preparation.

The T LS detects: unless other explanation is arranged, utilized anti-CD3 and the crosslinked analysis of carrying out the TCR differential stimulus of anti-CD28 in 1-12 month from transplanting the back.For the TCR differential stimulus, incubated cell is 48 hours on the plate of using anti-CD3 of purification (1-10 μ g/ml) and anti-CD28 (10 μ g/ml) coating in advance.(48-72 hour) adds 1 μ Ci's in each hole after forming plaque ³The H-thymidine, and plate hatched 16-24 hour again.Plate collected on the bag filter and (Packard-Coulter USA) goes up and determines with liquid scintigraphy at the β calculating instrument ³The degree of mixing of H-thymidine.

The I.LHRH-A administration has strengthened the responsiveness to the TCR differential stimulus behind the allogeneic stem cell transplantation.The patient of LHRH-A treatment compare according to the patient all time points all show the propagation that has strengthened reply (as ³The H-thymidine mixes to be estimated), except at 6th month and 9 months, because patient's number that analyzed this moment is few; (Figure 57 A-B).In with the allotransplantation patient that LHRH-A treated, after transplanting 4 and the responsiveness that stimulates of the antagonism CD3/CD28 that observed comparison 5 months the time and significantly increased according to the patient.Though control patients is after transplanting 6 and demonstrated replying of having strengthened in 9 months, and the patient of LHRH-A treatment demonstrated stronger responsiveness in back 12 months in transplanting.After transplanting 6 and 9 months, control patients have and treatment before the similar responsiveness of numerical value.But on all other times points, they are significantly lower.On the contrary, the patient of LHRH-A treatment on all time points, all have with treat before identical responsiveness, except transplanting back 6 months.The patient of LHRH-A treatment after transplanting 1,3 and demonstrated in 4 months the responsiveness that comparison strengthened according to the patient (as ³The H-thymidine mixes to be estimated).This has pointed out the direct acting result of periphery T cell, because up to transplanting the back 1-2 month, new CD4+T cell is still unconspicuous (Figure 57 A-B).

The II.LHRH-A administration has strengthened the responsiveness to the TCR differential stimulus behind the autologous stem cell transplantation.Also observed similar the replying of in the allotransplantation receptor, being seen (Figure 57 C) with autoplastic receptor.Those patients with LHRH-A treatment have confirmed after transplanting 4 and had the propagation that TCR is stimulated that has strengthened in 9 months and reply.The patient of LHRH-A treatment all time points all demonstrate comparison according to the stronger propagation of patient reply (as ³The H-thymidine mixes to be estimated), except transplanting back 5 months.In transplanting back 12 months, in contrast and LHRH-A treatment patient, all observe and returned to the preceding numerical value of treatment.

The III.LHRH-A administration has strengthened the responsiveness of the TCR differential stimulus behind the chronic cancer patient treatment.

In this research, be selected in and suffered from chronic hematologic malignancies and for the immunosuppressant patient, its immune state is determined by following: have that data confirms with CD4＜0.4 * 10 ⁹The severe infections that/L is relevant; Or lymphocytic hyperplasia disease (for example, CLL, myeloma, lymphoma) and the conventional preventative intravenous immunoglobulin (the low gamma globulinemia that has data to confirm) of acceptance; Or the treatment of fludarabine, deoxycorfomycin and 2-CdA arranged in previous 4 years; Or in previous 2 years allogeneic stem cell transplantation or autologous stem cell transplantation are arranged.Obtain ethics permission (test number 01/006) from Alfred human research Ethics Committee.Followingly give the patient LHRH-A, show until the result in 6 weeks after the LHRH-A administration.

D-1 (or before): signature informed consent, and treat preceding investigation;

D0: grant Zoladex (male 10.8mg, women 3.6mg);

D+28: women-grant Zoladex 3.6mg

D+56: women-grant Zoladex 3.6mg

D+84: women and male-grant Zoladex 3.6mg

After the administration the 7th day, with anti-CD3 and the crosslinked analysis of carrying out the TCR differential stimulus of anti-CD28.With level before the treatment relatively, the patient of LHRH-A treatment demonstrate the enhancing that the propagation of " circulation " mode replys (as ³The H-thymidine mixes to be estimated) (Figure 63).That is to say that in the increase of just observing the T cell proliferation same day of injection, and this propagation shows as slight minimizing in next time before the injection.This also illustrates the probability of LHRH-A to the direct influence of existing periphery T cell.This has reflected with the administration that stored the agonist of remover liquid injection in every month.These results suggest to the direct influence of periphery T cell.But, in the back enhancing of being seen in 12 months of treatment the variation of replying the T cell that has reflected the thymus source because all patients have just stopped the administration of agonist from 4 months.

Conclusion

Because early just observed the responsiveness to the TCR differential stimulus that increases on the 35th day to transplanting the back, this mainly is because of existing T cell, because the T cell in new source only just begins to leave thymus in this stage.Only just observing the influence of the T cell that the thymus in new source generates behind this time point, is because the raising of existing T cell rather than derive from the T cell of regeneration thymus so the conclusion that can draw is the T cell function that increased.

Embodiment 21: sex steroid is removed and has been strengthened the mitogenesis stimulation after the hematopoietic stem cell transplantation

In order to estimate the functional characteristic of regeneration T cell, analyzed patient's responsiveness to the mitogenesis stimulation.

Material and method

The patient: the patient for as above be selected in those patients of No.01/006 clinical trial protocol.Before stem cell transplantation, give patient LHRH-A (before 3 weeks).The patient who does not accept agonist is used as control patients.

The preparation of PBMC: the lymphocyte of purification is used for detection of T cytositimulation and TREC analysis, and as above preparation.

Mitogen stimulate to detect: from transplanting the back 1-12 month, carry out analysis to the mitogenesis responsiveness with pokeweed mitogen (PWM) (PWM) and tetanus toxin (TT).Stimulate for mitogen, with PBMC with in the 100 μ l RPMI-FCS 1 * 10 ⁵The density of individual cells/well is tiled in the 96 hole circle base plates.With cell and TT (2LFAU/ml) or PWM (10 μ g/ml) at 37 ℃, 5%CO ₂Under hatch.After plaque forms (48-72 hour), in each hole, add 1 μ Ci's ³The H-thymidine, and plate hatched 16-24 hour again.Plate collected on the bag filter and (Packard-Coulter USA) goes up and determines with liquid scintigraphy at the β calculating instrument ³The degree of mixing of H-thymidine.

The I.LHRH-A administration has strengthened the responsiveness that behind the allogeneic stem cell transplantation mitogenesis is stimulated.The analysis of mitogenesis responsiveness is shown that the allogene patient that carries out the LHRH-A treatment compare photograph and has the responsiveness to PWM (Figure 58 A) that has strengthened on time points all after the transplanting.That is to say, on all time points, all demonstrate the responsiveness that PWM is stimulated that comparison has strengthened according to the patient with the patient of LHRH treatment prior to stem cell transplantation.

After the responsiveness of analyzing TT, similar result is conspicuous (Figure 58 B).The patient of LHRH-A treatment compare the photograph patient and has the responsiveness that has strengthened on all time points, except in transplanting back 12 months.

The II.LHRH-A administration has strengthened the responsiveness that behind the autologous stem cell transplantation mitogenesis is stimulated.On most of time points of being studied, all demonstrate the responsiveness that PWM is stimulated that comparison strengthened according to the patient (3 months time P≤0.001) (Figure 59 A) prior to stem cell transplantation with the patient of LHRH-A treatment.Arrived when transplanting back 12 months, the patient that LHRH-A treated has returned to responsiveness the preceding level of treatment, and control patients is still suitable attenuating.

Similar to above-mentioned allogene patient (Figure 58 B), when giving LHRH-1 before treatment, the autotransplantation patient also demonstrates the replying TT that has strengthened.On most of time points of being studied, all demonstrate the responsiveness (Figure 59 B) that TT is stimulated that comparison has strengthened according to the patient prior to stem cell transplantation with the patient of LHRH-A treatment.Arrived when transplanting back 12 months, the patient that LHRH-A treated has returned to responsiveness the preceding level of treatment, and control patients is still suitable attenuating.

Conclusion

Because morning, this mainly was because of existing T cell, because the T cell in new source only just begins to leave thymus in this stage to transplanting the responsiveness to the mitogenesis stimulation that the back was just observed on the 35th day to be increased.Only behind this time point, just observe the influence of the T cell that the thymus in new source generates.

Embodiment 22: sex steroid is removed the speed that has strengthened the implantation among the hematopoietic stem cell transplantation patient

Material and method:

The material and the method that are used for these tests have been described above.Additional material and method are as follows:

Allogene and total WBC and total granulocyte or neutrophilic granulocyte number behind the HSCT of body patient (contrast or LHRH-A treatment) have been analyzed.3 weeks were treated the patient with LHRH-A before HSCT.The patient who does not accept agonist is the patient in contrast.Determine total leukocyte (WBC) counting, granulocyte (G) or the neutrophilic granulocyte counting of every μ l blood, until transplanted back 35 days.Or repeat twice with Cell-Dyn 1200 automatic cytological calculating instruments (Abbott) or hematimeter and analyze the full periphery blood specimen.This allows full leukocyte, lymphocyte and the granulocyte number that calculates after the transplanting.From transplanting the analysis of carrying out implanting in back the 14th to 35 day.

The result

I. the autologous stem cell transplantation patient who carries out LHRH-A treatment prior to transplanting has strengthened the speed of implanting.After transplanting the 14th, 28 and 35 day, determine total leukocyte (WBC) counting and the granulocyte (G) of every μ l blood and count.Shown in Figure 60 A-D, that accepts LHRH-A treatment demonstrated comparison on the 14th day according to remarkable higher WBC number (Figure 60 B) (P≤0.05) from the body patient after transplanting, wherein on this time point, compare with 45% of contrast, 87% demonstrates granulocyte implants (〉=500 cells/μ l blood) (P≤0.05).Accept LHRH-A treatment from the body patient after transplanting, also demonstrated in 10-12 days comparison according to the remarkable neutrophilic granulocyte of higher number (Figure 60 C, data are represented as meansigma methods ± 1SEM of 8-20 example patient, ^*P≤0.05).In addition, although there is not significant difference, what LHRH-A treated all has higher lymphocyte count (Figure 60 D) than matched group from the body patient at whole time point.The lymphocyte level of this explanation LHRH-A treatment having increased significantly after the stem cell transplantation.

II. the allogeneic stem cell transplantation patient who carries out LHRH-A treatment prior to transplanting has strengthened the speed of implanting.Shown in Figure 61 A, C and D, the allogene patient who accepts LHRH-A treatment demonstrated comparison on the 14th day according to remarkable higher WBC number (Figure 61 A) (P≤0.05) after transplanting, wherein on this time point, compare with 44% of contrast, 64% demonstrates granulocyte implants (〉=500 cells/μ l blood).In addition, accept LHRH-A treatment from the body patient after transplanting, also demonstrated in the 9th, 12 and 19 day comparison according to the remarkable neutrophilic granulocyte of higher number (Figure 61 C, data are represented as meansigma methods ± 1SEM of 8-20 example patient, ^*P≤0.05).In addition, the patient's that carries out autologous peripheral blood stemcell transplant analysis is confirmed when treating with LHRH-A that the remarkable increase (transplant afterwards the 10th, 12,13 and P≤0.05 17-21 days the time) (Figure 61 D) of lymphocyte count is arranged before allotransplantation.

Conclusion

In allogene and autoplastic model, the 14th day WBC and granulocyte number will be higher than contrast (Figure 60 and 61) significantly all to have observed LHRH-A treatment patient after transplanting.The speed of this implantation that has strengthened is important for the patient's of the agranulocytosis (≤200 neutrophilic granulocytes/ml blood) that has the indication infection rate to increase general mortality rate.Therefore, the early recovery of WBC and granulocyte number has shown LHRH-A treatment patient's better survival rate.The inhibition of sex steroid has been strengthened complete thymus reactivate or implantation and reconstruction before the new T cell that caused of thymus reactivate discharges fully.

Embodiment 23: the sex steroid removing has increased the T cell proliferation and has replied in 1 week.

Whether carry out these researchs removes early to the castrating back just to strengthen to breed in 3 to 7 days with the sex steroid of determining mice and replys.

