CN100406025C - Disease prevention by reactivation of the thymus - Google Patents

Abstract

The present disclosure provides methods for gene therapy utilizing hematopoietic stem cells, lymphoid progenitor cells, and/or myeloid progenitor cells. The cells are genetically modified to provide a gene that is expressed in these cells and their progeny after differentiation. In a preferred embodiment the cells contain a gene or gene fragment that confers to the cells resistance to HIV infection and/or replication. The cells are administered to a patient in conjunction with treatment to reactivate the patient's thymus. The cells may be autologous, syngeneic, allogeneic or xenogeneic, as tolerance to foreign cells is created in the patient during reactivation of the thymus. In a preferred embodiment the hematopoietic stem cells are CD34+. The patient's thymus is reactivated by disruption of sex steroid mediated signaling to the thymus. In a preferred embodiment, this disruption is created by administration of LHRH agonists, LHRH antagonists, anti-LHRH receptor antibodies, anti-LHRH vaccines or combinations thereof.

Description

The pharmaceutical applications of sex steroid antagonist and sex steroid agonist

The technical field of the invention

The present invention relates to the disease prevention field.Particularly, the present invention relates to activate again by thymus stimulates patient's immune system, and uses hematopoietic stem cell (HSC), hemopoietic progenitor cell, epithelial stem cell or bone marrow to carry out gene therapy alternatively.

Background technology related to the present invention

Immune system

Immune major function is that " dissident " antigen is distinguished mutually with " self " antigen, and correspondingly replys, and protects body to avoid infecting.In normal immunne response, the sequence of incident relates to special antigen presenting cell (APC) catches dissident's antigen and it is processed as little peptide segment, and the latter is rendered as the sliver (clefts) of main histocompatibility complex (MHC) molecule on the APC surface.This MHC molecule can be I type (expressing, by cytotoxic T cell (Tc) identification) or II type (mainly expressing through immune system cell, by helper T cell (Th) identification) on all nucleated cell.MHCII/ peptide complexes on these cell recognitions APC is also replied; The factor of these cells releases promotes Tc cell or the activation that produces the antibody (it has specificity to specific antigen) of B cell then.The importance of Th cell in all immunne response in fact obtains best embodiment in HIV/AIDS, wherein HIV/AIDS is exactly because virus damage Th cell causes the Th cell to lack, thereby causes serious immunodeficiency and finally cause death.The undesired growth of Th cell (and Tc, but degree is lighter) and can cause multiple other diseases, as anaphylaxis, cancer and autoimmune disease.

The cell-membrane receptor of T lymphocyte and bone-marrow-derived lymphocyte has the antigenic ability of identification.These receptors are produced at random by many complicated retracing sequences that may genes, like this each independently T or B cell have unique antigen receptor.This huge potential multiformity means, to any single antigen that body may run into, multiple lymphocyte can be discerned it and carries out replying in various degree with different bond strength (affinity).Because the specificity of antibody receptor is accidental the generation, thereby can propose following problem, promptly why body does not carry out " autoclasia " by the lymphocyte of antagonism autoantigen.Fortunately, have that several T of prevention and B cell do like this mechanism-they have built the environment of immune system to self tolerance jointly.

The most effective form of self tolerance is that all lymphocytes with potential reaction are removed to physical property (killing) from the position (thymus produces the T cell, and bone marrow produces the B cell) that they produce.This is called maincenter tolerance (central tolerance).The another kind of important method of tolerance is that it can directly or more likely suppress the id reaction sexual cell by cytokine by regulation and control Th cell.In view of in fact all immunne response all need the startup and the regulation and control of t helper cell, therefore the main target of any tolerance-induced scheme is at these cells.Similarly, because of the Tc cell is very important effector lymphocyte,, produce the main target that this type of cell is these therapeutic strategies therefore for for example anticancer and antiviral therapy.

Thymus

Someone thinks that thymus is immune major organs, because of it is to produce the lymphocytic main position of T.Its effect is to attract the suitable precursor that derives from bone marrow from blood, and induces their differentiating into T cell system, comprises producing the necessary gene rearrangement of T cell antigen receptor (TCR).What interrelate therewith is the tangible cell division of degree, the quantity of T cell is increased, thereby improve the probability that each exotic antigen is identified and removes.Yet different with the B cell is, the antigenic extremely odd feature of T cell recognition be TCR only discern with the MHC molecular physics on the fragments of peptides that links to each other; This is self MHC under the normal condition, and this ability has only thymus just to have.This process is called positivity to be selected, and is the exclusive feature of cortex epithelial cell.If TCR can't combine with self MHC/ peptide complexes, the T cell will be because of " ignoring " ripe needs of continuation the signal to a certain degree of death-T cell via TCR conduct.

Although thymus is indispensable for a functional immune system, be discharged into 1% of T cell-volume in the blood flow its every day, mammiferous one of obviously unusual be that because the generation of sex steroid, thymus can serious atrophy.This in addition just begun the pubarche aggravation of not associating in the Childhood.Concerning individual in normal health, always the forfeiture of generation and this function of release new T cell does not bring clinical consequences (although the sickness rate of immune diseases associated and yet increase thereupon of the order of severity with advancing age).When a large amount of forfeiture T cell, for example suffer from after AIDS, chemotherapy or the radiotherapy, because immunity is suppressed, the patient is to disease height susceptible.

Yet many T cells can continue to grow, and it can (have higher affinity) by accident and discern self MHC/ peptide complexes.This T cell thereby id reaction and can cause serious autoimmune disease may take place is such as multiple sclerosis, arthritis, diabetes, thyroiditis, systemic lupus erythematosus (sle) (SLE).Fortunately, if the affinity of TRC and self MHC/ peptide complexes is too high in the thymus, the thymocyte cell of growing will be committed suiside active and death by apoptosis through inducing to produce, and this process is called the negativity selection.This is called the maincenter tolerance.This T cell can be dead and can not reply because in thymus they that is that all right is ripe.The most effective inducer that this negativity is selected in thymus is the APC that is called dendritic cell (DC).As APC, they pass to the T cell to the most intensive signal; This can cause elimination in thymus, and in the more sophisticated peripheral lymphoid organs of T cell, DC can activated T cell.

Atrophy of thymus gland

Thymus is subjected to it oneself to influence (Kendall, 1988) with the neuroendocrine system two-phase is alternative to a great extent.The influence of the interaction partners thymus function between hypophysis, adrenal gland and the gonad is even more important, comprise nutrition (thyrotropin or TSH, with growth hormone or GH) and influence (metakentrin or the LH of atrophy, follicle stimulating hormone or FSH, with thyroliberin or ACTH) (Kendall, 1988; Homo-Delarche, 1991).In fact the physiological notable attribute of thymus is the sexual involution that carries out of its 26S Proteasome Structure and Function, itself and the increase proportional (Hirokawa and Makinodan, 1975 that produce at adolescence circulation blood neutral steroids; Tosi etc., 1982 and Hirakawa etc., 1994).The precision target of these hormones and induce the mechanism of atrophy of thymus gland to be still waiting to understand.Because thymus is generation and keeps the main place in periphery T cell storehouse, is the main cause that senectitude immune disease sickness rate raises so generally believe this atrophy.Especially, the decline of the immunologic function (such as the vigor and the mitogenic response of molten cell T cell) that relies on by the T cell and the immune system defect that illustrates, can raise from the morbidity of senectitude immunodeficiency, autoimmune and tumor (tumor load) and obtain reflection (Hirokawa, 1998).

The influence of atrophy of thymus gland is reflected in periphery, along with thymus is discharged into the minimizing of the cell number of T cell bank, causes the multiformity in the immune storehouse of TXi Baoshouti (TCR) to reduce.Also observe change (profile) (Hobbs etc., 1993 of cytokine; Kurashima etc., 1995), CD4 ⁺And CD8 ⁺The change of subgroup and by the transformation (Mackall etc., 1995) of naive T cell to memory cell.And thymus generates the efficient of (thymopoiesis) along with the age descends, so that finally loses (Mackall etc., 1995) in the ability that the back immune system of T cell exhaustion regenerates normal quantity T cell.Yet, recently Douek etc. (1998) even studies show that the mankind in one's old age thymus output may still take place.The circumscribed DNA product that tcr gene is reset is used to confirm that gerontal patient HIV infects the naive T cell that existence produces again in the back circulation blood.This output rating and the regeneration of periphery T cell storehouse subsequently also need further research, because through after the chemotherapy, postpubertal patient and prepuberal patient compare, the regeneration rate of T cell bank significantly descends, especially CD4 ⁺T cell (Machall etc., 1995).The research (1999) of Timm and Thoman has recently further proved this point, although they studies show that CD4 ⁺The T cell can be regenerated after Aged Mice bone marrow transplantation (BMT), but because periphery microenvironment aging, it shows the tendentiousness to memory cell, and it is also relatively poor that thymus produces the ability of naive T cell.

Thymus mainly is made up of the thymocyte cell of growing, these thymocyte cells are dispersed in the different stromal cell (mainly being the epithelial cell subgroup), and these stromal cells constitute microenvironment and best necessary somatomedin of growth of T cell and cell interaction are provided.Thymocyte cell and control its differentiation and sophisticated epithelial cell subgroup between the growth relation (Boyd etc., 1993) of symbiosis mean that sex steroid suppresses to occur in any cell type, the latter influences the other types cell subsequently.Thymocyte cell self has the probability of latent defect smaller, because adopted radiation chimera studies show that in the past, bone marrow (BM) stem cell is not subjected to influence (Hirokawa, 1998 at age; Mackall and Gress, 1997) and with young BM cell has thymus regeneration (repopulation) potential of similarity degree.And the thymocyte cell of geriatric animals has kept differentiation capability (Mackall and Gress, 1997 at least on some degree; George and Ritter, 1996; Hirokawa etc., 1994).Yet, up-to-date studies show that of Aspinall (1997), the defective of precursor CD3-CD4-CD8-three feminine genders (TN) colony betides TCR γ chain gene and resets the stage.

The invention summary

The invention provides prophylactic method, this method is by causing that patient's thymus activates again and strengthens the periphery T cell functional status, infecting or immune challenge thereby the patient can be resisted better.As employed at this paper, " prevention " and " preventing " refers to and completely or partially protects the patient to avoid the disease that infector causes.Optionally gene therapy (adopting genetically modified HSC, lymph sample CFU-GM, bone marrow CFU-GM or epithelial stem cell or its combining form (this group and each member are referred to herein as " GM cell ")) can give just again activated thymus, to produce specific immunity.In preferred specific embodiment, old (after the adolescence) patient's atrophy thymus is activated again.Again activated thymus becomes and can absorb HSC and medullary cell from blood, and they are converted into new T cell and DC in thymus.

One aspect of the present invention provides prevention or reduces the method for the ill danger of patient, and the method comprises the signal of the biography of cuttability steroid mediation to patient's thymus.

In a specific embodiment, go back bone marrow or HSC in patient's body.

In a specific embodiment, this disease has the special genes basis.

In preferred specific embodiment, this disease is the T cellular disorder, be selected from by viral infection (such as by HIV (human immunodeficiency virus) (HIV) infection), T cell function imbalance and other directly or indirectly reduce T cell quantity or the disease of function or group that state is formed.

On the other hand, the invention provides the method for the former infection of protecting from infection.Carried out the GM cell of genetic modification (resist or infection that prevention infection is former, activate, duplicate etc. and combination),, be injected in patient's body in the activated again while of thymus.

In preferred specific embodiment, HSC is genetically modified, thereby produces the HIV resistance in the T cell that forms after the activated again process neutralization of thymus activates again.For example, comprise a kind of gene thereby HSC is modified, the product of this gene can disturb HIV in the T cell infection, function and/or duplicate.This gives the resistance to a certain degree at virus, the disease of preventing this virus to cause thus.

In yet another aspect, the invention provides the method that activates thymus by the signal of barrier steroid mediation again.In a specific embodiment, utilize castrating to come the signal of barrier steroid mediation.In preferred specific embodiment, adopt chemical castration.In another specific embodiment, adopt the operation castrating.Castrating returns to preadolescence with thymic state, and thymus is activated again.

In particular specific embodiment, be agonist by giving LHRH or antagonist, estrogen antagonist antibody, androgen antagonist antibody, passive (antibody) or initiatively anti-LHRH vaccination (vaccinations) or its combination (" blocker ") of (antigen) realize to passing blocking-up to the signal of the sex steroid mediation of thymus.

In preferred specific embodiment, blocker gives with the peptide slow release formulation.The example of peptide slow release formulation is provided in WO98/08533, and its content is hereby expressly incorporated by reference.

In preferred specific embodiment of the present invention, genetically modified HSC before thymus activates again, simultaneously or be transplanted to subsequently in patient's body, produce new genetically modified T cell mass.

Accompanying drawing is briefly described

Figure 1A and B: the variation of thymocyte cell number before and after the castrating.Atrophy of thymus gland causes the thymocyte cell number obviously to reduce with the age.In castrating 2 weeks of back, cell number has increased to the level of young adult.In castrating 3 weeks of back, cell number is apparently higher than young adult, and the castrating back settled out in 4 weeks. ^{* *}=compare with young (February) thymus of growing up remarkable different, p＜0.001.

Fig. 2 A-C:(A) splenocyte quantity is not subjected to the influence of age and castrating.Periphery B: the T cells ratio is also kept stable (B), yet CD4: the CD8 ratio obviously reduces (p＜0.001) with the age, and returns to normal level in petticoats 4 weeks after castrating.

Fig. 3: cell sorting device (FACS) figure of fluorescence-activation shows age and the castrating influence to CD4 and CD 8 thymocyte cell groups.The percentage ratio of each quadrant provides on figure.The thymocyte cell subgroup does not change with the age, after the castrating, and the thymocyte cell synchronous amplification.

Fig. 4: the propagation of thymocyte cell, as by surveying in conjunction with the BrdU pulse.The ratio of the thymocyte cell of breeding does not change with age and castrating.

Fig. 5 A-D: age and castrating are to the influence of thymocyte cell subgroup propagation.(A) constitute the ratio that integral body is bred each subgroup of group: the ratio of CD8+T cell significantly raises among the propagation group.Propagation when (B) percent of each subgroup of breeding: TN and the CD8 subgroup propagation in the time of 2 years significantly is less than 2 months.In castrating two weeks of back, the TN subgroup returns to normal increment level at an early age, and the CD8 subgroup is significantly bred.After castrating for 4 weeks, its level equals normal level when young.(C) propagation of whole TN does not change with age and castrating.Yet (D) propagation of TN1 subgroup significantly descended with the age, and 4 weeks did not return to normal level yet after castrating. ^{* *}=highly significant, p＜0.001, ^*=significantly, p＜0.01.

Fig. 6: injection FITC in the mouse thymus.Calculated FITC+ cell quantity in the periphery in 24 hours later on.Although it is constant that the ratio of up-to-date thymus migration (RTE) keeps, account for 1% of thymocyte cell number, 2 weeks significantly reduced after castrating, and the RTE cell quantity significantly reduces (p＜0.01) with the age.After the castrating,, but still significantly be lower than the level of castrating young mice during 2 weeks though these values rise.Along with the increase at age, can see CD4+: CD8+RTE ratio significantly raises, but after castrating a week, just recovers normal.

Fig. 7 A-C: after cyclophosphamide (a kind of chemotherapeutics) treatment, the variation of thymus (A), spleen (B) and lymph node (C) cell quantity.Attention: 1 week and 2 weeks after treatment, the castrating treated animal with do not castrate treated animal (only adopt cyclophosphamide treat) and compare thymus generation rapid amplifying.In addition, and only compare with cyclophosphamide treatment group, castrating group spleen and lymph-node cell number obviously increase.In the time of around the, it is normal that cell quantity recovers.(n=3-4 of each treatment group and time point).

Fig. 8 A-C: after one week of surgery castrating, irradiation (625 rad) was also followed in the back, the variation of thymus (A), spleen (B) and lymph node (C) cell quantity.Attention: in 1 week and 2 weeks after treatment, castrate treated animal and do not castrate treated animal (only adopting irradiation) and compare thymus generation rapid amplifying.(n=3-4 of each treatment group and time point).

Fig. 9 A-C: after carrying out irradiation and castrating on the same day, the variation of thymus (A), spleen (B) and lymph node (C) cell quantity.Attention: compare 2 whens week after treatment, the thymus rapid amplifying in the neuter with not castrating group.Yet its difference is castrated so significantly (Fig. 7) not as treatment preceding 1 all mices.(n=3-4 of each treatment group and time point).

Figure 10: on the same day with after chemotherapeutics cyclophosphamide, surgery or the chemical castration treatment, the variation of thymus (A), spleen (B) and lymph node (C) cell quantity.Attention: 1 week and 2 weeks after treatment, the castrating treated animal with do not castrate treated animal (only adopt cyclophosphamide treat) and compare thymus generation rapid amplifying.In addition, and only compare with cyclophosphamide treatment group, castrating group spleen and lymph-node cell number obviously increase.(n=3-4 of each treatment group and time point).In with the cyclophosphamide treatment immune regeneration in back, the effect of chemical castration can be compared with the surgery castrating.

Figure 11: constitute (cellularity) with the lymph-node cell after I type herpes simplex virus (HSV-1) the sufficient sole of the foot immunity inoculation (foot-padimmunization).Attention: old castrating group with old age not castrating group compare cell quantity and rise.CD25 is to the total activated cell number of CD 8 cell fluorescence active cell separator doorizations in figure below explanation.

After Figure 12 A-C:HSV-1 inoculation, in activated LN (lymph node), the expression of V β 10 on cytotoxic T lymphocyte (CTL).Attention: cloning reaction reduces in the older mice, and the recovery of castrating back anticipation reaction.

Figure 13 A-C: castrating recovers the reaction to the HSV-1 immunity.(a) compare with castrating back mice with young, lymph-node cell digital display work total behind the older mouse infection descends.(b) activated (CD8 in the lymph node of HSV-1 infecting mouse ⁺CD25 ⁺) the typical FACS figure of cell.The ratio regular meeting of not seeing activated CTL is along with age or castrating back and different.(c) the remarkable decline of activated CTL quantity has reflected the decline of older mouse lymph nodal cell quantity.Can recover immunne response after the older mice castrating, and CTL quantity is suitable with young mice to HSV-1.The result represents with the mean value 1SD (standard deviation) of 8-12 mice. ^*=compare p≤0.01 with young mice (2 months); Compare ^=p≤0.01 with old mice (not castrating).

Figure 14: popliteal nest lymph node is taken from the mice immunized through HSV-1, and cultivates three days.Use without mice immunized and carry out CTL (cytotoxic T cell) detection, (measure with contrast as cracked background level ⁵¹Cr-discharges).The result represents with the meansigma methods of 8 mices, three measured value ± 1SD.Old mice shows the active obvious reduction of CTL, and (p≤0.01, *), E: T ratio is 10: 1 and 3: 1, shows the reduction of the percent that special CTL expresses in the lymph node.Older mice castrating back ctl response returns to the young level of growing up.

Figure 15 A and 15B: analyze CD4 ⁺The reaction that the T cell is auxiliary and V β TCR infects HSV-1.It is cut and in analyzed in vitro (a) CD25, CD8 and specificity TCR V β label and (b) expression of CD4/CD8T cell that popliteal nest lymph node infects back the 5th day at HSV-1.(a) activatory (CD25 of expression V β 10 or V β 8.1 ⁺) CD8 ⁺The percentage rate of T cell is represented with the mean value 1SD of every group of 8 mices.The result is not with the age or castrating does not change.(b) in tranquillization LN group the CD4/CD8 ratio along with the age reduces.Recover the castrating back.The result represents with the mean value 1SD of every group of 8 mices. ^{* *}=compare p≤0.001 with young with the castrating mice.

After Figure 16 A-D:Ly5 mouse bone marrow cells of the same race is transplanted, the variation of cell quantity in thymus (A), spleen (B), lymph node (C) and the bone marrow (D).Attention: compare with not castrating group, castrating treated animal all time point thymus after treatment all increases rapidly.In addition, and only compare with ring phosphinylidyne ammonia group, the spleen of castrating group and lymph footing obviously increase.(each treatment group and each time point n=3-4).Compare with neuter not, castrating mice allogenic cell (Ly5.2) significantly increases.(not having video data).

Figure 17 A and B: fetus liver is rebuild the back castrating and is not castrated the variation of thymocyte cell number in the mice.Each test group has 3-4 mice.(A) in 2 whens week,, the thymocyte cell number of castrating mice is in normal level, be significantly higher than do not castrate mice ( ^*P≤0.05).All around, can see that the thymus of castrating mice is loose.The cell number of not castrating mice still is lower than control level.(B) CD45.2 ⁺Cell: CD45.2+ is the labelling that shows donor source.Rebuild two weeks of back, castrate and do not castrate the cell that all occurs coming from donor in the mice.Around the treatment back, about 85% cell derives from donor in the castrating mouse thymus.In not castrating mouse thymus, do not derive from the cell of donor.

Figure 18: rebuild at lethal irradiation and fetus liver, with and subsequent the surgery castrating after, the thymocyte cell group's of CD4 and CD8 donor source FACS figure.The percentage ratio of each quadrant provides on the right of figure.Agematched contrast figure is eight months big Ly5.1 mouse thymus of the same race.Castrate and do not castrate mice and use CD45.2 ⁺The cell doorization only shows the cell that derives from donor.Rebuild two weeks of back, the castrating mice does not have different with the thymocyte cell subgroup of not castrating mice.

Figure 19 A and B: after lethal irradiation, fetus liver are rebuild and are castrated, bone marrow and lymph sample dendritic cell (DC) number.Each test group has 3-4 mice.Contrast (marking oblique line thereon) bar figure is based on the positive constant of the dendritic cell of finding in the untreated agematched mice among the figure.(A) derive from the bone marrow dendritic cell of donor: rebuild two weeks of back, do not castrate that DC is in normal level in the mice.At identical time point, the castrating mice in DC significantly more ( ^*P≤0.05).In the time of all around, the DC number still is higher than control level in the castrating mice.(B) derive from the lymph sample dendritic cell of donor: rebuild two weeks of back, the DC number of castrating mice is the twice of not castrating mice.Around the treatment back, the DC number still is higher than control level.

Figure 20 A and B: after fetus liver is rebuild, castrate and do not castrate total cell number and CD45.2 in the mice ⁺The variation of bone marrow cell.Each test group has 3-4 mice.(A) total cell number: rebuild two weeks of back, it is normal that bone marrow cell recovers, and castrating and the cell number of not castrating mice do not have significant difference.Rebuild around the back, castrating and do not castrate the cell number of mice there were significant differences ( ^*P≤0.05).(B) CD45.2 ⁺Cell number.Rebuild two weeks of back, castrating and the CD45.2 that does not castrate in the mouse bone marrow cells ⁺Cell number does not have significant difference.In the time of all around, the CD45.2 in the castrating mice ⁺Cell number is still higher.At identical time point, do not castrate the cell that does not derive from donor in the mice body.

Figure 21 A-C: after fetus liver is rebuild, the variation of castrating and the dendritic cell (DC) of not castrating T cell in the mouse bone marrow cells, bone marrow and lymph source.Each test group has 3-4 mice.Contrast (marking oblique line thereon) bar figure is based on the T cell found in the untreated agematched mice and the normal quantity of dendritic cell among the figure.(A) T cell number: rebuild two weeks of back with all around, castrating and the cell number average of not castrating in the mice reduce.(B) derive from the bone marrow dendritic cell of donor: rebuild two weeks of back, castrating and the DC cell number of not castrating in the mice are all normal.Point at this moment, castrating and do not castrate between the cell number of mice and do not have significant difference.(C) derive from the lymph sample dendritic cell of donor: rebuild two weeks of back and all around, the cell number average is in normal level.2 whens week, castrating and do not castrate between the cell number of mice and do not have significant difference.

Figure 22 A and B: after fetus liver is rebuild, castrating and do not castrating total cell number and donor (CD45.2 in the mice ⁺) variation of spleens cell number.Each test group has 3-4 mice.(A) total cell number: rebuild two weeks of back, cell number descends, and castrating and do not castrate between the cell number of mice and do not have significant difference.After around rebuilding, the cell number of castrating mice is near normal level.(B) CD45.2 ⁺Cell number: rebuild two weeks of back, castrating and the CD45.2 that does not castrate in the mice spleen ⁺Cell number does not have significant difference.In the time of all around, the CD45.2 of castrating mice ⁺Cell number is still higher.At identical time point, do not castrate the cell that does not derive from donor in the mice body.