Material and method

8 week of castrating big mices, and the T cell proliferation of analyzing the anti-CD28 of anti-CD3/ after the operation 3 days (Figure 62 A, C and E) and 7 days (Figure 62 B, D and F) and being stimulated is replied.Stimulate and stimulated periphery (cervical region, axillary fossa, humerus and groin) lymph node (Figure 62 A and B), mesenteric lymph node (Figure 62 C and D) and splenocyte (Figure 62 E and F) altogether 48 hours with the anti-CD3 of variable concentrations with the anti-CD28 of constant density 10 μ g/ml.Use tritiated thymidine active cell 18 hours then, and be that 3H-T mixes proliferation assay.Control mice is false castrating, n=4, ^*P≤0.05 (non-parametric, non-matching, Mann-Whitney statistical test).

The result

The 3rd day (Figure 62 A, C and E) and 7 days (Figure 62 B, D and F) just strengthened the propagation of the T cell of CD28/CD3 stimulation after the mice that sex steroid is removed was presented at and castrates.The T cell that the 3rd day (the anti-CD3 of 10 μ g/ml) and 7 days (2.5 μ g/ml and the anti-CD3 of 1.25 μ g/ml) is separated to from periphery LN after castrating demonstrates the remarkable increase that propagation is replied.

In addition, the T cell that was separated to from mesenteric lymph node (Figure 62 C and D) and spleen (Figure 62 E and F) in the 3rd day after the castrating remarkable increase that also demonstrates the propagation that anti-CD3 stimulates has surpassed false castrating mice.

Conclusion

Early, the increase of the responsiveness of T cell antagonism CD3 and the crosslinked stimulation of CD28 is just arranged to 3-7 days (the moving out from thymus) in castrating back prior to new T cell.These Notes of Key Datas are before the thymus reactivate, and sex steroid is removed has direct effect to peripheral blood T cell pool functional.

In order to determine the direct acting degree of LHRH-A, study the periphery T cell of human patients.The T cell of control patients is hatched with the LHRH-A of various dosage, and be compared to the propagation level of contrast specimen (only using culture medium) different time point (D3, D7 and D14) analysis.This allows to determine whether LHRH-A directly acts on these cells by the activation that causes existing T cell, as viewed in the mouse model.

Embodiment 24: the hemoposieis behind sex steroid removing enhancing homology HSCT

The result:

I. castrating has strengthened the implantation of the BM behind the HSCT, thymus and spleen.Homology HSCT castrating in preceding 1 day mice.With 5 * 10 ⁶The intravenous injection of individual Ly5.1+BM cell is in the C57/BL6 mice of irradiation (800Rad).Different time points after transplanting (2-6 week) is analyzed BM, spleen and thymus (Figure 35).Shown in Figure 35 B, in 2 weeks behind castrating and HSCT, when comparing, significantly more cell is arranged in the BM of castrating mice with the vacation castrating.Similarly, shown in Figure 35 C, in 2,4 and 6 weeks after transplanting, the castrating mice has the thymocyte cell number of remarkable increase than false castrating contrast.Shown in Figure 35 C, in periphery, the splenocyte number in castrating receptor's 4 and 6 weeks after transplanting also is higher than contrast significantly.

II. castrating has strengthened the implantation of HSC in BM behind the homology HSCT.Homology HSCT castrating in preceding 1 day mice.With 5 * 10 ⁶The intravenous injection of individual Ly5.1+BM cell is in the C57/BL6 mice of irradiation (800Rad).When transplanting for 2 weeks, with the lin-c-kit+sca-1+HSC (Figure 36) of flow cytometry BM.Transplant and castrating 2 weeks of back at BMT, the HSC in significantly more donor source is arranged in the false castrating contrast of the BM internal ratio of castrating mice.

III. castrating has strengthened homology HSCT (2.5 * 10 ⁶) after the implantation of HSC in BM.Homology HSCT castrating in preceding 1 day mice.With 2.5 * 10 ⁶The intravenous injection of individual Ly5.1+BM cell is in the C57/BL6 mice of irradiation (800Rad).When transplanting for 2 weeks, with the lin-c-kit+sca-1+HSC (Figure 37 A-B) of flow cytometry BM.Figure 37 A has described the percentage ratio of the common lymph CFU-GM in the BM.Figure 37 B has described the number of the common lymph CFU-GM in the BM.BMT transplants and castrating 2 weeks of back, and the false castrating contrast of the BM internal ratio of castrating mice has increased the ratio of the HSC in donor source significantly.

IV. castrating has strengthened homology HSCT (5 * 10 ⁶) after the implantation of HSC in BM.Homology HSCT castrating in preceding 1 day mice.With 5 * 10 ⁶The intravenous injection of individual Ly5.1+BM cell is in the C57/BL6 mice of irradiation (800Rad).When transplanting for 2 weeks, with the lin-c-kit+sca-1+HSC (Figure 37 C-D) of flow cytometry BM.Figure 80 A has described the percentage ratio of the common lymph CFU-GM in the BM.Figure 37 D has described the number of the common lymph CFU-GM in the BM.BMT transplants and castrating 2 weeks of back, and the false castrating contrast of the BM internal ratio of castrating mice has increased the ratio of the HSC in donor source significantly.

V. castrating has strengthened the speed of the DC in the donor source behind the homology HSCT in intrathymic implantation.With 5 * 10 ⁶The intravenous injection of individual Ly5.1+BM cell is in the C57/BL6 mice of irradiation (800Rad).When transplanting for 2 weeks, use the flow cytometry thymocyte cell.The DC in donor source be defined as CD45.1+CD11c+MHC I type+CD11b+ or-.2 whens week behind BMT, compare with the vacation castrating, increased the CD11b+ and the CD11b-DC (Figure 38) in the intrathymic donor source of castrating mice significantly.

VI. castrating has strengthened the speed of the implantation of B cell in spleen in the donor source behind the homology HSCT.With 5 * 10 ⁶The intravenous injection of individual Ly5.1+BM cell is in the C57/BL6 mice of irradiation (800Rad).When transplanting for 2 weeks, use the flow cytometry splenocyte.2 whens week behind homology BMT, compare with the vacation castrating, significantly more B220+B cell (Figure 39) is arranged in the spleen of castrating mice.

Embodiment 25: in having reduced after the chemotherapy with G-CSF and/or GM-CSF to cancer patient's treatment The sickness rate of agranulocytosis

This embodiment for example understands the usage that G-CSF and/or GM-CSF are used to increase the neutrophilic granulocyte level and reduce the incidence of infection of the patient who accepts chemotherapy.

Material and method

In 100 routine small cell lung cancer patients, carried out a double blinding, placebo-controlled study.

The G-CSF administration: according to product description, 4-17 days subcutaneous Neupogen_ that grant 4-8 μ g/kg/d dosage after the chemotherapy (Amgen, Thousand Oaks, CA).

GM-CSF administration: in first research, according to product description, the patient is at the 6mg Neulasta_ (Amgen that accepted single SC dosage on the 2nd day of each chemotherapy cycles, ThousandOaks, CA) begin to accept 5 subcutaneous μ g/kg/d Filgrastim_ (Amgen or from the 2nd day of each cycle, Thousand Oaks, CA).In second research, the object random packet is accepted as accepted single subcutaneous injection 100 μ g/kg Neulasta_ at the 2nd day or begin to accept 5 μ g/kg/d Filgrastim_ from the 2nd day of each chemotherapy cycles.

The patient: all patients have been diagnosed as small cell lung cancer and have given the treatment of cyclophosphamide, amycin and the etoposide of normal period.GM-CSF patient is diagnosed as metastatic breast cancer.

The result

The treatment of G-CSF by this way (Neupogen_) has caused as heat generation neutrophilic granulocyte deficiency disease, infection rate, the patient is admitted to hospital and antibiotic uses the clinical of determined the incidence of infection and statistics ground significantly to reduce.The application of the III clinical trial phase report G-CSF that a plurality of the said firms (www.neupogen.com) are reported has caused the increase of measuring of neutrophilic granulocyte, supports the clinical practice of G-CSF treatment in the cancer patient who accepts the inhibitive ability of immunity chemotherapy in view of the above.

Seen similar result in the randomized, double-blind ACTIVE CONTROL research that utilizes GM-CSF (Neulasta_), this research has been adopted in the treatment of metastatic breast cancer and had been given 60mg/m in per 21 days ²Amycin and 175mg/m ²Paclitaxel, the scheme in totally 4 cycles (www.neulasta.com).

The persistent period of selecting the neutrophilic granulocyte deficiency disease is as main terminal point (obviously having obtained the FDA permission).The patient who does not accept Neulasta_ has the sickness rate of 100% serious neutrophilic granulocyte deficiency disease, and average duration is 5-7 days, and the sickness rate of 30-40% heat generation neutrophilic granulocyte deficiency disease.

In two researchs, all used Neulasta_ (c.f.Neupogen_ wherein granted medicine at the 4th day) to the patient at the 2nd day.

Two researchs (for Neulasta_, a research is fixed dosage, and another is the dosage that weight is adjusted) confirm that all every kind of medicine does not have difference on the main terminal point of the average natural law of serious neutrophilic granulocyte deficiency disease.In two kinds of situations, average natural law all approaches 1.7 days (the untreated 5-7 of c.f. days).The sickness rate of the heat generation neutrophilic granulocyte deficiency disease in two research is suitable, all approaches 10-20%.

Embodiment 26: remove treatment and G-CSF (and/or GM-CSF) to the cancer patient's with sex steroid Treatment has reduced the sickness rate and the infection rate of the neutrophilic granulocyte deficiency disease after the chemotherapy

In a double-blind study at random, 80 routine small cell lung cancer patients by random packet to accept G-CSF (Neupogen_) or accept G-CSF and the 1-4 of Lupron_ group in; Preceding 4 day every day of chemotherapy cycles and all patients that monitored in all groups in per three days after this.Utilize all patients' of technical monitoring well known in the art CBC, neutrophilic granulocyte counting, packed cell volume and T cell divide typing.In addition, body temperature that the patient is arranged and other side effect are searched in twice monitoring every day.

Group #1: only use G-CSF

First group comprises one group at cyclophosphamide (1g/m ²/ sky), amycin (50mg/m ²/ day) and etoposide (120mg/m ²/ day x3) accepted the patient (n=20) of G-CSF after the standard chemotherapy in 4-17 days.According to administration in the related packaging description and dosage explanation, intravenous (I.V.) is granted all medicines.All patients accept this therapeutic scheme in 21 day cycle.In this group, the subcutaneous G-CSF that gives 5 μ g/kg/d dosage of dosage instructions that sets according to the package insert (range of options is 0-10 μ g/kg/d) of Neupogen_.Randomly give subsequently chemotherapy cycles according to persistent period of ANC minimum and severity.According to oncology's technology of routine, if ANC increases towards 10 000/mm ³, the treatment clinicist also can randomly reduce used dosage, reaches 10 at ANC, 000/mm ³The time G-CSF that stops using.

Group #2:G-CSF adds " heavy dose " Lupron_ that chemotherapy is preceding 21 days

Second group comprise one group chemotherapy gave in preceding 21 days " heavy dose " Lupron_ (3 months constant release products, dosage 22.5mg, S.C.) and also according to aforementioned schemes from cyclophosphamide (1g/m ²/ sky), amycin (50mg/m ²/ day) and etoposide (120mg/m ²/ day x3) gave G-CSF (5 μ g/kg/d) patient (n=20) of (range of options is 0-10 μ g/kg/d) in 4-17 days after the chemotherapy.All patients accept this therapeutic scheme in 21 day cycle.Randomly give subsequently chemotherapy cycles according to persistent period of ANC minimum and severity.According to oncology's technology of routine, if ANC increases towards 10 000/mm ³, the treatment clinicist also can randomly reduce used dosage, reaches 10 at ANC, 000/mm ³The time G-CSF that stops using.Grant all medicines according to administration in the corresponding package description and dosage explanation.