Figure 23 A-C: the splenic t-cell after fetus liver is rebuild and derive from bone marrow and the dendritic cell of lymph (DC).Each test group has 3-4 mice.Contrast (marking oblique line thereon) bar figure is based on the T cell found in the untreated agematched mice and the normal quantity of dendritic cell among the figure.(A) T cell number: rebuild two weeks of back with all around, the cell number average of castrating and not castrating mice reduces.(B) derive from (CD45.2 of donor ⁺) the bone marrow dendritic cell: rebuild two weeks of back with all around, the DC number of castrating and not castrating mice is all normal.In 2 whens week, castrating and do not castrate between the cell number of mice and do not have significant difference.(C) derive from (CD45.2 of donor ⁺) lymph sample dendritic cell: rebuild two weeks of back and all around cell number be in normal level.2 whens week, castrating and do not castrate between the cell number of mice and do not have significant difference.

Figure 24 A and B: after fetus liver is rebuild, castrate and do not castrate total cell number and donor (CD45.2 in the mice ⁺) variation of lymph-node cell number.Each test group has 3-4 mice.(A) total cell number: rebuild two weeks of back, cell number is in normal level, and castrating and do not castrate between the cell number of mice and do not have significant difference.Rebuild around the back, the cell number of castrating mice is in normal level.(B) CD45.2 ⁺Cell number: rebuild two weeks of back, castrating and the donor CD45.2 that does not castrate in the mouse lymph knot ⁺Cell number does not have significant difference.In the time of all around, the CD45.2 cell number in the castrating mice is still higher.At identical time point, do not castrate the cell that does not derive from donor in the mice body.

Figure 25 A-C: after fetus liver was rebuild, castrating and not castrating in the mice mesenteric lymph node, derived from the variation of the dendritic cell (DC) of bone marrow and lymph at the T cell.Each test group has 3-4 mice.Contrast (marking oblique line thereon) bar figure is the T cell found in untreated agematched mice and the quantity of dendritic cell.(A) rebuild two weeks of back with all around, the T cell number average of castrating and not castrating mice reduces.(B) castrate and do not castrate in the mice, the bone marrow dendritic cell number average that derives from donor is normal.In the time of all around, they all reduce.In 2 whens week, castrating and do not castrate between the cell number of mice and do not have significant difference.(C) derive from the lymph sample dendritic cell of donor: rebuild two weeks of back and all around cell number be in normal level.2 whens week, castrating and do not castrate between the cell number of mice and do not have significant difference.

Figure 26: after LHRH agonist treatment carcinoma of prostate, the phenotype composition of patient (all patients were greater than 60 years old) peripheral blood lymphocyte is analyzed.Four months the time, analyzed patient samples before the treatment and behind the beginning LHRH agonist treatment.Before the treatment, in every milliliter of blood of all patients total lymphocyte number all be control value than low side.After the treatment, 6/9 significantly rising (some cases have increased by a times) of patient's total lymphocyte counting.Total T cell number of relevant therewith is 6/9 patient increases.At CD4 ⁺In the subgroup, this increase is more obvious, and 8/9 patient's cd4 t cell level raises.And at CD8 ⁺In the subgroup, trend is not clearly, has 4/9 patient's level to raise, though in general the degree of Sheng Gaoing also less than CD4 ⁺The T cell.

Figure 27: patient's haemanalysis shows before and after the LHRH agonist treatment: T cell, CD4 or cd8 t cell toatl proportion significantly do not change, and treatment back CD4: CD8 ratio has indefinite variation.This explanation has minimum therapeutical effect to homeostatic the keeping of T cell subsets, though the total T cell number in treatment back significantly raises.All values all compare with control value.

Figure 28: behind the LHRH agonist treatment, the proportion grading of B cell and bone marrow sexual cell (NK, NKT and macrophage) is presented at the variation that has in the subgroup in various degree in patient's peripheral blood.Though the ratio of treatment back NK, NKT and macrophage is relative constant, the B cell proportion descends in 4/9 patient.

Figure 29: after the treatment, total cell number analysis of B cell and bone marrow sexual cell shows in patient's peripheral blood, and NK (5/9 patient), NKT (4/9 patient) and macrophage (3/9 patient) quantity obviously rise after treatment.The B cell number is not seen visible trend: 2/9 patient's level raises, and 4/9 patient does not change, and 3/9 patient's level descends.

Seen main variation is in the T of peripheral blood cell mass behind Figure 30 A and the B:LHRH agonist treatment.Especially pure (CD45RA ⁺) the ratio selectivity of CD4+ cell raises CD4 ⁺Pure (CD45RA in the T cell subsets ⁺) and memory (CD45RO ⁺) ratio in 6/9 patient, raise.

Figure 31: the stopping effect that reduces skin with different pulsed laser energies.With being low to moderate 10mJ energy irradiation skin, the skin stopping effect then descends, and utilizes in the matched curve and pushes away numerical value.

Figure 32: the percutaneous infiltration of medicine.As the sample medicine, after laser irradiation, the permeability of skin increases greatly insulin.

Figure 33: after applying 5-amino-laevulic acid (ALA) and individual pulse transient state to skin, SF over time.The peak value of intensity comes across about 640nm place, and after treating 210 minutes the highest (dotted line).

Figure 34: do not add the pulse transient state, only add 5-amino-laevulic acid (ALA) after, SF is over time.Almost there is not any variation in different time point intensity.

Figure 35: apply 5-amino-laevulic acid (ALA) with after the single pulse transient state under the different peak stress to skin, the comparison that SF changes.Cuticular permeable degree depends on peak stress.

Detailed Description Of The Invention

The present invention includes the method that the patient prevents disease. As mentioned above, old (puberty Afterwards) thymus gland causes body immune system function reduction (comparing with peak level). The present invention The method that utilization reactivates thymus gland strengthens function of immune system, thereby builds up one's resistance to disease, And the infection that prevents multiple external factor to cause. In some cases, fail to prevent to infect, But in these cases, more powerful strong immune system can help body to reduce infection Range and the length of the course of disease.

In some cases, method of the present invention utilize genetically modified hematopoiesis stem cell, Lymph sample ancestral cell, marrow ancestral cell, epithelial stem cell or its combination (GM cell), Come specific antigen is produced the immune system resistance. This kind embodiment is in common unsettled U.S. that proposes Have a detailed description in state's patent application 09/758,910. A kind of suitable can produce or induce right Gene or polynucleotides that one or more infect former resistance, be added into do and/or the ancestral thin Among the born of the same parents. Modified injection cell is reactivated with method of the present invention to its thymus gland Patient's body in. T cell, tree are absorbed and are converted in modified doing with ancestral's cell by thymus gland Prominent shape cell and other cell that in thymus gland, produces. Every kind of new cell all contains parent to be done The heredity of/ancestral cell is modified, and therefore has anti-to the former infection that causes of one or more infection The property. After castrating in two weeks, marrow, blood and periphery lymph organ (such as spleen and lymph node) The quantity of middle B cell also increases.

The thymus gland of acceptor can be by blocking-up sex steroid mediation biography to the signal of thymus gland and quilt Reactivate. This kind blocking-up has reversed the hormone state of acceptor. Produce the preferred side of blocking-up Method is by castrating. The method of castrating includes but not limited to chemical castration and operation castrating. In the castrating process or the castrating after, the GM cell is implanted in patient's body. These cells Admitted by patient's thymus gland, become the new T cell of thymus gland generation and the part of DC. To T cell group contain the heredity that is inserted in the ancestral cells and modify.

A kind of method of thymus gland that preferably reactivates is by blocking LHRH to hypophysis Spread effect directly and/or indirectly, this can cause losing of promoting sexual gland hormone FSH and LH Lose. These promoting sexual gland hormone usually act on sexual gland and discharge sex hormone, particularly the women Estrogen and the male sex's testis ketone; This release is blocked because of losing of FSH and LH. Directly The result who causes is the rapid decline of steroids blood plasma level, thereby causes thymus gland continuous Discharge Inhibitory signal. The degree of thymus gland regeneration and dynamics can be by injection CD34⁺Hematopoiesis Cell (preferably from body) and being enhanced.

The present invention can be used for anyly being subjected to sex steroid to order about maturation and having the moving of immune system Species belong to (comprising the mankind), and for example a mammality and bag animal is arranged is preferably large-scale mammality, Be preferably the mankind.

Term " regeneration ", " reactivating " and " reconstruction ", and their derivative at this Use can be replaced in literary composition, refers to that the thymus gland of atrophy returns to its active state.

As used herein, " castrating " refers to generation and the distribution of sex steroid in the body Obvious minimizing or disappearance. When the function of thymus gland is recovered fully, just can effectively make disease The people returns to the state before puberty. Operation castrating excision patient's sexual gland.

A kind of non-persistent castrating method is by giving chemical medicine in one period, basis Literary composition is called " chemical castration ". The chemical medicine of many kinds has this kind effect. At chemical medicine Between the operating period and following period of time subsequently, patient's hormone produces and stops. Preferably, After dispenser stopped, this castrating effect meeting reversed.

Cut off the signal that passes to the sex steroid mediation of thymus gland

Very easy understanding, biography can be cut with various ways to the signal of the sex steroid mediation of thymus gland Disconnected, these modes are known by the professional and technical personnel, and a part is wherein retouched at this paper State. For example, generation or interior one or more sex steroids of blocking-up thymus gland of suppressing sex steroid Acceptor will be finished desirable cut-out, as can give sex steroid activator or anti-dose short of money, Or the initiatively resistance steroids vaccine inoculation (vaccinations) of (antigen) or passive (antibody). Give the generation that the similar thing of one or more sex steroids also can suppress sex steroid. At some In the clinical case, may be utilized by the permanent excision of physics castrating sexual gland.

In a preferred embodiment, it is logical passing the signal that mediates to the sex steroid of thymus gland Cross and give the similar thing of sex steroid, be preferably luteinising hormone-releasing hormo (LHRH) Similar thing is cut off. The similar thing of sex steroid and the application in treatment and chemical castration thereof Well-known. This type of similar thing includes but not limited to following LHRH acceptor (LHRH-R) activator: Buserelin (Hoechst company), plug holder Rayleigh (Cystorelin (Hoechst company)), Decapepthl (trade name Debiopharm; Ipsen/Beaufour company), Deslorelin (Deslorelin) (Balance Pharmaceuticals company), that Rayleigh of dagger-axe (Ayerst company), dagger-axe house Rayleigh (commodity Name Zoladex Nuo Leide; Zeneca company), stand group ammonia Rayleigh (Orth company), bright third Moral (the sharp Pu'an of trade name Lupron; Abbott/TAP company), the bright third Rayleigh (Plosker In company), corpus luteum Rayleigh (Wyeth company), beautiful for Rayleigh (Meterelin) (WO 9118016), that method Rayleigh (Syntex company) and bent Pu Ruilin (United States Patent (USP) 4,010, No. 125). P-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2 includes but not limited to that also the short of money of following LHRH-R resists Agent: abarelix (Abarelix) (trade name Plenaxis; Praecis company) and the west bent Rui Ke (Cetrorelix) (trade name; Zentaris company). The combination of activator, short of money anti-The combination of agent, and in activator is also included within combination short of money anti-dose. Above-mentioned list of references Disclosed content is hereby expressly incorporated by reference. Preferred similar thing is Deslorelin at present (Deslorelin) (at United States Patent (USP) the 4th, 218,439 in disclose). More catalogue can be joined See Vickery etc., 1984.

In a preferred embodiment, give patient LHRH-R antagonist, give the LHRH-R agonist afterwards.This method can stop or limit any owing to use agonist and may produce the peak by inductive sex steroid before sex steroid output reduce.In another specific embodiment, adopt only to cause seldom or not cause and to give before or not give the LHRH-R antagonist by the LHRH-R agonist on sex steroid production peak.

Though stimulate thymus to activate again mainly, comprise that some other material that strengthens thymus function also is useful based on inhibition to the direct effect of the effect of sex steroid and/or LHRH analog.This compounds includes but not limited to interleukin-22 (IL2), interleukin 7 (IL7), interleukin 15 (IL15), epithelium and fibroblast growth family member, stem cell factor, granulocyte colony-stimulating factor (granulocyte colony stimulatingfactor, GCSF) and keratinocyte growth factor (keratinocyte growthfactor, KGF).Can imagine that these additional chemical compounds can only use once when the LHRH analog is used for the first time.Yet, a kind of or its combination in these materials can be in office when Hou Zengjia uses, further stimulate thymus.In addition, but development and application based on

Usually thymus and the improved key character of immune system are that after the barrier steroid, marrow function also has enhancing.This includes but not limited to the increase that HSC produces and/or discharges.These HSC can move to thymus and the auxiliary thymus that increases generates (thymopoiesis).