Group #3:G-CSF adds " low dose " Lupron_ that chemotherapy is preceding 21 days

The 3rd group comprises that one group gave " low dose " Lupron_ in preceding 21 days in chemotherapy (3 months constant release products, dosage 11.25mg is S.C.) and from cyclophosphamide (1g/m ²/ sky), amycin (50mg/m ²/ day) and etoposide (120mg/m ²/ day x3) gave G-CSF the patient (n=20) of (5 μ g/kg/d) in 4-17 days after the chemotherapy.All patients accept this therapeutic scheme in 21 day cycle.Randomly give subsequently chemotherapy cycles according to persistent period of ANC minimum and severity.According to oncology's technology of routine, if ANC increases towards 10 000/mm ³, the treatment clinicist also can randomly reduce used dosage, reaches 10 at ANC, 000/mm ³The time G-CSF that stops using.Grant chemotherapeutics, G-CSF and Lupron_ according to the package insert of these medicines.

Group #4:G-CSF adds " heavy dose " Lupron_ that chemotherapy is preceding 14 days

The 4th group comprises that one group gave " heavy dose " Lupron_ in preceding 14 days (patient S.C.) (n=20) is from cyclophosphamide (1g/m for 3 months constant release products, dosage 22.5mg in the beginning chemotherapy ²/ sky), amycin (50mg/m ²/ day) and etoposide (120mg/m ²/ day x3) gave these patient G-CSF (5 μ g/kg/d) in 4-17 days after the chemotherapy.All patients accept this therapeutic scheme in 21 day cycle.Randomly give subsequently chemotherapy cycles according to persistent period of ANC minimum and severity.According to oncology's technology of routine, if ANC increases towards 10 000/mm ³, the treatment clinicist also can randomly reduce used dosage, reaches 10 at ANC, 000/mm ³The time G-CSF that stops using.Grant chemotherapeutics, G-CSF and Lupron_ according to the package insert of these medicines.

The result

Expection is when with G-CSF treatment group (group 1) relatively the time, the treatment of G-CSF and " heavy dose " Lupron_ (group 2) will cause as infection rate, antibiotic use, WBC counting, serious neutrophilic granulocyte shortage (ANC＜500/mm ³) and the clinical and statistics of the incidence of infection of being confirmed of heat generation neutrophilic granulocyte deficiency disease reduce significantly.Expect that also the treatment (group 3) of G-CSF and " low dose " Lupron_ will produce the result who does not have significant difference with the result who treats the patient of (group 2) from more heavy dose of Lupron_.At last, be expected at chemotherapy and gave Lupron_ constant release in 3 months, 22.5mg S.C. dosage will produce does not have significant difference with group those patients' of 2 result data in preceding the 14th day.But it may be useful for some patients that expection has a plurality of data point explanations to grant Lupron_ in the earlier stage of treatment cycle.

Embodiment 27: remove the treatment for the treatment of the bone marrow transplantation patient with sex steroid and reduced the WBC meter The incidence rate of number, serious neutrophilic granulocyte deficiency disease, heat generation neutrophilic granulocyte deficiency disease and infection

In a non-at random blind research, 20 routine patients had given the treatment of GnRH analog before accepting BMT.This patient group of also carrying out BMT but not accepting the GnRH analog is matched group (n=19).

According to the Medical Technology of approval, all patients have accepted the allograft (Lincz et al., (2001) Leuk.and Lymph.40:373) from the coupling donor.

All patients in the treatment group all accepted 4 every month dosage the GnRH analog (Lupron_7.5mg, S.C.).Granted Lupron_ in preceding 21 days at BMT at first.

Before transplanting, the BM (Segerenet al., (1999) .Br.J.Haem.105:127-130) that removes all patients with the removing medicine and the technology of standard.

Extracted each patient's blood sample at the-21 ,-2,0,1,2,3,7,10,14 and 21 days.Utilize then other local methods described in detail of this description and the known method of those skilled in the art estimate blood sample WBC, neutrophilic granulocyte, T cell (through facs analysis, as top described in detail), granulocyte level and hematocrit value.

In addition, monitor all patients' infection rate (comprising viral infection), antibiotic use, heat generation neutrophilic granulocyte deficiency disease and serious neutrophilic granulocyte deficiency disease (ANC＜500/mm ³) average natural law.These are analyzed as top listed carrying out, from 1 month, 3 months, 6 months of transplanting that the date begins and 9 months with examining the patient.

The result

Expection is until after transplanting the 21st day, and when with no GnRH agonist treatment group relatively the time, the treatment of GnRH agonist (Lupron_) will cause WBC counting, serious neutrophilic granulocyte deficiency disease (ANC＜500/mm ³) average natural law and the clinical and statistics of heat generation neutrophilic granulocyte deficiency disease reduce significantly.

Also expection is because patient's quantity is few, and up to 1 month, the virus between two groups and the sickness rate of other infection rates will not have statistically-significant difference.But, being expected at and transplanting back 3,6 and 9 months, these infection rates between two groups will have significant difference, and wherein the patient of GnRH agonist treatment will treat better (that is, easier processing and recover faster).

Embodiment 28: with sex steroid remove treatment to cancer patient's treatment increased hemoposieis and in The property granulocyte count

In a non-at random blind research, the male patient of metastatic prostate cancer (n=30) has accepted GnRH agonist (Lupron_, injection (7.5mg s.c. every month once)) in their normal therapeutic process.All patients have also accepted the androgen antagonist medicine.Also can give Cosudex_ (5mg/ days or 50mg/ days oral) 1 month.

All patients carry out whole blood (FBA) before begin treatment.FB comprises the pathological analysis to the standard of lymphocyte, leukocyte, hematocrit value and erythrocyte content.In addition, carried out the analysis of amynologic parameter is comprised that T cytositimulation, cytokine generation, immunocyte subgroup and TREC analyze.

In addition, at the-2,0,1,2,3,7,10,14,21 and 28 days and after this blood sample that extracted each patient every month totally in 6 months.Utilize then other local methods described in detail of this description and the known method of those skilled in the art estimate blood sample WBC, neutrophilic granulocyte, T cell (through facs analysis, as top described in detail), granulocyte level and hematocrit value.

Be expected in initial 14 days of GnRH agonist treatment, when with baseline values (the-2 and 0 days) relatively the time, Most patients (nearly 70%) all will have the increase of hemoposieis and neutrophilic granulocyte counting.

Embodiment 29: sex steroid is removed therapy has increased influenza vaccinations to patient's treatment curative effect

In a non-at random blind research, the male patient of metastatic prostate cancer (n=45) has accepted GnRH agonist (Lupron Depot_, injection (7.5mg s.c. every month once)) in their normal therapeutic process.All patients have also accepted the androgen antagonist medicine.Also can give Cosudex_ (5mg/ days or 50mg/ days oral) 1 month.

All patients carry out whole blood (FBA) before begin treatment.

The patient who accepts GnRH agonist and Cosudex_ is by following random packet wherein a group in three groups of every group 15 routine patient:

Group #1:

The 1st group comprise 15 examples according to product description accepted at the 0th day influenza vaccinations (for example, Fluarix_ (GlaxoSmithKline, Australia), 0.5ml, patient i.m).

Group #2:

The 2nd group comprise 15 examples according to product description accepted at the 21st day influenza vaccinations (for example, Fluarix_ (GlaxoSmithKline, Australia), 0.5ml, patient i.m).

Group #3:

The 3rd group comprise 15 examples according to product description accepted in the 8th week influenza vaccinations (for example, Fluarix_ (GlaxoSmithKline, Australia), 0.5ml, patient i.m).

Group #4: contrast:

The 4th group is matched group, comprises that 15 examples do not have prostatosis not accept additional group of male at the similar age of medicine.Matched group according to product description accepted at the 0th day influenza vaccinations (for example, Fluarix_ (GlaxoSmithKline, Australia), 0.5ml, i.m).

In whole research (the-2,0,1,2,3,5,7,14,21,28 days and after this 6 totally months every month), utilization other local methods described in detail of this description and the known method of those skilled in the art estimate blood sample WBC, neutrophilic granulocyte, T cell (through facs analysis, as top described in detail), granulocyte level and hematocrit value.At each time point, also utilize nasopharynx part swab and FLU OIA (Thermo BioStar) monitoring patient whether to have influenza (A and Type B) virus.

Whether all time points after the-2 days and influenza immunity inoculation also utilize the patient in every group of the technical monitoring well known in the art to exist the blood coagulation of influenza H1N1 strain to suppress antibody.

The result:

When having 1 routine swab influenza virus positive relatively the time with the control patients of group in 4, being expected in arbitrary group of group 1-3 does not all have a routine patient to report the infectious formula part that any influenza is arranged.

Expect that also the patient in all groups will have than hemagglutination inhibition antibody titre higher before the GnRH agonist treatment.Patient in the expection group 2 and 3 has produced higher antibody titer than the patient in those groups 1 and 4.But, expect that since all patients will have similar antibody titer in 12 weeks.

In addition, expection protective rate (influenza H1N1 strain is had the blood clotting mortifier greater than 40% patient) is the highest in group 2, and is minimum in group 4.

Serum testosterone has been exhausted in embodiment 30:LHRH-A treatment effectively

Material and method

Utilize ^125I-testosterone radioimmunoassay (RIA) carries out the detection to the sex steroid level among the patients serum.Before detecting, all reagent, specimen and contrast all are placed on room temperature.Control tube or have independent buffer-non-specific bond (NSB) to manage or 0ng/ml standard testosterone (B0) arranged.Independent buffer, titer (0-10ng/ml testosterone) or test specimen are joined in each pipe, and adding property haptoglobin inhibitor (SBGI) is to limit the non-specific binding of radiolabeled testosterone subsequently.In each pipe, add ¹²⁵The I-testosterone adds anti-testosterone antibody (except the NSB pipe) subsequently.Then pipe was hatched 2 hours under 37 ℃.Afterwards, add second antibody in all pipes, vortex was also hatched 60 minutes again.To manage centrifugal (1000g _Max) 15 minutes, remove supernatant, on the automatic beta calculating instrument of Packard Cobra, count precipitate.Average three cpm results also use in conjunction with testosterone percentage ratio (B/B ₀) formula construct standard curve:

%B/B ₀=(specimen-NSB)/B ₀-NSB

The average cpm of specimen=fc-specific test FC specimen

The average cpm of NSB=non-specific binding pipe

B ₀The average cpm of=0ng/ml titer (total binding pipe)

From standard curve, determine the testosterone levels of each test specimen.

The result

Grant the serum testosterone that LHRH-A has caused the castrating level to patients with prostate cancer.In order to determine the effectiveness of LHRH-A treatment, before treatment and LHRH-A treatment in the time of 4 months, analyzed all patients' level of serum testosterone.Utilize ¹²⁵The radioimmunoassay of I-testosterone (RIA) is analyzed.Before hormone therapy, the concentration of serum testosterone is at the 1-3ng/ml testosterone (within the scope of meansigma methods=2.3ng/ml) (Figure 46 A).In treatment 4 months the time, the patient does not have the serum testosterone that can be measured to, the successful removing that prompting discharges sex steroid basically.

The LHRH-A administration does not influence the percentage ratio of the lymphocyte subgroup in the peripheral blood.After 4 months, do not observing any variation of the ratio of any lymphocyte subgroup with the LHRH-A treatment with numeric ratio before the treatment.These numerical value are in normal range (data do not show) all.With facs analysis the ratio and the cell number of (NK and macrophage) in the T of peripheral blood lymphocyte, B and medullary system source.After the LHRH-A administration, do not observe any variation of the ratio of any cell subgroup.In addition, the ratio of all lymphocyte subgroups (Figure 84 C) (Hannet et al., 1992 in the normal range of whole age group all; Xu et al., 1993).

Embodiment 31: laggard to the temporary transient removing of sex steroid with goserelin acetate (Zoladex_) Row is done thin dried flank meat transplant patient's thymus reactivate from body or allogene

In this embodiment, giving goserelin acetate (Zoladex_) before from body or allogeneic peripheral blood stem cell transplantation (PBSCT).Main terminal point is the thymus reactivate of measuring as vitro detection.The patient after transplanting with examining 6 months.20 routine patients (10 routine allotransplantations and 10 routine autotransplantations) will enter research.This embodiment has inquired into the effect that produces at the horizontal inhibition steroid of LHRH, utilizes the agonist desensitization hypophysis of LHRH and has therefore stoped the release of LH and FSH.Conversely, this has caused the androgen of gonad and the blocking-up of estrogen production, and this has just removed the inhibitory action to thymus function.The group that is detected in this test is the patient who carries out heavy dose of chemoradiotherapy (HDT) and PBSCT.