Pharmaceutical composition

The used chemical compound of the present invention can be carried on any pharmaceutical carrier or not use carrier.Object lesson comprises coating, solvent and the diluent of physiological compatibility.Be used for that gastrointestinal tract is outer, subcutaneous, the medicine of approach such as vein and intramuscular injection should be protected, such as adopting encapsulated mode (encap sulation).Replacedly, these pharmaceutical compositions can adopt can protect the carrier format of effective ingredient to give, and allows the slow release of these compositions simultaneously.Known in the present technique field have multiple polymer and copolymer to can be used for preparing time release formulation, such as the lactic acid/glycolic copolymer of various ways (versions).Example is seen United States Patent (USP) the 5th, 410, and No. 016, its modification polymer that utilizes Polyethylene Glycol (PEG) is as biodegradable capsule.

Be used for oral dosage form and can be made into liquid, capsule, tablet etc.These compositionss can comprise, for example, and excipient, diluent and/or prevent the encrusting substance (coverings) that effective ingredient decomposes.These dosage forms are well known.

In any preparation, can comprise that activity to the LHRH analog does not have other chemical compound of negative effect.Object lesson is various somatomedin described herein and other cytokine.

Dosage

The LHRH congener can disposablely give, and its effect can continue for some time.Preferably, the effect duration of preparation is one or two month, more preferably one to three months.When using agonist, the time that the blood neutral steroids is reduced to minimum to be needed was approximately for 3 weeks, then needed 1 day when using antagonist.In a preferred embodiment, dose should be able to be kept an epiphytotics cycle.For example, " influenza season " usually occur in the some months in winter.Can prepare the LHRH analog formulations and also use method afford described herein, so that can protect the patient not infected in two or more months after influenza season begins, per two months or more a plurality of months boosts reduce or disappearance until the danger of infecting.

Standard dose is different according to the kind of used analog.In general, dosage is preferably between about 0.01mg/kg and the about 5mg/kg between about 0.01 μ g/kg and about 10mg/kg.Dosage is according to the kind of used LHRH analog or vaccine and different.For example, to the male, under present administration condition, when the 21st day (before thymus is strengthened day) disposable (3 months effect duration) give 10.8mg goserelin, inject 3.6mg once more when the 63rd day (after thymus is strengthened day), it can keep effect about 28 days again.To the women, gave 3/6mg (28 days effect duration) respectively at the 1st, 7,35 and 63 day.

This preparation can be used for the enhance immunity system.Replacedly, said preparation can be prepared into enhance immunity system when preventing influenza infection especially.A kind of preparation in back can comprise the GM cell that also can produce resistance (seeing below) through genetic modification (engineered) to influenza virus.The GM cell can give or give respectively (comprise on the space and/or on the time) simultaneously with the LHRH agonist formulation.As the situation of non-GM cell, according to the length in influenza season, can append multidose to the patient, protect and avoid the infection of influenza virus.

Using of chemical castration agent

The administration of chemical compound of the present invention can adopt many methods well known to those skilled in the art to finish.Giving the standard method that chemical inhibitor suppresses to pass to the signal of the sex steroid mediation of thymus is, disposablely gives LHRH agonist, and its curative effect can be kept 3 months.To this, simple disposable vein injection or intramuscular injection are not enough, because this agonist will be disposed in patient's body before three months finish.As an alternative, can adopt durative action preparation injection or implant, perhaps adopt the method for the slow release inhibitor of other any energy.Equally, can adopt the method that prolongs the inhibitor half-life in vivo,, and keep required function herein simultaneously for example by chemical compound is modified.

The example of more efficiently administration mechanism includes but not limited to the laser irradiation of skin and produce high-voltage pulse transient state (impulse transients) (being also referred to as stress wave or pulse transient state) on skin, when every kind of method is implemented or after it is implemented, place the chemical compound that has or do not have carrier at same skin area.Preferred laying method is the employing paster and is affixed on the skin in therapeutic process always.

A kind of mode of administration is to adopt laser beam (distinguishingly focus on and produce laser in suitable wavelengths), to produce little perforation or alteration on patient's skin.Referring to United States Patent (USP) the 4th, 775, No. the 6th, 056,738, No. 361, No. the 5th, 643,252, United States Patent (USP), No. the 5th, 839,446, United States Patent (USP) and United States Patent (USP), all these are hereby expressly incorporated by reference document.In preferred specific embodiment, the wavelength of laser beam is between 0.2 and 10 micron.Better, wavelength is approximately between the 1.5-3.0 micron.Best, wavelength is about 2.94 microns.In a specific embodiment, laser beam is to focus on lens, to produce the irradiation speckle by epiderm skin on skin.In another specific embodiment, laser beam is focused on, thereby only percutaneous horny layer produces the irradiation speckle.

As used herein, " melting " and " punching " is meant the hole that produces on skin.Such hole can have the different degree of depth; For example it can only see through horny layer, can penetrate the capillary layer of skin always, perhaps can end at this two-layer between Anywhere.As used herein, " alteration " refers to the change of skin texture under the situation that does not produce the hole, and it makes the permeability of skin increase.As the situation of punching, skin can be by alteration (altered) to any thickness.

In the regulation laser beam, can consider several factors, comprise the size of wavelength, energy fluence, burst length (pulse temporal width) and irradiation speckle.In preferred specific embodiment, the scope of energy fluence is 0.03-100,000J/cm ²More preferably, the scope of energy fluence is 0.03-9.6J/cm ²Light beam wavelength depends in part on laser material, for example erbium: yttrium-aluminium-garnet.The burst length width is the result of pulse width, and this pulse width is determined by for example a series of capacitors, flash lamp and laser bar material (laser rod material).This pulse width optimum is between 1fs (dirt second) and 1000 μ s.

According to the method, the perforation or the alteration that are produced by laser needn't be realized by the pulse from laser.In preferred specific embodiment, seeing through cuticular perforation or alteration is to utilize a plurality of laser pulses to produce, and each pulse is only bored a hole or the part of the alteration target tissue degree of depth.

For this reason, the energy of using pulse just can a plurality of pulses of rough estimate form perforation or alteration energy needed divided by desirable umber of pulse on horny layer.For example, if if the speckle of specific size needs the energy of 1J to produce passes whole cuticular perforation or alteration, so available 10 pulses produce qualitative similar perforation or the alteration of, and the energy of each pulse is 1/10.Because wish that the patient is at running target tissue (people's response time is about 100ms) not during the irradiation, and wish that the heat that each pulse produces can obviously not spread, so, in preferred specific embodiment, whole perforation procedure is finished in less than 100ms from the pulse recurrence rate of laser.Alternatively, mechanical fixation can be carried out in the location of target tissue and laser, thereby the change of target position also can not take place in longer exposure time.

To cause that in order adopting method seldom hemorrhage or that do not cause bleeding carries out punch skin, can to bore a hole or alteration to skin,, but be not deep into capillary layer such as horny layer by outer surface (outer surface).Laser beam can accurately focus on skin, produces the light beam that diameter is about 0.5 μ m-5.0cm on skin.Alternatively, hot spot can be a shape of slit, and width is approximately 0.05-0.5mm, and length can reach 2.5mm.Width can be a virtually any size, is decided by the anatomical features and the needed infiltration rate that remains to be removed the medicine that liquid maybe will bestow of irradiation zone.But the focal length random length of condenser lens, but in a specific embodiment, be 30mm.

(it is laser output energy (unit is a joule) and focus beam size (cm by change wavelength, pulse width, energy fluence ²) function) and irradiation speckle size, can melt (perforation) and non-melting between the change (alteration) with cuticular influence is controlled at.Cuticular melting with non-melted alteration and all can be increased the permeability of drug administration subsequently.

For example, under the situation that other variable remains unchanged, by reducing pulse energy, the effect that tissue is produced can melt and non-melting between change.Utilize the erbium of pulse width about 300 μ s: YAG laser, adopt pulse or radiant energy and the big speckle of irradiation 2mm on skin, the pulse energy that surpasses about 100mJ can cause partly or entirely melting, and any pulse energy that is lower than about 100mJ then causes cuticular part to melt or non-ly melts alteration.Alternatively, by adopting the multiple-pulse mode, the required threshold pulse energy (threshold pulse energy) of permeability that strengthens the body fluid or the medicine of giving then descends, and the coefficient of decline approximates pulse number greatly.

Replacedly, keep under the constant situation of other variable, by reducing spot size, change to skin is in melt and non-ablation tissue effect between.For example, the facula area that reduces by half will cause producing the required energy in half of same effect.Can obtain little irradiation to 0.5 micron, for example, be coupled in by radiation output (radiant output) in a plurality of object lens of microscope objective (for example, can available from Nikon company, Melville, New York) laser instrument.In this case, the hot spot that laser beam focus on to form may the little order of magnitude to microscopical resolution limit, and the latter is approximately 0.5 micron the order of magnitude.In fact, if laser beam is Gauss distribution, be subjected to the area size that irradiation influences can be, and can surpass microscopical imaging resolution less than the beam sizes of measuring.In this case, for non-ablative ground alteration tissue, adopt 3.2J/cm ²Energy fluence may be proper, it will need the pulse energy of about 5nJ for half micron big hot spot.So low pulse energy can be easily obtains from diode laser, also can, for example, with erbium: the laser beam that YAG laser produces is through absorption filter (as glass) decay back acquisition.

Alternatively, keep its dependent variable constant, by changing the wavelength of radiant energy, change to skin is in melt and non-ablative organizational effect between.For example, utilize holmium: yttrium-aluminium-garnet (Ho:YAG; 2.127 micron) replace erbium: yttrium-aluminium-garnet (Er:YAG; 2.94 laser instrument micron) can make tissue that the absorption of energy is reduced, and produces lighter perforation or alteration.

Psec and dirt pulse per second (PPS) that laser instrument produces also can be used for producing the alteration of skin or melting.This available synthetic diode or relevant micro-slice laser are finished, its output time width (temp oral widths) 1 dirt second to the pulse between the 1ms.(referring to D.Stern etc., " be used in 532 and nanosecond, psec and dirt laser second at 625nm place carry out corneal ablative ", " cornea laser ablation ", (" Corneal Ablation byNanosecond, Picosecond, and femtosecond Lasers at 532and 625nm; " Corneal Laser Ablation) Vol.107, pp.587-592 (1989), it is hereby expressly incorporated by reference document, this list of references has disclosed the application of the pulse width that was low to moderate for 1 dirt second).

Another kind is passed the prescription method and is adopted the high-voltage pulse transient state to produce permeability on skin.Referring to United States Patent (USP) the 5th, 614, No. the 5th, 658,892, No. 502 and United States Patent (USP), both are hereby expressly incorporated by reference document.The high-voltage pulse transient state, for example, stress wave (for example, when stress wave is a laser stress wave (LSW) when being produced by laser, have specific rise time and peak stress (or pressure), can be safely and influence the transhipment of chemical compound (such as those chemical compounds disclosed in the present invention) by epithelial tissue layer (for example horny layer and mucosa) effectively.These methods can be used for the very administration of the chemical compound of large scale scope, and are irrelevant with its net charge.In addition, used pulse transient state can be avoided tissue injury in this method.

Before being exposed to the pulse transient state, epithelial tissue layer (for example, horny layer) may be impermeable to foreign compound; This stops the cellular invasion of this chemical compound below epithelial layer.Epithelial layer is exposed to the pulse transient state can make chemical compound spread by epithelial layer.Usually, the speed of diffusion depends on the character of pulse transient state and the size of the chemical compound that quilt is bestowed.

Transmission rate by specific epithelial tissue layer (for example keratodermatitis), also depend on other several factors, comprise metabolism, the cuticular outside of pH value, dermal matrix tissue (cutaneous substratetissue) and pressure inside is poor and the anatomy position and the physical condition of skin.The physical condition of skin depends on health status, age, sex, race, skin nursing and contact history again.For example, contacted before organic solvent or surfactant can cutaneous physical conditions.

Amount by epithelial tissue layer administered compound depends on that also epithelial layer keeps the penetrating time and the size of permeable epithelial layer surface area that becomes.

The character of pulse transient state and characteristics produce the energy by it and are controlled.Referring to WO98/23325, it is hereby expressly incorporated by reference document.Yet, their characteristics because of its propagate the linearity and the nonlinear characteristic of coupling medium of process change.The radio-frequency component of the main decaying pulse transient state of the linear attenuation that coupling medium causes.This makes bandwidth reduce along with the increase of corresponding rise time of pulse transient state.On the other hand, the nonlinear characteristic of coupling medium shortens the rise time.The shortening of rise time is the result that sound and particle rapidity counter stress (pressure) rely on.Along with stress raises, the velocity of sound and particle rapidity also improve.This makes the forward position of pulse transient state become steeper.The relative intensity of linear attenuation, nonlinear factor and peak stress has determined how far ripple need be propagated, and just can make the steepness increase of rise time become remarkable.

Select the persistent period of rise time, intensity (magnitude) and pulse transient state, nondestructive to produce (that is, non-shock ripple) pulse transient state, it can increase the permeability of epithelial tissue layer temporarily.Usually the rise time is at least 1ns, is preferably about 10ns.

To different epithelial tissue or cellular layer, the peak stress or the pressure of pulse transient state are different.For example, for transmitting chemical compound by horny layer, the peak stress of pulse transient state or pressure should be arranged at least 400 crust; Preferably at least 1,000 crust clings to but can not surpass about 2,000.

Concerning the epithelium mucous layer, surge pressure should be located between 300 crust and 800 crust, is preferably between 300 crust and 600 crust.Preferably, the persistent period of pulse transient state is tens ns, therefore only interacts very short time with epithelial tissue.After the interaction of pulse transient state, epithelial tissue is not subjected to permanent damage, but keeps permeability to reach about 3 minutes most.

In addition, these methods relate to the patient are only applied several discrete high-amplitude pulses.The quantity that imposes on patient's pulse transient state generally is less than 100, better is less than 50, and most preferably less than 10.When a plurality of light pulses were used to produce the pulse transient state, the interval between the adjacent pulse was 10 to 120 seconds, and this is sufficiently long to the permanent damage of being avoided epithelial tissue.

The characteristic of pulse transient state can be measured with the standard method in present technique field.For example, peak stress or pressure and rise time can measure with the method (being described in Doukas etc., ultrasound wave biomedicine (Ultrasound Med.Biol.), 21:961 (1995)) of Kynoar (PVDF) transducer.

Available various energy resources produces the pulse transient state.The physical phenomenon that produces the pulse transient state is selected from following three kinds of different mechanism usually: (1) thermoelasticity produces; (2) optical breakdown; Or melt (3).

For example, melt target material by adopting high energy laser sources, but the excitation pulse transient state is coupled to epithelial tissue or cellular layer by coupling medium with the pulse transient state then.This coupling medium can be that for example, liquid or gel are as long as it is non-linear.Therefore, water, oil (for example Oleum Ricini), etc. ooze medium (as phosphate-buffered saline (PBS)) or gel (as collagen gel), can be used as coupling medium.

In addition, this coupling medium can comprise the surfactant that can strengthen transhipment, for example, after the pulse transient state takes place, by prolonging horny layer chemical compound is kept the penetrating time.This surfactant can be, for example, therefore ionic detergent or non-ionic detergent can comprise, for example, and sodium lauryl sulfate, cetyl trimethyl ammonium bromide and N, N-dimethyl-N-dodecyl amine oxide (lauryl dimethyl amine oxide).

The absorbing target material is taken on the role that optics triggers transducer.Along with the absorption to light, target material experience Rapid Thermal expands, or is melted, thus the transmitted pulse transient state.Usually, metal and polymeric film have high absorption coefficient at visible light and ultraviolet spectra district.

The material of many types can be with laser beam as target material, as long as they are in used Wavelength of Laser absorbing light fully.Target material can be metal (as aluminum or a copper); Plastics (as polystyrene, for example black polystyrene); Pottery; Or high concentration dye solution.The size of target material must be bigger than the sectional area of used laser energy.In addition, target material must be thicker than the light penetration depth to guarantee that light can not reach skin surface.Target material also must be enough thick in mechanical support to be provided.When target material is when being made of metal, typical thickness can be that 1/32 inch (0.79375mm) is to 1/16 inch (1.5875mm).Concerning the plastics target material, thickness is that 1/16 inch (1.5875mm) is to 1/8 inch (3.175mm).

Also can utilize the restricted intensifier pulse transient state that melts.In restricted melting, the laser beam transparent material as quartzy optical window, is placed on the position of tight contact target material.The restriction of plasma (utilize transparent material that target material is melted and produce) has improved the coefficient of coup (Fabro etc., applied physics magazine (J.Appl.Phys), 68:775,1990) on the order of magnitude.This transparent material can be quartz, glass or transparent plastic.

Because of allowing plasma, the space between target material and the restricted transparent material expands, therefore reduced the momentum (momentum) of passing to target, so transparent material preferably adopts originally for liquid binding agent (such as carbonaceous epoxy material) is bonded in target material, to prevent this class space.

Laser beam can produce with the standard light modulation technique of knowing in the industry, as using Q-switch or mode-locked laser, utilizes, for example, electro-optical device or acousto-optic device.What standard commercial purchased can adopt the laser instrument of impulse form comprise Nd:YAG laser instrument, Nd:YLF laser instrument, CO at infrared ray, visible light and/or infrared spectrum ₂Laser instrument, excimer laser, dye laser, Ti: sapphire laser, diode laser, holmium (and other rare earth material) laser instrument and metallic vapor laser.The pulse width of these light sources is adjustable, and can change between hundreds of microseconds at tens of psecs (ps).In an application of the invention, light impulse length can change between 100ps and about 200ns, preferably approximately between 500ps and the 40ns.

The pulse transient state can also be produced (example can be referring to Coleman etc., ultrasound wave biomedicine (Ultrasound Med.Biol.), 15:213-227,1989) by the external stone crushing device.The rise time of these pulse transient states is 30 to 450ns, and it is longer than pulse transient state that laser produces.In order to form pulse transient state (for utilizing the new method of external stone crushing device), allow the pulse transient state in non-linear coupling medium (as, water), propagate certain distance (calculate and get) with aforementioned equation (1) with suitable rise time.For example, when the pulse transient state that adopts lithotrite to produce had the surge pressure of the rise time of 100ns and 500 crust, with before epithelium layer contacts, the distance that the pulse transient state should be walked in coupling medium was approximately 5mm.

Another benefit of the pulse transient state that is produced by lithotrite being repaired (shaping) is that as the result who propagates in non-linear coupling medium, the tensile product of ripple (tensile component) will broaden and decay.This propagation distance should be adjusted and produce such pulse transient state, the pressure of its tensile product only be ripple compressed component surge pressure about 5% to 10%.Like this, trimmed pulse transient state can damaged tissue.

The type of used lithotrite does not have strict restriction.Can adopt the lithotrite of electro-hydraulic, electromagnetic or piezoelectricity.

The pulse transient state also can be produced by transducer, as piezoelectric transducer.Preferably, this transducer directly contacts with coupling medium, and displacement rapidly after applying light field, thermal field or electric field, thereby produces the pulse transient state.For example, can adopt dielectric to puncture, and adopt usually high-voltage spark or piezoelectric transducer (with use in some extracorporeal lithotiptor similar, Coleman etc., ultrasound wave biomedicine (Ultrasound Med.Biol.), 15:213-227,1989) bring out.Under the situation that adopts piezoelectric transducer, this transducer stands rapid expanding after applying electric field, thereby causes fast offset in coupling medium.

In addition, pulse transient state can produce under fiberoptic help.The special easy operating of optical fiber feed system, and can be used for the irradiation target material adjacent with the epithelial tissue layer, thus produce the pulse transient state in the position that is difficult to arrive.When on the optics during with the laser instrument coupling, the feed system of these types is preferred, because it can be incorporated in conduit and the respective flexible device, and is used for most organs in the irradiation human body.In addition, in order to launch the rise time with expection and the pulse transient state of peak stress, the wavelength of light source can easily carry out shaping, to produce suitable absorption in the particular target material.

Replacedly, response firing pulse (detonating impulse), high energy material can produce the pulse transient state.Detonator can be ignited high energy material by producing discharge or electric spark.

Hydrostatic pressure and the coupling of pulse transient state can be passed the chemical compound transhipment of epithelial tissue layer with enhancing.Because the effect that the pulse transient state causes continues several minutes, apply the method for hydrostatic pressure by after the pulse transient state on epithelial tissue layer (for example, keratodermatitis) surface, medicine is complied with the transport speed that its Concentraton gradient passes epithelium layer and can be accelerated.

The genetic modification of stem cell or CFU-GM

Gene

The useful gene of the present invention and genetic fragment (polynucleotide) are comprised that infection that those encode T cells cause specific infector has the gene and the genetic fragment of resistance.This class infective agent includes but not limited to that HIV, T chronic myeloid leukemia virus and other cause the virus of lympahadenism.For HIV/AIDS, can use several genes and genetic fragment, include but not limited to: nef transcription factor (nef transcription factor); The coding specificity cuts off the gene of the ribozyme of HIV gene, as tat and rev (Bauer G. etc., 1997); The anti-dominant mutant of HIV-1rev gene (trans-dominant mutant form), RevM10, it has shown that can suppress HIV duplicates (Bonyhadi etc., 1997); The expression structure (Kohn etc., 1999) excessively of HIV-1rev response element (RRE); Coding RNA or proteic any gene, its expression can suppress cell HIV infection or duplicate; And fragment and combination.

These genes or genetic fragment are used with the form of stably express.As employed at this paper, term " stably express form " is meant, after gene or genetic fragment (" function fragment ") changed host cell over to, its product (RNA and/or albumen) can be expressed to not a half in the filial generation after this cell and division and/or the differentiation consistently.Whether this needs this gene or genetic fragment, no matter be contained in the carrier, all has the signal sequence of suitable DNA to rna transcription.In addition, when gene or genetic fragment encoded protein are when influencing the bioactive molecule of patient's states, the DNA translation signals of also will encoding.

In most of the cases, gene or genetic fragment are included in the carrier.One of ordinary skill in the art is known and be can be used for expressing required RNA or proteic expression vector.Expression vector is to guide the carrier of transcribing wherein contained DNA sequence and can guide special translating purpose RNA (resulting RNA).Expression vector can duplicate in the cell that will carry out genetic modification, and comprises plasmid, phage, virus and mini-chromosome.Replacedly, this gene or genetic fragment can become the integrated part of cell chromosome DNA.Recombinant vector and methodology are well known in the art.

Be used to express proteic expression vector of the present invention and comprise origin of replication.Suitably the expression vector that makes up comprise can be in cell the origin of replication of self-replicating, maybe can be incorporated in the host cell chromosome.Such carrier also can comprise useful restriction endonuclease sites, high copy number and the strong promoter of selected marker thing, limited quantity.Promoter is a DNA sequence, and its guiding RNA polymerase is attached to DNA and causes RNA synthetic; Strong promoter can cause this initial by high frequency.Expression vector of the present invention is the DNA that can be operatively connected in coding RNA or albumen (being used for the present invention), that is, this carrier can guide and adhere to duplicating of dna molecular, can guide the RNA or the proteic expression of dna molecule encode again.Therefore, for albumen, this expression vector must have the suitable transcription initiation signal upstream fragment of adhering to dna molecular, keeps correct reading frame, thereby allows expressible dna under the regulation and control of regulating and controlling sequence; Molecule and produce desired albumen by dna molecule encode.Expression vector can include but not limited to the cloning vehicle of cloning vehicle, modification and the plasmid or the virus of particular design.Preferably, adopt evoked promoter, thereby can control the quantity and the time of inserting gene or polynucleotide expression.

Cell

Hematopoietic stem cell (HSC) is the preferred cell that is used for genetic modification.These cells can derive from bone marrow, peripheral blood or umbilical cord or any other hematopoietic stem cell source, and can be from body or non-from body.Lymph sample and bone marrow CFU-GM and epithelial stem cell also are useful, and also can be from body or non-from body.

If use non-ly, the toleration of these cells is then produced in the activated again step of thymus from body (donor) cell.Pass in the initiating process of the signal of the sex steroid mediation of thymus or thereafter, relevant genetically modified donorcells is implanted in the receptor body in blocking-up.These cells are admitted by donor thymus, and become the new T cell of thymus generation and the part of DC.Therefore the T cell mass that is obtained all is identified as autogenous cell with receptor and donorcells, can produce the toleration from the graft of donor.Referring to the common pending application application U.S.S.N.09/ that proposes,, it is hereby expressly incorporated by reference document.