Goserelin acetate (Zoladex_) is effective, the synthetic decapeptide analog of a kind of LHRH.When acute administration, goserelin acetate will discharge LH from hypophysis.But behind chronic administration, goserelin acetate is the effective inhibitors that a kind of short gonadal hormone hormone produces, and has caused the gonad inhibition and has finally caused sexual organ's degeneration.At animal and human's apoplexy due to endogenous wind, after the of short duration raising to the initial stimulation of hypophysis, LH secretion and serum testosterone, chronic administration has caused the inhibition to gonadotrophin secretion.The result is that lasting inhibition and the level of serum testosterone among the male to the hypophysis LH that occur within nearly 3 weeks of initial treatment have reduced to as the normal scope of seeing in operation castrating male.Keep the same long time of this inhibition then with continued treatment.

The patient is a sex, and the age is more than or equal to 18 years old, because of heavy dose of treatment (HDT) and PBSCT have been carried out in malignant disease or BM depletion.10.8mg the implant dosage form of Zoladex_ (male with) is dispersed in the cylindrical bar of poly-acetic acid lactic polyester of biodegradable and bio-compatible, continues to discharge behind subcutaneous injection to surpass for 12 weeks.3.6mg implant (women with) is dispersed in the cylindrical bar of poly-acetic acid lactic polyester of biodegradable and bio-compatible and continues to discharge above 28 days behind subcutaneous injection.Implant is commercial, places to have the giving in the sample device of 14-16 syringe needle with the design of special-purpose purpose.

Sex steroid in the blood reduces to minima and spends several weeks possibly.Therefore, preceding 21 days of PBSC infusion (the 0th day), remove treatment for the sex steroid of patient infusion LHRH agonist Zoladex_ (implant) form.For the male, gave the 10.8mg goserelin (effective 3 months) of single dose at the-21 days and injected 3.6mg (effective 28 days) at the 63rd day once more.For the women, granted 3.6mg (effective 28 days) at the-21 days, the 7th, 35 and 63 day.This should be effectively (expectation behind PBSCT 4 months) for reducing the sex steroid level to the level that is enough to reactivate thymus.Therefore, in this embodiment, only give 4-6 month treatment.Those skilled in the art determine easily can received other dosage.

At the 0th day infusion PBPC.The thymus of reactivate absorbs the precursor of institute's infusion and they is converted into new T lymphocyte and epithelium thymocyte cell.Maximum sex steroid " removing " is in the PBSCT infusion, so the PBSC of institute's infusion can help thymus to rebuild.3-4 is in week behind PBSCT in expectation, and initial new T cell will appear in the blood flow, but treatment should be kept 3 months to allow immune complete normalization behind PBSCT.

Generation by flow cytometry, external t cell response and TREC is determined thymus function to the evaluation of T cell.

Before beginning one's study, carried out the preceding investigation of conventional HDT, FBE, electrolyte and the LFT on record basis.Analyze before the other treatment and comprise serum HCG (women), thymus CT, Study of bone mineral density, protein electrophoresis and immunoelectrophoresis, hormone study: TFT, FSH, LH, estrogen, progestogen, testosterone.In addition, carried out the T cell detection of various baselines.From 50ml blood, be purified into leukocyte, and following detection:

(a) flow cytometry

Originally the T cell is to memory T cell

CD27-FITC, CD45RA-PE, CD45RO-PerCP, CD4-or CD8-APC

CD27-FITC、CD45RO-PerCP、CD4/CD8-APC、Ki-67-PE

CD62L、CD45RO-PerCP、CD103、CD4/CD8-APC

T cell subgroup

CD4-FTTC、CD8-APC、αβTCR-PE、γδTCR-B/S-PerCP

CD25-PE、CD69-CyChrome、CD4-FITC、CD8-APC

CD28-CyChrome、αβTCR-PE、CD4-FITC、CD8-APC

B cell/myeloid cell

CD19-FITC、CD3-PerCP、CD56-PE、CD34-APC

CD11b-CyChrome、CD11c-PE、CD4-FITC、CD8-FITC

Cytokine

IL-4-PE、IFNγ-APC、CD4-FITC、CD8

Other labels

CD11a、CD95、HLA-DR、CD2、CD5

All patients are as internal reference because before treatment and after all check them.Dyeing specificity contrast will comprise the isoreagent contrast of FITC/PE/APC etc., and block FcR before dyeing.

(b) The T cell function

With the vitro examination blood lymphocyte to the crosslinked ability of replying of CD3.

(c) TREC analyzes

Separation is also detected the existence of the TXi Baoshouti deletion ring of T cell originally, and it is because above-mentioned tcr gene is reset formed.Their existence is the very strong prompting (because this is unique source of the T cell generation of main flow) of thymus output.Because the thymus development after the rearrangement of cell division and tcr gene is relevant, the TREC level may have been underestimated the moving out of thymus (practical level about 10%).

Embodiment 32: to the treatment of pernicious anemia

An adult female patient (for example, 35 years old) suffers from pernicious anemia, a kind of autoimmune disease.After 3 days, her CD34+ hematopoietic stem cell (HSC) is collected the blood from her in G-CSF treatment (every day twice, totally 3 days, each 10 μ g/kg).Can from blood, be purified into her HSC with CD34.In order to collect the CD34+ cell, collect the peripheral blood of donor (being about to provide the people of his/her organ or skin) to the receptor, from peripheral blood, isolate the CD34+ cell according to standard method.Nonrestrictive method with peripheral blood and the antibody that is incorporated into people CD34 specifically (for example is, available from Abcam Ltd., Cambridge, mouse monoclonal antihuman CD 34+antibody of UK) hatch, with anti-mouse antibodies (for example anti-mouse antibodies of FITC labelled goat) the secondary staining cell of detectable labelling, and isolate the CD34+ cell of FITC labelling through fluorescent activation cell sorting art (FACS).Because have only the CD34+ cell of minority in the circulation peripheral blood, may need donor is carried out collection and cell sorting to this.CD34+ can be frozen preservation, up to the amount of collecting abundant use.

Because the antigen of pernicious anemia, transfection patient's the HSC that is gathered is to express this antigen (being called the stomach proton pump) in any way.By utilize various technology including but not limited to electroporation, viral vector, based on laser the pressure wave technology, lipid infusion (seeing, for example the method described in Bonyhadi etc. 1997) can transfection HSC.In an example, with her HSC of C1 chain transfection of H/K-ATP enzyme proton pump, II type MHC promoter is used for expressing.

For the autoimmune disease in stopping making progress, the patient will carry out that the T cell is exhausted and/or other immunocytes are exhausted.She also will carry out the thymus reactivate replacing these T cells, and therefore overcome immune deficiency state.In order to reach this point, the injection that she will accept 4 mensal Lupron (7.5mg) is allowed the reactivate of her thymus in view of the above to exhaust sex steroid (to 3 weeks time).This will allow that also picked-up HSC also sets up the maincenter tolerance to relevant autoantigen.Though why not clear autoimmune disease can take place, be a kind of probability to the cross reaction of microorganism; Therefore exhaust that all T cells will remove the cell of these cross reactions.Start if disease is these cross reactions, may there is no need so with nominal autoantigen transfection HSC.Exhaust the T cell simply, and then the thymus reactivate by barrier steroid signal pathway may be enough.A kind of method of standard that is used to remove the T cell is as follows.The patient has accepted to inject every day 15mg/kg Atgam (the anti-T cytoglobin of xenogenesis, Pharmacia Upjohn) anti-T-cell antibody of form is totally 10 days, and the inhibitor cyclosporin A of uniting a kind of T cell activation, 3mg/kg continuous infusion 3-4 week, be subsequently as required every day 9mg/kg tablet.This treatment does not influence the intrathymic earlier T cell development of patient, can not carry the amount with so necessary antibody of effect because of the size of people's thymus and configuration.Keeping this to treat nearly 4-6 week rebuilds with the disappearance of admissibility steroid and thymus subsequently.Also can unite in the secondary signal level inhibitor of interleukin, accessory molecule (blocker, for example CD28), signal pathway molecule or cell adhesion molecule for example the prevention of t cell responses, the T cell is removed or other immunocytes are exhausted to strengthen.

Embodiment 33: to type i diabetes patient's treatment

The type i diabetes patient has accepted and the similar methods described in the embodiment 32.Remove T cell (on seeing) with broad-spectrum exhaustion method, 4 months Lupron treatment promotion thymus rejuvenation and the proinsulin gene institute transfection through using II type MHC promoter of gathering in advance by injection strengthened the patient's immune system recovery from body HSC.HSC will enter thymus, be divided into DC (with all thymocyte cells), and proinsulin is pass developmental T cell.Apoptosis will kill all those may with the T cell of proinsulin reaction, only stay the T cell bank of attacking external infectious pathogen.

For with the cross reaction of infecting or " mishap " caused autoimmune disease just, using from body HSC is enough to help to strengthen the thymus reactivate.If have the genetic predisposition (family member often suffers from autoimmune disease) to disease, the most handy heterogenic highly purified HSC carries out thymus and recovers, with the graft versus host disease of prevention via the T cell of passing by on one's way (passenger T cell).Cord blood also is a kind of good HSC source, does not have or have only very small amount of allogenic reaction T cell usually.Although umbilical blood does not have high-caliber CD34+HSC, they for set up microchimera be enough-even 10% hemocyte finally just can be enough to set up in the abundant thymus dendritic cell to the toleration of self antigen.

Embodiment 34: to suffering from patient's hypersensitive treatment

For allergy, can adopt the principle similar with 33 to embodiment 32.As above exhaust autopath's T cell.In the serious case that is increased the weight of by the B cell (plasma cell) that produces IgE or IgG, it may be necessary utilizing the bone marrow removing of chemotherapy.Perhaps, can use total irradiation (for example 6Gy).GnRH by using 3-4 month and intravenous injection HSC (by be required to be heterogenic or from the HSC of body) with the whole immune system of rejuvenation.For situation with hereditism's susceptibility hypersensitive, can use heterogenic, otherwise just use mobilized from body HSC.

Embodiment 35: castrating is to the effect of NOD and NZB mice

Non-obese diabetes mice (NOD mice) is that type i diabetes a kind of has distinctive model very much.Big quantity research has confirmed that the pathology of this disease is because destroy to the unusual T cellular infiltration of pancreas with to the islet cells autoimmunity of insulin-producing.The structure of the thymus in these animals is unusual, ectopic expression, a large amount of B cell folliculus of medullary epithelial cell (mAb MTS 10 is identified) is arranged here and lack epithelial existence of being rich in the thymocyte cell zone.

For of the influence of inspectability steroid to these mices, with 20 3 week big female NOD mice excision ovaries, and 20 carried out sham-operation.Selecting this stage is because of its generation early than disease.Since 10 weeks monitoring blood glucose greatly.To 21 weeks when big, surpass 60% vacation castrating mice and developed into diabetes, but＜20% castrating group mice suffers from diabetes.After operation castrating, the normalization of thymus defective is also arranged, comprise the increase that cortex that boundary is clear and definite and medullary substance, the follicular disappearance of B cell and CD25+ regulate cell.The increase of regulating the T cell is very important, because they can change the amount of the pathogenic cytokine of the thymocyte cell of moving out.Therefore, castrating all has remarkable influence to the generation and the progress of the diabetes of NOD mice.

Checked the diabetes of NOD mice of 16 OO and 16 sham-operations and the generation of the insulitis (blood sugar level that has improved; BGL) 21 weeks.In becoming celestial, also checked the appearance that thymus structure is unusual.As shown in figure 45, wherein the NOD mice of 60% sham-operation suffers from diabetes, is less than 20% castrating group mice and suffers from diabetes.This has clearly illustrated and has delayed or even prevented diabetes.

Shown in Figure 64, castrating NOD mice has the remarkable increase of total thymocyte cell number, but does not have difference on total splenocyte.In the castrating mice of diabetes, the remarkable minimizing of total thymocyte cell number is arranged, they may make these mices easily suffer from this disease and diabetes triggering thing had appearred in prompting before castrating.