The method of genetic modification

The recombination method of standard can be used for mediating the genetic modification thing and enters in the cell that is used for gene therapy.For example, the HSC of retrovirus vector transduction cultivation is a kind of successful method (Belmont and Jurecic, 1997, Bahnson, A.B. etc., 1997).Other carrier includes but not limited to that adenovirus is derived or deutero-carrier of slow virus and the deutero-carrier of Moloney murine leukemia virus.

Also can adopt following method: the gene transfer of microgranule mediation (particle-mediated) is as using particle gun (Yang and Ziegelhoffer, 1994), liposome-mediated gene transfer (Nabel etc., 1992), the co-precipitation of calcium phosphate and genetic modification carrier (Graham and Van Der Eb, 1973), electricity punching (Potter etc., 1984), and microinjection (Capecchi, 1980), and any other can be stably be transferred to HSC with gene or oligonucleotide (preferably in carrier) and will carry out in other cell of genetic modification, so that the method expressed in part-time at least of gene.

Prevention

The invention provides by activating the method for patient's thymus prevention infection or enhancing anti-infection ability again.This is to realize to the signal of thymus by the biography that the cuttability steroid mediates.In this stage, patient's immune system is restored, and has strengthened its responsibility to exotic antigen (as virus and antibacterial).Shown in Figure 10-15, it has illustrated that thymus activates the influence to the mouse immune system again under the situation that virus (HSV) is invaded.Carry out the activated again mice of thymus in advance and show the resistance that HSV is infected, and those do not carry out the mice that thymus activates (old thymus) again and have more serious HSV to infect.As everyone knows, the mouse immune system is very similar with human immune system, and the result of the test of mice can reflect human situation.This is further confirmed by the above-mentioned human thymus test data of activated effect again.

A further step promptly replenishes natural (native) hematopoietic cell to the patient in the activated again process of thymus, increased patient's body's immunological function.Particularly, by giving the cell of genetic modification to receptor, immune system can produce specific reaction to synantigen not.Genetically modified cell can be hematopoietic stem cell (HSC), epithelial stem cell or hemopoietic progenitor cell.Preferably, this genetically modified cell is CD34 ⁺HSC, lymph CFU-GM or bone marrow CFU-GM.Preferably, this genetically modified cell is CD34 ⁺HSC.Genetically modified cell is bestowed in patient's body, and passes through the peripheral blood system migration to thymus.Lacking under the situation of sex steroid, thymus obviously strengthens the picked-up of these hemopoietic forebody cells.These cellular integration are gone into thymus, and produce dendritic cell and the T cell that has from the genetic modification of these altered cells.The result is, circulates in to be subjected to have in the peripheral body T cell mass that expection heredity changes and to activate the increase of the corresponding cell mass, tissue and the organ that cause again by patient's thymus, and it can resist rapid, the special reaction of former generation.

Influence to bone marrow and HSC

The invention provides increases patient's bone marrow output, comprises the method that increases HSC output.This can be used for multiple situation.For example, no matter one of serious side reaction of chemotherapy is to be used for cancer or other purpose, can be its negative effect to patient's bone marrow (negative impact).The dosage that depends on chemotherapy, bone marrow may be destroyed, and the generation of hemocyte may be prevented from.According to the present invention, after chemotherapeutic treatment, give the LHRH analog of doses, can help to recover destructive bone marrow of chemotherapy and hemocyte.Replacedly, before chemotherapy, give the quantity that the LHRH analog can increase HSC and other hemocyte, thereby can reduce the influence of chemotherapy.

In some chemotherapy situations (regiment), for example treat the high-dose chemotherapy of any leukemia, to the destruction of bone marrow the consequence of anticipation.Method of the present invention can be used after destroying generation immediately, stimulates bone marrow and increases HSC and the output of its offspring's hemocyte, thereby shorten patient's convalescent period.After chemotherapy, allow the time of 1 day or several days that chemotherapeutics is removed in patient's body usually, according to method as herein described, the LHRH analog of doses can give the patient.This can use with merging such as factors such as stem cell factors from body or allosome (heterologous) bone marrow or hematopoietic stem cell or CFU-GM and other.

In addition, the patient can have " asthenia " bone marrow, and may not produce HSC and other hemocyte of the sufficient amount of keeping normal function.This can be caused by multiple situation, comprise the infection, chemotherapy of usual aging, prolongation after, after the radiotherapy, chronic disease state (comprising cancer), gene unconventionality and transplant the immunosuppressant that causes.Further, radiation (for example whole body radiation) can produce bigger influence to bone marrow output.These situations also can be by advanced processing at utmost reducing untoward reaction (for example to chemotherapy and/or radiotherapy), or can handle after taking place and reverse this effect.

Small animal research

Material and method

Animal

CBA/CAH and C57B16/J male mice available from animal service centre of Monash university are raised under normal condition.Thoughtful 26 months age at 4-6, and correlation circumstance carried out labelling.

Castrating

Contain 0.3mg xylazine (xylazine hydrochloride to animal intraperitoneal injection 0.3ml; Bayer Australia company, Botany NSW, Australia) and 1.5mg ketalar (Ketalar (Ketalar); Parke-Davis company, Caringbah, NSW, Australia) normal saline anaesthetize.Carrying out of surgery castrating is as follows: scrotum cuts, exposes testis, hitches testis, removes testis in company with fatty tissue on every side then with suture.

Bromodeoxyribouridine (BrdU) mixes

Mice is accepted twice intraperitoneal injection BrdU (Sigma chemical reagents corporation, St.Louis, the Missouri State) (the 100mg/kg body weight is in 100 μ l PBS), and interval is 4 hours.Control mice receives only the injection of excipient.Injected back one hour for the second time, cut thymus, perhaps make cell suspension and carry out facs analysis, perhaps in TissueTek (O.C.T. chemical compound, Miles company, the state of Indiana), carry out embedding immediately, in liquid nitrogen freezing rapidly and be stored in-70 ℃ stand-by.

Flow cytometry is analyzed

Use CO ₂Smother play is put to death mice, takes out thymus, spleen and mesenteric lymph node.Organ is in cold PBS/1%FCS/0.02% azide, and the filter screen by one 200 μ m gently filters, and centrifugal (650g, 5 minutes, 4 ℃) are resuspended in any PBS/FCS/ azide.Splenocyte was hatched 10 minutes in erythrocyte splitting buffer (8.9 grams per liter ammonium chloride) under 4 ℃, washed and was resuspended in the PBS/FCS/ azide.The concentration of cell and survival degree measure twice with blood counting instrument and ethidium bromide/acridine orange, observe (Axioskop company under fluorescence microscope; Carl Zeiss, Oberkochen, Germany).

Detect for carrying out trichroism immunofluorescence, with thymocyte cell with anti-α β TCR-FITC or anti-gamma delta T CR-FITC, anti-CD4-PE and anti-CD8-APC (all available from Pharmingen company, Santiago, California) carry out conventional labelling, carry out flow cytometry analysis then.Spleen and lymph node suspension carry out labelling with α β TCR-FITC/CD4-PE/CD8-APC or B220-B (Sigma company) together with CD4-PE and CD8-APC.B220-B develops the color with the trichroism conjugate of streptavidin (available from Caltag laboratory company, Burlingame, California).

Detect for carrying out BrdU, cell carries out surface markers with CD4-PE and CD8-APC, fixes and the permeability processing (Carayon and Bord, 1989) as discussed previously subsequently.Briefly, painted cell with the 1%PFA/0.01% tween 20 at 4 ℃ of fixing O/N.Washed cell is hatched 30 minutes in 500 μ l DNA enzymes (100 hole Ni Zi units, Boehringer Mannheim company, West Germany) under 37 ℃, so that the DNA degeneration.At last, cell is hatched with anti-BrdU-FITC (Becton-Dickinson company).

Detect for carrying out four color immunofluorescences, thymocyte cell carries out labelling with CD3, CD4, CD8, B220 and Mac-1, detects with the Mus Ig-Cy5 of the Chinese People's Anti-Japanese Military and Political College (Amersham company, Britain) is common, and the negative cells of doorization (TN) is used for analyzing.Then use CD25-PE (Pharmingen company) and CD44-B (Pharmingen company) dyeing, then with the trichroism conjugate of streptavidin (Caltag company, California) colour developing, (Godfrey and Zlotnik, 1993) as previously mentioned.Carrying out BrdU according to preceding method then detects.

Sample is analyzed with FacsCalibur (Becton-Dickinson company).The lymphocyte of survival carries out a change according to 0 ° and 90 ° of light scattering diagrams, and data analysis adopts cell searching (quest) software (Becton-Dickinson company).

SABC

Freezing thymus section (4 μ m) is cut into slices with household freezer (Leica), and uses 100% acetone fixed immediately.

Detect for carrying out the dichromatism immunofluorescence, section is carried out double labelling: MTS6,10,12,15,16,20,24,32,33,35 and 44 (Godfrey etc., 1990 with one group of monoclonal antibody of this Laboratory Production; Table 1), and with the coexpression of multivalence rabbit anti-cell keratin antibody (Dako company, Carpinteria, California) assessment epithelial cell determiner (determinants).Bonded monoclonal antibody (mAb) is then used the conjugated goat anti-rabbit immunoglobulin of TRITC (Silenus laboratory) colour developing with the conjugated sheep anti rat immunoglobulin of FITC (Silenus laboratory) colour developing, anti-cell keratin.

Detect for carrying out BrdU, will cut into slices and dye with anti-rabbit-TRITC then, or, use the Mus Ig-C γ of the Chinese People's Anti-Japanese Military and Political College 3 (Amersham company) colour developing then with special mAb dyeing with the anti-cell keratin.Carry out BrdU according to preceding method then and detect (Penit etc., 1996).Briefly, section is fixed 30 minutes in 70% ethanol.Half-dried section is hatched in 4M hydrochloric acid, washs with neutralization with borate buffer (Sigma company), afterwards with twice of PBS flushing.Detect BrdU with anti-BrdU-FITC (Becton-Dickinson company).

Detect section specificity MTS mAb and anti-cell keratin labelling together for carrying out trichroism immunofluorescence.Carrying out BrdU according to preceding method then detects.

With Leica fluorescence microscope and Nikon confocal microscope section is analyzed.

Migration research

Contain 0.3mg xylazine (xylazine hydrochloride to animal intraperitoneal injection 0.3ml; Bayer Australia company, Botany NSW, Australia) and 1.5mg ketalar (Ketalar; Parke-Davis company, Caringbah, NSW, Australia) normal saline anaesthetize.

The details of FITC labelling thymocyte cell technology and other place (Scollay etc., 1980; That Berzins etc., 1998) describes is similar.Briefly, expose lobes of thymus, and every leaf is injected the 350 μ g/ml FITC (in PBS) of about 10 μ m (μ l).Wound is through sewing up with operation needle, and the insulation mice is up to recovering from narcotism.The about 24h in injection back, mice is used CO ₂Smother play is put to death, and takes off lymphatic organ and is used for analyzing.

Behind the cell counting, sample is analyzed with flow cytometry then with anti-CD4-PE and anti-CD8-APC dyeing.The cell of migration is considered to express (live-gated) FITC+ cell (with eliminating autofluorescence cell and doublet (doublets)) of doorization of the work of CD4 or CD8.The percentage rate that adds FITC+CD4 and cd8 cell obtains the gross migration of lymph node and spleen respectively.Every day, output rating calculated with the method for (1998) such as Berzins.

The azygous t of The data check or non-parametric graceful-the Whitney check analyzes, and is used for determining the statistical significance between contrast and the experimental check result, experiment repeats 3 times at least.The significant difference that test data is compared with contrasting data is represented with following manner: ^*P≤0.05, ^*P≤0.01 and ^{* *}P≤0.001.