The remarkable increase (Figure 66 A) of all thymocyte cell hypotypes is arranged, but their ratio does not change (data do not show) here.What is interesting is that compare with vacation castrating mice (Figure 66 C), total T of B cell and spleen or B cell all do not have to change (Figure 66 B).

Increasing parallel with the total thymocyte cell in castrating back is that CD25+ regulates the obvious increase (data do not show) of cell.In spleen, there is not such variation.

Also checked the effect of castrating to the NZB mice, it is the model of a kind of systemic lupus erythematosus (sle) (SLE).The NZB mice has tangible thymus abnormalities, and it appears at disease and takes place to be closely related before and with disease.These defectives comprise indefinite cortex-medullary substance boundary and unusual B cell cluster (seeing Takeoka et al., (1999) Clin.Immunol.90:388).

In 4-7 week when big with mice castrating or false castrating, and 4 week the back check.

The obvious increase of total thymocyte cell (Figure 67 A) and splenocyte (Figure 67 B) is arranged.Also there is thymus to regulate the obvious increase of cell (CD25+ and NKT cell).May influence effector T cell and regulate and control that they are possible pathogenic from the cytokines of these mices.By immuno-chemical method, the castrating mice has normal thymus structure and the follicular disappearance of B cell (data do not show).

Embodiment 36: castrating is to the effect of the immunity inoculation of tumor specific antigen

Human papillomavirus (HPV) infects and causes the reproductive tract herpes, and this may cause some women's cervical cancer.In fact, all contain HPV DNA above in all cervical cancers of 90%.Papillomavirus is the double-stranded DNA virus of skin infection and mucomembranous surface.So far 80 polytype HPV have been identified.HPV16 is wherein a kind of main type relevant with cervical cancer.

E7 is the main carcinogenic protein relevant with the inductive cervical cancer of HPV16.Other groups have shown that the expression of the E7 open reading frame with activation ras is enough to the elementary epithelial cell in the culture is converted into malignant phenotype (Lin et al. (1996) Cancer Res.56:21).Therefore, E7 is a kind of attracting tumor specific antigen that is used for the immunization therapy and/or the vaccination of cervical cancer and precancerous lesion.In fact; other groups shown with the E7-GST fusion rotein of 50 μ g/ml optimal doses and Qiul A as the protected property of adjuvant institute mice immunized resisted with the secondary of the tumor cell line of HPV16E7 institute transfection and attacked (Fernando et al., (1999) Clin.Exp.Immunol.115:397).

Carry out this test to determine whether can strengthen the curative effect of vaccination (as preventative vaccine) and/or the immunization therapy (as therapeutic vaccine) of the HPVE7 of suboptimal dosage to the castrating of mice.

Material and method

Adult (＞9 months) C57BL/6 mice has been accepted subcutaneous injection and has derived from the male homology E7+TC1 tumor cell of elementary epithelial E7 through the C57BL/6 mice of HPV-16E6 and E7 and c-Ha-ras oncogene cotransformation.After 5 days, the operation castrating mice as above-mentioned through scrotal incision, appears testis,, comes along with the periphery fatty tissue then and removes its ligation with suture.Order with operation then and close wound.By top method but do not take out the false castrating of testis preparation mice, and with it as negative control.Behind tumor challenge 7 days, give the E7GST fusion rotein of castrating or false castrating injected in mice suboptimal dosage (5 μ g/ml).Positive control has been accepted the E7GST fusion rotein of optimal dose 50 μ g/ml.After 25 days, tumor (perusal and tumor weight) and the t cell response of analyzing mice (utilize the ELIspot method of standard to analyze INF γ output and the target cell warp that utilizes HPV16E7 to stimulate as mentioned above, ⁵¹Cr discharges check and analysis CTL lytic activity).

The result

Shown in Figure 68, with 47% (9/19) comparing of being occurred in the suboptimal dosage contrast of vacation castrating, only 22% (4/18) the castrating mice of having accepted suboptimal dosage E7GST has tumor.Ratio (78%) that it should be noted that the shielded castrating mice of accepting suboptimal dosage E7GST vaccine is suitable with the ratio of shielded positive control, and positive control has accepted to exceed the vaccine (3/4,75%) of 10 multiple doses (the best).All castrating mices of not accepting vaccine all have tumor.

Shown in Figure 69, to compare with other all groups, the castrating mice of accepting suboptimal dosage E7GST has significantly higher cell number (Figure 69 A) (P 〉=0.01) at Intradermal.But, to compare with other all groups of being tested, activation (CD25+) CD4+ (Figure 69 B) in the spleen of the suboptimal E7GST group of castrating and the total number of CD8+ (Figure 50 C) T cell have kept identical number.

Shown in Figure 70 A, the positive control group that accepts the non-specific IFN γ output of splenocyte of castrating mice of suboptimal dosage GST-E7 and the vaccine that acceptance exceeds 10 multiple doses (the best) is suitable.The vacation castrating mice of accepting suboptimal dosage vaccine has the IFN γ output of medium level, and wherein the negative control animal has seldom to the non-specific IFN γ product that does not have splenocyte.These data and parallel (the seeing Figure 68) seen in tumor incidence illustrate that the IFN γ output of splenocyte is directly parallel with the tumor incidence of these animals.This is N/R, supposes that replying that the Th1 cell mediated is mainly to act on tumor protection.

Shown in Figure 70 B, the level that the E7 specificity produces is being followed the above-mentioned identical trend of discussing about non-specific IFN γ generation.Though accept the level that level that the E7 specificity IFN γ of the castrating mice of suboptimal GST-E7 dosage produces will be lower than the positive control mice of the vaccine of accepting to exceed 10 times of (the best) dosage slightly, it still exceeds viewed level in accepting the vacation castrating mice of suboptimal dosage.Again, the negative control animal has only seldom to the IFN γ that does not have E7 specificity splenocyte to produce.

At last, analyzed the E7 specific CTL lethal effect of mice to the target cell that HPV16E7 infected.Shown in Figure 71 A, when the positive control mice of vaccine that exceeds 10 times of (the best) dosage with acceptance relatively the time, the castrating mice of accepting suboptimal E7GST dosage has the quite E7 specific CTL of level.The vacation castrating mice of accepting suboptimal dosage vaccine has the E7 specific CTL of medium level replys, and wherein negative control has only seldom to there not being the E7 specific CTL.These data are parallel with the data of being seen in tumor incidence (seeing Figure 49) and IFN γ output (seeing Figure 70).Figure 71 B shows that the E7 specific CTL is active antiparallel with tumor weight.That is to say that the active mice of CTL with top level does not have tumor, have more weak E7 specific CTL lytic activity and there is the minority mice of tumor to demonstrate.

Conclusion

These tests show that castrating can increase patient's vaccine curative effect effectively.When with the castrating mice of accepting suboptimal dosage vaccine relatively the time, do not realized the IFN γ output that equates in the mice castrating of the HPV16-E7 vaccine of accepting to exceed 10 times of (the best) dosage, CTL is active and to the protection of tumor challenge.

Can carry out similar test and add that to determine castrating E7 vaccination is in the curative effect aspect the vaccination of immunization therapy.In this case, will be as above-mentioned castrating mice.In 6 whens week, given injected in mice TCl cell after castrating, and 1 week back GSTE7 vaccine immune mouse.Tumor (perusal and tumor weight) and the t cell response of analyzing mice then (utilize the ELIspot method of standard to analyze INF γ output and utilize the target cell warp that is added with HPV16E7 as mentioned above, ⁵¹Cr discharges check and analysis CTL lytic activity).The mice that this therapeutic vaccine vaccination regimen is accepted in expection will have than vacation castrating contrast tumor tumor still less.In addition, expection castrating mice will have more low dose of vaccine than the copy of vacation castrating.

Embodiment 37: other viral vaccine vaccination strategies

By the inhibitor of using the sex steroid signal pathway for example GnRH analog (or other castrating method) can improve the curative effect of the viral vaccine of various this areas approval with method of the present invention before, afterwards or simultaneously.Except the top embodiment that provides, the embodiment of nonrestrictive viral vaccine is as follows:

I. Hepatitis B virus vaccine

An example of viral vaccine and recombinant DNA vaccine is the Hepatitis B virus vaccine that Glaxo Smith Kline (EngerixB_) and Merck Sharpe and Dohme (HBVaxII_) develop respectively.Engerix B_ bacterin preparation is the dosage of every 1ml 20 μ g, presses the product description administration.The HBVaxII_ bacterin preparation is the dosage of 1ml 10 μ g/ml, presses the product description administration.Also can obtain the various department of pediatrics dosage forms of these and other vaccines.These vaccines can maybe cannot contain for example thimerosal (Australian Immunization Handbook, 8th edition) of antiseptic.Vaccine dose is normally being granted 0.5ml in the scope of 1ml through intramuscular injection.The general course of treatment of vaccination can be different, but generally include single, initial immunization, are the nearly reinforced immunological inoculation more than 1 month of at least one minor tick afterwards.

II. Hepatitis A virus vaccine

The example of inactivated virus vaccine is those Aventis Pasteur (Avaxim_), Glaxo SmithKline (Havrix 14400 and Havrix Junior_) and other vaccines of developing that is used for the treatment of hepatitis A.For Avaxim_, every 0.5ml dosage contains hepatitis A (GBM strain) virus antigen of 160ELISA unit.For Havrix 1440_, every 0.5ml dosage contains the hepatitis A virus (HM 175 strains) of 1440ELISA unit.The vaccine dose of these univalent vaccines be through the 0.5ml of IM injection in the scope of 1ml.The general course of treatment of vaccination can be different, but are usually included in three times interior at interval vaccinations in 6 months.

III. hepatitis A and hepatitis B polyvalent vaccine

Perhaps, can use polyvalent vaccine, it contains and surpasses a kind of virus antigen.For example, the Twinrix_ of GlaxoSmith Kline contains the hepatitis A viral antigen and the 20 μ g recombinant DNA hepatitis B B surface antigen proteins of 720ELISA unit, 0,3 and 6 months the time through administered intramuscular.Another kind of polyvalent vaccine is the Vivaxim_ of Aventis Pasteur.Every 1ml dosage contains the deactivation hepatitis A viral antigen of 160ELISA unit and the typhoid fever capsular polysaccharide of 25 μ g purification, provides through dual chamber syringe, and muscle is granted this polyvalent vaccine of 2 or 3 dosage.

IV. cytomegalovirus vaccine

Vical company has developed the vaccine based on DNA of the immunotherapeutical of a kind of anti-CMV.By what provided in the product description, by 1 or three dosage of 5mg grant vaccine.Plasmid-encoded CMV phosphoprotein 65 of DNA (pp65) and Glycoprotein B (gB), and make vaccine with Poloxamer.

The V.EBV vaccine

Medlmmune, GlaxoSmithKline and Henogen have developed the solubility reorganization subunit vaccine of a kind of anti-EBV jointly.Express EBVgp220/350, and shown the protective effect of having given primates and the negative baby of EBV with the recombinant vaccine vector of living.In addition, carried out clinical trial (for the summary of this viral vaccine and other vaccines to the EBNA-3A peptide in Australia, see for example THEJORDAN REPORT 2000:ACCELERATEDDEVELOPMENT OF VACCINES, published by the Division ofMicrobiology and Infectious Diseases, National Institute of Allergy andInfectious Diseases, National Institutes of Health (can Http:// www.niaid.nih.gov/publications/pdf/Jordan.pdf-last visited Apr.2004Obtain)).

Embodiment 38: other cancer vaccine strategy

By the inhibitor of using the sex steroid signal pathway for example GnRH analog (or other castrating method) can improve the curative effect of the cancer vaccine of various this areas approval with method of the present invention before, afterwards or simultaneously.Except the top embodiment that provides, the embodiment of nonrestrictive cancer vaccine is as follows:

I. Melacine

By for example GnRH analog (or other castrating method) before, afterwards or simultaneously can be with the curative effect of method raising Melacine of the present invention at the inhibitor of using the sex steroid signal pathway.An example of such Melacine be AVAX company (Philadelphia, PA) developed from the autologous melanoma cell vaccine.The excision metastatic carcinoma remains on 4 ℃, and is transported to laboratory in the 48h of excision.By the enzyme separation and Extraction tumor cell of collagenase and DNA enzyme, packing and in the household freezer of controlled speed culture medium freezing and that contain human albumin and 10% dimethyl sulfoxide be stored in together in the liquid nitrogen when needs.On treatment patient's the same day, melt a part of cell, washing is also shone through 2500cGy.Wash tumor cell then, and hatched 30 minutes, use salt water washing (Miller et al. (1976) J.Immunol 117:1519) then with dinitrofluorobenzene.After washing, counting cells also is resuspended in the Hanks solution that 0.2ml contains human albumin, and remain in 4 ℃ up to administration.