The result

Age is to thymocyte cell group's influence

(i) thymic weight and thymocyte cell number

With advancing age, thymic weight (Figure 1A) and total thymocyte cell number (Figure 1B) all significantly reduce (p≤0.0001).Relative thymic weight (mg thymus/g body weight) meansigma methods of young adult is 3.34, then reduces to 0.66 (because lipidosis can't accurate Calculation) in the time of 18-24 month.The reduction of thymic weight is attributable to the minimizing of thymocyte cell sum: the thymus of the 1-2 month contains～and 6.7 * 10 ⁷Thymocyte cell, in the time of 24 months cell number then be reduced to～4.5 * 10 ⁶Castrating makes sex steroid eliminate the effect of thymus, then thymus regeneration, and after castrating 4 weeks, the weight of thymus and cell number all reach the level (Figure 1A and 1B) suitable with young adult.What is interesting is 2 whens week after castrating, thymocyte cell number (～1.2 * 10 ⁸Individual) show a marked increase (p≤0.001), 4 weeks the time returned to normal level (Figure 1B) at an early age after castrating.

The minimizing of the T cell number that thymus produces is not reflected in periphery, and spleens cell number is with age growth remain unchanged (Fig. 2 A).The homeostasis mechanism of periphery is significantly, because the influence (Fig. 2 B) that ratio is not subjected to the age and the T cell number of the periphery of arriving soon after reduces of B cell and T cell in spleen and the lymph node.Yet CD4+ obviously reduces (p≤0.001) with the CD8+T cell proportion with age growth, 1: 1 (Fig. 2 C) when during from February 2: 1 are reduced to 2 years old.After castrating increased with the T cell number that enters periphery subsequently, do not observe the variation of periphery T cell number: the B in splenic t-cell number and spleen and the lymph node: the T cell proportion did not change (Fig. 2 A and B) because of castrating.CD4 in the periphery that causes with age growth: CD8 ratio to be reduced in the castrating back still obvious during 2 weeks, but 4 weeks of castrating back just recover (Fig. 2 C) fully.

(ii) α β TCR, gamma delta T CR, CD4 and CD8 express

For determining whether the minimizing with age growth thymocyte cell number is the result of specific cells group loss, and thymocyte cell carries out labelling with particular marker, to analyze different subgroups.In addition, this allows the kinetics of castrating back thymus regeneration (repopulation) is analyzed.The ratio of main thymocyte cell subgroup compares (Fig. 3) with those normal young thymus, finds that it is not with change of age.In addition, thymocyte cell is segmented by express alpha β TCR and gamma delta T CR, the ratio that shows these cell masses is not with change of age (data not shown).In 2 week and 4 weeks after castrating, the thymocyte cell subgroup keeps same ratio, and because thymocyte cell quantity rises to after castrating up to 100 times, this shows the synchronous amplification of all thymocyte cell subgroups, and is not the progressively amplification of evolved.

Therefore it seems that the minimizing of cell number be the balanced result who reduces of all cells phenotype in this geriatric animals thymus, and do not see the remarkable change of T cell colony.Thymus regeneration is carried out with the method for synchronization, replenishes all T cell subsets simultaneously, rather than replenishes in order.

The propagation of thymocyte cell

As shown in Figure 4, the thymocyte cell of 15-20% is bred during week at 4-6.Most of (～80%) is DP, TN subgroup take second place (～6%) (Fig. 5 A).Correspondingly, by SABC, the most cells division sees under the tunicle and cortex (data not shown).Can see some cell divisions in the medullary substance district with facs analysis, display part SP cell (9% cd4 t cell, 25% cd8 t cell) division (Fig. 5 B).

Although cell number obviously reduces in older thymus, it is constant that the propagation of thymocyte cell still keeps, and reduced to 12-15% (Fig. 4) in the time of 2 years old, the thymus similar (Fig. 5 A) when breeding the phenotype of colony and February simultaneously.Cell division when SABC discloses 1 years old reflects the sight of young adult; Yet in the time of 2 years old, propagation is mainly seen in (data not shown) around outer cortex and the vasculature.In 2 weeks after castrating, although the thymocyte cell number obviously reduces, the constant rate of the thymocyte cell of propagation shows the synchronous amplification (Fig. 4) of cell once more.The scope that SABC has disclosed value-added location of thymocyte cell and somatoblast is similar to the situation (data not shown) in 2 months thymus 2 weeks after castrating.When the ratio of each subgroup of analyzing representative propagation group, (when February and 2 years old 1%, after castrating, be increased in 2 weeks～6%) (Fig. 5 A) that obviously raise (p＜0.001) of the cd8 t cell percentage ratio in the propagation colony.

Fig. 5 B shows young, older and castrates in the mice of back the multiplication nursery of each subgroup.The propagation of DN subgroup has tangible decay (p≤0.001) (4% during during February 35% to 2 years old).The propagation of CD8+T cell also obviously descends (p≤0.001), has reflected result's (data not shown) of SABC, i.e. no tangible cell division in the medullary substance of older thymus.In 4 weeks after castrating, the decline of DN propagation does not return to normal level at an early age.Yet the propagation of CD8+T cell subsets 2 weeks after castrating the time obviously increase (p≤0.001), and return to the normal year light water 4 weeks in the castrating back flat.

Further the propagation in the DN subgroup is descended with CD44 and CD25 labelling and to analyze.Also comprise α β TCR+CD4-CD8-thymocyte cell in the DN subgroup except that containing the thymocyte cell precursor, the latter is considered to down two kinds of coreceptors (co-receptor) (Godfrey and Zlotnik, 1993) of tuning SP cell conversion.By these mature cells being carried out a change (gating), just can analyze real TN compartment (compartment) (CD3 ^-CD4 ^-CD8 ^-), these do not show difference (Fig. 5 C) with age or castrating on the rate of increase.Yet, the subgroup of expressing CD44 and CD25 is analyzed, show TN1 subgroup (CD44 ⁺CD25 ^-) propagation obviously descend (p＜0.001), from normal 20% about 6% (Fig. 5 D) when dropping to 18 months at an early age, its 4 weeks after castrating recover.The propagation of TN1 subgroup descends by TN2 subgroup (CD44 ⁺CD25 ⁺) the obvious increase (p≤0.001) of propagation compensates, the latter returns to the normal year light water 2 weeks after castrating and puts down (Fig. 5 D).

Age is to the influence of thymus microenvironment

Use is used immunofluorescence from MTS series, with the monoclonal antibody of polyclone anti-cell keratin antibody double labelling, observes the variation of thymus microenvironment with the age.

Antigen by these monoclonal antibodies (MAbs) identification can be divided into 3 groups: thymus epithelial subgroup, blood vessel related antigen and those antigen that all exists on stromal cell and thymocyte cell.

(i) epithelial antigen

2 years old big mouse thymus carried out anti-keratin dyeing (general epithelium), show the forfeiture of overall thymus structure, the forfeiture of following serious epithelial cell disordering to be connected with tangible cortex-medullary substance.Further analyze with monoclonal antibody MTS 10 (medullary substance) and MTS 44 (cortex), show that the cortex size obviously reduced with the age, with the not really significantly minimizing (data not shown) of medullary substance epithelium.No epithelial cell zone, or the negative zone of keratin (KNA ' s, van Ewijk etc., 1980; Godfrey etc., 1990; Bruijntjes etc., 1993) more obvious in older thymus, and increase, as anti-cell keratin labelling finding.In older thymus, also see thymus epithelial " cryptomere " structure, in the medullary substance district particularly evident (data not shown).By the anti-cell keratin stain, clearly show lipidosis, thymus volume seriously dwindle and cortex-medullary substance connects the decline (data not shown) of integrity.Begin regeneration thymus 2 weeks after castrating.The increase of the cortex epithelium that this obviously shows the size of lobes of thymus, show by MTS44 and the location of medullary substance epithelium.The medullary substance epithelium detects with MTS10, and when 2 weeks, still useful MTS 10 painted secondary capsules (subpockets) are dispersed in the cortex.Just there are clearly medullary substance and cortex in castrating 4 weeks of back, and recognizable cortex-medullary substance connects (data not shown).

Labelling MTS20 and 24 is considered to can be used for detecting primary epithelial cell (primordialepithelial cells) (Godfrey etc., 1990), and further shows the degeneration of older thymus.They are a lot of at E14, can detect isolated medullary epithelial cell group 4-6 week, but intensity increase (data not shown) once more in older thymus.After the castrating, all these antigens are the horizontal expression (data not shown) to be equivalent to young adult thymus all, and MTS20 and MTS24 rotate back into the dispersive secondary capsule (subpockets) that is arranged in cortex-medullary substance bonding pad.

(ii) blood vessel related antigen

Blood-thymus barrier is considered to be responsible for the migration of T cell precursors to thymus, and mature T cells is from the migration of thymus to periphery.

15 pairs of thymus blood vessel endotheliums of MAb MTS have specificity, present the colored graph picture (Godfrey etc., 1990) of graininess, disperse.In older thymus, the expression of MTS 15 significantly increases, and reflects the increase (data not shown) with volume of increasing of blood vessel and perivascular space.

The thymocyte cell epimatrix comprises important structure and cell adhesion molecule, for example collagen protein, laminin and fibrinogen, and available mAb MTS 16 detects.MTS 16 is expressed in the normal young thymus and disperses, and more spreads and is connected with each other and become in old thymus.2 weeks of castrating back that are expressed in of MTS16 further increase, and after castrating 4 weeks, the situation of this expression reflection is similar to 2 monthly age thymus (data not shown).

(iii) total antigen

The MHC II in normal young thymus that detects with MAb MTS 6 is expressed in and is strong positive (granule) (Godfrey etc., 1990) in the cortex epithelium, a little less than the medullary substance epithelium then dyes.Older thymus shows the reduction that MHC II expresses, and its expression of 2 weeks obviously increases after castrating.In castrating 4 weeks of back, expression reduces once more and seems and big thymus similar (data not shown) in February.

A little less than.Older thymus shows the reduction that MHC II expresses, and its expression of 2 weeks obviously increases after castrating.In castrating 4 weeks of back, expression reduces once more and seems and big thymus similar (data not shown) in February.

Thymocyte cell is moved out

In the young mice body, the T cell of every day nearly 1% thymus (Scollay etc., 1980) of moving out.We found 14 months and even 2 years old mice, although quantitatively obviously reduce (p≤0.0001), the ratio of its migrating cell and normal young mice be (Fig. 5) quite.The ratio of CD4: CD8 increases to some extent in the thymocyte cell that move out recently in (school: take care), during from 2 months～when being raised to 26 month at 3: 1～7: 1.In castrating one week of back, the cell number that migrates to periphery obviously increases, and gross migration still remains on 1-1.5%.

Embodiment

The following examples provide the specific embodiment of method of the present invention, and it describes the restriction that does not constitute content of the present invention.For convenience's sake, these embodiment have described and have given the LHRH agonist and block biography to the signal of the sex steroid of thymus mediation and the prevention that HSV is infected that produced.

Embodiment 1

The sex steroid ablation

If relate to unmatched donor, then give patient's immunosuppressant therapy (for example cyclosporin treatment), to prevent repulsion to donorcells.

Employing is carried out the sex steroid ablation to the mode that the patient uses the LHRH agonist.With Leucrin (leuprorelin acetate, durative action preparation injection; 22.5mg) or goserelin acetate (inculcate; 10.8mg) form administration, any single dose can be kept curative effect 3 months.This can effectively reduce the sex steroid level, thereby is enough to activate again thymus.In some cases, also need to suppress the inhibitor that the adrenal gland produces sex steroid, for example during the sex steroid ablation, take a slice Cosudex (5mg/ days) every day.The sex steroid that the adrenal gland produces accounts for the 10-15% of human body steroid.

Sex steroid in the blood is reduced to minimum approximately needs 1-3 week, and corresponding with it is the activation again of thymus.In some cases, be necessary the extended treatment time, carry out second trimestral injection/inculcate the course of treatment.

Embodiment 2

Other medications

Can adopt other method to substitute the medication that the LHRH agonist was injected or inculcated to this 3 months durative action preparation.In one embodiment, patient's skin laser may is (as erbium: YAG laser) carry out irradiation, melt or alteration skin, thereby reduce cuticular stopping effect (impeding effect).

A. laser ablation or alteration: the iraser pulses of radiation are by solid-state, pulsed erbium: YAG laser produces, and this laser instrument is by forming as the lower part: two planar resonant cavity reflection mirrors (flat resonator mirrors), as the erbium of active medium: the mode of yag crystal, power supply and focussed laser beam.The wavelength of laser beam is 2.94 microns.Adopt pulse.