Vaccine dose is in 0.5-25.0 * 10 ⁶In the scope of individual cell.Before injecting, added in the vaccine in the past 0.1ml BCG (Tice, Organon Teknika, Durham, N.C.).Can progressively weaken BCG dosage, comprise the focal reaction of the inflammatory pimple of no ulcer with generation.The mixture intradermal injection is arrived one or more positions, for example upper arm.(for example every month once 12 totally months, or 6-12 week altogether) once in a week carry out a plurality of dosage vaccinations in the different time periods, and can comprise and grant low dosage (300mg/m ²) cyclophosphamide, when when the suitable time relevant with immunity inoculation gives, cyclophosphamide can increase cell-mediated immunity paradoxically.

II. lung cancer vaccine

By for example GnRH analog (or other castrating method) before, afterwards or simultaneously also can be with the curative effect of method raising lung cancer vaccine of the present invention at the inhibitor of using the sex steroid signal pathway.A dna vaccination that example is the nonsmall-cell lung cancer of Corixa company of such lung cancer vaccine.Vaccine is a kind of recombinant DNA vaccine of granting with the recombinant adenovirus adjuvant.(Bioject, Tualatin OR) are used to vaccine administration to ABiojector_ equipment, and it is the needleless injector of a kind of reusable compression CO2 tube energy supply.

Operable other the nonrestrictive lung cancer vaccines of method of the present invention comprise L523S (Wang et al. (2003) Br.J.Cancer 88:887) that is used for nonsmall-cell lung cancer and BEC-2, GM2, Globo H, fucosyl GM1 and the poly-silicic acid (Krug (2004) Sem.Oncol.31:112) that is used for small cell lung cancer.

III. carcinoma of prostate

By for example GnRH analog (or other castrating method) before, afterwards or simultaneously also can be with the curative effect of method raising carcinoma of prostate vaccine of the present invention at the inhibitor of using the sex steroid signal pathway.A non-limiting instance of a kind of vaccine like this is Provenge_ (Dendreon Corp.), and it is a kind of vaccine that is used for the androgen independence carcinoma of prostate.This vaccine has caused the formation to the T cellullar immunologic response relevant with antigen prostate acid phosphatase (PAP) of carcinoma of prostate.From the patient, obtain the DC precursor through the leukocyte exclusion, and itself and other leukocyte are separated with cell separation equipment.The delivery cartridge that the PAP of these cells and reorganization is merged was cultivated about 36 hours altogether then, to allow the DC maturation.Sophisticated then DC is used to vaccine.In the interval in two weeks, 30 minutes intravenous infusion transporting P rovenge vaccines.The carcinoma of prostate vaccine that is used for other candidates of the present invention is known (seeing, for example Shaffer andScher (2003) Lancet Ocol.4:407) in this area.

IV. colorectal cancer vaccine

By for example GnRH analog (or other castrating method) before, afterwards or simultaneously also can be with the curative effect of method raising colorectal cancer vaccine of the present invention at the inhibitor of using the sex steroid signal pathway.A non-limiting instance of a kind of vaccine like this is Trovax ^TM(OxfordBioMedica).This vaccine is a kind of solid tumor related antigen 5T4 that is transported by the vaccinia virus Ankara (MVA) that modifies through intramuscular injection.Give vaccine in 0,4 and 8 weeks simultaneously with chemotherapy.The peptide vaccine that is used for other cytotoxic T lymphocyte precursor guiding of colorectal cancer patients is known (seeing, for example Sato et al. (2004) Br.J.Cancer 90:1334) in this area.

V. ovarian cancer vaccine

By for example GnRH analog (or other castrating method) before, afterwards or simultaneously also can be with the curative effect of method raising ovarian cancer vaccine of the present invention at the inhibitor of using the sex steroid signal pathway.A non-limiting instance of a kind of vaccine like this is the M-FP (CancerVac Pty.Ltd.) that is used for the treatment of the transitivity ovarian cancer.It is a kind of autogenetic cell vaccine, wherein subcutaneous injection DC.The MUC1 of reorganization is fused in the mannan with the purification dendron shape precursor that promotes the patient antigenic picked-up.Present the activation DC of MUC1 peptide for then patient's subcutaneous injection.

Embodiment 39: castrating is to the effect of BM and the spleen of thymusectomy mice

Carrying out these trial tests is in order to determine whether castrate among the patient (mice) who acts on thymusectomy is tangible.Presentation of results has direct or indirect effect to the blocking-up of sex steroid signal pathway to immune system (for example BM and intrathymic immunocyte), no matter whether has the thymus of reactivate.

Material and method

Utilize conventional method known in the art with mice castrating and thymusectomy.Mice is assigned in the following group: treatment (be natural, " untreated "), false castrating (" sham-cx ") and castrating (" cx "), and this each group of three groups always has 6 analyzed groups all by thymusectomy (" tx ") or false thymusectomy (" shtx ").When bone marrow removing and 2 weeks of BMT (method above seeing) back and 4 weeks, analyze each group in 6 groups.

The result

I. thymusectomy does not influence the effect of sex steroid inhibition to BM

Shown in Figure 72 A, in 4 whens week behind BMT, the Tx/Cx mice has the common lymph CFU-GM of BM (CLP) that number increases, this number and ShTx/Cx mice suitable.

Shown in Figure 72 B, in 4 whens week behind BMT, the Tx/Cx mice has the BM B cell that total number increases, and this number is suitable with the ShTx/Cx mice.Compare with ShamCx/Tx or ShamCx/ShTx, Tx/Cx mice and ShTx/Cx mice mice also have the BM B cell that number increases.

Shown in Figure 72 C, in 4 whens week behind BMT, the Tx/Cx mice also has the BM immature B cells that total number increases, and this number is suitable with the ShTx/Cx mice.Compare with ShamCx/Tx or ShamCx/ShTx, Tx/Cx mice and ShTx/Cx mice mice also have the BM immature B cells that number increases.

Therefore, the result of Figure 72 A-C supports such conclusion, and the number and functional the comprising of castrating exactly increasing the BM cell increase the thymus that the effect of implanting does not need reactivate, the substitute is because of the direct effect to BM and immune other cells.

II. thymusectomy does not influence the effect of sex steroid inhibition to spleen

Shown in Figure 72 D, in 4 whens week behind BMT, the Tx/Cx mice also shows the increase of the total cellular score with spleen, and this is suitable with the ShTx/Cx mice.When comparing with ShamCx/Tx or ShamCx/ShTx control mice, Tx/Cx mice and ShTx/Cx mice also have the splenocyte that has increased total number.

Shown in Figure 72 E, in 4 whens week behind BMT, the Tx/Cx mice also shows the increase of the B total cellular score of spleen, and this is suitable with the ShTx/Cx mice.Compare with ShamCx/Tx or ShamCx/ShTx, Tx/Cx mice and ShTx/Cx mice also have the B cell of the spleen of number increase.

Therefore, the result of Figure 72 D-E supports such conclusion, the number and functional the comprising of castrating exactly the immunocyte that increases spleen strengthen the thymus that the effect of rebuilding does not need reactivate, the substitute is because to the direct effect of BM and immune other cells.

Together incorporate into reference to all mentioned documents in the description in the above at this.Only otherwise break away from scope of invention and spirit, various modifications and the variation to described method and system of the present invention is conspicuous for those skilled in the art.Although, be understood that desired invention should exceedingly be limited to these special embodiments in conjunction with specifically preferred embodiment having described invention.In fact, it is evident that the various modifications of the described mode that carries out an invention all in below the scope of claims to the personnel of biology or association area.

Those skilled in the art utilize routine test will recognize, maybe can determine multiple with this special embodiment embodiment of equal value mutually of special description.These equivalence are included in the scope of following claims.

Claims (154)

1. functional method that is used to strengthen patient's bone marrow, the signal pathway that comprises the sex steroid mediation of blocking the patient, wherein said bone marrow functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate.

2. one kind is used to prevent patient's the pathological changes or the method for disease, the signal pathway that comprises the sex steroid mediation of blocking the patient, patient's bone marrow functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate wherein, and wherein compare with the symptom that the patient occurred before the signal pathway of patient's sex steroid mediation is blocked, the clinical symptoms of described disease is alleviated.

3. claim 1 or 2 method wherein strengthen the HSC hemoposieis.

4. claim 1 or 2 method wherein strengthen the HSC output of bone marrow.

5. claim 1 or 2 method, the wherein signal pathway by the mediation of castrating barrier steroid.

6. the method for claim 5 is wherein castrated the signal pathway that the barrier steroid mediates by operation.

7. the method for claim 6 is wherein passed through the signal pathway that chemical castration barrier steroid mediates.

8. claim 1 or 2 method are wherein by using the signal pathway of one or more medicine barrier steroid mediation.

9. the method for claim 8, wherein one or more medicine is selected from the group of being made up of LHRH agonist, lhrh antagonist, anti-LHRH vaccine, androgen antagonist, estrogen antagonist, SERM, SARM, SPRM, ERD, aromatase inhibitor, gestation and combination thereof.

10. the method for claim 9, wherein the LHRH agonist is selected from the group of being made up of Eulexin, goserelin, the bright third sharp moral, Dioxalan derivant, triptorelin, meterelin, buserelin, histrelin, nafarelin, lutrelin, leuprorelin, deslorelin, Cystorelin, Decapeptyl, gonadorelin and combination thereof.

11. the method for claim 9, wherein lhrh antagonist is selected from the group of being made up of 1: PN: WO02056903 PAGE: 25 claimed protein, cetrorelix and combination thereof.

12. the method for claim 9, wherein androgen antagonist is Cosudex_.

13. the method for claim 1 or 2, wherein patient's thymus is to the small part atrophy.

14. the method for claim 13, wherein the patient suffers from disease or the pathological changes that causes patient's atrophy of thymus gland to small part.

15. the method for claim 13, wherein the patient has accepted the treatment to disease or pathological changes, and this is treated to small part and causes patient's atrophy of thymus gland.

16. the method for claim 15, wherein treatment is immunosuppressant, chemotherapy or radiotherapy.

17. the method for claim 13, wherein the patient is postpubertal.

18. the method for claim 14 also comprises the dosed cells to the patient, wherein said cell is stem cell, CFU-GM or its combination.

19. the method for claim 18, wherein stem cell is selected from the group of being made up of HSC, epithelial stem cell and combination thereof.

20. the method for claim 18, wherein CFU-GM is selected from the group of being made up of lymph CFU-GM, medullary system CFU-GM and combination thereof.

21. the method for claim 19, wherein CFU-GM is selected from the group of being made up of lymph CFU-GM, medullary system CFU-GM and combination thereof.

22. the method for claim 19, wherein said cell is HSC.

23. the method for claim 22, wherein HSC is CD34+.

24. the method for claim 22, wherein HSC is from body.

25. the method for claim 22, wherein HSC is not from body.

26. the method for claim 22 is wherein beginning to use HSC when the signal pathway of sex steroid mediation blocked.

27. the method for claim 2, wherein disease or pathological changes are by being selected from virus, antibacterial, fungus, parasite, Protein virus, cancer, anaphylactogen, asthma evocator and causing that the oneself protein of autoimmune disease and the cause of disease in the antigen are caused.

28. the method for claim 27, wherein the cause of disease is a virus.

29. the method for claim 28, wherein virus is selected from the group of being made up of Retroviridae, Picornaviridae, Calciviridae, Togaviridae, flaviviridae, coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, orthomyxoviridae family, Bungaviridae, Arenaviridae, Reoviridae, birnavirus section, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, herpetoviridae, Poxviridae and Iridoviridae.Section

30. the method for claim 28, wherein virus is selected from the group of being made up of influenza virus, HIV (human immunodeficiency virus) and herpes simplex virus.

31. the method for claim 27, wherein the cause of disease is an antibacterial.

32. the method for claim 31, wherein antibacterial is selected from by helicobacter pylori, the Bai Shi spirillum, the pneumonia legionella, mycobacterium tuberculosis, mycobacterium avium, Mycobacterium intracellulare, mycobacterium kansasii, the Ge Dengshi mycobacteria, the mycobacteria zygoblast, staphylococcus aureus, Diplococcus gonorrhoeae, Neisseria meningitidis, listerisa monocytogenes in mjme, micrococcus scarlatinae, streptococcus agalactiae, streptococcus faecalis, bargen's streptococcus, streptococcus pneumoniae, pathogenic Campylobacter zygoblast, the enterococcus zygoblast, hemophilus influenza, Bacillus anthracis (Bacillus antracis), corynebacterium diphtheriae, rod bacillus zygoblast, erysipelothrix rhusiopathiae, bacillus perfringens, clostridium tetanus, clostridium perfringen, klebsiella pneumoniae, multocida, the bacteroid zygoblast, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponoma palladium, superfine treponema, leptospira, the group of forming with actinomyces israelii.

33. the method for claim 31, wherein antibacterial is a mycobacteria.

34. the method for claim 27, wherein the cause of disease is a parasite.

35. the method for claim 32, wherein parasite is selected from the group of being made up of Plasmodium falciparum, Plasmodium yoelii and toxoplasma gondii.

36. the method for claim 34, wherein parasite is a plasmodium.

37. the method for claim 27, wherein the cause of disease is infectious fungus.

38. the method for claim 37, wherein infectious fungus is selected from the group of being made up of Cryptococcus histolyticus, Histoplasma capsulatum, Blastomyces coccidioides, Blastomyces dermatitidis, chlamydia trachomatis, Candida albicans.

39. the method for claim 27, wherein the cause of disease is a cancer.

40. the method for claim 40, wherein cancer is selected from the group of being made up of the brain cancer, pulmonary carcinoma, ovarian cancer, breast carcinoma, carcinoma of prostate, colon cancer and hematologic cancers.

41. the method for claim 27, wherein the cause of disease is an anaphylactogen.

42. the method for claim 41, wherein anaphylactogen causes the anaphylaxis pathological changes that is selected from the group of being made up of eczema, allergic rhinitis, allergia nose's mucositis, Hay Fever, bronchial asthma, rubella (urticaria) and food anaphylaxis.

43. the method for claim 27, wherein before the signal pathway of the sex steroid mediation of blocking the patient, the patient is exposed to the cause of disease.

44. the method for claim 27, wherein before the signal pathway of the sex steroid mediation of blocking the patient, the patient is not exposed to the cause of disease.

45. the method for claim 27 also comprises the combination of using at least a cytokine, at least a somatomedin or at least a cytokine and at least a somatomedin to the patient.

46. the method for claim 45, wherein cytokine is selected from the group of being made up of interleukin-22 (IL-2), interleukin 7 (IL-7), interleukin 15 (IL-15), stem cell factor (SCF) and combination thereof.

47. the method for claim 45, wherein somatomedin is selected from the group of being made up of member, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), keratinocyte growth factor (KGF), granulocyte-macrophage colony stimutaing factor (GM-CSF) and the combination thereof of the member of epithelium growth factor family, fibroblast growth family.

48. the method for claim 46, wherein somatomedin is selected from the group of being made up of member, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), keratinocyte growth factor (KGF), granulocyte-macrophage colony stimutaing factor (GM-CSF) and the combination thereof of the member of epithelium growth factor family, fibroblast growth family.

49. one kind is used to strengthen the method that HSC implants receptor patient, comprises:

The signal pathway of blocking-up patient's sex steroid mediation;

Use HSC to the patient;

Allow that HSC implants patient's bone marrow,

The wherein implantation of HSC or be enhanced under the situation that does not have the thymus reactivate or prior to the thymus reactivate or in the thymus reactivate.

50. a method that is used to prevent disease of patient or pathological changes comprises:

The signal pathway of blocking-up patient's sex steroid mediation;

Use HSC to the patient;

Allow that HSC implants patient's bone marrow,

The wherein implantation of HSC or be enhanced under the situation that does not have the thymus reactivate or prior to the thymus reactivate or in the thymus reactivate, and wherein compare with the symptom that the patient occurred before the signal pathway of patient's sex steroid mediation is blocked, the clinical symptoms of described disease or pathological changes is alleviated.

51. the method for claim 49 or 50, wherein said HSC is from body.

52. the method for claim 49 or 50, wherein said HSC is not from body.

53. the method for claim 49 or 50, the wherein signal pathway that mediates by castrating barrier steroid.

54. the method for claim 53, the wherein signal pathway that mediates by operation castrating barrier steroid.

55. the method for claim 54, the wherein signal pathway that mediates by chemical castration barrier steroid.

56. the method for claim 49 or 50 is wherein by using the signal pathway of one or more medicine barrier steroid mediation.

57. the method for claim 56, wherein one or more medicine is selected from the group of being made up of LHRH agonist, lhrh antagonist, anti-LHRH vaccine, androgen antagonist, estrogen antagonist, SERM, SARM, SPRM, ERD, aromatase inhibitor, gestation and combination thereof.

58. the method for claim 57, wherein the LHRH agonist is selected from the group of being made up of Eulexin, goserelin, the bright third sharp moral, Dioxalan derivant, triptorelin, meterelin, buserelin, histrelin, nafarelin, lutrelin, leuprorelin, deslorelin, Cystorelin, Decapeptyl, gonadorelin and combination thereof.

59. the method for claim 57, wherein lhrh antagonist is selected from the group of being made up of 1: PN: WO02056903 PAGE: 25 claimed protein, cetrorelix and combination thereof.

60. the method for claim 57, wherein androgen antagonist is Cosudex_.

61. the method for claim 49 or 50, wherein patient's thymus is to the small part atrophy.

62. the method for claim 61, wherein the patient suffers from disease or the pathological changes that causes patient's atrophy of thymus gland to small part.

63. the method for claim 61, wherein the patient has accepted the treatment to disease or pathological changes, and this is treated to small part and causes patient's atrophy of thymus gland.

64. the method for claim 63, wherein treatment is immunosuppressant, chemotherapy or radiotherapy.

65. the method for claim 61, wherein the patient is postpubertal.

66. the method for claim 62 also comprises to the patient and uses lymph CFU-GM, medullary system CFU-GM, epithelial stem cell or its combination.

67. the method for claim 49 or 50, wherein HSC is CD34+.

68. the method for claim 49 or 50 is wherein beginning to use HSC when the signal pathway of sex steroid mediation blocked.

69. the method for claim 50, wherein disease or pathological changes are by being selected from virus, antibacterial, fungus, parasite, Protein virus, cancer, anaphylactogen, asthma evocator and causing that the oneself protein of autoimmune disease and the cause of disease in the antigen are caused.

70. the method for claim 69, wherein the cause of disease is a virus.

71. the method for claim 70, wherein virus is selected from the group of being made up of Retroviridae, Picornaviridae, Calciviridae, Togaviridae, flaviviridae, coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, orthomyxoviridae family, Bungaviridae, Arenaviridae, Reoviridae, birnavirus section, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, herpetoviridae, Poxviridae and Iridoviridae.

72. the method for claim 70, wherein virus is selected from the group of being made up of influenza virus, HIV (human immunodeficiency virus) and herpes simplex virus.

73. the method for claim 69, wherein the cause of disease is an antibacterial.

74. the method for claim 73, wherein antibacterial is selected from by helicobacter pylori, the Bai Shi spirillum, the pneumonia legionella, mycobacterium tuberculosis, mycobacterium avium, Mycobacterium intracellulare, mycobacterium kansasii, the Ge Dengshi mycobacteria, the mycobacteria zygoblast, staphylococcus aureus, Diplococcus gonorrhoeae, Neisseria meningitidis, listerisa monocytogenes in mjme, micrococcus scarlatinae, streptococcus agalactiae, streptococcus faecalis, bargen's streptococcus, streptococcus pneumoniae, pathogenic Campylobacter zygoblast, the enterococcus zygoblast, hemophilus influenza, Bacillus anthracis, corynebacterium diphtheriae, rod bacillus zygoblast, erysipelothrix rhusiopathiae, bacillus perfringens, clostridium tetanus, clostridium perfringen, klebsiella pneumoniae, multocida, the bacteroid zygoblast, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponoma palladium, superfine treponema, leptospira, the group of forming with actinomyces israelii.

75. the method for claim 73, wherein antibacterial is a mycobacteria.

76. the method for claim 69, wherein the cause of disease is a parasite.

77. the method for claim 74, wherein parasite is selected from the group of being made up of Plasmodium falciparum, Plasmodium yoelii and toxoplasma gondii.

78. the method for claim 76, wherein parasite is a plasmodium.

79. the method for claim 69, wherein the cause of disease is infectious fungus.

80. the method for claim 79, wherein infectious fungus is selected from the group of being made up of Cryptococcus histolyticus, Histoplasma capsulatum, Blastomyces coccidioides, Blastomyces dermatitidis, chlamydia trachomatis, Candida albicans.

81. the method for claim 69, wherein the cause of disease is a cancer.

82. the method for claim 81, wherein cancer is selected from the group of being made up of the brain cancer, pulmonary carcinoma, ovarian cancer, breast carcinoma, carcinoma of prostate, colon cancer and hematologic cancers.

83. the method for claim 69, wherein the cause of disease is an anaphylactogen.

84. the method for claim 83, wherein anaphylactogen causes the anaphylaxis pathological changes that is selected from the group of being made up of eczema, allergic rhinitis, allergia nose's mucositis, Hay Fever, bronchial asthma, rubella (urticaria) and food anaphylaxis.

85. the method for claim 69, wherein before the signal pathway of the sex steroid mediation of blocking the patient, the patient is exposed to the cause of disease.

86. the method for claim 69, wherein before the signal pathway of the sex steroid mediation of blocking the patient, the patient is not exposed to the cause of disease.

87. the method for claim 56 also comprises the combination of using at least a cytokine, at least a somatomedin or at least a cytokine and at least a somatomedin to the patient.

88. the method for claim 87, wherein cytokine is selected from the group of being made up of interleukin-22 (IL-2), interleukin 7 (IL-7), interleukin 15 (IL-15), stem cell factor (SCF) and combination thereof.

89. the method for claim 87, wherein somatomedin is selected from the group of being made up of member, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), keratinocyte growth factor (KGF), granulocyte-macrophage colony stimutaing factor (GM-CSF) and the combination thereof of the member of epithelium growth factor family, fibroblast growth family.

90. the method for claim 88, wherein somatomedin is selected from the group of being made up of member, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), keratinocyte growth factor (KGF), granulocyte-macrophage colony stimutaing factor (GM-CSF) and the combination thereof of the member of epithelium growth factor family, fibroblast growth family.

91. functional method that is used to strengthen patient's immunocyte, the signal pathway that comprises the sex steroid mediation of blocking the patient, wherein said immune cell function or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate.

92. method that is used to prevent disease of patient or pathological changes, the signal pathway that comprises the sex steroid mediation of blocking the patient, patient's immunocyte functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate wherein, and wherein compare with the symptom that the patient occurred before the signal pathway of patient's sex steroid mediation is blocked, the clinical symptoms of described disease or pathological changes is alleviated.

93. the method for claim 91 or 92, wherein said immunocyte are selected from the group of being made up of T cell, B cell and dendritic cell.

94. the method for claim 93, wherein said immunocyte are the T cells.

95. the method for claim 91 or 92, the wherein signal pathway that mediates by castrating barrier steroid.

96. the method for claim 95, the wherein signal pathway that mediates by operation castrating barrier steroid.

97. the method for claim 96, the wherein signal pathway that mediates by chemical castration barrier steroid.

98. the method for claim 91 or 92 is wherein by using the signal pathway of one or more medicine barrier steroid mediation.

99. the method for claim 98, wherein one or more medicine is selected from the group of being made up of LHRH agonist, lhrh antagonist, anti-LHRH vaccine, androgen antagonist, estrogen antagonist, SERM, SARM, SPRM, ERD, aromatase inhibitor, gestation and combination thereof.