Operating parameter is as follows: the energy of each pulse is 40,80 or 120mJ, and the beam size of focal position is 2mm, and the energy fluence of generation is 1.27,2.55 or 3.82J/cm ²The burst length width is 300 μ s, and the energy fluence rate of generation (energy fluence rate) is 0.42,0.85 or 1.27 * 10 ⁴W/cm ²

Subsequently, the LHRH agonist of doses spreads on skin, and is scattered in the irradiation position.The form of this LHRH agonist can be an ointment, and it can remain on the irradiation position like this.Alternatively, sealing paster (occlusive patch) can place on the agonist, thereby it is remained on the irradiation position.

My god.

B. pressure wave: the LHRH agonist of doses is placed in suitable container and (in deformable plastic washer (washer) (diameter about 1 inch (25.4mm), thickness about 1/16 inch (1.5875mm)), is put in the skin place that will produce pressure wave.The skin at this position covers with target material (as the thick black polystyrene sheet of about 1mm).(pulse persistance 20ns, each pulse production capacity reaches 2 joules) produces laser beam with the solid-state ruby laser of Q-switch, and the latter hits target material and produces the pulse transient state.Black polystyrene target material absorbs laser emission fully, so skin only is exposed to the pulse transient state, and can not be exposed to laser emission.This process can not cause pain.But this operation repetition every day, or carry out as required, the level of agonist in the blood circulation kept.

Embodiment 3

Applying HSC strengthens thymus and activates again

From the patient, or preferably collect HSC from donor.Alternatively, these cells can increase by In vitro culture under the condition that stem cell factor (SCF) arranged.Give behind the LHRH agonist approximately 1-3 week, when thymus begins to activate again or before this, give patient infusion HSC, optimal dose is about 2-4 * 10 ⁶Cell/kilogram.Alternatively, G-CSF also can be injected in patient's body, helps the amplification of HSC.Again activated thymus picked-up HSC and donor HSC is converted into the T cell and the dendritic cell of donator type is converted into patient's HSC the T cell and the dendritic cell of patient's type.By inducing removing (passing through cell death), or by inducing tolerance (passing through immunity regulatory cell), the donor dendritic cell can tolerate any T cell that receptor is had potential reaction.

Embodiment 4

HSC is carried out genetic modification prevents HIV to infect

In practice, before cell harvesting, injected granulocyte colony-stimulating factor (G-CSF) 2-5 days, strengthen the HSC level in patient or the donor blood by dosage with 10 μ g/kg.CD34 ⁺Donorcells purification from donor or patient's blood or in the bone marrow obtains, and preferably adopts flow cytometry or immunomagnetic beads.HSC is confirmed as CD34 by the flow cytometry analysis ⁺Alternatively, these HSC can increase at external use SCF.

Retrovirus vector is built as the anti-dominant mutant that contains the HIV-1rev gene, RevM10, and it can suppress HIV and duplicate (Bonyhadi etc., 1997).The supernatant that contains both sexes (Amphotropic) carrier is to be obtained by the filtering supernatant infection of having a liking for Producer cell (ecotropic producer cells) through the carrier transfection.The CD34 that collects ⁺Cell is placed in pre-the stimulation 24 hours in the LCTM culture medium that is added with IL-3, IL-6 and SCF (every kind of 10ng/ml), enters cell cycle with inducing cell.Then, in cell, repeat to add supernatant 2-3 days that contain this carrier, realize that carrier transcribes to intracellular.

About 1-3 week after using the LHRH agonist, when thymus begins to activate again or before, give the genetically modified HSC of patient infusion, optimal dose is about 2-4 * 10 ⁶Cell/kilogram.Alternatively, G-CSF also can inject in the receptor body, helps the amplification of HSC.Again activated thymus absorbs genetically modified HSC, and they are converted into the T cell and the dendritic cell of donator type, simultaneously the HSC of receptor is converted into the T cell and the dendritic cell of receptor type.By inducing removing (passing through cell death), or by inducing tolerance (passing through immunity regulatory cell), the donor dendritic cell can tolerate any T cell that receptor is had potential reaction.

Embodiment 5

Immunosuppressant termination when using donorcells

When the thymus chimera has been set up and new mature T cells group when beginning to discharge from thymus, gather patient's blood, and the reactivity of donorcells is lacked (lack ofresponsiveness) with standard lymphocyte hybrid reaction vitro detection T cell.If not reaction, immunosuppressant therapy then reduces gradually, to set up the resistance to infecting.If there is not rejection,, then finally stop immunosuppressant therapy fully as by existing activating T cell part ground to show in the blood.Because HSC has very strong self renewal ability, so the hemopoietic chimera (chimera) that forms in theory the patient all be in life stable (as for normally, not for toleranceization and the Nonimplantation crowd).

Embodiment 6

Come activation of human thymus again with the LHRH agonist

In order to show that human thymus can activate again by method of the present invention, these methods are used for those patients through the carcinoma of prostate chemotherapy.In April before the sex steroid ablation and after the treatment, the patient assesses to carcinoma of prostate.The results are summarized among Figure 23-27.Generally, data show that the T cell state all has improvement in many patient's bodies on quality and quantity.

LHRH treats the influence to total lymphocyte number and T cell subsets thereof:

Analyzed the patient's's (equal＞60 years old) who accepts LHRH agonist treatment carcinoma of prostate peripheral blood lymphocyte phenotype and formed (Figure 23).Patient's specimen analyzed in 4 months before treatment and behind the beginning LHRH agonist treatment.Before the treatment all patients' the total lymphocyte number of every milliliter of blood be control value than low side.After the treatment, 6/9 patient's total lymphocyte counting obviously increases (in some cases, total cell number can be double).Relevant therewith is total T cell number increase of 6/9 patient.At CD4 ⁺In the subgroup, this growth is more obvious, has 8/9 patient to show the CD4 of rising ⁺The T cellular level.The trend of CD8+ subgroup does not clearly have 4/9 patient's level to increase to some extent, though in general the degree of Sheng Gaoing also less than CD4 ⁺The T cell.

LHRH treats the influence to T cell subsets ratio:

Blood samples of patients before and after the LHRH agonist treatment be the analysis showed that T cell, CD4 ⁺Or CD8 ⁺The toatl proportion of T cell does not have significant change, and CD4 ⁺: CD8 ⁺Ratio has different change (Figure 24) after treatment.This shows, although the T total cellular score is significantly increased after the treatment, treatment does not influence homeostatic the keeping of T cell subsets.All data compare with control value.

LHRH treats the influence to B cell and bone marrow sexual cell ratio:

Ratio to B cell in patient's peripheral blood of accepting the LHRH agonist treatment and bone marrow sexual cell (NK, NKT and macrophage) is analyzed, and shows that there is change (Figure 25) in various degree in each subgroup.Though the ratio of NK, NKT and macrophage is relative remaining unchanged after treatment, the ratio of B cell then descends in 4/9 patient.

The LHRH agonist treatment is to the influence of B cell and bone marrow sexual cell sum:

The B cell and the bone marrow sexual cell sum for the treatment of in the peripheral blood of back are analyzed clear treatment back NK (5/9 patient), the NKT (4/9 patient) of showing of analysis result. and the cell number of macrophage (3/9 patient) obviously increases (Figure 26).The B cell quantity does not have significant change trend: 2/9 patient's level increases, and 4/9 patient's no change also has 3/9 patient's level to reduce.

LHRH treatment is to the influence with respect to the pure cellular level of memory cell:

In main variation seen behind the LHRH agonist treatment is in peripheral blood T cell mass.Particularly, the ratio of pure (CD45RA+) CD4+ cell optionally raises, and the pure cell (CD45RA+) in the CD4+T cell subsets increases (Figure 27) to the ratio of memory cell (CD45RO+) in 6/9 patient simultaneously.

Conclusion

Therefore such conclusion can be drawn,, the activation again of thymus can be induced with LHRH agonist treatment animal the mankind of atrophy thymus (as have).The patients with prostate cancer of acceptance steroid ablation, the lymphocytic state of its blood T demonstrates overall improvement.Though judge accurately whether these cells only come from thymus and be difficult to, and this is the result who carries out logical reasoning, because also there is not description about main flow (CD8 α β chain) the T cell in other sources.Gastrointestinal tract T cell mainly is TCR γ δ or CD8 α α chain.

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Claims (28)

1. sex steroid antagonist and sex steroid agonist are used for preventing or improving the application that is caused the medicine of patient disease by infector in preparation.

2. application according to claim 1, wherein said patient's thymus are to the small part inactivation.

3. application according to claim 2, wherein said suffer from or once suffered from disease or treat disease, and it makes described patient's thymus inactivation to small part.

4. application according to claim 3, wherein said disease are bacillary and viral.

5. application according to claim 4, wherein said virus are influenza virus or herpes simplex virus.

6. sex steroid antagonist and sex steroid agonist are used for improving the application of the medicine of patient's marrow function in preparation.

7. application according to claim 6, wherein said bone marrow productivity ratio are by the generation that increases hematopoietic stem cell and/or discharge enhanced.

8. according to claim 6 or 7 described application, immunosuppressant or gene unconventionality that wherein said patient suffers from long-term infection, cancer, caused by transplanting.

9. according to claim 3,6 or 7 described application, wherein said patient maybe will carry out chemotherapy, radiation treatment or transplanting to disease.

10. application according to claim 9, wherein said transplanting is a bone marrow transplantation.

11. according to the described application of arbitrary claim among the claim 1-5,6 or 7, wherein said patient is postpubertal.

12. according to the described application of arbitrary claim among the claim 1-5,6 or 7, wherein said sex steroid antagonist blocking-up comprises one or more sex steroid receptors of retardance.

13. according to the described application of arbitrary claim among the claim 1-5,6 or 7, wherein said medicine causes described patient's chemical castration.

14. according to the described application of arbitrary claim among the claim 1-5,6 or 7, wherein said medicine comprises LHRH-R agonist and LHRH-R antagonist.

15. application according to claim 14, wherein said sex steroid agonist are selected from the group of being made up of buserelin, plug holder Rayleigh, Decapepthl, deslorelin, gonadorelin, goserelin, histrelin, leuproside, leuprorelin, lutrelin, meterelin, nafarelin, triptorelin and leuprorelin acetate.

16. application according to claim 14, wherein said sex steroid antagonist is selected from the group of being made up of flutamide, 1: PN: WO02056903 PAGE: 25 claimed protein and cetrorelix.

17. according to the described application of arbitrary claim among the claim 1-5,6 or 7, the compositions that wherein said medicine further comprises, maybe will give described patient comprises: be selected from one or more pharmaceutical preparatioies in the group that the inhibitor of the sex steroid that is produced by immunosuppressant and adrenal gland forms.

18. according to the described application of arbitrary claim among the claim 1-5,6 or 7, the compositions that wherein said chemical compound further comprises, maybe will give described patient comprises: be selected from one or more medicines in the group of being made up of at least a cytokine, at least a somatomedin or at least a cytokine and at least a combinations of. growth factors.

19. application according to claim 18, wherein said cytokine are to be selected from the group that is situated between element 2, interleukin 7 and interleukin 15 and forms by certainly.

20. application according to claim 19, wherein said somatomedin are to be selected from by epithelium growth factor family member, fibroblast growth family member, stem cell factor, granulocyte colony-stimulating factor, keratinocyte growth factor and the group formed thereof.

21. according to the described application of arbitrary claim among the claim 1-5,6 or 7, the compositions that wherein said medicine further comprises, maybe will give described patient comprises: be selected from the group of being made up of hematopoietic stem cell HSC, CD34+ hematopoietic stem cell, hemopoietic progenitor cell, bone marrow CFU-GM, lymph sample CFU-GM, epithelial stem cell.

22. application according to claim 21, wherein said cell is carried out genetic modification.

23. application according to claim 22, wherein said genetic modification produce the resistance of the infection that causes at infector in described cell and filial generation thereof.

24. application according to claim 23, wherein said infector are virus.

25. application according to claim 24, wherein said virus are to be selected from the group of being made up of HIV, T chronic myeloid leukemia virus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus.

26. application according to claim 23, wherein said genetically modified cell is to by the active of infector and/or duplicate the infection that causes and have to the small part resistance.

27. application according to claim 23, but wherein said genetically modified cell comprises the polynucleotide that can cause or induce the resistive stably express of one or more infectors.

28. application according to claim 22, but wherein described cell is modified with comprise be selected from by nef transcription factor, HIV gene specific ribonucleic acid constitute the anti-dominant mutant, RevM10, HIV-1 rev response element of enzyme, HIV-1 rev gene, RNA inhibitor that HIV duplicates and the group formed in the polynucleotide of stably express.

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