100. the method for claim 99, wherein the LHRH agonist is selected from the group of being made up of Eulexin, goserelin, the bright third sharp moral, Dioxalan derivant, triptorelin, meterelin, buserelin, histrelin, nafarelin, lutrelin, leuprorelin, deslorelin, Cystorelin, Decapeptyl, gonadorelin and combination thereof.

101. the method for claim 99, wherein lhrh antagonist is selected from the group of being made up of 1: PN: WO02056903 PAGE: 25 claimed protein, cetrorelix and combination thereof.

102. the method for claim 99, wherein androgen antagonist is Cosudex_.

103. the method for claim 91 or 92, wherein patient's thymus is to the small part atrophy.

104. the method for claim 103, wherein the patient suffers from disease or the pathological changes that causes patient's atrophy of thymus gland to small part.

105. the method for claim 103, wherein the patient has accepted the treatment to disease or pathological changes, and this is treated to small part and causes patient's atrophy of thymus gland.

106. the method for claim 105, wherein treatment is immunosuppressant, chemotherapy or radiotherapy.

107. the method for claim 103, wherein the patient is postpubertal.

108. the method for claim 104 also comprises the dosed cells to the patient, wherein said cell is stem cell, CFU-GM or its combination.

109. the method for claim 108, wherein stem cell is selected from the group of being made up of HSC, epithelial stem cell and combination thereof.

110. the method for claim 108, wherein CFU-GM is selected from the group of being made up of lymph CFU-GM, medullary system CFU-GM and combination thereof.

111. the method for claim 109, wherein CFU-GM is selected from the group of being made up of lymph CFU-GM, medullary system CFU-GM and combination thereof.

112. the method for claim 109, wherein said cell is HSC.

113. the method for claim 112, wherein HSC is CD34+.

114. the method for claim 112, wherein HSC is from body.

115. the method for claim 112, wherein HSC is not from body.

116. the method for claim 112 is wherein beginning to use HSC when the signal pathway of sex steroid mediation blocked.

117. the method for claim 102, wherein disease or pathological changes are by being selected from virus, antibacterial, fungus, parasite, Protein virus, cancer, anaphylactogen, asthma evocator and causing that the oneself protein of autoimmune disease and the cause of disease in the antigen are caused.

118. the method for claim 117, wherein the cause of disease is a virus.

119. the method for claim 118, wherein virus is selected from the group of being made up of Retroviridae, Picornaviridae, Calciviridae, Togaviridae, flaviviridae, coronaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, orthomyxoviridae family, Bungaviridae, Arenaviridae, Reoviridae, birnavirus section, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, herpetoviridae, Poxviridae and Iridoviridae.

120. the method for claim 118, wherein virus is selected from the group of being made up of influenza virus section, HIV (human immunodeficiency virus) and herpes simplex virus.

121. the method for claim 117, wherein the cause of disease is an antibacterial.

122. the method for claim 121, wherein antibacterial is selected from by helicobacter pylori, the Bai Shi spirillum, the pneumonia legionella, mycobacterium tuberculosis, mycobacterium avium, Mycobacterium intracellulare, mycobacterium kansasii, the Ge Dengshi mycobacteria, the mycobacteria zygoblast, staphylococcus aureus, Diplococcus gonorrhoeae, Neisseria meningitidis, listerisa monocytogenes in mjme, micrococcus scarlatinae, streptococcus agalactiae, streptococcus faecalis, bargen's streptococcus, streptococcus pneumoniae, pathogenic Campylobacter zygoblast, the enterococcus zygoblast, hemophilus influenza, Bacillus anthracis, corynebacterium diphtheriae, rod bacillus zygoblast, erysipelothrix rhusiopathiae, bacillus perfringens, clostridium tetanus, clostridium perfringen, klebsiella pneumoniae, multocida, the bacteroid zygoblast, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponoma palladium, superfine treponema, leptospira, the group of forming with actinomyces israelii.

123. the method for claim 121, wherein antibacterial is a mycobacteria.

124. the method for claim 117, wherein the cause of disease is a parasite.

125. the method for claim 122, wherein parasite is selected from the group of being made up of Plasmodium falciparum, Plasmodium yoelii and toxoplasma gondii.

126. the method for claim 124, wherein parasite is a plasmodium.

127. the method for claim 117, wherein the cause of disease is infectious fungus.

128. the method for claim 127, wherein infectious fungus is selected from the group of being made up of Cryptococcus histolyticus, Histoplasma capsulatum, Blastomyces coccidioides, Blastomyces dermatitidis, chlamydia trachomatis, Candida albicans.

129. the method for claim 117, wherein the cause of disease is a cancer.

130. the method for claim 129, wherein cancer is selected from the group of being made up of the brain cancer, pulmonary carcinoma, ovarian cancer, breast carcinoma, carcinoma of prostate, colon cancer and hematologic cancers.

131. the method for claim 117, wherein the cause of disease is an anaphylactogen.

132. the method for claim 131, wherein anaphylactogen causes the anaphylaxis pathological changes that is selected from the group of being made up of eczema, allergic rhinitis, allergia nose's mucositis, Hay Fever, bronchial asthma, rubella (urticaria) and food anaphylaxis.

133. the method for claim 117, wherein before the signal pathway of the sex steroid mediation of blocking the patient, the patient is exposed to the cause of disease.

134. the method for claim 117, wherein before the signal pathway of the sex steroid mediation of blocking the patient, the patient is not exposed to the cause of disease.

135. the method for claim 98 also comprises the combination of using at least a cytokine, at least a somatomedin or at least a cytokine and at least a somatomedin to the patient.

136. the method for claim 135, wherein cytokine is selected from the group of being made up of interleukin-22 (IL-2), interleukin 7 (IL-7), interleukin 15 (IL-15), stem cell factor (SCF) and combination thereof.

137. the method for claim 135, wherein somatomedin is selected from the group of being made up of member, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), keratinocyte growth factor (KGF), granulocyte-macrophage colony stimutaing factor (GM-CSF) and the combination thereof of the member of epithelium growth factor family, fibroblast growth family.

138. the method for claim 136, wherein somatomedin is selected from the group of being made up of member, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), keratinocyte growth factor (KGF), granulocyte-macrophage colony stimutaing factor (GM-CSF) and the combination thereof of the member of epithelium growth factor family, fibroblast growth family.

139. one kind is used to improve the method for patient to the immunne response of vaccine antigen, comprises:

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's bone marrow functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate; And

Use a kind of vaccine to the patient, described vaccine comprises a kind of vaccine antigen;

Wherein the patient produces the immunne response at described vaccine antigen, compares with the immunne response that the patient who does not have barrier steroid signal pathway will occur, and its immunne response improves.

140. one kind is used for the method that the hereditism changes the patient, comprises:

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's bone marrow functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate;

External genetic modification cell, wherein cell is selected from the group of being made up of stem cell, CFU-GM and combination thereof; And

Use the cell of genetic modification to the patient;

Wherein the patient is by genetic modification.

141. a method that is used to prevent or treat patient's HIV (human immunodeficiency virus) infection comprises:

Exhaust patient's immunocyte;

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's bone marrow functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate;

With the infection of HIV (human immunodeficiency virus) inhibiting, duplicate or the outer genetic modification cell of genosome of function, wherein cell is selected from the group of being made up of stem cell, CFU-GM and combination thereof, and

Use the cell of genetic modification to the patient;

Wherein prevent or treated HIV (human immunodeficiency virus) infection.

142. a method that is used for the treatment of patient's autoimmune disease comprises:

Exhaust patient's immunocyte; With

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's bone marrow functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate;

Wherein compare with the not treatment patient who suffers from autoimmune disease, the patient who is treated has the prognosis of the autoimmune disease of improvement.

143. a method hypersensitive that is used for the treatment of the patient comprises:

Exhaust patient's immunocyte; With

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's bone marrow functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate;

Wherein do not compare with treating the patient, the patient who is treated has the prognosis of improvement.

144. one kind is used to improve the method for patient to the immunne response of vaccine antigen, comprises:

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's bone marrow functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate;

Use a kind of vaccine to the patient, described vaccine comprises a kind of vaccine antigen;

Wherein the patient produces the immunne response at described vaccine antigen, compares with the immunne response that the patient who does not have barrier steroid signal pathway will occur, and its immunne response improves.

145. one kind is used to improve the method for patient to the immunne response of vaccine antigen, comprises:

The signal pathway of blocking-up patient's sex steroid mediation;

Use HSC to the patient;

Allow that HSC implants patient's bone marrow, wherein the implantation of HSC or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate; And

Use a kind of vaccine to the patient, described vaccine comprises a kind of vaccine antigen;

Wherein the patient produces the immunne response at described vaccine antigen, compares with the immunne response that the patient who does not have barrier steroid signal pathway will occur, and its immunne response improves.

146. one kind is used for the method that the hereditism changes the patient, comprises:

The signal pathway of blocking-up patient's sex steroid mediation;

Use HSC to the patient;

Allow that HSC implants patient's bone marrow;

External genetic modification cell, wherein cell is selected from the group of being made up of stem cell, CFU-GM and combination thereof;

Use the cell of genetic modification to the patient;

The wherein implantation of HSC or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate.

147. a method that is used to prevent or treat patient's HIV (human immunodeficiency virus) infection comprises:

Exhaust patient's immunocyte;

The signal pathway of blocking-up patient's sex steroid mediation;

Use HSC to the patient;

Allow that HSC implants patient's bone marrow;

With the infection of HIV (human immunodeficiency virus) inhibiting, duplicate or the outer genetic modification cell of genosome of function, wherein cell is selected from the group of being made up of stem cell, CFU-GM and combination thereof; And

Use the cell of genetic modification to the patient;

The wherein implantation of HSC or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate.

148. a method that is used for the treatment of patient's autoimmune disease comprises:

Exhaust patient's immunocyte;

The signal pathway of blocking-up patient's sex steroid mediation;

Use HSC to the patient;

Allow that HSC implants patient's bone marrow;

The wherein implantation of HSC or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate, and wherein compare with the not treatment patient who suffers from autoimmune disease, the patient who is treated has the prognosis of the autoimmune disease of improvement.

149. a method hypersensitive that is used for the treatment of the patient comprises:

Exhaust patient's immunocyte;

The signal pathway of blocking-up patient's sex steroid mediation;

Use HSC to the patient;

Allow that HSC implants patient's bone marrow;

The wherein implantation of HSC or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate, and wherein do not compare with treating the patient, the patient who is treated has the prognosis of improvement.

150. one kind is used to improve the method for patient to the immunne response of vaccine antigen, comprises:

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's immunocyte functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate; And

Use a kind of vaccine to the patient, described vaccine comprises a kind of vaccine antigen;

Wherein the patient produces the immunne response at described vaccine antigen.

151. one kind is used for the method that the hereditism changes the patient, comprises:

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's immunocyte functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate;

External genetic modification cell, wherein cell is selected from the group of being made up of stem cell, CFU-GM and combination thereof;

Use the cell of genetic modification to the patient;

Wherein the patient is enhanced the immunne response of vaccine antigen.

152. a method that is used to prevent or treat patient's HIV (human immunodeficiency virus) infection comprises:

Exhaust patient's immunocyte;

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's immunocyte functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate;

With the infection of HIV (human immunodeficiency virus) inhibiting, duplicate or the outer genetic modification cell of genosome of function, wherein cell is selected from the group of being made up of stem cell, CFU-GM and combination thereof;

Use the cell of genetic modification to the patient;

Wherein prevent or treated HIV (human immunodeficiency virus) infection.

153. a method that is used for the treatment of patient's autoimmune disease comprises:

Exhaust patient's immunocyte;

The signal pathway of blocking-up patient's sex steroid mediation, wherein patient's immunocyte functional or be enhanced under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or in patient's thymus reactivate;

Wherein compare with the not treatment patient who suffers from autoimmune disease, the patient who is treated has the prognosis of the autoimmune disease of improvement.

154. a method hypersensitive that is used for the treatment of the patient comprises:

Exhaust patient's immunocyte;

The signal pathway of blocking-up patient's sex steroid mediation is wherein or under the situation that does not have patient's thymus reactivate or prior to patient's thymus reactivate or increase patient's immunocyte functional in patient's thymus reactivate;

Wherein do not compare with treating the patient, the patient who is treated has the prognosis of improvement.

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